MASARYK
U N I V E R S I T Y
F A C U L T Y O F M E D I C I N E
Understanding of tooth
developmental trajectories as a novel
approach for dental regeneration
Doctoral thesis
Mgr. Josef Lavický
Supervisor: doc. Mgr. Jan Křivánek, Ph.D.
Department of Histology and Embryology
Programme Biomedical sciences
Brno 2025
MUNI
M E D
U N D E R S T A N D I N G O F T O O T H D E V E L O P M E N T A L T R A J E C T O R I E S A S A N O V E L
A P P R O A C H F O R DENTAL R E G E N E R A T I O N
Bibliographic record
Author:
Title of Thesis:
Mgr. Josef Lavický
Faculty of Medicine
Masaryk University
Department of Histology and Embryology
Understanding of tooth developmental
trajectories as a novel approach for dental
regeneration
Degree Programme: Biomedical sciences
Supervisor:
Year:
Number of Pages:
Keywords:
doc. Mgr. Jan Křivánek, Ph.D.
2025
223
Tooth development, dentin, dentinogenesis,
odontoblasts, odontogenesis, stem cells, cell
differentiation bioengineering, mineralization,
scRNA-seq, transcriptomics
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U N D E R S T A N D I N G O F T O O T H D E V E L O P M E N T A L T R A J E C T O R I E S A S A N O V E L
A P P R O A C H F O R D E N T A L R E G E N E R A T I O N
Annotation
Teeth are hard tissue structures used for food processing and social interactions.
While their importance cannot be overstated, their regeneration
potential is limited. One of the emerging possibilities that would
enable dental regeneration is the artificial recapitulation of development.
This approach is propelled by complex developmental trajectories
generated by contemporary bioinformatic approaches. However, the
success of developmentally inspired approaches is predicated on indepth
knowledge of the histological microstructure of specific dental tissues,
as well as an understanding of their origins. In summary, this thesis
provides insight into dental development and highlights potential avenues
for mediating dental regeneration using fundamental knowledge of
the development.
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A P P R O A C H F O R D E N T A L R E G E N E R A T I O N
Abstract
Introduction
Teeth are hard tissue structures located at the start of the gastrointestinal
system of most vertebrates. They are used for food processing and
social interactions. This makes them indispensable for the survival of the
individual and also significantly influences the quality of life. While their
importance cannot be overstated, their regeneration potential is limited.
Moreover, no targeted biomedical treatments for mediating dental regeneration
are currently available. One of the emerging possibilities that
would enable dental regeneration is the artificial recapitulation of development.
This approach is propelled by complex developmental trajectories
generated by contemporary bioinformatic approaches. However,
the success of the developmentally-inspired approaches is predicated on
in-depth knowledge of the histological microstructure of specific dental
tissues as well as an understanding of their origins.
Methods
The publications included in this thesis utilize a wide range of methodologies.
These include brightfield and fluorescence microscopy with advanced
imaging techniques like confocal and lightsheet microscopy. Various
histological staining techniques were utilized, ranging from conventional
stainings, immunohistochemistry to in situ hybridization. Approaches
for studying gene expression like RT-qPCR, were utilized as
well. Moreover, pluripotent stem cell culture, as well as the generation
of induced pluripotent stem cells, was performed. In addition, extensive
experiments using transgenic mouse strains were performed. Finally,
approaches for studying mineralized tissues were utilized ([J.CT, LIBS).
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U N D E R S T A N D I N G O F T O O T H D E V E L O P M E N T A L T R A J E C T O R I E S A S A N O V E L
A P P R O A C H F O R D E N T A L R E G E N E R A T I O N
Results
The results include the development of a new protocol for isolating single
cells from dental pulps for the purposes of performing scRNA-seq, as
well as a new methodology for monitoring the spatiotemporal dynamics
of mineralized tissues. Moreover, we also shed light on how several features
of teeth emerge during development. These include how the dentin
microstructure is controlled by the type of adjacent dental epithelium,
as well as how the enamel decussation patterns are formed. Finally, we
directly utilize the knowledge of the odontoblast developmental trajectory
to guide the differentiation of pluripotent stem cells into odontoblast-like
cells via the overexpression of selected transcription factors.
Conclusion
We have generated new approaches for investigating dental development.
Furthermore, we have expanded the current understanding of
dental development by elucidating mechanisms driving the emergence
of specific dental features. Finally, we have performed a proof of concept
study for the direct utilization of developmental trajectories as a means
of guiding the differentiation of pluripotent stem cells.
In summary, this thesis provides insight into dental development
and highlights potential avenues for mediating dental regeneration using
fundamental knowledge of the development.
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A P P R O A C H F O R D E N T A L R E G E N E R A T I O N
Acknowledgements
I would like to thank all who helped me along this PhD journey. First
and foremost, I thank my supervisor, Jan Křivánek, for his expert and
passionate guidance throughout these years. I will always cherish our
creative sessions, during which we had excitedly discussed fresh data
and planned further experiments. Moreover, I will always appreciate
both the life and scientific lessons he provided me with, as they have
contributed to the person I am today.
I would also like to thank all the members of the Křivánek Lab. Particularly
to Marcos, Vladislav and Míša for the help and kindness they
have shown me.
Similarly, I would like to thank both the current and past members
of the Department of Histology and Embryology, in which I had the
pleasure of working for no less than ten years. I will always look back on
this time with many fond memories, as the community of people working
there was always exceptionally friendly and kind. I hope that during
my time there, I was able to give at least a portion of this kindness back.
On the same note, I would like to thank the head of the department,
Aleš Hampl, for cultivating this positive atmosphere. I am thankful for
his very kind support over the years, as well as for reminding me to visit
a barber from time to time.
Next, I would like to thank Kuba for his early mentorship and aptitude
for absurd humour; Mario for his quick wit and no-nonsense attitude;
Vašek for his good-heartedness and for competing with me in the
contest of who can make the silliest joke, though there never were any
winners in this contest. Moreover, I would like to thank Denisa, Vincent,
Klára, Andrea and Fran for letting me gobble up their sweets. I would
also like to thank Matea for her fabulous general support. And above all,
I am grateful for your friendship, which I will always treasure.
Šablona DP 3.5.2-MED-dis-english-B5 (2025-04-14) © 2014, 2016, 2018-2025 Masarykova univerzita 7
U N D E R S T A N D I N G O F T O O T H D E V E L O P M E N T A L T R A J E C T O R I E S A S A N O V E L
A P P R O A C H F O R D E N T A L R E G E N E R A T I O N
More importantly, I would like to thank my parents, Josef and Hana,
as well as my sister Petra, who have always supported me through thick
and thin. I am forever grateful to them for always being there for me
when I needed someone to confide in, whatever the occasion. Moreover,
I am thankful for the wisdom and aid they have offered me over the
years. I could not have done it without you.
Most importantly, I would like to thank my significant other,
Kateřina, for her limitless compassion that gave me the strength to push
through the darker days and for helping to put everything into perspective.
It is hard for me to express how much I adore you. I would also like
to thank you for telling me that the idea of putting some quotes from my
favourite books to the end of this acknowledgement is silly and a little
bit cringe. However, while I agree with that, you should know by now
that I am silly and more than a little bit cringe. Therefore, the only things
that are left to be said are that Not all who wander are lost. And that during
my PhD, I have learned to put Life before death, strength before weakness,
journey before destination. Since it is important to remember that:
The shore gives way to the sea. And the sea, my friends, does not dream of
you. Finally, there is nothing more left to do than to Let the Dragon ride
again on the winds of time.
Thank you all.
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T A B L E O F C O N T E N T S
Table of Contents
Abstract 4
List of Figures 11
Glossary 12
List of Appendices 14
1 Introduction 15
1.1 Anatomical and histological structure of the tooth 16
1.2 Tooth development 33
1.3 In vitro differentiation towards odontogenic cell lines using the
conventional knowledge of development 38
1.4 Utilization of developmental trajectories in (re)generation of
dental tissues 41
2 Goals of the dissertation 46
3 Results 47
3.1 Commentary for: Rapid isolation of single cells from mouse
and human teeth 47
3.2 Commentary for: Spatiotemporal monitoring of hard tissue
development reveals unknown features of tooth and bone
development 48
3.3 Commentary for: The Development of Dentin Microstructure Is
Controlled by the Type of Adjacent Epithelium 50
3.4 Commentary for: Unraveling the Transcription Factor Code of
Odontoblast Differentiation 53
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T A B L E O F C O N T E N T S
4 Discussion 56
5 Conclusion 61
Summary of publications and research activities 63
Publications 63
Research activities related to the thesis 67
Bibliography 71
Appendix A Rapid isolation of single cells from mouse and
human teeth 90
Appendix B Spatiotemporal monitoring of hard tissue
development reveals unknown features of tooth and bone
development 103
Appendix C The Development of Dentin Microstructure Is
Controlled by the Type of Adjacent Epithelium 121
Appendix D Unraveling the Transcription Factor Code of
Odontoblast Differentiation 142
Appendix E Front page image of Journal of Bone and Mineral
Research, volume 37, February 2022 219
Appendix F Front page image of Cell Reports, volume 42,
November 2023 221
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LIST O F F I G U R E S
List of Figures
Figure 1: Anatomical overview of brachydont, hypselodont and
hypsodont teeth 17
Figure 2: Overview of the apical area of the mouse incisor and its stem
cell areas 19
Figure 3: Microstructure of murine ameloblasts and enamel 21
Figure 4: An overview of dentin microstructure 24
Figure 5: The comparison of enamel- and cementum-facing dentins in
the molar and incisor from DSPPCeruluean
;DMPlcherr
y mouse strain 26
Figure 6: Overview of the differences between the peritubular and
intertubular dentins 28
Figure 7: An example use of the developed methodology for monitoring
the dynamics of mineralized tissues 30
Figure 8: Comparison of reactionary (A) and reparative dentin (B) 32
Figure 9: Simplified overview of mouse dental development 35
Figure 10: Developmental similarities between various ectodermal
appendages 37
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G L O S S A R Y
Glossary
H.CT Micro-computed tomography
BEE-ST Bones and tEEth Spatio-Temporal growth monitoring
BMP Bone morphogenetic protein
CL Cervical loop
CreRT2 Cre recombinase fused to a mutant estrogen ligandbinding
domain
DAPI 4',6-diamidino-2-phenylindole
DEJ Dentin enamel junction
DPSCs Dental pulp stem cells
E Embryonic day
EAs Ectodermal appendages
EDA Ectodysplasin A
EK Enamel knot
ERM Epithelial rests of Malassez
FACS Fluorescence-activated cell sorting
FGF Fibroblast growth factor
HERS Hertwig's epithelial root sheath
HH Hedghehog
hPSC Human pluripotent stem cells
IHC Immunohistochemistry
LaCL Labial cervical loop
LIBS Laser induced breakdown spectroscopy
LiCL Lingual cervical loop
MD Mandibular
mPSCs Mouse pluripotent stem cells
MX Maxillary
NCSCs Neural crest stem cells
OP(s) odontoblast processes]
ORF Open reading frame
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G L O S S A R Y
PDL Periodontal ligament
R26 ROSA26 locus
scRNA-seq Single cell RNA sequencing
sEK Secondary enamel knot
SEM scanning electron microscopy
TF(s) Transcription factor(s]
TGFß Tumor growth factor ß
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LIST O F A P P E N D I C E S
List of Appendices
Appendix A Rapid isolation of single cells from mouse and human
teeth 90
Appendix B Spatiotemporal monitoring of hard tissue development
reveals unknown features of tooth and bone development
103
Appendix C The Development of Dentin Microstructure Is
Controlled by the Type of Adjacent Epithelium 121
Appendix D Unraveling the Transcription Factor Code of
Odontoblast Differentiation 142
Appendix E Front page image of Journal of Bone and Mineral
Research, volume 37, February 2022 219
Appendix F Front page image of Cell Reports, volume 42, November
2023 221
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INTRODUCTION
1 Introduction
Teeth are hard tissue structures located at the start of the gastrointestinal
system of most vertebrates. While being initially used predominantly
in food processing, their function has evolved to also include social interactions,
and in some specific cases, even digging1 2
. Their function,
therefore, makes them indispensable for the survival of the individual in
most vertebrate species and significantly influences the quality of life in
humans. Furthermore, the wide range of requirements for teeth's function
has resulted in specific adaptations of teeth among different species.
While their morphology, structure, and composition can vary between
the different adapted species, the molecular pathways controlling their
development are highly conserved3 - 5
. This makes the knowledge concerning
their development, regeneration, and homeostasis obtained using
model organisms generally applicable to human biology and medicine,
which is currently unable to resolve the limited regenerative potential
of human teeth by targeted biomedical therapies6
The model organism of choice for studying teeth is the house mouse
(Mi/5 musculus). Apart from the general benefits of this model organism
(e.g., short reproductive time, wide range of genetically modified strains,
etc.), the mouse also possesses a continuously growing (hypselodont]
incisor. Due to its ever-renewing characteristic, this incisor recapitulates
most stages of the tooth development along its sagittal axis at any moment7
- 9
. This makes it ideal for studying tooth development as all the
different stages contained within the incisor would otherwise require
multiple specimens from discrete embryonic timepoints. Furthermore,
the renewing nature of the incisor also results in a continuous cytodifferentiation
of both mesenchymal and epithelial stem cells into their specialized
tooth-forming progeny7 9
. The continuous cytodifferentiation
can then be exploited by contemporary transcriptomic and bioinformatic
approaches to generate developmental trajectories of the separate
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INTRODUCTION
cell populations, which provide an unprecedented insight into the inner
workings and interactions that take place during tooth development9
-10
.
These developmental trajectories can then be utilized as a resource
for guiding the differentiation of stem cells into specific cell types, with
the potential for facilitating dental regeneration. Nevertheless, the success
of the developmentally-inspired approaches is predicated on the indepth
knowledge of the histological microstructure of the corresponding
dental tissues as well as the understanding of their origins.
1.1 Anatomical and histological structure of the tooth
Although the morphology of teeth among different species and even
tooth types in one given species can take various forms, the general anatomical
structure remains mostly the same. Human teeth and mouse
molars are both termed brachyodont (Figure 1A). This reflects the fact
that their crowns are shorter than their roots in comparison to the highcrowned
(hypsodont) teeth found, for example, in ruminants (Figure
1C). For the purposes of this thesis, the anatomical structure of a noncontinuously
growing (brachydont) molar and a continuously growing
(hypselodont) incisor (Figure IB) is described.
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INTRODUCTION
A Brachydont B Hypselodont C Hypsodont
Figure 1: Anatomical overview of brachydont (A), hypselodont (B) and hypsodont
(C) teeth. Original image; modified from Lavický etal. (2022)1 1
. The visualization
of the hypsodont tooth was inspired by an equine tooth diagram from Dacre et al.
(2008)1 2
.
Facing the oral cavity, the crown of the tooth provides a series of
occlusal surfaces and dental cusps that are directly used for processing
food. The crown is covered with enamel - a hard tissue that serves as
a protective and functional layer of the tooth1
. Embedded into the dental
alveolus, the tooth's root anchors it into the jaw. The root is covered with
cementům, which mediates the tooth's fixation into the dental socket by
its interactions with the periodontal ligaments1 3
. Furthermore, the root
also provides an entry point into the dental pulp for blood vessels and
nerves1 4
. Between the crown and the root, the cemento-enamel junction
is located. While the dental crown is covered with enamel and the root
with cementům, the majority of the tooth is made up of dentin. Inside
the tooth is a pulpal cavity filled with dental pulp. The dental pulp, along
with dentin form the dentin-pulp complex, which is responsible for the
maintenance of homeostasis of the tooth1 4
.
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INTRODUCTION
In addition to brachydont molars, the mouse also possesses continuously
growing (hypselodont) incisors, which, while having a different
morphology, still reflect some aspects of the brachydont teeth (Figure
1). In the mouse incisor, the concepts of dental crown and root are not
applicable due to its shape and orientation. Nevertheless, separate areas
covered with either enamel or cementum are present in the incisor. To
reflect their functions and developmental origins, the terms crown analogue
and root analogue have been generally used (Figure I B ) 8 1 1
. The
crown analogue, also covered in enamel, is located on the outward-facing
portion of the incisor and, similarly to the brachydont teeth, serves
to provide a strengthened occlusal area used for gnawing. The root analogue
then mediates the fixation of the tooth inside the dental alveolus.
The emergence of the continuously growing nature of the mouse incisor
is connected to a specific dietary strategy that focuses on the processing
of hard foods using gnawing. Due to the constant gnawing, the
tips of the incisors get continuously abraded, which creates the need for
the continuous regrowth of the tooth. The tooth's renewal is driven by
both mesenchymal and epithelial stem cell niches located in the proximal
(apical) part of the tooth (Figure 2)8
.
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INTRODUCTION
Enamel
Figure 2: Overview of the apical area of the mouse incisor and its stem cell areas.
LaCL - labial cervical loop, LiCL - lingual cervical loop. Original image; partially modified
from Lavický etal. (2022)1 1
, visualization of apical area inspired by Křivánek et al.,
(2020)9
.
Nevertheless, the critical function of both continuously and noncontinuously
growing teeth, apart from their morphology, is also enabled
by the intricate inner structure of the specific dental tissues.
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INTRODUCTION
1.1.1 Enamel
This tissue provides the tooth with a protective layer, which helps to
shield the sensitive dentin from mechanical stimuli, as well as chemical
and biological agents such as changes in pH and bacteria1
. Epithelial cells
of ectodermal origin termed ameloblasts, produce enamel during embryonic
development, making it the only mineralized tissue produced by
epithelial cells1 5
. During the tooth's eruption, the ameloblastic layer is
lost and, therefore, enamel cannot be naturally regenerated or repaired
after the tooth's eruption. Enamel itself is the hardest mineralized matrix
found in vertebrate bodies, composed of more than 95% inorganic
material (mostly hydroxyapatite), with the rest being organic material
(structural, calcium-binding phosphoproteins1 6
) and water1 5
.
Barring the high amounts of mineralized material, it is the inner microstructure
of mammalian enamel that enhances its unique mechanical
properties. Enamel in amphibians and the majority of reptiles displays
a simple structure of hydroxyapatite crystallites that are oriented perpendicularly
to the enamel's surface. This type of enamel is named
aprismatic1 5
'1 7
. Conversely, in mammals, the majority of the enamel is
prismatic, apart from a thin outer layer of aprismatic enamel (Figure
3)1 8
. The prismatic nature of the mammalian enamel originates from the
fact that during amelogenesis, mammalian ameloblasts form Tomes's
processes, which enable the formation of discrete enamel rods
(prisms) (Figure 3A)1 5
. Remarkably, by intertwining these enamel
prisms, complex decussation patterns can be formed (Figure 3B). The
presence of these intertwined prisms in the mammalian enamel has
been widely accepted to have evolved to minimize the formation and
propagation of fractures. Thus, making the prismatic enamel more durable
when compared to the aprismatic enamel1 9 2 1
.
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INTRODUCTION
Figure 3: Microstructure of murine ameloblasts and enamel. Morphology of secretory
ameloblasts (A) on a phalloidin-stained section showing the presence of Tomes'
processes (arrowheads). (B) Scanning electron microscopy image of mature enamel
and ameloblasts incisor, displaying the differences in the microstructure of the
aprismatic and prismatic enamel. SEM - scanning electron microscopy. The incisor diagram
shows approximated locations of the images in panels A and B. All scale bars =
20 |im. Original data; the incisor visualization modified from Lavický etal. (2022)1 1
.
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INTRODUCTION
Interestingly, the decussation patterns among species display significant
differences20
-22
, suggesting their adaptation to different dietary
strategies or other ethological specifics. While the manner in which the
formation of these patterns is achieved has been theorized upon2 3
, its
precise mechanism has not yet been described.
Relation to my research activities
Using a lineage tracing of rare dental epithelial stem cells, we were
able to, for the first time, prove the mechanism behind the emergence of
the enamel decussation patterns. The genetic lineage tracing revealed
that the decussation pattern emerges as a result of organized shuffling
of the individual ameloblasts during enamel production (Rakultsev et al.,
under review). Our results, together with the existence of species-specific
enamel decussation patterns, suggest that this intra-epithelial
movement of individual ameloblasts is precisely regulated by molecular
signalling that still awaits uncovering.
1.1.2 Dentin
In contrast to enamel, dentin is a living tissue even after the tooth erupts.
Odontoblasts, cells of neural crest origin, start to produce dentin during
embryonic development and continue to do so, while the tooth remains
viable1 4
. The odontoblasts form an organized layer of polarized cells located
on the border between dentin and dental pulp, forming the socalled
dentin-pulp complex (Figure 4A,B)2 4
. Despite the epithelium-like
morphology of their layer, odontoblasts are mesenchymal and therefore,
do not express epithelial markers (CDH1, KRT14, etc.)9 1 0
and do not
have a basal lamina2 5
. During dentin production, each odontoblast
leaves behind a single cellular process (also known as Tomes' fibre]
which persists embedded inside dentin tubules (Figure 4B,D). These
processes then serve a key function in maintaining tooth homeostasis1
4
'2 4
.
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INTRODUCTION
Dentin itself is a hard matrix containing around 70% of mineral content,
20% of organic components, and water1 4
'2 6
. It is produced by
a gradual mineralization of a protein matrix secreted by the odontoblasts.
This precursor matrix, termed as predentin, is mostly composed
of collagen type I and several non-collagenous proteins (Figure 4B,C,D).
Dentin acidic matrix protein 1 (DMP1) and dentin sialoprotein (DSP, encoded
by the Dspp gene) are two major non-collagenous proteins found
in dentin1 4
. While DMP1 is expressed in several other cell types known
for producing hard tissues (osteoblasts, cementoblasts etc.), DSP is specific
predominantly to odontoblasts. Since these two proteins participate
in the mineralization of the secreted predentin, the co-expression
of these two proteins (apart from typical morphology) serves as a reliable,
widely used marker of the odontoblast(-like) phenotype (Figure
4A)2 7
.
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INTRODUCTION
Figure 4: An overview of dentin microstructure. Image of dentin from DSPPCeru,ean
;
DMPlCherr
y mouse strain (A) illustrating the expression of marker genes DSPP and
DMP1 in odontoblasts. (B) Phalloidin-stained section from a wildtype animal illustrating
the difference in the morphology of odontoblast processes between predentin and
dentin. Highlighted Areas approximate the location of the following SEM images. Scanning
electron microscopy (SEM) images of predentin (C) and dentin (D) illustrating the
differences in the microstructure of the (pre)dentin matrix. All scale bars = 30 |im. Original
data, panel A is adapted from Lavický et al. (2022)1 1
.
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INTRODUCTION
Dentin can be divided into several types based on its location, structure,
and the timing of its production. Dentin is produced in an inward
manner, towards the centre of the pulpal cavity. Furthermore, dentin is
not remodelled, which results in the (developmental) history of the
tooth being recorded from the outermost to the innermost part of dentin.
This reality makes the morphology of the dentin tubules and the
odontoblast processes within them an interesting subject for studying
hard matrix development.
Based on the location of the dentin, it can be divided into the
enamel-facing (crown) dentin and cementum-facing (root) dentin1 1
.
These two types of dentin differ in several parameters, such as phosphoprotein
composition, mineralization, and mechanical properties1 1
. Interestingly,
while it could hold important information concerning the differences
in development of two dentins, the morphology of odontoblast
processes located within has been generally poorly investigated.
Relation to my research activities
To fill this research niche, we have described the differences between
the enamel- and cementum-facing dentins. Using the DSPPCe
-
ruiuean.DMPlcherr
y mouse strain, we have also described the similarities
between the crown dentin in the molar and labial dentin in the incisor,
as well as the root and lingual side dentins. Furthermore, this highlighted
their analogous nature (Figure 5). In addition, this suggests that
the dentin microstructure is controlled by the type of adjacent epithelium
during development. This research is discussed further in the text,
and the original publication is included as Appendix C.
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INTRODUCTION
Dentin structure in molar and incisor: root (analogue) vs. crown (analogue)
G Enamel-facing dentin
Crown (analogue)
Enamel
Cementum-facing dentin
Root (analogue)
Figure 5: The comparison of enamel- (B,E) and cementum-facing (C,F) dentins in
the molar (A) and incisor (D) from DSPPCeruluean
;DMPlCherr
y mouse strain.
26
INTRODUCTION
Arrowheads highlight the presence of DMPl-expressing cementoblasts. (G) Graphical
summary of the findings. Ab - alveolar bone; De - dentin; En - enamel; Ob - odontoblasts;
P - pulp. Scale bars = 300 |im (A, D), 100 |im (B, C, E, F). Reproduced from
Lavický et al. (2022)1 1
Apart from this, the enamel-facing dentin then displays distinct
functional features that are not present in the cementum-facing dentin.
1.1.2.1 Features exclusive to enamel-facing dentin
The outermost and ontologically first produced part of crown dentin, directly
adjacent to enamel, is termed the dentin-enamel junction (DEJ). It
is a distinct, few microns-thick layer whose function is to attach enamel
to dentin2 8 2 9
. For this purpose, numerous collagen fibrils bridge the interface
to connect them3 0
. Furthermore, the DEJ has been shown to contain
grooves (scallops) that increase the contact surface between the
two layers, which likely further improves the connection between
them3 1
. Interestingly, the density of these scallops was, in humans, found
to be dependent on the type of tooth, suggesting at a molecular mechanism
driving these differences31
.
Directly underneath the DEJ, the mantle dentin is located. It represents
an early product of odontoblasts, as it generally does not contain
dentinal tubules yet, only their secondary branches. Furthermore, unlike
the rest of the dentin, mantle dentin contains a large amount of collagen
III and reduced amounts of phosphoproteins3 2 3 3
. Interestingly, this does
not seem to lead to reduced mineralization3 4 3 5
. Nevertheless, mantle
dentin displays lower microhardness than the underlying circumpulpal
dentin3 6 3 7
. This reality might be explained by a lower number of dentinal
tubules3 8
, which then results in the lower amount of a harder, peritubular
dentin. Overall, it has been hypothesized that the lower microhardness
of mantle dentin serves to mitigate the mechanical forces applied
to teeth and therefore helps to prevent the formation of fractures
in both enamel and dentin2 8
.
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INTRODUCTION
1.1.2.2 Circumpulpal dentin
The underlying so-called circumpulpal dentin forms the remainder of
the crown and all of the root. It displays the typical dentin characteristics
as described above. It can be further divided into the peritubular dentin,
which is in direct vicinity of the dentinal tubules, and intertubular dentin,
which is located between the tubules (Figure 6). Due to its contact
with the odontoblast processes, peritubular dentin displays a different
composition of protein components3 9
and increased microhardness
than the intertubular dentin3 7
.
Figure 6: Overview of the differences between the peritubular and intertubular
dentins. (A) SEM image of a coronal dentin section. Reproduced from Wagermaier and
Fratzl (20 12)4 0
. (C) Detail SEM image of a sagittal dentin section highlighting the differences
between the two dentins. Reproduced from Berg et al. (2021)4 1
.1 - intertubular
dentin; P - peritubular dentin. Scale bars = 20 |im (A); 5 |im (B)
The circumpulpal dentin can be further divided into primary, secondary,
and tertiary dentin. The precise distinction between primary
and secondary dentin is a matter of scientific discussion2 8
. Nevertheless,
the type of dentin that determines the shape of the tooth during tooth
development is termed the primary dentin2 8
. The production of the
28
INTRODUCTION
secondary dentin is slower. Generally, it starts after the tooth's eruption
and continues while the tooth remains viable.
In the past, the measurements of the speed to dentin production
were limited by the available methodologies. These are methodically arduous
and do not support performing the analysis in three dimensions,
which makes them susceptible to section plane orientation bias4 2 - 4 5
. The
lack of suitable methodologies hindered research focusing on the developmental
dynamics of the tooth as well as of other mineralized tissues.
Relation to my research activities
To expand the repertoire of current approaches for monitoring the
dynamics of mineralized tissues, we have developed a new methodology
by combining the usage of mineralized tissue-intercalating dyes and a
protocol for optical clearing of mineralized tissues. This new methodology
is significantly less laborious than the previous approaches. Furthermore,
it supports the imaging of wholemount samples, which enables
the visualization of the dynamics in three dimensions. In addition, it is
non-destructive, which enables the combination with conventional volumetric
analyses via [J.CT and others 4 6
. Using this methodology, we have,
for example, analysed the speed of primary and secondary dentin production
in all three mouse molars (Figure 7)4 6
. This research is discussed
further in the text, and the original publication is included as Appendix
B.
29
INTRODUCTION
C AC
Harvest
-o
P12 P15 P18 P21 P24 P27 P30 P31
Mandible Whole-mount projection
Figure 7: An example use of the developed methodology for monitoring the dynamics
of mineralized tissues. Whole mount images of isolated P30 mouse molars
injected with calcein and alizarin at several timepoints starting from P12. Image reproduced
from Gonzalez-Lopez et al. (2 02 3)4 6
.
In addition, recent evidence suggests that the production of secondary
dentin is accelerated in specific, occlusal areas of the tooth, likely to
compensate for increased mechanical stress4 7
. It is important to note
that this acceleration happens independently of the tooth's attrition and
is therefore distinct from the last type of dentin - the tertiary dentin4 7
.
1.1.2.3 Tertiary dentin
In contrast to enamel, dentin can be repaired by the odontoblasts, albeit
only to a limited degree. Reactionary, tertiary dentin is produced by
odontoblasts when their processes detect signals signifying damage to
30
INTRODUCTION
the dentin in the specific area (e.g., shallow dental caries). This leads to
a thickening of the dentin wall in the compromised area. This is to prevent
a possible breach of the dentin enclosure, which would result in the
inflammation of the dental pulp and subsequently the loss of the
tooth(Figure 8A)4 8
. The initiation of tertiary dentin production is enabled
by the fact that odontoblasts express a wide range of receptors capable
of detecting both mechanical (e.g., Piezo and TRPV receptors)4 9 - 5 1
,
chemical signals (e.g., changes in pH via TRPA1)5 2
, as well as pathogens
(e.g., Toll-like receptors)5 3 5 4
.
In the case that the damage to the dentin is significant enough to
result in the death of a portion of the odontoblast layer (e.g., deep dental
caries), reparative dentin is formed instead, to block pathogens from entering
the pulp6 2 8
. It is a subtype of tertiary dentin produced by odontoblast-like
cells that differentiate from mesenchymal stem cells found in
the dental pulp6
'5 5
'5 6
. The resulting new thin layer of dentin (also known
as dentin bridge) lacks dentinal tubules (Figure 8B)6 2 6
. Nevertheless,
this reparation is very limited and capable of sealing only small lesions.
Currently, there are no treatments capable of mediating tooth repair
apart from filling the lesion with inorganic materials5 7
. No specific biomedical
treatments are available.
31
INTRODUCTION
Figure 8: Comparison of reactionary (A) and reparative dentin (B). Staining images
reproduced from Neves et al. (2017a)4 8
and Neves at al. (2017b)6
.
1.1.3 Dental Pulp
Odontoblasts, while they produce and maintain dentin, are technically
part of the dental pulp. It is a specialized tissue located in the inner compartment
of the tooth, termed the dental cavity. Odontoblasts and pulp
are often grouped together as the dentin-pulp complex, since this correctly
highlights the fact that they are functionally integrated. The pulp
is a soft tissue that is highly vascularized and densely innervated via the
apical foramina located on the tips of the dental roots5 8
. In general, the
pulp maintains homeostasis of dentin via providing nutrients, immune
32
INTRODUCTION
support5 9
, and providing stem cells to mediate dentin repair as was mentioned
above6 5 6
. Furthermore, due to its innervation, dental pulp assures
that the signals (nociception, pain, etc.) from odontoblasts as well
as sensory neurons is properly processed5 8
.
In the mouse incisor, the populations of resident stem cells are significantly
expanded, as the apical part of the tooth contains several stem
cell niches that drive the tooth's renewal via continuous cytodifferentiation,
recapitulating tooth development (Figure 2)7 - 9
.
1.2 Tooth development
The development of teeth is a process driven by the reciprocal interaction
between ectodermal dental epithelium and the underlying, neural
crest-derived, ectomesenchyme (Figure 9). Teeth primordia emerge on
the oral sides of the first branchial arch6 0
. The oral epithelium determines
the odontogenic potential of the ectomesenchyme by expressing
ligands belonging to several distinct families. These are Fibroblast
growth factor (FGF), Tumor growth factor beta/bone morphogenetic
protein (TGFp/BMP), Hedgehog (HH), Wnt, and EDA6 1
. The details of the
intricate interplay of these signalling pathways are reviewed elsewhere
61-65 For this dissertation, only those relevant to the presented research
are commented on.
One of the initial signals influencing odontogenesis is FGF8. Its expression
originates in the oral epithelium and determines the oral-aboral
polarity of the mandibular and maxillary processes6 0
. This polarity is
might be established by an upregulation of FGF8-downstream effector
Etv466
and downregulation of Gsc60
. In mice, dental development starts
as early as embryonic day 11 ( E l l ) when the oral epithelium produces
localized thickenings known as dental placodes 6 2 6 7
. Interestingly, the
tissue-specific deletion of FgjB at this stage proved to be sufficient to arrest
the formation of dental placodes and, therefore, stop tooth development6
8
. In normal development, the thickened oral epithelium
33
INTRODUCTION
invaginates into the ectomesenchyme and stimulates its cells to condense
around the developing tooth bud. The expression of several transcription
factors, including Salll69
and Runx270
, is typical for this condensed
ectomesenchyme (dental papilla). Later, the dental lamina further
invaginates around the condensed ectomesenchyme, resulting in
the cap stage. This condensed ectomesenchyme later gives rise to odontoblasts
and the dental pulp. The cap stage is followed by the early and
late bell stages, during which the shape of the developing tooth is determined
by the formation of primary and secondary enamel knots, which
are important signalling centres7 1
. At this stage of the tooth development,
the dental lamina stimulates the adjacent dental papilla cells to
differentiate into pre-odontoblasts and later odontoblasts. Pre-odontoblasts,
in turn, stimulate the epithelial cells to differentiate into ameloblasts.
As a result, the crown of the tooth is formed. This process is regulated
by a wide range of ligands and their downstream transcription
factors7 2
.
After the crown's formation, the dental epithelium elongates into
the mesenchyme and forms Hertwig's epithelial root sheath (HERS).
This unique epithelium then controls the formation of the tooth's roots
by stimulating the adjacent ectomesenchyme to also differentiate into
odontoblasts. Later, the HERS disintegrates into epithelial rests of
Malassez (ERM)5
(Figure 9). This means that the odontoblasts forming
the dentin in the crown and root of the tooth have different interacting
partners, which mediate their differentiation via somewhat different signalling
cascades. Those are reviewed elsewhere5
'7 3
'7 4
.
34
INTRODUCTION
E11 E11.5 E12.5
Initiation Epithelial placode Early bud
Epithelium
Mesenchyme
mesenchyme
E13.5 E14.5 E15.5
Late bud Cap Early bell
CL CL
E18.5
Late bell
Figure 9: Simplified overview of mouse dental development. ERM - epithelial rests
of Malassez; CL - cervical loop; HERS - Hertwig's' epithelial root sheath; EK - primary
enamel knot; sEK - secondary enamel knot. Original image, visualization inspired by
Yuand Klein (2020)6 1
.
35
INTRODUCTION
Relation to my research activities
We have shown that these differences in signalling present during dentin
development result in differences between the enamel and cementum-facing
dentins1 1
. These findings are further described in the commentary
section and in the respective publication, which is included as
Appendix C.
The development of the tooth is, most intriguingly, highly similar to
the development of several organs. Those are named ectodermal appendages
- a term that expresses the fact that all of these organs emerge
as a result of an interaction between ectodermal epithelium and underlying
mesenchyme. In mammals, this group consists of the teeth, hairs,
mammary, salivary, and sweat glands7 5
'7 6
.
1.2.1 Similarities with other ectodermal appendages
The development of all ectodermal appendages goes through very similar
initial phases. First, the epithelium forms a placode, which stimulates
the underlying mesenchyme to condense. Subsequently, the Placode invaginates
into the mesenchyme, forming an appendage bud. From this
point, the development diverges based on the concrete type of the developing
organ (Figure 10). More importantly, apart from the morphogenetic
similarities, the development of the appendages is also generally
governed by the same molecular pathways, with shared signalling molecules7
6
'7 7
and even regulatory transcription factors7 8 - 8 1
.
36
INTRODUCTION
Figure 10: Developmental similarities between various ectodermal appendages.
Original image, visualization adapted from Jiménez-Rojo etal. (2012)7 7
.
Due to these similarities, some examples of developmental plasticity
between various types of appendages and their corresponding cell
types have been reported8 2 - 8 6
. The majority of examples were conversions
of the ameloblastic epithelium in the adult mouse incisor to a hair
forming epithelium. These have been induced by several approaches, including
the knockouts of Meal and Sox2184r
-85
. Moreover, this inherent
plasticity was also highlighted while generating mammary gland epithelium
by inducing a cell fate switch in dental epithelial stem cells by recombining
them with mammary mesenchyme8 2
. Most interestingly,
a similar switch from a hair follicle epithelium to a mammary gland epithelium
was also observed in K14-Cre; Smof/f mutant mice with impaired
hedgehog signalling in epithelial cells8 6
. In addition, Sahu et. al.
(2024)8 7
have adapted a protocol for generating hair follicle organoids
from pluripotent stem cells8 8
to generate mammary organoids instead.
Intriguingly, they have done so by modulating the activity of just a few
signalling pathways8 7
.
37
INTRODUCTION
Overall, this suggests that the switch from one ectodermal appendage
fate to another can be achieved by adjusting the expression of singular
genes or only a few external signals. However, when it comes to almost
spontaneous generation of specific cell lines as observed with the
generation of hair follicle and mammary gland lineages, the lack of similar
findings suggests thattransdifferentiation into odontogenic lineages
is not as accessible. Nevertheless, attempts to generate odontogenic cell
lines, most often ameloblast and odontoblasts, are quite numerous as
they hold the promise of facilitating the repair of tissues that otherwise
display very limited natural regenerative potential.
1.3 In vitro differentiation towards odontogenic cell lines
using the conventional knowledge of development
The majority of currently used protocols for differentiating stem
cells were initially generated to reflect some aspects of the development
of corresponding tissues8 9 - 9 3
. This has been done by using specific
growth factors as well as small molecules to modulate the signalling
pathways responsible for stem cell fate decisions. By optimizing the
composition of the culture media to reflect the development more
closely, the protocols were incrementally improved over time9 1
'9 3
.
These utilized insights into development were generally based on
studies of the expression of a few development-associated genes. Moreover,
by performing gene knockouts, the roles of some of the genes were
outlined. Doubtlessly, using this approach, the bedrock of knowledge of
development, as well as artificial differentiation, was laid. In this thesis,
this approach to studying development is referred to as "conventional"
due to its long history and wide usage.
38
INTRODUCTION
1.3.1 Generation of odontoblasts
The first attempts at generating odontoblasts in vitro originate in the differentiation
of isolated dental pulp stem cells (DPSCs) using a cocktail of
small molecules usually used for driving the differentiation of mesenchymal
stem cells into osteogenic lineages9 4
. This cocktail is most often
composed of general pro-mineralization signals like ascorbic acid and
glycerophosphate, as well as synthetic glucocorticoid dexamethasone,
which has been shown to upregulate Runx2, which is an osteogenic master
regulator9 5
. Nevertheless, since DPSCs display a tendency to differentiate
towards odontoblastic lineages rather than osteogenic94
, a population
of odontoblast-like cells is more likely to be generated than osteoblast-like
cells. Albeit the resulting population is likely to contain a significant
fraction of cells with an osteoblast-like phenotype. This uncertainty
can be partially attributed effects of dexamethasone which has
been shown to promote adipogenic differentiation as w e l l 9 6 9 7
. Furthermore,
this uncertainty can be further obscured because the distinction
between osteoblast- and odontoblast-like phenotypes is generally made
based on differing cellular morphology and a specific expression ofDspp
(generally along with Dmpl). However, most publications rely on bulk
expression analysis methods (RT-qPCR and western blot) instead of
methods capable of analysing the co-expression on a cell-to-cell basis
(e.g., IHC, FACS)9 8 - 1 0 0
. In addition, this co-expression can display incomplete
penetrance even in odontoblasts in vivo1 1 2 7
. Due to this, current
publications often label this approach to differentiation of DPSCs as osteogenic/odontogenic9
8 _ l o a
Therefore, attempts to more closely mimic the natural processes
when generating odontoblasts in vitro were conceived. An example of
this involved the usage of specific molecular signals like FGF2 to mimic
the interactions typical for odontoblast development1 0 1
'1 0 2
.
In parallel, other groups have also utilized even more developmentinspired
approaches by initiating the odontoblast differentiation in neural
crest stem cells (NCSCs)1 0 3 - 1 0 5
. Apart from generating odontoblastlike
cells in vitro, these have also validated the role of various signalling
molecules (e.g., FGF8, BMP4, and WNT3A) as well as their interplay
39
INTRODUCTION
during odontoblast differentiation1 0 3 - 1 0 5
. Moreover, while using this approach,
the first odontoblast-like cells from pluripotent stem cells were
generated, albeit not directly1 0 4
'1 0 5
.
1.3.2 Generation of ameloblasts
Instead of utilizing stem cells and their differentiation into ameloblasts,
the scientific community has initially utilized several immortalized ameloblastic
cell l i n e s 1 0 6 - 1 0 8
and primary dental epithelium cultures5
'1 0 9 - 1 1 1
to investigate the mechanisms responsible for the production of enamel
as well as tooth development in vitro.
Interestingly, the first successful attempt at generating ameloblastlike
cells in vitro was performed by directly co-cultivating mouse pluripotent
stem cells (mPSCs) with dental epithelial cells serving as
a feeder layer1 1 2
. This was later streamlined by differentiating pluripotent
stem cells first into ectodermal progenitors and subsequently culturing
them either in ameloblast-conditioned m e d i u m 1 1 3 1 1 4
and/or coculturing
them with dental mesenchyme1 1 3
'1 1 5
.
While providing important leaps in understanding of the differentiation
into dental cell types, all of the above-mentioned differentiation
protocols were based only on approximations of real developmental
events. Therefore, these differentiation protocols likely generate cells
with an incomplete target phenotype and generally struggle with the
generation of cell populations with unwanted heterogeneity93
.
In recent years, a new strategy for guiding the differentiation of
stem cells has emerged. This approach utilizes current fundamental
knowledge of development by harnessing developmental trajectories of
the target cell types generated by transcriptomic approaches.
40
INTRODUCTION
1.4 Utilization of developmental trajectories in
(re)generation of dental tissues
As was previously mentioned, the attempts at producing odontogenic
cell lines have generally utilized the conventional knowledge concerning
the roles of various signalling molecules and transcription factors. These
findings have usually been obtained using knockout mouse strains.
Those studies have utilized the fact that the absence of the specific genes
would impress their importance on (tooth) development by resulting in
arrests or aberrations1 1 6
. This approach, while being essential for obtaining
the basis for understanding tooth development, may also have
obscured significant information. This possibility is predicated on the
presence of potentially hidden, deep effects of the gene knockouts on the
intricate inner workings, which were then not completely extrapolated
from the observed phenotype.
Nevertheless, after the advent of current transcriptomic methods,
the inner machinery driving tooth development and cell differentiation
has been completely unraveled9
'1 0
'1 1 7 - 1 1 9
. However, the unprecedented
volume of gene expression data introduced a new type of problem, as
the complexity of the gene expression patterns is difficult to interpret,
and key regulators are hard to determine.
Nevertheless, this also introduced new possibilities. Cell differentiation
is driven by distinct transcription programs, which are defined by
specific gene expression patterns. During differentiation/development,
the gene expression patterns gradually change. This enables the reconstruction
of developmental trajectories in silico based on the similarity
of gene expression patterns within the individual cells9
'1 2 0
'1 2 1
. The resulting
predicted developmental trajectories can be an essential resource
for investigating the dynamic changes in the transcriptional programs
driving the stem cell fate decisions9
'1 1 9
'1 2 0
. Furthermore, compared
to the conventional methods, the developmental trajectory-based
41
INTRODUCTION
approaches might promise improved differentiation efficiency as well as
a more complete target phenotype9 3
.
During recent years, several attempts at generating specific cell
types using their specific developmental trajectories have been made.
These attempts, however, have been predicated on the ability to isolate
single cells from specific tissues1 2 2
. This, especially in hard tissues, is
a significant hurdle to overcome to generate datasets with the highest
possible quality1 2 3
.
1.4.1 Performing scRNA-seq on dental tissues
Single-cell RNA sequencing (scRNA-seq) is a method used to obtain both
quantitative and qualitative transcriptomic data from individual cells,
isolated from the tissue of interest. To minimize potential gene expression
artifacts caused by either the disassociation process1 2 4
or ischemia1
2 5
, the isolation process needs to be gentle, as well as swift. When it
comes to the isolation of single cells from dental pulp, the process is even
more arduous due to the presence of mineralized tissues (alveolar bone,
enamel, and dentin) that need to be removed before the disassociation
can be performed1 2 6
. For these reasons, the isolation of single cells from
dental pulps needs a highly optimized protocol for both tissue isolation
and following enzymatic disassociation.
Relation to my research activities
To enable the scRNA-seq analysis or other experiments on dental
tissues, we have developed a protocol for isolating single cells from both
human and mouse teeth1 2 6
. The resulting transcriptomic data can be
a powerful resource not only for understanding development but also
for guiding stem cell fate by recapitulating it in vitro. This research is
discussed further in the text, and the original publication is included as
Appendix A.
42
INTRODUCTION
1.4.2 Guiding differentiation using the scRNA-seq dataobtained
knowledge of morphogen expression
One of the generated resources can be the temporal expression patterns
of signalling molecules and their receptors. Based on the interactions between
the ligands and their receptors at different stages of tooth development,
Alghadeer etal. (2023) were able to predict the signalling pathways
responsible for ameloblast differentiation1 2 7
. By targeting the predicted
signalling pathways, they were able to generate ameloblast-like
cells from hPSC-derived ectodermal epithelium. Organoids generated
from these cells then displayed the presence of rosette-like structures
containing the polarized ameloblast-like cells with apically-localized
Ameloblastin (AMBN) and DSP - mirroring the phenotype of secretory
ameloblasts. Apart from generating a protocol for obtaining ameloblasts
in vitro from hPSCs, the authors have also dissected the effects of the
predicted pathways on ameloblast differentiation, further highlighting
the potential of this approach.
Using the identical methodology, Hanson-Drury et al. (2023) were
able to map the signalling during odontoblast differentiation1 2 8
. By activating
FGF, BMP, and HH signalling in a specific temporal pattern, the
authors were then able to enhance the differentiation of hPSC-derived
NCSCs into a population of odontoblast-like cells with a higher expression
of marker genes. More importantly, by successfully replicating the
odontoblast differentiation in vitro, the authors have validated the role
of the signalling pathways, as well as their temporal pattern. Moreover,
by further leveraging the transcriptomic data, authors have described
minute differences in the activity of these pathways in different types of
teeth - likely highlighting their role in tooth morphogenesis1 2 8
.
While cell signalling pathways are instrumental in both development
and cytodifferentiation, these processes are, most importantly,
governed by the activity of specific transcription factors (TFs).
43
INTRODUCTION
1.4.3 Guiding differentiation using the targeted
overexpression of transcription regulators
This approach is based on the inherent capacity of transcription regulators
to guide all manner of cell behaviour, including differentiation. The
core idea is then to overexpress the TFs that are enriched in the target
cell types or are present during their development.
In the past, this approach has been utilized to generate several different
cell types of all germ layers directly from hPSCs1 2 9 - 1 3 2
. This includes
neurons by overexpressing Neurogenin-2 [NGN2]131
-132
, skeletal
myocytes by overexpressing Myoblast determination protein 1
[MYOD1)131
, and hepatocytes by overexpressing four liver-enriched TFs
[HNF1A, HNF6, FOXA3, and RORc)130
. Nevertheless, lineage-specific external
signalling cues were still needed to maintain cell viability and to
complete the differentiation1 2 9 - 1 3 2
. Moreover, the selection of the utilized
TFs was made based on conventional knowledge of the develop-
ment.
In the context of the odontogenic cell lineages, Seki et al. (2015}
have generated odontoblast-like cells by overexpressing Bmp4 and Pax9.
Nevertheless, the authors have selected these two genes based on the
conventional approach as well. Furthermore, the overexpression was initiated
after differentiating mPSCs into NCSCs 1 3 3
.
Nevertheless, the developmental trajectories offer deep insight into
the TFs and the sequence in which they are activated during differentiation.
Therefore, these trajectories are likely an essential tool for selecting
genes to replicate the specific TF code driving tissue development to
guide the stem cell fate into that of the target cell type.
Relation to my research activities
To explore the possibilities of the utilization of developmental trajectories,
we have set out to generate odontoblasts directly from pluripotent
stem cells using the overexpression of specific TFs. We have determined
four transcription regulators [Gsc, Etv4, Salll, and Nuprl).
44
INTRODUCTION
These were selected based on an odontoblast developmental trajectory
that was previously constructed using a mouse incisor scRNA-seq da-
taset9
. By overexpressing these four TFs directly in mPSCs, we were able
to generate cells displaying an odontoblast-like phenotype, without the
use of any growth factors or dexamethasone69
. The details of this research
are discussed further in the text, and the publication is included
in this thesis as Appendix D.
Overall, the referenced research highlights the power of the developmental
trajectory-based approaches for mediating cell differentiation.
These can be used for dissecting the developmental processes as well as
potentially enabling development-inspired therapies for tissue regener-
ation.
45
G O A L S O F T H E DISSERTATION
2 Goals of the dissertation
The goals of this dissertation can be divided into three general topics,
with all of them being focused on improving the understanding of dental
development.
1. The generation of new or refined methodologies that would contribute
to the study of dental development.
2. The investigation of tooth development for understanding the
mechanisms behind the emergence of features critical for the
tooth's function.
3. Direct utilization of the developmental trajectories of odontogenic
cell types to guide the differentiation of pluripotent stem
cells.
46
R E S U L T S
3 Results
During my PhD, I had the opportunity to work on several different projects.
The following section contains the details and commentaries of
publications relevant to the topic and goals of this thesis. The commented
papers are also included in this thesis as appendices
A through D.
3.1 Commentary for: Rapid isolation of single cells from
mouse and human teeth
Citation
Jan Křivánek, Josef Lavický. Thibault Bouderlique, Igor Adameyko.
Rapid Isolation of Single Cells from Mouse and Human Teeth. / Vis
Exp. 2021 Oct28;(176). doi: 10.3791/63043. PMID: 34779437.
Journal of Visualized Experiments (JoVE), IF (2021): 1.424; Q3; WoS
category: MULTIDISCIPLINARY SCIENCES; Citations: 2 (WoS)
This manuscript is enclosed in this thesis as Appendix A.
Commentary
In this publication, we present an optimized protocol for isolating single
cells from dental pulp cells from mouse incisor, molars and human teeth.
This protocol covers the isolation of the murine teeth, the recovery of
the dental pulps, and their subsequent enzymatic digestion. This is followed
up by a general guide for FACS processing for filtering out specific
cell populations.
47
R E S U L T S
The goal of this protocol is to provide readers with an entry point
for performing single-cell RNA sequencing on dental samples, but it can
be used for other types of analyses requiring the preparation of single
cell suspensions from dental tissues. Moreover, it outlines the general
requirements for performing successful scRNA-seq experiments and
may therefore serve as a general outline for researchers planning similar
experiments on different tissues. In addition, the protocol contains
a video guide for performing the whole procedure, making it more accessible
and easier to reproduce.
Overall, the presented protocol is a powerful resource for researchers
interested in performing scRNA-seq on dental tissue or other exper­
iments.
My contribution
I have co-written the first draft of the manuscript, as well as critically
revised it. Furthermore, I have cooperated on preparing the figures.
3.2 Commentary for: Spatiotemporal monitoring of hard
tissue development reveals unknown features of
tooth and bone development
Citation
Marcos Gonzalez Lopez, Barbora Huteckova, Josef Lavický. Nikodém
Zezula, Vladislav Rakultsev, Vendula Fridrichova, Haneen Tuaima, Cita
Nottmeier, Julian Petersen, Michaela Kavkova, Tomas Zikmund, Jozef
Kaiser, Rupali Lav, Haza Star, Vítězslav Bryja, Petr Henyš, Miroslav
Vořechovský, Abigail S Tucker, Jakub Harnos, Marcela Buchtová, Jan
Křivánek. Spatiotemporal monitoring of hard tissue development
reveals unknown features of tooth and bone development. Sci Adv.
48
R E S U L T S
2023 Aug 2;9(31):eadi0482. doi: 10.1126/sciadv.adi0482. Epub 2023
Aug 2. PMID: 37531427; PMCID: PMC10396306.
Science Advances, IF (2023): 11.7; Ql; WoS category: MULTIDISCIPLINARY
SCIENCES; Citations: 12 (WoS)
This manuscript is enclosed in this thesis as Appendix B.
Commentary
In this publication, we have developed a new method for monitoring the
dynamics of mineralized tissues over time. Using fluorescent dyes capable
of being incorporated into a newly produced mineralized matrix and
combining them with a method for optical clearing of mineralized tissues,
we were able to visualize their dynamics using confocal and lightsheet
microscopy4 6
.
While the specific fluorescent dyes have been used before, the only
approaches for visualizing them inside the mineralized tissues were extremely
laborious. This previously involved either sectioning the mineralized
tissue without decalcification or generating ground sections4 2 4 4 4 5
.
By combining the usage of the dyes with the compatible tissue clearing
protocol1 3 4
, we have made the process significantly less demanding and
not destructive. Furthermore, it also enables the usage of wholemount
imaging, which provides a previously unattainable 3D view of the mineralization
dynamics in hard tissues. Therefore, the strength of this approach
also lies within its flexibility, as it enables to combine the analysis
of the dynamics (e.g., confocal or lightsheet microscopy) with precise microCT-enabled
analyses of the final state (e.g., volumetrics, wall thickness
analysis.
Moreover, the publication contains optimized conditions for both
staining and optical clearing of various samples from mouse [Mus musculus),
Zebrafish [Danio rerio), chicken [Gallusgallus), xenopus [Xenopus
laevis), and chameleon [Chamaeleo calyptratus). The inclusion of
49
R E S U L T S
optimized parameters for a wide range of experimental models makes
the presented methodology experiment-ready for most applications.
Importantly, by using this approach, we have also quantified the
growth dynamics of several types of teeth in unprecedented detail. Overall,
this new methodology is indispensable for the exploration of the developmental
dynamics of both teeth and bones.
My contribution
I have co-developed the methodology. I have performed the initial experiments
as well as acquired the first proof of concept results. I have
acquired some of the presented data, analysed them, and subsequently
visualized them. In addition, I have critically revised the figures and the
manuscript.
3.3 Commentary for: The Development of Dentin
Microstructure Is Controlled by the Type of Adjacent
Epithelium
Citation
fosef Lavický. Magdalena Kolouskova, David Procházka, Vladislav
Rakultsev, Marcos Gonzalez-Lopez, Klara Steklikova, Martin Bartos,
Anushree Vijaykumar, Jozef Kaiser, Pavel Pořízka, Maria Hovorakova,
Mina Mina, Jan Křivánek, The Development of Dentin Microstructure
Is Controlled by the Type of Adjacent Epithelium, Journal of Bone and
Mineral Research, Volume 37, Issue 2, 1 February 2022, Pages 323-
339, https://doi.org/10.1002/jbmr.4471
Journal of Bone and Mineral Research, IF (2022): 6.2; Ql; WoS category:
ENDOCRINOLOGY & METABOLISM; Citations: 21 (WoS)
This manuscript is enclosed in this thesis as Appendix C.
50
R E S U L T S
Commentary
In this publication, we have for the first time analysed and quantified the
differences in the morphology of odontoblast processes (OPs) between
the enamel- and cementum-facing dentins using DSPPCeruluean
;DMPlcherr
y
mouse strain. Moreover, we provide evidence that most of the differences
in the morphology of the OPs between the two dentins originate
during development as a result of divergent signalling, dependent on the
type of adjacent epithelium. This was achieved by utilizing a Sprouty2+/;Sprouty4-/-
mouse strain, which has an ectopic ameloblastic epithelium
on the lingual side of the incisor. Therefore, the odontoblasts that form
the cementum-facing dentin in wildtype animals are, in the mutants, in
contact with the same type of epithelium as the odontoblasts forming
the enamel-facing dentin. The fact that the dentin facing the ectopic
enamel displayed an OP morphology similar to the canonical enamelfacing
dentin suggests that it is the adjacent epithelium that determines
the microstructure.
It is important to note that the altered morphology of the lingual
dentin was present only in teeth where the Sprouty2+/-;Sprouty4-/formed
the ectopic ameloblastic epithelium and not in the mutants without
it (data not shown). Therefore, the mutations in the Sprouty genes
most likely did not influence the dentin microstructure themselves.
We have also performed an expression analysis of several genes derived
from the odontoblast developmental-trajectory in an attempt to
elucidate possible signalling that controls the differences in the two
types of dentin. We have observed that in wildtype mouse incisors' labial
side, the expression of Dkkl, Wispl, and SALL1 is upregulated. To provide
an expanded context on these observations: while Dkkl is a Wnt
pathway inhibitor1 3 5
, Wispl is a Wntl downstream effector1 3 6
. Wnt signalling
has been shown to promote odontoblast differentiation. Either
up- or downregulation of Wnt results in an altered tooth development1
3 5
. When downregulated, reduced dentin formation can be observed1
3 7
. Conversely, increased dentin production is present when the
51
R E S U L T S
Wnt pathway is upregulated in the mesenchyme1 3 8
. In both cases, the
resulting dentin is hypomineralized1 3 5 1 3 9
. Interestingly, the last upregulated
protein on the enamel-facing dentin, SALL1, is a positive regulator
of dentin mineralization1 4 0
. Altogether, this suggests that normal dentin
development is a result of a delicately balanced speed of odontoblast differentiation
and their mineralization potential. This balance is then regulated
by an intricate interplay of signalling, with the Wnt pathway likely
at the forefront. The differences in dentin microstructure between the
two dentins might therefore be influenced by this balance. Nonetheless,
an analysis of the OP morphology in the mutants with altered Wnt signalling
would need to be performed to investigate this hypothesis.
The differences in the mineralization potential between the two
types of dentin, suggested by the contrasting SALL1 expression, were
further supported by laser induced breakdown spectroscopy (LIBS) and
microCT. Using LIBS, we were able to determine the spatial differences
in the elemental composition of the tooth, which elucidated the differences
between the two types of dentin. The results showed that the
enamel facing dentin displays higher amounts of calcium. This, together
with an observed higher X-ray density of the enamel-facing dentin, suggested
a higher degree of mineralization.
Overall, this paper provides a multifaceted view into the differences
between the enamel- and cementum-facing dentins and does so at an
unprecedented detail. Furthermore, it investigates the origin of these
observed differences and provides compelling new evidence concerning
the effects of the type of facing epithelium on dentin development.
My contribution
I have acquired a majority of the presented data, as well as designed and
performed their analyses. I have visualized the data, cooperated on conceptualizing
the conclusions, and written the first draft of the manu-
script.
52
R E S U L T S
3.4 Commentary for: Unraveling the Transcription Factor
Code of Odontoblast Differentiation
Citation
fosef Lavický. Marcos Gonzalez Lopez, Václav Chochola, Petra Kompaníková,
Vladislav Rakultsev, Jan Raška, Emma Wentworth Winchester,
Haneen Tuaima, Lucie Englmaier, Jan Verner, Eva Švandová, Marcela
Buchtová, Justin Cotney, Vítězslav Bryja, Dáša Bohačiaková, Tomáš Bárta,
Jan Křivánek; Unraveling the Transcription Factor Code of Odontoblast
Differentiation./Dentites. 2025 Jul 12:220345251348148. doi:
10.1177/00220345251348148. Epub ahead of print. PMID: 40650465.
Journal of Dental Research, IF (2024): 5.9; Dl; WoS category: DENTISTRY,
ORAL SURGERY & MEDICINE; Citations: 0
This manuscript is enclosed in this thesis as Appendix D.
Commentary
In this final paper, we have set out to investigate the capacity of several
odontoblast-specific TFs to guide the differentiation of mPSCs into odontoblasts.
To do so, we have reanalysed previously obtained transcriptomic
data from the mouse incisor9
and expanded it by performing
the analysis of their regulons. Based on the bioinformatic analyses of the
odontoblast developmental trajectory, we have selected four transcription
regulators: Gsc, Etv4, Salll, and Nuprl. To validate the in silico predictions,
we have mapped the expression of the four genes in the adult
mouse incisor and during molar development. The observed expression
patterns mirrored the predicted developmental trajectory.
To investigate the capacity of the TFs to guide odontoblast differentiation,
we have generated mPSC lines capable of inducible overexpression
of pairwise combinations of the genes. The combinations of genes
53
R E S U L T S
were made to reflect their expression in the developmental trajectory.
Genes expressed during early phases of odontoblast differentiation [Gsc
and Etv4) and genes expressed in mature odontoblasts [Salll and
Nuprl). We have elucidated that conditions overexpressing the early
genes [Gsc and Etv4) display a significant upregulation of DSP, DMP1,
and Runx2 as well as elevated mineralization, which suggested an odontoblast
like phenotype. Conversely, the conditions overexpressing only
the late genes [Salll and Nuprl) did not display the presence of cells with
a similar phenotype.
To further investigate the capacity of the engineered cells to selforganize,
we have implanted cell spheroids inside the kidney capsules of
immunodeficient mice. The resulting implants also displayed the presence
of odontoblast-like cells expressing DSP and DMP1. Since these
cells formed regions with an epithelium-like morphology, the expression
of epithelial marker CDH1, as well as a keratinocyte marker KRT10,
was performed. This analysis revealed that both the kidney implants, as
well as in vitro cultured cell spheroids, contain regions in which the expression
of odontoblast markers (DSP, DMP1) is conjoined with the expression
of keratinocyte markers (CDH1, KRT10). This, along with other
analyses, strongly suggested that the observed odontoblast-like cells
also displayed a keratinocyte-like phenotype. Interestingly, the presence
of this dual phenotype most likely stems from the function of Etv4,
which has been previously implicated in keratinocyte differentiation7 9
and the formation of different ectodermal appendages (EAs)7 8
. This suggests
that the observed dual phenotype of the engineered cells might be
the result of the TF overexpression initiating a shared transcription program
responsible for the formation of EAs. This notion was also further
supported by the gene ontology of the regulons of both Gsc and Etv4.
These findings may, therefore, highlight the cell fate plasticity between
the different types of EAs.
Altogether, our results suggest that the overexpression of TFs expressed
at the beginning of the developmental trajectory is paramount
for the initiation of differentiation towards the target cell type, albeit an
54
R E S U L T S
overexpression of a suitable TF is required for maintaining the correct
cell fate.
One of the limitations of this study was the system for assuring the
overexpression of the four TFs. Since it was composed of three individual
lentiviral vectors, a certain degree of population heterogeneity was
present. This was generally due to the fact that antibiotic resistance was
used to select the transductants. Even though this approach is generally
very robust, it has been shown that it is impossible to obtain a 100%
transduced mPSC population1 4 1
.
Moreover, due to the choice of promotor, the transgenes were most
probably expressed only during a limited window early during the differentiation.
This aspect presumably diminished the effects of the late
TFs [Salll, Nuprl), which were likely not expressed at their correct differentiation
phase. On a similar note, this system did not support the
temporal separation of the overexpression of the early [Gsc, Etv4) and
late TFs [Salll, Nuprl). It is likely that the sequential activation of specific
TFs at specific timepoints using discrete induction techniques will
be important to for the future of this TF-driven differentiation approach.
In conclusion, despite some limitations, this research demonstrates
that the differentiation of mPSCs into cells with an odontoblast-like phenotype
can be achieved directly. Moreover, it can be done by only overexpressing
several TFs derived from the odontoblast developmental trajectory.
Nevertheless, since the development is a complex process and
the activity of TFs is dependent on the cellular context, which can dynamically
change, more research is required to effectively generate specific
cell types for possible clinical applications.
My contribution
I have designed and performed all of the experiments. I have acquired
and analysed the majority of the presented data. I have visualized the
data and conceptualized the conclusions. I have written the first draft of
the manuscript and critically revised it.
55
D I S C U S S I O N
4 Discussion
During my PhD, I had to opportunity to work on several projects.
Some were aimed at developing new methodologies for advancing the
research of dental development, while others were original research focused
on unravelling the emergence of dental features. Finally, we have
utilized current knowledge of odontoblast developmental trajectories to
produce a proof-of-concept study for driving the differentiation of odontoblasts
directly from pluripotent stem cells.
Firstly, we have published an optimized protocol for isolating single
cells from the dental pulps of both murine and human teeth1 2 6
. This protocol
may serve as a solid entry point for isolating single cells for scRNAseq
or other experimental purposes. Nevertheless, one of the limitations
of this protocol is the isolation of viable odontoblasts9
. Since odontoblasts
are embedded in the dentin matrix, their isolation is problematic.
This might be accentuated by their complex, elongated morphology,
which is likely not compatible with FACS or microfluidic encapsulation
of individual cells during scRNA-seq library preparation. Furthermore,
this protocol uses a short incubation at 37°C for the enzymatic digestion.
Therefore, more work needs to be done to compare this process with the
long digestions at 6°C using cold proteases1 4 2
, to investigate which of the
dissociation approaches produces single cells with lower stress-related
transcriptional changes.
The other paper concerning the methodological aspects of developmental
research was focused on advancing the analyses of the dynamics
of mineralized tissues4 6
. The protocol, referred to as BEE-ST (Bones and
tEEth Spatio-Temporal growth monitoring), offers a powerful and convenient
option for analysing the growth dynamics of teeth and bones. Its
power has been recently demonstrated by Rico-Llanos et al. (2024)1 4 3
,
who used it to monitor the effects of potential achondroplasia treatment
on the dynamics of bone growth in mutant mice.
56
D I S C U S S I O N
While the BEE-ST protocol is generally robust, it is potentially limited
by the efficiency of the optical tissue clearing protocols and the utilized
imaging approaches. These stem from the fact that both confocal
and lightsheet microscopy are limited by the optical parameters of the
cleared tissues. These restrict the imaging of large samples due to diminished
excitation and emission of their innermost parts. Nonetheless,
these can be, at least partially ameliorated in time by improving the optical
clearing protocols, which are being actively worked on1 4 4
'1 4 5
. Furthermore,
imaging of both teeth and bones can be restricted by the autofluorescence
of the dental pulp or bone marrow. Nevertheless, these
can be quenched by several approaches as reviewed elsewhere1 4 6
.
Overall, BEE-ST is a flexible and robust methodology opening new
avenues of analysing the effects of gene mutations and/or treatments on
mineralized tissue dynamics. In addition, its uses might be expanded in
the future as the tissue clearing methodologies advance.
The third paper was focused on elucidating the intricacies of dental
features and their origin. More specifically, on the differences in the microstructure
of enamel- and cementum-facing dentins. Moreover, it provides
compelling evidence concerning the origin of the observed differences
between the two types of dentin. One of the limitations of this
study was the incomplete exploration of the signalling responsible for
the observed differences. Albeit, due to the complexity of the signalling
taking place during odontogenesis, this would have been a significant
undertaking, likely amounting to another paper. Nonetheless, it is likely
that the differences in cell signalling, determining the dentin microstructure,
could be unravelled using the same approach Alghadeer et al.
(2023)1 2 7
used to predict signalling pathways involved in ameloblast
and odontoblast differentiation. Nevertheless, the investigation of similar
interactions using scRNA-seq is somewhat limited, as all of the individual
analysed cells are removed from their histological context1 4 7
.
Therefore, the interplay of adjacent cell populations and the resulting
dynamic changes in their transcriptional profiles can be only
57
D I S C U S S I O N
approximated in silico. However, this particular hurdle is being actively
resolved by the advancements of spatial transcriptomics that provide
both the histological and transcriptomic context for each of the analysed
cells/spots with increasing resolution1 4 7
. Moreover, the spatial transcriptomic
approaches are not limited by the problematic isolation of
odontoblasts or other cells with similarly complex morphology like regular
scRNA-seq, as highlighted previously1 2 6
.
Finally, in the last paper, we have generated cells with an odontoblast-like
phenotype using the overexpression of several odontoblastspecific
transcription regulators. This was done directly from mPSCs
without the use of any growth factors or dexamethasone, making it the
first such attempt to our knowledge. Intriguingly, our results also describe
a potential plasticity between the cell fates of teeth and hair follicles
that is likely regulated by Etv4. These findings go in line with similar
research describing the plasticity between the cell fates of various ectodermal
appendages8 2
'8 3
'8 5
'8 7
.
While some limitations of this study have already been mentioned,
it is likely that the differentiation may have achieved improved differentiation
specificity if external, pro-odontogenic signals had been utilized.
This assumption is based on the other studies utilizing a similar TFdriven
approach1 3 0
'1 3 1
. Conversely, improved differentiation may have
also been achieved by first generating neural crests stem cells to align
the differentiation more closely to the in vivo development, similarly to
Seki et al. (2015)1 3 3
. Nevertheless, that would go against one of the research
goals of this project, which was aimed at investigating whether
the differentiation is achievable with minimal external cues. Altogether,
we highlight that this approach is likely to be suitable for generating
even cell types with complex developmental origins, like odontoblasts.
In broader terms, the concept of guiding differentiation via the
overexpression of selected TFs is being actively explored as a tool for
generating specific cell types. Our research highlights the importance of
using TFs that are involved in early phases of the developmental
58
D I S C U S S I O N
trajectory (e.g., Gsc and Etv4) rather than TFs present in later
phases(e.g., Salll and Nuprl). Generally, these findings complement previous
discoveries concerning the TF-driven differentiation of hepatocytes
as reviewed elsewhere1 4 8
. This reality is also reflected in other research,
which suggests that the utilization of early inductors is instrumental
for the generation of any cell type9 3
. Moreover, recently, Joung et
al. (2023)1 4 9
have systematically mapped the effects of the overexpression
of all annotated human TFs and their isoforms (approximately 3500
ORFs) on early differentiation of hPSCs. This TF atlas might be an important
resource for selecting the correct TFs for closely aligning the initial
phases of artificial differentiation to the developmental trajectory of
the target cell type9 3
.
Overall, this suggests that this approach might serve as an important
tool for studying the importance of various TFs on the cell differentiation,
disease modelling, as well as regenerative applications9
3
'1 3 0
.1 4 9
.
Nevertheless, in the context of recapitulating tooth development,
several fundamental hurdles remain unanswered. One might be the recapitulation
of all aspects of the interactions between the ameloblastic
epithelium and the mesenchyme. While the signalling can be emulated
by adding specific signalling ligands or small molecules1 2 7 1 2 8
, the natural
development is also significantly influenced by the physical contact of
these two cell populations. As a result of this contact, important features
of both enamel and dentin are formed. These range from the general
shape of the tooth to the DEJ, whose emergence is conditioned by a direct
contact between ameloblasts and odontoblasts2 8
'2 9
. In addition, for
example, little is known about the mechanisms driving the formation of
the scallops on the DEJ, or the signalling controlling the emergence of
enamel decussation patterns. Needless to say, without understanding
the origin of several key dental features outlined in this thesis, they can
hardly be recreated for regenerative or other purposes.
59
D I S C U S S I O N
The general complexity of the dental features and their microstructure
makes their controlled "manual" recapitulation for regenerative
purposes a daunting task. Therefore, the future may favour approaches
striving to initiate tooth morphogenesis and then support the natural interactions
driving the tooth's formation. Conversely, approaches aimed
at stimulating the limited regeneration potential of dental tissues, as
seen in Neves et al. (2017)6
, are likely to come into use sooner due to
their inherent simplicity.
Overall, while many aspects of dental development and regeneration
have been answered, there are still proverbial puzzle pieces missing.
Large volume of developmental and applied research is still required
to elucidate the origin of some of the mentioned structural features
of teeth until they can be artificially recreated. Nevertheless, our
results may be a step towards accomplishing that goal.
60
C O N C L U S I O N
5 Conclusion
While working on this thesis, I have fulfilled the set goals. Firstly, we
have generated new approaches for investigating dental development.
First being the optimized protocol for isolating single cells from dental
pulps for usage in scRNA-seq analyses. The second was the BEE-ST
methodology, which enables to study the dynamics of mineralized tissues
in unprecedented detail.
Furthermore, we have elucidated mechanisms driving the emergence
of specific dental features. These were that the origin of the differences
in the morphology of odontoblast processes in the crown (analogue)
and root (analogue) dentins lies within the effects of the type of
adjacent dental epithelium. Moreover, we have also proved that the formation
of enamel decussation patterns is dependent on the organized
intra-epithelial shuffling of individual ameloblasts.
Finally, by utilizing a previously constructed developmental trajectory
of odontoblasts, we have selected four transcription regulators. By
overexpressingthem in mPSCs, we have guided their differentiation into
cells displaying an odontoblast-like phenotype. Albeit, while doing so,
we have also highlighted the plasticity between the cell fates of teeth and
those of a different ectodermal appendage.
Altogether, this thesis and the works within provide insight into
dental development as well as delineate potential avenues for mediating
dental regeneration by utilizing fundamental developmental knowledge.
61
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
Summary of publications and research
activities
Publications
1. Jan Křivánek, Josef Lavický. Thibault Bouderlique, Igor
Adameyko. Rapid Isolation of Single Cells from Mouse and Human
Teeth. J Vis Exp. 2021 Oct 28;(176). doi: 10.3791/63043.
PMID: 34779437.
Journal of Visualized Experiments (JoVE), IF (2021): 1.424; Q3;
WoS category: MULTIDISCIPLINARY SCIENCES; Citations: 2
(WoS)
My contribution
I have co-written the first draft of the manuscript and critically revised
it. Furthermore, I have cooperated on preparing the figures.
2. Marcos Gonzalez Lopez, Barbora Huteckova, Josef Lavický. Nikodém
Zezula, Vladislav Rakultsev, Vendula Fridrichova, Haneen
Tuaima, Cita Nottmeier, Julian Petersen, Michaela Kavkova, Tomas
Zikmund, Jozef Kaiser, Rupali Lav, Haza Star, Vítězslav Bryja,
Petr Henyš, Miroslav Vořechovský, Abigail S Tucker, Jakub Harnos,
Marcela Buchtová, Jan Křivánek. Spatiotemporal monitoring
of hard tissue development reveals unknown features of
tooth and bone development. Sci Adv. 2023 Aug
2;9(31):eadi0482. doi: 10.1126/sciadv.adi0482. Epub 2023 Aug
2. PMID: 37531427; PMCID: PMC10396306.
Science Advances, IF (2023): 11.7; Ql; WoS category: MULTIDISCIPLINARY
SCIENCES; Citations: 12 (WoS)
63
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
My contribution
I have co-developed the methodology. I have performed the initial experiments
and acquired the first proof-of-concept results. I have acquired
some of the presented data, analysed and subsequently visualized
them. In addition, I have critically revised the figures and the man­
uscript.
3. Josef Lavický. Magdalena Kolouskova, David Procházka,
Vladislav Rakultsev, Marcos Gonzalez-Lopez, Klara Steklikova,
Martin Bartos, Anushree Vijaykumar, Jozef Kaiser, Pavel Pořízka,
Maria Hovorakova, Mina Mina, Jan Křivánek, The Development
of Dentin Microstructure Is Controlled by the Type of Adjacent
Epithelium, Journal of Bone and Mineral Research, Volume
37, Issue 2, 1 February 2022, Pages 323-
339, https://doi.org/10.1002/jbmr.4471
Journal of Bone and Mineral Research, IF (2022): 6.2; Ql; WoS
category: ENDOCRINOLOGY & METABOLISM; Citations: 21
(WoS)
Awarded the annualprize of the Czech Anatomical Society in 2022
My contribution
I have acquired a majority of the presented data as well as designed and
performed their analyses. I have visualized the data, cooperated on conceptualizing
the conclusions, and written the first draft of the manu­
script.
4. Josef Lavický. Marcos Gonzalez Lopez, Václav Chochola, Petra
Kompaníková, Vladislav Rakultsev, Jan Raška, Emma Wentworth
Winchester, Haneen Tuaima, Lucie Englmaier, Jan Verner, Eva
Švandová, Marcela Buchtová, Justin Cotney, Vítězslav Bryja, Dáša
64
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
Bohačiaková, Tomáš Bárta, Jan Křivánek; Unraveling the Transcription
Factor Code of Odontoblast Differentiation. J Dent
Res. 2025 Jul 12:220345251348148. doi:
10.1177/00220345251348148. Epub ahead of print. PMID: 40650465.
Journal of Dental Research, IF (2024): 5.9; Dl; WoS category:
DENTISTRY, ORAL SURGERY & MEDICINE; Citations: 0
My contribution
I have designed and performed all of the experiments. I have acquired
and analysed the majority of the presented data. I have visualized the
data and conceptualized the conclusions. I have written the first draft
of the manuscript and critically revised it.
5. Pernille Bogetofte Thomasen, Alena Salasova, Kasper KjaerSorensen,
Lucie Woloszczuková, Josef Lavický. Hande Login,
Jeppe Tranberg-Jensen, Sergio Almeida, Sander Beel, Michaela
Kavková, Per Qvist, Mads Kjolby, Peter Lund Ovesen, Stella Nolte,
Benedicte Vestergaard, Andreea-Cornelia Udrea, Lene Niemann
Nejsum, Moses V Chao, Philip Van Damme, Jan Křivánek, Jeremy
Dasen, Claus Oxvig, Anders Nykjaer. SorCS2 binds progranulin
to regulate motor neuron development. Cell Rep. 2023 Nov
28;42(11):113333. doi: 10.1016/j.celrep.2023.113333. Epub
2023 Oct 27. PMID: 37897724.
Cell Reports, IF (2023): 7.5; Dl; WoS category: CELL BIOLOGY;
Citations: 9 (WoS)
My contribution
I have optically cleared the mouse embryo samples and imaged all of
them using lightsheet microscopy. I have cooperated on designing their
analysis. I have visualized the results of the imaging and critically revised
the manuscript.
65
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
6. Cita Nottmeier, Josef Lavický. Marcos Gonzalez Lopez, Sarah
Knauth, Bärbel Kahl-Nieke, Michael Amling, Thorsten Schinke, Jill
Helms, Jan Křivánek, Till Koehne, Julian Petersen. Mechanicalinduced
bone remodeling does not depend on Piezol in dentoalveolar
hard tissue. Sei Rep. 2023 Jun 12;13(1):9563. doi:
10.1038/s41598-023-36699-9. PMID: 37308580; PMCID:
PMC10261143.
Scientific Reports, IF (2023): 3.8; Ql; WoS category: MULTIDISCIPLINARY
SCIENCES; Citations: 11 (WoS)
My contribution
I have cooperated on performing the mouse experiments and prepared
the resulting samples. I have critically revised the manuscript.
7. Vladislav Rakultsev; fosef Lavický: Marcos Gonzalez Lopez;
Klara Cigosova; Igor Adameyko; Jan Křivánek. Enamel Decussation
Pattern Originates from Directional Sliding of Amelo-
blasts.
(UNDER REVIEW)
My contribution:
I have performed initial proof-of-concept experiments. I have provided
mentoring to the first author. I have cooperated on the acquisition of
some of the data. I have critically revised the manuscript.
66
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
Research activities related to the thesis
Conference presentations
1. LAVICKÝ, Josef*, Magdaléna KOLOUŠKOVÁ, David PROCHÁZKA,
Vladislav RAKULTSEV, Marcos GONZALEZ LÓPEZ, Klara STEKLIKOVA,
Martin BARTOŠ, Anushree VIJAYKUMAR, Jozef KAISER,
Pavel POŘÍZKA, Marie HOVORAKOVA, Mina MINA and Jan
KŘIVÁNEK. The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium. Oral presentation at
Imaging Principles of Life 2023 - Czech-Bioimaging Scientific Conference,
Hradec Králové, Czech Republic, 2023
• Invited speaker
2. LAVICKÝ, Josef*, GONZALEZ LOPEZ, Marcos, RAKULTSEV, Vladislav,
WENTWORTH WINCHESTER, Emma, TUAIMA, Haneen, PEŠKOVÁ,
Lucie, VERNER, Jan, ŠVANDOVÁ, Eva, COTNEY, Justin, BÁRTA,
Tomáš, BUCHTOVÁ, Marcela, KŘIVÁNEK, Jan. Differentiation of
pluripotent stem cells using odontoblasts-specific transcription
factors. Poster presentation at 3r d
Visegrád Group Society for Developmental
Biology meeting, Warsaw, Poland, 2023.
3. LAVICKÝ, Josef*, Magdaléna KOLOUŠKOVÁ, David PROCHÁZKA,
Vladislav RAKULTSEV, Marcos GONZALEZ LOPEZ, Klara STEKLIKOVA,
Martin BARTOŠ, Anushree VIJAYKUMAR, Jozef KAISER,
Pavel POŘÍZKA, Marie HOVORAKOVA, Mina MINA and Jan
KŘIVÁNEK. The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium. Oral presentation at
the 14t h
Tooth Morphology and Differentiation conference, Prague,
Czech Republic, 2022.
67
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
4. LAVICKÝ, Josef*, BÁRTA, Tomáš, Vladislav Rakultsev, Marcos Gonzáles-López,
FRIDRICHOVA, Vendula, PEŠKOVÁ, Lucie, HAMPL, Aleš,
KŘIVÁNEK, Jan. Differentiation of pluripotent stem cells using
odontoblasts-specific transcription factors. Poster presentation
at the 14t h
Tooth Morphology and Differentiation conference, Prague,
Czech Republic, 2022.
• Received the Best Poster Award
5. LAVICKÝ, Josef*, Magdaléna KOLOUŠKOVÁ, David PROCHÁZKA,
Vladislav RAKULTSEV, Marcos GONZALEZ LOPEZ, Klara STEKLIKOVA,
Martin BARTOŠ, Anushree VIJAYKUMAR, Jozef KAISER,
Pavel POŘÍZKA, Marie HOVORAKOVA, Mina MINA and Jan
KŘIVÁNEK. The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium. Oral presentation at
EFEM Student Symposium, Dublin, Ireland (online), 2022
• Awarded 3r d
place in the Classical Research Talk category
6. LAVICKÝ, Josef, Magdaléna KOLOUŠKOVÁ, David PROCHÁZKA,
Vladislav RAKULTSEV, Marcos GONZALEZ LOPEZ, Klara STEKLIKOVA,
Martin BARTOŠ, Anushree VIJAYKUMAR, Jozef KAISER,
Pavel POŘÍZKA, Marie HOVORAKOVA, Mina MINA and Jan
KŘIVÁNEK. The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium. Poster presentation at
Biologické dny, Brno, Czech Republic, 2021
• Poster prize
7. LAVICKÝ, Josef*, BÁRTA, Tomáš, FRIDRICHOVA, Vendula,
PEŠKOVÁ, Lucie, HAMPL, Aleš, KŘIVÁNEK, Jan. Differentiation of
pluripotent stem cells using odontoblasts-specific transcription
factors. Poster presentation at 2n d
Visegrád Group Society for Developmental
Biology meeting, Szeged, Hungary, 2021.
68
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
8. LAVICKÝ, Josef, Magdaléna KOLOUŠKOVÁ, David PROCHÁZKA,
Vladislav RAKULTSEV, Marcos GONZALEZ LÓPEZ* Klara STEKLIKOVA,
Martin BARTOŠ, Anushree VIJAYKUMAR, Jozef KAISER,
Pavel POŘÍZKA, Marie HOVORAKOVA, Mina MINA and Jan
KŘIVÁNEK. The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium. Poster presentation at
2n d
Visegrád Group Society for Developmental Biology meeting, Szeged,
Hungary, 2021.
• Poster prize
* presenting author
Other achievements
• Author of a cover image for Cell Reports, Volume 42, November
28, 2023 (Included as Appendix F)
• Winner of CiteAb's 2023 contest for the best published image
• Author of a cover image for Journal for Bone and Mineral Research,
Volume 37, February 2022 (Included as Appendix E)
• Awarded 2n d
place in Best Scientific Picture Contest at EFEM Student
Symposium, Ireland (online), 2022.
• Awarded student grant funding for one year from the Internal
Grant Agency of Masaryk University for the project Investigation
of the role ofFoxDl+
stem cells in the development of the craniofacial
region (2021).
• Brno Ph.D. Talent Scholarship holder (awarded 2019)
69
S U M M A R Y O F PUBLICATIONS A N D R E S E A R C H ACTIVITIES
Internship
Medical University of Vienna (2024, Duration: 9 weeks)
• Supervision: Igor Adameyko
70
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Bibliography
1. Nanci A, ed. Chapter 1 - Structure of the Oral Tissues. In: Ten Gate's
Oral Histology (Eighth Edition). Mosby; 2013:1-13.
doi:10.1016/B978-0-323-07846-7.00001-X
2. Berkovitz BKB, Faulkes CG. Eruption rates of the mandibular incisors
of naked mole-rats (Heterocephalus glaber)./Zool.
2001;255(4):461-466. doi:10.1017/S0952836901001546
3. Hulsey CD, Cohen KE, Johanson Z, et al. Grand Challenges in Comparative
Tooth Biology. Integr Comp Biol. 2020;60(3):563-580.
doi:10.1093/icb/icaa038
4. Tucker AS, Fraser GJ. Evolution and developmental diversity of
tooth regeneration. Semin Cell Dev Biol. 2014;25-26:71-80.
doi:10.1016/j.semcdb.2013.12.013
5. Hermans F, Hemeryck L, Lambrichts I, Bronckaers A, Vankelecom
H. Intertwined Signaling Pathways Governing Tooth Development:
A Give-and-Take Between Canonical Wnt and Shh. Front Cell Dev
Biol. 2021;9:758203. doi:10.3389/fcell.2021.758203
6. Neves VCM, Babb R, Chandrasekaran D, Sharpe PT. Promotion of
natural tooth repair by small molecule GSK3 antagonists. Sci Rep.
2017;7(1):39654. doi:10.1038/srep39654
7. Krivanek J, Buchtova M, Fried K, Adameyko I. Plasticity of Dental
Cell Types in Development, Regeneration, and Evolution./ Dent Res.
2023;102(6):589-598. doi:10.1177/00220345231154800
8. Krivanek J, Adameyko I, Fried K. Heterogeneity and Developmental
Connections between Cell Types Inhabiting Teeth. Front Physiol.
2017;8. doi:10.3389/fphys.2017.00376
71
B I B L I O G R A P H Y
9. KřivánekJ, Soldatov RA, Kastriti ME, et al. Dental cell type atlas reveals
stem and differentiated cell types in mouse and human teeth.
NatCommun. 2020;11(1):4816. doi:10.1038/s41467-020-18512-7
10. Chiba Y, Saito K, Martin D, etal. Single-Cell RNA-Sequencing From
Mouse Incisor Reveals Dental Epithelial Cell-Type Specific Genes.
Front Cell Dev Biol. 2020;8. doi:10.3389/fcell.2020.00841
11. Lavický J, Kolouskova M, Procházka D, etal. The Development of
Dentin Microstructure Is Controlled by the Type of Adjacent Epithelium.
J Bone Miner Res. 2022;37(2):323-339.
doi:10.1002/jbmr.4471
12. Dacre IT, Kempson S, Dixon PM. Pathological studies of cheekteeth
apical infections in the horse: 4. Aetiopathological findings in 41 apically
infected mandibular cheekteeth. Vet J. 2008;178(3):341-351.
doi:10.1016/j.tvjl.2008.09.028
13. Nanci A, ed. Chapter 9 - Periodontium. In: Ten Gate's Oral Histology
(Eighth Edition). Mosby; 2013:205-232. doi:10.1016/B978-0-323-
07846-7.00009-4
14. Nanci A, ed. Chapter 8 - Dentin-Pulp Complex. In: Ten Gate's Oral
Histology (Eighth Edition). Mosby; 2013:165-204.
doi:10.1016/B978-0-323-07846-7.00008-2
15. Lacruz RS, Habelitz S, Wright JT, Paine ML. Dental Enamel Formation
and Implications for Oral Health and Disease. Physiol Rev.
2017;97(3]:939-993. doi:10.1152/physrev.00030.2016
16. Sire JY, Davit-Béal T, Delgado S, Gu X. The Origin and Evolution of
Enamel Mineralization Genes. Cells Tissues Organs. 2007;186(1):25-
48. doi:10.1159/000102679
72
B I B L I O G R A P H Y
17. Diekwisch TGH, Jin T, Wang X, et al. Amelogenin Evolution and Tetrapod
Enamel Structure. Published online September 23, 2009.
doi:10.1159/000242395
18. Ripa LW, Gwinnett AJ, Buonocore MG. The "prismless" outer layer
of deciduous and permanent enamel. Arch Oral Biol. 1966;11(1):41-
IN5.doi:10.1016/0003-9969(66)90116-6
19. Bajaj D, Arola D. Role of prism decussation on fatigue crack growth
and fracture of human enamel. Acta Biomater. 2009;5(8):3045-
3056. doi:10.1016/j.actbio.2009.04.013
20. Renteria C, Fernandez-Arteaga JM, Grimm J, Ossa EA, Arola D. Mammalian
enamel: A universal tissue and diverse source of inspiration.
Acta Biomater. 2021;136:402-411.
doi:10.1016/j.actbio.2021.09.016
21. Beniash E, Stifler CA, Sun CY, et al. The hidden structure of human
enamel. Nat Commun. 2019;10(1):4383. doi:10.1038/s41467-019-
12185-7
22. Marsico C, Grimm JR, Renteria C, et al. Characterizing the microstructures
of mammalian enamel by synchrotron phase contrast
microCT.^lcta Biomater. 2024;178:208-220.
doi:10.1016/j.actbio.2024.02.038
23. Cox BN. How the tooth got its stripes: patterning via strain-cued
motility. JRSoc Interface. 2013; 10(84):20130266.
doi:10.1098/rsif.2013.0266
24. Kawashima N, Okiji T. Odontoblasts: Specialized hard-tissue-forming
cells in the dentin-pulp complex. Congenit Anom.
2016;56(4):144-153.doi:10.1111/cga.l2169
25. Thesleff I, Lehtonen E, Saxen L. Basement Membrane Formation in
Transfilter Tooth Culture and Its Relation to Odontoblast
73
B I B L I O G R A P H Y
Differentiation. Differentiation. 1978; 10(1):71-79.
doi:10.1111/j.l432-0436.1978.tb00948.x
26. Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: Structure,
Composition and Mineralization. Front Biosci Elite Ed. 2011;3:711-
735.
27. Vijaykumar A, Ghassem-Zadeh S, Vidovic-Zdrilic I, et al. Generation
and characterization of DSPP-Cerulean/DMPl-Cherry reporter
mice.genesis. 2019;0(0):e23324. doi:10.1002/dvg.23324
28. Tjaderhane L, Carrilho MR, Breschi L, Tay FR, Pashley DH. Dentin
basic structure and composition—an overview. Endod Top.
2009;20(l):3-29. doi:10.1111/j.l601-1546.2012.00269.x
29. Gallagher RR, Demos SG, Balooch M, Marshall Jr. GW, Marshall SJ.
Optical spectroscopy and imaging of the dentin-enamel junction in
human third molars. J Biomed Mater Res A. 2003;64A(2):372-377.
doi:10.1002/jbm.a.l0436
30. Lin CP, Douglas WH, Erlandsen SL. Scanning electron microscopy of
type I collagen at the dentin-enamel junction of human teeth. J Histochem
Cytochem. 1993;41(3):381-388. doi:10.1177/41.3.8429200
31. Brauer DS, Marshall GW, Marshall SJ. Variations in human DEJ scallop
size with tooth type./Dent. 2010;38(7):597-601.
doi:10.1016/j.jdent.2010.03.010
32. Takagi Y, Fujisawa ,Ryuichi, and Sasaki S. Identification of Dentin
Phosphophoryn Localization by Histochemical Stainings. Connect
Tissue Res. 1986;14(4):279-292.
doi:10.3109/03008208609017471
33. Nakamura 0, Gohda E, Ozawa M, etal. Immunohistochemical studies
with a monoclonal antibody on the distribution of
74
B I B L I O G R A P H Y
phosphophoryn in predentin and dentin. Calcif Tissue Int.
1985;37(5):491-500. doi:10.1007/BF02557832
34. Tjäderhane L, Hietala EL, Larmas M. Mineral Element Analysis of
Carious and Sound Rat Dentin by Electron Probe Microanalyzer
Combined with Back-scattered Electron Image./ Dent Res.
1995;74(11):1770-1774. doi:10.1177/00220345950740110901
35. Stratmann U, Schaarschmidt K, Wiesmann HP, Plate U, Höhling HJ,
Szuwart T. The mineralization of mantle dentine and of circumpulpal
dentine in the rat: an ultrastructural and element-analytical
study. AnatEmbryol (Berl). 1997;195(3):289-297.
doi:10.1007/s004290050048
36. Wang RZ, Weiner S. Strain-structure relations in human teeth using
Moire fringes./Biomech. 1997;31(2):135-141. doi:10.1016/S0021-
9290(97)00131-0
37. Kinney JH, Balooch M, Marshall SJ, Marshall GW, Weihs TP. Hardness
and young's modulus of human peritubular and intertubular
dentine. Arch Oral Biol. 1996;41(1):9-13. doi:10.1016/0003-
9969(95)00109-3
38. Chu CY, Kuo TC, Chang SF, Shyu YC, Lin CP. Comparison of the microstructure
of crown and root dentin by a scanning electron microscopic
study. J Dent Sei. 2010;5(l):14-20. doi:10.1016/S1991-
7902(10)60003-7
39. Bertassoni LE, Stankoska K, Swain MV. Insights into the structure
and composition of the peritubular dentin organic matrix and the
lamina limitans. Micron. 2012;43(2):229-236. doi:10.1016/j.mi-
cron.2011.08.003
40. Wagermaier W, Fratzl P. 9.03 - Collagen. In: Matyjaszewski K, Möller
M, eds. Polymer Science: A Comprehensive Reference. Elsevier;
2012:35-55. doi:10.1016/B978-0-444-53349-4.00247-8
75
B I B L I O G R A P H Y
41. Berg C, Unosson E, Engqvist H, Xia W. Comparative Study of Technologies
for Tubule Occlusion and Treatment of Dentin Hypersensitivity.
J Fund Biomater. 2021;12(2):27. doi:10.3390/jfbl2020027
42. Wennberg A, Mjor IA, Heide S. Rate of formation of regular and irregular
secondary dentin in monkey teeth. Oral Surg Oral Med Oral
Pathol. 1982;54(2):232-237. doi:10.1016/0030-4220(82)90222-5
43. Stanley HR, White CL, McCray L. The rate of tertiary [reparative]
dentine formation in the human tooth. Oral Surg Oral Med Oral
Pathol. 1966;21(2):180-189. doi:10.1016/0030-4220(66)90240-4
44. Emken S, Witzel C, Kierdorf U, Frolich K, Kierdorf H. Characterization
of short-period and long-period incremental markings in porcine
enamel and dentine—Results of a fluorochrome labelling study
in wild boar and domestic pigs./i4nat. 2021;239(5):1207-1220.
doi:10.1111/joa.l3502
45. Pautke C, Vogt S, Tischer T, et al. Polychrome labeling of bone with
seven different fluorochromes: Enhancing fluorochrome discrimination
by spectral image analysis. Bone. 2005;37(4):441-445.
doi:10.1016/j.bone.2005.05.008
46. Gonzalez Lopez M, Huteckova B, Lavický J, et al. Spatiotemporal
monitoring of hard tissue development reveals unknown features
of tooth and bone development. SciAdv. 2023;9(31):eadi0482.
doi:10.1126/sciadv.adi0482
47. Nudel I, Pokhojaev A, Bitterman Y, etal. Secondary Dentin Formation
Mechanism: The Effect of Attrition. Int J Environ Res Public
Health. 2021;18(19):9961. doi:10.3390/ijerphl8199961
48. Neves VCM, Sharpe PT. Regulation of Reactionary Dentine Formation.
J Dent Res. 2018;97(4):416-422.
doi:10.1177/0022034517743431
76
B I B L I O G R A P H Y
49. Ohyama S, Ouchi T, Kimura M, et al. Piezol-pannexin-l-P2X3 axis in
odontoblasts and neurons mediates sensory transduction in dentinal
sensitivity. Front Physiol. 2022;13.
doi:10.3389/fphys.2022.891759
50. Son AR, Yang YM, Hong JH, Lee SI, Shibukawa Y, Shin DM. Odontoblast
TRP Channels and Thermo/Mechanical Transmission. J Dent
Res. 2009;88(11):1014-1019. doi:10.1177/0022034509343413
51. Won J, Vang H, Kim JH, Lee PR, Kang Y, Oh SB. TRPM7 Mediates
Mechanosensitivity in Adult Rat Odontoblasts./Dent Res.
2018;97(9):1039-1046. doi:10.1177/0022034518759947
52. Kimura M, Sase T, Higashikawa A, et al. High pH-Sensitive TRPA1
Activation in Odontoblasts Regulates Mineralization./ Dent Res.
2016;95(9):1057-1064. doi:10.1177/0022034516644702
53. Pääkkönen V, Rusanen P, Hagström J, Tjäderhane L. Mature human
odontoblasts express virus-recognizing toll-like receptors. IntEndodj.
2014;47(10):934-941. doi:10.1111/iej.12238
54. Yumoto H, Hirao K, Hosokawa Y, et al. The roles of odontoblasts in
dental pulp innate immunity. Jpn Dent Sei Rev. 2018;54(3): 105-117.
doi:10.1016/j.jdsr.2018.03.001
55. Feng J, Mantesso A, De Bari C, Nishiyama A, Sharpe PT. Dual origin
of mesenchymal stem cells contributing to organ growth and repair.
Proc Natl Acad Sei. 2011;108(16):6503-6508.
doi:10.1073/pnas.l015449108
56. Kaukua N, Shahidi MK, Konstantinidou C, et al. Glial origin of mesenchymal
stem cells in a tooth model system. Nature.
2014;513(7519):551-554. doi:10.1038/naturel3536
57. Song M, Yu B, Kim S, etal. Clinical and Molecular Perspectives of Reparative
Dentin Formation: Lessons Learned from Pulp-Capping
77
B I B L I O G R A P H Y
Materials and the Emerging Roles of Calcium. Dent Clin North Am.
2017;61(1):93-110. doi:10.1016/j.cden.2016.08.008
58. Yu CY, Abbott PV. Pulp microenvironment and mechanisms of pain
arising from the dental pulp: From an endodontic perspective. Aust
EndodJ. 2018;44(2):82-98. doi:10.1111/aej.12257
59. Nagaoka S, Miyazaki Y, Liu HJ, Iwamoto Y, Kitano M, Kawagoe M.
Bacterial invasion into dentinal tubules of human vital and nonvital
teeth. JEndod. 1995;21(2):70-73. doi:10.1016/S0099-
2399(06)81098-8
60. Tucker AS, Yamada G, Grigoriou M, Pachnis V, Sharpe PT. Fgf-8 determines
rostral-caudal polarity in the first branchial arch. Development.
1999;126(1):51-61. doi:10.1242/dev.l26.1.51
61. Yu T, Klein OD. Molecular and cellular mechanisms of tooth development,
homeostasis and repair. Development. 2020;147(2).
doi:10.1242/dev.l84754
62. Cobourne MT, Sharpe PT. Tooth and jaw: molecular mechanisms of
patterning in the first branchial arch. Arch Oral Biol. 2003;48(1):1-
14. doi:10.1016/S0003-9969(02)00208-X
63. Thesleff I. Epithelial-mesenchymal signalling regulating tooth morphogenesis./
CellSci. 2003;116(9):1647-1648.
doi:10.1242/jcs.00410
64. Tummers M, Thesleff I. The importance of signal pathway modulation
in all aspects of tooth development. / Exp Zoolog B Mol Dev
Evol. 2009;312B(4):309-319. doi:10.1002/jez.b.21280
65. Nanci A, ed. Chapter 5 - Development of the Tooth and Its Supporting
Tissues. In: Ten Gate's Oral Histology (Eighth Edition). Mosby;
2013:70-94. doi:10.1016/B978-0-323-07846-7.00005-7
78
B I B L I O G R A P H Y
66. Roehl H, Ntisslein-Volhard C. Zebrafish pea3 and erm are general
targets of FGF8 signaling. Curr Biol. 2001;ll(7):503-507.
doi:10.1016/s0960-9822(01)00143-9
67. Kim R, Green JBA, Klein OD. From snapshots to movies: Understanding
early tooth development in four dimensions. Dev Dyn.
2017;246(6):442-450. doi:10.1002/dvdy.24501
68. Procházka J, Procházkova M, Du W, et al. Migration of Founder Epithelial
Cells Drives Proper Molar Tooth Positioning and Morphogenesis.
Dev Cell. 2015;35(6):713-724.
doi:10.1016/j.devcel.2015.11.025
69. Lavický J, Lopez MG, Chochola V, et al. Unraveling the Transcription
Factor Code of Odontoblast Differentiation./ Dent Res. Published
online July 12, 2025:220345251348148.
doi:10.1177/00220345251348148
70. Áberg T, Wang XP, Kim JH, et al. Runx2 mediates FGF signaling from
epithelium to mesenchyme during tooth morphogenesis. Dev Biol.
2004;270(l):76-93. doi:10.1016/j.ydbio.2004.02.012
71. Thesleff I, Mikkola M. The role of growth factors in tooth development.
IntRev Cytol. 2002;217:93-135. doi:10.1016/s0074-
7696(02)17013-6
72. Zhang YD, Chen Z, Song YQ, Liu C, Chen YP. Making a tooth: growth
factors, transcription factors, and stem cells. Cell Res.
2005;15(5):301-316. doi:10.1038/sj.cr.7290299
73. Huang XF, Chai Y. Molecular regulatory mechanism of tooth root
development. IntJ Oral Sci. 2012;4(4):177-181.
doi:10.1038/ijos.2012.61
79
B I B L I O G R A P H Y
74. LiJ, Paráda C, Chai Y. Cellular and molecular mechanisms of tooth
root development. Dev Camb Engl. 2017;144(3):374-384.
doi:10.1242/dev.l37216
75. Cui CY, Schlessinger D. Eccrine sweat gland development and sweat
secretion. Exp Dermatol. 2015;24(9):644-650.
doi:10.1111/exd.l2773
76. Biggs LC, Mikkola ML. Early inductive events in ectodermal appendage
morphogenesis. Semin Cell Dev Biol. 2014;25-26:11-21.
doi:10.1016/j.semcdb.2014.01.007
77. Jiménez-Rojo L, Granchi Z, Graf D, Mitsiadis TA. Stem Cell Fate Determination
during Development and Regeneration of Ectodermal
Organs. Front Physiol. 2012;3:107. doi:10.3389/fphys.2012.00107
78. Kurpios NA, MacNeil L, Shepherd TG, Gludish DW, Giacomelli AO,
Hassell JA. The Pea3 Ets transcription factor regulates differentiation
of multipotent progenitor cells during mammary gland development.
Dev Biol. 2009;325(1):106-121. doi:10.1016/j.yd-
bio.2008.09.033
79. Finnegan A, Cho RJ, Luu A, et al. Single-Cell Transcriptomics Reveals
Spatial and Temporal Turnover of Keratinocyte Differentiation Regulators.
Front Genet. 2019;10:775. doi:10.3389/fgene.2019.00775
80. Porntaveetus T, Otsuka-Tanaka Y, Basson MA, Moon AM, Sharpe PT,
Ohazama A. Expression of fibroblast growth factors (Fgfs) in murine
tooth development. JAnat. 2011;218(5):534-543.
doi:10.1111/j.l469-7580.2011.01352.x
81. Naveau A, Seidel K, Klein OD. Tooth, hair and claw: Comparing epithelial
stem cell niches of ectodermal appendages. Exp Cell Res.
2014;325(2):96-103. doi:10.1016/j.yexcr.2014.02.003
80
B I B L I O G R A P H Y
82. Jimenez-Rojo L, Pagella P, Harada H, Mitsiadis TA. Dental Epithelial
Stem Cells as a Source for Mammary Gland Regeneration and Milk
Producing Cells In Vivo. Cells. 2019;8(10):1302.
doi:10.3390/cells8101302
83. Thaler R, Yoshizaki K, Nguyen T, et al. Mediator 1 ablation induces
enamel-to-hair lineage conversion in mice through enhancer dynamics.
Commun Biol. 2023;6(1):766. doi:10.1038/s42003-023-
05105-5
84. Yoshizaki K, Hu L, Nguyen T, et al. Ablation of Coactivator Medl
Switches the Cell Fate of Dental Epithelia to That Generating Hair.
PLOSONE. 2014;9(6):e99991. doi:10.1371/journal.pone.0099991
85. Saito K, Michon F, Yamada A, et al. Sox21 Regulates AnapclO Expression
and Determines the Fate of Ectodermal Organ. iScience.
2020;23(7):101329. doi:10.1016/j.isci.2020.101329
86. Gritli-Linde A, Hallberg K, Harfe BD, et al. Abnormal Hair Development
and Apparent Follicular Transformation to Mammary Gland
in the Absence of Hedgehog Signaling. Dev Cell. 2007;12(1):99-112.
doi:10.1016/j.devcel.2006.12.006
87. Sahu S, Sahoo S, Sullivan T, et al. Spatiotemporal modulation of
growth factors directs the generation of multilineage mouse embryonic
stem cell-derived mammary organoids. Dev Cell.
2024;59(2):175-186.e8.doi:10.1016/j.devcel.2023.12.003
88. Lee J, Böscke R, Tang PC, Hartman BH, Heller S, Koehler KR. Hair
Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin
Organoids. Cell Rep. 2018;22(l):242-254.
doi:10.1016/j.celrep.2017.12.007
89. Zaret KS. Genetic programming of liver and pancreas progenitors:
lessons for stem-cell differentiation. Nat Rev Genet. 2008;9(5):329-
340. doi:10.1038/nrg2318
81
B I B L I O G R A P H Y
90. Si-Tayeb K, Lemaigre FP, Duncan SA. Organogenesis and development
of the liver. Dev Cell. 2010;18(2):175-189.
doi:10.1016/j.devcel.2010.01.011
91. Lyra-Leite DM, Gutierrez-Gutierrez Ö, Wang M, Zhou Y, Cyganek L,
Burridge PW. A review of protocols for human iPSC culture, cardiac
differentiation, subtype-specification, maturation, and direct reprogramming.
STAR Protoc. 2022;3(3):101560.
doi:10.1016/j.xpro.2022.101560
92. Cai J, DeLaForest A, Fisher J, et al. Protocol for directed differentiation
of human pluripotent stem cells toward a hepatocyte fate. In:
StemBook. Harvard Stem Cell Institute; 2008. Accessed August 16,
2025. http://www.ncbi.nlm.nih.gov/books/NBK133278/
93. Fowler JL, Ang LT, Loh KM. A critical look: challenges in differentiating
human pluripotent stem cells into desired cell-types and organoids.
Wiley Interdiscip Rev Dev Biol. 2020;9(3):e368.
doi:10.1002/wdev.368
94. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human
dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad
Sei. 2000;97(25):13625-13630. doi:10.1073/pnas.240309797
95. Komori T. Roles of Runx2 in Skeletal Development. Adv Exp Med
Biol. 2017;962:83-93. doi:10.1007/978-981-10-3233-2_6
96. Zilberfarb V, Siquier K, Strosberg AD, Issad T. Effect of dexamethasone
on adipocyte differentiation markers and tumour necrosis
factor-alpha expression in human PAZ6 cells. Diabetologia.
2001;44(3):377-386. doi:10.1007/s001250051630
97. Delia Bella E, Buetti-Dinh A, Licandro G, et al. Dexamethasone Induces
Changes in Osteogenic Differentiation of Human Mesenchymal
Stromal Cells via S0X9 and PPARG, but Not RUNX2. IntJMol
Sei. 2021;22(9):4785. doi:10.3390/ijms22094785
82
B I B L I O G R A P H Y
98. SabbaghJ, Ghassibe-Sabbagh M, Fayyad-Kazan M, et al. Differences
in osteogenic and odontogenic differentiation potential of DPSCs
and SHED./Dent. 2020;101:103413.
doi:10.1016/j.jdent.2020.103413
99. Teti G, Salvatore V, Focaroli S, et al. In vitro osteogenic and odontogenic
differentiation of human dental pulp stem cells seeded on carboxymethyl
cellulose-hydroxyapatite hybrid hydrogel. Front Physiol.
2015;6. doi:10.3389/fphys.2015.00297
100. Gopinath VK, Soumya S, Jayakumar MN. Osteogenic and odontogenic
differentiation potential of dental pulp stem cells isolated
from inflamed dental pulp tissues (I-DPSCs) by two different methods.
Acta OdontolScand. 2020;78(4):281-289.
doi:10.1080/00016357.2019.1702716
101. Madan AK, Kramer B. Immunolocalization of fibroblast growth
factor-2 (FGF-2) in the developing root and supporting structures
of the murine tooth./Afo/ Histol. 2005;36(3):171-178.
doi:10.1007/sl0735-005-2684-l
102. Sagomonyants K, Kalajzic I, Maye P, Mina M. Enhanced Dentinogenesis
of Pulp Progenitors by Early Exposure to FGF2./ Dent Res.
2015;94(11):1582-1590. doi:10.1177/0022034515599768
103. Jiang HB, Tian WD, Liu LK, Xu Y. In vitro odontoblast-like cell differentiation
of cranial neural crest cells induced by fibroblast
growth factor 8 and dentin non-collagen proteins. Cell Biol Int.
2008;32(6):671-678. doi:10.1016/j.cellbi.2008.01.293
104. Zhang M, Zhang X, Luo J, et al. Investigate the Odontogenic Differentiation
and Dentin-Pulp Tissue Regeneration Potential of Neural
Crest Cells. Front Bioeng Biotechnol. 2020;8.
doi:10.3389/fbioe.2020.00475
83
B I B L I O G R A P H Y
105. Takada K, Odashima A, Onodera S, et al. The effect of BMP4,
FGF8 and WNT3a on mouse iPS cells differentiating to odontoblastlike
cells. Med Mol Morphol. 2022;55(3):199-209.
doi:10.1007/s00795-022-00318-9
106. Sarkar J, Simanian EJ, Tuggy SY, et al. Comparison of two mouse
ameloblast-like cell lines for enamel-specific gene expression. Front
Physiol. 2014;5:277. doi:10.3389/fphys.2014.00277
107. Nakata A, Kameda T, Nagai H, et al. Establishment and characterization
of a spontaneously immortalized mouse ameloblast-lineage
cell line. Biochem Biophys Res Commun. 2003;308(4):834-839.
doi:10.1016/S0006-291X(03)01467-0
108. Zou T, Ma L, Gu L, Xi S, Zhang K, Guo X. Role of Wnt/ß-catenin
signaling pathway in ameloblast differentiation in relevance to dental
fluorosis. Chem Biol Interact. 2022;367:110145.
doi:10.1016/j.cbi.2022.110145
109. Chen J, Zhang Y, Mendoza J, DenBesten P. Calcium-mediated differentiation
of ameloblast lineage cells in vitro. J Exp Zoolog B Mol
DevEvol. 2009;312B(5):458-464. doi:10.1002/jez.b.21279
110. Tabata MJ, Matsumura T, Fujii T, Abe M, Kurisu K. Fibronectin
accelerates the growth and differentiation of ameloblast lineage
cells in vitro. J Histochem Cytochem Off J Histochem Soc.
2003;51(12):1673-1679. doi:10.1177/002215540305101211
111. Hemeryck L, Hermans F, Chappell J, et al. Organoids from human
tooth showing epithelial sternness phenotype and differentiation
potential. Cell Mol Life Sei. 2022;79(3):153. doi:10.1007/s00018-
022-04183-8
112. Arakaki M, Ishikawa M, Nakamura T, et al. Role of EpithelialStem
Cell Interactions during Dental Cell Differentiation*. J Biol
Chem. 2012;287(13):10590-10601. doi:10.1074/jbc.M111.285874
84
B I B L I O G R A P H Y
113. Kim EJ, Yoon KS, Arakaki M, et al. Effective Differentiation of Induced
Pluripotent Stem Cells Into Dental Cells. Dev Dyn.
2019;248(1):129-139. doi:10.1002/dvdy.24663
114. He B, Kram V, Furusawa T, et al. Epigenetic Regulation of Ameloblast
Differentiation by HMGN Proteins./Dent Res. 2024;103(1):51-
61. doi:10.1177/00220345231202468
115. Cai J, Zhang Y, Liu P, et al. Generation of tooth-like structures
from integration-free human urine induced pluripotent stem cells.
Cell Regen. 2013;2(1):2:6. doi:10.1186/2045-9769-2-6
116. Bei M. Molecular Genetics of Tooth Development. Curr Opin
Genet Dev. 2009;19(5):504-510. doi:10.1016/j.gde.2009.09.002
117. Pagella P, Roditi L de V, Stadlinger B, Moor AE, Mitsiadis TA. A
single-cell atlas of human teeth. [Science. 2021;24(5).
doi:10.1016/j.isci.2021.102405
118. Sharir A, Marangoni P, Zilionis R, et al. A large pool of actively
cycling progenitors orchestrates self-renewal and injury repair of
an ectodermal appendage. Nat Cell Biol. 2019;21(9):1102-1112.
doi:10.1038/s41556-019-0378-2
119. Qiu C, Cao J, Martin BK, et al. Systematic reconstruction of cellular
trajectories across mouse embryogenesis. Nat Genet.
2022;54(3):328-341.doi:10.1038/s41588-022-01018-x
120. Farrell JA, Wang Y, Riesenfeld SJ, Shekhar K, Regev A, Schier AF.
Single-cell reconstruction of developmental trajectories during
zebrafish embryogenesis. Science. 2018;360(6392):eaar3131.
doi:10.1126/science.aar3131
121. La Manno G, Soldatov R, Zeisel A, et al. RNA velocity of single
cells. Nature. 2018;560(7719):494-498. doi:10.1038/s41586-018-
0414-6
85
B I B L I O G R A P H Y
122. Haque A, EngelJ, Teichmann SA, Lönnberg T. A practical guide to
single-cell RNA-sequencing for biomedical research and clinical applications.
Genome Med. 2017;9(1):75. doi:10.1186/sl3073-017-
0467-4
123. Greenblatt MB, Ono N, Ayturk UM, Debnath S, Lalani S. The Unmixing
Problem: A Guide to Applying Single-Cell RNA Sequencing to
Rone. J Bone Miner Res. 2019;34(7):1207-1219.
doi:10.1002/jbmr.3802
124. Machado L, Relaix F, Mourikis P. Stress relief: emerging methods
to mitigate dissociation-induced artefacts. Trends Cell Biol.
2021;0(0).doi:10.1016/j.tcb.2021.05.004
125. Ferreira PG, Muňoz-Aguirre M, Reverter F, et al. The effects of
death and post-mortem cold ischemia on human tissue transcriptomes.
NatCommun. 2018;9(1):490. doi:10.1038/s41467-017-
02772-x
126. Křivánek J, Lavický J, Bouderlique T, Adameyko I. Rapid Isolation
of Single Cells from Mouse and Human Teeth. JoVEJ Vis Exp.
2021;(176):e63043.doi:10.3791/63043
127. Alghadeer A, Hanson-Drury S, Patni AP, et al. Single-cell census
of human tooth development enables generation of human enamel.
DevCell. 2023;58(20):2163-2180.e9.
doi:10.1016/j.devcel.2023.07.013
128. Hanson-Drury S, Patni AP, Lee DL, et al. Single Cell RNA Sequencing
Reveals Human Tooth Type Identity and Guides In Vitro
hiPSC Derived Odontoblast Differentiation (iOB). Front Dent Med.
2023;4. doi:10.3389/fdmed.2023.1209503
129. Ding Y, Tamhankar S, Du F, et al. ETV2 Overexpression Promotes
Efficient Differentiation of Pluripotent Stem Cells to Endothelial
86
B I B L I O G R A P H Y
Cells. BiotechnolBioeng. 2025;122(7):1914-1928.
doi:10.1002/bit.28979
130. Tomaz RA, Zacharis ED, Bachinger F, et al. Generation of functional
hepatocytes by forward programming with nuclear receptors.
Quinn MA, Zaidi M, eds. eLife. 2022;ll:e71591.
doi:10.7554/eLife.71591
131. Pawlowski M, Ortmann D, Bertero A, et al. Inducible and Deterministic
Forward Programming of Human Pluripotent Stem Cells
into Neurons, Skeletal Myocytes, and Oligodendrocytes. Stem Cell
Rep. 2017;8(4):803-812. doi:10.1016/j.stemcr.2017.02.016
132. Zhang Y, Pak C, Han Y, et al. Rapid Single-Step Induction of Functional
Neurons from Human Pluripotent Stem Cells. Neuron.
2013;78(5):785-798. doi:10.1016/j.neuron.2013.05.029
133. Seki D, Takeshita N, Oyanagi T, et al. Differentiation of Odontoblast-Like
Cells From Mouse Induced Pluripotent Stem Cells by
Pax9 and Bmp4 Transfection. Stem Cells Transl Med.
2015;4(9):993-997. doi:10.5966/sctm.2014-0292
134. Hong S, Lee J, Kim JM, Kim SY, Kim HR, Kim P. 3D cellular visualization
of intact mouse tooth using optical clearing without decalcification.
IntJ Oral Sei. 2019;ll(3):l-8. doi:10.1038/s41368-019-
0056-z
135. Tokavanich N, Wein MN, English JD, Ono N, Ono W. The Role of
Wnt Signaling in Postnatal Tooth Root Development. Front Dent
Med. 2021;2. doi:10.3389/fdmed.2021.769134
136. Pennica D, Swanson TA, Welsh JW, et al. WISP genes are members
of the connective tissue growth factor family that are up-regulated
in Wnt-1-transformed cells and aberrantly expressed in human
colon tumors. Proc Natl Acad Sei. 1998;95(25):14717-14722.
doi:10.1073/pnas.95.25.14717
87
B I B L I O G R A P H Y
137. Han XL, Liu M, Voisey A, et al. Post-natal effect of overexpressed
DKK1 on mandibular molar formation./ Dent Res.
2011;90(11):1312-1317. doi:10.1177/0022034511421926
138. Kim TH, Lee JY, Baek JA, et al. Constitutive stabilization of fscatenin
in the dental mesenchyme leads to excessive dentin and cementum
formation. Biochem Biophys Res Commun.
2011;412(4):549-555.doi:10.1016/j.bbrc.2011.07.116
139. Kim TH, Bae CH, Jang EH, et al. Collal-cre mediated activation of
(3-catenin leads to aberrant dento-alveolar complex formation. Anat
Cell Biol 2012;45(3):193-202. doi:10.5115/acb.2012.45.3.193
140. Lin Y, Xiao Y, Lin C, et al. SALL1 regulates commitment of odontoblast
lineages by interacting with RUNX2 to remodel open chromatin
regions. Stem Cells. 2021;39(2):196-209.
doi:10.1002/stem.3298
141. Nakatake Y, Fujii S, Masui S, et al. Kinetics of drug selection systems
in mouse embryonic stem cells. BMC Biotechnol.
2013;13(1):64. doi:10.1186/1472-6750-13-64
142. O'Flanagan CH, Campbell KR, Zhang AW, et al. Dissociation of
solid tumor tissues with cold active protease for single-cell RNAseq
minimizes conserved collagenase-associated stress responses.
Genome Biol. 2019;20(1):210. doi:10.1186/sl3059-019-1830-0
143. Rico-Llanos G, Spoutil F, Blahova E, et al. Achondroplasia: aligning
mouse model with human clinical studies shows crucial importance
of immediate postnatal start of the therapy. J Bone Miner
Res. 2024;39(12):1783-1792. doi:10.1093/jbmr/zjael73
144. Jing D, Zhang S, Luo W, et al. Tissue clearing of both hard and
soft tissue organs with the PEGASOS method. Cell Res.
2018;28(8):803-818. doi:10.1038/s41422-018-0049-z
88
B I B L I O G R A P H Y
145. Jin BH, Woo J, Lee M, et al. Optimization of the optical transparency
of bones by PACT-based passive tissue clearing. Exp Mol Med.
2023;55(10):2190-2204. doi:10.1038/sl2276-023-01089-8
146. Jing D, Yi Y, Luo W, et al. Tissue Clearing and Its Application to
Bone and Dental Tissues./Dent Res. 2019;98(6):621-631.
doi:10.1177/0022034519844510
147. Wang H, Cheng P, Wang J, et al. Advances in spatial transcriptomics
and its application in the musculoskeletal system. Bone
Res. 2025;13(1):54. doi:10.1038/s41413-025-00429-w
148. Rombaut M, Boeckmans J, Rodrigues RM, Grunsven LA van,
Vanhaecke T, Kock JD. Direct reprogramming of somatic cells into
induced hepatocytes: Cracking the Enigma code./ Hepatol.
2021;75(3):690-705.doi:10.1016/j.jhep.2021.04.048
149. Joung J, Ma S, Tay T, et al. A transcription factor atlas of directed
differentiation. Cell. 2023;186(l):209-229.e26.
doi:10.1016/j.cell.2022.11.026
150. Thomasen PB, Salasova A, Kjaer-Sorensen K, et al. SorCS2 binds
progranulin to regulate motor neuron development. Cell Rep.
2023;42(11). doi:10.1016/j.celrep.2023.113333
89
R A P I D ISOLATION O F SINGLE C E L L S F R O M M O U S E A N D H U M A N T E E T H
Appendix A Rapid isolation of single cells
from mouse and human teeth
This section contains the corresponding paper.
90
jpve
Rapid Isolation of Single Cells from Mouse and Human
Teeth
2,3
Jan Krivanek1
, Josef Lavičky1
, Thibault Bouderlique2
, Igor Adameyko
1 2
Department of Histology and Embryology, Faculty of Medicine, Masaryk University Department of Molecular Neuroimmunology, Centre for Brain
3
Research, Medical University of Vienna Department of Physiology and Pharmacology, Karolinska Institute
Corresponding Authors
Jan Krivanek
jan.krivanek@med.muni.cz
Igor Adameyko
igor.adameyko@meduniwien.ac.at
Citation
Krivanek, J., Lavický, J . , Bouderlique, T.,
Adameyko, I. Rapid Isolation of Single
Cells from Mouse and Human Teeth. J.
Vis. Exp. 0, e63043, doi:10.3791/63043
(2021).
Date Published
October 27, 2021
DOI
10.3791/63043
URL
jove.com/video/63043
Mouse and human teeth represent challenging organs for quick and efficient cell
isolation for single-cell transcriptomic or other applications. The dental pulp tissue,
rich in the extracellular matrix, requires a long and tedious dissociation process that
is typically beyond the reasonable time for single-cell transcriptomics. For avoiding
artificial changes in gene expression, the time elapsed from euthanizing an animal until
the analysis of single cells needs to be minimized. This work presents a fast protocol
enabling to obtain single-cell suspension from mouse and human teeth in an excellent
quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on
accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation
of final single-cell suspension. This enables fast and gentle processing of tissues and
allows using more animal or human samples for obtaining cell suspensions with high
viability and minimal transcriptional changes. It is anticipated that this protocol might
guide researchers interested in performing the scRNA-seq not only on the mouse or
human teeth but also on other extracellular matrix-rich tissues, including cartilage,
dense connective tissue, and dermis.
Introduction
Single-cell RNA sequencing is a powerful tool for deciphering
in vivo cell population structure, hierarchy, interactions, and
homeostasis1
'2
. However, its results strongly depend on
the first step of this advanced analysis - the preparation
of a single-cell suspension of perfect quality out of the
complex, well-organized tissue. This encompasses keeping
cells alive and preventing unwanted, artificial changes in gene
expression profiles of the c e l l s 3 , 4
. Such changes might lead
to the inaccurate characterization of population structure and
misinterpretation of the collected data.
Specific protocols for the isolation out of a wide range
of tissues have been d e v e l o p e d 5
' 6
' 7 , 8
. They usually
employ mechanical dissociation in combination with further
incubation with various proteolytic enzymes. These typically
include trypsin, collagenases, dispases, p a p a i n 6
' 7
' 8 , 9
, or
commercially available enzyme mixtures such as Accutase,
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 • • e63043 • Page 1 of 12
jpve
Tryple, etc.5
. The most critical part affecting the transcriptome
quality is enzymatic digestion. It was shown that prolonged
incubation with enzymes at 37 °C influences the gene
expression and causes the upregulation of many stressrelated
g e n e s 1 0 , 1 1 , 1 2
' 1 3
. The other critical parameter of the
isolation process is its overall length, as it has been shown
that cell transcriptomes change after the tissue ischemia1 4
.
This protocol presents an efficient protocol for gentle isolation
of single cells from mouse and human teeth, fasterthan other,
previously utilized protocols for isolation of cells from complex
t i s s u e s 5
' 6
' 9
' 1 1
' 1 3
' 1 5
' 1 6
.
This protocol presents how to quickly dissect soft tissue from
the hard tooth and prepare a single-cell suspension suitable
forscRNA-seq. This method employs only one centrifugation
step and minimizes the effect of unwanted transcriptional
changes by reducing the tissue handling and digestion time
and keeping the tissue and cells at 4 °C most of the time.
The procedure showcases the isolation of cells from mouse
incisors, molar, and human wisdom teeth as an example, but
principally should work for other teeth in various organisms.
The complete protocol is schematically visualized in Figure
1. This protocol has been recently used to generate a dental
cell type atlas obtained from mouse and human teeth1
.
Protocol
All animal experiments were performed according to
the International and local regulations and approved
by the Ministry of Education, youth and sports,
Czech Republic (MSMT-8360/2019-2; MSMT-9231/2020-2;
MSMT-272/2020-3). This protocol was tested with both
male and female wildtype C57BL/6 and CD-1 mice and
with genetically modified Sox10::iCreERT 2
mice1 7
(combined
with various reporter systems) on a C57BL/6 background.
Experiments with human samples were performed with the
approval of the Committees for Ethics of the Medical Faculty,
Masaryk University Brno & St. Anne's Faculty Hospital in
Brno, Czech republic.
1. Experimental set-up and preparation of
solutions
1. Instrument set-up
1. Cool down the centrifuge to 4 °C.
2. Heat the incubation chamber to 37 °C.
3. Start the Fluorescence-activated cell sorting (FACS)
machine and set all the temperatures of the sorter
(including collection tube holder) to 4 °C. Perform the
instrument quality control, set the drop delay.
4. Set the preliminary gating strategy and perform the
test sorting.
NOTE: When using FACS, use the 100 urn nozzle.
2. Prepare the solutions (steps 1.2.1-1.2.3).
1. Wash solution: Prepare fresh 2% FBS (Fetal Bovine
Serum) in HBSS (Hanks' Balanced Salt Solution).
2. Digestion mixture: Prepare fresh collagenase P (3
U/mL) fully dissolved in HBSS.
3. Optional: Prepare fresh HBSS + BSA (0.04%) and
chill analytical grade methanol in a -20 °C freezer for
storing single-cell suspension at -80 °C.
NOTE: Step 1 needs to be performed before the start
of the experiment. The composition of the utilized
solutions is summarized in Supplementary Table 1.
2. Preparation of experimental animal/s and
human tooth
1. Prepare the experimental animals (mouse).
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1. Euthanize the mouse according to the local
regulations; e.g. by anaesthetics overdose as
described previously1
.
CAUTION: Regulations for humane euthanizing of
experimental animals varies locally. Always follow
valid local regulations.
2. Immediately proceed to the tissue dissection step
(step 3).
NOTE: If the tissue from the experimental animals
cannot be dissected immediately (e.g., because
of transfer from animal housing facility), place the
experimental animals on ice and perform tissue
dissection as soon as possible. To obtain more cells
from mouse molar pulps, use younger (6 weeks and
less) animals. With increasing age, the size of dental
pulp decreases. Mouse incisors mostly keep their
structure with increasing age so animals of various
ages can be used for tissue dissection.
2. Prepare the human tooth
NOTE: Human teeth were extracted for a clinically
relevant reason. Every diagnosis was treated
individually, and an experienced dental surgeon always
performed the tooth extraction.
1. Put the freshly extracted tooth immediately into a 50
ml_ tube with ice-cold HBSS and keep the tube on
ice until further processing.
NOTE: Using retained wisdom teeth from patients
until age 25-30 is recommended for the highest cell
yield.
3. Tissue dissection
1. Hold the experimental animal behind its head, looking on
the ventral aspect of its head so that the tail points away.
2. Using small, sharp scissors, quickly remove the skin
from the mandible to expose the mandibular arch, the
soft tissue between each half of the mandibles, and the
adjacent facial muscles.
1. Make a deep cut from each side of the mandible;
firstly, through m. masseter along the buccal side
of the mandible up to the temporomandibular joint,
and then along the inner part of each half of the
mandible through the base of the oral cavity (see
Supplementary Figure 1).
3. Cut all the muscles and ligaments along the mandible
up to the temporomandibular joint from both outside and
inside of the oral cavity.
NOTE: Avoid cutting bones. This might damage the most
apical part of the incisor.
4. Grasp the mandible using bent tip tweezers and remove
it. Then, split the dissected mandible into two halves with
scissors by cutting through mandibular symphysis (see
Supplementary Figure 1).
5. Use an industrial low lint wipe to chafe the remaining soft
tissue from each half of the mandible. After both parts of
the mandible are cleaned, place them into a pre-prepared
Petri dish with ice-cold HBSS.
NOTE: From this point forward, work on ice. Further
dissection of mouse incisors and molars is performed
under a stereomicroscope with a black background.
6. Mouse mandibular incisors
1. For the dissection of mandibular incisors, remove
the alveolar ridge with all three molars and
transversally crack the mandibular arch in the place
corresponding to the position between the first and
second molar.
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NOTE: A sharp scalpel blade no. 11 and tweezers
are used to perform this step (see Table of
Materials).
2. Carefully pull the incisor out of the rest of the dental
socket.
NOTE: If successful, the extracted incisor will
contain intact apical parts, including epithelial tissue
with complete cervical loops.
3. If needed, remove the remaining fragments of bone
still attached to the incisor with tweezers and a
scalpel.
4. Place the dissected incisors into fresh, ice-cold
HBSS and dissect the tissue of interest: cervical
loop, dental pulp, or other parts of the tooth.
5. Place the dissected soft tissue into a droplet of fresh,
ice-cold HBSS in the middle of a 10 cm Petri dish.
Keep on ice.
7. Mouse mandibular molars
1. For dissecting mandibular molars, completely
remove the alveolar ridge from the rest of the
mandible using a scalpel blade.
2. Move the dissected alveolar crest into fresh, ice-cold
HBSS in a Petri dish and carefully remove all the
remaining fragments of alveolar bones attached to
the roots.
3. Place the dissected molars without alveolar bone
into fresh, ice-cold HBSS. To expose the pulp, start
to remove parts of the dentin from the apical side
using fine, sharp tip tweezers until you reach the pulp
cavity.
4. Once the pulp cavity is reached, carefully dissect
dental pulp using a pair of sharp tip tweezers and
place the soft tissue of the dental pulp into a droplet
of fresh, ice-cold HBSS in the middle of a 10 cm Petri
dish kept on ice.
NOTE: Since mouse molar pulps are extremely
small, adapt magnification on the stereomicroscope
accordingly.
8. Human tooth
1. Wash human tooth once again in ice-cold HBSS to
remove the remaining blood.
2. Place the tooth into three thick-walled sterile plastic
bags and use a cast iron benchtop engineer's vise
to crack the tooth. Use vise jaws with a flat surface
to avoid penetrating the bags.
3. Slowly tighten the vise until you hear the tooth
cracking; remove it from the bags and place it into
fresh, ice-cold HBSS in a Petri dish.
4. Using two tweezers, take out the dental pulp, clean
it from all the remnants of hard tissue and place it
into one droplet of ice-cold HBSS in the middle of a
10 cm Petri dish kept on ice.
CAUTION: Human tissue might potentially be
infectious. When working with human tissue, use
protective equipment to avoid direct contact with the
tissue.
4. Preparation of single-cell suspension
1. Prepare a 15 ml_ tube with 2.5 ml_ of digestion mixture
composed of Collagenase P (3 U/mL) fully dissolved in
HBSS. Keep the solution on ice until use.
NOTE: Always use freshly prepared Collagenase P; do
not freeze the aliquots. Collagenase P activity varies from
batch-to-batch. Check the activity of your batch before
diluting. Collagenase P is activated by calcium, whose
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amount is already sufficient in the lyophilized powder.
Therefore, it is unnecessary to enrich the enzyme
mix with calcium. Collagenase P is not inactivated
by FBS but can be inactivated by chelating agents
(e.g., Ethylenediaminetetraacetic - EDTA). Stopping
the dissociation process is ensured by diluting the
Collagenase P in Wash solution (2% FBS in HBSS). It
has been shown that adding FBS increases cell viability
and the final number of cells after sorting1 8
.
2. Using a round-shaped scalpel blade no. 10, cut the
tissue in all directions into the smallest possible pieces.
Reaggregate the tissue pieces in the middle of the droplet
and repeatedly cut the tissue aggregates using a roundshaped
(No. 10) scalpel blade. Repeat this process
several times until the material is sufficiently minced.
3. Transfer the shredded tissue pieces using a 1 ml_ pipette
tip into the prepared digestion mixture.
NOTE: In the case of a larger number of animals being
processed at once, split the tissue into several tubes
with Collagenase P. The maximum amount per tube are
pulps obtained from approximately 10 mouse incisors. In
the case of molars, where pulps are much smaller, the
number of processed pulps can be increased adequately.
When using human teeth, use a maximum of 2-3 pulps
per tube, depending on the pulp size.
4. Place the tube into a 37 °C preheated incubator with a
shaker. Tilt the tube inside the shaker to an angle of 60°
and set the speed to 150-200 rpm to ensure constant
suspension movements inside the tube.
5. Vigorously triturate the suspension every 3-4 min with a
1 mL pipette tip to disintegrate all the clumps.
NOTE: Wth time, the clumps will become smaller and
softer until they almost disappear.
6. In total, incubate for 15-20 min. At the end of the
incubation, triturate for the last time, and then slowly add
ice cold Wash solution to a final volume of 12 mL.
7. Remove the remaining clumps or pieces of calcified
tissue by filtering the suspension using a 50 urn cell
strainer.
8. Take 10-20 uL of the filtered cell suspension and count
the cells during centrifugation using a cell counting
chamber (Hemocytometer). Centrifuge for 5 min at 300
x g at 4 °C Remove the supernatant using a 10 mL
serological pipette.
NOTE: If the number of cells is limited that cannot be
counted in diluted suspension, skip the cell counting and
use all the cells for further processing.
9- Optional: Proceed for fixation and storage2 1
.
1
• Re-suspend the pellets (up to 107
cells) in 100 uL of
HBSS + BSA (0.04%).
2. Add 400 uL of chilled methanol and mix slowly.
3. Incubate the cell suspension for 15 min on ice.
4. Store at -80 °C until processed (no longer than 1
month).
10. Resuspend the pellet in the Wash solution. Aim for
700-1200 cells/uL of the Wash solution.
11. Keep the tube on ice until further processing.
NOTE: If necessary, perform the fixation and storage
at -80 °C However, immediate processing of the cell
suspension for scRNA-seq is recommended. Further
steps will depend based on the single-cell RNA
seq protocol. When the scRNA-seq protocol uses a
microfluidic system (some sc-RNA-seq companies do),
load the cells based on the manufacturer's guidelines
either directly or after cell sorting.
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jpve
12. For FACS, proceed to step 5.
5. Fluorescence-activated cell sorting (FACS)
1. Before sorting, prepare the cell sorting instrument.
1. Cool down the whole system to 4 °C, perform the
instrument quality control, set the drop delay and the
voltage of the deflection plates. Put collection tubes
in the collection tube holder.
NOTE: To avoid additional centrifugation steps
resulting in an inevitable cell loss, sort cells into a
microtube (1.5 ml_) with a small amount (25-50 ul_)
of the Wash solution.
2. Optional: perform viability staining before FACS.
1. Add Propidium iodide into cell suspension before
FACS to a final concentration 0.5 ug/mL and
incubate for 15 min at 4 °C.
3. Load the sample into the cell sorter. Set a strict gating
strategy to remove cell debris, doublets, and dead cells.
Sort the cells into a prepared tube or multi-well plates. An
example of a gating strategy is shown in Figure 2.
NOTE: To minimize manipulation steps and accelerate
the protocol, applying a strict gating strategy is
recommended (suggested in Figure 2) rather than using
a viability staining. To separate the immune cells from
the isolated population, CD45 antibody staining can be
performed. To perform this, resuspend cell suspension
in 100 uL of staining solution (PBS + 2% FBS + antiCD45-APC
conjugated antibody 1/100 dilution). Incubate
for 15 min on ice protected from light and perform FACS
directly.
Representative Results
Exemplary isolation of single cells was performed from
two mandibular incisors from one 6-week-old C57BL/6
mouse male. Following this protocol, a single-cell suspension
was prepared, and subsequently, single-cell sequencing
was performed. The prepared single-cell suspension was
analyzed and sorted using FACS (Figure 2). Firstly, the FSCA
(forward scatter, area) and SSC-A (side scatter, area)
plotting was applied, and an appropriate gating strategy was
used to select a population with expected size and granularity
to filter our cell debris and cell doublets or aggregates (Figure
2A). This selected population (P1), counting 38% of all
events, was further used, and FSC-A and FSC-H (forward
scatter, height) parameters were applied to remove the
remaining cell doublets (Figure 2B). The population without
cell doublets (P2) counting 95% of P1 can be subsequently
used for scRNA-seq. Alternatively, additional gating can be
used to select the population of interest (e.g., expression
of fluorescent proteins or live/dead staining). To check the
number of live/dead cells in the final suspension, the PI
(propidium iodide) staining was performed (Figure 2C). The
P3 population containing PI" (living) cells was 98.4% out of
the parent P2 population and 35.5% out of the total events.
The total number of filtered out, dead (Pl+
) cells was 1887.
The final number of cells obtained without viability staining
suitable for RNA-seq (P2) counted 118,199 cells from two
mouse incisor pulps. This means the numberof almost 60,000
living cells from one mandibular incisor.
To clarify the number of immune cells in the final singlecell
suspension, two approaches were used. Firstly, the
CD45 antibody staining and subsequent FACS analysis were
used. As a complementary method, the total number of
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immune cells (CD45+) in scRNA-seq data was analysed.
FACS analysis showed 14.44% of CD45+ cells (13.20% alive
and 1.24% dead) (Figure 3A). Analysis of scRNA-seq data
showed 10.90% of CD45-expressing cells (Figure 3B). The
decrease of CD45+ cells in scRNA-seq data can be caused
by additional thresholding during scRNA-seq analysis.
These representative data on the example of mouse incisor
show that the given protocol in combination with strict gating
strategy is efficient in obtaining a high number of cells out of
a single mouse tooth without the necessity of additional use
of viability staining. The ratio of immune (CD45+) cells was
minor (13.2%). Moreover, it was previously shown that the
immune cells are essential in maintaining tooth homeostasis,
so removing them from scRNA-seq analysis during the FACS
would be counterproductive in some applications.
Figure 1: Schematic representation of the protocol. Different steps, including temperature conditions and expected time,
are represented to prepare single-cell suspension from mouse and human teeth. Please click here to view a larger version of
this figure.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 • • e63043-Page7of 12
jpve
B
I 150
6
c
3
D
50 100 150
F S C - A
200 250 50 100 150 200 250
F S C - A
1V 10- 10*
PI
101
-
Population
• A l Events
• E w r t »
318
124.380
113.199
ar«it%1c*ai
##*# 1D0D
33.0 39 0
95.0 36 1
• P 3 I 116.312 98.4 35 5 I
Figure 2: Example of the gating strategy. FSC-A and SSC-A gating was used to produce the P1 gate, reflecting the cell
population with expected size and granularity and filtering out the cell and extracellular matrix debris and most large events
(A). Subsequently, the P1 population was plotted in FSC-H and FCS-A plot, which filtered out cell doublets (B). This P2
population was then analyzed for the presence of dead cells by propidium iodide (C). The number of events/cells per gate
are represented in (D). (FSC-A - forward scatter, area; SSC-A - side scatter, area; FSC-H - forward scatter, height; PI propidium
iodide). Please click here to view a larger version of this figure.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 • • e63043-Page 8 of 12
jpve
A C D 4 5 * cells: FACS analysis B C D 4 5 * cells: scRNA-seq
o 1
CD45- Dead CD45+
Dead
2.20 % 1 24 %
•-x:y
~.ir-\ • t \
' ':~':i.r*
CD45
Live CD45+
Live
83.3 % 13.2%
I" 1
• I 1
•1
^ ? ISM Jilfelfl
i f c A Expression
10.9%
0 10
C D 4 5 - A P C
t - S N E embedding
Figure 3: Quantification of the immune cells. Quantification of the immune cells was performed by FACS analysis of
cells stained with anti-CD45 antibody and Live/Dead analysis using propidium iodide staining (A). Further quantification of
immune cells was performed during scRNA-seq analysis (B). (CD45-APC - anti-CD45 allophycocyanin conjugated antibody;
PI - propidium iodide; t-SNE - t-distributed stochastic neighbor embedding). Please click here to view a larger version of this
figure.
Supplementary Figure 1: Overview of the mandible
dissection process. Dashed lines illustrate suggested cuts.
TMJ - temporomandibular joint, m. masseter - musculus
masseter. Please click here to download this File.
Supplementary Table 1: The compositions of the
solutions used in the study. Please click here to download
this Table.
Discussion
Studying teeth and bones on the cellular or molecular level
is generally challenging since cells forming these tissues
are surrounded by different kinds of hard matrices1 9
. One
of the main goals for performing single-cell RNA-seq on
dental tissue is the need to obtain cells of interest fast
and without any artificial changes in their transcriptomes.
To accomplish this, a highly efficient protocol suitable for
isolating cells from mouse and human tooth pulps was
developed, which allows for quick generation of single-cell
suspensions for all transcriptomic applications. This was
ensured by fast tissue isolation, minimizing the steps of tissue
and cell manipulations, and streamlining the mechanical and
enzymatic digestion.
The most critical steps of this protocol are fast
tissue processing and adequate single-cell suspension
preparation8
'9
. A manual approach is used to obtain dental
pulps without utilizing a dental drill or other heat-generating
devices. Overheating may cause an artificial expression of
heat shock proteins and other genes, ultimately leading to the
analyzed gene expression patterns being unrepresentative
of the original tissue2 0
. Manual tissue harvesting may
be a challenging step that will likely need some training
beforehand. The pulp is then cut into small pieces and
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 • • e63043-Page 9 of 12
jpve
enzymatically digested at 37 °C. Except for the 15-20 min of
enzymatic digestion, the whole protocol is performed at 4 °C.
The tissue processing and especially the enzymatic digestion
were minimized to the shortest possible time since more
prolonged incubation at 37 °C can cause changes in gene
expression patterns1 0
. Mechanical removing of the dentin is
recommended before enzymatic digestion. Dentin and the
pulp-attached predentin contain a large amount of collagen,
and its excessive presence might decrease the effectiveness
of the digesting solution. After being removed from the body
(or death of organisms), it was shown that cells start to
modify their gene expression patterns quickly1 2
. Therefore,
cell isolation and processing should be carried out as fast as
possible. The current protocol reduces the processing time
to 35-45 min from isolating the tissue (euthanizing animal) to
preparing single-cell suspension.
One alternative modification of this technique is cell
preservation for later use. This is achieved by methanol
fixation. Methanol-fixed cell suspension can be stored for up
to 1 month at-80 °C, as described in the protocol2 1
. However,
whenever possible, perform scRNA-seq directly, since it was
shown that the single-cell data from methanol-fixed singlecell
suspensions might suffer from increased expression of
stress-related genes and contamination with ambient R N A 2 2
.
This step might need additional modification according to the
manufacturer's protocols.
Before the first application of this protocol, performing several
validation steps are recommended to test the technique.
From our experience, we suggest testing the aforementioned
critical steps of the protocol. Additionally, we suggest testing
the effectiveness of the collagenase P solution and testing the
handling of the tissue dissociation step. Specifically, around
the first 5 min after the initiation of collagenase P incubation,
the pieces of tissue should aggregate together. This is a
common situation. Aggregates are disintegrated every 3-4
min using a 1 ml_ pipette, and with increasing time, they
should become smaller until barely visible.
Furthermore, it is recommended to perform cell counting in a
cell counting chamber before centrifugation and before and
after filtering to detect possible cell losses due to suboptimal
supernatant removal. If the final single-cell suspension needs
to be purified, FACS can be used. Cell sorting enables not
only to remove debris or dead cells but importantly enables to
enrich final suspension with fluorescently labeled c e l l s 1 3
' 1 9
.
To avoid shear stress or clogging of the cell sorter, a wide
nozzle (85 urn or 100 urn) is used. This will further improve
the viability of the sorted cells.
This technique was designed and tested on both mouse and
human teeth. The major limiting factor is the small number of
cells in the reduced dental pulps of the teeth of older mice
(molars) and humans. Suppose a larger number of cells need
to be obtained or cells from the teeth of older patients are to be
acquired. One possible solution is to process a higher number
of teeth and merge them into a single batch, subsequently
processed as one sample.
Living cells of human dental pulp were firstly isolated
more than twenty years ago using an enzyme mixture of
collagenase I and dispase2 3
. Since then, isolations of dental
pulp cells became widely utilized, and several techniques
have been u s e d 5
' 6
' 7
' 8
. The critical significance of the
method presented here is the adaption of all isolation steps to
make the isolation fast and gentle to ensure the high quality of
the final cell suspension forscRNA-seq. Higher cell yield can
be obtained by more prolonged incubation with enzymes. This
protocol provides an efficient solution for quickly obtaining
single cells from mouse and human teeth of suitable quality
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 - - e63043 - Page 10 of 12
jpve
for single-cell RNA-sequencing. This technique is expected to
be widely used for other tissues or organisms with just slight
technical modifications.
Disclosures
Authors declare no conflicts of interest.
Acknowledgments
J.K. was supported by the Grant Agency of Masaryk
University (MUNI/H/1615/2018) and by funds from the
Faculty of Medicine MU to junior researcher. J.L. was
supported by the Grant Agency of Masaryk University, (MUNI/
IGA/1532/2020) and is a Brno Ph.D. Talent Scholarship
Holder - Funded by the Brno City Municipality. T.B. was
supported by the Austrian Science Fund (Lise Meitner grant:
M2688-B28). We thank to Lydie Izakovicova Holla and
Veronika Kovar Matejova for their help with the obtaining of
human teeth. Finally, we thank Radek Fedr and Karel Soucek
for their kind assistance with FACS sorting.
References
1. Krivanek, J . et al. Dental cell type atlas reveals stem
and differentiated cell types in mouse and human teeth.
Nature Communications. 11 (1), 4816 (2020).
2. Soldatov, R. et al. Spatiotemporal structure of cell fate
decisions in murine neural crest. Science. 364 (6444)
(2019).
3. Machado, L, Relaix, F., Mourikis, P. Stress relief:
emerging methods to mitigate dissociation-induced
artefacts. Trends in Cell Biology. 0 (0) (2021).
4. Nguyen, Q. H., Pervolarakis, N., Nee, K., Kessenbrock,
K. Experimental considerations for single-cell RNA
sequencing approaches. Frontiers in Cell and
Developmental Biology. 6 (2018).
5. Chiba, Y. et al. Single-cell RNA-sequencing from mouse
incisor reveals dental epithelial cell-type specific genes.
Frontiers in Cell and Developmental Biology. 8 (2020).
6. Debnath, S. et al. Discovery of a periosteal stem cell
mediating intramembranous bone formation. Nature. 562
(7725), 133-139 (2018).
7. Kanton, S., Treutlein, B., Camp, J. G. Chapter 10
- Single-cell genomic analysis of human cerebral
organoids. Methodsin Cell Biology. 159,229-256 (2020).
8. Price, F. D. et al. Inhibition of JAK-STAT signaling
stimulates adult satellite cell function. Nature Medicine.
20 (10), 1174-1181 (2014).
9. van den Brink, S. C. et al. Single-cell sequencing
reveals dissociation-induced gene expression in tissue
subpopulations. Nature Methods. 14 (10), 935-936
(2017).
10. O'Flanagan, C. H. et al. Dissociation of solid tumor
tissues with cold active protease for single-cell RNAseq
minimizes conserved collagenase-associated stress
responses. Genome Biology. 20 (1), 210 (2019).
11. van Veithoven, C. T. J., de Morree, A., Egner, I. M., Brett,
J. O., Rando, T. A. Transcriptional profiling of quiescent
muscle stem cells in vivo. Cell Reports. 21 (7), 1994-2004
(2017).
12. Miyawaki-Kuwakado, A. et al. Transcriptome analysis of
gene expression changes upon enzymatic dissociation
in skeletal myoblasts. Genes to Cells. 26 (7), 530-540
(2021).
13. Mattei, D. et al. Enzymatic dissociation induces
transcriptional and proteotype bias in brain cell
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 • • e63043-Page 11 of 12
jpve
populations. International Journal of Molecular Sciences.
21 (21), 7944 (2020).
14. Ferreira, P. G. et al. The effects of death and postmortem
cold ischemia on human tissue transcriptomes.
Nature Communications. 9 (1), 490 (2018).
15. He, W., Ye, J., Xu, H., Lin, Y., Zheng, Y. Differential
expression of a6 and p1 integrins reveals epidermal
heterogeneity at single-cell resolution. Journal of Cellular
Biochemistry. 121 (3), 2664-2676 (2020).
16. Machado, L. et al. In situ fixation redefines quiescence
and early activation of skeletal muscle stem cells. Cell
Reports. 21 (7), 1982-1993 (2017).
17. Laranjeira, C. et al. Glial cells in the mouse enteric
nervous system can undergo neurogenesis in response
to injury. The Journal of Clinical Investigation. 121 (9),
3412-3424 (2011).
18. Song, X. et al. Improved strategy for jet-in-air cell sorting
with high purity, yield, viability and genome stability.
FEBS Open Bio. 11 (9), 2453-2467 (2021).
19. Greenblatt, M. B., Ono, N., Ayturk, U. M., Debnath, S.,
Lalani, S. The unmixing problem: A guide to applying
single-cell RNA sequencing to bone. Journal of Bone and
Mineral Research. 34 (7), 1207-1219 (2019).
20. Charlebois, D. A., Hauser, K., Marshall, S., Balázsi, G.
Multiscale effects of heating and cooling on genes and
gene networks. Proceedings of the National Academy of
Sciences. 115 (45), E10797-E10806 (2018).
21. Chen, J . et al. PBMC fixation and processing for
Chromium single-cell RNA sequencing. Journal of
Translational Medicine. 16 (1), 198 (2018).
22. Denisenko, E. et al. Systematic assessment of tissue
dissociation and storage biases in single-cell and singlenucleus
RNA-seq workflows. Genome Biology. 21 (1),
130 (2020).
23. Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi,
S. Postnatal human dental pulp stem cells (DPSCs) in
vitro and in vivo. Proceedings of the National Academy
of Sciences of the United States of America. 97 (25),
13625-13630 (2000).
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com October 2021 • • e63043 • Page 12 of 12
S P A T I O T E M P O R A L M O N I T O R I N G O F H A R D T I S S U E D E V E L O P M E N T R E V E A L S
U N K N O W N F E A T U R E S O F T O O T H A N D B O N E D E V E L O P M E N T
Appendix B Spatiotemporal monitoring of
hard tissue development reveals
unknown features of tooth and
bone development
This section contains the corresponding paper.
103
*H) Check for updates
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
DEVELOPMENTAL BIOLOGY
Spatiotemporal monitoring of hard tissue development
reveals unknown features of tooth and bone
development
Marcos Gonzalez Lopez1
, Barbora Huteckova2,3
+, Josef LavickyH, Nikodém Zezula2
,
Vladislav Rakultsev1
, Vendula Fridrichova1
, Haneen Tuaima1
, Cita Nottmeier4
, Julian Petersen4
,
Michaela Kavkova1,5
, Tomas Zikmund5
, Jozef Kaiser5
, Rupali Lav6
, Haza Star6
, Vítězslav Bryja2
,
Petr Henyš7
, Miroslav Vořechovský8
, Abigail S. Tucker6 , 9
, Jakub Harnos2
, Marcela Buchtová2
'3
,
Jan Křivánek1
*
Mineralized tissues, such as bones or teeth, are essential structures of all vertebrates. They enable rapid movement,
protection, and food processing, in addition to providing physiological functions. Although the development,
regeneration, and pathogenesis of teeth and bones have been intensely studied, there is currently no tool
to accurately follow the dynamics of growth and healing of these vital tissues in space and time. Here, we
present the BEE-ST (Bones and tEEth Spatio-Temporal growth monitoring) approach, which allows precise quantification
of development, regeneration, remodeling, and healing in any type of calcified tissue across different
species. Using mouse teeth as model the turnover rate of continuously growing incisors was quantified, and role
of hard/soft diet on molar root growth was shown. Furthermore, the dynamics of bones and teeth growth in
lizards, frogs, birds, and zebrafish was uncovered. This approach represents an effective, highly reproducible,
and versatile tool that opens up diverse possibilities in developmental biology, bone and tooth healing, tissue
engineering, and disease modeling.
Copyright © 2023 The
Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
License 4.0 (CC BY).
INTRODUCTION
Most of hard tissues in vertebrate bodies are centered on the generation
of calcium-based crystalline compounds. This calcium-based
biomineralization provides hard tissues, such as bones and teeth,
with essential mechanical properties. Therefore, these tissues are responsible
for providing a strong supporting body structure, which
allows for efficient movement, protects the body surface and internal
organs, or, in the case of teeth, ensures hard food processing or
predation (2,2).
Having precise information about growth, regeneration, and biomineralization
dynamics in teeth and bones, is one of the basic requirements
for research in many fields including developmental
biology, tissue healing, bone remodeling, tissue engineering, and
disease modeling. One of the major obstacles in current studies is
the absence of methodological approaches that enable observation
of these dynamic processes, which require high-resolution three-dimensional
imaging and temporal information. Current techniques
such as micro-computed tomography (microCT), magnetic
'Department of Histology and Embryology, Faculty of Medicine, Masaryk University,
Brno, Czech Republic, d e p a r t m e n t of Experimental Biology, Faculty of
Science, Masaryk University, Brno, Czech Republic, institute of Animal Physiology
and Genetics, Czech Academy of Sciences, Brno, Czech Republic. 4
Department of
Orthodontics, University of Leipzig Medical Center, Leipzig, Germany.5
Central European
Institute of Technology, Brno University of Technology, Brno, Czech Republic.
6
Centre for Craniofacial and Regenerative Biology, King's College London,
London, UK. institute of New Technologies and Applied Informatics, Faculty of
Mechatronics, Informatics and Interdisciplinary Studies, Technical University of
Liberec, Liberec, Czech Republic, institute of Structural Mechanics, Faculty of
Civil Engineering, Brno University of Technology, Czech Republic, institute of Histology
and Embryology, First Faculty of Medicine, Charles University, Prague, Czech
Republic.
•Corresponding author. Email: jan.krivanekiamed.muni.cz
+These authors contributed equally to this work.
resonance, or ground sectioning provide only limited end-point information,
missing the spatiotemporal dynamics occurring during
the growth, regeneration, and remodeling of calcified structures (3-
6).Thus, these approaches do not provide the information uncovering
the hard tissue dynamics.
Here, we have developed an approach that enables monitoring of
the dynamics of development, growth, healing, and remodeling of
any calcified hard tissues in a three-dimensional context and over
long periods of time (Fig. 1) This technique was adapted for selected
model species across the vertebrates (Mas musculus representing
mammals, Chamaeleo calyptratus representing reptiles, Gallus
gallus representing birds, Danio rerio representing fish, and
Xenopus laevis representing amphibians) (Fig. 2A).
Our approach is based on the sequential controlled administration
of dyes with the natural ability to incorporate into any currently
calcifying tissues at designated time points, followed by the clearing
of the nondecalcified hard tissue and high-resolution three-dimensional
imaging. The ability of substances to incorporate into
forming calcified tissues has been known for more than 50 years,
first observed in tetracycline antibiotics (7, 8). These substances
not only incorporate into newly forming bone tissues but also incorporate
into dentin, enamel, and cementum, if administered
during their formation (9,10). Their fluorescence enables the tracking
of the exact site of matrix deposition during the period of their
presence after in vivo administration. Dozens of these compounds
with different binding efficiencies and fluorescence parameters have
been described (10). The most commonly used include calcein
(green), alizarin (red), xylenol orange, calcein blue, or oxytetracycline
(11). In contrast to tetracycline antibiotics, these substances
have been reported to be harmless in different animal species,
such as mice, guinea pigs, amphibians, rabbits, or dogs at effective
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 1 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
Bones and Teeth S patio-Temporal growth monitoring (BEE-ST)
Species versatility
Mammals Reptiles Birds Amphibians Fish
Sequential administration
of fluorescent dyes
Any calcified tissue
Whole-mount 3D imaging
Measuring
151.88 urn
130.09 |jm
Precise quantification
Quantification
Time (days/weeks)
Various applications
Micrometer-scale monitoring of
tooth and bone growth in vivo
Developmental biology
Bone and tooth healing
Bone remodeling
Tissue engineering
Disease modeling
Fig. 1. Possibilities of the BEE-ST growth monitoring technique application. We present the BEE-ST approach for tracking the dynamics of hard tissue growth in space
and time. Our approach is applicable to all main groups of vertebrates represented by their model organisms: mammals (M. musculus), reptiles (C. calyptratus), birds (G.
gallus), amphibians (X. faew's), and fish (D. rerio). It is based on the sequential administration of calcium-incorporating fluorescent dyes, followed by optical tissue clearing
of fully calcified hard tissues, whole-mount imaging, and subsequent precise quantification of the growth in a micrometer scale. BEE-ST has many potential applications
in fundamental biology, tissue engineering, or disease modeling. Scale bars, 100 um. 3D, three-dimensional.
dosages (12-16). Their sequential administration enables observation
of the dynamics of hard tissue production in a very precise
manner by generating stripes, or regions, of fluorescently labeled
calcified matrix corresponding to the matrix produced in the
short time window after the respective dye was administered. The
rapid development of tissue optical clearing techniques in recent
years has enabled whole-mount imaging of complex structures
and organs, including hard tissues such as bones and teeth (17-
21). In most of these protocols, decalcification step is necessary
before the optical clearing of hard tissues. Unfortunately, the decalcification
process also removes incorporated dyes from the tissue
and, thus, makes it incompatible with the use of aforementioned
dyes. However, in addition, there were developed methods that
enable the optical clearing of fully calcified tissues allowing the parallel
application of dyes incorporating into calcifying hard tissues
with three-dimensional whole-mount imaging (22, 23).
Here, we developed a versatile technique named BEE-ST (Bones
and tEEth Spatio-Temporal growth monitoring) approach, which
takes advantage of whole-mount hard tissue optical clearing that
does not require prior decalcification steps to uncover and quantify
the dynamics of bone and tooth growth or repair. To image the
calcium-binding dyes incorporated at designated time points
(Fig. 2B), we adapted modified Murray's clearing method. This
method is based on the usage of deperoxidized mixture of benzyl
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 2 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
BEE-ST across the species
A Mammals Reptiles Birds Amphibians Fish
Fluorochrome
administration
calcein green
alizarin red
Intraperitoneal injection In ovo injection Dissolved in water
Tissue
collection
Teeth (incisors, molars)
Bones (long, short, or flat)
Bones
(long, short, or flat)
Whole mount
Tissue
fixation
Yes No
Tissue
dehydration
Yes Yes No
Tissue
clearing
Murray's clearing (BABB deperoxidated solution) No
Imaging
Data
analysis
Fluorescence microscopy, confocal microscopy, light sheet microscopy
Image analysis and quantification (ZEN, IMARIS, ImageJ, etc.)
Clearing efficiency on the example of mouse mandible
Not cleared (dyes injected) B E E - S T
Not
cleared (dyes injected) B E E - S T
-I 1 -
O
o
3
O
t f
O
I
C
>5
o
'15
Fig. 2. The step-by-step BEE-ST protocol across the species. A detailed protocol summarizing the key steps of the BEE-ST approach in the representative animal models
for vertebrates is presented in (A). The most effective administration of the dyes is optimized for each species (mammals and reptiles, intraperitoneally; birds, in ovo;
amphibians and fish, dissolved in water). For nonaquatic species, it is required to fix and optically clear the dissected calcified tissue, while for aquatic species, this should
be omitted. The different samples can be visualized according to the specific needs: wide-field fluorescence microscopy for general scans of larger samples, confocal
microscopy for smaller samples and high-resolution three-dimensional imaging, or light sheet microscopy for three-dimensional imaging of larger samples. In (B), the
efficiency of clearing after using BEE-ST in a mouse mandible taken as an example is shown. The efficiency of BEE-ST staining and clearing is visualized using transmitted
light and fluorescent microscope (blue box, not cleared mandible; orange box, cleared mandible). Scale bars, 1 mm.
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 3 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
alcohol and benzyl benzoate (peroxide-free BABB) capable of optically
clearing even fully calcified tissue (22,24,25). This was further
followed by a nontoxic and device-friendly ethyl-cinnamate for safe
imaging. The combination of the fluorescent dyes and calcified
tissue clearing techniques enables the precise measuring of the distances
between the individual stripes of fluorescendy labeled mineralized
tissue with reference to the precise time of their injection.
Together, this allows for precise quantification of bone and tooth
growth and healing in three dimensions on a micrometer scale.
The great advantage of the BEE-ST approach is the nontoxicity at
standard concentrations and the versatility of the selected dyes.
Moreover, we show that even repetitive administration of selected
dyes does not influence calcium deposition or hard tissue morphology.
Another advantage is the possibility to obtain three-dimensional
information about the hard tissue growth over time
without the need for physical destruction of the sample by sectioning
or grinding. This method allows whole-mount imaging and,
thus, prevents the loss of three-dimensional information and the introduction
of artifacts resulting from the physical processing of the
tissue before imaging.
In summary, here, we introduce a robust, fast, versatile, and reproducible
methodology that enables the quantification of the spatiotemporal
dynamics of hard tissues in micrometer scale by a
nondestructive approach. This approach was successfully adapted
for various model organisms across all major vertebrate phylogenetic
groups. Using the example of mouse teeth, unexpected differential
regenerative dynamics in continuously growing incisors in
young and older animals were uncovered, and the influence of
hard and soft food diet on molar root growth was elucidated. We
expect that this universal method will find application in fields
ranging from developmental biology and tissue repair and regeneration
to bone and dental engineering applications.
RESULTS
Analysis of the growth dynamics of mouse incisor and
molars during aging or hard/soft diet
Continuously growing rodent teeth represent a valuable model to
study the development and growth of a complex organ (26, 27).
However, analysis of the dynamics of incisor growth throughout
an animal's lifespan, or visualization of sex-dependent growth
rate differences, requires more sophisticated methodological
approaches.
The BEE-ST approach was used to provide a precise and absolute
measurement of incisor growth rate (elongation) and dentin ingrowths
(thickening of dentin wall). Calcein and alizarin dyes
were alternatingly administered every 168 hours to uncover the dynamics
of the renewal rate of the maxillary and mandibular incisors
(Fig. 3, A and B). Calcification of dentin occurs in a conical manner,
allowing for the growth of the incisors together with the thickening
of the dentin wall (Fig. 3C). Daily (precisely every 24 hours) administration
of calcein together with weekly alizarin administration (as
weekly checkpoints) enabled precise quantification of the incisor
growth rate in length, as well as the thickening of the dentin wall
using optical cross sections through whole-mount samples (Fig. 3,
C to F). Although there were no significant differences observed
between the daily growth rates of female and male maxillary incisors
(young females: 160.2 ± 27.0 [im versus young males: 138.8 ± 20.9
[im; older females: 116.0 ± 17.5 [im versus older males: 134.5 ± 16.8
\im) or mandibular incisors (young females: 243.0 ± 41.0 \im versus
young males: 232.9 ± 53.3 \im; old females: 194.1 ± 42.5 \im versus
older males: 188.3 ± 34.2 [im), the growth rate of both sexes decreased
significantly during aging. The average mandibular
incisor turnover rate was 6.4 weeks in young adults (3 to 5
months) and 8.1 weeks in older adults (13 to 15 months); meanwhile,
the maxillary incisor turnover rate in young adults was 5.5
weeks compared to 6.8 weeks required in older mice (Fig. 3B). Analysis
of incisor dentin wall thickening after daily calcein administration
using optical cross sections uncovered three stages of
odontoblast activity throughout incisor development: acceleration,
production, and termination (Fig. 3D). Using the mouse incisor, we
were able to demonstrate that repetitive use (administered every 24
hours for 56 consecutive days) of selected dyes did not have any
adverse effect on the deposition of dentin and enamel (Fig. 4).
It was uncovered that alizarin and calcein have different
windows of activity after in vivo administration (Fig. 5). Therefore, Q
to quantify daily growth, we used the administration and measure- 1
ment of only calcein. There was a significant shift (a ratio difference o
of 0.876, i.e., 2.98 hours in 1 day between the injections) of lines 8"
observed in the incisor after the same interval (24 hours) of administration.
This difference in incorporation is especially relevant for 3
applications using a daily injection approach and is less significant Jfor
longer periods, where distinguishing between different time £.
points by red and green colors brings more benefits. |
Because of the lack of methodological possibilities, the spatio- ?•
temporal dynamics of root and crown development in mouse §•
molars over time have not been precisely characterized yet. To de- p
scribe the growth dynamics of molars during development, we se- S
quentially administered alizarin and calcein at different 2- and 3- S
day intervals during their development (Fig. 6). Our results show g
the progression of molar root growth in M l (the first molar), M2, 8j
and M3 during an interval between postnatal day 12 (P12) to P30 £
(Fig. 6A). Before root elongation begins, the dental crown develops. 3.
To analyze crown development and early root patterning of the §
mandibular and maxillary M l , we selected 2-day intervals for the •§
administration of alizarin and calcein (Fig. 6, B and C). Our o
results reveal that dentin fusion and the formation of two (in the J"
case of mandibular Ml) and three (in the case of maxillary Ml) £
roots take place after P10 (Fig. 6C). Using the mandibular M l as ui
an example of the potential of combining BEE-ST with three-di- g
mensional confocal imaging was shown to allow generation of
virtual optical sections to demonstrate growth from different
points of view throughout the whole sample (movies SI and S2).
These analyses provide detailed information not only about tooth
morphology but also about the dynamics of dentin deposition at
selected time points. This allows differences in growth dynamics
to be measured under various conditions or in animals of diverse
genetic backgrounds. To test our approach, we functionally tested
the manipulation of growth dynamics by comparing the effect of
hard and soft diets on mandibular molar root growth (Fig. 6, D
and E). Both mesial and distal roots showed slower elongation
under the soft food diet (Fig. 6E). Therefore, the hardness of the
food after the lactation period influences the pace of root growth
and, thus, direcdy affects root length.
Growth and healing dynamics of mouse long and flat bones
Uncovering the process of development and healing of long and flat
bones is key not only for understanding many developmental
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 4 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
C A C A C A C A C A
T T
•
• T T T • T
• .WO W1 W2 W3 W4 W5 W6 W7 W8 W9 Harvest
B Maxillary incisor Mandibular incisor
Daily Turnover Daily Turnover
growth rate growth rate
urn Weeks urn Weeks
Sex Mean ±SD Mean± SD
Young
<-? 138.8 232.9
adults ±20.9
5.5
± 53.3
6.4
(3-5
9 160.2
5.5
243.0
6.4
months) ±27.0 ± 41.0
Older c? 134.5 194.1
adults ± 16,8
6 8
±42.5
8 1
(13-15
Q 116.0 188.3
months) + ± 17.5 ± 34.2
A Growth of maxillary and mandibular incisor - weekly (W) injected
m
wo Y^f *
C Thickening of dentin wall - daily injected
pay lines {calcein)
Week lines {alizarin)
Transverse view
Pulp
A C C C C C C C A C C C C C C C A C C
T T T T T T T T T T T T T T T T .Continue until harvest
1 15 29 43 1 15 29 43
Time (days)
E Effect of ageing and sex on the incisor turnover - daily injected
D40 D42 D44 D46 D48 D35 D37 D39 D41
Pace of incisor growth
Maxillary incisor Mandibular incisor
* * * *
Young Older
adults adults
Young Older
adults adults
Fig. 3. Growth dynamics of the continuously growing mouse incisor. To analyze the growth rate of maxillary (MX) and mandibular (MD) incisors, alizarin (A) and
calcein (C) were administered in weekly intervals over 9 weeks (W) (A). Quantification of mandibular and maxillary incisors growth revealed that the turnover rates significantly
differ between the young adults (3 to 5 months old) and older adults (13 to 15 months old) mice, but no significant alterations were observed when comparing
males and females (B and D to F). To analyze the thickening of the dentin wall over time (C and D) and the daily incisor growth rate (C, E, and F), daily calcein administration
(with weekly alizarin checkpoints) was performed. Thickening of dentin wall in mandibular and maxillary was demonstrated on the longitudinal and transversal
sections (C) and measured from the longitudinal optical sections of both the lingual side (maxillary) and labial side (mandibular) (D). Quantification of the incisor dental
pulp narrowing indicates decreased rate of dentin deposition in older mice when compared to young adults (D),n = 4 young adult mice (32 stripes); n = 4 older adults (45
stripes). This analysis uncovered three stages of dentin wall thickening: (1) acceleration, (2) production, and (3) termination phase that correspond to the distance from
dentin-enamel junction and cementum-dentin junction respectively (D). Representative images of daily ingrowths of dentin (incisor growth) shows reduced rate of
incisor growth in older animals (E). The reduction of growth rate is more prominent in mandibular incisors (E and F). Data are means ± SD (unpaired Student's t test).
* * * * P < 0.0001. Scale bars, 1 mm (A) and 250 urn (B to H).
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 5 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
A Schematic visualization of mouse incisor
Dental pulp
Distal
Distal-mid
Apical
Cementum
Dental pulp
Cementum-facing
dentin (CFD)
Enamel-facing
dentin (EFD)
Enamel
Apical-mid
Sagittal section Cross section
B Spot measurement of mean gray value
Distal Distal-mid Apical-mid Apical
• 0
I ^
C Differences in mean gray values of dentin and enamel in noninjected and daily-injected animals
500 -i
4 0 0 %
3 0 0 ,
200
1 0 0 Daily
injected
(A-C for 56 days)
QLi_
Not injected
LU
Daily injected
ns
Q
LL
— Not injected
I
X
u Daily injected
"05
E
Not injected
c
LU
Daily injected
Distal Distal-mid Apical-mid
Incisor position
Apical
O
o
i f
o
I
C
>5
o
'15
Fig. 4. Repeated long-term administration of calcein and alizarin does not affect the mineralization process of hard tissues. Experimental mice were daily alternatively
administered by calcein and alizarin for 56 consecutive days (the whole incisor was completely regenerated). Subsequently, the analysis of dentin and enamel
density was performed and compared with control mice. (A) Longitudinal and transversal sections of a continuously growing mouse incisor and (B) the sites of the
analysis (distal, distal-middle, apical-middle, and apical regions). The results (C) show that daily administration of calcein and alizarin does not cause any significant
difference in dentin and enamel density; n = 5 per each location, conditions, and the type of analyzed tissue type (in total, 120 measurements). CFD, cementumfacing
dentin; EFD, enamel-facing dentin; au, arbitrary units; ns, not significant. Data are means ± SD (unpaired Student's ttest). Scale bar, 200 um.
Gonzalez Lopez ef a/., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 6 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
A Delay in alizarin vs. calcein deposition rate
Scheme of fluorochrome deposition
Oh 24h 48h 72h 96h 120h
C A C A C A
II II IIShifted
0 0.5 1
Rate of deposition
A - Alizarin
C - Calcein
B Quantification of the vital dyes
delay deposition
1.5-1
cg
:
55 1.0HoQ.
CD
"O
H—
O
B 0 . 5 -
0.0- —I 1 1—
C-C A - A C - A
t?
o
Fig. 5. Calcein and alizarin have different kinetics of action in vivo. Alizarin and calcein differ in the duration of deposition in newly formed tissue and the peak of
deposition rate (A). Calcein provides broader lines than alizarin, indicating its longer duration of activity. (A) schematically shows how alternating applications of these
dyes create a shift between administrations. This delay was quantified in (B) measured in maxillary incisors of daily administered mice;n = 32 data points. Data are means
± SD (unpaired Student's ttest). * * * * P < 0.0001. Scale bar, 1 mm.
abnormalities [e.g., achondroplasia or craniosynostosis (28,29)] but
also for the development of future strategies in tissue engineering
(30, 31). First, to enable precise monitoring of the development of
long and flat bones in mice, we adapted the BEE-ST approach for
use in newborn mice and pups (Fig. 7, A to G, and table SI). Using
the example of the bones that build the forelimb and hindlimb, the
development of bones in a single animal over time was analyzed,
showing that the rate of bone growth is a nonlinear function of
time (Fig. 7, C to F). To achieve this, we selected sequential administration
of alizarin and calcein during the time points P8 and P10
and measured daily ingrowths in the radius, ulna, metacarpal bones,
and phalanges (Fig. 7, A to F). The BEE-ST approach can also be
used to monitor the development of flat bones using the example
of cranial bone fusions in newborns, when administered in the P3
to P10 period as shown in Fig. 7G. Furthermore, the dynamics of
the fusion of the parietal bones with the occipital bone, forming
the sagittal and lambdoid sutures, were elucidated using BEE-ST
(Fig. 7G). This is particularly important for understanding the dynamics
of some congenital malformations where the cranial sutures
fuse prematurely, such as in the case of craniosynostosis (29). Last,
the BEE-ST approach was adapted to enable the monitoring of
cranium healing after skull drilling at weekly time points and established
a method for the quantification of the lesion healing (Fig. 7,
H to L). The method enables quantification of the progress of bone
healing over space and time (Fig. 7, K to L). This was achieved by the
measurement of weekly growth lines from optical sections taken by
three-dimensional confocal imaging (Fig. 7J). The results highlight
that the healing of flat bones after injury slows down over time
(Fig. 7, K and L). This approach will be instrumental for study of
the bone healing process and for the rapidly growing field of
bone tissue engineering enabling to evaluate the dynamics and
the efficiency of the hard tissue-forming process on scaffolds
both in vitro and in vivo.
Monitoring the development of teeth and bones in reptiles
and amphibians
Compared to M, musculus (representing mammals), little is known
about the development of bones and teeth in other major vertebrate
groups. Here, the versatility of the BEE-ST approach using C. calyptratus
(representing reptiles) and X. laevis (representing amphibians)
is demonstrated (Figs. 8 and 9). To observe the development
of chameleon bones and teeth, we administered the dyes at 2-week
intervals: calcein at P14, alizarin at P28, and both dyes to obtain a
pseudo-orange color at P42. Confocal imaging of optically cleared
hard tissues then enabled us to observe and measure multiple
aspects of the development of various bones (Fig. 8, A to N) and
teeth (Fig. 8, O to T). Monitoring of tooth development uncovered
a joint mineralization of dentin in several neighboring teeth
(Fig. 8S). Quantification of the dentin growth rate in the cusp and
interdental region revealed that the dentin deposition rate increased
slightly during the traced weeks 4 to 6 compared to the interval from
weeks 2 to 4 of postnatal development (Fig. 8T). The three-dimensional
analysis uncovered unreported parameters not only of the dynamics
of tooth growth but also of the process of ankylotic fusion of
the tooth and bone (Fig. 8, O to S, and movie S3).
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 7 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
A Elongation of roots of mouse molars
A C A
T
D Hard/soft diet influence on the development
of roots of mouse molars
A
T
C
T V T Harvest
P 1 2 P 1 5 P 1 8 P 2 1 P 2 4 P 2 7 P 3 0 P31
Mandible Whole-mount projection
A
T
C
•
A
•
C
T
A
T Y Harvest
P 1 2 P 1 5 P 1 8 P21 P 2 4 P 2 7 P 3 0
B Crown molar formation C Root patterning
A
T
C
• Harvest
A
T
C
• Harvest
P 4 P 6 P 7 P 8 P 1 0 P11
Mandibular M1 Mandibular M1
Lateral projection Apical projection
Quantification of molar roots elongation
Maxillary M1
Apical projection
300-1
(LUri)
250-
days
200-
CO
per
150-
c
g 100-
ongat
50-
LU
0 M1
m e s i a l root M1 distal root
• Hard food(n = 5)
j
O Soft food (n = 5)
\ \ ns
^ \ \ ns
Lact. Diet Lact. Diet
IP > ^ ^ ^ <j
Time (postnatal days)
o
o
==
3
O
>5
o
>
C
'15
Fig. 6. Tracing the dynamics of mouse molar development in time and under the influence of hard and soft diet. (A) The elongation of roots of mandibular molars
M l , M2, and M3 during an early postnatal (PI 2 to P30) development visualized by alternating administration of alizarin and calcein in 72-hour intervals. To follow the
molar crown (B) and root (C) patterning, alizarin and calcein were administered in 48-hour intervals at P4 to P6 and P8 to P10, respectively. Whole-mount confocal
imaging of cleared sample demonstrates the dynamics of the crown mineralization (B) and the fusion of dentin during root pattering of mandibular (two roots) and
maxillary (three roots) first molars (C). (Dand E) The influence of the hard and soft food diet on root elongation during the main period of mandibular first molar growth
(PI 2 to P27);n = 5 M l mandibular molars from hard food mice;n = 5 M l mandibular molars from soft food mice. A hard diet induces faster molar root growth than a soft
diet, which causes a slower growth of the roots. M, medial; V, ventral; L, lateral; D, dorsal. Data are means ± SD (paired Student's f test). ***p< 0.001; * P < 0.05. Scale bars,
250 urn.
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 8 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
A Forelimb development D Hindlimb development
Jizarin Calcein
B
Dynamics of forelimb bones growth
. . Tracing - P 1 0
1 mm im n T H
I. Tracing P8-P9 II. Tracing P8-P10
Radius j j ^ ^
Ulna f
_
C Bone growth rate
(forelimb)
400-1
Ě 3 0 0 S>
2 0 0 -
5 100Radius
Ulna
T
i—
Dynamics of hindlimb bones growth
^ ^ ^ ^ ^ P h a l a n g e s ^
Metacarpals
_
• " . : - í I• " . : - í I
F Bone growth rate
(hindlimb)
400-1
300D)
2 0 0 Metacarp.
| Phalange
TO
4*>
—I—
cfsi
G Development of cranium H Healing of cranium (after drilling of the skull)
J Optical cross sections from "i"
A C
T T Harvest
A
T
Drill
V
A
•
A C A C
T T • T Harvest
Harvest
P3 P6
_ . . . . Sagittal suture
Occipital |
y bone Corona
suture
P4 P10
W0 W1 W2 W3 W4 W5 W6
I Lesion after healing period (top view)
\
* 100 |iin
c
... Hole '"'
d . . . - 5 7 . . . - ; , Ho* ts^j. • •
Parielal bone
K Quantification of healing process L Lesion closing dynamics
a
60-
E
f 40-
9 20-
1 T
H V <o'
o
3
O
C
o
>
#
Fig. 7. Development and healing of long and flat bones in the mouse. (A and D) An overview of optically cleared forelimb and hindlimb paws after a series of alizarin
and calcein injections during its postnatal development. Quantification of the growth dynamics in forelimb bones (radius and ulna used as example) is shown in (B) and
(C), and that in hindlimb bones (metacarpal bones together with the first phalanges of second, third, and fourth fingers used as an example) is shown in (E) and (F);n = 3
mice traced from P8 to P9;n = 3 mice traced from P8to PI 0. The BEE-ST approach was also used to trace the development of cranium to observe the dynamics of parietal
and occipital bone fusion and the formation of sutures (G). The BEE-ST approach was adapted for the quantification of the healing progress after skull injury (H to L). (I)
The partially healed skull 5 weeks after drill (top view). (J) Optical cross sections of the different planes of (I), showing a rounded pattern of bone healing. Quantification
(weeks 3 to 5) of the healing progress was performed on the optical cross sections (J) and graphically visualized (K and L). Data are means ± SD. Scale bars, 500 um.
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 9 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
Teeth and bones growing dynamics in reptiles (Chamaeleo calyptratus)
A Craniofacial bone development
A c ACT T T
P14 P28 P42
B Prefrontal bone C External naris
B R
D Maxilla E Tooth (maxilla)
F Ulna elongation tracing J Femur elongation tracing N Quantification of bone
elongation
o
3
O
0 Development of teeth T Dentin growth rate
Cusp region
IC
>5
o
'15
P14-P28 P28-P42
Fig. 8. Dynamics of chameleon (C. calyptratus) skeletogenesis and odontogenesis. (A) The progress bone formation in a whole-mount chameleon with a focus on
the craniofacial bones: prefrontal bone (B), external naris (C), maxilla (D), the tooth (E);and long bones (Fto N). Quantification of long bone elongation (ulna and femur) is
shown in (N);n = 3 chameleons (six ulnae and six femurs). The growth rate of dentin was analyzed using 14-day intervals and showed dentin deposition over time across
teeth from the mandible (O to T).The dentin mineralization is coordinated along the jaw (O to S), but the rate of dentin growth showed different deposition patterns in
the cusp and intercuspal regions in time (T); n = 1 chameleon (six teeth cusps regions and six interdental regions). Data are means ± SD (unpaired Student's ttest). **P<
0.01; *P < 0.05. Scale bars, 1 m m (A) and 100 urn (B to S).
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 10 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
Teeth and bones growing dynamics in amphibians
HarvestSt 65 S t 6 6
2 days 5 days 2 days
A Overview whole-mount imaging
2 days
B Craniofacial bone development
(Xenopus laevis)
C Nasal bone area
Skull Ingrowth
J J J direction
D Hindlimb bone elongation E Metacarpal growth
(hindlimb)
"55 0.5F
Vertebra development H Tooth development
Dentition (exclusively located in r
o
3
O
t f
O
I
C
>5
o
'15
Fig. 9. The versatility of BEE-ST shown in amphibians' (X. laevis) bone and tooth growth. (A) Whole-mount frog images in separate and merged channels from both
ventral and dorsal views after alizarin andcalcein administration into the water. The dynamics of the formation of craniofacial bones (B and C) visualized the growth of the
nasal bone in high detail; n = 1 frog (two nasal bones). (Dand E) The dynamics of hindlimb bone elongation that enables the detection of the ossification center and the
direction of growth of long and short bones shown in the tibia and metacarpal bones; n = 1 frog (four metacarpal bones). Comparison of microCTand BEE-ST approaches
used for tracing of the development of vertebrae (Fand G) and maxillary dentition (H and I). Subset (a) represents panoramic fluorescence imaging of the upper jaw and
(b) shows a spatiotemporal reconstruction of tooth mineralization (confocal imaging). Data are means ± SD. Scale bars, 5 mm (A), 2 mm (B and D), 500 um (F and G), 100
um (H, I, and a), and 25 um (b).
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 11 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
Amphibians have traditionally been used in developmental
biology especially for the study of their specific features of early embryogenesis
(32, 33). However, not much is known about the dynamics
of amphibian bone and tooth development. The BEE-ST
approach was adapted for use with these animals by adding alizarin
or calcein to the water tanks for 2 days, alternating with a 5-day
period with no dye present in the water during the postmetamorphosis
developmental stages 65 to 66 (Fig. 9 and table SI) (34). Subsequent
whole-body fluorescence imaging showed bone growth at
different locations, which revealed the directions of bone growth
and the thickening of the bone wall. Furthermore, it enabled us to
quantify these processes within each individual animal (Fig. 9, A to
F). The bodies of vertebrae were shown to dynamically fuse, which
could be compared to data from end-point microCT analysis. This
illustrates the possibility of combining the two methods to obtain
both the information about the bone growth dynamics via the
BEE-ST and precise volumetric data via microCT (Fig. 9, F and
G) , therefore enabling the generation of comprehensive sets of
data from a single sample. Last, we focused on one of the least
studied structures of X. laevis—the teeth, which are located exclusively
in the maxilla (Fig. 9, H and I) (35). The analysis uncovered
similarities in the dentin growth process in different rows of teeth
that differ in size. Together with microCT analysis, the BEE-ST approach
provides a comprehensive view of the complex organization
and dynamics of development of amphibian teeth (Fig. 9, H and I).
Bone development and remodeling in ray-finned fish
and birds
In the past decade, zebrafish (D. rerio) has become an important
model organism used for the study of bone development and
disease modeling (36). To enable the study of dynamics of bone
growth and development in this model organism, we further
adapted the BEE-ST approach (Fig. 10, A to F). In zebrafish, most
bony tissues arise from condensed mesenchymal precursors that
differentiate directly into osteoblasts, which generate the bone by
intramembranous ossification. Craniofacial bones start to form
around 3 to 4 days postfertilization (DPF), and this process continues
until adulthood (around 90 DPF) (36). As the zebrafish body is
already transparent, in contrast to other tested organisms, zebrafish
larvae were imaged as living organisms without the need for tissue
fixation and optical clearing steps (Fig. 2A). To observe craniofacial
bone development, we selected a 48-hour period of exposure to alizarin
(7 to 9 DPF). This was followed by a 48-hour period without the
presence of a dye, followed by a 48-hour period (11 to 13 DPF) with
the presence of calcein in the water. Deeply anesthetized animal was
then imaged from lateral and ventral perspectives as a whole-mount
in three dimensions using a confocal microscope (Fig. 10, A and D),
and the dynamics of growth of bones in the head including the
mandible, hyomandible, and operculum were analyzed (Fig. 10,
B, C, E, and F). In principle, this approach would also be applicable
for monitoring the development of long bones (e.g., ribs), spine, or
fin bones, simply by choosing different time points of exposure to
the dyes. Anesthetizing the experimental animal without the need of
fixation and subsequent optical clearing provides the potential to
allow the experimental animals mature after the initial imaging, reanalyze
it at later stages, and, thus, determine the fate of the deposited
tissues at future time points.
Birds (Aves) represent the last class of vertebrates whose bone
development dynamics were investigated. The BEE-ST approach
was further adapted to be usable for studying bone development
during in ovo embryogenesis. Chicken (G. gallus) was used, as it
is the most used species in avian developmental biology (Fig. 10,
G to T) (37). To achieve optimal signal in the experimental
embryo, we injected dyes in ovo direcdy into the abdomen of the
embryo. Time points of administration of alizarin and calcein at
embryonic day 13 (E13) and E16, respectively, were selected to
observe the development of the head and selected long bones
such as femur and radius. The embryos were then collected at
E17. This enabled to observe the growth of selected bones including
the nature of their bone wall thickening (Fig. 10, M to T).
Overall, we have adapted the newly developed BEE-ST approach
to all major vertebrate groups that represent the most used model
organisms in research. The results demonstrate the versatility of the
approach and open diverse avenues for the analysis and precise
quantification of bone and tooth growth dynamics during development,
remodeling, or repair under physiological or pathological D
conditions. §
DISCUSSION t*
Here, we introduce a versatile approach (BEE-ST), which enables 3
the tracing of the processes of in vivo mineralization in space and Jtime.
This approach opens a variety of possibilities in multiple fields £.
focused on bone or tooth development, regeneration, and healing. |
In addition, its use can be extended for bone and tooth tissue ^
engineering. §•
Current methods used for the imaging of the hard tissues are in p
the vast majority based on microCT imaging (3). This imaging pro- Jj
vides a three-dimensional perspective of observed objects with the B
spatial resolution dependent on the size of the observed object (38). |F
Although microCT imaging can provide relatively high-quality S
spatial resolution, it enables to scan only one time point of the ob- £
served animal. To trace the dynamics of the process, more experi- 3.
mental subjects must be analyzed, which brings the individual §
variability into the measurement. In the case of bone remodeling •§
or continuous tooth growth, a standard microCT analysis does o
not provide the information needed to uncover the tissue growth j=
dynamics. However, in some specific cases, it has been shown |
that microCT or high-resolution synchrotron radiation-based pi
microCT analysis can answer some questions about hard tissue dy- g
namics by following their gradual ingrowths. This has been particularly
demonstrated, for example, in the animal fossils when
focusing on the cementum growth lines that correlate with metabolic
rate (39).
The BEE-ST approach uncovers the mineralization dynamics at
multiple selected time points within a single experimental animal in
a single scan. The data are obtained by whole-mount, nondestructive
three-dimensional confocal or light sheet imaging providing excellent
spatial and temporal resolution. Thus, BEE-ST can be used
to uncover the dynamics of the hard tissue growth and remodeling
or even to investigate healing patterns after the injury. This is
ensured by the sequential administration of harmless fluorescent
dyes that incorporate into hard matrices that are being mineralized
in that specific moment. This is followed by a nondecalcified tissue
clearing and whole-mount three-dimensional imaging that enables
the quantification on a micrometer scale.
The use of calcium-binding fluorochromes in hard tissue research
has been known for several decades and has been used in
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 12 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
Bone development and remodeling in fish and birds
Harvest
7-9 DPF 11-13 DPF 14 DPF
A Lateral overview
B Mandible
Eye Eye v ! Eye
Mandible
Calcein
> .
Merged
C Operculum
\ Hyomandible
Eye I /
/ 4 Operculum
Eye i JDPF
/ I Jk \__13DPF
y
- I P
Branchiostegal ray
(Danio rerio)
D Ventral overview
E Mandible
-rf
— V Calcein Merqed
F Operculum
Hyomandible
• Operculum
Branchiostegal ray
o
3
O
i f
O
Harvest
E13 E16 E17
G Craniofacial bones
(Gallus gallus)
IH Mandible I Mesethmoid
Nasal Nasal
aperture
I K
J Mesethmoid K Nasal bone L Z y g . arch
VI R a d i u s ossification dynamics 3 F e m u r ossification dynamics
R Femur
| ^ Femur g Femur
s
Single plane
T
j Femur
Single plane
Single plane
j Femur
Single plane
I
c
>5
o
'15
Fig. 10. Adaptation of BEE-ST for use in zebrafish (D. rerio) and chicken (G. gallus). Sequential administration of calcein and alizarin into the water of experimental
zebrafish larvae enables us to trace the development of different bones forming the head: lateral whole-mount overview (A), lateral view of the mandible (B) and operculum
(C), ventral whole-mount overview (D), and ventral detail of the mandible (E) and operculum (F). In the example of the chicken (G. gallus) embryo, we show the
possibility of using the BEE-ST approach also to following the development of bones of birds by in ovo administration of calcein and alizarin. (G) The development of
craniofacial bones (lateral whole-mount skull overview), the mandible (H), mesethmoid (I), mesethmoid—single plane (J), nasal bone—single plane (K), zygomatic arch—
single plane (L), radius ossification dynamics (M), details of radius single planes (N to P), femur ossification dynamics (Q), and femur single planes details (R to T). Scale
bars, 250 Mm (A, D, H, I, N, and R), 50 Mm (B, C, E, F, J, K, L, O, P, S, and T), 2 mm (G), and 100 Mm (M and Q).
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 13 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
various adaptations (40, 41). Traditionally, they were used for the
visualization of the calcified tissue after its isolation from the experimental
animal or in the cell culture for uncovering signs of mineralization
(42). Later, in larger mammals (pig and sheep), these dyes
were administered in vivo, and the samples were subsequendy processed
by destructive ground sectioning to observe the development
of mineralized tissues in single plane (43, 44). Recendy, calcein and
xylenol orange administration followed by fluorescent imaging was
also used to follow the natural replacement dynamics of gecko teeth
(reptiles) (45). In bone field, the administration of hard tissue-incorporating
fluorochromes has been used to observe bone healing
after the injury and to assess the integration of artificial implants
using wide-field fluorescence imaging (46, 47). In addition to
these studies, BEE-ST represents a universal approach broadly applicable
in variety of species providing a tool to precisely measure
the dynamics of hard tissue growth and healing in a three-dimensional
perspective.
Although, nowadays, a variety of different dyes with the abovementioned
features were described, not all of them have optimal
properties. To achieve optimal results, the sequential administration
of two carefully selected dyes, alizarin red and calcein green, was
used. Both are in different fluorescent spectra and provide a
strong and highly specific fluorescence signal with low background
(48). In addition, in some cases, a third pseudo-orange signal can be
obtained while using the combination of calcein and alizarin via
their parallel administration. This is specifically useful while studying
calcified tissues that are not being remodeled over time. Moreover,
it was shown that the selected dyes do not disrupt the process
of biomineralization even during long-term daily administration.
Furthermore, at the selected concentrations, all dyes are nontoxic
for the animals (49).
An unknown fact is that fluorescent dyes capable of incorporating
into currendy calcifying tissues have different in vivo kinetics.
We revealed that even alizarin and calcein exhibit a slighdy shifted
pattern in the forming dentin of mouse incisor that grows at stable
pace. Via the use of continuously growing incisors, this shift was
demonstrated and quantified in detail. This is particularly important
to consider when the analysis needs to be performed while
using 24 hours or in even shorter intervals for dye administration.
When the intervals are 48 hours or more, the differences are negligible,
and it might be more beneficial to use a combination of dyes.
Thus, it is crucial to carefully select dyes and consider their combinations
accordingly for each type of experiment.
Using continuously growing mouse incisor as a model, the BEEST
approach was used to precisely quantify several features of its
growth. We used both weekly and daily administrations of dyes to
quantify the speed of incisor growth, turnover, and dentin wall
thickening speed in groups of animals with different ages and
sexes. This was achieved by daily calcein administrations together
with combined alizarin and calcein weekly administrations used
as checkpoints. Compared to previous studies, we were able to
quantify the growth of the incisor with several orders of magnitude
more precise manner in real numbers (50). This approach can be
used for a precise quantification of incisor growth acceleration
after its injury (shortening by the tooth clipping). Previously, it
was only possible to observe this by making notches in the
enamel and subsequently following the changes in their location
as the tooth regrows (51). These measurements unfortunately did
not provide real values but only a relative comparison to the
neighboring tooth. Moreover, notches in teeth cause damage to
the enamel and sometimes even dentin that might influence the experiment.
In the example of mouse molars (teeth more similar to
the human dentition), the BEE-ST approach can be applied to describe
and quantify root patterning and elongation during their
postnatal development. The speed of root growth was found to
depend on the type of diet (soft or hard food) given to the pups
during the posdactation phase. These results may have implications
for human medicine and may explain the mechanisms controlling
root elongation.
We have shown that the BEE-ST approach can be independendy
used in all major groups of both terrestrial and aquatic vertebrates to
study processes such as bone development, formation of ankylosis,
or wound healing. We present a complex, efficient, and very precise
approach that not only is widely applicable in vivo in various
animals but also has potential to be used in tissue engineering
and can be combined with other techniques. D
o
==
3
O
MATERIALS AND METHODS I
Vital dyes preparation For
all animal models, the dyes were prepared as follows: calcein (0.9 3
mg/ml; C0875, Sigma-Aldrich) and alizarin red S complex (4.5 mg/ Jml;
A5533, Sigma-Aldrich). Solutions were prepared in 2% £.
NaHC03 (71628, Honeywell Fluka) in MilliQ water, filtered using f
a syringe filter 22 \im polyethersulfone membrane (99722, TPP) ?•
under sterile conditions and stored at 4°C in the dark until its use §•
(up to 1 month). The volume and ratios of injections for the differ- p
ent animal models are specified in table SI. S
Hard tissue clearing z
BABB peroxide-free clearing solution was prepared by mixing 8j
benzyl benzoate (B6630, Sigma-Aldrich) and benzyl alcohol £
(10880, Penta) in a 2:1 ratio and adding activated neutral Brock- 3.
mann Grade I 58 A aluminum oxide (11502.A1, VWR) at a concen- §
tration of 0.25 g/ml. The mixture was agitated for at least 1 hour up •<
to overnight at room temperature (RT). Then, the clearing solution 3
was centrifuged at 2000g for 10 min. The supernatant was used in c
the optical clearings. Protocol was adapted from (25). |
Mouse [M. musculus) g
All experiments with mice were approved by the Ministry of Education,
Youth and Sports, Czech Republic (MSMT-8360/2019-2,
MSMT-9231/2020-2, and MSMT-6379/2022-4). Mice were kept
in a 12-hour:12-hour light/dark cycle, 18° to 23°C, and 40 to 60%
humidity. Animals had access to food and water ad libitum. No exclusion
criteria were applied for experimental animals.
Hard/soft food-type experiment
A soft diet (Nutra-Gel, Bio-Serv) or hard diet (regular 10 mm pellet
food, 1414, Altromin) was given to the newborn mice since P10 in a
specialized pyramid feeder located inside of the cage. The dam was
removed from the cage at PI7. Food was routinely changed every
3 days.
Tissue preparation and imaging
Mouse molars and incisors were carefully isolated under stereomicroscope
and fixed in 4% paraformaldehyde (PFA) (18070, Penta)
prepared at pH 7.4 for 2 hours at RT. Bones were fixed in 4% PFA
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 14 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
overnight. Fixed tissues were subsequently dehydrated in an ethanol
gradient (30 to 50 to 80 to 100%) diluted in MilliQ water (15 min in
each concentration for teeth and 1 hour for bones) and stored overnight
in 100% ethanol at RT. Excess ethanol was removed, and
samples were transferred to the prepared BABB peroxide-free clearing
solution. The incisor, molar, or bone samples were incubated for
72 hours at RT and slowly shaken in the prepared BABB solution,
until reaching complete transparency. For imaging, samples were
transferred to ethyl cinnamate (W243000, Sigma-Aldrich) and
imaged in a suitable vessel: glass-bottom dish for a fluorescence microscope
or (i-slide ibidi dish (80826 and 80281) for confocal
microscope.
Skull drilling
Six-month-old mice were anesthetized by an intraperitoneal administration
of a mixture of ketamine/xylazine (90 and 12.5 mg/kg, respectively)
in 0.9% saline. Fur was removed from the head, and the
area was swabbed with 70% ethanol. The skin was subsequently cut,
and the injury was generated in the middle of the parietal bone
using a drill machine without damaging the underlying tissue. Carboxymethylcellulose
(Ocugel) drops were topically applied to the
eyes of the mice before and after surgery. Ibuprofen (40 mg/kg)
was dissolved in the water tank as analgesia for a week after surgery.
X-ray microtomography
Before the scanning the samples were placed in either a 2 or 15 ml
tube (based on sample size) and mounted in 1% agarose gel to
prevent the motion of the sample during the scan. The microCT
measurement was performed using the GE Phoenix v|tome|x L
240 laboratory system equipped with a 180 kV/15 W nanofocus
x-ray tube and a 4000 x 4000 pixel flat panel detector with a pixel
size of 100 um. The scan conditions were as follows for both jaw and
spine samples: The samples were scanned at 60 kV acceleration
voltage and 200 uA tube current, and the exposure time of 500
ms and three images were averaged to reduce the noise. Over the
360° rotation of the sample, 2700 projections were taken in the
case of the sample of the jaw (with a 3.4 [im voxel resolution of
the scan), and, in the case of the sample of the spine, 2700 projections
were taken, and the final scan resolution was set to the 15 urn
voxel size. The tomographic reconstruction was performed by the
GE phoenix datos|x 2.0 software. The reconstructed data were imported
into the VG Studio MAX 2022.1 software where all the data
visualizations were created.
Chameleon (C. calyptratus)
All experiments on chameleon juveniles were approved by the Ministry
of Education, Youth and Sports, Czech Republic (MSMT-
10946/2021-5) and followed national regulations. Juvenile animals
were obtained from private and commercial breeders. All chameleons
were kept in a 12-hour: 12-hour light/dark cycle, 25° to
30°C, and 55 to 65% humidity. Animals had access to food and
water ad libitum. No exclusion criteria were applied for experimental
animals. At selected time points, animals were euthanized, decapitated,
and fixed in 4% PFA for at least 48 hours. Jaws, skull,
and limbs were isolated. Fixed tissues were processed through a dehydration
ethanol gradient (30 to 50 to 80 to 90 to 95 to 100%) at RT
(each for 1 hour) and then placed directly into the prepared BABB
solution. Teeth and bones were kept separately depending on the
size of the sample and achieved complete clearing for 24 to
72 hours.
Frog (X. laevis)
The work with X. laevis was carried out according to the Czech
animal use and care law and approved by the local authorities and
committees (MSMT-30784/2022-1; Animal Care and Housing Approval:
45055/2020-MZE-18134, Ministry of Agriculture of the
Czech Republic; and Animal Experiments Approval: CZ
62760214, State Veterinary Administration/Section for South Moravian
Region). No exclusion criteria were applied for experimental
animals. The generation and cultivation of Xenopus embryos were
performed following general protocols. Briefly, testes from males
under anesthesia (20% MS-222, A5040, Sigma-Aldrich) were
removed surgically from the body cavity and transferred to cold
lx Marc's Modified Ringers (MMR; 100 mM NaCl, 2 mM KC1, 1
mM MgS04 , 2 mM CaCl2 , and 5 mM Hepes, buffered to pH 7.4) 0
supplemented with gentamycin (50 (ig/ml; G3632, Sigma- |
Aldrich). To induce egg laying, fully mature Xenopus females o
were injected with 260 U of human chorionic gonadotropin g(Merck,
Ovitrelle 250G) into the dorsal lymph sac for about 12 to b>
16 hours before use and kept overnight at 18°C. For fertilization, 3
eggs were squeezed from an induced female directly into a petri Jdish
and mixed with a piece of testes in O.lx MMR. Then, £.
embryos were cultivated in O.lx MMR at 18° to 21°C. Once the |
embryos reached the desirable stage to trace the calcium deposition, ?•
they were transferred to a water tank containing the fluorochrome §•
dyes dissolved in the water. At the selected time point, the frogs were p
euthanized and sacrificed. Skin, inner soft organs, and blood vessels S
were carefully removed using scissors and tweezers under stereomi- B
croscope. Frogs were staged after the normal table of Nieuwkoop fF
and Faber (34). " I
c
Chicken (G. gallus) 3.
All experiments on chicken embryos were performed according to §
national regulations. The project complies with Act No. 246/1992 •?
Coll. on the Protection of animals against cruelty, as amended. Ex- o
periments with chicken embryos were approved by the expert com- c
mittee for ensuring the welfare of experimental animals at the |
Breeding facility for laboratory animals of lower vertebrates at the pi
Faculty of Science, Masaryk University (CZ 62760214). Fertilized g
ISA Brown chicken eggs were obtained from Integra farm ">
(Zabcice, Czech Republic). Eggs were incubated in a humidified incubator
at 37.8°C for 48 hours, and then all eggs were opened. Next,
2 ml of egg white was extracted for easier handling, and holes in the
eggshell were secured by tape. No exclusion criteria were applied for
experimental animals. At the selected time point, embryos were decapitated
and fixed in 4% PFA overnight. Skulls and limbs were isolated
from the embryos. Fixed tissues were processed through a
dehydration ethanol gradient (30 to 50 to 80 to 90 to 95 to 100%)
at RT (each for 1 hour) and then placed directly into the prepared
BABB solution for 24 hours to achieve complete tissue clearing.
Embryos were staged according to the method of Hamburger and
Hamilton (52).
Fish (D. rerio)
All work with zebrafish juveniles were approved by the Ministry of
Agriculture, Czech Republic (45052/2020-MZE-18134 and 45055/
2020-MZE-18134) and experimental exposure to dye by the
Gonzalez Lopez ef al., Sci. Adv. 9, eadi0482 (2023) 2 August 2023 15 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
Ministry of Education, Youth and Sports (MSMT-34667/2022-1)
and State Veterinary Administration/Section for South Moravian
Region (Animal Experiments Approval: CZ 62760214). Embryos
were produced by natural spawning of AB zebrafish strain in breeding
chambers after the preceding overnight separation of females
and males. After spawning, embryos were collected and kept in
an E3 medium with daily medium changes. No exclusion criteria
were applied for experimental animals. From 1 DPF, 0.003% 1phenyl-2-thiourea
(P7629, Merck) was added to the medium to
block the pigmentation process. Since 5 DPF, embryos were fed
daily with dry food (ZebraFeed, Sparos). Once the embryos
reached the desirable stage, they were transferred to a tank where
the fluorochrome dyes were diluted in an E3 medium. Before
imaging, embryos were anesthetized using buffered tricaine solution
[tricaine-ethyl 3-aminobenzoate methane sulfonate salt (0.17
mg/ml) in 0.09 mM tris-HCl (pH 7.0)] (A5040, Merck). Wholemount
zebrafish were embedded in 1% TopVision Low Melting
Point Agarose (R0801, Thermo Fisher Scientific).
Microscopy and image analysis
Wide-field fluorescence overview images of specimens were taken
on Axio Zoom V.16—Apotome (Zeiss). Detailed confocal images
were obtained using the laser scanning confocal microscope (LSM
880, Zeiss), and overview visualizations of chameleon jaws were
scanned using light sheet fluorescence microscopy (Lightsheet 7,
Zeiss). The data were rendered and analyzed using Imaris (Bitplane)
and ZEN (Zeiss) software.
Statistical analysis
All the statistical analyses use the unpaired t test and were performed
using the GraphPad Prism 8 (GraphPad Software, United
States). Data are shown as the means ± SD. Statistically significant
differences are presented in the figures as follows: *P < 0.05, **P <
0.01, ***P < 0.001, ****P < 0.0001, and "ns," which is not significant.
Supplementary Materials
This PDF file includes:
Table S1
Legends for movies S1 to S3
Other Supplementary Material for this
manuscript includes the following:
Movies SI to S3
REFERENCES AND NOTES
1. E. F. Morgan, L. C. Gerstenfeld, Chapter 2 - The bone organ system: Form and function, in
Marcus and Feldman's Osteoporosis, D. W. Dempster, J. A. Cauley, M . L. Bouxsein, F. Cosman,
Eds. (Academic Press, ed. 5, 2021), pp. 15-35; www.sciencedirect.com/science/article/pii/
B9780128130735000022.
2. M . Kobayashi, Y. Masuda, M . Kishino, T. Ishida, N. Maeda, T. Morimoto, Characteristics of
mastication in the anodontic mouse. J. Dent. Res. 81, 594-597(2002).
3. Y. Kim, M . D. Brodt, S. Y. Tang, M . J. Silva, MicroCT for scanning and analysis of mouse
bones. Methods Mol. Biol. 2230, 169-198(2021).
4. T. Kubíková, M . Bartoš, Š. Juhas, T. Suchý, P. Sauerová, M . Hubálek-Kalbáová, Z. Tonar,
Comparison of ground sections, paraffin sections and micro-CT imaging of bone from the
epiphysis of the porcine femur for morphometric evaluation. Ann. Anat. 220,
85-96 (2018).
5. L. Zhao, T. Dodge, A. Nemani, H. Yokota, Resonance in the mouse tibia as a predictor of
frequencies and locations of loading-induced bone formation. Biomech. Model. Mechanobiol.
13, 141-151 (2014).
6. K. A. Apaza Alccayhuaman, P. Heimel, J. S. Lee, S. Tangl, U. Kuchler, J. Marchesan,
L. Panahipour, S. Lettner, E. Matalová, R. Gruber, FasL is a catabolic factor in alveolar bone
homeostasis. J. Clin. Periodontol. 50, 396-405 (2023).
7. W. H. Harris, R. H.Jackson, J.Jowsey, The in vivo distribution of tetracyclines in canine bone.
J. Bone Joint Surg. Am. 44-A, 1308-1320(1962).
8. R. A. Milch, D. P. Rail, J. E. Tobie, Fluorescence of tetracycline antibiotics in bone. J. Bone
Joint Surg. Am. 40, 897-910(1958).
9. J. Wang, A. M . Muir, Y. Ren, D. Massoudi, D. S. Greenspan, J. Q. Feng, Essential roles of bone
morphogenetic protein-1 and mammalian tolloid-like 1 in postnatal root dentin formation.
J.Endod. 43, 109-115(2017).
10. H.-H. Hong, T.-A. Chou, A. Hong, Y.-F. Huang, T.-H. Yen, C.-H. Liang, A. H o n g , H.-Y. Hsiao, C Y.
Nien, Calcitriol and enamel matrix derivative differentially regulated cemento-induction
and mineralization in human periodontal ligament-derived cells. J. Periodontol. 93,
1553-1565 (2022).
11. S. M . van Gaalen, M . C. Kruyt, R. E. Geuze, J. D. de Bruijn, J. Alblas, W. J. A. Dhert, Use of
fluorochrome labels in in vivo bone tissue engineering research. Tissue Eng. Part B Rev. 16,
209-217 (2009).
12. J. Schmidt, K. Lumniczky, B. D. Tzschaschel, H. L. Guenther, A. Luz, S. Riemann, W. Gimbel,
V. Erfle, R. G. Erben, Onset and dynamics of osteosclerosis in mice induced by Reil ly-FinkelBiskis
(RFB) murine leukemia virus. Am. J. Pathol. 155, 557-570(1999).
13. M . Takumida, D. M . Zhang, K. Yajin, Y. Harada, Polychromatic labeling of otoconia for the
investigation of calcium turnover. ORL J. Otorhinolaryngol Relat. Spec. 59, 4 - 9 (1997).
14. T. Jinno, V. M . Goldberg, D. Davy, S. Stevenson, Osseointegration of surface-blasted implants
made of titanium alloy and cobalt-chromium alloy in a rabbit intramedullary model.
J. Biomed. Mater. Res. 42, 2 0 - 2 9 (1998).
15. C. Paddock, T. Youngs, E. Eriksen, R. Boyce, Validation of wall thickness estimates obtained
with polarized light microscopy using multiple fluorochrome labels: Correlation with
erosion depth estimates obtained by lamellar counting. Bone 16, 381-383 (1995).
16. H. Francillon, J. Castanet, Experimental demonstration of the annual characteristic of the
lines of arrest of skeletal growth in Rana esculenta (Amphibia, anura). C. R.Acad. Sei. Ill 300,
327-332 (1985).
17. D.Jing, Y.Yi, W. Luo, S.Zhang, Q. Yuan, J.Wang, E. Lachika, Z. Zhao, H.Zhao, Tissue clearing
and its application to bone and dental tissues. J. Dent. Res. 98, 621-631 (2019).
18. N. Renier, Z. Wu, D. J. Simon, J. Yang, P. Ariel, M . Tessier-Lavigne, iDISCO: A simple, rapid
method to immunolabel large tissue samples for volume imaging. Cell 159,
896-910 (2014).
19. R. Tomer, L. Ye, B. Hsueh, K. Deisseroth, Advanced CLARITY for rapid and high-resolution
imaging of intact tissues. Nat. Protoc. 9, 1682-1697(2014).
20. K. Matsumoto, T. T. Mitani, S. A. Horiguchi, J. Kaneshiro, T. C. Murakami, T. Mano,
H. Fujishima, A. Konno, T. M. Watanabe, H. Hirai, H. R. Ueda, Advanced CUBIC tissue clearing
for whole-organ cell profiling. Nat. Protoc. 14, 3506-3537(2019).
21. W. Luo, Y. Yi, D. Jing, S. Zhang, Y. M e n , W.-P. Ge, H. Zhao, Investigation of postnatal craniofacial
bone development with tissue clearing-based three-dimensional imaging. Stem
Cells Dev. 28, 1310-1321 (2019).
22. S. H o n g , J. Lee, J. M . Kim, S.-Y. Kim, H.-R. Kim, P. Kim, 3D cellular visualization of intact
mouse tooth using optical clearing without decalcification. Int. J. Oral Sei. 11, 25 (2019).
23. Y. Yi, Y. M e n , D. Jing, W. Luo, S. Zhang, J. Q. Feng, J. Liu, W.-P. Ge, J. Wang, H. Zhao, 3-dimensional
visualization of implant-tissue interface with the polyethylene glycol associated
solvent system tissue clearing method. Cell Prolit 52, e12578 (2019).
24. K. Becker, N. Jährling, S. Saghafi, H.-U. Dodt, Immunostaining, dehydration, and clearing of
mouse embryos for ultramicroscopy. Cold Spring Harb. Protoc. 2013, 743-744 (2013).
25. M . Acar, K. S. Kocherlakota, M . M . Murphy, J. G. Peyer, H. Oguro, C. N. Inra, C. Jaiyeola,
Z. Zhao, K. Luby-Phelps, S. J. Morrison, Deep imaging of bone marrow shows non-dividing
stem cells are mainly perisinusoidal. Nature 526, 126-130(2015).
26. J . Křivánek, R. A. Soldatov, M . E. Kastriti, T. Chontorotzea, A. N. Herdina, J. Petersen,
B. Szarowska, M . Landova, V. K. Matějova, L. I. Holla, U. Kuchler, I. V. Zdrilic, A. Vijaykumar,
A. Balic, P. Marangoni, O. D. Klein, V. C. M . Neves, V. Yianni, P. T. Sharpe, T. Harkany,
B. D. Metscher, M . Bajénoff, M . Mina, K. Fried, P. V. Kharchenko, I. Adameyko, Dental cell
type atlas reveals stem and differentiated cell types in mouse and human teeth. War.
Commun. 11, 4816 (2020).
27. J . Lavický, M . Kolouskova, D. Procházka, V. Rakultsev, M . Gonzalez-Lopez, K. Steklikova,
M. Bartos, A. Vijaykumar, J . Kaiser, P. Pořízka, M . Hovorakova, M . Mina, J . Křivánek, The
development of dentin microstructure is controlled by the type of adjacent epithelium.
J. Bone Miner. Res. 37, 323-339 (2022).
28. R. M . Pauli, Achondroplasia: A comprehensive clinical review. Orphanet J. Rare Dis. 14,
1 (2019).
29. F. Di Rocco, A. Rothenbuhler, V. Cormier Daire, J . Bacchetta, C. A d a m s b a u m , G . Baujat,
M. Rossi, A. Lingart, Craniosynostosis and metabolic bone disorder. A review. Neurochirurgie
65, 258-263 (2019).
Gonzalez Lopez ef al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 16 of 17
S C I E N C E A D V A N C E S R E S E A R C H A R T I C L E
30. L. Guo, Z. Liang, L. Yang, W. Du, T. Yu, H. Tang, C. Li, H. Qiu, The role of natural polymers in
bone tissue engineering. J. Control. Release 338, 571-582(2021).
31. Z . W a n g , Y . W a n g , J. Yan, K. Zhang, F. Lin, L. Xiang, L. Deng, Z. Guan, W. Cui, H. Zhang,
Pharmaceutical electrospinning and 3D printing scaffold design for bone regeneration.
Adv. Drug Deliv. Rev. 174, 504-534(2021).
32. L. N. Borodinsky, Xenopus laevis as a model organism for the study of spinal cord formation,
development, function and regeneration. Front. Neural Circuits 11, 90 (2017).
33. L. Medina-Cuadra, A. H. Monsoro-Burq, Xenopus, an emerging model for studying pathologies
of the neural crest. Curr. Top. Dev. Biol. 145, 313-348 (2021).
34. P. D. Nieuwkoop, J. Faber, Normal Table of Xenopus Laevis (Daudin). A Systematical and
Chronological Survey of the Development from the Fertilized Fgg Till the Fnd of Metamorphosis
(Garland Pub., 1994).
35. H. Katow, Structure and formation of ankylosis in Xenopus laevis. J. Morphol. 162,
327-341 (1979).
36. K. Dietrich, I. A. Fiedler, A. Kurzyukova, A. C. Löpez-Delgado, L. M . McGowan, K. Geurtzen,
C. L. H a m m o n d , B. Busse, F. Knopf, Skeletal biology and disease modeling in zebrafish.
J. Bone Miner. Res. 36, 4 3 6 ^ 5 8 ( 2 0 2 1 ) .
37. J. M. Bahr, The chicken as a model organism, in Sourcebook of Models for Biomedical Research,
P. M. Conn, Ed. (Humana Press, 2008), pp. 161-167; https://doi.org/10.1007/978-1-
59745-285-4_18.
38. J. Rueckel, M. Stockmar, F. Pfeiffer, J. Herzen, Spatial resolution characterization of a x-ray
microCT system. Appl. Radiat. Isot. 94, 230-234(2014).
39. E. Newham, P. G. Gill, P. Brewer, M. J. Benton, V. Fernandez, N. J. Gostling, D. Haberthür,
J. Jernvall, T. Kankaanpää, A. Kallonen, C. Navarro, A. Pacureanu, K. Richards, K. R. Brown,
P.Schneider, H.Suhonen, P.Tafforeau, K. A. Williams, B. Zeller-Plumhoff, I.J. Corfe, Reptilelike
physiology in early Jurassic stem-mammals. Nat. Commun. 11, 5121 (2020).
40. C. Pautke, S. Vogt, T. Tischer, G. Wexel, H. Deppe, S. Milz, M. Schieker, A. Kolk, Polychrome
labeling of bone with seven different fluorochromes: Enhancing fluorochrome discrimination
by spectral image analysis. Bone 37, 441-445 (2005).
41. T. Nishikawa, K. Masuno, K. Tominaga, Y. Koyama, T. Yamada, K. Takakuda, M . Kikuchi,
J. Tanaka, A. Tanaka, Bone repair analysis in a novel biodegradable hydroxyapatite/collagen
composite implanted in bone. Implant Dent. 14, 252-260(2005).
42. V. Oralova, E. Matalova, M . Killinger, L. Knopfova, J. Smarda, M. Buchtova, Osteogenic
potential of the transcription factor c-MYB. Calcif. Tissue Int. 100, 311-322 (2017).
43. C. Witzel, U. Kierdorf, K. Frölich, H. Kierdorf, The pay-off of hypsodonty - timing and dynamics
of crown growth and wear in molars of Soay sheep. BMCFvol. Biol. 18, 207 (2018).
44. S. Emken, C. Witzel, U. Kierdorf, K. Frölich, H. Kierdorf, Characterization of short-period and
long-period incremental markings in porcine enamel and dentine—Results of a fluorochrome
labelling study in wild boar and domestic pigs. J. Anat. 239, 1207-1220 (2021).
45. K. S. Brink, J. I. Henriquez, T. M . Grieco, J. R. Martin del Campo, K. Fu, J. M. Richman, Tooth
removal in the leopard gecko and the de novo formation of replacement teeth. Front.
Physiol. 12, 576816 (2021).
46. M . Lingner, R. Seidling, L. J. Lehmann, E. Mauermann, U. Obertacke, M . L. R. Schwarz, Osseointegrative
effect of rhBMP-2 covalently bound on a titan-plasma-spray-surface after
modification with chromosulfuric acid in a large animal bone gap-healing model with the
Gottingen minipig. J. Orthop. Surg. Res. 13, 219(2018).
47. K. Gurzawska, K. Dirscherl, B. Jorgensen, T. Berglundh, N. R. Jorgensen, K. Gotfredsen,
Pectin nanocoating of titanium implant surfaces - An experimental study in rabbits. Clin.
Oral Implants Res. 28, 298-307(2017).
48. F. Bashey, A comparison of the suitability of alizarin red S and calcein for inducing a
nonlethally detectable mark in juvenile guppies. Trans. Am. Fish. Soc. 133,
1516-1523 (2004).
49. A. J. Stuart, D. A. Smith, Use of the fluorochromes xylenol orange, calcein green, and
tetracyclineto document bone deposition and remodeling in healing fractures in chickens.
Avian Dis. 36, 4 4 7 ^ 4 9 ( 1 9 9 2 ) .
50. M. Goldberg, O. Kellermann, S. Dimitrova-Nakov, Y. Harichane, A. Baudry, Comparative
studies between mice molars and incisors are required to draw an overview of enamel
structural complexity. Front. Physiol. 5, 359(2014).
51. Z. A n , M . Sabalic, R. F. Bloomquist, T. E. Fowler, T. Streelman, P. T. Sharpe, A quiescent cell
population replenishes mesenchymal stem cells to drive accelerated growth in mouse
incisors. Nat. Commun. 9, 378(2018).
52. V. Hamburger, H. L. Hamilton, A series of normal stages in the development of the chick
embryo. J. Morphol. 88, 49-92(1951).
Acknowledgments: We acknowledge the core facility CELLIM supported by the CzechBioimaging
large Rl project (LM2023050 funded by MEYS CR) for their support with obtaining
scientific data presented in this paper. We would like to thank A. Lochmanova for help with
taking care of the mice colonies and K. Mareckova for help with sample preparation and
laboratory assistance. Funding: This work was supported by Czech Science Foundation (22-
02794S) to J.KR. and M.B., Czech Science Foundation (22-06405S) to J.H., Czech Science
Foundation (23-04974S) to M.V., CzechNanoLab Research Infrastructure-Ministry of
Education,Youth and Sports of Czech Republic (LM2018110) to T.Z. and J.KA., CzechBiolmaging-Ministry
of Education, Youth, and Sports of Czech Republic (LM2023050) to M.G.L.,
and National Institute for Cancer Research (Programme EXCELES, LX22NPO5102)-European
Union-Next Generation EU to V.B. Author contributions: Conceptualization, funding
acquisition, supervision, and writing the manuscript: J.KR. and M.B. Methodology, investigation,
and writing the manuscript: M.G.L. Methodology, investigation, and review and editing the
manuscript: B.H., J.L., V.R., C.N., J.P., and A.S.T. Methodology, investigation, review and editing
the manuscript, and funding acquisition: J.H. Methodology and investigation: N.Z., V.F., H.T.,
M.K., R.L., H.S., P.H., and M.V. Funding acquisition:T.Z., J.KA., and V.B. Competing interests: The
authors declare that they have no competing interests. Data and materials availability: All
data needed to evaluate the conclusions in the paper are present in the paper and/or the
Supplementary Materials.
Submitted 31 March 2023
Accepted 28 June 2023
Published 2 August 2023
10.1126/sciadv.adi0482
Gonzalez Lopez er al., Sei. Adv. 9, eadi0482 (2023) 2 August 2023 17 of 17
T H E D E V E L O P M E N T O F D E N T I N M I C R O S T R U C T U R E IS C O N T R O L L E D B Y
T H E T Y P E O F A D J A C E N T EPITHELIUM
Appendix C The Development of Dentin
Microstructure Is Controlled by
the Type of Adjacent Epithelium
This section contains the corresponding paper including its appendix.
121
11) Check for updates
ORIGINAL ARTICLE JBMR
The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium
Josef Lavický,1
Magdalena Kolouskova,1
David Procházka,2
Vladislav Rakultsev,1
Marcos Gonzalez-Lopez,1
Klara Steklikova,3 , 4
Martin Bartoš,5 , 6
Anushree Vijaykumar,7
Jozef Kaiser,2
Pavel Pořízka,2
Maria Hovorakova,3
Mina Mina,7
and Jan Křivánek1
1
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
2
A d v a n c e d Instrumentation and Methods for Materials Characterization, CEITEC Brno University of Technology, Brno, Czech Republic
i n s t i t u t e of Histology and Embryology, First Faculty of Medicine, Charles University, Prague, Czech Republic
d e p a r t m e n t of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic
i n s t i t u t e of Dental Medicine, First Faculty of Medicine, Charles University, Prague, Czech Republic
i n s t i t u t e of Anatomy, First Faculty of Medicine, Charles University, Prague, Czech Republic
'Department of Craniofacial Sciences School of Dental Medicine, University of Connecticut, Farmington, CT, USA
ABSTRACT
Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in
dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental
epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown
and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP-cerulean/DMP1-cherry)
mouse strain and mice with ectopic enamel (Spry2+ / _
;Spry4_ / _
), we show that the different microstructure of dentin is initiated in
the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon
is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we
introduce more generalized terms for two distinct types of dentins: cementům versus enamel-facing dentin. In the odontoblasts,
which produce enamel-facing dentin, we identified uniquely expressed genes {Dkk1, Wispl, and So//7) that were either absent or
down regulated in odontoblasts, which form cementum-facing dentin. This suggests the potential role of Wnt signalling on the dentin
structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different
types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element
composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken
together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts
and the possible role of Wnt signalling which together results in formation of dentin of different quality. © 2021 The Authors. Journal
of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research
(ASBMR).
KEY WORDS: TEETH; DENTIN; MICROSTRUCTURE; ODONTOBLAST; PROCESSES; ODONTOGENESIS; DENTINOGENESIS; INCISOR; MOLAR; W N T SIGNALING;
LIBS
T
Introduction well. Apart from the soft dental pulp, which is responsible for the
maintenance of tooth viability, teeth are composed of three difeeth
are highly specialized structures with an intricate inner ferent types of calcified tissues: enamel, dentin, and cementum.
architecture, positioned at the very beginning of the diges- The general shape, mechanical properties, and functions of teeth
tivesystem.They areimportantforfood processing and essential are determined by dentin, the most abundant and ontogenetifor
social interactions not only in humans but in other species as cally first developing hard dental tissue. Enamel, as the hardest
This is an open access article under the terms of the Creative Commons Attribution-Noncommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
Received in original form June 5, 2021; revised form October 12, 2021; accepted November 8, 2021.
Address correspondence to: Jan Krivanek, PhD, Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
E-mail: jan.krivanek@med.muni.cz
Additional Supporting Information may be found in the online version of this article.
JL and MK contributed equally to this work.
Journal of Bone and Mineral Research, Vol. 37, No. 2, February 2022, pp 323-339.
DOI: 10.1002/jbmr.4471
© 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research
(ASBMR).
323 •
and the most calcified dental tissue, protects the most exposed
part of the tooth — the crown. Cementum covers the root surface
and, together with periodontal ligaments, maintains the
anchoring of teeth in the adjacent alveolar bone. Dentin, in contrast
to enamel, is a living tissue, which, alongside pulp, forms
dentin-pulp complex.0 1
This integrated functional unit maintains
tooth homeostasis and enables the tooth to react to the outer
environment.'2
"61
Odontoblasts are responsible for dentin development and
maintenance of its viability and sensitivity. They are polarized,
long-living cells positioned on the interface between the dental
pulp and dentin itself.(7)
Odontoblasts differentiate from ectomesenchyme
after an orchestrated interaction with oral epithelium.'8
, 9 1
During tooth development, two basic types of oral
epithelium can be distinguished: the enamel-forming epithelium
and the epithelium, which plays a role in the development of the
root. Enamel-forming oral epithelium in the crown gives rise to
enamel-producing ameloblasts, while oral epithelium in the root
forms Hertwig's epithelial root sheath (HERS), which does not
produce any hard matrix and serves as an interactive partner
for differentiating odontoblasts. After initiation of dentin production,
HERS disintegrates into epithelial cell rests of Malassez
(ERM).'8 , 1 0
"1 3 1
This means that odontoblasts forming the crown
and root dentin have a different interactive epithelial partner.
The influence of oral epithelium on the determination of the different
character of the adjacent dentin's microstructure and elemental
composition has not yet been studied.
As odontoblasts lay down the dentin matrix, they are pushed
back from the dental epithelium toward the dental pulp and
leave behind a single, long process, which then persists inside
the dentin matrix.'141
Odontoblasts' processes (also known as
Tomes' fibers) are the key structural and functional components
of dentin and make this tissue sensitive and able to react to the
outer environment. This is particularly important in the process
of tertiary dentinogenesis, a process that can be referred to as
dental healing.'1 5 , 1 6 1
Odontoblasts contribute to sensing and
reacting to increased tooth wear or dental caries by thickening
the dental wall and thus prolonging the life span of teeth.'1 5 1
Interestingly, although dentin is a living tissue (similar to bone),
once dentin is formed, it is not remodelled as it occurs in bone;
therefore, dentin inner structure is determined during its
formation.'1 7 1
On the molecular level, the dentin matrix is formed by the calcification
of the protein network, which is synthesized by the
odontoblasts. This protein network is predominantly composed
of collagen I fibrils but also several other non-collagenous proteins.
Among the most abundant non-collagenous proteins
belongs dentin sialophosphoprotein (DSPP) and dentin matrix
acidic phosphoprotein 1 (DMP1), which serve as key mineralization
centers.'81
Through the expression and phosphorylation of
these proteins, odontoblasts regulate the rate of dentin matrix
calcification.'1 8 1 9 1
Both proteins are highly expressed by odontoblasts.'2
0 1
Although, in contrast to DMP1, which is also expressed
by other hard tissue-forming cells (osteoblasts/-cytes,
cementoblasts/-cytes),'2 1 , 2 2 1
DSPP is specifically expressed
within odontoblasts, which makes DSPP a unique molecular
marker suitable for odontoblast detection and characterization.
In this work, we focus on the differences between the microstructure
of dentin in the crown (labial aspect of incisor) and
the root (lingual aspect of incisor). Because we used genetically
altered organisms, which have an ectopic enamel on the lingual
aspect of mouse incisors, we are introducing more generalized
terms: cementum-facing dentin and enamel-facing dentin.
Here we show, using different genetically altered animals and
various methodological approaches, an unanticipated sitedependent
microstructure of dentin in high detail. Our results
show that the lingual and labial aspects of mouse incisors fully
reflect the crown and root aspect in brachyodont teeth in the
perspective of the microstructure of dentin and also the presence
of cementum-forming cells in the periodontium. Further,
we show a different gene expression pattern in odontoblasts
on the labial and lingual aspect as well as the elemental composition
and X-ray density in each type of dentin. Finally, using a
mouse strain with ectopic enamel on the lingual aspect of the
incisor, we confirm that the microstructure of dentin is dependent
on the interaction with a different type of adjacent epithelium
during development. Taken together, we hypothesize that
the dentin microstructure is initially induced by the type of adjacent
dental epithelium present during the beginning of odontoblast
differentiation and influenced by Wnt signalling.
Materials and Methods
Animals
Animals used in this study included DSPPCerulean
/DMP1cheny
reporter mouse strain on CD-1 genetic background,'2 3 1
Spry2+/
^;Spry4^ mice'2 4 , 2 5 1
obtained by crossing the strains
Spry2/ORF-null allele and Spry4/ORF-null allele (originally a kind
gift from Dr Ophir Klein, San Francisco, UCSF'2 6
'2 7 1
) on C57BL/6
genetic background, and C57BL/6 wild-type animals, which were
used as controls. All animal experiments were approved by the
Ministry of Education, Youth and Sports, Czech Republic
(MSMT-8360/2019-2; MSMT-9231/2020-2; MSMT-272/2020-3)
and performed according to international and local regulations.
All mice were kept in a 12-hour light/dark cycle, 18°C to 23°C
and 40% to 60% humidity. Animals had access to food and water
ad libitum. Animals were genotyped using endpoint PCR utilizing
transgene/mutation-specific primers. Both male and female
mice were used. Adult (2-month-old) animals with suitable genotype
were euthanized by isoflurane overdose (KDG9623, Baxter,
Deerfield, IL, USA) and cervical dislocation and further utilized
for experiments.
Histological analysis
Mandibles and maxillae were carefully dissected, briefly washed
in PBS, and fixed in 4% paraformaldehyde (pH 7.4) for 5 hours at
4°C. Fixed tissue was then decalcified in 10% EDTA (pH 7.4) for
10 days at 4°C. Samples were then processed either for cryosectioning
or paraffin processing. Briefly, for cryosectioning, samples
were incubated in 30% sucrose (Sigma-Aldrich, St. Louis,
MO, USA; 16104) overnight at 4°C, washed twice in Tissue-Tek
OCT Compound (Sakura Finetek Europe B.V., Alphen aan den
Rijn, Netherlands; 4583) and embedded in fresh OCT. Samples
used to obtain detail images were then sectioned to a thickness
of 14 um on cryostat Leica CM1860 (Leica Biosystems, Wetzlar,
Germany). To obtain overview images of the mandible and maxilla
of DSPPCerulean
/DMP1cherry
l samples were sectioned to a thickness
of 40 um using Kawamoto's tape (SECTION-LAB Co. Ltd.,
Hiroshima, Japan) on cryostat Leica CM1860 (Leica Biosystems).
Samples embedded in paraffin were sectioned on a microtome
(Leica SM2000R) to a thickness of 2 \im and processed as
described further.
• 324 LAVICKÝ E T A L . Journal of Bone and Mineral Research
In situ hybridization Process width analysis
For the in situ hybridization, RNAscope 2.5 HD Assay - RED
(Advanced Cell Diagnostics, Newark, CA, USA; 322350) was utilized
as described before.'2 8 1
The following probes were used:
Dkk1 (501921) and Wispl (402521). Slides were counterstained
with hematoxylin for 1 minute and mounted in Pertex medium
(Histolab, Brea, CA, USA; 00801). Sections were imaged using
Axio Imager 2 (Carl Zeiss AG, Jena, Germany).
For the analysis of odontoblast process width, confocal images of
the phalloidin-stained sagittal sections were used. Images were
analyzed using Imaris software. The width of the processes was
measured using the measure distance tool in the slice mode.
The measurement was performed at the beginning of dentin
(above the predentin) at the unified distance of 40 um from
the dentin-pulp edge. For each experimental condition, 85 individual
processes were measured.
Immunostaining, confocal imaging, and image analysis
All cryosections were washed three times in PBS with 0.1%
Tween 20 (Sigma-Aldrich, P1379). Sections from DSPPC e r
"t e o
7
DMP1cherry
animals were stained with DAPI (Carl Roth, Karlsruhe,
Germany; 6335.1) and mounted in VECTASHIELD Antifade
mounting medium (Vector Laboratories, Burlingame, CA, USA;
H-1000-10). Sections from wild-type and Spry2+/
~;Spry4~'~ animals
were stained with Phalloidin-Alexa Fluor 488 (Cell Signaling
Technology, Danvers, MA, USA; 8878S) diluted 1:100 in PBS
(pH 7.4) overnight at 4°C. Sections were then subsequently
stained with DAPI and mounted in 87.5% Glycerol (Penta CZ,
Katovice, Czech Republic) diluted in PBS. For the detection of
periostin (POSTN), sections from DSPPCerulear
'/DMP1cherry
animals
were stained with anti-POSTN antibody (Novus Biologicals, Littleton,
CO, USA; NBP1 -30042) diluted 1:200 overnight at 4°C. Subsequently,
the sections were stained with donkey anti-rabbit
secondary antibody conjugated with Alexa Fluor 647 (Thermo
Fisher Scientific, Waltham, MA, USA; A31573) diluted 1:500 at
room temperature for 2 hours. Sections were then stained with
DAPI and mounted using VECTASHIELD Antifade mounting
medium. For the analysis of the expression of odontoblastspecific
markers, SALL1 antibody (Abeam, Cambridge, MA, USA;
ab31526) and NFIC antibody (Novus, NBP2-37935) were used.
SALL1 staining was performed on paraffin sections after pH 9
antigen retrieval (Agilent Dako, Santa Clara, CA, USA; S2367) at
95°C for 15 minutes. NFIC staining was performed on cryosections
from wild-type animals without antigen retrieval. Sections
were incubated with antibodies overnight at 4°C. Subsequently,
donkey anti-rabbit secondary antibody conjugated with Alexa
Fluor 568 (Thermo Fisher Scientific, A10042) diluted 1:800 was
used, and nuclei were counterstained with DAPI (Carl Roth,
6335.1). Slides were then mounted in Fluoromount Aqueous
Mounting Medium (Sigma-Aldrich, F4680). Sections were
imaged using LSM880 confocal microscope (Carl Zeiss AG,
Germany). Obtained data were then analyzed and exported
using Imaris (Oxford Instruments, Abingdon, UK) and ZEN software
(Carl Zeiss AG, Germany).
Process number density analysis
The number density of processes was analyzed using coronal
sections of dentin and from Z projections of sagittal sections of
dentin obtained in the section mode of Imaris software. Subsequently,
the processes were manually counted using ImageJ
software, utilizing the point tool. The number of processes was
counted on four unique regions of interest. All of the regions of
interest used for the analysis contained at least 75 unique
processes.
Analysis of dentin fraction without the presence of the
main process
The distance from the furthermost point of processes main filament
up to the enamel-dentin or cementum-dentin junction
(= height of dentin without the presence of the main process)
and total height of dentin in the corresponding place was measured
using the measure distance tool in the slice mode of Imaris
software. To obtain the fraction of dentin without the presence
of the main process, the height of dentin without main OP was
divided by the total dentin height. A total of 33 unique processes
per experimental condition were analyzed.
Analysis of the distribution of processes in dentin
The unified 10-um-thick Z-stacks were created using ZEN Blue
software's (Carl Zeiss AG) employing the image subset function.
Z-stacks were exported as maximum-intensity projections using
Imaris software. Subsequently, the images were analyzed in ImageJ
software.'2 9 1
Binary single-channel images were produced
using the threshold function. A 10x10 grid was overlayed over
the image using the grid overlay plugin. Using the rectangle
selection tool and the overlayed grid, 10 equal sectors (parallel
with the dentin-pulp edge) were analyzed using the measure
function. The area fraction parameter was then used for the final
analysis. The analysis was performed on three images per experimental
condition.
Process straightness analysis
Segmentation was performed in Imaris using semiautomatic
approach. Each of the randomly selected odontoblast processes
(OPs) were traced using a single polyline. Dendrite straightness
parameter was chosen for the quantification. This parameter is
defined as the ratio between the length of the whole segmented
polyline (OP) and the radial distance between the beginning and
the end of the polyline. The value of the function is always equal
to or smaller than 1 (a completely straight line has the straightness
of 1). We compared 10 randomly chosen processes per each
condition.
Analysis of the distance from cervical loop to the first
reporter expression
Sagittal sections containing the cervical loop region of incisors
from maxillae and mandibles of the DSPPCerulea
'VDMPlcheny
animals
were analyzed. The distance from the apex of the cervical
loops to the first odontoblast displaying either cerulean, cherry,
or both signals above the background level was measured using
the measure polyline tool in the slice mode in the Imaris software.
The distance was measured both on the labial and lingual
sides of the incisors. A total of eight sections (four animals, two
sections each) were analyzed.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 325 •
Calcified matrix analysis
For the analysis of the elemental composition of the incisor and
micro-CT analysis, wild-type BI/6 animals were overdosed by isoflurane
(KDG9623, Baxter), the mandibles and maxillae were then
carefully dissected, briefly washed in PBS, and fixed in 4% paraformaldehyde
(pH 7.4) overnight at4°C. Fixed tissues were then
dehydrated in ethanol (30%, 50%, 70%, 80%, 90%, 96%, 100%) in
each solution three times for 10 minutes and subsequently
embedded into epoxy adhesive LR White (Sigma-Aldrich,
L9774). The blocks containing the embedded mandibles and
maxillae were cut using a diamond saw and sections were
polished using abrasive papers P1500 and P3000.
Laser-induced breakdown spectroscopy (LIBS) analysis
The polished samples were placed in the interaction chamber,
which is a part of the LIBS Discovery device (CEITEC BUT, Brno,
Czech Republic). As the laser source Nd:YAG laser CFR-400
(Quantel, Lannion, France; 20 Hz, 532 nm, 10 ns) was employed.
The laser pulse energy was controlled by a motorized attenuator
(Eksma Optics, Vilnius, Lithuania) and was kept constant (30 mJ/
pulse) throughout the experiment. The laser beam was focused
on the sample surface with a glass triplet (Sill optics, DE,
f = 32 mm). The resulting spot size diameter was 30 um. Plasma
radiation was collected using two UV-grade lenses with a total
focal length of 75 mm and led via optical cable (400 um, Thorlabs,
Newton, NJ, USA) to the entrance of a spectrometer
(HR2000, Ocean Optics, Orlando, FL, USA). The precise timing of
the experiment was controlled by the digital pulse generator
(SyncRay, Lightigo, Brno, Czech Republic). The gate delay was
set to 1.5 us and gate width to 50 us. All measurements were
controlled by a computer equipped with LIBS control software
(Lightigo). The spatial resolution for all measurements was determined
by the laser spot size diameter and was set at 30 um in
both directions. Obtained data were processed using the R software
(R Foundation for Statistical Computing, Vienna, Austria).
Micro-CT evaluation of dentin structure
Selected specimens (sectioned teeth in PMMA block) were
scanned using desktop micro-CT SkyScan 1272 (Bruker microCT,
Kontich, Belgium). Specimens were fixed on specimen holder
and scanned under the following scanning parameters: 0.5 um
pixel size, source voltage 60 kV, source current 166 uA,
0.25 mm Al filter, rotation step of 0.1°, frame averaging = 2,
180° rotation, scanning time ~9 hours for each specimen. The
flat-field correction was updated before each acquisition. Projection
images were reconstructed into cross-section images using
NRecon software (Bruker). 2D visualizations were achieved using
DataViewer (Bruker).
The X-ray density of dentin was analyzed using ImageJ software.
Briefly, five square, regularly distributed selections per
enamel, enamel- and cementum-facing dentins, were analyzed
using the measure function. Mean grey value was then used
for the final analysis. A total of 25 selections per tissue type were
analyzed (five images, five selections per tissue type each).
Statistical analysis
Data were analyzed using Graph Pad Prism software (GraphPad
Software, La Jolla, CA, USA). First, the normality of the data was
assessed using Anderson-Darling (A2*), D'Agostino-Pearson
omnibus (K2), Shapiro-Wilk(W), Kolmogorov-Smirnov (distance)
tests, and QQ plots. If the data passed normality tests and Ftest
to determine whether they have equal variances, a two-tailed ttest
was used for the analysis. If the data did not pass normality
tests, a two-tailed Mann-Whitney test was used to analyze the
data. Subsequently, box plots and violin plots were constructed
to visualize the obtained data. Box plots were made to display
the interquartile range (25th to 75th percentile). Whiskers were
used to indicate upper and lower extremes (1.5 x interquartile
range). Outliers were shown in red. When the number of data
was less than 20, all points were shown. To visualize the data
obtained from the analysis of the distribution of processes in
dentin, the trend was traced through the medians of the respective
regions with whiskers showing the whole range of values.
The trends were tested using Pearson's linear correlation of
medians from the individual data sets. This analysis was performed
in MATLAB (MathWorks, Natick, MA, USA) programming
language using a built-in linear of rank correlation (corr) function
(testing a null hypothesis of no correlation against the alternative
hypothesis of a nonzero correlation). The critical p-value for statistical
significance was set to p = 0.05.
Results
The microstructure of odontoblasts' processes in the
crown and roots of molars is reflected by the labial and
lingual aspects of incisors
To investigate the microstructure of OPs in different parts of
mouse molars, we performed confocal microscopy analysis of
the first molars of DSPferulean
/DMP1cherry
mice.'2 3 1
This mouse
strain enables the visualization of odontoblast bodies and processes
through the dual expression of fluorescent proteins constitutively
expressed under the DSPP (cerulean) and DMP1
(cherry) promotors. Three-dimensional confocal analysis of the
first molars showed an entirely diverse structure of OPs in the
crown and root part of the tooth (Fig. 1A-C). Processes in tooth
crown dentin displayed smooth and straight morphology with
thin and short secondary processes (branches of the main process).
On the other hand, odontoblasts' processes in root dentin
displayed complicated irregular morphology with longer and
more pronounced secondary processes (Fig. 18, Q. To uncover
if this phenomenon is also mirrored in the crown and root analogue
of continuously growing teeth, we performed the same
type of analysis in mouse incisors. Our analysis confirmed a consistent
trend between processes found in both molar and incisor
(Fig. 1D-F), supporting the crown analogue/root analogue theory
(Fig. 1G). Mandibular incisors are in contrast to maxillary incisors
more widely used in research.'2 8 , 3 0 , 3 1 1
To assess if different sitespecific
microstructure is reflected in dentin in both morphologically
different incisors, we compared both incisors via the same
approach and found an identical microstructural pattern (Fig. 2).
Enamel-forming epithelium controls the morphology of
OPs in adjacent dentin
To investigate the induction role of different kinds of dental epithelium
during early stages of hard-tissue formation, we took
advantage of Spry2+/
^;Spry4^ mice, which are known for the
presence of ectopic enamel on the lingual side of the incisors.'2
4 , 2 5 1
In contrast to the wild-type mouse in which theameloblast
layer is only present on the labial side, Spry2+/
~/Spry 4~7
~
mice display ameloblast layer on both labial and lingual sides.
Additionally, the lingual cervical loop (LiCL) displays similar
• 326 LAVICKÝ E T A L . Journal of Bone and Mineral Research
Dentin structure in molar and incisor: root (analogue) vs. crown (analogue)
Crown (analogue)
Enamel
Cementum-facing dentin
Root (analogue)
Fig 1. Dentin microstructure in molar and incisor: root (analogue) versus crown (analogue). Confocal images from thick, cleared histological sections from the
DSPPC e r u , M
"/DMP/c
', e r
'y
mouse reporter strain show details of the microstructure of dentin with the focus on odontoblasts' processes in different parts of teeth.
The sagittal section of the first molar (A) and transversal section of the mandibular incisor (D) show the structural similarities between these two types of teeth
and the distribution of fluorescent proteins in the dental tissue and its close surroundings. Note the expression of cherry (DMP1) in odontoblasts but also boneforming
osteoblasts(cytes) and cementum-forming cementoblasts(cytes) on the surface of roots, while cerulean (DSPP) is expressed specifically by odontoblasts.
Subsets of A and 6 show details of enamel-facing (crown/crown-analogue) dentin (6, £) and cementum-facing (root/root analogue) dentin (C, F), respectively.
Arrowheads highlight the presence of DMP1-expressing cementum-forming cells located around the root of molar and root analogue of incisors. Schematic
visualization of the microstructure of dentin showing the same pattern in enamel-facing and cementum-facing aspects in both molars and incisors (G). Scale
bars = 300 a m (A, D), 100 am (6, C, E, F). A b = alveolar bone; De = dentin; en = enamel; O b = odontoblasts; P = pulp.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 327 •
A Maxillary incisor (sagittal) B Mandibular incisor (sagittal)
QSPflCerulean
DMP1
D A P I ''.JM
Fig 2. Mandibular and maxillary incisor dentin microstructure. Both upper and lower continuously growing incisors [A, B) show the same pattern in the
labial (enamel-facing) (C, E) and lingual (cementum-facing) (D, F) odontoblast processes, respectively. White asterisks mark different microstructure of
odontoblast processes in the lingual (cementum-facing) aspect of both mandibular and maxillary incisors. Scale bars = 1000 urn (A, B) and 50 urn (CF)
for details. En = enamel; PDL = periodontal ligaments.
morphology to the labial cervical loop (LaCL) (Fig. 6C, D); however,
wild-type mice have an ameloblast layer only on the labial side and
the lingual dental epithelium is disintegrated.011
To visualize the morphology
of OPs, we used the staining with Phalloidin-Alexa488,
which specifically binds to actin'3 2 1
and enables the visualization of
all cellular compartments in mineralized tissues. Confocal analysis
of the phalloidin-stained sections from wild-type animals further confirmed
the different structure of odontoblasts' processes in the labial
and lingual aspects of the incisor (Fig. 3A), which coincided with our
previous analysis of the DSPPCeruleon
/DMP1che
"y strain (Fig. 1D-F;
Fig. 2). However, in the Spry2+/
^;Spry4^ animals, where the ectopic
ameloblast layer and enamel are also developed on the lingual
aspect, the microstructure of OPs was the same as on the labial
aspect of the incisor (Fig. 3A, B), displaying the same smooth and
straight morphology. Their appearance, therefore, reflected the morphology
of the OPs in the crown dentin of the molar (Fig. 1A 8). To
further analyze differences in the morphology of OPs and specifically
their terminal branching regions,041
which are in contact with adjacent
enamel or cementum, we first performed detailed microscopy
focusing on the terminal part of OPs (Fig. 4A). Furthermore, we performed
the segmentation of the processes and quantified their
straightness (Fig. 4S, Q.
Our results show similarities in the pattern among the OP endings
(Fig. 4A) and whole-process morphology (Fig. 48) in the
enamel-facing dentin and cementum-facing dentin. These
results are consistent in wild-type molars, maxillary, and mandibular
incisors, using DSPPCeru
'ean
/DMP1cherry
animals or phalloidin
staining in wild-type and Spry2+/
^;Spry4^ animals (Fig. 4).
The morphology of OPs in all enamel-facing dentins was highly
similar. In the case of cementum-facing dentins, OPs in both
maxillary and mandibular incisors displayed highly similar morphology.
In the molars, OPs in the cementum-facing dentin displayed
thinner and more twisting morphology when compared
with the incisors (Fig. 4A).
To uncover the morphology and distribution of cementumforming
cherry+
(DMP1-expressing) cells in maxillary and mandibular
incisors and to compare this pattern to molar roots, we
analyzed the periodontal space on the lingual aspects of both
types of incisors using DSPPCerulean
/DMP1cheny
mouse strain
(Fig. S1). Our data show that the structure of periodontal space
on the labial aspect of the incisor resembles the situation in the
molar. However, in the more distal parts, just a seldom presence
of DMP1-expressing cementoblasts was observed. This enabled
us to establish universal terms cementum-facing and enamelfacing
dentin.
Quantifications of labial and lingual dentin differences
To quantify these differences, we performed segmentation and
analysis of the individual processes. Quantification of processes'
straightness shows that in the DSPPCerulean
/DMP1cheny
strain's
molar crown dentin, OPs display the mean straightness of
0.96 ± 0.02 (a straight line has the straightness of 1), while the
OPs in the root dentin display the straightness of 0.90 ± 0.04
(Fig. 4Q. OPs in the labial (with enamel) dentin of mandibular
incisor display the straightness of 0.99 ± 0.01 and the OPs on
the lingual (without enamel) side display 0.98 ± 0.01. In the maxillary
incisor, the OPs of the labial (enamel-facing) side showed
the straightness of 0.98 ±0.01, while on the lingual (cementum-facing)
side, OPs displayed 0.95 ± 0.02. In the wild-type
maxillary incisor stained with phalloidin, OPs of the labial side
displayed the straightness of 0.98 ± 0.01. The OPs on the lingual
side displayed, on the other hand, the straightness of
0.90 ± 0.05. Finally, the OPs of the Spry2+/
~;Spry4^ animals
displayed on the labial (enamel-facing) side the straightness of
0.99 ± 0.01 and 0.99 ± 0.00 on the lingual (ectopic enamelfacing)
side.
• 328 LAVIČKY ET AL. Journal of Bone and Mineral Research
To further highlight the differences in the features of odontoblast
processes and corresponding dentins, we analyzed
confocal images of labial (enamel-facing) and lingual (cementum-facing)
dentins of wild-type animals and labial (enamel-facing)
and lingual (ectopic enamel-facing) dentins of Spry2+/
~;
Spry4^ animals (Fig. 5). The analysis of OP's width showed that
processes localized on the labial-side dentins of wild-type mice
(both mandibular and maxillary) and maxillary Spry2+/
~ ;Spry4~y
~
incisors were significantly thinner (p < 0.0001) than the processes
located in the corresponding lingual-side dentins
(Fig. 5A). Interestingly, the width of theOPs located in the lingual
(ectopic enamel-facing) dentin of the Spry2+/
~;Spry4^ maxillary
incisor was significantly lower compared with its lingual
wild-type counterpart (Fig. 5A).
Analysis of the number density of the processes showed that
the processes on the labial sides of the incisors have significantly
higher density than processes on the lingual side of all analyzed
conditions, including the ectopic enamel-facing lingual dentin of
Spry2+/
~;Spry4~'~ animals (Fig. 58). Comparison of wild-type and
Spry2+/
~;Spry4~y
~ process density showed no significant change
(p = 0.0532) between the lingual sides of the incisors.
To quantify the changes of the dentin quality in relation to the
OPs, we analyzed the relative fraction of dentin height without
the presence of the main process (Fig. 5C] and the distribution
of all processes in different types of dentins (Fig. 5D). The fraction
of the dentin without the presence of the main process was significantly
larger (p < 0.0001) in the lingual (cementum-facing)
dentin than in the labial (enamel-facing) dentins of wild-type
mandibular and maxillary incisors. In contrast to this, the fraction
of the labial and lingual dentins of the maxillary incisor of the
Spry2+/
~;Spry4^ animals showed similar values (p < 0.7462),
which fully reflects the altered situation in the lingual (ectopic
enamel-facing) dentin and show high similarity induced by the
changed type of dental epithelium. The differences between
the lingual sides of maxillary incisors of wild-type and Spry2+/
~;
Spry4^ animals were also significant (Fig. 5Q.
To quantify the density and distribution of all OPs (including side
branches) in the enamel-facing and cementum-facing dentins, we
analyzed the OP density in 10 different regions, beginning at the
predentin up to the outermost dentin (Fig. 5D; Fig. S2). Pearson's
correlation between the lingual and labial dentins was calculated
using medians of each of the data points (Fig. 5D). Our analysis
shows distinct trends of the area fraction in labial and lingual dentin
of mandibular (Ft = 0.6165, p = 0.0802) and maxillary (R = 0.2612,
p = 0.46) incisors of wild-type animals. In contrast to wild types,
the analyses of labial and lingual dentins in Spry2+/
~;Spry4^ maxillary
incisor show correlating trends (R = 0.8697, p = 0.0011), which
further confirms our hypothesis about the inductive function of the
type of attached dental epithelium during development. The plots
of Pearson's correlations are shown in Fig. S3.
All performed quantifications further support our hypothesis
of the different microstructure of cementum-facing and
enamel-facing dentin. Most interestingly, the lingual, ectopic
enamel-facing dentin of the Spry2+/
^;Spry4^ animal displayed
similar patterns of OPs compared with its labial counterpart. This
confirms a different inductive role of ameloblast-forming dental
epithelium and non-ameloblast-forming dental epithelium on
adjacent dentin microstructure.
A Wildtype B Sprouty2+/
- Sprouty4~'
Fig 3. Dentin adjacent to ectopically developed enamel shows altered microstructure. Confocal images of the labial and lingual aspect of maxillary incisors
stained with Phalloidin-Alexa488 show the different microstructure of OPs on the labial and lingual aspect of incisor in wild-type animals (A) and similar
microstructure on both aspects in maxillary incisor of Spry2+/
~;Spry4 animals (6) having ameloblasts and enamel on both sides of the tooth. (C)
Schematic drawing reflecting the influence of different dental epithelium on dentin development. Red asterisk shows the altered morphology of OPs
in dentin adjacent to ectopic enamel on the lingual aspect of the maxillary incisor in Spry2+/
^;Spry4^/
^. Scale bars = 50 u m . En = enamel space; LaCL
= labial cervical loop; LiCL = lingual cervical loop; PDL = periodontal ligament.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 329 •
Microstructure of odontoblast processess of enamel-facing and cementum-facing dentin
Crown / Labial aspect Root / Lingual aspect
B C
Cr, R o , Process straightness
p=0.0001
I
Cr. Ro.
p=0.0019
rjjri
Lab. Ling.
p<0.0001
Lab. Ling.
u . i B Lab. Ling.
Fig 4. Analysis of odontoblasts' terminal branching regions and processes straightness in enamel-facing and cementum-facing dentin. Confocal images
of histological sections from DSPPc
"u
'e
',n
/DMP1ch
"ry
, wild-type, and Spry2+/
^;Spry4^ animals stained with Phalloidin-Alexa488 with a special emphasis
on the terminal branching regions relating to the first formed dentin adjacent to enamel and c e m e n t u m , respectively (A). Details on the process morphology
are shown in (6) and quantifications straightness of the processes in (Q. Red asterisks s h o w the altered morphology of OPs in dentin adjacent to
ectopic enamel on the lingual aspect of the maxillary incisor in Spry2+/
~;Spry4 . Scale bars = 20 iim.Cr = crown (analogue); en = enamel/enamel space;
Lab. = labial side; Ling. = lingual side; PDL = periodontal ligament; Ro = root (analogue).
• 330 LAVIČKY ET AL. Journal of Bone and Mineral Research
Characterization of the features of processes and dentin
Mandibular incisor Maxillary incisor
Wildtype Wildtype Sprouty2v
Sprouty4' Lingual sides
_ 4 _ 4p<0.0001
p<0.0001
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p<0.0001
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p=0.0219
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ect. enamel
Wildtype Mandibular incisor Wildtype Maxillary incisor Spry2r/
- Spry4-A
Maxillary incisor
_100-
50Pearson's
correlation
R = 0.6165
p = 0.0802
• Labial
I Lingual
- i — I — I — I — I — I — I — r
1 2 3 4 5 6 7 8 9 10
Regions
Predentin -»Outermost dentin
Pearson's correlation
R = 0.2612
0.4600
• Labial
• Lingual
2 50~~i
i i i r
1 2 3 4 5
T 1 1 1 r—
6 7 8 9 10
Regions
Predentin —> Outermost dentin
Pearson s correlation
R = 0.8697
P = 0.0011
1 2 3 4 5 6 7 8 9 10
Regions
Predentin ->Outermost dentin
Fig 5. Characterization of t h e features of processes a n d d e n t i n . Quantification of different aspects of o d o n t o b l a s t processes a n d d e n t i n quality
in m a n d i b u l a r wild-type a n d maxillary wild-type a n d Spry2+/
~;Spry4~/
~ animals. Process w i d t h {A) m e a s u r e d as a w i d t h of individual o d o n t o blast
processes at t h e distance of 4 0 u.m f r o m t h e p r e d e n t i n - p u l p e d g e (n = 85). Process density (6) calculated as a n u m b e r of O P s per
100 u.m2
of d e n t i n (n = 4 ROIs). Fraction of d e n t i n height w i t h o u t t h e presence of t h e m a i n process (Q (n = 33). Analysis of t h e distribution
of all processes in d e n t i n ranging from predentin (1) to t h e o u t e r m o s t d e n t i n (10) is s h o w n in (D). Pearson's correlation coefficients (ft) were
calculated a n d t e s t e d , p < 0.05 expresses t h e fact that trends are correlated (n = 3 ROIs for e a c h data point). Ect. e n a m e l = e c t o p i c e n a m e l ;
Lab. = labial side; L i n g . = lingual side.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 331 •
Early polarization and differentiation of odontoblasts in
enamel and cementum-facing dentin
To further investigate an early odontoblast's differentiation and
dentin morphogenesis, we analyzed the (pre)odontoblastepithelium
interphase close to the labial and lingual cervical
loops in wild-type and Spry2+/
^;Spry4^ animals (Fig. 6). We
show that the presence of differentiating preameloblasts stimulates
adjacent preodontoblasts to synchronous polarization and
more consistent generation of OPs, resulting in the generation of
highly tubular dentin (Fig. 6A, C, D). Conversely, the emerging
dentin adjacent to the lingual aspect of the wild-type incisor
shows almost atubular structure, likely resulting from more asynchronous
polarization of odontoblasts suggested by the uneven
distribution of odontoblast nuclei (Fig. 68). These findings suggest
an early patterning effect of the adjacent dental epithelium
type on the future microstructure of the whole dentin matrix.
To investigate differences in the timing of odontoblast differentiation
between the labial and lingual side of the tooth, we
measured the distance of first DSPP- and/or DMP1-expressing
(cerulean and cherry fluorescent protein-expressing, respectively)
odontoblast from the apex of each cervical loop in
DSPPCerulean
/DMP1che
"y
animals (Fig. 7). In the mandibular incisor,
the distance to the first cerulean-positive, cherry-positive, and
both reporter-expressing odontoblast was significantly longer
on the lingual side when compared with the labial side (Fig. 7A,
B). In the maxillary incisor, the differences in the distances were
greatly reduced, but the distances on the lingual side were still
significantly longer for all three measured features (Fig. 7C, D).
These data suggest that the differentiation into functional odontoblasts
is faster on the labial side of the incisor.
Molecular differences of odontoblasts on the labial and
lingual aspect of mouse incisor
To address molecular differences of odontoblasts on the labial
and lingual aspect, we performed in situ hybridization (ISH)
and immunohistochemical (IHC) expression analysis of selected
odontoblast-specific genes. First, we analyzed the expression of
two Wnt-signaling related genes, Dkk1 and Wispl, using ISH.
Organization of dental epithelium in wildtype and Spry2+
/~ Spry4'/~
Fig 6. Early polarization of odontoblast and dentin patterning. Confocal images of histological sections from maxillary incisors of wild-type and Spryz' ;
Spry4~'~ animals stained with Phalloidin-Alexa488 (green) and DAPI (blue). Each panel contains an overview image of the early differentiating area where
preodontoblasts polarize and differentiate. Highlighted subsets show details of differentiation. In the wild-type mouse, epithelium at the labial aspect
gives rise to (pre)ameloblasts (highlighted with white arrowheads A), whereas at the lingual aspect, preameloblasts are not formed and the epithelium
is being disintegrated (6). In contrast to this, the enamel-forming ameloblast epithelium is formed by both labial and lingual cervical loops of Spry2+/
~;
Spry4_/
~ incisors. Ectopic, (pre)ameloblastic epithelium is marked with red arrowheads. Asterisks highlight the difference between the structure of
wild-type and Spry2+/
~;Spry4^/
^ lingual cervical loop highlighting different interacting partners of the respective (pre)odontoblasts. Note faster polarization
of odontoblasts induced by adjacent epithelium with ameloblastsatthe labial aspect of wild-type and both aspects of Spry2+/
^ ;Spry4^^ incisors {A, C,
D). Scale bars = 100 u.m (cervical loop tile scans), 25 u.m (details). A b = ameloblasts; p A b = preameloblasts; De = dentin; En = enamel space; LaCL = labial
cervical loop; LiCL = lingual cervical loop; O b = odontoblasts; p O b = preodontoblasts; P = pulp; PDL = periodontal ligament.
• 332 LAVICKÝ E T A L . Journal of Bone and Mineral Research
Strong expression of Dkkl was localized in preodontoblasts
exclusively on the labial side of the incisors (Fig. 8A, B). On the lingual
side, only weak spots of positive signal were found. Wispl
expression was also located predominantly on the labial side of
the incisors with the expression being consistent even in older
odontoblasts (Fig. 8C, D). On the lingual side, a weaker signal
was found only close to the cervical loop. Similar to this, we
observed a specific and strong nuclear signal of SALL1 (IHC) in
the odontoblasts on labial sides of the incisors (Fig. 8/5, F). On
the lingual side, the nuclear signal of Sal 11 was only slightly
above the level of the background and started further from the
cervical loop apex. Nuclear signal of NFIC was found in the pulp
cells surrounding both cervical loops (Fig. 8G, H). NFIC was also
expressed in odontoblasts and subodontoblastic cells on both
sides of the incisors. The signal found in odontoblasts did, however,
diminish further from the cervical loops.
Analysis of element composition and dentin density
To further investigate whether the differences in the morphology
of the OPs are connected to the different composition of
key elements of the dentin matrix, we performed laser-induced
breakdown spectroscopy (LIBS) analysis on the established
model of mouse incisor. The spectroscopic analysis provided
semiquantitative information on the spatial distribution of calcium
and magnesium in mandibular and maxillary mouse incisors
on transversal sections. The methodology of elemental
mapping by LIBS has been previously described.'3 3 , 3 4 1
The
analysis was performed on the ground, polished sections from
the distal part of the maxillary and mandibular incisors (Fig. 9A).
The relative contents of calcium and magnesium were plotted
as a function of the measurement position. It was observed that
calcium was most abundant in the enamel in contrast to magnesium,
which was detected in higher amounts in dentin (Fig. 9A).
Focusing on the dentin, we detected a higher amount of calcium
in the dentin facing the enamel in both mandibular and maxillary
incisors. Magnesium showed uneven distribution within the
dentin matrix being slightly more present in the enamel-facing
dentin in the mandibular incisor.
Additionally, principal component analysis (PCA) of the LIBS
data was performed to further highlight more complex relationships
of the elemental composition of incisors. The utilization of
PCA in LIBS has already been thoroughly described previously.'3
5 1
Principal component (PC) scores were plotted on a
map with respect to the original spectra position. The resulting
patterns were afterwards compared with the original teeth structure
knowing the correlation between PC scores and the original
samples. The respective PC loadings were investigated to yield
information about the contribution of individual elements. Most
interesting information was observed in PC1 and PC3 presented
in Fig. 9/3 and Figs. S3 and S4. Analysis of loadings (Fig. S4)
showed the highest contribution of Ca followed by Mg to PC1.
In other words, the higher the PC score means the higher the
intensity of thespectral lineand hence the content of the respective
element. Conversely, in PC3, higher values resulted from the
high intensity of calcium and low intensity of magnesium. PCA
suggested a trend of enamel-facing dentin being different in
Lingual cervical loop DSPPc
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Distance from C L to reporter expression
2500-
3 2000p
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• Distance to DSPP
• Distance to DMP1
H Distance to both
LaCL
—I
LiCL
Q Distance from C L to reporter expression
2500-1 i
• Distance to DSPP
• Distance to DMP1
• Distance to both
» 1500-
_1
O
£ 1000p
= 0.0008
p = 0.0047
p = 0.0413
_ 3
T T
Fig 7. Reporter expression distance analysis. Analysis of images taken from DSPPCenjlean
/DMP1cherr
' mandibular (A) and maxillary (O incisor's apical
regions. The distance from the apex of the cervical loop to the first cell expressing fluorescent protein: DSSP (cerulean), DMP1 (cherry), or both were measured
and results were compared between the lingual and labial aspects (6, D). Scale bars = 200 um. Arrows indicate cementoblasts. A b = alveolar bone;
LaCL = labial cervical loop; LiCL = lingual cervical loop; PDL = periodontal ligament.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 333 •
elemental composition from the opposing, cementum-facing
dentin (Fig. 98; Fig. S4). This phenomenon was more pronounced
in the case of the mandibular incisor.
Based on the above-mentioned results, the PC1 and PC3 scores
were cross plotted (Fig. S4) and data points clustered by using kmeans
clustering (5 clusters). Each cluster was assigned a color
and the respective colors were subsequently presented in the
picture as a function of position (Fig. 9Q. Cluster 1 corresponds
to enamel, clusters 2 and 3 correspond to both cementum-facing
dentin and alveolar bone. Cluster 5 corresponds to resin in which
the samples were embedded and soft tissues. Finally, cluster 4 specifically
designates enamel-facing dentin.
Finally, to assess the dentin X-ray density in different regions
of dentin, we utilized the micro-CT analysis of wild-type maxillary
Molecular differences in odontoblasts on the labial and lingual aspect of mouse incisors
Mandibular incisor Maxillary incisor
Fig 8. Molecular differences of odontoblasts on the labial and lingual aspect of mouse incisor. Analysis of selected odontoblast-specific molecular
markers shows a different expression pattern on the labial and lingual aspect of both mandibular and maxillary incisors. In situ hybridization of Dkkl
(A,B) and Wispl (C,D) shows a specific expression only in odontoblasts on the labial aspect of the incisor. Similar to this, the immunohistochemical staining
of SALL1 shows the same expression pattern (E, F), while the immunohistochemical staining of previously shown NFIC (G, H) did not show a different
expression on labial and lingual aspects. Scale bars = 200 am (overview images) and 50 am (details). LaCL = labial cervical loop; LiCL = lingual cervical
loop; A b = ameloblasts; p A b = preameloblasts; O b = odontoblasts; p O b = preodontoblasts; PDL = periodontal ligament.
• 334 LAVICKÝ E T A L . Journal of Bone and Mineral Research
LIBS analysis of element composition of wildtype incisors
A Bright field Calcium Magnesium
B P C I P C 3 C Clustering
Micro-CT analysis of X-ray density of wildtype incisors
D uCT crossection Maxillary incisor Mandibular incisor
Fig 9. Analysis of elemental composition and dentin density. Brightfield images (left) of ground sectioned, polished teeth sections and subsequent analyses
(right) represent heat maps showing the relative amount of calcium and magnesium on the transversal section from the distal part of the mandibular
and maxillary incisor (A). The function of position plots of principal c o m p o n e n t analysis (6) and /c-means clustering from the P C A (D). Micro-CT analysis of
dentin density in EFD and C F D of mandibular and maxillary incisors (C) (n = 25). Graphs also plot the grey mean values of enamel to enable the comparison
between dentin and enamel. Scale bars = 500 urn. AB = alveolar bone; C F D = cementum-facing dentin; EFD = enamel-facing dentin; En = enamel;
PDL = periodontal ligament.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 335 •
and mandibular incisor (Fig. 9D). Although the differences
between the enamel-facing (EFD, labial) and cementum-facing
(CFD, lingual) dentins are faint, the analysis of mean gray value
of these two regions has shown significantly higher values in
EFD (p < 0.0001) in both mandibular and maxillary incisor.
Discussion
Microstructure, chemical composition, and development of dentin
and odontoblasts have been extensively studied topics for
many decades. This tissue gives the tooth its shape and specific
mechanical and sensory properties. Here we expand the knowledge
about dentin microstructure and composition and provide
evidence for differences in the microstructure of dentin secreted
by odontoblasts facing dental epithelium during early hardtissue
formation. Furthermore, we show differential expression
of several odontoblast-specific genes that may control the
observed differences in dentin and odontoblast structure.
Because dentin is not remodelled, the complete developmental
history of dentin is sealed in the inner structure of this hard
matrix. The morphology of dentinal tubules then reflects the
development of odontoblasts and visualization of dentin microstructure
provides us with a detailed history of the dentinogenesis
itself.'1 4 3 6 1
Our data show that the type of dental
epithelium influences the adjacent preodontoblasts in a different
manner, which results in different microstructure and elemental
composition of the adjacent dentin controlled by the
very beginning of its formation.
It was shown before that dentin in root and crown differs on
several levels including the composition of phosphoproteins,
levels of calcification, speed of crystal growth, and mechanical
properties.'1 8 1 9 , 3 7 1
However, only a limited number of research
studies focus on the morphology of the dentinal tubules and
their functional components—odontoblast processes.'1 4 , 3 6 , 3 8 1
Using DSPPCerulean
/DMP1cheny
mouse strain, we were able to
map the three-dimensional morphology of odontoblast processes
in an unprecedented detail. First, we described a significantly
different OPs morphology within the crown and roots of
mouse molars. Furthermore, our results show that this phenomenon
is reflected in the incisors, which are structurally and developmentally
different. Importantly, we show that the inner
microstructure of dentin is changed when the type of adjacent
epithelium changes its function. Our findings suggest that the
predetermination of dental epithelium controls the structure of
OPs by controlling the differentiation of the adjacent odontoblasts.
Our results are consistent when comparing different types
of teeth (both molars and incisors) and different inbred strains
(CD1 and BI/6 background). Functional analysis on animals with
ectopic enamel on the lingual aspect of maxillary incisors (using
Spry2+/
~'\Spry4~'~ animals) enabled us to make a conclusion of
enamel-facing and cementum-facing predetermination of dentin
microstructure. Important to note is that the phenotype of
Spry2+/
~;Spry4~'~ animals is highly variable with incomplete
penetrance. We observed the presence of ectopic enamel only
in a few maxillary incisors (which were used for the analysis),
but no mandibular incisors with ectopic enamel were observed.
Comparing the microstructure of maxillary and mandibular incisors
using DSPPCeru
'ean
/DMP1chenv
animals provided us with identical
results regarding the microstructure of OPs. This, together
with the results obtained from the analysis of Spry2+/
~;Spry4~'~
maxillary incisor, serves as adequate proof of principle.
It had been shown that cementum is present on the lingual
aspect of rodent incisors.'3 9 1
The development of cementoblasts
and cementum in continuously growing incisors is still not fully
understood.'3 9
"4 1 1
Our results confirm the presence of
DMP1 -expressing cementoblasts in the periodontal space
attached to the lingual side of dentin. Periodontal ligaments in
periodontal space were visualized by POSTN staining for greater
clarity (Fig. S1).'2 8 )
The structure of the cementoblast layer and
periodontal space in the incisors displayed the same pattern as
in molar roots. This enabled us to establish universal terms
cementum-facing and enamel-facing dentin. DMP1 -expressing
cementoblasts were preferentially found in the more apical
region on the incisor lingual aspect. In the more distal parts, just
a seldom presence of DMP1 -expressing cementoblasts was
observed. This might show that the activity of cementum-forming
cells is limited mostly to the apical region and later their
activity decreases.
To quantify the observed differences between the OPs found
in enamel-facing and cementum-facing dentin, we at first chose
straightness. This parameter was chosen because of its independence
on the length of the process, which naturally varies
because of changing dentin height. The observed trend showed
that in the enamel-facing dentin, the OPs have straighter
(straightness value closer to 1) morphology when compared
with the cementum-facing dentin. Although the general trend
of cementum-facing and enamel-facing microstructure of OPs
was similar in all observed conditions and samples, minor differences
can be observed in OP morphology comparing incisors
and molars.
The overall larger differences in the straightness of the two
different classes of OPs in the molar may be caused by the temporal
separation of the production of the crown and root dentin.
Conversely, the smaller differences in the incisors may be caused
by synchronous deposition of the dentin matrix, which has to
accommodate for the abrasion of the tip of the incisor. Furthermore,
the small, though still statistically significant, differences
between the OPs in the mandibular incisor may be in part caused
by less pronounced curvature of the incisor when compared
with the maxillary incisor in which the OPs have a larger difference
between the two sides.
Moreover, in mouse incisors, we analyzed the OP's width,
number density, the dentin quality by measuring the fraction
of dentin height without the presence of the main process and
the analysis of the distribution of all processes in different
regions of dentin. All these data have consistently shown statistically
significant differences in all aspects between the labial
and lingual sides of wild-type mandibular and maxillary incisors.
Additionally, the analysis of dentin in Spry2+/
^;Spry4^ maxillary
incisors has shown that dentin facing an ectopically present
enamel on the lingual aspect of the incisor is in most of the
observed aspects more similar to dentin on the labial side. This
further confirms the inductive role of different kinds of dental
epithelium on dentin microstructure. Interestingly, the number
density of processes in Spry2+/
^;Spry4^ displayed the same
trend as observed in wild-type animals. This suggests that this
feature of dentin is determined by a different mechanism, which
is independent of the inductive role of the ameloblastic
epithelium.
Previous studies performed on rat incisors report that in mandibular
incisors the enamel-facing odontoblasts display production
of predentin that exceeds that of the cementum-facing
odontoblasts.'1 8 1
Furthermore, it has been reported that the conversion
of cementum-facing predentin into dentin is faster than
• 336 LAVICKÝ ET AL. Journal of Bone and Mineral Research
in the enamel-facing dentin. Thus, the speed of dentin growth
might cause a change of OP morphology as a secondary effect.
Based on these previous observations, we decided to investigate
the possible differences in the early odontoblast differentiation
and dentin production. To do so, we focused on the
cervical regions of the incisors. Our results show that the distinct
morphology of OPs on labial and lingual sides of wild-type incisors
is established during the early odontoblast differentiation.
Interestingly, on the lingual side, the odontoblasts polarize further
from the cervical loop and only a few of them leave their
processes in the newly formed dentin. This paired with the analysis
of the relative fraction of dentin height without the presence
of the main process suggests a more asynchronous differentiation
of odontoblast without the presence of ameloblastic epithelium.
This is further supported by the fact that in both analyses
the Spry2+/
~;Spry4^^ lingual dentin displays the same trends
as the labial dentin.
To further investigate the rate of differentiation of
odontoblasts, we analyzed the expression of the reporters in
DSppCerulean/DMpiCherry m j c e g Q j n | j n e ^ ( h e a f o r e _
mentioned trends. Odontoblasts on the lingual side start to
express Dspp and Dmp1 further from the cervical loop, which
suggests slower odontoblast differentiation in the absence of
ameloblastic epithelium.
Although we observed a consistent phenotype in all analyzed
features and conditions, the precise molecular signalling responsible
for a different dentin production and microstructure is,
however, still unknown. One of the mechanisms that might have
a role in this process is RUNX2 and Wnt signalling, which has
been shown to have a differential role and effect on adjacent
dental mesenchyme in the crown and root dental epithelium.( 4 2 )
Our results support these findings and suggest that Wnt signalling
likely plays an important role in dentin patterning. Two
Wnt pathway-related genes {Dkk1 and Wispl) showed robustly
different expression patterns in odontoblasts on the labial and
lingual aspects of mouse incisor. Moreover, SALL1, a newly
identified transcription factor present during odontoblast
development,( 2 8 )
showed the same pattern and was predominantly
expressed in odontoblasts on the labial side. Recently,
SALL1 has been found to be an interacting partner of RUNX2
and was shown to increase the accessibility of cis-regulatory elements
inside the Runx2 locus.'4 3 1
Interestingly, the knockdown of
SalU in murine dental pulp cells in vitro have reduced their odontoblastic
differentiation and also inhibited calcification.'4 3 1
This
might coincide with the slower odontoblast differentiation and
reduced amount of calcium in the lingual dentin and also its
lesser X-ray density in vivo.
Even though it was previously shown that NFIC plays an
important role in dental mesenchyme during the growth of
molar roots,'4 4
"4 6 1
we did not observe a clear difference in the
expression of this protein on the labial and lingual side of mouse
incisor, suggesting it has a different role in incisor renewal than
in the extensively studied molar development.'4 4
"4 6 1
Further
investigation needs to be performed to answer precise molecular
pathways controlling divergent dentin development.
To investigate the elemental composition of the calcified
matrix in each type of dentin, we performed LIBS analysis
focused on calcium and magnesium content. We uncovered
characteristic regionalization of these two elements distinguishing
a cementum-facing and enamel-facing dentin on the elemental
level. Previous studies, which used microindentation as
one of the methodologies, suggested that in the rat incisor, the
enamel-facing dentin is harder, which is caused by a higher
amount of calcium and lower amount of magnesium and vice
versa for the cementum-facing dentin.'4 7 1
It has been further
connected to the fact that the enamel-facing dentin matrix displays
larger calcospherites, which result from slower calcification,
which is likely regulated by the larger ratio of highly
phosphorylated phosphoproteins. Cementum-facing dentin
then displays smaller calcospherites, resulting from faster calcification
likely regulated by a larger ratio of slightly phosphorylated
phosphoproteins.'4 7 1
Authors then attribute the higher amount
of magnesium in the cementum-facing dentin as a result of faster
calcification, which produced apatite with lower purity (polluted
with magnesium).'1 8 , 1 9 , 3 7 1
Contrary to the research
performed on the rat model, we have found that in the mouse
mandibular incisor, the intensity of magnesium signal is slightly
elevated in the enamel-facing region of the tooth, and thus
reflecting the distribution of calcium, which was, however, much
more distinct. In the case of the maxillary incisor, magnesium
was more evenly distributed, and its signal displayed lesser
intensity compared with the mandibular incisor. The calcium distribution
in the maxillary incisor, however, precisely reflected the
calcium distribution in the mandibular incisor and therefore was
more concentrated in the enamel-facing dentin. Additionally, we
employed PCA analysis and /c-means clustering of the PCA
scores. PCA can reveal differences in multidimensional data,
express the difference numerically (by scores of respective principal
components), and describe the importance of each part of
multidimensional data for a particular principal component (PC).
In our case, by employing the PCA, we were able to discriminate
specific parts of the transversal section of mouse teeth not only
based on the relative change of selected elements but also on
their complex relationship. The PCA plots and the /c-means clustering
obtained from the PCA data, therefore, highlight the fact
that the labial and lingual dentins are distinct when it comes to
the composition of the two important elements. Our findings
concerning the X-ray density of labial and lingual dentins go in
line with previous work on the topic, as it was previously shown
that root dentin is less dense than the average value of dentin
density.'4 8 , 4 9 1
Taken together, we summarize that the distinct inner structure
of dentin is, from the very beginning of its development,
controlled by the type of the adjacent epithelium, and we
hypothesize that it is responsible for the different mechanical
properties of dentin. Our data also suggest that the different
dentin microstructure is controlled by Wnt signalling in odontoblasts.
This new insight into the connection between dentin
development, function, structure, and composition will be
important for future research focused on dental patterning or
organogenesis based on epithelial-mesenchymal interactions.
Disclosures
All authors state that they have no conflicts of interest.
Acknowledgments
JK was supported by the Grant Agency of Masaryk University
(MUNI/H/1615/2018) and by funds from the Faculty of Medicine
MU to junior researcher. JL was supported by the Grant Agency
of Masaryk University (MUNI/IGA/1532/2020) and is a Brno PhD
Talent Scholarship Holder, funded by the Brno City Municipality.
MH and KS were supported by the Grant Agency of the Czech
Republic (21-04178S). PP gratefully acknowledges the support
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 337 •
of the Czech Grant Agency under the project 20-19526Y. MH, KS,
and MB were supported by Charles University and the Czech
Ministry of Education, Youth and Sports (Progres Q25 and Q29).
We thank Adela Lochmanova for her help taking care of the
mice colony and Katarina Mareckova for her help with histological
analysis. Our special thanks go to Karel Novotny (Faculty of
Science, Masaryk University) and Anna Konecna (CEITEC, Brno
University of Technology), who helped with the initial LIBS analysis,
and Petr Zaunstock (Faculty of Science, Masaryk University)
for his help with the polishing of samples used for LIBS analysis.
Furthermore, we also thank Daniel Kovac for his help with statistical
analysis. We acknowledge the core facility CELLIM of CEITEC
supported by the Czech-Biolmaging large Rl project (LM2018129
funded by MEYS CR) for their support with obtaining scientific
data presented in this paper.
Authors' roles: JKr planned and performed experiments, analyzed
the data and wrote the manuscript. JL performed experiments,
analyzed the data and wrote the manuscript. MK, DP,
VL, MGL, KS, MB, AV, PP, MH, MM performed experiments and
analyzed the data. JKa analyzed data.
Peer Review
The peer review history for this article is available at https://
publons.com/publon/10.1002/jbmr.4471.
Data Availability Statement
The data that supports the findings of this study are available in
the supplementary material of this article
References
1. Pashley D H . Dynamics of the pulpo-dentin complex. Crit Rev Oral Biol
M e d . 1996;7(2):104-133.
2. C h o YS, Ryu CH, W o n JH, et al. Rat odontoblasts may use glutamate to
signal dentin injury. Neuroscience. 2016;29(335):54-63.
3. Lee Y-L, Liu J, Clarkson BH, Lin C-P, Godovikova V, Ritchie H H . Dentinpulp
c o m p l e x responses to carious lesions. Caries Res. 2006;40(3):
256-264.
4. Lin M , Luo ZY, Bai BF, X u F, Lu TJ. Fluid mechanics in dentinal microtubules
provides mechanistic insights into the difference between
hot and cold dental pain. PLOS O n e . 2011;6(3):e18068.
5. Veerayutthwilai O, Byers MR, P h a m T-TT, Darveau RP, Dale BA. Differential
regulation of i m m u n e responses by odontoblasts. Oral Microbiol
Immunol. 2007;22(1):5-13.
6. W o n J, V a n g H, Kim J H , Lee PR, Kang Y, O h SB. TRPM7 mediates
mechanosensitivity in adult rat odontoblasts. J Dent Res. 2018;
97(9):1039-1046.
7. Arana-Chavez VE, Massa LF. Odontoblasts: the cells forming and
maintaining dentine. Int J Biochem Cell Biol. 2004;36(8):1367-1373.
8. Balic A, Thesleff I. Chapter seven—tissue interactions regulating
tooth development and renewal. Curr Top Dev Biol. 2015;115:
157-186.
9. Thesleff I, Sharpe P. Signalling networks regulating dental development.
M e c h Dev. 1997;67(2):111 -123.
10. Bae C-H, Kim T-H, C h u J-Y, C h o E-S. N e w population of odontoblasts
responsible for tooth root formation. G e n e Expr Patterns. 2013;13(5):
197-202.
11. Xiong J, Gronthos S, Bartold P M . Role of the epithelial cell rests of
Malassez in the development, maintenance and regeneration of
periodontal ligament tissues. Periodontol. 2000;63:217-233.
12. YamashiroT, Z h e n g L, Shitaku Y, et al. Wnt10a regulates dentin sialophosphoprotein
m R N A expression and possibly links odontoblast
differentiation and tooth morphogenesis. Differentiation. 2007;
75(5):452-462.
13. Křivánek J, A d a m e y k o I, Fried K. Heterogeneity and developmental
connections between cell types inhabiting teeth. Front Physiol.
2017;8:376.
14. Khatibi Shahidi M , Křivánek J, Kaukua N, et al. Three-dimensional
imaging reveals n e w compartments and structural adaptations in
odontoblasts. J Dent Res. 2015;94(7):945-954.
15. Smith A J , Cassidy N, Perry H, Begue-Kirn C, Ruch J-V, Lesot H. Reactionary
dentinogenesis. Int J Dev Biol. 1995;39(1):273-280.
16. Smith A J , Scheven BA, Takahashi Y, Ferracane JL, Shelton R M ,
Cooper PR. Dentine as a bioactive extracellular matrix. Arch Oral Biol.
2012;57(2):109-121.
17. C o u v e E, Osorio R, Schmachtenberg O. The amazing odontoblast:
activity, autophagy, and aging. J Dent Res. 2013;92(9):765-772.
18. Beertsen W, Niehof A. Root-analogue versus crown-analogue dentin:
a radioautographic and ultrastructural investigation of the mouse
incisor. Anat Rec. 1986;215(2):106-118.
19. Steinfort J, van den Bos T, Beertsen W. Differences between enamelrelated
and cementum-related dentin in the rat incisor with special
emphasis on the phosphoproteins. J Biol Chem. 1989;264(5):2840-2845.
20. Simon S, Smith A J , Lumley PJ, et al. Molecular characterization of
young and mature odontoblasts. Bone. 2009;45(4):693-703.
21. Sun Y, G a n d h i V, Prasad M , et al. Distribution of small integrinbinding
ligand, N-linked glycoproteins (SIBLING) in the condylar cartilage
of rat mandible. Int J Oral Maxillofac Surg. 2010;39(3):272-281.
22. Toyosawa S, Okabayashi K, Komori T, ljuhin N. m R N A expression and
protein localization of dentin matrix protein 1 during dental root formation.
Bone. 2004;34(1):124-133.
23. Vijaykumar A, Ghassem-Zadeh S, Vidovic-Zdrilic I, et al. Generation
and characterization of DSPP-cerulean/DMP1-cherry reporter mice.
Genesis. 2019;57:e23324.
24. Boran T, Peterkova R, Lesot H, Lyons DB, Peterka M , Klein O D . T e m p o ral
analysis of ectopic enamel production in incisors from sprouty
mutant mice. J Exp Z o o l o g B M o l Dev Evol. 2009;312B(5):473-485.
25. Klein O D , Lyons DB, Balooch G, et al. A n FGF signaling loop sustains
the generation of differentiated progeny from stem cells in mouse
incisors. Development. 2008;135(2)377-385.
26. Klein O D , M i n o w a d a G, Peterkova R, et al. Sprouty genes control diastema
tooth development via bidirectional antagonism of epithelialmesenchymal
FGF signaling. Dev Cell. 2006;11(2):181-190.
27. Shim K, M i n o w a d a G, Coling DE, Martin GR. Sprouty2, a mouse deafness
gene, regulates cell fate decisions in the auditory sensory epithelium
by antagonizing FGF signaling. Dev Cell. 2005;8(4):553-564.
28. Křivánek J, SoldatovRA, Kastriti ME, et al. Dental cell type atlas reveals
stem and differentiated cell types in mouse and h u m a n teeth. Nat
Commun.2020;11(1):4816.
29. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years
of image analysis. Nature Methods. 2012;9(7):671-675. https://doi.
org/10.1038/nmeth.2089
30. A n Z, Sabalic M, Bloomquist RF, Fowler TE, Streelman T, Sharpe PT. A quiescent
cell population replenishes mesenchymal stem cells to drive
accelerated growth in mouse incisors. Nat C o m m u n . 2018;9(1):1-9.
31. Harada H, Kettunen P, Jung H-S, M u s t o n e n T, W a n g YA, Thesleff I.
Localization of putative stem cells in dental epithelium and their
association with notch and Fgf signaling. J Cell Biol. 1999;147(1):
105-120.
32. Bleckert A, Photowala H, Alford S. Dual pools of actin at presynaptic
terminals. J Neurophysiol. 2012;107(12):3479-3492.
33. Limbeck A, Brunnbauer L, Lohninger H, et al. M e t h o d o l o g y and applications
of elemental m a p p i n g by laser induced breakdown spectroscopy.
Anal C h i m Acta. 2021;22(1147):72-98.
34. Modlitbová P, Pořízka P, Kaiser J. Laser-induced breakdown spectroscopy
as a promising tool in the elemental bioimaging of plant tissues.
TrAC Trends in Analytical Chemistry, 2020;122:115729. https://doi.
org/10.1016/j.trac.2019.115729
35. Pořízka P, Klus J, Képeš E, Procházka D, Hahn DW, Kaiser J. O n the utilization
of principal c o m p o n e n t analysis in laser-induced breakdown
spectroscopy data analysis, a review. Spectrochim Acta Part B Atomic
• 338 LAVICKÝ E T A L . Journal of Bone and Mineral Research
Spectroscopy. 2018;148:65-82. https://doi.Org/10.1016/j.sab.2018.
05.030
36. Li C, Jing Y, W a n g K, et al. Dentinal mineralization is not limited in the
mineralization front but occurs along with the entire odontoblast
process. Int J Biol Sei. 2018;14(7):693-704.
37. Mishima H, Kozawa Y, S a k a e T . T w o patterns of calcification in rat and
rabbit incisor dentin. In Suga S, Nakahara H, eds. Mechanisms and
phylogeny of mineralization in biological systems. Tokyo: Springer
Japan; 1991 pp 223-227.
38. Shuhaibar N, Hand AR, Terasaki M . Odontoblast processes of the
mouse incisor are plates oriented in the direction of growth. Anat
Ree. 2020;304(8):1820-1827.
39. Foster BL, Soenjaya Y, Nociti F H , et al. Deficiency in acellular c e m e n tum
and periodontal attachment in Bsp null mice. J Dent Res. 2013;
92(2):166-172.
40. Imhof T, Balic A, Heilig J, et al. Pivotal role of tenascin-W (-N) in postnatal
incisor growth and periodontal ligament remodeling. Front
Immunol. 2021 ;11:608223.
41. Tummers M , Thesleff I. Observations on continuously growing roots
of the sloth and the K14-Eda transgenic mice indicate that epithelial
stem cells can give rise to both the ameloblast and root epithelium
cell lineage creating distinct tooth patterns. Evol Dev. 2008;10(2):
187-195.
42. Wen Q, Jing J, Han X, et al. Runx2 regulates mouse tooth root develo
p m e n t via activation of W N T inhibitor N O T U M . J Bone Miner Res.
2020;35(11 ):2252-2264.
43. Lin Y, Xiao Y, Lin C, et al. SALL1 regulates c o m m i t m e n t of odontoblast
lineages by interacting with RUNX2 to remodel o p e n chromatin
regions. Stem Cells. 2021 ;39(2):196-209.
44. Huang X, Xu X Bringas P, Hung YP, Chai Y. Smad4-Shh-Nfic Signaling
Cascade-Mediated Epithelial-Mesenchymal Interaction is Crucial in
Regulating Tooth Root Development. J Bone Miner Res. 2010;25(5):
1167-1178. https://doi.Org/10.1359/jbmr.091103
45. Kim T-H, Bae C - H , Yang S, Park J-C, C h o ES. Nfic regulates tooth root
patterning and growth. Anat Cell Biol. 2015;48(3):188-194.
46. Liu Y, Feng J, Li J, Z h a o H, Ho T-V, Chai Y. A n Nfic-hedgehog signaling
cascade regulates tooth root development. Development. 2015;
142(19):3374-3382.
47. Steinfort J, Deblauwe B M , Beertsen W. The inorganic c o m p o n e n t s of
c e m e n t u m - and enamel-related dentin in the rat incisor. J Dent Res.
1990;69(6):1287-1292.
48. Gradl R, Zanette I, Ruiz-Yaniz M, et al. Mass density measurement of
mineralized tissue with grating-based X-ray phase tomography.
PLOS O n e . 2016;11 (12):e0167797.
49. Manly RS, H o d g e HC, A n g e LE. Density and refractive index studies of
dental hard tissues: II. Density distribution curves 1,2. J Dent Res.
1939;18(3):203-211.
Journal of Bone and Mineral Research DENTIN MICROSTRUCTURE CONTROLLED BY EPITHELIUM 339 •
A p p e n d i x f o r :
The Development of Dentin Microstructure Is Controlled by the
Type of Adjacent Epithelium
D S P P c e r u i e a n / D M P 1 c h e r r y Periostin staining Merged
Fig. SI. Cementoblasts in molars and incisors. Confocal analysis of cherry (DMP1) expressing
cells show cherry"1
" odontoblasts, osteoblasts (-cytes), and dentin-attaching cementoblasts
located in the periodontal space of the molar root (A), lingual side of the maxillary (B), and
mandibular (C) incisor. Periostin (POSTN) immunohistological staining marks the periodontal
ligament. Scale bars = 100 urn. Cb and arrowheads = cementoblasts; De = dentin; Ob =
odontoblasts; Osc = osteocytes; PDL = periodontal ligament.
1
Wildtype Mandibular incisor Wildtype Maxillary incisor
o
0
o
o
-
o
oo
Pearson's correlation
R = 0.6165o
Pearson's correlation
R = 0.6165
0
p = 0.0802
0
60 60 70
Area fraction (%)
Labial dentin
c —
n S
— u
03 to
3 *0 5
S
c £
0
o
o
o
—0 —
o
o
o
o
r e a r s o n s correiatior
R = 0.2612
o
o
p = 0.4600 0
o
0
50 60 70
Area fraction (%)
Labial dentin
Fig. S2. Plots of linear regression (red line) of the relation between corresponding median
values measured on the labial and lingual dentins of wild-type mandibular and maxillary
incisors and Spry2+/-;Spry4-/- maxillary incisor. Pearson's correlation coefficient (R) of the
two trends and p values.
2
Labial side Lingual side
Fig. S3. Representative, thresholded images were used for the analysis of the density of all OPs
and their distribution in dentin (measured data shown in Fig. 5D). The numbers 1 to 10 signify
the specific sector of the image from which the corresponding area fraction was calculated. The
images were generated from confocal data of the same z dimensions (z = 10 Lim).
3
Principal component 1 loadings Principal component 3 loadings R /(-means clustering
Fig. S4. Loadings of principal component analysis of the LIBS data and subsequent clustering
analysis. Based on loadings (A), the highest contribution to PCI scores has ionic Ca II spectral
lines (393.36 nm and 396.85 nm) followed by atomic spectral lines of Ca I (321.5 nm, 322.6,
and 422.67). Less significant is Mg II (279.55 nm and 280.27 nm) and Mg I (285.21 nm). The
highest contribution to PC3 scores has atomic Ca I spectral lines (321.5 nm, 322.6, and 422.67)
together with Mg II (279.55 nm and 280.27 nm) and Mg I (285.21 nm). Higher values of PC3
scores means the higher intensity of Ca together with low intensity of Mg and vice versa. Scatter
plots of PCI versus PC3 (B), data points clustered into 5 clusters by using &-means clustering
applied to PCI and PC3. Showing clustering of characteristic spectra of individual matrices.
Values of the PC scores in &-means clustering graphs were shortened by the factor of 1000.
Scale bars = 500 um.
4
U N R A V E L I N G T H E T R A N S C R I P T I O N F A C T O R C O D E O F O D O N T O B L A S T
DIFFERENTIATION
Appendix D Unraveling the Transcription
Factor Code of Odontoblast
Differentiation
This section contains the corresponding paper including its appendix.
142
*H) Check for updates
Research Reports: Biological
Unraveling the Transcription Factor
Code of Odontoblast Differentiation
Journal of Dental Research
l-l I
© International Association for Dental, Oral,
and Craniofacial Research and American
Association for Dental, Oral, and Craniofacial
Research 2025
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/00220345251348148
journals.sagepub.com/home/jdr
J. Lavický1
, M. Gonzalez Lopez1
, V. Chochola1
,
P. Kompaníková2
, V. Rakultsev1
, J.Raška1
,
E. Wentworth Winchester3
, H. Tuaima1
,
L. Englmaier1
, J. Verner4
, E. Švandová1
, M. Buchtová5
'6
©,
J. Cotney7
, V. Bryja2
, D. Bohačiaková1
, T. Bárta1
, and J. Křivánek1
Abstract
The molecular mechanisms controlling the differentiation of stem cells into specialized cells and tissues are enormously complex.
Deciphering the mechanisms behind this precisely regulated process is essential not only for understanding ontogenesis but also for
interpretations of evolutionary dynamics. A deep understanding of differentiation processes in various cell types would open the way
for safe and targeted tissue engineering. This was previously limited by technical constraints, but the recent development of multiomic
approaches has overcome these barriers, providing unprecedented insight into the intricate workings of this molecular machinery.
Based on single-cell RNA-seq analyses of continuously growing mouse teeth, 4 transcription factors (Etv4, Gsc, Salll, and Nuprl) were
selected. Their role during tooth development has not been previously evaluated. Here, we provide evidence about their function in the
differentiation of odontoblasts—dental cells responsible for dentin production. Our results revealed that controlled overexpression of
specific transcription factors directly in mouse-induced pluripotent stem cells is sufficient to guide their differentiation into cells with an
odontoblast-like phenotype, both in vitro and in vivo. The differentiated cells exhibited upregulated expression of odontoblast-specific
molecular markers (DMPI and DSP) as well as production of collagenous and mineralized tissue. We demonstrate that a deep insight
into fundamental developmental events can provide a powerful basis for innovative cell differentiation approaches. Taken together, this
research might serve as a proof of concept for using large-omics data in the generation of specific, differentiated cell types by controlled
expression of a particular transcription factor code. This may represent a turning point in both developmental biology and regenerative
medicine fields.
Keywords: tooth development, odontogenesis, dentin, bioengineering, cell differentiation, stem cell(s)
Introduction
Odontoblasts are highly polarized cells that produce dentin.
They originate from neural crest, which migrates during early
embryonic development into the first pharyngeal arch. Here,
at the prospective site of dental germ formation, a complex
reciprocal interaction of the migrated ectomesenchyme with
the odontogenic epithelium subsequently occurs. These interactions
involve the activation of a number of transcription factors
(TFs) that control and regulate this process (Thesleff
2003; Kim et al. 2017; Balic 2018). Odontoblasts not only
produce the main hard dental matrix during tooth development
but are also able to respond to tooth damage and contribute
to the reparative processes that are essential for the
longevity of dentition (Neves and Sharpe 2018). Thus, they
are an important target for biologically based regenerative
dental medicine (Balic 2018).
To investigate the process of gradual odontoblast differentiation
from their mesenchymal progenitors, continuously
growing teeth of rodents are traditionally used as a
model system (Křivánek et al. 2020; Lavický et al. 2022).
Their continuous growth is secured by permanently active
mesenchymal and epithelial stem cell niches in their apical
Department of Histology and Embryology, Faculty of Medicine, Masaryk
University, Brno, Czech Republic
d e p a r t m e n t of Experimental Biology, Faculty of Science, Masaryk
University, Brno, Czech Republic
^Department of Genetics and Genome Sciences, U C O N N Health,
Farmington, C T , U S A
4
Central European Institute of Technology, Masaryk University, Brno,
Czech Republic
l a b o r a t o r y of Molecular Morphogenesis, Institute of Animal Physiology
and Genetics, Czech Academy of Sciences, Brno, Czech Republic
'Department of Experimental Biology, Faculty of Science, Masaryk
University, Czech Republic
' C e n t e r for Craniofacial Innovation, Children's Hospital of Philadelphia,
Philadelphia, P A U S A
A supplemental appendix to this article is available online.
Corresponding A u t h o r :
J. Krivanek, Department of Histology and Embryology, Faculty of
Medicine, Masaryk University, Brno, 625 00, Czech Republic.
Email: jan.krivanek@med.muni.cz
2 Journal of Dental Research 00(0)
region. The incisor constantly recapitulates all stages of odontoblast
(and ameloblast) differentiation along its apical-distal
axis. This enables investigation of the continuous differentiation
of all dental cell lineages including transient cell states in
a single organ at any time during the lifetime (Křivánek et al.
2017). Furthermore, it was shown that molecular mechanisms
driving the formation of the tooth are highly conserved across
species, including humans and mice (Tucker and Fraser 2014;
Hulsey et al. 2020; Hermans et al. 2021).
Several recent omics-based technological advances accelerated
research in developmental biology and opened an unexpected
range of possibilities. In continuously growing teeth,
comprehensive works including expression databases have been
published. These have analyzed the sequential differentiation
steps of tooth-forming cells at an unprecedently detailed molecular
level (Sharir et al. 2019; Chiba et al. 2020; Křivánek et al.
2020). Importantly, recent works suggest how to apply these
fundamentaldentaldevelopment-related largedatatoobtaintoothforming
cells. This is the basic prerequisite for bridging the fields
of developmental biology and regenerative dentistry (Alghadeer
et al. 2023; Hanson-Drury et al. 2023). Based on single-cell transcriptomics
data, these studies proposed a strategy for manipulating
different signaling pathways in multiVpluripotent cells using
specific proteins and small molecules to differentiate them into
odontoblasts or ameloblasts in vitro. However, the potential of
TFs to mediate this process remains largely unrecognized.
Therefore, the aim of this study was to elucidate whether information
about TFs' activity during odontoblast development can
be used to obtain a code of TFs capable of controlling the differentiation
of odontoblasts directly from pluripotent cells.
Previous work done by Křivánek et al. (2020) had mapped
the developmental trajectory of odontoblasts at a detailed transcriptional
level. Analyzing these data, 4 transcription factors/
transcriptional regulators (TFs) were selected: ETV4, GSC,
SALL1, and NUPR1 (detailed information concerning functions
of selected genes can be found in the Appendix). These
were predicted to have a high potential for controlling the differentiation
of dentin-forming cells—odontoblasts. While
these TFs play important roles in a wide variety of developmental
processes, only one of them (SALL1) was recently
found to be involved in tooth development and odontoblast
differentiation (Lin et al. 2021). The role of the remaining TFs
during odontoblast differentiation, however, remains unclear.
This work demonstrates that the controlled overexpression
of the selected development-related TFs guides the differentiation
of mouse induced pluripotent stem cells into lineages
capable of expressing late odontoblast markers and possessing
the capacity of generating mineralized matrix.
Materials and Methods
Details concerning the methodology can be found in the
Appendix Materials and Methods. The article complies with
the ARRIVE 2.0 checklist.
Results
Selection and Validation of Odontoblast-Specific
TF Code
The selected odontoblast-specific TFs were selected based on
a secondary analysis of previously published scRNA-seq data
(Krivanek et al. 2020). First, the (pre)odontoblast cluster containing
odontoblasts and their progenitors was defined within
the mouse incisor dataset using the expression of known
marker genes and their consensual expression patterns (Fig.
1A). Subsequently, based on the consensual expression pattern
similarity search, an unbiased analysis of potential target
genes was performed (Appendix Figs. 3, 4). Subsequently,
based on their expression specificity (Fig. IB) and regulon
activity analysis (Fig. 1C) genes Etv4, Gsc, Salll, and Nuprl
have been selected to be used. Moreover, we have also used
the current knowledge concerning their function as well as the
bioinformatic analysis of their regulon members to support
this selection (Appendix Figs. 5-9).
The identity of four subgroups of cells within the odontoblast
differentiation trajectory was defined (Fig. 2A). The
expression pattern of the four selected TFs was incorporated
into an expression heatmap of the used marker genes to elucidate
during which phases of odontoblast differentiation
they are being expressed (Fig. 2B). To validate our scRNAseq-based
gene expression pattern predictions, the expression
of the selected TFs was mapped onto the odontoblast
trajectory, and in situ gene expression analysis of the TFs in
continuously growing incisor (Fig. 2C) and during molar
development (Appendix Fig. 10) was performed. The developmentally
earliest expressed TF was Etv4, located in the
near-cervical loop area of the incisor. This area has been
previously identified as a progenitor region (Krivanek et al.
2020, 2023). In addition, the expression of Etv4 was also
observed in pre-odontoblasts and adjacent ameloblastic epithelium
(Fig. 2C). The second TF, Gsc, was highly expressed
in the preodontoblastic region on the labial aspect of the
incisor and continued up to mature odontoblasts (Fig. 2C).
The expression of the third TF, Salll, was initiated in the
preodontoblastic region in both the labial and lingual sides
of the incisor. However, contrary to the Etv4 and Gsc, the
Salll expression in mature odontoblasts continued further in
the odontoblast layer on the labial side (Fig. 2C). The last
transcriptional regulator Nuprl was expressed exclusively
in mature odontoblasts on both the labial and lingual sides
(Fig. 2C). While selected genes start at different stages of
odontoblast differentiation, a natural expression overlap of
these genes exists in mature odontoblasts expressing Dspp
and Dmpl (Fig. 2B, C). Moreover, in parallel to the incisor,
we mapped the expression pattern of the selected TFs also
during molar development. It reflected the expression pattern
observed in continuously growing incisor (Appendix
Fig. 10).
Transcription Factor Code of Odontoblast Differentiation 3
Selection of potential odontoblast-specific TFs for guided differentiation of pluripotent stem cells
A Determination of (pre)odontoblast cluster within t h e m o u s e incisor s c R N A s e q d a t a s e t
Ameloblasts
Endothelial cells;nai ceiis i
Clusters C o n s e n s u s «
Dental pulp
..Od
Dkk1
Smpd3 .
0 lH*
Křivánek et al. 2020
Dental follice
. Osteocytes ^
Immune cells 'J ;
§>
Perivascular cells
F 9 f 3
5 Notum «
B Expression pattern of the odontoblast markers and selected transcription factors L o w S r ^ T i H i g h ^ k j s t e r s
H i
I I 111
I
Smpd3
Frmd5
Fgf3
Thy1
Mppedl
Etv4 -
SalHGsc
-
Notum
Dkk1
Nupn -
Dmp1
Dspp
(pre)odontoblasts (pre)ameloblasts
Nuprl
C Activity of regulons of G s c , Etv4 and SalH in the mouse incisor dataset
C o n s e n s u s
(pre)odontoblasts
Gsc
0 >*
Em
§
SalH
If(pre)cdontoblasts
Figure I. Analysis of the odontoblast differentiation trajectory—background for the selection of the utilized transcription factors. t-SNE visualization
of the whole mouse incisor s c R N A dataset with simplified cluster labels. Using the consensus expression pattern of previously established (Krivanek
et al. 2020) odontoblast markers (Frmd5, Dkkl, Smpd3, Fgf3, and Notum) the (pre)odontoblast subpopulation was defined within the dataset (A).
(B) Heatmap of the expression of the odontoblast markers and selected odontoblast-specific transcription factors and t-SNE plots of Gsc, Etv4 Sail I,
Nuprl, and their consensus expression pattern further illustrate their specificity to the (pre)odontoblast subpopulation. (C) Heatmap showing the
activity of SCENIC-inferred regulons of Gsc, Etv4, and Salll in the whole incisor dataset and U M A P visualizations of the activity of selected regulons
within the mouse incisor dataset. Each cell is colored according to the activity of the corresponding regulon, as quantified by A U C e l l scores.
4 Journal of Dental Research 00(0)
A Definition of odontoblast differentiation trajectory Lowl ^ S
iHiah
Clusters Mature odontoblasts •
• • Dspp Early odontoblasts
Dmp1 O ^-p
Nuprl o n
Mature odontoblasts o °q0 Dmp1
consensus ° •
Near-CL area
Tnc
Moxdl
Twist2
o o o o
<h°G O
Wispl
Col24ct1
Slc8a3
Křivánek etal. 2020
9 Pre-odontoblasts
Sal!1
Notum
Dkk1
N e a r - C L
consensus
Ea'ly odontoblasts
consensus
Pre-odontoblasts
consensus
B H e a t m a p of o d o n t o b l a s t s u b t y p e m a r k e r s a n d s e l e c t e d T F s
Expression Clusters
Low ^Hl^Zl^ZHHigh
•
Etv4 -
Twist2
Moxdl
Tnc
Notum
SalU Gsc
-
Dkk1
Slc8a3
W i s p l
Col24a1
Nupn
Dspp
Dmp1
Validation of expression of the selected TFs in the mouse incisor
C Differentiation trajectory Overview Labial aspect Lingual aspect
Expression
Low High
Mature ^ * Co)
odontoblasts q " ~q
Etv4 i
LaCL
Near-CL area
\0k
ocoCD
c 0 c n
° c
G s c
LaCL
° i
)»o o
L i C L ^ b J v 0 d
Pulp
Od
L l C L - - ' " " " f T J " Od
Pulp
Etv4
Pulp
Od r
Gsc,1
V 7 ' Pulp
Etv4 PDL
--Epithelium;
pOd
\
\
- Pulp
*• r **• r * •
G s c PDL
Epithelium _ _
"pOd-
Pulp
CO00
[b SalU . . . i-'-i ^
LaCL
• o o o / ;> Pulp
SalU PDL
e p i t h e l i u m f - "
" p o d
• v.
• • LaCL
Od r
l.iCL Od i
. l i f t • •
^ ; ; . ; ; ; ' puip
• % 0 ' 0 2
' i '
Figure 2. Visualization of the expression of the four selected genes in the odontoblast differentiation trajectory and its in situ validation in the
continuously growing mouse incisor. The definition of the odontoblast differentiation trajectory including its subgroups using the consensus patterns of
known marker genes (A). Heatmap of the color-coded subgroup-specific marker genes and the selected genes illustrates the predicted spatiotemporal
expression of Etv4, Gsc, Sail I, and Nuprl during odontoblast differentiation (B). The t-SNE embedding of these four genes further illustrates their
expression pattern within the odontoblast differentiation trajectory (C). In situ detection of selected transcription factor (TF) genes was performed on
sections from the mandibular incisor (C). The expression of the corresponding genes is highlighted with black arrows. Representative areas demonstrating
the expression of the corresponding genes are displayed in detailed images focusing on both labial and lingual aspects of the incisor. A m , ameloblasts; LaCL,
labial cervical loop; LiCL, lingual cervical loop; O d , odontoblasts; PDL, periodontal ligament; pOd, preodontoblasts. Scale bars = 200 urn.
Transcription Factor Code of Odontoblast Differentiation 5
Overexpression ofEtv4, Gsc, Sail I, and Nuprl
in Induced Pluripotent Stem Cells Results in the
Upregulation of Odontoblast Molecular Markers
and Induction of Mineralization In Vitro
To understand the role of selected TFs during odontoblast differentiation,
we performed an odontogenic differentiation
assay on freshly isolated dental pulp stem cells (DPSCs) (Fig.
3A). The expression of Gsc was stable during the differentiation.
Etv4's expression displayed a trend with the highest mean
at D7 and significantly upregulated expression at D14. The
expression of Salll mirrored that of Runx2 with low expression
at D3 and D7 and with stable significant upregulation at D14
and D21. Finally, Nuprl displayed a steadily growing expression
over the whole culture period.
The mouse induced pluripotent stem cell line derived from
the DSPP-Cerulean/DMP 1-Cherry reporter strain was transduced
with lentiviral vectors to enable the inducible overexpression
of the selected TFs (Appendix Fig. 11). To test the
effects of the overexpression of the selected TFs, in vitro differentiation
experiments were performed (Fig. 3B). The growth
dynamics, proliferation, and apoptosis rates were analyzed in
cultured spheroids during the differentiation. The analysis
showed their growth and formation of necrotic centers
(Appendix Fig. 13). The expression of DSP and DMP1 was
significantly upregulated in spheroids generated from cell lines
containing the GsclEtv4 vector (Fig. 3C). Both DSP and DMP1
formed granular aggregates inside the spheroids (Fig. 3C).
Co-staining illustrating their overlap is in Appendix Fig. 15.
Observed trends were quantified as a positive relative area for
the given protein. Interestingly, due to the background activity
of the TET3G transactivator (Appendix Fig. 11), spheroids
generated from TG and TGS cell lines displayed a significant
upregulation of marker proteins even without added doxycycline
(this known phenomenon is further discussed in Appendix
Figs. 11,12). For this reason, the T-Dox condition was used as
a negative control instead of conditions without added
doxycycline.
To further investigate the expression of Dspp and Dmpl,
reverse transcriptase quantitative polymerase chain reaction
(RT-qPCR) was performed (Fig. 3C). The analysis of Dmpl
expression mirrored the results of the immunohistochemistry
(IHC) analysis (Fig. 3C). While a significant upregulation was
observed in the TG and TGS conditions, low or no expression
was detected in the remaining conditions. The expression of
Dspp was less robust, mirroring the protein expression trends
in only the TG-Dox condition.
To demonstrate the mineralization capability of the engineered
cells, von Kossa staining was performed (Fig. 3D). This
approach highlighted the presence of mineralized areas in TS,
TG, and TGS conditions. Interestingly, mineralized particles
exhibited similar size and distribution to the DSP and DMP1positive
particles identified by IHC (Fig. 3C, D). Quantification
of the von Kossa stain uncovered the same trends as seen in the
IHC analysis of DSP and DMPL Both TG-Dox and TGS-Dox
conditions were significantly more mineralized than the T-Dox
condition. To complement the mineralization analysis, an
RT-qPCR analysis of Runx2 was performed (Fig. 3C). Its
expression mirrored the mineralization pattern, in which Runx2
was upregulated in TS, TG, and TGS conditions, while the
control condition displayed no expression of Runx2 (further
discussed in Appendix Fig. 14). Taken together, the features of
the odontoblast-like phenotype observed after the TFs' overexpression-driven
differentiation of miPSCs mirrored those
observed in the odontogenic differentiation of DPSCs.
Overexpression of Selected TFs In Vivo Results
in an Odontoblast-like Phenotype
To achieve more natural growth of engineered cells and to
show their full differentiation potential, doxycycline-primed
miPSC spheroids were implanted under the kidney capsule of
immunodeficient mice 5 d after seeding (Fig. 4A). Based on
the previous in vitro experiments and to minimize the number
of experimental mice (3R), 2 borderline experimental groups
were used: the T-Dox as a control group and the TGS-Dox as
an experimental condition. IHC analysis of the TGS-Dox condition
uncovered the presence of distinct structures expressing
both DSP and DMP1 (Fig. 4B, C) Their co-staining showed
their stratification and overlap in the structures (Appendix Fig.
15). In contrast, the T-Dox condition displayed no such organized,
epithelium-like features. Quantifications of the DSP-land
DMP1+ area have shown significantly upregulated DSP
and DMP 1 expression in the TGS-Dox condition. The quantification
of the IHC has revealed a certain level of cell population
heterogeneity within implants (Fig. 4B; Appendix Fig.
19). To validate the observed trends on the transcriptional
level, the expression of the fluorescent reporters DSPPCerulean/DMP
1-Cherry was investigated. The expression pattern
of the Dspp and Dmpl reporters was generally comparable
with the protein expression observed via IHC detection (Fig.
4C). Similar to the in vitro experiments, the expression of Dspp
quantified by RT-qPCR was not significantly upregulated in
the TGS-Dox condition. The expression of Dmpl was more
robust, displaying significant upregulation in the TGS-Dox
condition (Fig. 4C).
To uncover whether any mineralization occurs inside the
DSP/DMP1-positive structures, von Kossa staining was performed
(Fig. 4D). The TGS-Dox condition displayed mineralized
nodules inside the DSP and DMPl-positive structures in
contrast to the control T-Dox condition. Moreover, RT-qPCR
analysis of Runx2 expression was performed, and it revealed
significant upregulation of Runx2 in the TGS-Dox condition
(Fig. 4D).
Dual Nature of Odontoblast-like Cells
To further investigate whether collagenous matrix, characteristic
of (pre)dentin, is produced, Masson's trichrome staining was
performed on the kidney implant sections (Fig. 5A). The staining
revealed that the DSP and DMPl-positive structures in the
TGS-Dox condition display the presence of collagenous matrix
in their centers. However, it also revealed a red-stain-positive
matrix being present in the TGS-Dox implants, suggesting the
6 Journal of Dental Research 00(0)
A If) vitro differentiation of DPSCs into odontoblasts reveals distinct expression patterns of the selected TFs
D 3 D 7 D 1 4
Plating medium Proliferation medium Mineralization med
0
D21
D3 07 014 D21 03 07 D14 D21
. H a i l
Nuprl
03 D7 D14 D21 03 07 D14 021
B Overview of transcription factor-driven /n vrtro differentiation of transduced mPSCs
D 5
Matrigel embedding
until D21
Organoid cell culture
D21
LIF M e d i u m B a s e medium + A A B a s e medium + A A + B G P |
Q IHC and qPCR reveal upregulated expression of late odontoblast markers in cells overexpressing Gsc and Etv4
T Dox T G S Dox DSP* area at D21 Dspp expression
1
0.2. , *
T TS TG T G S
DMP1* area at D21
rli
Day 0 21 0 21 0 21 0 21
T TS T G T G S
Dmp7 expression
TS TG T G S Da/ 0 21 0 21 0 21 0 21
T TS T G T G S
D The combination of odontoblast-specific transcription factors results in elevated mineralization
Von Kossa4
area at D21 Runx2 expression
Von Kossa stain D21 Von Kossa stajn r D2T _
. '-i i s . , r,. - i
• *?\p% -V-, r
n.s. '
TS TG TGS Day 0 21 0 21 0 21 Ö 21
T TS T G T G S
Figure 3. The expression of Etv4, Gsc, So///, and Nuprl displays distinct expression patterns during in vitro differentiation of DPSCs and their
overexpression in miPSCs leads to the upregulation of odontoblast molecular markers and initiation of mineralization in vitro. Results of in vitro
odontogenic differentiation of DPSCs (A) show, that over the 21 days of culture, matrix mineralization and the expression of odontoblast markers
increased. The expression of the four selected genes displayed distinct expression patterns during the differentiation (n = 3). The overview of the
in vitro differentiation experiments (B) summarizes the differentiation protocol. I H C and R T - q P C R (C) reveal the upregulated expression of the
odontoblast markers DSP (Dspp) and D M P I ( D m p l ) in cell lines containing the Gsc/Etv4 vector in cell spheroids cultivated for 21 days (n = 5-6 for
IHC quantification and n = 3 for RT-qPCR). The overexpression of the chosen TFs results in the initiation of mineralization process (n = 5-6) and
increased expression of the mineralization-associated, preodontoblast-specific TF Runx2. (D) (n = 3). A A , ascorbic acid; |3GP, beta-glycerophosphate;
DPSCs, dental pulp stem cells; IHC, immunohistochemistry; miPSCs, mouse induced pluripotent stem cells; n.d., not detected; n.s., not significant;
RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; T, cell line containing T e t 3 G / G F P vector; TF, transcription factor; T G , cell line
containing T e t 3 G / G F P and Gsc/Etv4 vectors; T G S , cell line containing T e t 3 G / G F P , Sail I / N u p r l , and Gsc/Etv4 vectors; TS, cell line containing T e t 3 G /
EGFP and Salll/Nuprl vectors. Scale bars = I00urn. Data are presented as meanistandard deviation. *P<0.05; * * P < 0 . 0 l ; * * * P < 0 . 0 0 l ; ****P<0.0001.
Transcription Factor Code of Odontoblast Differentiation 7
A Overview of in vivo differentiation experiments
G F P +
Tet3G+ DO C \ D2
Gsc/Etv4+ Seeding \\Doxycycline
Sall1/Nupr1+ <\ (D°x)
D5
Kidney capsule implantation D35
Implant recovery
LIF Medium
D2 D5
Base medium + A A
D35
Kidney capsule implant |
Overexpression of the selected TFs results in the formation of structures expressing D S P and DMP1
B T Dox T G S Dox C T G S Dox
DSP+
area at D35
< 10
EEfl
T TGS
DMP1* area at D35
s
a o
*
T TGS
Dspp expression
Ctrl
Dox
n.d. rfl n.d. [IDay 0 35 0 35
T TGS
Dmp1 expression
it
Day 0 35 0 35
T TGS
D T Dox TGS Dox
Von Kossa stain D35 Von Kossa stain D35 Runx2 expression
4.' i
Ctrl
D o x
n.d. r + i n.d.
Day 0 35 0 35
T TGS
Figure 4. The overexpression of Etv4, Gsc, Salll, and Nuprl leads to acquisition of odontoblast characteristics in vivo. The process of the in vivo
experiment is summarized in (A). The IHC analysis of DSP and D M P I in the control (T Dox) and experimental (TGS Dox) conditions reveals their
significant upregulation in the experimental condition (B). The IHC was quantified as a relative area of positive signal (n = 3). The m R N A expression
analysis of Dspp and Dmp I was performed using fluorescent reporters of DSPP-Cerulean/DMP I-Cherry and RT-qPCR (n = 3) (C). The analysis of both
reporters shows mostly the same expression pattern as the I H C analysis. The RT-qPCR analysis of the expression showed that D m p / was robustly
upregulated in the T G S D o x condition, while the expression of Dspp was not. Nevertheless, the data displays a trend suggesting upregulation of the
odontoblast markers in the T G S D o x condition that mirrors the previous findings. Von Kossa staining revealed mineralization in the T G S D o x condition
in contrast to the T D o x condition, in which no mineralization was observed. The RT-qPCR analysis of Runx2 revealed a significant upregulation in the
T G S D o x condition (n = 3) (D). Red lines for displaying statistical significance are connecting conditions compared in the representative images. Red
data points in the R T - q P C R graphs show data points in which no specific P C R product was detected. A A , ascorbic acid; IHC, immunohistochemistry;
n.d., not detected; n.s., not significant; RT-qPCR, reverse transcription quantitative polymerase chain reaction; T, cell line containing Tet3QEGFP vector;
T G , cell line containing Tet3GIEGFP and GsdEt/4 vectors; T G S , cell line containing Tet3GIEGFP, SalllINuprl, and GsdEt/4 vectors; TS, cell line containing
Tet3GIEGFP and Salll INuprl vectors. Scale bars = 100 urn. Data are represented as the mean istandard deviation. *P<0.05.
8 Journal of Dental Research 00(0)
Figure 5. The structures expressing odontoblast markers also demonstrate epithelial characteristics and express keratinocyte markers. Masson's
trichrome staining of the kidney capsule implants (A) revealed that the structures expressing odontoblast markers also contain collagen-rich regions
(blue arrowheads). Alongside the collagen, the staining revealed that the structures might also contain keratinized regions (red arrowheads). IHC
demonstrated that these structures express an epithelial marker C D H I and a keratinocyte marker K R T I 0 (A). Representative images showing that
the C D H I -positive areas are also D M P I -positive (B). The expression of C D H I was quantified as a relative area (C); although an apparent trend is
present, the upregulation of C D H I in T G S D o x condition was not statistically significant (n = 3). The expression of keratinocyte markers Krtl 0 and
Transcription Factor Code of Odontoblast Differentiation 9
presence of either enamel (Cho et al. 2013), keratin (Yang et al.
2016), or muscle tissue (Chang and Kessler 2008). Since some
DSP+/DMP1+ regions displayed an epithelium-like arrangement,
IHC for epithelial (CDH1) and keratinocyte (KRT10)
markers was performed to further characterize the nature of the
engineered tissue. The staining revealed CDHl-positive epithelial
structures in the TGS-Dox condition (Fig. 5B). The centers
of these regions were positive for the Masson's trichrome red
stain and generally expressed KRT10 (Fig. 5A). Co-staining
with odontoblast markers (DMP1, DSP, Nestin) further revealed
that these structures display both epithelial and odontoblast
markers (Fig. 5B, Appendix Figs. 16, 17). Quantification
showed that while the expression of CDH1 was not significantly
upregulated in the TGS-Dox condition, 2 of the 3 replicates
displayed approximately 10 times higher relative area
(mean=36.4%+ 1.9%) than those in the T-Dox condition
(mean=3.5%+2.1%) (Fig. 5C). To confirm the expression of
keratinocyte markers, RT-qPCR was performed and showed
upregulated expression of KrtlO and Ivl (Involucrin).
To investigate a possible mechanism behind the observed
keratinocyte-like phenotype, SOX21 expression was analyzed
(Fig. 5D, Appendix Fig. 18). It revealed that SOX21 is
expressed predominantly on the periphery of the epithelial
structures. KRT10 co-staining showed that centrally located
KRT10+ cells display generally lower or no SOX21 expression.
Taken together, this suggests that the unknown matrix in
the TGS-Dox condition is keratinous.
Similarly, in the in vitro-generated cell spheroids, the
Masson's trichrome staining uncovered the presence of a collagenous
matrix in cell lines overexpressing Gsc and Etv4 (TG
and TGS), albeit without any signs of keratinous matrix (Fig.
5E). These collagenous regions were surrounded by cells
expressing CDH1 and contained KRTlO-positive cells similarly
to the kidney implants. Subsequently, IHC staining
revealed that some of the epithelial areas displayed DMP1 and
DSP expression (Fig. 5F, Appendix Fig. 15). Quantification of
the CDHl-positive area revealed significant upregulation of
CDH1 in the Gsc and Etv4 overexpressing conditions (TG and
TGS) (Fig. 5G). Furthermore, the RT-qPCR results for KrtlO
and Ivl mirrored the CDH1 expression as well as the previous
DSP, DMP1, and Runx2 quantifications (Fig. 3C, D). Both
KrtlO and Ivl were significantly upregulated in conditions
overexpressing Gsc and Etv4 when compared with the T-Dox
condition.
Taken together, the expression trends and localization of the
odontoblast markers DSP and DMP1 (Fig. 5B, E; Appendix
Fig. 14) mirrored those of CDH1, KrtlO, and Ivl (Fig. 5). This
suggests a potential developmental connection or a partially
shared transcriptional program between odontoblasts and keratinocytes.
This is especially apparent in the kidney implants, as
both the keratinocyte and odontoblast markers were generally
more overlapping in the same structures.
Discussion
The boom of modern single-cell-based transcriptomics
approaches has provided unprecedented insight into the complex
transcriptomic landscape that regulates cell behavior during
development (Sharir et al. 2019; Krivanek et al. 2020;
Pagella et al. 2021). This in-depth knowledge serves as an
important resource for elucidating how organs and tissues are
formed, cells interact, differentiate, and fulfill their function.
Previously, Krivanek et al. (2020) generated a dental cell-type
atlas using scRNA-seq of the continuously growing mouse
incisor. The analysis of the ever-growing incisor enabled the in
silico gene expression-based reconstruction of a differentiation
trajectory of one of the main tooth hard-tissue-forming
cells: odontoblasts. This trajectory, mapping their continuous
differentiation from mesenchymal progenitors, was used as a
basis for the selection of potential regulatory TFs driving
odontoblast differentiation.
Suitable TFs have been selected by a combination of bioinformatic
approaches as well as previously obtained knowledge
on the function of the potential candidate genes. The expression
patterns suggested by the differentiation trajectory closely
resembled those found in the labial side of the incisor. Based on
in vivo localization and regulon analyses, newly uncovered TFs
in teeth Etv4 and Gsc are likely essential for early stages of
odontoblast differentiation. In addition, the localization of Etv4
expression suggested its importance for the differentiation of
(pre)ameloblastic epithelium. The observed expression pattern
of Salll aligned with recent studies (Lin et al. 2021; Lavicky
et al. 2022). This, along with bioinformatic analysis, suggests
its significance for both odontoblast differentiation and function.
Finally, transcriptional regulator Nuprl displayed striking
specificity to cells expressing odontoblast markers Dspp and
Dmpl. Its strong expression, specific for mature odontoblasts,
suggests its important role in dentin production.
-<
M was analyzed using R T - q P C R (n = 3) (C). The expression of Krtl 0 was detected in only 2 of 3 biological replicates of the T G S D o x conditions.
The upregulation of Ivl in the T G S D o x conditions was significant compared with T D o x . The co-staining of S O X 2 I and K R T I 0 revealed a possible
mechanism behind the apparent keratinocyte-like phenotype of the epithelial structures (D). The Masson's trichrome and I H C staining for C D H I
and K R T I 0 was performed on samples from in vitro experiments (E). Representative images show that the C D H I-positive areas were again shown
to contain D M P I-positive areas (F). The analysis (G) revealed that conditions overexpressing Gsc and Etv4 displayed upregulated expression of
C D H I (n = 5-6), Krtl 0, and Ivl (n = 3) when compared with negative control and the condition overexpressing only Salll and Nuprl. Red lines indicate
statistical significance between the conditions compared in the representative images. Red data points in the R T - q P C R graphs indicate instances in
which no specific P C R product was detected. IHC, immunohistochemistry; n.d., not detected; n.s., not significant; RT-qPCR, reverse transcriptase
quantitative polymerase chain reaction; T, cell line containing Tet3G/EGFP vector; T G , cell line containing Tet3G/EGFP and Gsc/Etv4 vectors; T G S , cell
line containing Tet3G/EGFP, Salll/Nuprl, and GsdEtv4 vectors; TS, cell line containing Tet3G/EGFP and Salll/Nuprl vectors. Scale bars= 100 urn. Data are
represented as mean istandard deviation. *P<0.05; * * P < 0 . 0 l ; ***P<0.00l.
1 0 Journal of Dental Research 00(0)
The in vitro differentiation of transduced miPSCs uncovered
the formation of mineralized DSP and DMP1-positive
particles in the conditions overexpressing Gsc and Etv4. The
formation of these mineralized particles, along with the formation
of structures producing collagen, suggested the differentiation
of the engineered cells into those with an odontoblast-like
phenotype. Conversely, the overexpression oiSalll and Nuprl
caused a downregulation of odontoblast markers. Intriguingly,
it also slightly increased the formation of mineralized particles
and the upregulation of Runx2. These findings mirrored odontogenic
differentiation of DPSCs and are consistent with previous
studies (Lin et al. 2021). Taken together, our data suggest
that Salll and Nuprl are important for the mineralization capability
of odontoblasts but are not sufficient to trigger direct
odontogenic differentiation from miPSCs alone without Gsc
and Etv4.
The in vivo differentiation experiments highlighted the
same trends observed in the in vitro experiments. The expression
of DSP, DMP1 was significantly upregulated in the TGSDox
conditions. Moreover, the expression of both DSP and
DMP1 was localized into distinct structures that produced a
collagenous matrix, strongly suggesting their odontoblast-like
phenotype. This further supported our hypothesis that overexpression
of selected TFs leads to the acquisition of an odontoblast-like
phenotype in a portion of the cell population.
The dual nature of a subpopulation of transduced cells was
found in the conditions overexpressing Gsc and Etv4 both in
vivo and in vitro. The epithelial structures that formed in the
kidney implants displayed an inward stratification, similar to
ameloblast epithelium organoids generated by Hemeryck et al.
(2022). The outer cells displayed DSP, DMP1, CDH1 as well
as rare Nestin expression. This, apart from the CDH1 expression,
suggested an odontoblast-like phenotype. The inner cells
displayed a keratinocyte-like phenotype suggested by the
KRT10 and CDH1 expression (Wang et al. 2020) and formation
of keratinous matrix. Interestingly, the inner, keratinocytelike
cells also displayed DMP1 but no DSP expression. This
downregulation of DSP might be explained by a change in the
transcriptional landscape as the cells move toward the keratinocyte-like
phenotype while Dmpl remains transcriptionally
active. Nonetheless, the incomplete overlap oiDspp andDmp\
expression is present during regular tooth growth as well
(Appendix Fig.4) (Lavický et al. 2022).
To our knowledge, no naturally occurring cell types exhibit
this combination of odontoblast and keratinocyte molecular
hallmarks in vivo. We hypothesize that via an artificial overexpression
of Gsc and Etv4, we have generated cells with a dual
phenotype that partially display the characteristics of both
odontoblasts and a keratinocyte-like epithelium. The presence
of keratinocyte markers can be explained by the function of
Etv4, which is also involved in keratinocyte differentiation
during hair formation (Finnegan et al. 2019). Therefore, Our
results may highlight the developmental similarities between
different ectodermal appendages: hair follicles and teeth. Their
embryonic development shares many molecular and morphological
similarities, including an Etv4 expression in the
epithelium (Biggs and Mikkola 2014). The co-expression of
odontoblast and keratinocyte markers may indicate the initiation
of an odontogenic transcriptional program in conjunction
with a hair-forming program. A similar remarkable level of cellular
plasticity has been suggested before in studies highlighting
the change of stem cell fate from one ectodermal appendage
to another (Yoshizaki et al. 2014; Jimenez-Rojo et al. 2019).
This notion is further supported by the presence of SOX21 on
the KRTlO-negative periphery of the epithelial structures. This
suggests that the conversion between the DSP-expressing outer
cells and KRTlO-expressing inner cells occurred via the mechanism
described previously by Saito et al. (2020). Presented
data, together with the research of other teams (Kurpios et al.
2009), highlight the importance of Etv4 and Gsc in the development
of ectodermal appendages. This is supported by the
performed bioinformatic analysis of their regulons, which suggests
their involvement in appendage and epithelial morphogenesis.
In addition, we show that even relatively short
overexpression of the selected genes is enough to determine
the fate of stem cells toward specific cell types, which is in line
with the currently used approaches of generating neuronal lineages
in vitro (Ng et al. 2021).
Taken together, our results demonstrate that controlled
overexpression of a few select TFs is sufficient to guide the
differentiation of miPSCs into matrix-producing cells with an
odontoblast-like phenotype. However, because TFs usually
serve multiple roles during differentiation, which is dynamically
and precisely regulated, further research is needed to reliably
generate single, specific cell types using this approach.
This work shows how to utilize new knowledge arising from
the rapidly advancing field of -omics research, bringing it
closer to precise and reliable applications in tissue engineering
and regenerative medicine.
Author Contributions
J. Lavický, J. Křivánek, contributed to conception, design, data
acquisition, analysis, and interpretation, drafted and critically
revised the manuscript; M.G. Lopez, V. Rakultsev, V. Bryja, D.
Bohačiaková, T. Bárta, contributed to data acquisition and analysis,
critically revised the manuscript; V. Chochola, J. Raška, H.
Tuaima, J. Cotney, contributed to data analysis, critically revised
the manuscript; P. Kompaníková, E.W. Winchester, contributed to
data analysis and interpretation, critically revised the manuscript;
L. Englmaier, J. Verner, E. Švandová, contributed to data acquisition,
critically revised the manuscript; M. Buchtová, contributed
to data acquisition and interpretation, critically revised the manuscript.
All authors gave final approval and agree to be accountable
for all aspects of the work.
Acknowledgments
We would like to sincerely thank Katarína Marečková and Lucie
Vrlíková for their help with histological sample preparation.
Furthermore, we would like to sincerely thank Dr. Květoslava
Matulová from the Department Pathology at the University
Hospital Brno for her assistance with sample imaging. We would
like to sincerely thank Zdeněk Andrysik for his kind help with
Transcription Factor Code of Odontoblast Differentiation
exploring bioinformatic analyses. We would like to thank Kateřina
Syrová for her kind help with proofreading the manuscript.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: The
research of JK was supported by Czech Science Foundation (23-
06160S) and MB by the Ministry of Education, Youth and Sports
of the Czech Republic from the Operational Programme Research,
Development and Education (CZ.02.1.01/0.0/0.0/15_003/0000460).
We acknowledge funds from the Faculty of Medicine of Masaryk
University (MUNI/A/1738/2024). PK and VB have been supported
by Czech Science Foundation (grant No. GA25-17360S).
We acknowledge the core facility CELLIM supported by the
Czech Bioimaging large RI project (LM2023050 funded by
MEYS CR) for their support with obtaining scientific data presented
in this paper. JL is a Brno PhD Talent Scholarship Holder,
funded by the Brno City Municipality.
ORCID iDs
P. Kompaníková © https://orcid.org/0000-0002-3742-4523
V. Rakultsev © https://orcid.org/0000-0002-2130-6071
E.W. Winchester ©https://orcid.org/0000-0003-2695-7938
H. Tuaima © https://orcid.org/0009-0008-1066-1511
M. Buchtová © https://orcid.org/0000-0002-0262-6774
J. Křivánek ©https://orcid.org/0000-0002-7590-187X
Data Availability Statement
All raw data are available upon request from the corresponding
author.
References
Alghadeer A , Hanson-Drury S, Patní A P , Ehnes D D , Zhao Y T , L i Z, Phal A ,
Vincent T, L i m Y C , O'Day D, et al. 2023. Single-cell census of human
tooth development enables generation of human enamel. Dev Cell.
58(20):2163-2180.
Balic A . 2018. Biology explaining tooth repair and regeneration: a mini-review.
Gerontology. 64(4):382-388.
Biggs L C , Mikkola M L . 2014. Early inductive events in ectodermal appendage
morphogenesis. Semin Cell Dev Biol. 25-26:11-21.
Chang JYF, Kessler HP. 2008. Masson trichrome stain helps differentiate myofibroma
from smooth muscle lesions in the head and neck region. J Formos
M e d Assoc. 107( 10) :767-773.
Chiba Y , Saito K, Martin D, Boger ET, Rhodes C, Yoshizaki K, Nakamura T,
Yamada A , Morell RJ, Yamada Y , et al. 2020. Single-cell RNA-sequencing
from mouse incisor reveals dental epithelial cell-type specific genes. Front
Cell Dev Biol. 8:841. doi:10.3389/fcell.2020.00841
Cho A , Haruyama N, Hall B, Danton M J S , Zhang L, Arany P, Mooney DJ,
Harichane Y , Goldberg M , Gibson C W , et al. 2013. T G F - p regulates
enamel mineralization and maturation through K L K 4 expression. PLoS
One. 8(ll):e82267. doi:10.1371/journal.pone.0082267
Finnegan A , Cho R J , Luu A , Harirchian P, Lee J, Cheng JB, Song JS. 2019.
Single-cell transcriptomics reveals spatial and temporal turnover of keratinocyte
differentiation regulators. Front Genet. 10:775. doi:10.3389/
fgene.2019.00775
Hanson-Drury S, Patni A P , Lee D L , Alghadeer A , Zhao Y T , Ehnes D D , V o
V N , K i m S Y , Jithendra D, Phal A , et al. 2023. Single cell R N A sequencing
reveals human tooth type identity and guides in vitro hiPSC derived odontoblast
differentiation (iOB). Front Dent Med. 4:1209503. doi:10.3389/
fdmed.2023.1209503
Hemeryck L, Hermans F, Chappell J, Kobayashi H, Lambrechts D, Lambrichts
I, Bronckaers A , Vankelecom H. 2022. Organoids from human tooth showing
epithelial sternness phenotype and differentiation potential. Cell M o l
Life Sei. 79(3):153. doi:10.1007/s00018-022-04183-8
Hermans F, Hemeryck L, Lambrichts I, Bronckaers A , Vankelecom H. 2021.
Intertwined signaling pathways governing tooth development: a give-andtake
between canonical Wnt and Shh. Front Cell Dev Biol. 9:758203.
doi: 10.3389/fcell.2021.758203
Hulsey C D , Cohen K E , Johanson Z, Karagic N, Meyer A , Miller CT, Sadier
A , Summers A P , Fräser GJ. 2020. Grand challenges in comparative tooth
biology. Integr Comp Biol. 60(3):563-580.
Jimenez-Rojo L, Pagella P, Harada H, Mitsiadis T A . 2019. Dental epithelial
stem cells as a source for mammary gland regeneration and milk producing
cells in vivo. Cells. 8(10):1302. doi:10.3390/cells8101302
K i m R, Green J B A , Klein OD. 2017. From snapshots to movies: understanding
early tooth development in four dimensions. Dev Dyn. 246(6):442^150.
Křivánek J, Adameyko I, Fried K. 2017. Heterogeneity and developmental
connections between cell types inhabiting teeth. Front Physiol. 8:376.
doi:10.3389/fphys.2017.00376
Křivánek J, Buchtová M , Fried K, Adameyko I. 2023. Plasticity of dental cell
types in development, regeneration, and evolution. J Dent Res. 102(6):
589-598.
Křivánek J, Soldatov R A , Kastriti M E , Chontorotzea T, Herdina A N , Petersen
J, Szarowska B, Landova M , Matějova V K , Holla LI, et al. 2020. Dental
cell type atlas reveals stem and differentiated cell types in mouse and
human teeth. Nat Commun. 11(1):4816. doi:10.1038/s41467-020-18512-7
Kurpios N A , MacNeil L, Shepherd T G , Gludish D W , Giacomelli A O , Hassell
JA. 2009. The Pea3 Ets transcription factor regulates differentiation of multipotent
progenitor cells during mammary gland development. Dev Biol.
325(1):106-121.
Lavický J, Kolouskova M , Procházka D, Rakultsev V , Gonzalez-Lopez M ,
Steklikova K, Bartos M , Vijaykumar A , Kaiser J, Pořízka P, et al. 2022. The
development of dentin microstructure is controlled by the type of adjacent
epithelium. J Bone Miner Res. 37(2):323-339.
Lin Y , Xiao Y , L i n C, Zhang Q, Zhang S, Pei F, L i u H, Chen Z. 2021. S A L L 1
regulates commitment of odontoblast lineages by interacting with R U N X 2
to remodel open chromatin regions. Stem Cells. 39(2):196-209.
Neves V C M , Sharpe PT. 2018. Regulation of reactionary dentine formation.
J Dent Res. 97(4):416^122.
Ng Y H , Chanda S, Janas JA, Yang N, Kokubu Y , Südhof TC, Wernig M . 2021.
Efficient generation of dopaminergic induced neuronal cells with midbrain
characteristics. Stem Cell Reports. 16(7): 1763-1776.
Pagella P, de Vargas Roditi L, Stadlinger B, Moor A E , Mitsiadis T A . 2021.
A single-cell atlas of human teeth. iScience. 24(5):102405. doi: 10.1016/j
.isci.2021.102405
Saito K, Michon F, Yamada A , Inuzuka H, Yamaguchi S, Fukumoto E,
Yoshizaki K, Nakamura T, Arakaki M , Chiba Y , et al. 2020. Sox21 regulates
AnapclO expression and determines the fate of ectodermal organ.
iScience. 23(7): 101329. doi:10.1016/j.isci.2020.101329
Sharir A , Marangoni P, Zilionis R, Wan M , Wald T, Hu JK, Kawaguchi K,
Castillo-Azofeifa D, Epstein L, Harrington K, et al. 2019. A large pool of
actively cycling progenitors orchestrates self-renewal and injury repair of
an ectodermal appendage. Nat Cell Biol. 21(9): 1102-1112.
Thesleff I. 2003. Epithelial-mesenchymal signalling regulating tooth morphogenesis.
J Cell Sei. 116(pt 9):1647-1648.
Tucker A S , Fraser G J . 2014. Evolution and developmental diversity of tooth
regeneration. Semin Cell Dev Biol. 25-26:71-80.
Wang S, Drummond M L , Guerrero-Juarez C F , Tarapore E, MacLean A L ,
Stabell A R , W u SC, Gutierrez G , That BT, Benavente C A , et al. 2020.
Single cell transcriptomics of human epidermis identifies basal stem
cell transition states. Nat Commun. 11(1):4239. doi:10.1038/s41467-020-
18075-7
Yang S, Geng Z, M a K, Sun X , Fu X . 2016. Comparison of the histological
morphology between normal skin and scar tissue. J Huazhong Univ Sei
Technolog Med Sei. 36(2):265-269.
Yoshizaki K, Hu L, Nguyen T, Sakai K, He B, Fong C, Yamada Y , Bikle D D ,
Oda Y . 2014. Ablation of coactivator M e d l switches the cell fate of dental
epithelia to that generating hair. PLoS One. 9(6):e99991. doi:10.1371/
journal.pone.0099991
Appendix for:
Unraveling the transcription factor code of odontoblast differentiation
Josef Lavický1
, Marcos Gonzalez Lopez1
, Václav Chochola1
, Petra Kompaníková2
, Vladislav Rakultsev1
,
Jan Raška1
, Emma Wentworth Winchester3
, Haneen Tuaima1
, Lucie Englmaier1
, Jan Verner4
, Eva
Švandová1
, Marcela Buchtová5
'6
, Justin Cotney7
, Vítězslav Bryja2
, DášaBohačiaková1
, Tomáš Bárta1
, Jan
Křivánek1
*
1
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech
Republic
2
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
3
Department of Genetics and Genome Sciences, UCONN Health, Farmington, CT USA
4
Central European Institute of Technology, Masaryk University, Brno, Czech Republic
5
Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, Czech Academy
of Sciences, Brno, Czech Republic
6
Department of Experimental Biology, Faculty of Science, Masaryk University, Czech Republic
7
Center for Craniofacial Innovation, Children's Hospital of Philadelphia, Philadelphia, PA USA
*Correspondence: i an.krivanek@med. muni, cz
1
1. Contents
1. Contents 2
2. List of Appendix Figures 4
3. Appendix materials and methods 6
3.1. Bioinformatic analysis 6
3.2. Animals 7
3.3. The detection of the four selected transcription factors utilizing in situ hybridization ...7
3.4. In vitro odontogenic differentiation of DPSCs and alizarin red staining of mineralized matrix
8
3.5. Preparation of MEFs from DSPP-Cerulean/DMPl-Cherry mouse strain 9
3.6. Generation of Pluripotent stem cells from DSPP-Cerulean/DMPl-Cherry mouse strain 10
3.7. The design and generation of lentiviral vectors 11
3.8. Transduction of reporter induced pluripotent stem cells 14
3.9. In vitro differentiation of transduced miPSCs 15
3.10. In vivo differentiation of transduced miPSCs 16
3.11. Conventional histological staining and brightfield microscopy 17
3.12. Immunohistochemistry and confocal microscopy 17
3.13. In situ detection of GFP/Cerulean 18
3.14. Image analysis 19
3.15. RNA isolation and Realtime quantitative PCR 20
3.16. Statistical analysis and graph preparation 21
4. Appendix Results and Discussion 22
4.1. Initial selection of potential TFs 22
4.2. Strategy behind the initial selection of mature odontoblast-specific TFs 25
4.3. Current knowledge concerning the function of the selected genes 33
2
4.4. The expression of the selected TFs in the developing mouse molar 35
4.5. Design of lentiviral vectors and the generation of miPSC line with inducible expression of
the TFs
38
4.6. Analysis of growth, proliferation and cell death during spheroid cell culture 44
4.7. The analysis of mineralization in the in vitro cell spheroids 46
4.8. Co-staining of DSP and DMP1 49
4.9. Co-staining of mature odontoblast markers and CDH1 51
4.10. Analysis of Nestin expression in the epithelial structures in vivo 52
4.11. Analysis of SOX21 expression in the epithelial structures in vivo 54
4.12. The differentiation of transduced cells driven by the upregulation of the selected genes results
in the silencing of the TET3G+GFP transgene 56
5. References 61
3
2. List of Appendix Figures
Appendix Figure 1: Morphology and the expression of pluripotency markers in the derived reporter
mouse pluripotent stem cell line 11
Appendix Figure 2: Maps of the utilized lentiviral vectors. (A) Tet3G/EGFP vector; (B) GsclEtv4
vector; (C) SallllNuprl vector 13
Appendix Figure 3: The strategy behind the initial selection of odontoblast-specific TFs 24
Appendix Figure 4: The strategy behind the initial selection of mature odontoblast-specific genes
26
Appendix Figure 5: The heatmap of top active regulons in the whole incisor dataset 28
Appendix Figure 6: Heatmap of the Gsc regulon expression within the odontoblast incisor dataset.
29
Appendix Figure 7: Heatmap of the Etv4 regulon expression within the odontoblast incisor dataset
30
Appendix Figure 8: Heatmap of the Salll regulon expression within the odontoblast incisor dataset
31
Appendix Figure 9: Dotplot of enriched Gene Ontology (GO) terms identified by OverRepresentation
Analysis (ORA) of genes in the Gsc (A), Etv4 (B) and Salll (C) in (pre)odontoblast
cluster 32
Appendix Figure 10: Expression of Etv4 (A), Gsc (B), Salll (C) and Nuprl (D) transcription factors
throughout mouse molar development 36
Appendix Figure 11: The generation and validation of pluripotent stem cell lines with inducible
overexpression of four selected transcription factors 40
Appendix Figure 12: The transduced cell lines display upregulated expression of odontoblast and
keratinocyte markers due to the background activity of the TET3G transactivator 43
4
Appendix Figure 13: Analysis of growth, proliferation and cell death during the cell spheroid cell
culture 45
Appendix Figure 14: Investigation of the identity of observed mineralization and its dependence on
the cCASP3+ relative area 48
Appendix Figure 15: Co-staining of DSP and DMP1 in both cell spheroids (A) and kidney implants
(B) 50
Appendix Figure 16: Co-staining of mature odontoblast markers and CDH1 in conditions with
upregulated expression of selected TFs 52
Appendix Figure 17: Analysis of Nestin expression in the epithelial structures in vivo 53
Appendix Figure 18: Analysis of SOX21 expression in the epithelial structures in vivo 55
Appendix Figure 19: The investigation of the area-specific reduction of the overexpression of the
selected genes as a result of differentiation-induced silencing of the TET3G+GFP transgene 58
5
3. Appendix materials and methods
3.1. Bioinformatic analysis
Raw fastqs were retrieved from GEO and aligned to the mmlO genome (GRCm39) using Kallisto/bustools
(kallisto vO.46.2, bustools vO.40.0) (Meisted et al. 2021). Kallisto/bustools were used in filtering and
Seurat object generation. Seurat (v3.2.0) (Yaylaoglu et al. 2005) was used to merge replicates. Cells were
filtered (<10% mtRNA, >900 nFeatureRNA, <20% rbRNA, <60000 nCount_RNA, >2500 nCount_RNA)
and log normalized. Cell cycle stages were assigned with mouse orthologs from known human cell cycle
genes. Data was scaled using the top 5000 variable genes. Marker genes were identified with DESeq2
using logfc>0.5, min.pct=0.05. SymlDs were converted to EntrezID with clusterProfiler. clusterProfiler
was used to calculate GO term enrichment against background. clusterProfiler "simplify" combined
similar GO terms, with z score cutoff of 0.5. The generation of the visualizations from the dental cell type
atlas is described elsewhere (Krivanek et al. 2020).
To generate an unbiased list of genes with similar expression patterns to the selected odontoblast-specific
marker genes, the SCDE package was used (Kharchenko et al. 2014; Fan et al. 2016). It is available as
a component of the previously generated, freely accessible online analysis platform of mouse incisor
scRNA-seq dataset (Krivanek et al. 2020; Dental Cell Atlas).
Gene regulatory network inference was performed using the pySCENIC package (vO.12.1) (Aibar et al.,
2017) with Python 3.8.20. The analysis began with the GRNBoost2 algorithm to construct an adjacency
matrix based on gene co-expression scores. Subsequently, cisTarget was employed to identify enriched
transcription factor binding motifs and infer regulons using mmlO refseqr80
10kb_up_and_down_tss.mc9nr.genes_vs_motifs.rankings.feather and mml 0 refseqr80
500bp_up_and_100bp_down_tss. The activity of the inferred regulons in each cell was quantified
using AUCell, which calculates the Area Under the Curve (AUC) scores for regulon enrichment. To
integrate and visualize the results, AUC scores were added to a Seurat object in R 4.4.1. Regulon activity,
along with the gene expression of their target genes, was visualized using UMAP projections and/or
heatmaps, generated with the Seurat package (v5.2.1) and ggplot2 (v3.4.4).
To integrate and visualize the results, AUC scores were added to a Seurat object in R 4.4.1. Top active
regulons for the given dataset were identified using Seurat's FindAllMarkers function, with the following
6
parameters: only positive regulons were included, a log fold-change threshold of 0.25 was applied, and
statistical significance was assessed using the Wilcoxon rank-sum test. Activity of top regulons, along
with the gene expression of their target genes, was visualized using UMAP projections and/or heatmaps,
generated with the Seurat package (v5.2.1) and ggplot2 (v3.4.4).
3.2. Animals
Mice (Mus musculus) used in this study included a DSPP-Cerulean/DMP 1-Cherry reporter mouse strain
on a CD-I genetic background (Vijaykumar et al. 2019), which was used for the validation of the
developmental trajectory and as a source for mouse embryonic fibroblasts. For the kidney implantation
experiments, an immunodeficient mouse strain NOD. Cg-PrkdcscidIl2rgtmlWjl/SzJ (NSG) was used as a
host for the implants. C57BL/6 wildtype animals were used for the detection of the expression of the four
chosen TFs in adult incisor and in the molar during embryonic development and at P14. Mice from the
CD-I strain were used as a source of dental pulp stem cells. All animal experiments were approved by
Ministry of Education, Youth and Sports, Czech Republic (MSMT-8360/2019-2; MSMT-9231/2020-2;
MSMT-6379/2022-4). Experiments were performed according to international and local regulations. Mice
were housed in 12/12 light/dark cycle, at temperature ranging from 18 to 23 °C and 40-60% humidity.
Food and water were accessible to the animals ad libitum. Genotyping of the DSPP-Cerulean/DMP 1Cherry
construct was performed using endpoint PCR utilizing transgene-specific primers (primer
sequence in Appendix Table 1). Immunodeficient mice were housed in individually ventilated cages (IVC)
in standard conditions. The animals were euthanized using anesthesia overdose (Isoflurane, KDG9623,
Baxter) and tissue was processed as further described.
3.3. The detection of the four selected transcription factors utilizing in situ hybridization
For the detection of Gsc, Etv4, Salll and Nuprl in the mouse incisor, mandibles from three adult (2 months
old) C57BL/6 wildtype animals were carefully dissected, briefly washed in PBS and fixed in 4%
paraformaldehyde (pH 7.4) for 48 hours at room temperature. Fixed tissue was then decalcified in 10%
EDTA (pH 7.4) for 14 days at 4 °C. For the detection in the developing molar, series of timed breedings
were performed utilizing the C57BL/6 wildtype animals. Four C57BL/6 wildtype pregnant females at
timepoints 12.5, 14.5, 16.5 and 18.5 (E12.5, E14.5, E16.5, E18.5) days post coitum were euthanized,
embryos were isolated, decapitated and fixed using 4% paraformaldehyde (pH 7.4) for 24 hours at room
temperature. In addition three, P14 (postnatal day 14) pups were euthanized, the mandibles were isolated
7
and fixed using 4% paraformaldehyde (pH 7.4) for 48 hours at room temperature. E16.5 and E18.5
embryos were decalcified in 10% EDTA (pH 7.4) for 3 days at 4 °C, while the P14 mandibles were
decalcified for 10 days at 4 °C. All samples were processed for paraffin sectioning.
Samples were sectioned on a microtome (Leica SM2000R) to a thickness of 5 urn. In situ hybridization
was performed utilizing the RNAscope 2.5 HD Assay - RED (322350, ACDBio) as described before
(Lavický et al. 2022). The following probes were used: Gsc (504821), Etv4 (458121), Salll (469661) and
Nuprl (434811). Subsequently, slides were stained with Haematoxylin and mounted in Pertex medium
(00801, Histolab). Sections were later imaged using Axio Imager 2 (Carl Zeiss AG, Germany) and
AxioScan Z. 1 (Carl Zeiss AG, Germany). Images were subsequently exported in ZEN Blue 3.4 (Carl Zeiss
AG, Germany) as 8-bit .png images.
3.4. In vitro odontogenic differentiation of DPSCs and alizarin red staining of
mineralized matrix
Dental pulp stem cells from the continuously growing incisors were isolated as described before (Křivánek
et al. 2021) from CD-I mice. The total of 5 mice were used to generate cells for each of the three biological
replicates used in the experiments.
The used odontoblast differentiation protocol was adapted from previous works (Balic et al. 2010;
Sagomonyants et al. 2015; Sagomonyants and Mina 2015). Briefly, the isolated cells from each biological
replicate were plated in cell culture-treated well plates. For qPCR, 4.9* 104
cells per well were plated in
12-well plates, and for alizarin red staining, 2.5* 104
cells per well were plated in 24-well plates. Cells
were cultured in plating medium (days 0-3), proliferation medium (days 3-7) and mineralization medium
(days 7-21). Fresh medium was changed every other day. Plating medium consisted of DMEM (10313021,
Gibco™), 20% Fetal Bovine Serum Ultra low Endotoxin (FB-1101/500, Biosera), 1% Glutamax™
Supplement (35050061, Gibco™) and l%ZellShield (13-0050, Minerva Biolabs). Proliferation medium,
consisting of DMEM, 5% FBS, 1% GlutaMAX, 20 ng/ml FGF-2 (450-33-10, Peprotech) and 1%
ZellShield. Mineralization medium consisted of M E M a (22561021, Gibco™), 5% FBS, 1% GlutaMAX,
50 ug/ml ascorbic acid (A8960, Sigma-Aldrich), 4 mM P-Glycerophosphate (G9422, Sigma-Aldrich), 1%
ZellShield. Samples were harvested on days 3, 7, 14 and 21. Samples for qPCR were washed two times
with PBS and lysed in RNA Blue (R013, Top-Bio) and further processed as described below. For the
8
mineralization assay, cells were washed three times with PBS and subsequently fixed using 4% PFA (pH
7.4) for 20 minutes at RT.
For the mineralization assay, Alizarin Red S solution was used according to Stanford et al. (1995), with
minor modifications. Briefly, cells were washed 3 times with deionized water for 5 minutes at RT.
Subsequently, 2% Alizarin Red S (R 03482, Roth) dthO solution (pH adjusted to 4.2) was added to the
cell culture wells and was then incubated for 10 minutes at RT with gentle agitation. Wells were then
rinsed 5 times with dthO and incubated for 15 minutes with PBS to reduce non-specific Alizarin Red S
binding. Well's contents were subsequently imaged in dthO using an AxioZoom V16-Apotome2 (ZEISS,
Germany) stereomicroscope. Images were exported using ZEN Blue 3.4 (ZEISS, Germany).
3.5. Preparation of MEFs from DSPP-Cerulean/DMPl-Cherry mouse strain
Induced pluripotent stem cells from the odontoblast reporter strain DSPP-Cerulean/DMPl-Cherry were
generated from murine embryonic fibroblasts (MEFs). Briefly, timed breedings of a CD1 wildtype females
and a males heterozygous for the DSPP-Cerulean/DMPl-Cherry transgenes were performed. Pregnant
females 12.5 days post coitum were euthanized using anesthesia overdose (Isoflurane, KDG9623, Baxter).
The El2.5 embryos were then isolated in aseptic conditions under a stereo microscope. The embryos were
decapitated using a scalpel with a type 12 blade and the developing organs in the abdomen of the embryos
were dissected out using sharp tip tweezers. The remaining tissue was minced in 3 ml of of Trypsin-EDTA
solution (25200056, Gibco™) using surgical scissors and scalpel. The digestion was stopped by adding
10 ml of MEF medium (KnockOut™ D M E M [10829018, Gibco™], 10% Fetal Bovine Serum (South
America), Ultra low Endotoxin [FB-1101/500, Biosera], 1% Glutamax™ Supplement [35050061,
Gibco™], 1% M E M Non-Essential Amino Acids Solution (100X) [11140035, Gibco™], 0.1 mM 2Mercaptoethanol
[M3148, Sigma-Aldrich]). The cell suspension was centrifuged at 300g for 4 minutes
and the cell pellet was resuspended in fresh MEF medium. The isolated cells from each of the embryos
separately, were then plated on 10 cm tissue culture dishes (93100, TPP) with 10 ml of MEF medium.
The cell culture of primarily embryonic fibroblasts was then cultured for three more passages. The cells
not needed for plating were frozen using Synth-a-Freeze™ Cryopreservation Medium (Al 254201,
Gibco™) and kept in liquid nitrogen until further use. The heads of the embryos were used for the
genotyping of the embryos for the presence of DSPP-Cerulean/DMPl-Cherry transgenes. The cells from
9
the embryos positive for the DSPP-Cerulean/DMPl-Cherry transgenes were used for the generation of the
reporter induced pluripotent stem cells.
3.6. Generation of Pluripotent stem cells from DSPP-Cerulean/DMPl-Cherry mouse
strain
The induced pluripotent stem cells line from the odontoblast reporter strain was generated from the
corresponding mouse embryonic fibroblasts (MEFs) using Epi5™ Episomal iPSC Reprogramming Kit
(A15960, Gibco™). Briefly, the MEFs were transfected with the reprogramming plasmids using Neon™
Transfection System according to the manufacturer protocol. Subsequently, the transfected cells were
plated onto a six-well culture plate (92406, TPP), coated with Matrigel® Basement Membrane Matrix
Growth Factor Reduced Phenol Red Free (356231, Corning®). Day after transfection the medium was
changed to a Base medium (DMEM, high glucose, pyruvate, no glutamine [10313021, Gibco™], 15%
Tetracychne-free FBS [FB-1001T500, Biosera], 1% Glutamax™ Supplement [35050061, Gibco™], 1%
M E M Non-Essential Amino Acids Solution (100X) [11140035, Gibco™], 0.1 mM 2-Mercaptoethanol
[M3148, Sigma-Aldrich]) supplemented with 5 ng/ml Leukaemia inhibiting factor (LIF Recombinant
Mouse Protein, embryonic stem cell-qualified; A35933 Gibco™), 50 ug/ml L-Ascorbic acid 2-phosphate
sesquimagnesium salt hydrate (A8960, Sigma-Aldrich) and 3 uM CHIR99021 (SML1046; SigmaAldrich)
according to Bar-Nur et al. (2014). The manual selection and passaging of the reprogramming
cells were performed manually according to previously established criteria. The validation of the
expression of pluripotency markers was performed using immunocytochemistry (ICC) for markers Oct4
and Nanog (Appendix Figure 1). Briefly, the cells were seeded onto a Matrigel®-coated glass coverslips
and cultured for 3 days. Subsequently, the cells were washed with PBS and fixed for 20 minutes using 4%
PFA. Cells were then permeabilized for 10 minutes using 0.1% Triton™ X-100 (T8787, Sigma-Aldrich)
in PBS and blocked using 5% normal donkey serum (D9663, Sigma-Aldrich) in PBS with 0.1% Tween20
(P1379, Sigma-Aldrich) for 30 minutes. Primary antibodies for Oct4 (sc-5279, Santa Cruz) and Nanog
(PA1-097, Thermo Fisher) were then applied to the coverslips diluted in the blocking solution and
incubated overnight at 4°C. Secondary antibodies (A-21206 and A-21203, Thermo Fisher) diluted in the
blocking solution were then incubated on the slides for 2 hours at RT. The coverslips were counterstained
with DAPI (6335.1, Carl Roth) and mounted in Fluoromount-G™ Mounting Medium (00-4958-02,
Thermo Fisher).
10
The generated induced pluripotent stem cell line was subsequently maintained in the same culture medium
as used during the reprogramming process supplemented with ZellShield® (13-0050, Minerva Biolabs)
and passaged using StemPro™ Accutase™ Cell Dissociation Reagent ( A l l 10501, Gibco™). The cells
were routinely checked for the presence of mycoplasma.
f $ i F ~ DAPI OCT4
Merge
! # " •' J J « ^ ,
Appendix Figure 1: Morphology and the expression of pluripotency markers in the derived reporter mouse
pluripotent stem cell line. Scalebars = 200 um.
3.7. The design and generation of lentiviral vectors
Three lentiviral vectors Tet3G/EGFP (pLV[Exp]-EFl A>Tet3G(ns):P2A:EGFP), GsclEtv4 (pLV[TetOn]Puro-TRE3
G>mGsc[NM_010351.1 ](ns):P2A: m£/W[NM_001316365.1]) and SallllNuprl
(pLV[TetOn]-Bsd-TRE3G>m1S,
a//7[NM_021390.3](ns):P2A:mArwpr7[NM_019738.1]) were designed
using the VectorBuilder web application. These were subsequently constructed and packaged by
VectorBuilder. The IDs of the utilized vectors are VB191115-1031qfz for the Tet3G/EGFP vector;
VB191115-1065spd for the GsclEtv4 vector and VB191115-1040ykg for the SallllNuprl vector, which
can be used to retrieve detailed information about the vector on vectorbuilder.com. The maps of all three
lentiviral vectors are shown in Appendix Figure 2.
The supplied VB UltraStable strain of E. coli hosting the lentiviral vectors were cultured overnight at 42
°C in Luria Broth (LB) liquid growth media supplemented with Carbenicillin (10177012, Gibco™) to a
final concentration of 100 ug/ml. From the resulting bacterial cultures, corresponding lentiviral vectors
11
were isolated using QIAGEN Plasmid Midi Kit (12143, QIAGEN). The purity and concentration of vector
DNA was assessed using a NanoPhotometer® N50 (IMPLEN, Germany). Subsequently, lentiviral
particles were generated using the cloned vectors as described before (Barta et al. 2016). Briefly,
HEK293T cells cultured in a Lentivirus Production culture medium (Opti-MEM™ 31985062 Gibco™),
containing 1% Tetracycline-free FBS (FB-1001T500, Biosera), 1% M E M non-essential amino acid
solution (11140050, Gibco™), 1% Penicillin-Streptomycin (10378016, Gibco™), 4 mM caffeine (C0750,
Sigma-Aldrich) and 1 mM sodium butyrate (19-137, Sigma-Aldrich) were transfected with the vectors.
Medium containing the newly produced lentiviral particles was collected every 12 hours for a total of 36
hours with fresh Lentivirus Production medium added into the wells each time. Media containing the
lentiviral particles were centrifuged (4.500 g, 10 minutes, room temperature), filtered through a 0.45 um
low protein-binding filter and kept at -80°C until further use.
12
Maps of the utilized lentiviral vectors
A Tet3G/EGFP (pLV[Exp]-EF1A>Tet3G(ns):P2A:EGFP)
3SV promoter
5 L7R-AU3
5V40 e^ly r>A
3' LTRrAU3
C SalM/NupM (pLV[TetOn]-Bsd-TRE3G>mSall1[NM_021390.3](ns):P2A:mNupr1[NM_019738.1])
Appendix Figure 2: Maps of the utilized lentiviral vectors. (A) Tet3G/EGFP vector; (B) GsclEtv4 vector; (C)
SallllNuprl vector.
13
3.8. Transduction of reporter induced pluripotent stem cells
The reporter induced pluripotent stem cells were plated onto a Matrigel-coated six-well culture plate
(92406, TPP), into the "Lentivirus Production" medium containing the specific lentiviral particles. The
medium was supplemented with 5 ug/ml of Polybrene (TR-1003, Sigma-Aldrich) to enhance the
transduction efficiency. Three hours after seeding, the medium was exchanged for the LIF medium
(DMEM, high glucose, pyruvate, no glutamine (10313021, Gibco™), 15% Tetracycline-free FBS (FB-
1001T500, Biosera), 1% Glutamax™ Supplement (35050061, Gibco™), 1% M E M Non-Essential Amino
Acids Solution (100X) (11140035, Gibco™), 0.1 mM 2-Mercaptoethanol (M3148, Sigma-Aldnch), 5
ng/ml Leukemia inhibiting factor (LIF Recombinant Mouse Protein, embryonic stem cell-qualified;
A35933 Gibco™), 50 ug/ml L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (A8960, SigmaAldnch),
3 uM CHIR99021 (SML1046; Sigma-Aldnch), ZellShield® (13-0050, Minerva Biolabs)). The
transduced cells were then selected based on the antibiotic resistance gene contained in the specific vector.
The cells positive for the Tet3G/EGFP vector were selected 4-5 days after the transduction using
fluorescently activated cell sorting on BD FACSAria™ (BD Biosciences, USA) and further propagated.
The selection of cells positive for the GsclEtv4 vector was initiated 2 days after the transduction by adding
Puromycin dihydrochloride (P8833, Sigma-Aldrich) to the culture medium to a final concentration of 0.25
ug/ml. The selection of cells positive for the SallllNuprl vector was initiated 2 days after the transduction
by adding Blasticidin S (15205, Sigma-Aldrich) to the culture medium to a final concentration of 10
Ug/ml. In both cases the remaining EGFP+ colonies were mechanically cut and transferred into a fresh
Matrigel-coated cell culture dish containing fresh LIF medium 3-4 days after the start of the selection. The
total of four cell lines with different combinations of the lentiviral transgenes were generated by sequential
transduction. These were named "T" (containing the Tet3G/EGFP vector); "TS" (containing the
Tet3G/EGFP and SallllNuprl vectors); "TG" (containing Tet3G/EGFP and GsclEtv4 vectors) and "TGS"
(containing Tet3G/EGFP, Gsc/Etv4 and SallllNuprl vectors). The cell line resulting from each of the
transduction steps was cryopreserved using Synth-a-Freeze™ Cryopreservation Medium (Al 254201,
Gibco™) after achieving stable morphology and proliferation (regularly 3-4 passages after the first
mechanical passage of the selected cells). These cell stocks were then used either for further transductions
or experiments. To validate the success of the transductions, all cell lines were seeded at a concentration
of 3000 cells per cm2
to a Matrigel™-coated 12-well plate (92012, TPP). The cells were subsequently
cultured in LIF medium with and without 10 ug/ml of Doxycycline hyelate (D9891, Sigma-Aldrich).
Three replicates per each condition. Media were changed on the second day. Four days after seeding, the
14
cells were washed with PBS and lysed in RNA Blue (R013, Top-Bio). The samples were subsequently
stored at -80° until further processing.
Research done by Nakatake et al. (2013) highlights that no antibiotic selection is capable of producing
100% pure transduced cell population. Nevertheless, we have performed several procedures to minimize
the heterogeneity of the transduced cell lines. One of our approaches was to use a reduced number of
lentiviral vectors (rationale given in the Appendix Results and Discussion) as well as utilizing EGFP as a
selection gene. Additionally, we have closely optimized a dose of the selection antibiotics to achieve
maximum cell death over a 3-day period. Furthermore, to achieve the highest ratio of transduced cells
possible, we have performed a secondary antibiotic selection in addition to the initial selection following
the lentiviral transduction. This secondary selection was done using slightly increased antibiotic
concentrations and was done on cells plated at low densities.
3.9. In vitro differentiation of transduced miPSCs
To diminish the effects of leaky expression of the transgenes, the prepared transduced cell stocks were
thawed as close to the time of seeding of the experiments as possible (2-3 passages). To form cell
spheroids, the cells from the cell lines T, TS, TG and TGS were seeded in a concentration of 5000 cells
per well into Poly(2-hydroxyethyl methacrylate) (poly-FlEMA, P3932, Sigma-Aldrich) coated V-shape
96-well plate (277143, Thermo Scientific) with 150 ul of LIF medium. Two days after seeding, the
medium was discarded and 150 ul of fresh Base medium (DMEM, high glucose, pyruvate, no glutamine
(10313021, Gibco™), 15% Tetracycline-free FBS (FB-1001T500, Biosera), 1% Glutamax™ Supplement
(35050061, Gibco™), 1% M E M Non-Essential Amino Acids Solution (100X) (11140035, Gibco™), 0.1
mM 2-Mercaptoethanol (M3148, Sigma-Aldrich), 50 ug/ml L-Ascorbic acid 2-phosphate
sesquimagnesium salt hydrate (A8960, Sigma-Aldrich, ZellShield® (13-0050, Minerva Biolabs) was
added into the wells. Two days later, the medium was exchanged again. In addition, starting from day 4,
up until the end of the experiment, half of the cell spheroids were stimulated to overexpress the transgenes
by adding doxycycline to a final concentration of 1 ug/ml to the medium. The next day, the formed
spheroids were distributed into non-adherent 6-cm wide Petri dishes (10060, SPL) (20 cell spheroids per
Petri dish) and embedded into 5 ul Matrigel® Basement Membrane Matrix Growth Factor Reduced
Phenol Red Free (356231, Corning®). From this point onwards, the cell spheroids were cultured in a base
medium supplemented with [3-Glycerophosphate disodium salt hydrate (4 mM, G9422, Sigma-Aldrich).
15
The medium was changed every 3 days. From day 11, the Petri dishes were transferred onto a horizontal
shaker to enhance nutrient exchange. The cell spheroids were then processed for qPCR and
immunohistochemistry (IHC) on day 21 after seeding. Briefly, the spheroids were washed in PBS and
either lysed in RNA Blue or fixed in 4% paraformaldehyde (pH 7.4) for 1 hour at room temperature and
further processed. From each condition 5-6 spheroids were utilized for both approaches.
3.10. In vivo differentiation of transduced miPSCs
Cell spheroids were prepared similarly to the in vitro experiments from the T and TGS cell lines. The only
difference being the start of doxycycline treatment was at day 2 after seeding. The implantation itself was
performed at day 5. Briefly, the NSG mice were put under anesthesia utilizing Isoflurane (3% induction
and 2% for maintenance) with pure oxygen as a carrier. The fur was removed in the dorsal region of the
skin above the left kidney. Subsequently, the kidney capsule implantation was performed as shown
elsewhere (Szot et al. 2007). The only difference being the fact that the cell spheroids were implanted
using an intravenous G18 cannula Vasofix® Safety (4269136S, B. Braun) attached to a glass syringe
(1001 LT, Hamilton) and the incision was closed using absorbable sutures (MV0252, CFflRANA T.
Injecta). Following the surgery, the well-being of the experimental animals was monitored daily for signs
of pain and suffering, and analgesia was applied to the experimental animals for the following 3 days in
the form of ibuprofen dissolved in drinking water with a final concentration of 0.2 mg/ml. The drinking
water was also supplemented with 1% sucrose (16104, Sigma-Aldrich) and doxycycline (1 mg/ml)
(Leitner et al. 2014; Redelsperger et al. 2016). The drinking water was changed every 2 days. The cages
with experimental animals were kept in the same location. Twenty-eight days after the implantation, the
mice were euthanized, and the implant was recovered under a fluorescent stereo microscope (Ml 65 FC,
Leica Camera AG, Germany) based on the EGFP signal of transgene-expressing cells. The isolated
implant was washed in Ice cold Hanks' Balanced Salt solution (HBSS, H6648, Sigma-Aldrich) and put
into fresh ice-cold HBSS. Subsequently, the implant was cut into equal parts and 5 pieces of tissue from
different parts of the implant were collected, minced using a type 12 scalpel and lysed in RNA Blue.
Remaining tissue was fixed using 4% PFA for 24 hours at 4 °C and subsequently processed for either
cryo- or paraffin sectioning as described before (Lavický et al. 2022). For each condition, implants from
three mice from independent experiments were analysed using both RT-qPCR, IHC and conventional
histology (Total of 6 mice were used). Mice whose implant was not found in the kidney capsule were
excluded from the analysis, this criterium was set a priori. No animals were excluded. The number of
16
experimental animals was to the lowest permissible (3R). No specific method of randomization of
experimental animals was utilized to allocate them into experimental groups. Due to the nature of the
experiment design and analysis all researchers were aware of group allocation during the whole
experiment. Experimental protocol was not preregistered.
3.11. Conventional histological staining and brightfield microscopy
For the conventional histological staining, the samples were sectioned on a microtome (Leica SM2000R)
to a thickness of 2 um. Von Kossa and Masson's trichrome staining were performed. For the quantification
of mineralization of cell spheroids, sections from three distinct z-layers were used. The total of 5-6
spheroids, each on 9-10 sections, were analyzed per condition. The imaging for quantification was
performed on AxioScan Z.l (Carl Zeiss AG, Germany) using a lOx objective. The detailed images were
obtained using a 20x objective on Axio Imager 2 (Carl Zeiss AG, Germany).
3.12. Immunohistochemistry and confocal microscopy
For immunohistochemistry on the paraffin embedded tissue, the samples were sectioned on a microtome
(Leica SM2000R) to a thickness of 2 um. The sections were subsequently deparaffinized and antigen
retrieval was performed. For the staining of DSP, boiling in pH 6.1 Citrate buffer (SI69984-2, DCS) for
5 minutes was used. For the rest of the antibodies, boiling for 30 minutes in a pH 9 Tris/EDTA buffer
(S236784-2, DCS) was used. The sections were then blocked utilizing a 5% normal donkey serum (D9663,
Sigma-Aldrich) in PBS with 0.1% Tween20 (PI379, Sigma-Aldrich) for 30 minutes. Subsequently the
antibodies for DSP (ENH083-FP, Kerafast), DMP1 (abl03203, Abeam, PA5-47621, Thermo Fisher),
CDH1 (AF748, Bio-Techne), KRT10 (905404, BioLegend), Nestin (4760, Cell Signaling Technology)
and Sox21 (#AF3538, Bio-Techne) were applied to the sections diluted in the blocking solution and were
incubated overnight at 4°C Subsequently, the secondary antibodies (A-11055,A10042 and A-21202,
Invitrogen) were applied onto the sections diluted in the blocking solution and incubated for 2 hours at
RT. The sections were stained with DAPI (6335.1, Carl Roth) and mounted in Fluoromount-G™
Mounting Medium (00-4958-02, Thermo Fisher).
For the analysis of the expression of the reporter fluorescent proteins, cryosection were utilized. Briefly,
tissues were sectioned on a cryostat microtome (CM1860, Leica Biosystems, Germany) and further
processed and stained as described before (Lavický et al. 2022). The total of 3 adult (2 months old) reporter
17
mice was used. For the validation of the reporter expression, antibody staining for DSP and DMP1 was
utilized as described before, albeit without the antigen retrieval steps. The sections were imaged on LSM
880 confocal laser scanning microscope (Carl Zeiss AG, Germany) using a 5x objective for quantifications
and 25x objective for detailed images.
3.13. In situ detection of GFP/Cerulean
To verify the origin of the implanted cells, in situ hybridization was performed using a EGFP/Ceruleanspecific,
digoxigenin-labelled probe. The probe was synthesized using following, EGFP/Cerulean-specific
primers (Forward primer 5'-TCATCTGCACCACCGGCAAGC-3', Reverse primer 5'AGCAGGACCATGTGATCGCGC-3')
(Vijaykumar et al. 2019). Firstly, the specificity of both the
primers and their amplicon (probe) for the EGFP and Cerulean ORFs was verified using Primer BLAST
and BLASTn (Zhang et al. 2000) against the mouse genomic and transcript databases. No off-targets were
found. The primers were then used in a PCR reaction (M7505, Promega) using DSPP-Cerulean/DMPlCherry
mouse genomic DNA as template. Size of PCR product was validated using agarose gel
electrophoresis and the PCR product was purified using QIAquick PCR Purification Kit (28104, Quigene).
Subsequently, 5 ng of the synthesized amplicon was used to synthesize a digoxigenin labelled DNA probe
using HighFidelity Digoxigenin PCR Labeling Kit (APP-101-DIGX, Jena Bioscience) according to the
manufacturer's protocol.
For the hybridization itself, the slides were baked for 60°C for 1 hour. Slides were deparaffinized,
incubated with 3% hydrogen peroxide solution for 15 minutes at RT and washed in CLH2O. Subsequently,
sections were incubated with Protease Plus reagent (322331, Biotechne) for 15 minutes at 40°C and
washed in dH2 0. Slides were then incubated in pH 6.1 Citrate buffer (S169984-2, DCS) at 98°C for 30
minutes. Immediately after, the slides were briefly washed in CLH2O at 98°C, dipped into a washing buffer
(O.lx SSC, 0.03% lithium dodecyl sulfate) and denatured probe solution was added onto the sections.
Probe was diluted to a 3 ng/ul concentration in a hybridization buffer (6* SSC, 25% formamide, 0.2%
lithium dodecyl sulfate) and denatured at 98°C for 10 minutes right before being put onto the sections.
Probes were hybridized overnight at 42°C. The following day the slides were washed in the washing
buffer, PBS-T and blocked using 5% NDS solution in PBS-T for 1 hour at RT. Anti-Digoxigenin primary
antibody (700772, Invitrogen) diluted in the blocking buffer was applied to the sections and incubated
overnight at 4°C. Slides were washed in PBS and the detection was performed using Alexa Fluor™ 555
18
Tyramide SuperBoost™ Kit, goat anti-rabbit IgG (B40923, Invitrogen) according to the manufacturer's
protocol. Slides were counterstained with DAPI and mounted using Fluoromount G. The sections were
imaged on LSM 880 confocal laser scanning microscope (Carl Zeiss AG, Germany) using a 63x objective
for detailed images.
3.14. Image analysis
For the analysis of the expression of DSP, DMP1, CDH1 and GFP in the in vitro experiments, ImageJ
1.54g was used (Schneider et al. 2012). Briefly, confocal images were exported into single channel, 16bit,
grayscale jpeg images using ZEN Blue 3.4 (Carl Zeiss AG, Germany) using the Export Image
function. Subsequently, the images were imported into ImageJ, and individual cell spheroids were
manually segmented using the Polygon selection function using the images of the DAPI channel. The
selections were added into the overlay manager. The images of DSP and DMP1 channels were then made
binary using the threshold function (the same thresholds were used for all the images from each of the
stains). Subsequently, the relative DSP-positive and DMP1-positive area of individual spheroids was
measured as a fraction of the segmented spheroid area.
For the analysis of the Von Kossa-stained sections, QuPath 0.4.4 was utilized (Bankhead et al. 2017).
Briefly, for the segmentation of the spheroids and the Von Kossa-positive tissue within them, two
threshold-based pixel classifiers were used. Based on these pixel classifiers, annotations of corresponding
areas were generated. Subsequently, the relative mineralized area of individual spheroids was measured
as a fraction of the segmented spheroid area. These steps were automated using a built-in script function
and images of all the spheroids were analyzed using the same settings. For the analysis of the expression
of DSP, DMP1, CDH1 and GFP in the in vivo implant sections QuPath 0.5.0 was used. The areabelonging
to the implants was manually segmented. Pixel classifiers for each of the stained proteins were trained
using the same number of images. All the trained pixel classifiers used were based on the Random trees
(RTrees) model. Furthermore, the analysis was performed only on color channel corresponding to the
analyzed protein utilizing 1.0 and 2.0 scales. The total of four features were used: Gaussian, Laplacian of
Gaussian, Weighted deviation and Hessian determinant. Subsequently, relative area positive for the given
protein was measured and calculated as stated above. QuPath 0.5.1 was used to analyze the cCASP3 and
mKI-67 stainings on the in vitro cell spheroids. Briefly, the spheroids were manually segmented. The
cCASP3+ area was subsequently analysed using a single pixel tresholder. The absolute and relative
19
cCASP3-postive and negative areas were measured as described above. The number of mKI-67+ cells
was quantified using a built-in Detect Cells function. The number of mKI-67+ cells was then normalized
to the cCASP3-negative area obtained from the previous analysis. This was done to adjust for the
necrotic/apoptotic areas within the cell spheroids. The detail images were converted into .ims format
using the ImarisFileConverter 9.2.0 (Oxford Instruments) and exported into .png using ImarisViewer 9.7.0
as maximum intensity projections.
3.15. RNA isolation and Realtime quantitative PCR
The RNA from the samples lysed in RNA Blue was isolated using phenol/chloroform extraction according
to the manufacturer protocol. The concentration and purity of the RNA was assessed using a
NanoPhotometer® N50 (EVIPLEN, Germany). Reverse transcription was then performed using
Transcriptor First Strand cDNA Synthesis Kit (04897030001, Roche). Two sets of cDNA were prepared
for each of the samples, one using the Poly T primers (for short transcripts e.g. Nuprl and Etv4) and the
other using random hexanucleotides (for long transcripts e.g. Dmpl and Dspp). Subsequently, Realtime
quantitative PCR was performed using LightCycler® 480 SYBR Green I Master (4887352001, Roche,
Switzerland) on a LightCycler® 480 System (Roche, Switzerland) using the default SYBR green program.
The specificity of the PCR products was assessed using no reverse transcription (NRT) controls and
melting curve genotyping for each of the samples and genes. The expression of the genes of interest was
normalized to the expression Gapdh. Resulting Delta-Ct values were then converted into fold change by
converting them to a negative power of 2 (2A
-dCT) in Microsoft Excel. Fold change data were
subsequently further normalized to a geometric mean of replicates from T Day 0 (Gsc, Etv4, Salll and
Nuprl), TG Day 21 Ctrl (Dspp, Dmpl, Runx2, KrtlO and Ivl), TS Day 0 (Salll/P2A/Nuprl), TG Day 0
(Gsc/P2A/Etv4). For each replicate, the means of 3 reactions were used. The statistical analysis was
performed on the Delta-Ct values. For the relative quantification of the expression of odontoblast markers
and selected TFs during odontogenic differentiation of DPSCs the expression was normalized to the
expression Gapdh. Resulting Delta-Ct values were then converted into fold change by converting them to
a negative power of 2 (2A
-dCT). In the case that no specific PCR product was observed in the melting
curve analysis, the samples were labelled as non-detects (n.d.). In a case that the one or two replicates
from one condition were non-detects, 45 ct value (the total number of cycles in the used PCR program)
was attributed to the non-detect replicates to enable statistical analysis. These values are then shown as
20
red data points in the graphs. The sequences of the used primers are available in Appendix Table 1 (Ye
et al. 2012; Vijaykumar et al. 2019; Takada et al. 2022).
Target gene Primer sequence Reference
Gsc/P2A/Etv4
F: G A G G A G T C A G A A A A C G C C G A
Gsc/P2A/Etv4
R: C T C G C T G G T C C A A G T A T C C G
Gsc
F: G G A G G A G A A G G T G G A G G T C T G
Gsc
R: C T G T C C G A G T C C A A A T C G C T
Etv4
F: C G G A C A G T G A T G A G C A G T T T G
Etv4
R: G G A G T C A T A G G C A C T G G A G T A A
Salll/P2A/Nuprl F:
R:
G C G A G A A C G G A A C C A A C T T C
A G A G G T T G C T G G G A A G G G T T
Salll F:
R:
G C A A G C G A A G C C T C A A C A T T
C T T C T C T G T G T C C C C A T C T C G
Nuprl F: C C C T T C C C A G C A A C C T C T A A A PrimerBank ID: 9790145al;
R: T C T T G G T C C G A C C T T T C C G A (Spandidos et al. 2010)
Gapdh F: A G G T C G G T G T G A A C G G A T T T G PrimerBank ID: 6679937al
R: T G T A G A C C A T G T A G T T G A G G T C A (Spandidos et al. 2010)
Dspp F:
R:
A G C C A G T C A G A A G C A T G T C C
C C T T T G T T G G G A C C T T C A G T
(Takada et al. 2022)
Runx2 F:
R:
C G T G T C A G C A A A G C T T C T T T T
G G C T C A C G T C G C T C A T C T
(Takada et al. 2022)
Dmpl F:
R:
T C T G A A G A G A G G A C G G G T G A T
G A T T C A C T G C T G T C C G T G T G
Krt10 F:
R:
A C G A G A A G C A T G G C A A C T C A
T T G T C A G G G T G A G G A T C T G C
Ivl F:
R:
C C C T C C T G T G A G T T T G T T T G G
T G A G A G G T C C C T G A A C C A C A
Appendix Table 1: List of utilized primers containing the sequences and references.
3.16. Statistical analysis and graph preparation
Statistical analysis was performed in GraphPad Prism 8 software (Dotmatics, USA). The normality of the
data was assessed using Shapiro-Wilk (W), Anderson-Darling (A2*), Kolmogorov-Smirnov (distance),
D'Agostino-Pearson omnibus (K2) tests and QQ plots. Datasets that passed the normality tests and had
equal variances based on the results of F test were analyzed using either one or two-tailed t-test depending
on the null hypothesis. Datasets that did not pass the normality tests, were analyzed using either a one or
two-tailed Mann-Whitney test depending on the null hypothesis. For the analysis of DPSCs differentiation
into odontoblasts, paired t-test was used to conserve the continuity of the biological replicates.
Furthermore, outliers were identified using Grubbs' method at a = 0.1. To properly visualize the inner
variability within the datasets, SuperPlots were constructed as described elsewhere (Lord et al. 2020).
Otherwise, bar plots displaying all replicate values and their mean with standard deviation (whiskers) were
used. The critical p-value for statistical significance was set to p=0.05.
4. Appendix Results and Discussion
4.1. Initial selection of potential TFs
To find the potential odontoblast-specific TFs we have utilized interactive PAGODA visualization and
the statistical tools provided in the previously published, freely accessible online platform containing
scRNA-seq mouse incisor datasets (Krivanek et al. 2020). Firstly, the odontoblast lineage subpopulation
was determined in the whole mouse incisor dataset using the expression of seven, previously established
odontoblast marker genes (Appendix Figure 3A) (Krivanek et al. 2020). These seven genes were found
to be expressed in most of the odontoblast lineage. This enabled us to determine TFs specific to even the
earlier stages of odontoblast differentiation. Using the built-in, unbiased tools, one hundred genes
displaying the most similar expression pattern to the consensus expression pattern of the seven markers
were found (Appendix Figure 3B). These one hundred genes contained 12 transcription factors
(Appendix Figure 3C). From these 12 TFs, three genes displayed higher specificity to the established
odontoblast lineage than the rest. These were Etv4, Salll and Gsc, which were ultimately chosen for
further validation as they were deemed promising based on the concurrent knowledge of their function.
To further visualize the specificity of the individual stages of TF selection, we have generated t-SNE plots
of their consensus expression patterns (Appendix Figure 3D). The consensus expression of the marker
genes shows high specificity to the odontoblast lineage subpopulation. The one hundred genes displayed
an expression pattern somewhat more spread out in the dental pulp cluster. The twelve TFs then showed
very similar expression pattern to that of the 100 genes. The ultimately chosen TFs then showed an
expression pattern that was the most specific to the odontoblast lineage.
To further explore the potential roles of the transcription factors Gsc, Etv4, and Salll in odontoblast
differentiation, we applied pySCENIC—an independent Python implementation of the SCENIC pipeline
22
(Aibar et al. 2017) - which infers regulon activity based on gene co-expression and predicted cisregulatory
motif enrichment. This analysis revealed active Gsc, Etv4, and Salll regulons specifically
within odontoblast clusters, supporting their involvement in odontoblast lineage specification (Appendix
Figure 5). Furthermore, the heatmaps of all the members of the regulons were exported (Appendix Figure
6Appendix Figure 7Appendix Figure 8). In addition, gene ontology from the regulon enrichment of the
three selected genes was performed (Appendix Figure 9).
23
The strategy behind the initial selection of potential TFs
A Odontoblast markers used to determine the odontoblast lineage cluster
E x p r e s s i o n
. Of, • 1 H i g h
Krivanek et all. 2020
B Top 100 genes with expression pattern similar to the markers
(Pre)Odontoblasts
Smpd3
Frmd5
Fgf3
Mppedl
Thy1
Dkkl
Notum
I H.I.I J A W
m m i
i i n ' i t I» 'immtm
• I I I i
r I i M H I H H H i l M
I • » » IMMII II H I i n i M i l ii n I • I ii i u
a.
C Top transcription factors (Pre)Odontoblasts
IIIIII II I I i m • in i n i n iln in J J i I J .
II II llll I I I ' M i l l II I I I I 1 Hill III II um u m • I I i in i e r f *•
Sp6
Saltl*Gsc
-t»
n W l M i;,nil a TT In r i r M i l B i F F
III 11
e r f *•
Sp6
Saltl*Gsc
-tM
l lit n
1•' )
• i II1
i | l | i l
ML fiMpir». LiuCM
J * "
'HI II
DtaS
U M
Dlx4
Gi:j
Gill
Sal/3
Tb>;2
D t-SNE visualization of consensus patterns
U s e d odontoblasts markers (A) Top 100 similarly e x p r e s s e d g e n e s (B) Top transcription factors (C)
High a
<>• (preodontoblasts ,> (pie)Odontoblasts
EW4, SalH a n d Gsc (C)
<5jC> (preodontoblasts
Appendix Figure 3: The strategy behind the initial selection of odontoblast-specific TFs. Previously established
markers genes (Smpd3, Frmd5, Fg/3, Mppedl, Thyl, Dkkl, Notum) were used to determine the odontoblast lineage
subpopulation in the whole mouse incisor dataset (expression heatmap shown in A). Based on the consensus
24
expression pattern of these seven markers, 100 genes with the most similar expression patterns were found
(Expression heatmap shown in B). Transcription factors shown in orange and highlighted with an arrowhead. Of
these 100 genes 12 were transcription factors (expression heatmap shown in C). Ultimately chosen TFs, with the
highest specificity to the odontoblast lineage subpopulation shown in red, highlighted with an arrowhead. The
consensus expression patterns of each of the generated heatmaps is shown in (D).
4.2. Strategy behind the initial selection of mature odontoblast-specific TFs
Furthermore, apart from the initial strategy for determining the potential odontoblast-specific TFs using
marker genes specific for the whole odontoblast lineage, we have also used a strategy targeting the genes
expressed in mature odontoblasts. To do so we have firstly determined the mature odontoblasts within the
odontoblast lineage dataset using the dual expression of Dspp and Dmpl (Appendix Figure 4A). The
dual expression of Dspp and Dmpl was visualized within the odontoblast differentiation trajectory using
t-SNE embedding (Appendix Figure 4B). To further illustrate the presence of the Dspp and Dmplpositive
odontoblasts within the continuously growing mouse incisor and molar, histological sections from
DSPP-Cerulean/DMP 1-Cherry reporter mouse strain were analysed (Appendix Figure 4C,D). Using the
consensus expression pattern of these two mature odontoblast markers we have found fifty genes with the
most similar expression patterns using the same unbiased tools (Appendix Figure 4E). Of these 50 genes,
three were transcription factors (Appendix Figure 4F). Due to its high specificity to the mature
odontoblasts and information about its function in current literature, Nuprl was ultimately selected for
further validation.
25
The strategy behind the initial selection of mature odontoblast-specific TFs
Markers used to determine mature odontoblasts Mature odontoblasts
Expression
Low ^HT High
I Urv.y
I
B Differentiation trajectory C Mouse incisor (Labial side) D
Expression
Low High
Mouse adult molar
Mature
odontoblasts V
N e a r - C L a r e a
a _ » » » ' .
'fiT'-Vr~",i
'i
orT"
bxpression
[ I
Low High
o o
b" "Od
° o
o°
>Oo o
0SPPQ,
""""/DMP1 DSPPC
""""/DMP1
A m •
E Top 50 genes with expression pattern similar to the markers
^ f i - . l f . X
. • i ' .
JDmpI
|Creb3D«-
lAbndlS
I Sema3c
I RassdO
I Mapt
usrI Kcnn4
I Klt9
I Coro2b
I Pde5a
|ziP 764
1 S'..Mp3
I Ccn'C
|Rnd2
I Occii
Isic17a9
I
I Mapla
I Nupfl *j
r,',1Hd;:2
1 llgal 1
Set
Htra3
Pö2g5
Mho
Chst13
Dysf
Crtad
Coro 1a
Sytl2
Pstpipl
v„::i<i
Fxyd5
M prints
Wispl
Lgats3
Fam189a2
Col11a2
Wfdd
Tgml
Tnfaip2
5730508B09
Nfatcl
F Top transcription factors
Mature odontoblasts
Appendix Figure 4: The strategy behind the initial selection of mature odontoblast-specific genes. Mature
odontoblasts were determined using the dual expression ofDspp and Dmpl (A). This was further visualized using
26
t-SNE embedding (B) and validated using histological sections of incisor (C) and molar (D) from DSPPCerulean/DMP1-Cherry
reporter mouse strain. Using the consensus expression pattern, fifty genes with the most
similar expression pattern were found (E). Transcriptional regulators and TFs highlighted in red color and an
arrowhead (F). The ultimately chosen gene, Nuprl, is highlighted in red and with an arrowhead. The detected
expression of the reporters in the tissue sections is highlighted with white arrowheads. AB - alveolar bone, Am Ameloblasts,
Od - Odontoblasts, PDL - periodontal ligament, LaCL - labial cervical loop, LiCL - lingual cervical
loop. Scalebars=100 um.
27
Appendix Figure 5: The heatmap oftop active
regulons in the whole incisor dataset. Heatmap
showing the activity of SCENIC-inferred
regulons within the mouse incisor dataset. Color
intensity reflects regulon activity within the
given cell of the specific subcluster. The Gsc,
Etv4 and Salll regulons highlighted with red
arrowheads.
Appendix Figure 6: Heatmap of the Gsc regulon expression within the odontoblast incisor dataset. Utilized
markers and the selected genes expressed as a part of this regulon are highlighted in red {Etv4, Gsc, Dkkl, Frmd5,
Col24al, Fg/3, Scd.ll).
29
Appendix Figure 7: Heatmap of the Etv4 regulon expression within the odontoblast incisor dataset. Utilized
markers and the selected genes expressed as a part ofthis regulon are highlighted in red (Etv4, Notum, Gsc, Mppedl,
Dkkl, Fgft, Scdll, Smpd3)
30
31
G O analysis of the Gsc regulon m e m b e r s
morphogenesis
epithelial tube morphogenesis1
tore bra in development'
morphogenesis1
appendage development1
limb development1
morphogenesis of a branching,
developmental induction1
0 faciory bulb cove cprrert
olfactory lobe development'
cell fate specification
organ induction
O >
O <
Op
ad, li
I
B G O analysis of the Etv4 regulon m e m b e r s
Jtube morphogenesis
Wnt signaling pathway
ncll-cc- sigra inn l>v wnt
regulation of neurogenesis
DNA replication
DNA-templated DNA replication
m esenchyme development
DNA recombination
epithelial to mesench]
nuclear DNA replic;
tell cycle DNAreplic,
developmental
mitotic DNA n
O
O
O
o
o
O u
O "
O iDD
O <»
O «•
p.adjust
6- M
4:: ;)-
9 2c-04
G O analysis of the SalH regulon members D Embedding of regulon expression
slsnn c<?l difeierliyLiyri
sensory organ morphogenesis
muscle organ development
bi-omineral tissue development
calcium ion transmembrane
import into cylosol
ear development
cell fate commitment
t- " -jiycric oiLiar rnor jl'ogi=n=s.i!
ear morphogenesis
negative regulation o1
(..LliU-et •'.IK:!- Ll,Itrr
Bevel op menl
negative regulation of
muscle organ development
negative regulation of
muscle tissue development
asymmetric: eel d vis on
organ induction
regulation of anil
:>;i:lern iy
s elongation
•
O
O
O
o
O 2
O >
O-
op.dOJU
Gsc (A) m
A 3.1C
JJ.05
I
SalH (C) m
If
Appendix Figure 9: Dotplot of enriched Gene Ontology (GO) terms identified by Over-Representation
Analysis (ORA) of genes in the Gsc (A), Etv4 (B) and Salll (C) in (pre)odontoblast cluster. The y-axis lists the
32
top 23 enriched GO terms (Biological Process category), while the x-axis shows the GeneRatio, defined as the
proportion of xy regulon genes annotated to each term. Dot size represents the number of genes associated with
each term, and dot color indicates the statistical significance of enrichment (adjusted p-value). UMAP visualization
of Gsc, Etv4 and Salll regulon activity within the mouse incisor dataset. Each cell is colored according to the
activity of the corresponding regulon, as quantified by AUCell scores.
Our data show that the regulons of Gsc, Etv4 and Salll, belong among the most upregulated in the
(pre)odontoblast cluster of the mouse incisor dataset (Appendix Figure 5).
The results of the regulon analysis suggest that among the Selected TFs possible temporal or functional
hierarchy is present, due to the fact that both Gsc and Etv4 regulons contain Salll (Appendix Figure 6,
Appendix Figure 7). Furthermore, the importance of the selected TFs, for driving odontoblastdifferentiation
is likely suggested by the fact that several used odontoblast markers are contained in their
regulons. Intriguingly, none of the regulons contained the mature odontoblast markers Dspp and Dmpl
and neither Runx2 (Appendix Figure 6, Appendix Figure 7, Appendix Figure 8).
Moreover, the analysis of enriched GO terms of genes in the three regulons revealed their predicted
functions. Top enriched GO terms of the Gsc regulon were generally focused on epithelial tube and
appendage development (Appendix Figure 9A). Top enriched GO terms of the Etv4 regulon were
generally situated around epithelial tube morphogenesis, Wnt pathway regulation and mesenchyme
development (Appendix Figure 9B). Finally, the top enriched GO terms of the Salll regulon were
generally themed around stem cell differentiation, embryonic organ morphogenesis, mineralization and
odontogenesis (Appendix Figure 9C).
4.3. Current knowledge concerning the function of the selected genes
The most suitable TFs have been selected and confirmed by a combination of approaches. These ranged
from the analysis of differentially expressed genes (Appendix Table 2), qualitative analysis of the
expression patterns within the trajectory, to utilizing previously obtained knowledge on the function of
the potential candidate genes. Due to the limitations associated with the challenging isolation of
odontoblasts resulting in potentially biased statistical analysis due to small sample size, we have placed
greater emphasis on the qualitative data.
The first TF, ETV4 (ETS Variant Transcription Factor 4), is a downstream effector of FGF signaling
(Porntaveetus et al. 2011) and has been previously associated with Fgf8 signaling in zebrafish (Roehl and
33
Nusslein-Volhard 2001). This indicates its significant role in tooth development and odontoblast
differentiation, as suggested by the research conducted by Prochazka et al. (2015). Although its presence
in tooth development has been previously mapped (Porntaveetus et al. 2011) and discussed (Kim et al.
2017), its specific role in odontogenesis or odontoblast differentiation remains unclear.
When it comes to the effects of Etv4 knockdown or downregulation on odontoblast differentiation, no
previously attained experimental data are available. However, based on its function we can predict that its
downregulation during tooth development would likely disrupt the inductive effects of FGF signals from
the adjacent epithelium which has been shown to be essential for tooth morphogenesis (Thesleff 2003;
Porntaveetus et al. 2011). However, ETV4 likely acts synergistically with ETV5 (Zhang et al. 2009;
Herriges and Sun 2011; Herriges et al. 2015) and their expression pattern during molar development is
very similar (Porntaveetus et al. 2011). Therefore, the downregulation of ETV4 might not be sufficient to
induce a phenotype as its absence might be supplemented by ETV5.
The second TF, GSC (Goosecoid), is a homeobox protein previously identified as a regulator of the neural
crest specification. GSC is (apart from other regions) expressed in the first branchial arch, albeit only in
the regions not giving rise to teeth (Tucker et al. 1999). This was confirmed by studies performed on Gsc
knockout animals which displayed malformations of the Meckel's cartilage and mandible hypoplasia, but
not a phenotype in teeth (Rivera-Perez et al. 1995; Yamada et al. 1995; Rivera-Perez et al. 1999). Taken
together, Gsc has not previously been associated either with tooth development or odontoblast
differentiation.
The third TF, SALL1 (Spalt Like Transcription Factor 1), was recently shown to act as a positive regulator
of pre-odontoblast differentiation via the upregulation of Tgfb2 and Runx2 expression due to its capacity
to increase the chromatin accessibility of these genes (Lin et al. 2021). Moreover, a knockdown of SailI
in differentiating dental pulp stem cells resulted in a downregulation of the TGFB pathway and a reduction
of matrix calcification.
The last transcription coactivator, NUPR1 (Nuclear protein 1), has been categorized as a stress-related
protein implicated in tumor progression, cellular senescence, autophagy and apoptosis (Huang et al. 2021).
More fundamentally, Nuprl is upregulated by the TGFB pathway (Pommier et al. 2012; Murayama et al.
2023) and potentially might downregulate BMP signaling by attenuating SMAD1 activity. Furthermore,
Nuprl knockout also resulted in a reduction of age-related bone loss in mice (Murayama et al. 2023),
34
which might suggest its role in hard tissue homeostasis. Nevertheless, it has never been associated with
either tooth development or odontoblast differentiation.
4.4. The expression of the selected TFs in the developing mouse molar
To elucidate whether the selected four TFs also participate in the odontoblast differentiation in noncontinuously
growing teeth, in vivo validation in mouse molar was performed. To investigate the timing
of the expression of the TFs, the E12.5, E14.5, E16.5, E18.5 andP14 time points were chosen to cover all
main morphogenetic events of tooth development. The Etv4 was strongly expressed throughout the molar
development in the ectomesenchyme, dental papilla and preodontoblasts (Appendix Figure 10A).
Additionally, Etv4 was also robustly present in the dental epithelium facing the dental mesenchyme from
E14.5. While the expression pattern of both Salll (Appendix Figure IOC) mdNuprl (Appendix Figure
10D) closely mirrored those in the incisor, the expression pattern of Gsc was distinct (Appendix Figure
10B). Its expression was completely absent from the tooth-forming ectomesenchyme in the earlier stages
(E12.5 to E16.5). During the late bell stage of tooth development (E18.5), weak Gsc expression was
located in the preodontoblasts of the future pulp horn regions. During the postnatal tooth growth (P14),
the expression of Etv4 and Gsc (Appendix Figure 10A,B) was localized in the non-differentiated preodontoblastic
mesenchymal region adjacent to the Hertwig's epithelial root sheath (HERS), which is
responsible for mediating root growth. Conversely, the expression of Salll (Appendix Figure 2C) and
Nuprl (Appendix Figure 10D) was strongly expressed throughout the entire odontoblastic layer,
extending to apical regions of the growing root. Interestingly, the exact sequence of the expression of all
four TFs observed in the incisor was found also in the cervical loop region of the molar's root.
35
Expression of the selected transcription factors during mouse molar development
A Etv4 B Gsc C SalU D Nuprl
LD
LU
Etv4 Gsc SalU Nuprl
--QE-'
EM EM 4 EM -V
OE , '
EM
LO
LU
Etv4
V EO \
•' ''- 'EK
4 'V
Gsc Sa//f
EO
EK;.'
EO V
^EK_,
< v
/ V
Nuprl
EO
EK-
LO
CD
LU
Etv4 Gsc SalU : - r « « » : .-. • • Nuprl
DP DP DP DP
LO
co
Am;---J
; Odi I
Gsc
Am,
Or>
CL
Sa//7<-
Am.--
'od4
Nuprl
Arn - ;od•*r
n .
SalU Nuprl
• D
•
P
13 '
AB
D
AB
•
L7 '
AB
9>. v - . .
. : ' 0 D
, P
Od v
1
:'pd. p
1
:'pd. p
Od v
P
w
;6dv
,'6d*
P Od • - p Od v
eb--; v v
P v V
P
Appendix Figure 10: Expression of Etv4 (A), Gsc (B), Salll (C) and Nuprl (D) transcription factors
throughout mouse molar development. Expression of Etv4 (A) is strong throughout the embryonic molar
36
development, while being mostly limited to the non-differentiated pre-odontoblastic mesenchyme of the apical
region of the root at PI4. Gsc (B) is expressed in the mesenchyme of the mandibular process that is separated from
the odontogenic epithelium at the E12.5. At E18.5, Gsc is starting to be expressed in the forming preodontoblast
layer of future pulp horn regions. Postnatally (P14), Gsc expression is mostly limited to the pulp adjacent to HERS.
Salll (C) is expressed during the whole molar development and is present exclusively in the ectomesenchyme,
dental papilla and preodontoblasts. At P14, Salll is strongly expressed in the odontoblast layer and less in the
cervical loop. The expression of Nuprl (D) is absent from the embryonic molar development and is present only at
PI4, showing strong expression in the odontoblast layer. The detected expression of respective genes is highlighted
with arrowheads. AB - Alveolar Bone, Am - Ameloblasts, CL - Cervical Loop, EK - Enamel Knot, EM Ectomesenchyme,
EO - Enamel Organ, OE - Odontogenic Epithelium, Od - Odontoblasts, P - Pulp, PDL Periodontal
Ligament. Scale bars=200 urn.
Based on all the above-mentioned features, four TFs were chosen. Their expression throughout the
odontoblast differentiation was validated in both adult mouse incisor and during molar development. The
expression patterns predicted in the differentiation trajectory closely resembled those found in the labial
side of the incisor. Furthermore, the analysis of the C57BL/6 incisor revealed a region in which the
differentiating odontoblasts expressed all four TFs - mirroring the predicted developmental trajectory.
The in situ analysis determined strong expression of Etv4 throughout the whole molar development, first
in the ectomesenchyme, and later in the dental epithelium. These findings align with those presented by
Porntaveetus et al. (2011), albeit in more detail. The diminished expression in the P14 molar and its
restriction to the most proximal region in the incisor suggest that Etv4 is crucial for odontoblast
differentiation, but likely not for their secretory function. The expression of Gsc in the molar primordium
was not present until El8.5 when a weak expression was found in the pre-odontoblasts located in the
future pulpal horns. Interestingly, Tucker et al. (1999) present strong evidence that Gsc is negatively
regulated by FGF8 in the mandibular process. This, combined with the knowledge that FGF8 is most
likely a positive regulator of Etv4 expression (Roehl and Nusslein-Volhard 2001; Zhang et al. 2009),
suggests that Etv4 and Gsc are mutually exclusive during tooth development and odontoblast
differentiation. While all evidence suggests this is the case in molar development, our data from mouse
incisor indicate otherwise, as both genes are expressed simultaneously for a significant portion of the
odontoblast differentiation trajectory in the incisor. Furthermore, its vicinity to the epithelial cervical loops
in both the incisor and molar suggests that similarly to Etv4, Gsc plays a more important role in the
differentiation of odontoblast than in their function.
While the expression of the other genes has not previously been investigated in the incisor, the expression
of Salll closely aligns with findings from recently published studies (Lin et al. 2021; Lavický et al. 2022)
37
as it was expressed in the odontoblastic layer towards the tip of the incisor on the labial side. The
expression of Salll displayed a very similar pattern in both the incisor and molar. This suggests its
significance both for odontoblast differentiation and their function, as it is strongly expressed in mature
odontoblasts throughout the entire tooth, except in the root analogue (Lavický et al. 2022). The last of the
TFs, Nuprl, displayed striking specificity to cells expressing odontoblast markers Dspp and Dmpl in the
scRNA-seq data. Remarkably, it was completely absent during the early molar development. However,
strong and very specific expression of Nuprl was characteristic of mature odontoblasts in the adult incisor
and PI4 molar. This suggests an important role in dentin production.
Finally, the fact that the expression pattern of the four TFs in the progenitor mesenchyme in the pulp of
the developing molar root closely mirrored the pattern in the labial cervical loop might be used to draw
parallels between these two structures. Even though similarities between the root's cervical loop and
incisor's root analogue were more expected. We hypothesize that the observed similarities between the
two structures may indicate accelerated odontoblast differentiation - as both the HERS at P14 and labial
cervical loop are actively elongating the molar's root and driving the regrowth of the incisor (Gonzalez
Lopez et al. 2023).
4.5. Design of lentiviral vectors and the generation of miPSC line with inducible
expression of the TFs
Three lentiviral vectors were designed. Our system was composed of one driver vector (Tet3g/EGFP)
responsible for inducible expression of the selected genes and two vector carrying the selected genes in
pairs. The reasoning for this decision are as follows: Due to the complex developmental origin of
odontoblasts, we deemed it highly unlikely that overexpression of single gene (even TF) would result in
steering pluripotent stem cells into this specific cell type. In addition, the pairing of the genes reduced the
potential number of required rounds of lentiviral transduction from 5 (one gene per vector + one driver
vector) to 3 (2 genes per vector + one driver vector). This also ensured methodological feasibility, as each
of the vectors needs a selection gene (either an antibiotic resistance gene or a fluorescent protein (please
note that the used reporter cell line is already capable of producing Cerulean and Cherry fluorescent
reporters). Furthermore, since no antibiotic resistance-mediated selection of transduced cells is capable of
selecting 100% pure population (as illustrated by Nakatake et al. (2013)), by using 3 only vectors we have
therefore severely reduced the heterogeneity of the final, transduced cell populations.
38
The combinations of genes were chosen for two reasons. Expression of both Etv4 and Gsc was in the in
situ validations found to be expressed in the proximal area of the incisor in the vicinity of the cervical
loop. The expression of Salll and Nuprl was located more distally with more overlap. Therefore, the two
pairs of genes were made to reflect this. The second, more technical reason, was the fact that the used
lentiviral vectors have only a limited cargo capacity (around 6.4 kb). Based on the vector manufacturer's
experience, the insertion of larger constructs results in significantly reduced viral titer. Due to the large
size of the Salll ORF (3969 bp) it's only gene pairing, that would produce a viable lentiviral vector, was
with Nuprl ORF (243 bp). Please note that apart from the ORFs, the construct also needs to contain
regulatory sequences as well as antibiotic resistance that make up the rest of the vector cargo space.
These vectors were used to generate stable transductant cell lines from mouse induced pluripotent stem
cell line derived from DSPP-Cerulean/DMP 1-Cherry reporter strain (Vijaykumar et al, 2019) (Appendix
Figure 11A) (for details see the chapter Materials and methods). Four lines were generated: "T" (carrying
the "driver" Tet3GIEGFP vector), "TS" (carrying the Tet3G/EGFP and SallllNuprl vectors), "TG"
(carrying the Tet3GIEGFP and GsclEtv4 vectors) and finally, "TGS" which contained all three vectors.
To validate the successful transduction and effectiveness of designed vectors and engineered cell lines,
the total expression of the transgenes and individual genes was quantified. Generated cell lines were
treated with doxycycline for four days and the upregulation of the TF-carrying constructs was analyzed
using RT-qPCR (Appendix Figure 11B). The expression of the transgenes was only present in the
corresponding cell lines and the rate of transgene expression was significantly higher in doxycyclinetreated
cells compared to the untreated cells (Appendix Figure 11B). Moreover, the overexpression of
the GsclEtv4 transgene was higher than that of the SallllNuprl transgene in the doxycycline-treated
conditions (Appendix Figure 11B).
Following this, the total (transgene + endogenous) expression of the individual genes was evaluated
(Appendix Figure 11C). The doxycycline treatment resulted in a significant upregulation of Gsc in the
cell lines containing the GsclEtv4 vector (Appendix Figure 11C). The same pattern was observed in the
expression of Etv4, albeit with a higher level of expression (Appendix Figure 11C). The expression of
both Salll and Nuprl was less pronounced than that of the other two genes, yet still significant compared
to the cell line containing only the Tet3G/EGFP vector. Interestingly, the chosen genes were significantly
upregulated in the corresponding cell lines even in conditions not treated with doxycycline (Appendix
39
Figure 11C). This suggested that the system displays an overexpression of the transgenes that is
independent on the added doxycycline.
Generation of m P S C cell lines with Tet-ON driven overexpression of selected odontoblast-specific TFs
Gsc ,
E t v 4 .
m P S C s
Tet3G S r X
G F P + Salll
Tet3G+ N u
P r 1
G F P +
Tet3G+
Gsc/Etv4+ Doxycycline
Sall1/Nupii+ (Dox)
G F P +
Tet3G+
Gsc/Etv4+
Sall1/Nupr1 +
Expected unaltered expression of the selected odontoblast-specific transcription factors
TGsc tSalM
t E t v 4 a n d
' ° ' t N u p r 1
B Transgene expression
Gsc/Etv4 Vector
Ctrl
Dox
r i d . n d.
a 15T
TS TG TGS
Sall1/Nupr1 Vector
Ctrl
Dox
n n
n d . | M | n d . |
Overall expression of the selected transcription factors
Gsc Etv4
20|
15-
IN
I 10-
Ctrl
Dox
nn n n
TS TG
Sal/1
TGS
Ctrl
Dox
nn nil rTS TG TGS TS TG TGS
Appendix Figure 11: The generation and validation of pluripotent stem cell lines with inducible
overexpression of four selected transcription factors. (A) The cell lines were generated by sequential
transduction cells,firstwith the Tet3G/GFP vector, followed by either one or both TF-carrying vectors. (B) The RTqPCR
detection of the expression of the TF-containing transgenes was performed (n=3). In all combinations of the
vectors only the expected transgenes were expressed. Conditions stimulated with doxycycline displayed significant
upregulation of the given transgenes. (C) The detection of the expression of both endogenous and transgenic Gsc,
Etv4, Salll and Nuprl, indicating the real change in their transcription profile after the induction with doxycycline
40
(n=3). mPSCs - mouse pluripotent stem cells. T - Cell line containing Tet3G/GFP vector, TS - Cell line containing
Tet3G/GFP and SallllNuprl vectors, TG - Cell line containing Tet3G/GFP and GsclEtv4 vectors, TGS - Cell line
containing Tet3G/GFP, SallllNuprl and GsclEtv4 vectors. Data are represented as mean ± SD. N.d. - Not Detected,
n.s-not significant. * -p<0.05; ** -p<0.01; *** -p<0.001; **** - p<0.0001.
To determine the importance of picked TFs in odontoblast differentiation, we have generated four
transgenic pluripotent stem cell lines capable of their inducible overexpression. To validate the generated
cell lines, we have analyzed their overexpression capabilities. The observed differences in the relative
overexpression of Gsc/Etv4 and SallllNuprl constructs likely stemmed from the relative potency of the
utilized selection antibiotics Puromycin (Gsc/Etv4) andBlasticidin S {SallllNuprl) (Nakatake et al. 2013).
Along with that, the total expression of both Gsc and Etv4 was significantly higher than the expression of
Salll and Nuprl genes. While this can be partially explained by the difference in the expression level of
the vectors, the original expression profile and epigenetic regulation of the host cells might have also been
an important factor. Apart from being involved in tooth development, as we demonstrated previously
(Lavický et al. 2022), Salll is involved in the regulation of pluripotency in mouse embryonic stem cells,
leading to a potential relative upregulation in source cells (Karantzali et al. 2011). Similarly, Nuprl is
involved, among other processes, in the regulation of antioxidative stress response and apoptosis (Huang
et al. 2021), which might also suggest a higher degree of control of its expression. Conversely, both Etv4
and Gsc are TFs involved in the regulation of cell differentiation (Tucker et al. 1999; Lu et al. 2009; Sudou
et al. 2012; Akagi et al. 2015) and their expression in pluripotent stem cells is likely to be lower. Taken
together, these are likely the reasons standing behind the imbalance between the relative total expression
of genes carried by the GsclEtv4 and SallllNuprl constructs.
In addition to differences in the rate of overexpression among the chosen genes, we also uncovered the
fact that the cell lines exhibited overexpression of the contained genes without added doxycycline. This
occurred despite our efforts to minimize this background activity, for example by utilizing fetal bovine
serum (FBS) with reduced amounts of tetracycline (>10ng/ml) and keeping the cell cultures with and
without doxycycline separate. This background activity (leakiness) of the tet-inducible platform is a
phenomenon that was observed previously (Loew et al. 2010; Zhou et al. 2020) and most likely stems
from the fact that, the used Tet3G transactivator was generated to have the highest doxycycline-sensitivity
and transcriptional activity as possible (Zhou et al. 2006). This resulted in the transactivator being capable
of driving the upregulation of target gene expression in the presence of as little as 5 ng of doxycycline per
milliliter of culture media (Zhou et al. 2006). This concentration is on a similar level to the one most likely
41
present in the used cell culture media, since the detection limit for certifying the "Tetracycline-free nature"
of the used Tet-free FBS is >10 ng/ml. Furthermore, this background activity might seem more
pronounced when the target genes display very low or no expression in the given cell line. This way even
a small absolute increase in gene expression due to the background activity, results in a high relative
upregulation. For these reasons we have decided to use the cell line with only the Tet3GIEGFP vector (T)
as a negative control in the following differentiation experiments.
In Appendix Figure 12 we show the results of the analyses and quantifications shown in the main figures
with the conditions without added doxycycline (Ctrl) included.
42
• S P 4
area at D21 Dspp expression
t 0-4-
SL
a
0.2'
C S S _ * * 1
**
**
t
n.s.
i f
* *
- T
£ •
n.s.
i f
T TS TG TGS Day 0 21 21 0 21 21 0 21 21 0 21 21
T TS TG TGS
B DMPr area at D21 Dmp1 expression
E5H
IHSH .—
n.s.
*+
n.s.
** T
TS TG TGS 0 21 21 0 21 21 0 21 21 0 21 21
T TS TG TGS
Von Kossa* area at D21 Runx2 expression
EES
ram ****
**
* *
n.s.
0- n.s.
**
T TS TG TGS
w/o
Dox
n.s. n.s.
n.d.
ft
n dn d. rx
"| n d
Day 0 21 21 0 21 21 0 21 21 0 21 21
T TS TG TGS
D CDH1+
area at D21
>
2 10
o
n.s. n.s.
KrMO expression
***
o
g 1
2
O
n.s. n.s. ^j- i -J- 1
W / O
Dox
n.d. n.d. n.d
T TS TG T G S
Day 0 21 21 0 21 21 0 21 21 0 21 21
T TS TG TGS
o
a 1W
/ O
D o x
M expression
*
n.d. t n.d. T
i i T i i i T
Day 0 21 21 0 21 21 0 21 21 0 21 21
T TS TG TGS
Appendix Figure 12: The transduced cell lines display upregulated expression of odontoblast and
keratinocyte markers due to the background activity of the TET3G transactivator. Expression of DSP and
Dspp (A). Expression of DMP1 and Dmpl (B). Mineralization and Runx2 expression (C). Expression of CDH1,
KrtlO and Ivl (D). Red data points in the RT-qPCR graphs show data points in which no specific PCR product was
detected. W/O - without added doxycycline; T - Cell line containing Tet3GIEGFP vector, TS - Cell line containing
Tet3G/EGFP and SallllNuprl vectors, TG - Cell line containing Tet3GIEGFP and GsclEtv4 vectors, TGS - Cell
line containing Tet3GIEGFP, SallllNuprl and GsclEtv4 vectors. Data represented as mean ± SD. N.d. - not
detected, n.s - not significant, * -p<0.05, ** -p<0.01, *** -p<0.001, **** -p<0.0001.
43
4.6. Analysis of growth, proliferation and cell death during spheroid cell culture
The growth parameters of cell spheroids during the differentiation experiments were analysed. These
comprised of the size of spheroids, presence of apoptotic marker cCASP3 (cleaved Caspase 3) and the
expression of proliferation marker mKI-67. Representative images of the control T-Dox and the
experimental condition TGS-Dox are present in (Appendix Figure 13A). First, the size of the spheroids
was quantified at days 5, 10,15 and 20 after seeding. In general, the size of the spheroids increased during
the culture period. The cell lines T and TS were on average larger than the remaining cell lines (Appendix
Figure 13B). The relative cCASP3-positive area was calculated (Appendix Figure 13C). In the T and TS
cell lines the relative cCASP3-positive area increased during the culture period. The remaining cell lines
displayed a mostly stable relative amount of cCASP3-positive area. Furthermore, absolute cCASP3negative
area was also quantified (Appendix Figure 13D). This metric was included to show whether the
spheroids maintain "positive" growth (whether the amount of viable cells increases over time). The area
covered by viable cells increased in all cells lines during the culture period or stayed, on average, the same
between the last two timepoints (TGS-Dox). Furthermore, the density of mKI-67-positive cells was
calculated as the number of mKI-67+ cells normalized to absolute cCASP3-negative area (Appendix
Figure 13E). This was done to correctly normalize the amount of proliferating cells to the area of the
spheroid covered by viable cells. In both T and TS-Dox 0 conditions the density of proliferating cells
increased over the culture period. The remaining conditions displayed, mostly stable density of
proliferating cells. With the exception of a slight decrease in TS-Dox and a slight increase in TGS-Dox
conditions.
44
A Tet3G Dox 1 Tet3G+Sall1/Nupr1+Gsc/Etv4 Dox 1
D 2 0 | D 1 0
•
i
D15 niKi-67 D20
B Tet3G Tet3G+Sall1/Nupr1 Tet3G+Gsc/Etv4
Dox 0 D o x 1
Spheroid size Spheroid size
DOX 0 DOX 1
Spheroid size Spheroid size
DOX 0 DOX 1
Spheroid size Spheroid size
Tet3G+Sall1/Nupr1+Gsc/E1v4
D o x 0 D o x 1
Spheroid size Spheroid size
i
• 4-'
•S DIG D15 D2D
• * ^
D5 did D15 D20 D5 D10 D15 D2Q D5 D10 D1S D2C • 5 D1D D15 D2Q
*4*
DS DID D1S D20 D5 D10 D15 D20 D5 D10 D15 D2C
Relative cCasp3* Area Relative cCasp3+ Area Relative cCasp3+ Area Relative cCasp3+ Area Relative cCasp3+ Area Relative cCasp3+ Area Relative cCasp3+ Area Relative cCasp3+ Area
• 5 D10 D15 D2C D5 DIU D15 D20 D5 DID D15 D20 • 5 DIU D15 D2C D5 01Q D15 D20
4
D& Did D15 D20 • 5 D10 D15 Q2C • 5 DID DIE D20
D Absolute cCasp3- Area Absolute cCasp3- Area Absolute cCasp3- Area Absolute cCasp3- Area Absolute cCasp3- Area Absolute cCasp3- Area Absolute cCasp3- Area Absolute cCasp3- Area
+
DS DIU D1S D2C D5 D10 D15 D20 D5 D10 D1S D20 • 5 DID D1S D2C
•J
• 5 D10 D15 D20 • S D10 D15 D20 • 5 D10 D1S PJ2C • 5 D10 D1S D2C
E Density of mKi-67+ cells Density of mKi-67+ cells Density of mKi-67+ cells Density of mKi-67+ cells Density of mKi-67+ cells Density of mKi-67+ cells Density of mKi-67+ cells Density of mKi-67+ cells
t t t
• 5 D1Ö D15 DZD • 5 OT0 D15 D2Ü D5 Din D15 D20
u.a. ^
• 5 D10 D15 D2C • 5 DID D15 020 D6 Did D15 D20 • 5 D10 D15 D2C • 5 D10 D15 D2C
Appendix Figure 13: Analysis of growth, proliferation and cell death during the cell spheroid cell culture.
Representative brightfield and IHC images of T and TGS Dox conditions during the differentiation (A).
Quantification of spheroid size during differentiation (B). Quantification of the relative cCASPS-positive area (C).
Quantification of the absolute cCASPS-negative area (D). Quantification of the density of mKI-67-positive cells,
45
calculated as a number ofmKI-67-positive cells normalized to the absolute cCASPS-negative area (E). Size analysis
n = 11 - 48. IHC analysis n = 3 - 11. All scalebars = 250 urn. N.a. - not analysed.
The generally lower proliferation and growth of the TS-Dox, TG and TGS conditions might be caused by
the transgene overexpression-induced, accelerated cell differentiation. The generally, increased ratio of
cCASP3+ area in the T and TS cell lines might stem from phosphate toxicity (He et al. 2021) originating
from the added beta-glycerophosphate (40 mM concentration) in combination with differentiation to
sensitive cell lineages, that were different from those emerging in TG and TGS cell lines. In addition to
this, the increased cell death might be caused by increased acidosis, poorer nutrient exchange or hypoxia
due to larger size of the spheroids in T and TS cells lines.
Furthermore, the statistical analysis of the of relative cCASP3+ area (Appendix Figure 14B) shows that
the conditions overexpressing the selected TFs do not display significantly increased cCASP3+ area when
compared to the control counterparts (T-Dox). This suggests that overexpression itself is not toxic to the
cells.
4.7. The analysis of mineralization in the in vitro cell spheroids
The von Kossa staining further revealed that the overexpression of Gsc and Etv4 leads to an upregulated
formation of mineralized particles inside the spheroids. The upregulated mineralization was associated
with an upregulated expression of Runx2, which suggests an ongoing mineralization-associated
transcription program (Li et al. 2011; Liu and Lee 2013). Nevertheless, it could be also argued that the
mineralized particles formed passively. This passive formation might have been caused by the inherent
function of the detected odontoblast markers DSP and DMP1. In vivo, these glycoproteins likely serve as
mineralization centers for the creation of hydroxyapatite crystals (Deshpande et al. 2011; Suzuki et al.
2012). Therefore, the von Kossa-positive particles may have formed passively within the spheroids,
without a specific cellular process being involved. Conversely, the von Kossa+ area might have emerged
as a results of cell death. Interestingly, the distribution pattern of cCASP3+ area and that of von Kossa+
area is very similar (Appendix Figure 14A). However, the cCASP3+ area in the TGS-Dox, TG-Dox and
TS-Dox conditions was significantly lower than in the T-Dox. Therefore, the mineralization displays an
inverted trends to that of the relative cCASP3+ area (Appendix Figure 14B). Furthermore, the
morphology of the observed von Kossa+ particles in TG-Dox and TGS-Dox conditions is different to that
in T-Dox (Appendix Figure 14A). This, along with the fact that the upregulation of Runx2 mirrors the
46
increase in von Kossa-positive area, suggests that observed increase in mineralization in comparison to TDox
control occurred as a result of specific cellular process.
Furthermore, the presence of mineralized structures in the kidney implants suggests that the TGS cells are
capable of forming mineralized tissue even in an environment that does not contain specific signals
promoting mineralization (e.g., P-glycerophosphate).
47
Appendix Figure 14: Investigation of the identity of observed mineralization and its dependence on the
cCASP3+ relative area. Comparison of the expression of DMP1 and cCASPS as well as von Kossa positive area
among the in vitro cultured cell spheroids from all cell lines (A). In the overview images arrowheads were used to
highlight same areas displaying both DMP1 and mineralization. In the detail images, arrowheads highlight von
Kossa+ particles in the various cell spheroids. The quantification of DMP 1+ relative area, cCASP3+ relative area,
von Kossa+ relative area and Runx2 expression (B). T - Cell line containing Tet3GIEGFP vector, TS - Cell line
containing Tet3GIEGFP and SallllNuprl vectors, TG - Cell line containing Tet3GIEGFP and GsclEtv4 vectors,
48
TGS - Cell line containing Tet3G/EGFP, SallllNuprl and Gsc/Etv4 vectors. Scale bars=100 urn. Data represented
as mean ± SD. N.d. - not detected, n.s-not significant, * -p<0.05, ** -p<0.01, *** -p<0.001, **** -p<0.0001.
4.8. Co-staining of DSP and DMP1
The co-staining of DSP and DMP1 was performed in both cell spheroids and kidney implants (Appendix
Figure 15). The expression patterns of the marker protein are distinct with some overlap in highlighted
cells and areas.
49
TGS Dox
Appendix Figure 15: Co-staining of DSP and DMP1 in both cell spheroids (A) and kidney implants (B). The
expression patterns of the two marker proteins are distinct with some overlap in areas and cells highlighted with
arrowheads. The borders of epithelial structures are marked by dashed lines. All scalebars = 100 um.
50
4.9. Co-staining of mature odontoblast markers and CDH1
To validate that the epithelial markers are expressed in the same structures as the mature odontoblast
markers, co-staining was performed. The expression of CDH1, DMP1 and DSP was located in the same
structures in the kidney implants (Appendix Figure 16A). Due to different optimal antigen retrieval
conditions for the DSP and CDH1 antibodies, the CDH1 staining is of a lesser quality in their co-staining
than in the DMP1+CDH1 co-staining. However, the same expression pattern of CDH1 is still observable.
In the cell spheroids, the DMP1 expression was also located inside CDH1-positive areas (Appendix
Figure 16B). In the cell spheroids the co-staining of DSP and CDH1 was not performed. However, the
same area shown in the DMP1+CDH1 co-staining was also stained for DSP on a related section suggesting
that also DSP is expressed in the CDH1-positive area.
51
Appendix Figure 16: Co-staining of mature odontoblast markers and CDH1 in conditions with upregulated
expression of selected TFs. The expression of CDH1 was present in structures expressing DMP1 and DSP in the
kidney implants from the TGS condition (A). The expression of DMP1 showed similar pattern in the in vitro
cultured cell spheroids. In addition, the expression of DSP was present in the same area as the expression of both
CDH1 and DMP1 (B). All scale bars = 100 um.
4.10. Analysis of Nestin expression in the epithelial structures in vivo
The expression of Nestin in the epithelial structures present in the kidney implants of TGS Dox condition
was investigated using IHC (Appendix Figure 17). Nestin was generally expressed more in the
mesenchyme surrounding the epithelial structures. However, some Nestin-positive cells were located in
52
the epithelial structures. In these cells the expression of both Nestin and CDH1 was colocalized.
Furthermore, these double positive cells were found to be located on the periphery of the epithelial
structures.
Appendix Figure 17: Analysis of Nestin expression in the epithelial structures in vivo. The expression ofNestin
was investigated in kidney implants from TGS Dox condition. Nestin was expressed mostly in the mesenchyme,
while some Nestin-positive cells were present in the epithelial structures as well. Cells positive for both CDH1 and
Nestin are highlighted with arrowheads. Epithelial structures are highlighted by dashed lines, e - erythrocytes. All
scalebars =100 um.
53
4.11. Analysis of SOX21 expression in the epithelial structures in vivo
To further investigate the keratinocyte differentiation in the epithelial structures, co-staining of KRT10
and SOX21 was performed on TGS Dox kidney implants (Appendix Figure 18). Overview images
illustrate the general morphology of the kidney implant, with epithelial regions intertwined with
mesenchyme. The detail images then show the inner distribution of the two proteins. The KRT10
expression is generally located in the centers of the epithelial structures. The cells positive for SOX21
were predominantly located in the epithelial structures as well, while being located on their periphery.
Furthermore, the cells expressing KRT10 were also mostly negative for SOX21 and vice versa.
54
Overview Detail
SOX21 ; D35
t V i ? -'-38
f[
KRTjO, SOX21-. D35
'-* J
"i-'A-. .jr..
I v. .. f • - - . . ' • j i
Appendix Figure 18: Analysis of SOX21 expression in the epithelial structures in vivo. The expression of
SOX21 was investigated in kidney implantsfromTGS Dox condition. SOX21 was expressed predominantly in the
epithelial structures. Detail images further show that cells positive for SOX21 are mostly negative for the
keratinocyte marker KRT10. Epithelial structures are highlighted by dashed lines. All scalebars =100 urn.
55
4.12. The differentiation of transduced cells driven by the upregulation of the selected
genes results in the silencing of the TET3G+GFP transgene
The expression of the four selected genes was investigated in the TGS Dox kidney implants (Appendix
Figure 19A). The observed expression of Etv4, Gsc and SALL1 was mostly located outside the epithelial
structures, with some positive cells being present inside the structures. Nuprl however, displayed an
inverted pattern, with its expression being generally located inside the structures. To further elucidate the
reason behind the decrease in amount of transgene-overexpressing cells we performed IHC to stain for
GFP - the selection marker for the TET3G+EGFP vector (Appendix Figure 19B). In this vector, the
TET3G tetracycline-dependent transactivator is co-expressed with EGFP in a single ORF under the
control of EFla promotor (Appendix Figure 2A). The quantification of the IHC revealed that in both in
cell spheroids as well as kidney implants the GFP+ area is reduced from the expected majority. In cell
spheroids the GFP+ area ranged from 10-20 % in all conditions. The co-staining with odontoblast marker
DSP revealed that DSP-positive particles are mostly located in GFP-negative cells. Nevertheless, some
GFP+ cells with DSP+ particles were present as well and are highlighted in Appendix Figure 19B. In the
kidney implants, both T and TGS Dox conditions on average displayed 60 % of GFP+ area. The costaining
with odontoblast marker DMP1 showed that the DMP1-positive epithelial structures also display
a generally lower number of GFP-positive cells when compared to the surrounding mesenchyme.
To assure that the epithelial structures originate from the implanted, transduced, reporter cell line in situ
hybridization for GFP-specific nucleotide sequences was performed. The used probe was generated to
hybridize to GFP and Cerulean genomic sequences as well as their transcripts, to enable a distinction
between GFP/Cerulean-positive implanted cells and the host tissue from SCID mice that do not contain
any GFP related genes and transcripts (Appendix Figure 19C). The analysis revealed that the epithelial
structures as well as the surrounding mesenchyme indeed originate from the implanted cells, with only a
small number of cells with either weaker or no observable signal. Furthermore, no nuclear signal was
observed in the host-derived connective tissue enveloping the implant, as well as negative control without
the probe (Appendix Figure 19C). To investigate the loss of GFP in the transduced cell population over
time in more controlled conditions, we performed flow cytometry on cultured T and TGS cells lines at
three timepoints during differentiation with and without added doxycycline (Appendix Figure 19D). The
analysis revealed that without external, pluripotency-maintaining signals (LIF and CFHR-99021) both cell
lines display significantly reduced ratio of GFP+ cells over the culture period. Furthermore, the
56
visualizations of the cell populations showed that the GFP-positive and negative subpopulations that were
distinct at DO start to merge at D6 (Appendix Figure 19D). This suggested a gradual reduction of GFP
expression after the differentiation is initiated.
57
Appendix Figure 19: The investigation of the area-specific reduction of the overexpression of the selected
genes as a result of differentiation-induced silencing of the TET3G+GFP transgene. The expression of the
58
overexpressed genes was performed on the kidney capsule implants from the TGS Dox condition using in situ
hybridization and IHC (A). The centers of the epithelial structures are highlighted with green asterisks and borders
marked by dashed lines. The expression of the selected genes that is present inside the epithelial structures is
highlighted with arrowheads. The IHC staining of odontoblast markers and GFP on cell spheroids and kidney
capsule implants from the TGS Dox condition alongside the quantification of GFP+ area of TGS and T Dox
conditions (B). GFP+ cells expressing odontoblast markers (DSP or DMP1) highlighted with arrowheads. Borders
of the epithelial structures marked by dashed lines. In situ hybridization using a GFP-specific digoxigenin-labelled
probe on TGS Dox kidney implant (C). Random nuclei positive for the GFP and Cerulean transcripts and/or GFP
and Cerulean genes highlighted with arrowheads. Erythrocytes are marked with white asterisks. Borders of the
epithelial structures are marked with white dashed lines. The border of the TGS implant marked by a red dashed
line. FACS analysis of the loss of GFP expression after spontaneous and/or doxycycline induced differentiation in
T and TGS cell lines (D) (n = 3). In the FACS plots, every contour expresses 5 % increments of the cell population.
All scalebars =100 um. Data presented as mean ± SD.
The reduction in GFP+ ratio in the TGS conditions was more pronounced than in the T cell line. This
reality was almost assuredly caused by the overexpression of the selected genes in both Dox and Ctrl
conditions (the overexpression in the absence of doxycycline was due to the leakiness of the TET3G
transactivator as described in Appendix Figure 11B). This suggests that GFP is silenced in response to
the overexpression-induced differentiation of the cells. The mechanism behind this apparent silencing of
the Tet3g+GFP construct most likely lies with the promotor that drives the expression of this construct.
The EFla is a constitutive promotor that was in the past shown to provide high transgene expression in
mouse pluripotent stem cells (Suter et al. 2006; Hong et al. 2007). However, same authors also report a
reduction in i?F7a-driven transgene expression after differentiation into ectodermal lineages (Hong et al.
2007).
Furthermore, Qin et al. (2010) show that the activity of the EFla promotor varies significantly between
various cell types of different origin. Taken together, this strongly suggests that the reduction of GFP+
cell population after TF-driven differentiation is a result of epigenetic or transcriptional silencing of the
EFla promoter. This is likely further supported by the changes in the dynamics between GFP- and GFP+
subpopulation at DO and D6 of the in vitro differentiation. Furthermore, this might also explain the reduced
GFP loss in the T cell line over the 6-day period as the spontaneous differentiation (without the
overexpression of the selected genes) is likely to be slower and less specific when compared to the TFdriven
differentiation of the TGS cell line.
Therefore, this silencing of EFla is likely the cause behind the observed reduction of GFP+ area in both
cell spheroids and kidney implants, as well as the emergence of the spatial patterns of GFP and late
59
odontoblast marker expression. These patterns suggest that the epithelial structures mostly lost their GFP
expression due to differentiation-induced EFla silencing, while the surrounding mesenchyme did so in a
lesser degree. This is likely a symptom of the two cell populations differentiating into different lineages
with different epigenetic landscapes as suggested by the research on the different levels of EFla activity
in different cell types done by Qin et al. (2010).
The emergence of this pattern also explains the observed expression pattern of Gsc, Etv4 and SALL1, as
only the GFP+ cells are expressing the TET3G transactivator are therefore capable of upregulating the
selected genes. Interestingly, the expression of Nuprl was located predominantly inside the epithelial
structures and thus displayed a pattern not reminiscent of GFP's. This likely suggests that its expression
does not originate from the transgene.
Nevertheless, in practice, this also means that in the cells displaying odontoblast and/or keratinocyte
markers, the overexpression of the four genes was likely limited to several days. However, this was
expected. For this reason, we have decided to primarily use the overexpression of transcription factors to
modulate the stem cell fate decision and not some other signaling of signal-modulating genes. It was
hypothesized that the overexpression of specific transcription factors might determine or guide the stem
cell fate even during a short period. Furthermore, similar approach has already been used for initiating the
differentiation of human pluripotent stem cells into neurons by a 2-day long upregulation of Neurogenin
2 (NGN2) (Zhang et al., 2013; Schmid et al, 2021). It is important to note that the protocol in mention
consists of more than the NGN2 overexpression. Nevertheless, even during its short expression window,
it provides an essential initial push towards the neuronal lineage.
Our results suggest that the overexpression of Gsc and Etv4 pushes the pluripotent stem cells towards the
ectodermal appendage lineages. Furthermore, we hypothesize that the likely shorter time window, during
which the selected genes were overexpressed, might explain the incomplete odontoblast-like phenotype,
as well as the dual nature of the observed epithelial cells.
60
5. References
Aibar S, González-Blas CB, Moerman T, Huynh-Thu VA, Imrichova H, Hulselmans G, Rambow F,
Marine J-C, Geurts P, Aerts J, et al. 2017. SCENIC: single-cell regulatory network inference and
clustering. Nat Methods. 14(11): 1083-1086. doi:10.1038/nmeth.4463.
Akagi T, Kuure S, Uranishi K, Koide H, Costantini F, Yokota T. 2015. ETS-related Transcription Factors
ETV4 and ETV5 Are Involved in Proliferation and Induction of Differentiation-associated Genes in
Embryonic Stem (ES) Cells*. JBiol Chem. 290(37):22460-22473. doi:10.1074/jbc.M115.675595.
Balic A, Aguila HL, Mina M. 2010. Identification of cells at early and late stages of polarization during
odontoblast differentiation using pOBCol3.6GFP and pOBCol2.3GFP transgenic mice. Bone. 47(5):948-
958. doi:10.1016/j.bone.2010.08.009.
Bankhead P, Loughrey MB, Fernandez JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray
RT, Murray LJ, Coleman HG, et al. 2017. QuPath: Open source software for digital pathology image
analysis. Sci Rep. 7(1):16878. doi:10.1038/s41598-017-17204-5.
Bar-Nur O, Brumbaugh J, Verheul C, Apostolou E, Pruteanu-Malinici I, Walsh RM, Ramaswamy S,
Hochedlinger K. 2014. Small molecules facilitate rapid and synchronous iPSC generation. Nat Methods.
11(11): 1170-1176. doi: 10.1038/nmeth.3142.
Bárta T, Peškova L, Hampl A. 2016. miRNAsong: a web-based tool for generation and testing of miRNA
sponge constructs in silico. Sci Rep. 6(1):36625. doi:10.1038/srep36625.
Dental Cell Atlas, [accessed 2025 Apr 4]. https://pklab.med.harvard.edu/ruslan/dental.atlas.html.
Deshpande AS, Fang P-A, Zhang X, Jayaraman T, Sfeir C, Beniash E. 2011. Primary Structure and
Phosphorylation of Dentin Matrix Protein 1 (DMP1) and Dentin Phosphophoryn (DPP) Uniquely
Determine Their Role in Biomineralization. Biomacromolecules. 12(8):2933-2945.
doi: 10.1021/bm2005214.
Fan J, Salathia N, Liu R, Kaeser GE, Yung YC, Herman JL, Kaper F, Fan J-B, Zhang K, Chun J, et al.
2016. Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis.
Nat Methods. 13(3):241-244. doi:10.1038/nmeth.3734.
Gonzalez Lopez M, Huteckova B, Lavický J, Zezula N, Rakultsev V, Fridrichova V, Tuaima H, Nottmeier
C, Petersen J, Kavkova M, et al. 2023. Spatiotemporal monitoring of hard tissue development reveals
unknown features of tooth and bone development. Sci Adv. 9(31):eadi0482. doi:10.1126/sciadv.adi0482.
He P, Mann-Collura O, Fling J, Edara N, Hetz R, Razzaque MS. 2021. High phosphate actively induces
cytotoxicity by rewiring pro-survival and pro-apoptotic signaling networks in HEK293 and HeLa cells.
FASEB J. 35(l):e20997. doi:10.1096/fj.202000799RR.
Herriges J, Sun X. 2011. Investigating the role of FGF-regulated transcription factors ETV4 and ETV5 in
lung development and maturation. Dev Biol. 356(1):251. doi:10.1016/j.ydbio.2011.05.561.
61
Herriges JC, Verheyden JM, Zhang Z, Sui P, Zhang Ying, Anderson MJ, Swing DA, Zhang Yan,
Lewandoski M, Sun X. 2015. FGF-Regulated ETV Transcription Factors Control FGF-SHH Feedback
Loop in Lung Branching. Dev Cell. 35(3):322-332. doi:10.1016/j.devcel.2015.10.006.
Hong S, Hwang D-Y, Yoon S, Isacson O, Ramezani A, Hawley RG, Kim K-S. 2007. Functional Analysis
of Various Promoters in Lentiviral Vectors at Different Stages of In Vitro Differentiation of Mouse
Embryonic Stem Cells. Mol Ther. 15(9): 1630-1639. doi:10.1038/sj.mt.6300251.
Huang C, Santofimia-Castaflo P, Iovanna J. 2021. NUPR1: A Critical Regulator of the Antioxidant
System. Cancers. 13(15):3670. doi:10.3390/cancersl3153670.
Karantzali E, Lekakis V, Ioannou M, Hadjimichael C, Papamatheakis J, Kretsovali A. 2011. Salll
Regulates Embryonic Stem Cell Differentiation in Association with Nanog. J Biol Chem. 286(2): 1037.
doi: 10.1074/jbc.Ml 10.170050.
Kharchenko PV, Silberstein L, Scadden DT. 2014. Bayesian approach to single-cell differential
expression analysis. Nat Methods. 11(7):740-742. doi:10.1038/nmeth.2967.
Kim R, Green JBA, Klein OD. 2017. From snapshots to movies: Understanding early tooth development
in four dimensions. Dev Dyn. 246(6):442-450. doi:10.1002/dvdy.24501.
Křivánek J, Lavický J, Bouderlique T, Adameyko I. 2021. Rapid Isolation of Single Cells from Mouse
and Human Teeth. JoVE J Vis Exp.(176):e63043. doi: 10.3791/63043.
Křivánek J, Soldatov RA, Kastriti ME, Chontorotzea T, Herdina AN, Petersen J, Szarowska B, Landova
M, Matějova VK, Holla LI, et al. 2020. Dental cell type atlas reveals stem and differentiated cell types in
mouse and human teeth. Nat Commun. 11(1):4816. doi:10.1038/s41467-020-18512-7.
Lavický J, Kolouskova M, Procházka D, Rakultsev V, Gonzalez-Lopez M, Steklikova K, Bartos M,
Vijaykumar A, Kaiser J, Pořízka P, et al. 2022. The Development of Dentin Microstructure Is Controlled
by the Type of Adjacent Epithelium. J Bone Miner Res. 37(2):323-339. doi: 10.1002/jbmr.4471.
Leitner NR, Lassnig C, Rom R, Heider S, Bago-Horvath Z, Eferl R, Müller S, Kolbe T, Kenner L, Rülicke
T, et al. 2014. Inducible, Dose-Adjustable and Time-Restricted Reconstitution of Statl Deficiency In
Vivo. PLOS ONE. 9(l):e86608. doi:10.1371/journal.pone.0086608.
Li S, Kong H, Yao N, Yu Q, Wang P, Lin Y, Wang J, Kuang R, Zhao X, Xu J, et al. 2011. The role of
runt-related transcription factor 2 (Runx2) in the late stage of odontoblast differentiation and dentin
formation. Biochem Biophys Res Commun. 410(3):698-704. doi:10.1016/j.bbrc.2011.06.065.
Lin Y, Xiao Y, Lin C, Zhang Q, Zhang S, Pei F, Liu H, Chen Z. 2021. SALL1 regulates commitment of
odontoblast lineages by interacting with RUNX2 to remodel open chromatin regions. Stem Cells.
39(2): 196-209. doi: 10.1002/stem.3298.
Liu TM, Lee EH. 2013. Transcriptional Regulatory Cascades in Runx2-Dependent Bone Development.
Tissue Eng Part B Rev. 19(3):254-263. doi:10.1089/ten.teb.2012.0527.
62
Loew R, Heinz N, Hampf M, Bujard H, Gossen M. 2010. Improved Tet-responsive promoters with
minimized background expression. BMC Biotechnol. 10(1):81. doi:10.1186/1472-6750-10-81.
Lord SJ, Velle KB, Mullins RD, Fritz-Laylin LK. 2020. SuperPlots: Communicating reproducibility and
variability in cell biology. J Cell Biol. 219(6):e202001064. doi:10.1083/jcb.202001064.
Lu BC, Cebrian C, Chi X, Kuure S, Kuo R, Bates CM, Arber S, Hasseil J, MacNeil L, Hoshi M, et al.
2009. Etv4 and Etv5 are required downstream of GDNF and Ret for kidney branching morphogenesis.
Nat Genet. 41 (12): 1295-1302. doi:10.1038/ng.476.
Meisted P, Booeshaghi AS, Liu L, Gao F, Lu L, Min K H (Joseph), da Veiga Beltrame E, Hjörleifsson KE,
Gehring J, Pachter L. 2021. Modular, efficient and constant-memory single-cell RNA-seq preprocessing.
Nat Biotechnol. 39(7):813-818. doi:10.1038/s41587-021-00870-2.
Murayama M, Hirata H, Shiraki M, Iovanna JL, Yamaza T, Kukita T, Komori T, Moriishi T, Ueno M,
Morimoto T, et al. 2023. Nuprl deficiency downregulates HtrAl, enhances SMAD1 signaling, and
suppresses age-related bone loss in male mice. J Cell Physiol. 238(3):566—581. doi: 10.1002/jcp.30949.
Nakatake Y, Fujii S, Masui S, Sugimoto T, Torikai-Nishikawa S, Adachi K, Niwa H. 2013. Kinetics of
drug selection systems in mouse embryonic stem cells. BMC Biotechnol. 13(1):64. doi:10.1186/1472-
6750-13-64.
Pommier RM, Gout J, Vincent DF, Cano CE, Kaniewski B, Martel S, Rodriguez J, Fourel G, Valcourt U,
Marie JC, et al. 2012. The human NUPR1/P8 gene is transcriptionally activated by transforming growth
factor ß via the SMAD signalling pathway. Biochem J. 445(2):285-293. doi:10.1042/BJ20120368.
Porntaveetus T, Otsuka-Tanaka Y, Basson MA, Moon A M , Sharpe PT, Ohazama A. 2011. Expression of
fibroblast growth factors (Fgfs) in murine tooth development. J Anat. 218(5):534-543.
doi:10.1111/j. 1469-7580.2011.01352.x.
Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren B-Z, Lahn BT. 2010. Systematic Comparison of
Constitutive Promoters and the Doxycycline-Inducible Promoter. PLOS ONE. 5(5):el0611.
doi:10.1371/journal.pone.0010611.
Redelsperger IM, Taldone T, Riedel ER, Lepherd ML, Lipman NS, Wolf FR. 2016. Stability of
Doxycycline in Feed and Water and Minimal Effective Doses in Tetracycline-Inducible Systems. J Am
Assoc Lab Anim Sei JA ALAS. 55(4):467-474.
Rivera-Perez JA, Mallo M, Gendron-Maguire M, Gridley T, Behringer RR. 1995. Goosecoid is not an
essential component of the mouse gastrula organizer but is required for craniofacial and rib development.
Development. 121(9):3005-3012. doi:10.1242/dev.l21.9.3005.
Rivera-Perez JA, Wakamiya M, Behringer RR. 1999. Goosecoid acts cell autonomously in mesenchymederived
tissues during craniofacial development. Development. 126(17):3811—3821.
doi:10.1242/dev.l26.17.3811.
Roehl H, Nüsslein-Volhard C. 2001. Zebrafish pea3 and erm are general targets of FGF8 signaling. Curr
Biol. ll(7):503-507. doi:10.1016/s0960-9822(01)00143-9.
63
Sagomonyants K, Kalajzic I, Maye P, Mina M. 2015. Enhanced Dentinogenesis of Pulp Progenitors by
Early Exposure to FGF2. JDentRes. 94(11): 1582-1590. doi:10.1177/0022034515599768.
Sagomonyants K, Mina M. 2015. Stage-Specific Effects of Fibroblast Growth Factor 2 on the
Differentiation of Dental Pulp Cells. Cells Tissues Organs. 199(5-6):311-328. doi:10.1159/000371343.
Schneider CA, Rasband WS, Eliceiri KW. 2012. NTH Image to ImageJ: 25 years of image analysis. Nat
Methods. 9(7):671-675. doi:10.1038/nmeth.2089.
Spandidos A, Wang X, Wang H, Seed B. 2010. PrimerBank: a resource of human and mouse PCR primer
pairs for gene expression detection and quantification. Nucleic Acids Res. 38(suppl_l):D792-D799.
doi:10.1093/nar/gkpl005.
Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Mi dura RJ. 1995. Rapidly Forming Apatitic Mineral
in an Osteoblastic Cell Line (UMR 106—01 BSP) (*). J Biol Chem. 270(16):9420-9428.
doi: 10.1074/jbc.270.16.9420.
Sudou N, Yamamoto S, Ogino H, Taira M. 2012. Dynamic in vivo binding of transcription factors to cisregulatory
modules of cer and gsc in the stepwise formation of the Spemann-Mangold organizer.
Development. 139(9): 1651-1661. doi:10.1242/dev.068395.
Suter DM, Carrier L, Bettiol E, Tirefort D, Jaconi ME, Dubois-Dauphin M, Krause K-H. 2006. Rapid
Generation of Stable Transgenic Embryonic Stem Cell Lines Using Modular Lentivectors. Stem Cells.
24(3):615-623. doi:10.1634/stemcells.2005-0226.
Suzuki S, Haruyama N, Nishimura F, Kulkarni AB. 2012. Dentin sialophosphoprotein and dentin matrix
protein-1: Two highly phosphorylated proteins in mineralized tissues. Arch Oral Biol. 57(9): 1165-1175.
doi: 10.1016/j.archoralbio.2012.03.005.
Szot GL, KoudriaP, Bluestone JA. 2007. Transplantation of Pancreatic Islets Into the Kidney Capsule of
Diabetic Mice. J Vis Exp JoVE.(9):404. doi:10.3791/404.
Takada K, Odashima A, Onodera S, Saito A, Aida N, Furusawa M, Azuma T. 2022. The effect of BMP4,
FGF8 and WNT3a on mouse iPS cells differentiating to odontoblast-like cells. Med Mol Morphol.
55(3): 199-209. doi: 10.1007/s00795-022-00318-9.
Thesleff I. 2003. Epithelial-mesenchymal signalling regulating tooth morphogenesis. J Cell Sei.
116(9): 1647-1648. doi:10.1242/jcs.00410.
Tucker AS, Yamada G, Grigoriou M, Pachnis V, Sharpe PT. 1999. Fgf-8 determines rostral-caudal
polarity in the first branchial arch. Development. 126(1):51—61. doi:10.1242/dev.l26.1.51.
Vijaykumar A, Ghassem-Zadeh S, Vidovic-Zdrilic I, Komitas K, Adameyko I, Krivanek J, Fu Y, Maye
P, Mina M. 2019. Generation and characterization of DSPP-Cerulean/DMPl-Cherry reporter mice,
genesis. 0(0):e23324. doi:10.1002/dvg.23324.
64
Yamada G, Mansouri A, Torres M, Stuart ET, Blum M, Schultz M, Roberts EMD, Gruss P. 1995.
Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death.
Development. 121(9):2917-2922. doi:10.1242/dev.l21.9.2917.
Yaylaoglu MB, Titmus A, Visel A, Alvarez-Bolado G, Thaller C, Eichele G. 2005. Comprehensive
expression atlas of fibroblast growth factors and their receptors generated by a novel robotic in situ
hybridization platform. DevDyn. 234(2):371-386. doi:10.1002/dvdy.20441.
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. 2012. Primer-BLAST: A tool to
design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13(1): 134.
doi: 10.1186/1471-2105-13-134.
Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A Greedy Algorithm for Aligning DNA Sequences. J
ComputBiol. 7(l-2):203-214. doi: 10.1089/10665270050081478.
Zhang Z, Verheyden JM, Hassell JA, Sun X. 2009. FGF-Regulated Etv Genes Are Essential for
Repressing Shh Expression in Mouse Limb Buds. Dev Cell. 16(4):607-613.
doi:10.1016/j.devcel.2009.02.008.
Zhou X, Vink M, Klaver B, Berkhout B, Das AT. 2006. Optimization of the Tet-On system for regulated
gene expression through viral evolution. Gene Ther. 13(19): 1382—1390. doi:10.1038/sj.gt.3302780.
Zhou Y, Lei C, Zhu Z. 2020. A low-background Tet-On system based on post-transcriptional regulation
using Csy4. PLOS ONE. 15(12):e0244732. doi:10.1371/journal.pone.0244732.
65
F R O N T P A G E IMAGE O F J O U R N A L O F B O N E A N D M I N E R A L R E S E A R C H ,
V O L U M E 37, F E B R U A R Y 2022
Appendix E Front page image of Journal of
Bone and Mineral Research,
volume 37, February 2022
The following cover image, showing the phalloidin-stained odontoblasts
processes in the labial dentin, was generated by me while working on
Lavický etal. (2022)1 1
.
219
F R O N T P A G E IMAGE O F J O U R N A L O F B O N E A N D M I N E R A L R E S E A R C H ,
V O L U M E 37, F E B R U A R Y 2022
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F R O N T P A G E IMAGE O F C E L L R E P O R T S , V O L U M E 4 2 , N O V E M B E R 2 0 2 3
Appendix F Front page image of Cell
Reports, volume 42, November
2023
The following cover image, showing a E12.5 Hb9-EGFP mouse embryo,
was generated by me in cooperation with Alena Salasova (who generated
the imaged mouse embryo) while working on Thomasen et al.
(2 02 3)1 5 0
. The reporter is expressed in motor neurons.
Published with the following caption:
On the cover: The cover depicts a whole-mount light-sheet microscopy
image of developing motor neurons in a transparent mouse embryo at
embryonic day 12.5. Green fluorescent protein expression is coupled
with Hb9 transcription, allowing visualization of developing motor axons
in an intact specimen. Using this model, Thomasen et al. find that
SorCS2 deficiency specifically affects axon outgrowth of posterior brachial
motor neurons, their cell fate decisions, and macrophage-to-motoraxon-related
cell communication via SorCS2 interaction with progranulin.
Cover image by Josef Lavický and Alena Salasova.
TAKEN FROM:
https://www.cell.com/cell-reports/issue?pii=S2211-1247(23)X0011-
4#
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F R O N T P A G E IMAGE O F C E L L R E P O R T S , V O L U M E 4 2 , N O V E M B E R 2 0 2 3
Cell ReportsVolume 42
Number It
CelPress
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