-t**^»-"! erimentální biologie PřF MU iofyzikální ústav AV CR Writs (Wingless/Int) - family of ligands -19 members in human and mouse - glycosylated and palmitoylated extracellular proteins - short range of action, bind to extracellular matrix - only in multicellular animals / \ canonical non-canonical (eg. Wnt-1 or Wnt-3a) (e9- Wnt-5a) Wnt/ß-catenin dráha (= kanonická dráha) - např. Wnt-1 nebo Wnt-3a - induce axis duplication in Xenopus - induce transformation of mammary cell line C57mg -signal via nuclear translocation ofß-catenin [1] Moon-cel2[l].swf Příklady vývojových a fyziologických procesů regulovaných kanonickou Wnt dráhou Maternální Wnt/ß-cateninova dráha determinuje dorsální (horní) pól vyvíjející se zygoty a embrya Blailula, stage 10 " rirqmd Animal pole Nh-Iw.:? ions Early gaslrula. slag* 1OVj Gasliuli.Sfagell archenlernn Ute gasimia, «tage 12 yolk ; .3 VWtll|H>lf >ŮK« s i-sí-ůpc? l«lo(foííT. ■ r*5MřfTi ennossrm D< Fig 2.6 Gastrulation In amphibians. The bíastula (hrst panel) contains several thousand cells end ihere is a fluid-filled cavity, lhe Dlastocoel. benealn lhe cells at the animal pale. Gaslrulation begins (second panal) a I the blastopore, which forms on lhe-dorsal side of the embryo. Future mesoderm and endoderm of lhe marginal zona move inside at this site through the dorsal lip of the blastopore, lhe mesoderm ending up sandwiched Waüoüoel between lhe endoderm and ecloderm in the animal region (third panel). The «issue movemenis creale anew internal eavrty lhe archenteron which will become lhe gul. Endoderm in the ventral region also moves inside ihrough lhe ventral Itp of lhe blastopore (fourth panel) and will eventually completely hne the archenteron. At lhe end ol gaslrulalion the blastoccol has considerabty reduced in size. After Balinsky. 8.1 : 1975. Early Xetwptts embryo Lateral view Animal Marginal Vegetal Xcrwpiis blastula Zygotic Wnt pathway marginal zone, stage 9} Maternal Wnt pathway (dorsal aide, stage S) Ventral dorsal axis duplication assay: control Xwnt-8 dshADEP CKIe ß-catenin -DE-- 3lsl Wnt/ß-cateninova dráha určuje anterioro-posteriorní (AP, předozadní) osu těla během gastrulace -podporuje vznik zadních a blokuje vznik předních částí těla A ^m^ Animal ^^^ Xenopus b\as\u\a Early Xenopus A ^ ^ ^ ■ ^n-------- A V Zygotic Wnt pathway embryo H^"^ (Marginal | l(marginal zone, stagey Lateral View \ / \ ^F Maternal Wnt pathway >Jy' (dorsal side, stage 8) Ventral dorsal myší embryo po gastrulaci (E8.5) Cílové geny Wnt/ß-cateninove dráhy jsou exprimovány v zadní části těla. Wnt5a+/+;LRP6+/+ c a) D) O tn d) x u c Wnt5a"/-;LRP6+/- Deplece Wnt/ß-kateninove dráhy při gastrulaci = ztráta zadních částí těla wild type Wnt-3a knockout Deplece inhibitorů Wnt/ß- kateninové dráhy při gastrulaci = ztráta předních částí těla wild type vs. Dkk1 knockout _________________I RNAi |__________________ Control |icafen.'n-l rM-f;(M-P APC-1 3L Cün1m| I hr-..--- I vn-1 cM-7.cn/J-2 y j R c k' N -' r. V ( f p b- RNAi 1 Ucsíen/n- T Q . _ g4 hm |j f ' 1 E *»ílfl "ľa > ß-Catenin Defines Head Versus Tail Identity During Planarian Regeneration and Homeostasis Kyle A. Gurtey, Jochen C. Rink, Alejandro Sanchez ALvarado* SCIENCE VOL 319 18 JANUARY 2008 Wnt/ß-cateninova dráha je klíčovým regulátorem aktivace kmenových buněk jak v embryogenezi, tak v dospělých tkáních Wnt/ß-catenin dráha je velmi často deregulovaná u nádorů! according to Beachy et al., Nature 2004 Wnt patliway Galon Adenocarcinoma Tumorigsnesis by inacth/ationof APC. Axin; tumorigsnesis by stabilization of ß-catenin; epigenetic inactrvation of SFRPs Liver Hepatoblastoma Tumorigsnesis fln mouse) by inactrvation of APG and by stabilization of ß-catenin Blood Multiple myeloma Cell-gnowth inhibition by dominant negative TGF4; growth stimulation by Wnt ligand Hairfollide Rlornatricoma Tumorigsnesis ijn mouse) by overexpression of ß-catsnin ^■mwmwmwmwmwmp ^iwmwmwmwmwmwmwmwmwmwmwmľ ^iwmwmwmwmwmwmwmwmwmwmwmľ iiwmwmwmwmwmp Bone Osteosarcoma Dkk3 and LRPs expression inhibits tumour cell grov/th in /.:,.r-> JMHIMMHIMMHIMMHIMMHIMMHIMMMI1IMMI1IMMI1IMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHI1IMMI1IMMI1IMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMHIMMH Lung Non-small-cell carcinoma Apoptosis and cell-cjcwth inhbition by short intefering RNA and a blocking antibody against Wnt2 Pleura Mesothelioma Apoptosis and cell-cjcwth inhbiticn by transfection of SFHP Figure 3 Tissue anatomy of the colonic epithelium. Putative stem cells [dark blue) Figure 4 The hair follicle. Stem cells reside in the bulge niche. Cellscan migrate reside at the c rypt bottom. Prol iterating p roge nitor eel Is occu py two-th irds of the upwa ids from here to pop ulate the sebaceous g land and the i nte rfo llicu lar crypt. Differentiated cells (green) populate the remainder of the crypt and the flat epidermis Cells that migrate downwards enter the matrix where they rapidly surface epithelium, adapted from ref. Ů9.) proliferate and then differentiate to form the hair, adapted f ram -ef. 90.) Reya & Clevers 2005, Nature Důsledky aktivace ß-cateninu v epidermis (po depilaci) Lo Celso, C. L. et al. Development 2004;131:1787-1799 Aktivace kanonické Wnt dráhy indukuje de novo tvorbu vlasových kořínků ----■ Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding Mayumi lto\ Zaixiri Yarig\ Thomas Aridl]r Chunhua Cui\ Noori Kim\ Sarah E. Millar1 rk George Cotsarelis] Vcl W\ 17 Miy i007 Jwlfl.lOll/nHuriiSTrA LETTERS Aktivace ß-cateninu ve vyvíjející se mozkové trubici: midbrain (Brn4-promotor) cortex (nestin enhancer) d ÜM, ■ *;-0" ■■■''' ^^F^ ' %' ÉP (Pri:/ L, 1 1 9 ^o i.«í- ■■-.'.■ n Zechner et al., 2003: Dev. Biol.;258:406-418. Chenn & Walsh, 2002: Science;297:365-369. Aktivace beta-cateninu v kmenových buňkách zubu: ß_catAex3K14/+ ftátii »UM» ♦ MIM i • wild type Järvinenet al. (2006) Proc. Natl. Acad. Sei. USA 103, 18627-18632 Wnt/ß-cateninova dráha v regeneraci II. regenerace axolotl A A cs^, tjF epidermis r> C:;'i'.i-n..r-y -ŕ >j?" converger! ajfierigian Exencephaly Open neural tube Neura.1 fald fusion Shaping or inie^dorsal naural Issue Hamblet et al., 2002, Development Možnosti studia CE - Kellerovy explantáty (Xenopus) Lrp5/6 loading control control XLRP5 MO XLRP5 MO + ml_rp5 Non-canonical/PCP (Planar cell polarity) pathway: phenotypes in mouse Stereocilia orientation in inner ear hair cells Actin > - - < **S /"N ^\ 0+ Ft t t t t t t t t t t t t VangV ^^ t / t t • t Qian etal., 2007, Dev. Biol. Increased migration and metastatic potential! Known Wnt5a knockout phenotypes +/+ -/- Yamaguchi etal., 1999 control,' i Má Qian et al, 2007 Mutations in Ror2 cause dominant brachydactyly type B (BDB) and recessive robinow syndrome (RRS) ror2 1321-1325del(5) IVS8+3+5del3insl9 1398-1399insA W749X 2249delG Y755X Q760X I *~~ GRK2 CKl£ Dlxin WľlP C182X R189W R184C R205X R366W R396X Q502X 1740-1774del35 N620K W720X Komponenty nekanonické Wnt dráhy jsou zvýšeny u pacientů s chronickou lymfoidní leukémií (CLL) I. Vývoj neurální lišty: Ventral Wnt-3a _S" "Z_ • neural • crest ■v ° cel Is neural tube - neurální lišta je zdrojem periferního nervového systému, melanocytu, obličejových kostí a svalů, srdce a dalších control mutant E9.0 1 T> Neural crest progenitor X> ngn2-positive Sensory precursor t Melanocyte precursor v\ Neurat crest progenitor -p. ngn1-positive Sensory precursor J Sensory r neurons ■^ Melanocytes ENS E10.5 Hari, L. et al. J. Cell Biol. 2002:159:867-880 o Heart outflow tract development Henderson DJ et al., 2006, TrendsCard. Res. Purification of Wnt ligands Wnt proteins are lipid-modified and can act as stem cell growth factors Kari Wilier!, Jeffrey D. Brown , Esther Danenberg , Andrew W. Duncan-'-, Irving L Wetssma n I, Tannishtha Reya >,John R. Yates III> & Reel Müsse NATĽRE | VOL 423 \22. MAV2Ö03 |- Wnt-3a no detergent detergent added Wnt-conditioned medium i 4 liters of media conditioned by fibroblasts expressing HA tagged Wnt-5a Blue Sepharose column i Superdex gel filtration i Heparin column WB: Wnt-5a silver staining Fundamental question of Wnt signalling: How the specificity is achieved? b Planar Cell Polarity/JNK/Rock Rack JNK- Cascade \ / Cell Differentiation Cytoskeletal Polarity Vincan, 2004, Frontiers in Bioscience