•From last week… • •Visible mutations, Dominant mutations and balancers. •Read the genotype from the fly. • • • •Lecture 2. • • Genetic screens for Drosophila mutants affecting embryonic pattern formation Figure 10.3 Life cycle and early embryonic development of Drosophila melanogaster Figure 10.3 Life cycle and early embryonic development of Drosophila melanogaster. (A) Following fertilization, embryogenesis begins with the division of nuclei (cleavage) and their subsequent cellularization, followed by the cell and tissue movements of gastrulation and organ formation. The embryo hatches out as a first instar larva that grows, going through two molts to become a third instar larva. The third instar larva becomes a pupa, which metamorphoses into the adult fly. (B) The anterior-to-posterior patterning of segments in a Drosophila embryo are visualized with a fluorescent histone reporter in the live fly embryo. Figure 10.4 Laser confocal micrographs of stained chromatin showing syncytial nuclear divisions and superficial cleavage in a series of Drosophila embryos Figure 10.4 Laser confocal micrographs of stained chromatin showing syncytial nuclear divisions and superficial cleavage in a series of Drosophila embryos. The future anterior end is positioned upward; numbers refer to the nuclear division cycle. The early nuclear divisions occur centrally within a syncytium. Later, the nuclei and their cytoplasmic islands (energids) migrate to the periphery of the cell, creating the syncytial blastoderm. After cycle 13, the cellular blastoderm forms by ingression of cell membranes between nuclei. The pole cells (germ cell precursors) form in the posterior. Figure 10.6 Formation of the cellular blastoderm in Drosophila Figure 10.6 Formation of the cellular blastoderm in Drosophila. Nuclear shape change and cellularization are coordinated through the cytoskeleton. (A) Cellularization and nuclear shape change shown by staining the embryo for microtubules (green), actin filaments (blue), and nuclei (red). The red stain in the nuclei is due to the presence of the Kugelkern protein, one of the earliest proteins made from the zygotic nuclei. It is essential for nuclear elongation. (B) This embryo was treated with nocadozole to disrupt microtubules. The nuclei fail to elongate, and cellularization is prevented. (C) Diagrammatic representation of cell formation and nuclear elongation. (After A. Brandt et al. 2006. Curr Biol 16: 543–552.) Figure 10.7 Gastrulation in Drosophila Figure 10.7 Gastrulation in Drosophila. The anterior of each gastrulating embryo points upward in this series of scanning electron micrographs. (A) Ventral furrow beginning to form as cells flanking the ventral midline invaginate. (B) Closing of ventral furrow, with mesodermal cells placed internally and surface ectoderm flanking the ventral midline. (C) Dorsal view of a slightly older embryo, showing the pole cells and posterior endoderm sinking into the embryo. (D) Schematic representation showing dorsolateral view of an embryo at fullest germ band extension, just prior to segmentation. The cephalic furrow separates the future head region (procephalon) from the germ band, which will form the thorax and abdomen. (E) Lateral view, showing fullest extension of the germ band and the beginnings of segmentation. Subtle indentations mark the incipient segments along the germ band. Ma, Mx, and Lb correspond to the mandibular, maxillary, and labial head segments; T1–T3 are the thoracic segments; and A1–A8 are the abdominal segments. (F) Germ band reversing direction. The true segments are now visible, as well as the other territories of the dorsal head, such as the clypeolabrum, procephalic region, optic ridge, and dorsal ridge. (G) Newly hatched first instar larva. (D after J. A. Campos-Ortega and V. Hartenstein. 1985. The Embryonic Development of Drosophila melanogaster. Springer-Verlag: New York.) • • • • Figure 10.27 Expression of Gurken between the oocyte nucleus and the dorsal anterior cell membrane Nurse cells pour proteins and RNAs into the oocyte. Maternal contribution means many zygotic mutants will survive to hatching and die as larvae. Figure 10.27 Expression of Gurken between the oocyte nucleus and the dorsal anterior cell membrane. (A) The gurken mRNA is localized between the oocyte nucleus and the dorsal follicle cells of the ovary. Anterior is to the left; dorsal faces upward. (B) A more mature oocyte shows Gurken protein (yellow) across the dorsal region. Actin is stained red, showing cell boundaries. As the oocyte grows, follicle cells migrate across the top of the oocyte, where they become exposed to Gurken. • D:\Harish\CB32-online figure\CB32-online figure\CH-01\CB32-WieschausFig02-revised.tif • D:\Harish\CB32-online figure\CB32-online figure\CH-01\Nusslein-VollhardFig04.tif • D:\Harish\CB32-online figure\CB32-online figure\CH-01\CB32-WieschausFig05-revised.tif Genes on the same chromosome will recombine New pattern-forming mutants were mapped by recombination with visible markers • MGA2-06-15b PPT - Crazy Chromosomes! PowerPoint Presentation, free download - ID ... Expected 5000 lethal genes in flies, 1000/chromome arm and 1,000 on Chr 2 ; similar to number of bands in interphase polytene chromosomes. In fact there are about 13,700 genes but only 5,000 mutate to give lethals. Most lethals survive embryogenesis and die as larvae. 61 mutant genes affecting cuticle pattern were found on Chr 2. • D:\Harish\CB32-online figure\CB32-online figure\CH-01\Nusslein-VollhardFig10.tif Figure 10.16 Three types of segmentation gene mutations Figure 10.16 Three types of segmentation gene mutations. The left side shows the early-cleavage embryo (yellow), with the region where the particular gene is normally transcribed in wild-type embryos shown in blue. These areas are deleted as the mutants develop into late-stage embryos. (Original art based on M. P. Scott and P. H. O’Farrell. 1986. Annu Rev Cell Biol 2: 49–80 and C. Nüsslein-Volhard and W. E. Wieschaus. 1980. Nature 287: 795–801.) • • • • Figure 10.10 Generalized model of Drosophila anterior-posterior pattern formation Figure 10.10 Generalized model of Drosophila anterior-posterior pattern formation. Anterior is to the left; the dorsal surface faces upward. (A) The pattern is established by maternal effect genes that form gradients and regions of morphogenetic proteins. These proteins are transcription factors that activate the gap genes, which define broad territories of the embryo. The gap genes enable the expression of the pair-rule genes, each of which divides the embryo into regions about two segments wide. The segment polarity genes then divide the embryo into segment-sized units along the anterior-posterior axis. Together, the actions of these genes define the spatial domains of the homeotic genes that define the identities of each of the segments. In this way, periodicity is generated from nonperiodicity, and each segment is given a unique identity. (B) Maternal effect genes. The anterior axis is specified by the gradient of Bicoid protein (yellow through red; yellow being the highest concentration). (C) Gap gene protein expression and overlap. The domain of Hunchback protein (orange) and the domain of Krüppel protein (green) overlap to form a region containing both transcription factors (yellow). (D) Products of the fushi tarazu pair-rule gene form seven bands across the blastoderm of the embryo. (E) Products of the segment polarity gene engrailed, seen here at the extended germ band stage. Figure 10.11 Anterior-posterior specification in Drosophila originates with morphogen gradients Figure 10.11 Anterior-posterior specification in Drosophila originates with morphogen gradients. In the most anterior portion of the egg cytoplasm, bicoid mRNA is stabilized, while nanos mRNA is tethered to the posterior end. (The anterior can be recognized by the micropyle, a structure that permits sperm to enter.) Once the egg is laid and fertilized, the mRNAs are translated into proteins that activate transcription of genes that specify the distinct segment identities of both the larva and the adult fly. The two proteins form gradients, with the Bicoid gradient highest at the egg’s anterior end and the Nanos gradient highest at the posterior end. The coordinate system formed by these gradients distinguishes each position along the axis from any other position. When the nuclei divide, each nucleus is given its positional information by the ratio of the Bicoid and Nanos proteins present at that position. Figure 10.13 Caudal protein gradient of a wild-type Drosophila embryo at the syncytial blastoderm stage Figure 10.13 Caudal protein gradient of a wild-type Drosophila embryo at the syncytial blastoderm stage. Anterior is to the left. The protein (stained darkly) enters the nuclei and helps specify posterior fates. Compare with the complementary gradient of Bicoid protein in Figure 1 in Further Development 10.5, Bicoid mRNA Localization in the Anterior Pole of the Oocyte, online. Figure 10.14 Model of anterior-posterior pattern generation by Drosophila maternal effect genes Figure 10.14 Model of anterior-posterior pattern generation by Drosophila maternal effect genes. (A) The bicoid, nanos, hunchback, and caudal mRNAs are deposited in the oocyte by the ovarian nurse cells. The bicoid message is sequestered anteriorly; the nanos message is localized to the posterior pole. (B) Upon translation, the Bicoid protein gradient extends from anterior to posterior, while the Nanos protein gradient extends from posterior to anterior. Nanos inhibits the translation of the hunchback message (in the posterior), while Bicoid prevents the translation of the caudal message (in the anterior). This inhibition results in opposing Caudal and Hunchback gradients. The Hunchback gradient is secondarily strengthened by transcription of the hunchback gene in the anterior nuclei (since Bicoid acts as a transcription factor to activate hunchback transcription). (C) Parallel interactions whereby translational gene regulation establishes the anterior-posterior patterning of the Drosophila embryo. (C after P. M. Macdonald and C. A. Smibert. 1996. Curr Opin Genet Dev 6: 403–407.) Figure 10.15 Bicoid protein gradient in the early Drosophila embryo Figure 10.15 Bicoid protein gradient in the early Drosophila embryo. (A) Localization of bicoid mRNA to the anterior tip of the embryo in a steep gradient. (B) Bicoid protein gradient shortly after fertilization. Note that the concentration is greatest anteriorly and trails off posteriorly. Notice also that Bicoid is concentrated in the nuclei. (C) Densitometric scan of the Bicoid protein gradient. The upper curve (black) represents the Bicoid gradient in wild-type embryos. The lower curve (red) represents Bicoid in embryos of bicoid mutant mothers. (D) Phenotype of cuticle from a strongly affected embryo produced by a female fly deficient in the bicoid gene compared with the wild-type cuticle pattern. The head and thorax of the bicoid mutant have been replaced by a second set of posterior telson structures, abbreviated fk (filzkörper) and ap (anal plates). Figure 10.15 Bicoid protein gradient in the early Drosophila embryo (Part 1) Figure 10.15 Bicoid protein gradient in the early Drosophila embryo. (A) Localization of bicoid mRNA to the anterior tip of the embryo in a steep gradient. (B) Bicoid protein gradient shortly after fertilization. Note that the concentration is greatest anteriorly and trails off posteriorly. Notice also that Bicoid is concentrated in the nuclei. (C) Densitometric scan of the Bicoid protein gradient. The upper curve (black) represents the Bicoid gradient in wild-type embryos. The lower curve (red) represents Bicoid in embryos of bicoid mutant mothers. (D) Phenotype of cuticle from a strongly affected embryo produced by a female fly deficient in the bicoid gene compared with the wild-type cuticle pattern. The head and thorax of the bicoid mutant have been replaced by a second set of posterior telson structures, abbreviated fk (filzkörper) and ap (anal plates). Figure 10.15 Bicoid protein gradient in the early Drosophila embryo (Part 2) Figure 10.15 Bicoid protein gradient in the early Drosophila embryo. (A) Localization of bicoid mRNA to the anterior tip of the embryo in a steep gradient. (B) Bicoid protein gradient shortly after fertilization. Note that the concentration is greatest anteriorly and trails off posteriorly. Notice also that Bicoid is concentrated in the nuclei. (C) Densitometric scan of the Bicoid protein gradient. The upper curve (black) represents the Bicoid gradient in wild-type embryos. The lower curve (red) represents Bicoid in embryos of bicoid mutant mothers. (D) Phenotype of cuticle from a strongly affected embryo produced by a female fly deficient in the bicoid gene compared with the wild-type cuticle pattern. The head and thorax of the bicoid mutant have been replaced by a second set of posterior telson structures, abbreviated fk (filzkörper) and ap (anal plates). Figure 10.15 Bicoid protein gradient in the early Drosophila embryo (Part 3) Figure 10.15 Bicoid protein gradient in the early Drosophila embryo. (A) Localization of bicoid mRNA to the anterior tip of the embryo in a steep gradient. (B) Bicoid protein gradient shortly after fertilization. Note that the concentration is greatest anteriorly and trails off posteriorly. Notice also that Bicoid is concentrated in the nuclei. (C) Densitometric scan of the Bicoid protein gradient. The upper curve (black) represents the Bicoid gradient in wild-type embryos. The lower curve (red) represents Bicoid in embryos of bicoid mutant mothers. (D) Phenotype of cuticle from a strongly affected embryo produced by a female fly deficient in the bicoid gene compared with the wild-type cuticle pattern. The head and thorax of the bicoid mutant have been replaced by a second set of posterior telson structures, abbreviated fk (filzkörper) and ap (anal plates). Figure 10.19 Messenger RNA expression patterns of two pair-rule genes, even-skipped (red) and fushi tarazu (black), in the Drosophila blastoderm Figure 10.19 Messenger RNA expression patterns of two pair-rule genes, even-skipped (red) and fushi tarazu (black), in the Drosophila blastoderm. Each gene is expressed as a series of seven stripes. Anterior is to the left, dorsal is up. Figure 10.20 Specific promoter regions of the even-skipped (eve) gene control specific transcription bands in the embryo Figure 10.20 Specific promoter regions of the even-skipped (eve) gene control specific transcription bands in the embryo. (A) Partial map of the eve promoter, showing the regions responsible for the various stripes. (B–E) A reporter β-galactosidase gene (lacZ) was fused to different regions of the eve promoter and injected into fly embryos. The resulting embryos were stained (orange bands) for the presence of Even-skipped protein. (B–D) Wild-type embryos that were injected with lacZ transgenes containing the enhancer region specific for stripe 1 (B), stripe 5 (C), or both regions (D). (E) The enhancer region for stripes 1 and 5 was injected into an embryo deficient in giant. Here, the posterior border of stripe 5 is missing. (A after C. Sackerson et al. 1999. Dev Biol 211: 39–52.) Figure 10.21 Model for formation of the second stripe of transcription from the even-skipped gene Figure 10.21 Model for formation of the second stripe of transcription from the even-skipped gene. The enhancer element for stripe 2 regulation contains binding sequences for several maternal and gap gene proteins. Activators (e.g., Bicoid and Hunchback) are noted above the line; repressors (e.g., Krüppel and Giant) are shown below. Note that nearly every activator site is closely linked to a repressor site, suggesting competitive interactions at these positions. (Moreover, a protein that is a repressor for stripe 2 may be an activator for stripe 5; it depends on which proteins bind next to them.) B, Bicoid; C, Caudal; G, Giant; H, Hunchback; K, Krüppel; N, Knirps; T, Tailless. (After H. Janssens et al. 2006. Nat Genet 38: 1159–1165.) Figure 10.17 Parasegments in the Drosophila embryo are shifted one compartment forward in relation to the segments Figure 10.17 Parasegments in the Drosophila embryo are shifted one compartment forward in relation to the segments. Ma, Mx, and Lb are the mandibular, maxillary, and labial head segments; T1–T3 are the thoracic segments; and A1–A8 are the abdominal segments. Each segment has an anterior (A) and a posterior (P) compartment. Each parasegment (numbered 1–14) consists of the posterior compartment of one segment and the anterior compartment of the segment in the next posterior position. Black bars indicate the boundaries of ftz gene expression; these regions are missing in the fushi tarazu (ftz) mutant (see Figure 10.16B). (After A. Martinez-Arias and P. A. Lawrence. 1985. Nature 313: 639–642.) Figure 10.22 Model for transcription of the segment polarity genes engrailed (en) and wingless (wg) Figure 10.22 Model for transcription of the segment polarity genes engrailed (en) and wingless (wg). (A) Expression of wg and en is initiated by pair-rule genes. The en gene is expressed in cells that contain high concentrations of either Even-skipped or Fushi tarazu proteins. The wg gene is transcribed when neither eve nor ftz genes are active, but when a third gene (probably sloppy-paired) is expressed. (B) The continued expression of wg and en is maintained by interactions between the Engrailed- and Wingless-expressing cells. Wingless protein is secreted and diffuses to the surrounding cells. In those cells competent to express Engrailed (i.e., those having Eve or Ftz proteins), Wingless protein is bound by the Frizzled and Lrp6 receptor proteins, which enables the activation of the en gene via the Wnt signal transduction pathway. (Armadillo is the Drosophila name for β-catenin.) Engrailed protein activates the transcription of the hedgehog gene and also activates its own (en) gene transcription. Hedgehog protein diffuses from these cells and binds to the Patched receptor protein on neighboring cells. The Hedgehog signal enables the transcription of the wg gene and the subsequent secretion of the Wingless protein. For a more complex view, see Sánchez et al. 2008. (After M. S. Levine and K. W. Harding. 1989. In D. M. Glover and B. D. Hames [Eds.], Genes and Embryos. IRL, New York, pp. 39–94; M. Peifer and A. Bejsovec. 1992. Trends Genet 8: 243–249; E. Siegfried et al. 1994. Nature 367: 76–80.) Figure 10.23 Homeotic gene expression in Drosophila Figure 10.23 Homeotic gene expression in Drosophila. (A) Expression map of the homeotic genes. In the center are the genes of the Antennapedia and Bithorax complexes and their functional domains. Below and above the gene map, the regions of homeotic gene expression (both mRNA and protein) in the blastoderm of the Drosophila embryo and the regions that form from them in the adult fly are shown. (B) In situ hybridization for four genes at the extended germ band stage (a slightly later stage than the embryo shown in A). The engrailed (blue) expression pattern separates the body into segments; Antennapedia (green) and Ultrabithorax (purple) separate the thoracic and abdominal regions; Distal-less (red) shows the placement of jaws and the beginnings of limbs. (A after T. C. Kaufman et al. 1990. Adv Genet 27: 309–362; S. Dessain et al. 1992. EMBO J 11: 991–1002.) Figure 10.24 (A) Wings of the wild-type fruit fly emerge from the second thoracic segment and (B) a four-winged fruit fly constructed by putting together three mutations in cis-regulators of the Ultrabithorax gene Figure 10.24 (A) Wings of the wild-type fruit fly emerge from the second thoracic segment. (B) A four-winged fruit fly constructed by putting together three mutations in cis-regulators of the Ultrabithorax gene. These mutations effectively transformed the third thoracic segment into another second thoracic segment, thus transforming halteres into wings. Figure 10.25 (A) Head of a wild-type fruit fly. (B) Head of a fly with the Antennapedia mutation that converts antennae into legs Figure 10.25 (A) Head of a wild-type fruit fly. (B) Head of a fly with the Antennapedia mutation that converts antennae into legs. • • • Figure 10.28 Specification of cell fate by the Dorsal protein Figure 10.28 Specification of cell fate by the Dorsal protein. (A) Transverse sections of embryos stained with antibody to show the presence of Dorsal protein (dark area). The wild-type embryo (left) has Dorsal protein only in the ventralmost nuclei. A dorsalized mutant (center) has no localization of Dorsal protein in any nucleus. In the ventralized mutant (right), Dorsal protein has entered the nucleus of every cell. (B) Fate maps of cross sections through the Drosophila embryo at division cycle 14. The most ventral part becomes the mesoderm; the next higher portion becomes the neurogenic (ventral) ectoderm. The lateral and dorsal ectoderm can be distinguished in the cuticle, and the dorsalmost region becomes the amnioserosa, the extraembryonic layer that surrounds the embryo. The translocation of Dorsal protein into ventral, but not lateral or dorsal, nuclei produces a gradient whereby the ventral cells with the most Dorsal protein become mesoderm precursors. (C) Dorsal-ventral patterning in Drosophila. Following invagination of the mesoderm, the readout of the Dorsal gradient can be seen in the trunk region of this whole-mount stained embryo. The expression of the most ventral gene, ventral nervous system defective (blue), is from the neurogenic ectoderm. The intermediate neuroblast defective gene (green) is expressed in lateral ectoderm. Red represents the muscle-specific homeobox gene, expressed in the mesoderm above the intermediate neuroblasts. The dorsalmost tissue expresses decapentaplegic (yellow). (B after C. A. Rushlow et al. 1989. Cell 59: 1165–1177.) Figure 10.28 Specification of cell fate by the Dorsal protein (Part 3) Figure 10.28 Specification of cell fate by the Dorsal protein. (A) Transverse sections of embryos stained with antibody to show the presence of Dorsal protein (dark area). The wild-type embryo (left) has Dorsal protein only in the ventralmost nuclei. A dorsalized mutant (center) has no localization of Dorsal protein in any nucleus. In the ventralized mutant (right), Dorsal protein has entered the nucleus of every cell. (B) Fate maps of cross sections through the Drosophila embryo at division cycle 14. The most ventral part becomes the mesoderm; the next higher portion becomes the neurogenic (ventral) ectoderm. The lateral and dorsal ectoderm can be distinguished in the cuticle, and the dorsalmost region becomes the amnioserosa, the extraembryonic layer that surrounds the embryo. The translocation of Dorsal protein into ventral, but not lateral or dorsal, nuclei produces a gradient whereby the ventral cells with the most Dorsal protein become mesoderm precursors. (C) Dorsal-ventral patterning in Drosophila. Following invagination of the mesoderm, the readout of the Dorsal gradient can be seen in the trunk region of this whole-mount stained embryo. The expression of the most ventral gene, ventral nervous system defective (blue), is from the neurogenic ectoderm. The intermediate neuroblast defective gene (green) is expressed in lateral ectoderm. Red represents the muscle-specific homeobox gene, expressed in the mesoderm above the intermediate neuroblasts. The dorsalmost tissue expresses decapentaplegic (yellow). (B after C. A. Rushlow et al. 1989. Cell 59: 1165–1177.) Figure 10.29 Gastrulation in Drosophila Figure 10.29 Gastrulation in Drosophila. In this cross section, the mesodermal cells at the ventral portion of the embryo buckle inward, forming the ventral furrow (see Figure 10.7A,B). This furrow becomes a tube that invaginates into the embryo and then flattens and generates the mesodermal organs. The nuclei are stained with antibody to the Twist protein, a marker for the mesoderm. Figure 10.30 Cartesian coordinate system mapped out by gene expression patterns Figure 10.30 Cartesian coordinate system mapped out by gene expression patterns. (A) A grid (ventral view, looking “up” at the embryo) formed by the expression of short-gastrulation (red), intermediate neuroblast defective (green), and muscle segment homeobox (magenta) along the dorsal-ventral axis, and by the expression of wingless (yellow) and engrailed (purple) transcripts along the anterior-posterior axis. (B) Coordinates for the expression of genes giving rise to Drosophila salivary glands. These genes are activated by the protein product of the sex combs reduced (scr) homeotic gene in a narrow band along the anterior-posterior axis, and they are inhibited in the regions marked by decapentaplegic (dpp) and dorsal gene products along the dorsal-ventral axis. This pattern allows salivary glands to form in the midline of the embryo in the second parasegment. (B after S. Panzer et al. 1992. Development 114: 49–57.) •Reading • •Heidelberg screen, Wieschaus and Nusslein Vollhard • •Scot Gilbert textbook chapter of Drosophila