Drosophila melanogaster development How do Drosophila embryos develop? How can one use genetics to find genes that regulate embryo development?

DROSOPHILA LIFE CYCLE 4 days 1 day 2 days Life cycle of Drosophila egg larva 1st instar larva 2nd instar larva 3rd instar pupa embryogenesis female

OOGENESIS IN DROSOPHILA Germarium ring canal Germline Cyst Formation Cystoblast Pro-Oocyte (undergoes meiosis) germline stem cells follicle stem cells germline: stem cell > cystoblast > 1 oocyte + 15 nurse cells

OOGENESIS IN DROSOPHILA GermariumVitellarium nurse cell oocyte stalk follicle cells border cells germline stem cells follicle stem cells oocyte oocyte + nurse cells surrounded by (somatic) follicle cells

Drosophila oocyte and supporting cells Nurse cellsRing canalsOocyte Follicle cells (from Gonzalez-Reyes and St Johnston (1994) Science 266: )

Drosophila oocyte and supporting cells Nurse cell nucleiFollicle cell nuclei (from Gonzalez-Reyes and St Johnston (1994) Science 266: )

Nuclear divisions start without cell division in Drosophila (superficial cleavage) Fig. 9.1 Zygotic gene expression begins

eggblastodermfate map larva T A Larvae already have substantial patterning acron head thorax abdomen telson T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 (cuticle) “stripy” expression of segmentation gene fushi tarazu (ftz) anterior posterior ventral dorsal epithelium (6,000 cells)

The fruit fly body plan is assembled in 24 hours: How?

Christiane Nüsslein-Volhard and Eric Wieschaus used genetics to identify proteins that set up the embryonic body plan

Wieschaus and Nüsslein-Volhard looked for mutants that affect the fly body plan wildtype

Genes identified in a famous screen for Drosophila mutants with embryo patterning defects

Screen for developmental mutants (Drosophila) Lethal hits = 100% (essential genes - ca ) (efficiency of mutagenesis = number of hits per gene) embryonal-lethal mutants zygotic mutants 25 % with morphological defects3 % - segmentation defects (AP) 0.5 % - tissue types defective (DV) 0.5 % female-sterile mutants8 % 100 % with effects on embryogenesis2 % (= maternal-effect mutants) - antero-posterior pattern0.4 % - dorso-ventral pattern0.3 % ca. 2% of all genes involved in embryo pattern formation (male-sterile mutants) (ca. 100 of > protein-encoding genes, only essential genes)

Maternal-effect mutations Genes expressed during oogenesis (= before fertilization) or genes expressed in maternal cells (follicle) All progeny of heterozygous mother are normal. All progeny are affected only if mother is homozygous mutant Zygotic mutations Genes expressed during embryogenesis (= after fertilization) Only genetically mutant embryos are affected. (25% of progeny of heterozygous mother are affected.)

Drosophila axis detemination; dorsal/ventral polarity How does the embryonic dorsal-ventral axis get translated into differentiation of different tissue types?

amnio- serosa dorsal ectoderm neuro- ectoderm mesoderm Cell fate specification at the blastoderm stage mesoderm formation fate map dorsal ventral

Dorsal-Ventral fate map

Gurken protein specifies the Anterior-Posterior axis of the Drosophila embryo during oogenesis (Similar to EGF)

Localized maternal mRNA sets up anterior and posterior poles

Gurken also signals dorsal pole formation during oogenesis follicle cells anteriorposterior AP V D D V microtubules A gurken expression in the oocyte 10A gurken expression in the oocyte 1-6 migration of nucleus oocyte nucleus

Expression of the Gurken Message and Protein Between the Oocyte Nucleus and the Dorsal Anterior Cell Membrane

D ORSO-VENTRAL P ATTERN F ORMATION follicle cells Oocyte pipe expression Ventral follicle cell Pipe (Golgi?) X X Nucleus Wind (ER?) X X X X mod. from van Eeden & St.Johnston Gurken = Epidermal Growth Factor (EGF) Torpedo = EGF receptor (in follicle cells)

Toll Tl - membrane receptor cactus cact - cytoplasmic inhibitor of Dorsal nuclear translocation dorsal dl - transcription factor (morphogen) Zygotic mutations tube - cytoplasmic protein pelle - ser/thr protein kinase Maternal effect mutations ndl, pipe, wbl gd, snk, ea - serine proteases spz - ligand Dorsal protein dorsal RNA Toll protein Spätzle protein Dorsal protein nudel, pipe, wbl amnio serosa dorsal ectoderm neuro- ectoderm mesoderm Dl nuclear protein Dorso-ventral pattern formation dorsal

Wild type ventralized dorsal mutant cactus mutant dorsalized T1 T2 T3 A1A2 A3A4A5 A6 A7 A8 Dorso-ventral pattern formation dorsal ventral

Wild type toll mutant cactus mutant Translocation of Dorsal protein into ventral nuclei but not lateral or dorsal nuclei

Generation of Dorsal-Ventral Polarity in Drosophila

Wild type toll mutant Inject wild-type cytoplasm mesoderm neuro-ectoderm (denticle belts) dorsal ectoderm Dorso-ventral pattern formation pivotal role of Toll pathway into toll mutant eggs dorsalized local rescue ventral dorsal polarity reversal

Conserved pathway for regulating nuclear transport of transcription factors in Drosophila and mammals

Cells with highest nuclear Dorsal levels become mesoderm

Zygotically expressed genes

Action of Dorsal protein in ventral cells

High affinity for promoter, Not much Dorsal needed to activate

Action of Dorsal protein in ventral cells Lower affinity for promoter, More Dorsal needed to activate

twist dpp Dorsal protein dorsal RNA Toll protein Spätzle protein Dorsal protein nudel, pipe, windbeutel Dorso-ventral pattern formation: summary oocyte nucleus dorsal > repression of ventral fate in dorsal follicle cells ventral production of ligand > activation of Toll receptor > graded nuclear uptake of Dorsal morphogen > regulation of zygotic target gene expression > cell fates along DV axis

Use of a similar regulatory system to pattern insects and vertebrates

Patterns mesoderm in vertebrates Patterns ectoderm in Drosophila

Gastrulation in Drosophila

Schematic representation of gastrulation in Drosophila