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“ It is not birth, marriage or death, but gastrulation which is truly the most important time in your life.”

“ It is not birth, marriage or death, but gastrulation which is truly the most important time in your life.”. Lewis Wolpert. Germ layer formation: from blastula to animal. onset of zygotic transcription. Germ layer formation: from blastula to animal. Fate-mapping the early Xenopus embryo.

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“ It is not birth, marriage or death, but gastrulation which is truly the most important time in your life.”

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  1. “It is not birth, marriage or death, but gastrulation which is truly the most important time in your life.” Lewis Wolpert

  2. Germ layer formation: from blastula to animal onset of zygotic transcription

  3. Germ layer formation: from blastula to animal

  4. Fate-mapping the early Xenopus embryo animal blastula vegetal

  5. Fate-mapping the early Xenopus embryo animal ectoderm (epidermis, nervous system) mesoderm (blood, muscle, kidneys, bones) endoderm (digestive, respiratory systems) blastula vegetal

  6. Origin of mesoderm: Nieuwkoop experiments ectoderm ectoderm What happened to mesoderm? endoderm How is endoderm formed?

  7. Origin of endoderm: maternal factors animal • maternally supplied transcript • T-box transcription factor • activates endoderm cascade cell-autonomously • inhibition blocks endoderm (and mesoderm) formation maternal vegT vegetal

  8. Cell autonomous and non-autonomous effects Cell autonomy: genetically mutant cell/tissue exhibits phenotype associated with loss of gene product (gene product required by cell producing it) Cell non-autonomy: genetically mutant cell/tissue causes phenotypes in other (genetically wild-type) cells (gene product required by cells other than those producing it) X A B X X X

  9. Origin of mesoderm: Nieuwkoop experiments • mesoderm induced from ectoderm when apposed to endoderm • effect has limited range (4-5 cell diameters) • origin of “animal cap” assay

  10. Origin of mesoderm: Nieuwkoop experiments • induction event tolerant of small physical separation (filter or spacing between tissues): secreted molecule(s) responsible

  11. Origin of mesoderm: Nieuwkoop experiments • type of mesoderm induced depends on duration of contact with endoderm/signal

  12. Origin of mesoderm: Nieuwkoop experiments “young” • “heterochronic” transplants: competence of ectoderm to form mesoderm restricted to gastrula stages “old”

  13. What is the mesoderm-inducing signal? • secreted factor • act at a distance • expressed by endoderm at right time (late blastula/gastrula stages) • ectoderm competent to respond Finding the signal: • “cut and dump” cap assay: expose caps to candidate secreted factors, assay for response • functional cap screen: inject eggs with candidate genes, cut young caps, assay for response • expression screen: determine endoderm-specific transcripts • 30+ years of screening: TGFβ and FGF signaling

  14. What is the mesoderm-inducing signal?

  15. What is the mesoderm-inducing signal? FGF signaling pathway

  16. What is the mesoderm-inducing signal? • brachyury: founding member of T-box transcription factor family • expressed throughout mesoderm • crucial for posterior mesoderm development (similar to FGFs)

  17. Additional findings • Brachyury activates Fgf expression, leading to positive feedback loop • FGFs do not induce the entire mesoderm spectrum, only posterior fates • FGFs do not have unique targets involved in mesoderm induction

  18. Additional findings • VegT directly activates expression of Nodal • Nodals induce expression of their antagonist, Lefty (mechanism for restricting inductive properties)

  19. Making matters more complex… animal Spemann’sorganizer (dorsal blastopore lip) β-catenin vegetal • vegetal β-catenin moves dorsally after fertilization • induces high levels of Nodal expression • Nodal induces expression of “dorsal determinants” ventral dorsal

  20. Discovery of the dorsal organizer Spemann-Mangold experiment *Donor organizer will induce/respecify host tissue to more dorsal fates!*

  21. Spemann’s organizer: patterning the DV axis animal high Nodal vegetal ventral ventral dorsal dorsal

  22. Spemann’s organizer: patterning the DV axis Bra Gsc Nodal ventral dorsal

  23. Spemann’s organizer: patterning the DV axis

  24. Evolutionary conservation of mesoderm induction yolk • Main differences • holoblastic vs. meroblastic cleavage • endoderm formation is cell non-autonomous • no maternal VegT homologue (zygotic homologue does not activate Nodal expression)

  25. Evolutionary conservation of mesoderm induction • Conserved mechanisms • Nodal signaling is required • β-catenin induces high levels of dorsal nodal • dorsal organizer (shield) inhibits ventral signaling for DV patterning • brachyury expressed throughout mesoderm • Wnt, FGF, BMP signaling maintain and pattern mesoderm fates ectoderm neuro-ectoderm mesoderm ventral lateral dorsal organizer endoderm (gut) yolk ectoderm and tail somites neuro-ectoderm yolk “no Nodal”

  26. Recap: vertebrate germ layer formation • Endoderm origin varies between species • Cell autonomous in Xenopus: maternal VegT • Cell non-autonomous in zebrafish: maternal YSL signals • Mesoderm is induced by signals (e.g. Nodal) from endoderm and maintained by feedback loops (FGF-Wnt-Brachyury) • Concurrent with dorsal-ventral patterning • Non-dorsal mesoderm produces FGFs, Wnts, BMPs • Dorsal organizer inhibits ventral signals to form signaling gradients and pattern mesoderm

  27. Applying knowledge of embryonic tissue induction

  28. Applying knowledge of embryonic tissue induction mouse embryonic stem cells induced to cardiac muscle using FGF, BMP, and low activin; day 7 of induction protocol

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