“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 blastula animal vegetal

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

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

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

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) AB X X X X 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)

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

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

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

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

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

What is the mesoderm-inducing signal?

FGF signaling pathway

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)

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

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

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

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

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

Spemann’s organizer: patterning the DV axis dorsalventral Nodal GscBra

Spemann’s organizer: patterning the DV axis

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

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 yolk neuro- ectoderm organizer endoderm (gut) ectoderm yolk neuro- ectoderm tail somites and mesoderm ventrallateraldorsal Evolutionary conservation of mesoderm induction “no Nodal”

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

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

Nieuwkoop’s Classic Induction Experiments