1. Dorsal and Ventral Gene Expression Associated with Neural Induction in Xenopus embryos Notes from review paper by De Robertis and Kuroda, 2004 Jennifer Slade B.Sc (Hon), M.Sc Candidate

2. Overview Introduction to Xenopus –Life cycle and stages of development –Fate map of germ layers Discovery of Induction –Possible genes involved in induction Formation of signaling centers –Dorsal Nieuwkoop and BCNE Spemann –Ventral Dorsal Genes –BMP antagonists: Chordin, Noggin and Xnr3 –Wnt antagonists: Frzb-1, Crescent, sFRP-2 and Dickkopf –Cerberus Ventral Genes –BMP synexpression group: Crossveinless-2, Twisted Gastrulation, Xolloid- Related and Bambi –Sizzled Summary

4. Dorsal/Ventral (D/V) Axis Formation: –Sperm fertilizes egg at animal pole –Cortical rotation shifts vegetal pole 30º –Point of sperm entry becomes ventral –Point opposite of sperm entry becomes dorsal D/V axis gives rise to backbone and belly Dorsal ectoderm: Neural Plate and CNS (therefore want neural induction) Ventral ectoderm: Epidermis and its derivatives (therefore do NOT want neural induction) Mesoderm: Dorsal Ventral »Prechordal Plate, Notochord, Somites, Kidney, Lateral plate and ventral blood islands »Neural Induction Less Neural Induction Sperm entry Ventral Dorsal

5. Fate Map of Germ Layers in Blastula Animal Vegetal VentralDorsal VM DM Endoderm Mesoderm Ectoderm DEcVEc VEnd DEnd

7. Discovery of Induction in Mesoderm Spemann and Mangold, 1924 Transplanted small piece of dorsal mesoderm from one embryo into ventral mesoderm of another Changed differentiation of neighbouring ventral cells –dorsalized –Became CNS, somites rather than epidermis Twinned embryo

8. What genes are involved in induction? Transplant was from dorsal to ventral –Neural induction occurs at dorsal, and not at ventral Therefore genes that affect dorsal and ventral axis formation may be involved… Possible genes discovered through mutations observed in other model organisms –Seven genes in zebrafish –Seven genes in Drosophila –All associated with members from bone morphogenetic protein (BMP) signaling pathway

9. Another Gene Present After D/V Formation β-catenin accumulates in dorsal blastula after cortical rotation Moves from cytoplasm into nuclei to induce transcription of various genes –Siamois – dorsalizing gene –Nodal-related genes – specify Nieuwkoop center Partially regulated through Wnt signaling Important in the formation of signaling centers

10. Formation of Dorsal Signaling Centers Two signaling centers: –Nieuwkoop center (dorsal vegetal) Induces dorsal mesoderm in overlying cells –Nodal-related factors (Xnr1, 2, 4, 5 and 6) Nieuwkoop center cells themselves form dorsal anterior endoderm –Blastula Chordin and Noggin expression center (BCNE) (dorsal animal) Eventually gives rise to anterior neural tissue –Chordin, noggin and Xnr3 Both form simultaneously when zygotic transcription start Both require β-catenin signaling

11. “Combination” of Nieuwkoop and BCNE: Spemann Organizer Once blastula becomes gastrula, signals from Nieuwkoop center lead to the generation of the Spemann organizing center at the dorsal lip In addition, a ventral signaling center is established

12. Dorsal Gastrula Genes Expressed from Spemann Organizer Include: –BMP signaling antagonists: Chordin, noggin and Xnr3 –Wnt signaling antagonists Secreted Frizzled-related proteins (sFrps), and Dickkopf –Cerberus

13. BMP Signaling BMP’s are growth factors Binds to BMP receptors (BMPR) Interacts and phosphorylates SMAD1 leading to interactions with other SMAD proteins from TGF-β signaling pathway Complex enters nucleus to repress transcription of target neural genes Overall: BMP signaling reduces neural induction SMAD1 P P SMAD4 Neural Genes BMP

14. BMP Antagonist: Chordin Large protein, about 1000 amino acids Contains four cysteine-rich domains (CR1 – CR4) of about 70 residues each –Constitutes BMP binding Binding to BMP proteins prevents them from binding to their receptor –Reduces BMP signaling –Allows neural induction High concentrations of Chordin dorsally –Enough to block BMP signaling by itself When Chordin is knocked-out in mice: –Small percentage of embryos ventralized Therefore must be other anti-BMP genes present

15. BMP Antagonist: Noggin Binds to BMP as Chordin does –Contains cysteine-knot structural motif –Shares conserved protein fold as BMP Knocked-out Noggin in mice: –Still form normal gastrula and neural plate Both Chordin and Noggin knocked out in mice: –Embryos exhibit loss of neural development, thus both are required –Redundant function – same as Chordin

16. BMP Antagonist: Xenopus Nodal Related – 3 (Xnr3) Nodal-related protein –Similar to those expressed in Nieuwkoop center –Lacks mesoderm inducing activity When overexpressed in animal pole: –Induces neural differentiation Antagonizes BMP signal through amino-terminal proregion –Mechanism still unclear In Xenopus, after Chordin, gene most induced by early β-catenin signal

17. Wnt Signaling Wnt is a secreted glycoprotein that binds to Frizzled receptor Has 2 functions: –Activates Dishevelled (Dsh) which leads to neural induction –Deactivates a degradation complex to reduce degradation of β-catenin Increase β-catenin induced transcription of genes Can either have: –Blocked Wnt signaling –Just blocked Wnt/β-catenin signaling Dsh Degradation Complex Nucleus Wnt Neural Induction Βeta-catenin

18. Wnt Antagonist: sFRPs secreted Frizzled-Related Proteins Secrete forms of the amino-terminal cysteine-rich domain of Wnt receptor –Like Frizzled receptor without “bottom” –When Wnt binds, no signaling cascade is initiated –Binding of Wnt proteins lowers binding to normal Frizzled receptor and signaling High level of expression in Xenopus dorsal gastrula Include Frzb-1, Crescent and sFRP2 –Frzb-1 dorsalizes mesoderm

19. Wnt Antagonist: Dickkopf (Dkk-1) Encodes cysteine-rich secreted protein Antagonizes Wnt/β-catenin signaling only Dkk-1 binds Wnt coreceptor LRP6 –Binding of LRP6 to Wnt and Frizzled forms a ternary receptor complex –Draws one member of the β-catenin degradation complex to cell membrane, thereby reducing degradation of β-catenin Therefore Dkk-1 can selectively inhibit Wnt induced degradation of β-catenin without affecting rest of Wnt signaling Dkk-1 also binds transmembrane protein Kremen –Binds to LRP6 at same time –Endocytosed, depleting LRP6 Inactivation of Dkk-1: no head or prechordal plate formation

20. Cerberus Cytokine Antagonist of: –BMP –Wnt –Nodal signaling Responsible for head induction –Can induce ectopic heads Knocked-out: –No head formation –Only trunk and tail

21. Ventral Gastrula Genes Expressed at ventral signaling center –Ventral mesoderm and ectoderm –180º from Spemann Organizing center Ventral signaling was not viewed important as it lacked inductive activity, but now that loss of function mutations in ventral signaling centers are known, this may be an area of focus in the future Include: –BMP synexpression group: Crossveinless-2 (Cv-2) Twisted Gastrulation (Tsg) Xolloid-Related –Sizzled

22. BMP Synexpression: Crossveinless-2 (Cv-2) Originally discovered in Drosophila –Forms wing cross-veins Contains 5 cysteine-rich domains –Similar to those of Chordin Normal expression is upregulated by BMP4 –Represses Chordin expression –Allows BMP signaling When overexpressed, has a BMP antagonist activity

23. BMP Synexpression: Twisted Gastrulation (Tsg) and Xolloid-Related Tsg binds to both BMP and Chordin Forms ternary complex to prevent binding of BMP to its receptor Promotes cleavage of Chordin –Xolloid-Related acts as scissors to cut Chordin Tolloid metalloprotease Expression upregulated by BMP4 Plays critical role in inactivation of Chordin (in conjunction with Tsg) Releases BMP Allows BMP signaling

25. Sizzled Encodes sFRP-like molecule Has a ventralizing function: –Increases ventral mesoderm –Decreases neural plate formation Overexpression phenotype is indistinguishable from loss of Chordin phenotype Knock-down of Chordin leads to increase in Sizzled expression and increased BMP signaling Exact function still undetermined –Expected to be associated with BMP signaling

26. Summary Number of dorsal and ventral genes affect downstream neural induction Similarities: –Both sets of genes secrete related growth factor antagonists Differences: –Under opposite transcriptional control by BMP signaling Established communication between dorsal and ventral genes Now need to discover what it means and why it happens

27. References De Robertis and Kuroda, 2004. Dorsal- Ventral Patterning and Neural Induction in Xenopus Embryos. Annu. Rev. Cell. Dev. Biol. 20: 285-308 Wolpert, L., et al. 2002. Principles of Development, 2 nd Edition. Oxford University Press, New York. Chapters 2-4