Molecular Insights into Cleavage, Gastrulation & Axis Formation in Amphibians

1. Xenopus Cleavage and Gastrulation II: A Molecular Focus Gilbert - Chapter 10

2. Today’s Goals • Become familiar with the concepts of Cleavage, Gastrulation and Axis Determination • Become familiar with the types of cell movements in the embryo • Describe the processes of Cleavage and Gastrulation in Sea Urchin and Xenopus embryos • Become familiar with the types of genes that help guide gastrulation

3. As we move on, it will be important to remember • Differentiation: the development of specialized cell types • Commitment • Before the cell actually overtly differentiates, a period of cellular commitment occurs • Specification • Reversible • Autonomous and Conditional • Determination • Not reversible • Mosaic vs. Regulative Development

4. Amphibian Gastrulation • We’ll more closely examine some of the “regulative” aspects of Xenopus (and newt) gastrulation • Specifically • How cells interact with one another during cell migration • How cells signal to each other to determine cell fates

5. One more important concept before we begin . . .

6. Cell Signaling • One group of cells changes the behavior of an adjacent group of cells • (shape, mitotic rate, fate, gene expression) • This is called induction • The cells that will produce signal = inducer • Cells that receive signal = responder

7. Cell Signaling • For this to happen: • Inducer must produce signal molecule • Responder must be competent to receive that signal! (and process it) • Example: • Signaling molecule is a secreted growth factor • Responder must have receptors on the cell membrane specific to that growth factor to receive that signal

8. What if we waited until the next cleavage to transplant the cells? • Would back cells still be competent to receive the signal that they are now belly tissue? • Would the belly cells still be secreting that signal?

9. So - now back to Amphibian gastrulation • Let’s apply these concepts. . .

13. AXES ARE FORMED!

14. Amphibian Axis Formation and “The Organizer” • Amphibian gastrulation and axis formation are an example of regulative development • Inductive interactions occur between cells • This was demonstrated by Hans Spemann and Hilde Mangold (University of Frieburg, early 1900’s) • Nobel Prize winners

16. The Grey Crescent • If one blastomere does not contain a portion of the grey crescent, it will not form a normal embryo • Conclusion: grey crescent is essential for proper embryonic development • What is so special about the grey crescent? • Fate maps show that these cells form the Dorsal lip of the blastopore! • Dorsal lip cells initiate gastrulation • These cells must be committed when the grey crescent forms - but how?? • What is in the grey crescent that commits them?

17. Spemann and Mangold • Performed many types of transplants at the early gastrula and late gastrula stages in the newt embryo • High amount of technical difficulty! • Results were fascinating and sent many developmental biologists on a hunt for signaling molecules

19. These experiments showed that in most cases, the cells of the embryo are not committed until at least the late gastrula stage • But - There is ONE tissue from the early gastrula that is already committed. . .

22. The Organizer • Spemann dubbed the Dorsal lip of the blastopore as “the organizer” • Induced ventral cells to form neural tube and somites • Organized the axis of the embryo

23. The organizer: more questions than answers! • How did the organizer get its abilities? • Why is the dorsal blastopore lip different than rest of embryo? • What factors are secreted to cause the induction of the axes?

24. The Nieuwkoop Center • Pieter Nieuwkoop’s and Osamu Nakamura’s experiments of the 1970’s • Recombined parts of the blastula to examine cellular inductions • They discovered that the dorsal-most vegetal cells are capable of inducing the dorsal blastopore lip to begin • Dubbed these cells the “Nieuwkoop Center” • Remember: this area is formed by cytoplasmic rotation after fertilization!

26. Other findings by Niewkoop • Found that the vegetal cells that are destined to become endoderm • Can induce the ectoderm cells above to become mesoderm

27. The Organizer: Molecular mechanisms • The discovery of the Niewkoop center’s induction of the ectoderm (by endoderm) to form mesoderm sent scientists on a hunt for the signaling molecule/s involved . . . • Induction of mesoderm? • Induction of the organizer? • How does the organizer induce the series of events that lead to gastrulation and axis formation???

28. Hunting for Signaling Molecules • Needed to be able to screen cDNA libraries, clone the mRNA’s that could mimic these inductions • Choosing candidate molecules also became easier with help from Drosophila studies • Weischaus, Nusslein-Volhard, Lewis • Massive mutagenesis screen to find all genes essential for fly embryogenesis • Frog embryologists could try out some of those candidate molecules as well

30. Niewkoop Molecules • Xenopus brachyury (Xbra) • A transcription factor that helps trigger endoderm induction of the ectoderm to form mesoderm • Expressed throughout the vegetal hemisphere • NOT a good candidate for induction of the organizer

31. Inducing the Organizer • -catenin • Protein that accumulates in the dorsal portion of the egg after fertilization • 2 known functions: Cell adhesion, nuclear transcription factor in the WNT signaling pathway • A possible candidate

32. Adherens junction: ß- catenin

34. ß-catenin and organizer induction: The evidence • ß-catenin continues to accumulate in the dorsal most vegetal cells • SO: it’s in the right place at the right time! (expressed) • BUT: can it do the job?? (Overexpression?) • AND: Is it essential for getting the job done? (KO?) • Injection of ß-catenin on the ventral side of the embryo inducesa secondary axis • SO: it can do the job! • Depletion of ß-catenin using anti-sense oligonucleotides results in the lack of dorsal structures • SO: it is essential for getting the job done!

35. How does ß-catenin become localized to the Niewkoop center? • ß-catenin is initially synthesized from maternal mRNA’s throughout the embryo • In the ventral cells, GSK-3 degrades the ß-catenin • It is proposed that during cytoplasmic movements after sperm entry, an inhibitor of GSK-3 is specifically placed at the site opposite that of sperm entry (future organizer!)