Xenopus Cleavage and Gastrulation II A Molecular Focus

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

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

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

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

• One more important concept before we begin. . .

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

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

• 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?

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

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

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?

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

• 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. . .

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

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?

Hunting for Signaling Molecules • Needed to be able to screen c. DNA libraries, clone the m. RNA’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

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

Adherens junction: ß- catenin

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 induces a 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!