1. Antibody staining (Lecture 3, p.4)

2. In situ hybridization (Lecture 4 p.4)

3. mRNA depletion (“knockdown”) methods (Lecture 3, p.4)

4.  -catenin builds up Axin Wnt is absent.  -catenin mRNA is continuously translated and the protein is continuously degraded. Wnt is present.  -catenin protein is not degraded. It builds up, binds to Tcf/LEF and enters the nucleus. Wnt11 protein Outside cell Inside cell  -catenin mRNA

5. Wnt, beta-catenin, and organizer formation * * * * * * *

6. Endo-mesoderm induction. Pieter Nieuwkoop 1968

7. (Nieuwkoop center)

8. Nodal signaling pathway (belongs to TGF-beta family of pathways) phospho- Smad2

9. A dominant negative form of the Nodal (or Bmp) receptor can block Nodal (or Bmp) signaling

10. Deplete and replace vegT mRNA vegT depleted No mesoderm or endoderm, No blastopore vegT mRNA restored by injection into vegetal hemisphere vegT depleted and restored

11. VegT depleted embryos: rescue with mRNA for xnr1, 5, 6, or derriere, injected into vegetal pole cells Normal late blastula: xnr1 expressed in vegetal cells derriere rescue xnr5, xnr6 rescue xnr1 rescue xnr1