1. Chapter 10- Amphibians Large yolks- hence, animal pole cleavage dominates, while vegetal pole cleavage lags 123 4 micromeres + 4 macromeres (animal pole) 4 macromeres (vegital pole) Termed “morula”- 16-128 cells 4 5 8 Blastocoel Cleavage Fig. 10.1

2. Gastrulation Use Xenopus as a model Three tasks- 1.To bring inside those cells destined to be endodermal organs 2.To surround embryo with cells destined for ectoderm 3.To place cells cells “in between” destined to be mesoderm Ectoderm- skin/nerves Endoderm- Gut and associated organs Mesoderm- Blood, heart, kidney, bones VegT establishes animal/vegital pole VegT Antisense results in lack of endoderm Fig. 10.6

3. Gastrulation Gastrulation begins in marginal zone at dorsal lip, formed by bottle cells Cells migrate toward dorsal lip, then turn inward into interior Waves of cells continue to enter through dorsal lip Animal pole cells migrate downward and eventually encase embryo, except for yolk plug, termed epiboly (see fig. 8.6) Blastocoel function 1. permits cell migra- tion 2. Prevents premature interactions Animal Vegital Archenteron- formed by invading cells Fig. 10.7

4. Gastrulation Fig. 10.7 Order of cells entry through dorsal blastopore 1. Pharengeal endomesoderm (foregut) 2. Notochord and somite precursors 3. Heart, kidney and ventral mesoderm

5. Gastrulation Positioning the blastopore The point of sperm entry marks the ventral side The opposite side marks point of gastrulation initiation The cortical cytoplasm rotates 30 degrees toward sperm entry point Sperm entry Dorsal blastopore lip formation 90 min 0 min 30 o Fig. 7.35 Some details on Xenopus gastrulation

6. How do the invading cells know to crawl along inside of animal pole? Answer- fibronectin Cells on the blastocoel roof secrete fibronectin, which invading mesodem cells will track along Mesoderm receptors recognize the Arg-Gly-Asp peptide Evidence- if inject Arg-Gly-Asp peptide, disrupt involution Fig. 10.13

7. What dictates axis formation? Answer- the Spemann organizer – aka the dorsal lip Separate embryo vertically Separate embryo horizontally The dorsal lip is in the gray crescent Fig. 10.18

8. Of all tissues in early gastrula, only dorsal lip of blastopore has its cells fated Transplant dorsal lip onto an embryo, observe a second invagination and a second embryo Fig. 10.20 Thus, Dorsal lip cells dictate primary embryonic induction The Spemann organizer Evidence

9. Yeah, but what is the mechanism of axis formation by the Spemann Organizer?? Fig. 10.21 Answer- signals from vegetal cells Normal “layers” Animal- Ectoderm Marginal- Mesoderm Vegetal-Endoderm If place animal cap next to vegetal cells- animal becomes mesoderm (muscle, etc) Evidence Vegetal cells that induce the Spemann organizer are called the Nieukoop center Organizer Nieukoop center

10. A mechanistic model of axis formation: B-catenin accumulates in dorsal region to activate organizer genes, including goosecoid Fig. 10.23 B-catenin (orange) How does B-catenin accumulate? Hint- It is initially throughout embryo. Recall Wnt signaling pathway from ch. 6 Fig. 6.23 Dishevelled expression inhibits GSK-3, thus stablezing B-catenen

11. A mechanistic model of axis formation: Fig. 10.24 Dishevelled Cortical rotation Fertilization Discheveled released GSK-3 inhibited B-catenin stabilized only in dorsal region Siamois gene B-catenin protein Goosecoid gene Goosecoid protein TGF-  signaling B-catenin activates Samois expression, which activates Goosecoid expression

12. Organizer functions become dorsal mesoderm dorsalize surrounding ventral mesoderm into lateral mesoderm dorsalize ectoderm into neural ectoderm initiate gastrulation movements cause neural plate to become neural tube The ability to

13. Dishevelled GSK-3 B-catenin samois goosecoid But what is goosecoid function?? -- If increase organizer mesoderm, observe expanded region of goosecoid expression Inject goosecoid into 4-cell ventral blastomeres- progeny blastomeres induce cell migration and form dorsal lip Activates two groups of proteins- 1.Bone morphogenesis protein (BMP) inhibitors 2. Wnt inhibitors Examples Noggin, Chordin, Follistatin Cerberus, FRZB, DICCK Evidence of goosecoid’s importance Goosecoid also recruits neighboring cells into the dorsal axis Thus, goosecoid must activate genes involved in dorsal/ventral and anterior/posterior axes

14. Example- Noggin (a BMP inhibitor) If expose embryo to ultraviolet radiation- cortical rotation fails, and embryo lacks dorsal structures How discovered?- cDNAs from a plasmid library injected into UV treated embryos. Noggin mRNA injected Tail + belly Normal Head + belly Only head Fig. 10.29 Noggin functions by binding BMP4 and BMP2, thus preventing these proteins from binding their receptors Chordin and follistatin also act by inhibiting BMP proteins 1.Bone morphogenesis protein (BMP) inhibitors

15. Example- Frzb (a Wnt inhibitor) Fig. 10.36 How?? Frzb functions by binding Xwnt8, thus preventing it from binding the wnt receptor 2. Wnt inhibitors Frzb Only head Normal Fig. 10.36 Fig. 10.35- Xwnt is inhibited by Frzb (produced by Organizer) Thus, the Organizer secretes BMP and Wnt signaling inhibitors to direct head, belly and tail production

16. Thus, BMP4 is the inducer of ventral ectoderm (epidermis) BMP4 is the ventralizer of mesoderm (connective tissue, blood) Noggin, chordin and follistatin (secreted by the Organizer) inhibit BMP4

17. Regional specificity of induction The organizer not only induced the neural tube, but also specifies neural regions- forebrain, hindbrain, spinocaudel region 1 st organizer cells to enter embryo induce brain and head 2 nd organizer cells become spinal cord and tail Transplant young gastrula dorsal lip Transplant advanced gastrula dorsal lip Two heads Two bellies?

18. What molecules are involved in regional specification? Fibroblast growth factors (FGFs)- induces anterior neural tissue into posterior neural tissue Induces forebrain to express hind-brain-specific genes Retinoic Acid (RA)- Overexpression inhibits fore and mid-brain development as wells as prevent posterior structure formation Induces forebrain to express hind-brain-specific genes RA 3. What molecules are involved in left-right axis formation? Vg1 protein activates nodal only on left side, which activates Pitx2 Note that internal organs are not placed symmetrically- heart, gut tube