1. Developmental Biology – Biology 4361 Axis Formation and Mesoderm Induction October 27, 2005

2. Amphibian anteroposterior specification - polarized eggs – animal/vegetal - localized cytoplasmic components - pigment - yolk v. clear cytoplasm - mitochondrial cloud - germinal vesicle

3. Figure 8.25 RNA localization – Xenopus oocytes

4. Figure 9.7 Anteroposterior axis – VegT depletion normal

5. Figure 9.7 depletion of VegT = - shift from endoderm to mesoderm and ectoderm - mesoderm replaced with ectoderm - animal region forms only epidermis and no nervous system Anteroposterior axis – VegT depletion normalVegT - depleted

6. Figure 9.18 Dorsalization - Xenopus UV = ventralized

7. Figure 9.15 Transplantation of dorsalizing activity

8. Figure 9.19 Early Dorsoventral Determination

9. Gray crescent formation

10. Cortical rotation and Disheveled sperm Dsh Disheveled protein (Dsh) 1. Fertilization 2. Cortical rotation 3. Dorsal enrichment of Dsh

11. Figure 9.21 gylcogen synthase kinase-3 Disheveled activity

12. Figure 9.21 gylcogen synthase kinase-3 Disheveled protein blocks GSK-3 phosphorylation of  -catenin Disheveled activity Transcription factor

13. Molecular basis of dorsoventral axis  -catenin stabilized Repressed TGF-b signaling pathway  -catenin degraded Tcf-3 proteins siamois gene transcription goosecoid gene Goosecoid protein  -catenin proteins siamois gene Siamois protein Activated

14. Organizer transplant Spemann’s organizer – dorsal lip of the blastopore

15. Organizer transplant

16. Gilbert: Developmental Biology, 7 th ed (2003) Table 10.2. chordin noggin nodal-related proteins (several) XLim1 Xnot Otx2 XFD1 XANF1 Goosecoid Cerberus Follistatin Frzb Secreted ProteinsNuclear Proteins “Organizer” proteins - expressed almost exclusively in the organizer

17. goosecoid mRNA can induce a second dorsal axis: goosecoid mRNA injection causes formation of a second dorsal blastopore lip Gilbert: Developmental Biology, 7 th ed (2003) Fig 10.28. Organizer gene activity produces embryo with two dorsal axes and two sets of head structures

18. Rescue of dorsal structures by noggin protein: Gilbert: Developmental Biology, 7 th ed (2003) Fig 10.30. “overdose” of noggin mRNA causes formation of dorsal structures at the expense of ventral structures dose-dependent induction of dorsal structures by injection of noggin mRNA ventralized embryo without dorsal structures (UV-irradiated) Organizer gene activity noggin binds to bone morphogenic proteins (BMP2 & BMP4) - inhibits binding BMP receptor binding

19. chordin mRNA is localized in the ‘organizer’: Gilbert: Developmental Biology, 7 th ed (2003) Fig 10.32. Organizer gene activity - inhibition of BMP4 & BMP2 induces formation of the neural tube in adjacent ectoderm - chordin protein binds to BMP4 and BMP2 – inhibits receptor BMP-receptor binding - late in gastrulation, chordin is localized in the dorsal mesoderm of the notochord - chordin mRNA is found in the dorsal lip

20. Figure 9.8 Mesoderm induction - Xenopus

21. Figure 9.8 Mesoderm induction - Xenopus

22. Figure 9.8 Mesoderm induction - Xenopus

23. Figure 9.9a, b Mesoderm induction - Xenopus mesoderm inducers: bFGF Vg1 activin

24. Mesoderm induction, Organizer formation β-catenin VegT, Vg1 Nodal related high Nodal related low Organizer Ventral mesoderm 1. β-catenin acts with VegT and Vg1 to activate Xnr genes (Xenopus Nodal-related) 2. Organizer originates in the region where VegT & Vg1 and β-catenin overlap 3. Gradient of Xnr protein specifies mesoderm: low Xnr  ventral mesoderm 4. High Xnr levels activate goosecoid and other ‘organizer’ genes BMP4 high BMP4 low

25. Left-right asymmetry Most animals are bilaterally symmetrical (  Bilateria) - however, individuals deviate to some degree from true bilateral symmetry: - regular asymmetry or directed asymmetry: sidedness is fixed for a species or for a higher taxon e.g. in humans: - heart on left side - stomach curves to the left - liver & spleen on right side - fluctuating asymmetry: non-heritable minor left-right differences - antisymmetry: heritable morphological left-right differences - sidedness is randomly distributed (ca. 50% each)

26. - situs inversus: complete reversal of left-right symmetry in all organs - heterotaxis: some organs reversed - isomerism: normally asymmetrical organs duplicated or missing Left-right asymmetry Deviation from directed asymmetry is often lethal!

27. Left-Right Asymmetry Mechanistic basis for establishing asymmetry: - translated into left-right differences at the level of cells, tissues and the whole organism - chiral molecules may cause “symmetry-breaking” event (specific orientation of stereoisomeric molecules relative to the body axes) Candidate chiral molecule: Dynein - motor protein complex associated with axonemes, cilia

28. Fig 2.7. Dyneins - microtubule-associated motor protein complexes - chiral: curve clockwise (from base) = ‘handedness’ - mediate sliding between adjacent microtubules in cilia or flagella - cause cilia to rotate in a specific direction (clockwise) - monocilia (at Hensen’s node - mouse) generate oriented flow of signal molecules to the left side of the embryo - signal molecules activate or inhibit patterning genes on left side Axonemal dyneins: Left-Right Asymmetry

29. Iv + and Inv + - iv protein is a left-right dynein - iv - /iv - = no motility, no fluid flow - randomized L-R asymmetry (lethal) iv+: ‘situs inversus viscerum’ - wild type & heterozygous embryos turn clockwise inv+: “inversion of embryonic turning” - inv - /inv - turn counterclockwise in amniotic cavity - mechanism of inv action is unknown - 100 % of homozygotes for inv show situs inversus

30. nodal expression in mouse: Nodal activated by iv,inv wild typeectopic Nodal - nodal is involved in determining left-right asymmetry in mice, frogs, chicken & zebrafish - ectopic expression of nodal on right side randomizes location of the heart - mesoderm adjacent to nodal expression develops into asymmetrical organs - nodal protein synthesized in left lateral plate mesoderm - nodal gene activated by iv and inv genes - intracellular protein - TGF-β family

31. pitx2 injection in Xenopus: Pitx2 & lefty activated by iv, ivn, nodal pitx2+ and lefty+ genes : - pitx2 expression depends on iv, ivn and nodal genes - pitx2 and lefty encode homeobox transcription factors that regulate genes - both are expressed primarily on left side of vertebrate embryos have been found in all vertebrates studied - injection of ptx2 on right side of embryo - can cause a complete reversal of gut coiling and heart looping nodal, pitx2 and lefty form an evolutionary conserved signaling system that is involved in regulating left-right asymmetry in all vertebrates