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Developmental Biology – Biology 4361

Developmental Biology – Biology 4361. Axis Formation and Mesoderm Induction. October 27, 2005. - polarized eggs – animal/vegetal. - pigment. - yolk v. clear cytoplasm. - mitochondrial cloud. - germinal vesicle. - localized cytoplasmic components. Amphibian anteroposterior specification.

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Developmental Biology – Biology 4361

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

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

  3. RNA localization – Xenopus oocytes Figure 8.25

  4. Anteroposterior axis – VegT depletion normal Figure 9.7

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

  6. Dorsalization - Xenopus UV = ventralized Figure 9.18

  7. Transplantation of dorsalizing activity Figure 9.15

  8. Early Dorsoventral Determination Figure 9.19

  9. Gray crescent formation

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

  11. Disheveled activity gylcogen synthase kinase-3 Figure 9.21

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

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

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

  15. Organizer transplant

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

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

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

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

  20. Mesoderm induction - Xenopus Figure 9.8

  21. Mesoderm induction - Xenopus Figure 9.8

  22. Mesoderm induction - Xenopus Figure 9.8

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

  24. Mesoderm induction, Organizer formation Nodal related high BMP4 low β-catenin Organizer VegT, Vg1 Nodal related low BMP4 high 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

  25. Left-right asymmetry Most animals are bilaterally symmetrical (Bilateria) - however, individuals deviate to some degree from true bilateral symmetry: - fluctuating asymmetry: non-heritable minor left-right differences - antisymmetry: heritable morphological left-right differences - sidedness is randomly distributed (ca. 50% each) • - 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

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

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

  28. Left-Right Asymmetry Dyneins - microtubule-associated motor protein complexes Fig 2.7. Axonemal dyneins: - 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

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

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

  31. Pitx2 & lefty activated by iv, ivn, nodal pitx2+ and lefty+ genes : - pitx2 expressiondepends on iv,ivn and nodal genes - pitx2 and lefty encode homeoboxtranscription 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 leftyform an evolutionary conserved signaling system that is involved in regulating left-right asymmetry in all vertebrates pitx2 injection in Xenopus:

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