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Early Development in Amphibians Cleavage in Amphibians • Radial, holoblastic • Affected by yolk • First two through AV axis • Third “equatorial” • Blastula of ca 100,000 cells Xenopus Fate Map When Do Zygotic Genes Turn On? • Almost all zygotic expression occurs after 12th cycle in mid-blastula • = mid-blastula transition • Senses changing chromatin cytoplasm ratio When Do Axes Form? • Anterior-posterior axis develops from the animal-vegetal axis – Originates during oogenesis • Dorsal-ventral axis established after fertilization – Before first cleavage Animal-Vegetal Axis in Oocyte • Yolk protein (vitellin) accumulates in vegetal half • Cortical cytoplasm of animal half accumulates dark pigment What Else Localizes Along A-V? • Transport system (MTs) localizes some maternal mRNAs to different poles – ex. Vg1 and VegT mRNAs localize to the cortex of the vegetal hemisphere – Vg1 = TGF-b – VegT = transcription factor What Controls A-V Axis? • Asymmetric distribution of factors such as Vg1 and VegT imposes animalvegetal fates How Do We Know? VegT • VegT antisense RNA – Artificial depletion of VegT • Results in altered fate-map: How Do We Know? Vg1 • • • • Deplete Vg1 No endoderm No dorsal mesoderm (why?) Therefore, pre-localized cortical vegetal determinants specify germ layers Can Xenopus Regulate Along Animal-Vegetal Axis? • Fates along the A-V axis are specified through a series of cell interactions • Experiment: isolate blastomeres at 32-cell stage: Mesoderm not autonomous Can Xenopus Regulate Along Animal-Vegetal Axis? • Repeat same experiment on a later stage embryo: – isolate blastomeres at 128-cell stage: – now each produces proper structures in isolation – so induction of mesoderm between 32 and 128 by ? Regulative Specification Along Animal-Vegetal Axis • Which cells are competent to respond to induction by vegetal pole? • Nieuwkoop’s recombination experiments: mesoderm forms Regulative Specification Along Animal-Vegetal Axis • Recombination experiments: – Presumptive endoderm induces pre-ectoderm to form mesoderm Further Regulative Specification Along Animal-Vegetal Axis – Regionally specific • Ventral and lateral vegetal cells induce ventral and intermediate mesoderm (blood, kidney) • Dorsal vegetal cells induce dorsal mesoderm (somites, notochord) Model A-P Polarity • Oocyte early blastula mid-blastula Marginal zone (= region surrounding equator of blastula where animal and vegetal hemispheres meet) is induced by vegetal region early Bl DV Axis Dorsal-Ventral Axis • Arises from cytoplasmic rearrangements following fertilization – Unfertilized egg is still radially symmetrical – Fertilized egg is not D V How Does Dorsal-Ventral Axis Arise? • Cortical cytoplasm rotates 30o relative to internal cytoplasm – Reveals underlying gray pigment – “Gray crescent” – Bisected by first cleavage plane Importance of Gray Crescent Region • Hans Spemann • Artificially displace first cleavage, get – Bauchstuck – ecto, meso, and endoderm, but no dorsal structures (nervous system, notochord, somites) • Or by blocking rotation • So rotation related to DV What Determines Rotation and the Dorsal-Ventral Axis? • Greatest displacement of cortical cytoplasm occurs opposite point of sperm entry (SEP) • Gastrulation will begin in this region (blastopore lip) • Identifies future dorsal side Twins Produced by Rotation • Move SEP up just before first cleavage • Gravity displacement forms new axis