Axis determination in frog embryosSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Axis determination in frog embryosHow are embryonic axes set up?What signaling events regulate embryo development?Time-lapse videos of Xenopus gastrulation:By David Shook :http://faculty.virginia.edu/shook/ShowMovies/index.htmBy Mike Danilchik:http://worms.zoology.wisc.edu/frogs/gastxen/gastxen_sagview.htmlOverview of frog gastrulationFuture Dorsal side (back)Future Ventral Side (belly)Sperm entry in animal hemisphereVg1 mRNA(in situ hybridization)A 3’ UTR sequence element localizes Vg1 mRNA(From Mowry and Melton (1992) Science 255: 991-993)i) Inject RNA molecules into oocyteii) Isolate animal and vegetal RNAsiii) Northern blot using Vg1 probeA 3’ UTR sequence element localizes Vg1 mRNA(depends on microtubules, mRNA-binding proteins)(From Mowry and Melton (1992) Science 255: 991-993)Sperm entry provides the asymmetric cue to set up the dorsal-ventral axis of Xenopus embryosCortical rotation requires the microtubules – may be nucleated by the centriole delivered by sperm.UV irradiation on vegetal side prevents cortical rotation.No dorsal axis forms, no gastrulation occurs. Embryo is ventralized.Rotate zygote in a new plane at time of first cleavage: two blastopores formRotated zygotes after gastrulation and hatchingRotate zygote in a new plane at time of first cleavage: two blastopores formShows sufficiency.Prevent normal cortical rotation by UV-irradiating vegetal side of zygote: No dorsal axis or gastrulationShows necessity.Does cortical rotation induce the dorsal axis?Cortical rotation creates asymmetry of cytoplasmAsymmetric cell divisions – a general way to create distinct cell fates in daughter cells.(Relies on asymmetry in mother cell = cell polarity)Are cell identities determined (in developmental sense) as shown in the fate map?Fate map of late blastulaExperimental test of specification.Specification map: shows what the cells have been instructed to becomeExplant early blastula tissues: don’t get mesodermearlyTransplanting dorsal vegetal cells restores axis formation in embryos from UV-irradiated embryos(32-64 cell stage)Figure 10.11Transplanting dorsal vegetal cells to the ventral vegetal side causes a second axis to formTransplant organizer just before gastrulationTransplanted organizer~64-cell stage late blastulaMesoderm inductionSignals 3 and 4 induce further specialization of mesoderm cells.Different parts of the dorsal mesoderm signal differentlySource of transplant: Anterior mesodermPosterior mesodermDorsal blastopore lip signals differently at different times(Actually different cells at different times)Can responding cells acquire any fate?Transplanting presumptive epidermisCan responding cells acquire any fate?Transplanting presumptive neural ectodermTransplant later, get different result:Cells have become determined (or, perhaps an epidermal signal is missing
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