Early Development and Axis Formation in AmphibiansBiology 4361July 9, 2009The Amphibian Model1) How are the body axes established? - How do relatively homogeneous cells (i.e. zygotes) establish polarity (complexity from simplicity)?2) How are the germ layers determined?- How do zygotes send their cellular progeny (i.e. blastomeres) down pathways toward different fates?3) How is development organized in a regulative system?Common vertebrate (e.g. Rana, Xenopus)Manipulable, observable (i.e. large eggs and embryos)Amphibian Development - OverviewCleavageFertilization, cortical rotationGastrulationAxis and germ layer determinationThe “Organizer”MesodermDorsal/organizerInductions: EctodermAxes Summary(Photo by Harland lab/UC Berkeley)Left – Right AsymmetryCortical RotationFertilization – animal hemisphereMicrotubular network originates at fertilization pointCleavage PatternsMeroblastic (incomplete cleavage)Holoblastic (complete cleavage)cephalopod molluscsfish, reptiles, birdsmost insectsechinoderms, amphioxisannelids, molluscs, flatwormstunicatesmammals, nematodeamphibiansSpeciesSpiralBilateralRadialRotationalDisplaced radialBilateralDiscoidalSuperficialCleavageYolk classificationIsolecithalTelolecithalCentrolethicalMesolecithalUnequal Radial Holoblastic CleavageCell cycles regulated by mitosis-promoting factor (MPF)- no G phases… until mid-blastula transition- promoters demethylated (i.e. de-repressed)- transcription factors formed in vegetal cytoplasm- embryonic control of developmentMechanics of GastrulationFormation of the dorsal lip- vegetal rotation- invagination of bottle cells- involution of marginal zone cellsInvoluting Marginal ZoneMechanics of Gastrulation - 2motive force(D)Xenopus GastrulationCell Movements during GastrulationMovements- invagination- involution- epiboly (animal cap)- intercalation andconvergent extension- migrationXenopus GastrulationXenopus Gastrulation - BlastoporeNIMZAmphibian Development - OverviewCleavageFertilization, cortical rotationGastrulationAxis and germ layer determinationThe “Organizer”MesodermDorsal/organizerInductions: EctodermAxes Summary(Photo by Harland lab/UC Berkeley)2-cellstageDetermination of Amphibian AxesIf one blastomere received no gray crescent material, resulted in- “belly piece” – blood, mesenchyme, gut cells-no dorsal structures(e.g. notochord, somites)The gray crescent area is criticalfor proper developmentGray crescent = future dorsal lip of the blastoporeDorsal Lip TransplantDoral lip = “Organizer”- organizes secondary D-V axis- induced ventral cells to change fates(Spemann, Mangoldexperiment)Amphibian Development - OverviewCleavageFertilization, cortical rotationGastrulationAxis and germ layer determinationThe “Organizer”MesodermDorsal/organizerInductions: EctodermAxes Summary(Photo by Harland lab/UC Berkeley)Left – Right AsymmetryThe Organizer- head mesoderm (prechordal plate)- chordamesoderm (notochord)Transplantation experiments established the organizing propertiesof the dorsal blastopore lip, which …2) Establishes the dorsal-ventral axis3) Specifies multiple tissues, including…1) Self-differentiates (all other tissues conditionally specified)- dorsal mesoderm, which includes…4) Dorsalizes surrounding mesoderm into paraxial mesoderm 5) Induces the neural tube6) Initiates the movements of gastrulationHow is the dorsal lip specified?Dorsal Signal: β-Cateninβ-catenin is initially distributed throughout the embryo,- accumulates only in prospective dorsal cells.- concentrated in the Nieuwkoop center and organizerOrganizerNieuwkoopcenterβ-cateninDorsalization of β-catenin:a) protect β-catenin in dorsal area,b) degrade β-catenin everywhere else.Mechanism - cortical rotation …(in sea urchins, specifies micromeres, endomesoderm)nuclear transcription factor (in Wnt pathway)β-catenin(in Xenopus, specifies dorsal structures; e.g. organizer)Disheveled/β-Catenin/Cortical Rotationβ-catenin is initially distributed throughout the oocyteβ-catenin induces cells to dorsal fatesGSK3 marks β-catenin for degradationGlycogen synthase kinase 3 (GSK3)is also distributed throughout oocyteDishevelled (Dsh) blocks GSK3 activityDsh localizes in the cytoplasmic cortex at the vegetal poleDisheveled/β-Catenin/Cortical Rotationβ-catenin, GSK3 distributed throughoutGBP – GSK3 binding proteinDsh – Dishevelled Kinesin - motorprotein- anywhere Dsh exists, β-catenin survivesDishevelled - blocks GSK3-mediated β-catenin degradationDisheveled/GSK3/ β-Cateninbut Disheveled and GBP block GSK3;- resulting in β-catenin present onlyin the marginal area opposite the point of sperm entry (i.e. future dorsal lip)GSK3 mediates β-catenin destruction ……At the blastula stage, β-catenin is locatedexclusively in the future dorsal regionOrganizer Inductionβ-catenin acts with Tcf3 (transcriptionfactor); stimulates expression of dorsalizing genes:Siamois – TF; activatesXlim, goosecoid(dorsal determinants)Goosecoid protein –TF responsible for organizer propertiesGoosecoidalso plays a part inspecifying dorsal mesoderm;however, additional vegetal factors are needed: e.g. Vegetal TGF-β signalsMesoderm/Organizer Induction(mid-blastula)VegT, Vg1, Xnr(s) –Xenopusnodal related genes; TGF-β family(late blastula)(early gastrulation), dorsal and lateral mesoderm- somites, notochord- mesenchyme, bloodmuscle, kidney& intermediateDetermination of EctodermLater gastrula cells are determined- exhibit autonomous developmentEarly gastrulation stage cells are uncommitted- exhibit regulative developmentEstablishment of Axes - SummaryD-V axis – set up at fertilizationA-P axis – established by gastrulationmovements across the dorsal lip ofthe blastoporeL-R axis – Nodal expression on Left, not RightLeft – Right AsymmetryLeft–Right axis establishedby Xenopus nodal-related(Xnr1)Xnr1 expression is limited to the left side in a process involving cortical rotation and Vg1gut coiling – counter-clockwiseheart loops to the leftBlock Xnr1 expression = random gut coiling, heart loopingNodal expression: - common to all vertebrates- expressed on the left
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