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Fate Mapping Axolotl Embryos in Early Gastrulation
Background Sincedifferentamphibiansexecutegastrulationindifferentways,thestudyofamphibiangastrulationhasbeencomplex.Apopularmethodusedtoexamineandexploregastrulationanddeterminethefuturefateofcellsinamphibianembryosisthevisualizationofcellmovementbyapplicationofavital(non-toxic)dyetotheregionofinterestontheamphibianembryo.Thisprocessofobservingmorphogeneticcellmovementswithvitaldyeandcorrelatingwuthdevelopmentalfateisknownasfatemapping(Gilbert,2000andHamburger1960). In1929,WalterVogt,anembryologist,usedvitaldyestoconstructfatemapsofamphibianembryos.Hespreadanddrieddyeandagaronamicroscopeplateandcuttheendsofthedyedagartoapplytodesiredregionsoftheembryo.Heplacedthesmallvitaldyechipsonthesurfaceofamphibianembryosatvariousstagesofdevelopmenttostudythemovementsandfatesofvariousregionsoftheembryo.Vogt"sfatemappingstudiesatthegastrulastageintroducedembryologiststoaveryusefulmethodofdeterminingwhichportionsoftheembryobecomewhichlarvaloradultstructuresandtomoderninvestigationsofamphibiangastrulation(Hardin,1995). Objective Cellsoftheamphibianblastulahavedifferentfatesdependingonthepositionofthecells(dorsal-ventral,anterior-posterior,left-right)andhowdeeptheyareinthelayersoftheembryo(Gilbert,2000),Wewillobservethemovementofapopulationofcellstoseewhatthieirdevelopmentalfateisbydyingtheembryossuperficialregionsattwodifferentpositionswithrespecttotheblastoporelip(figure1). Procedure 1)WashalbinoAxolotlamphibiangastrulaeinadishfilledwith100%HEPES-bufferedModifiedSteinberg"sSolution(HBSt).UsingalbinoAxolotl"swillmakeiteasiertotrackthedye. 2)Placeembryosin10%HBStandremovejellycoatusingusingfineforceps. 3)Transportembryosand10%HBSttoblackagarosedishforcontrast. 4)Applyanilebluesulfatedyechiptooneend(animalpoleorvegetalpole)ofthegastrulaeandaneutralreddyechiptotheotherendofthegastrulae(animalpoleorvegetalpole)(figure1).Replace10%Steinberg"ssolutionwithfresh10%HBSt. 5)Observegastrulaeinmicroscopeandtakeapictureondayoneofexperiment.Takeapictureeachdayforthenextfewdaystoobservethemovementofthedyedcellsofthegastrulae.(Note:Todelaygrowth,placegastrulaeat14oC). ResultsandConclusions Dyewasappliedtooppositeendsof6Axolotlembryos(figure2).Overathreedayperiod,theelongationofthestainedmaterialwasnoted.Theembryojustbelowthegreenstar(figure2)wasdyedatoppositeends(leftandright)oftheblastopore.Adye-markedembryosimilartothestar-markedembryo(figure2)wouldbeexpectedtogiverisetotheleftandrightsideoftheneuralectoderm(figure3).Lookingdownontheday2dye-markedembryo,theblueandreddyeappearaslongitudinalstripesonoppositesides.Thebluedyecanbeseenontherightsideoftheneuralectodermandreddyeontheleftsideoftheneuralectoderm(figure3a).Theday3neurula,whichislyingonitsleftside(whichwouldbeexpectedtocontainreddyeontheneuralectoderm),showsthebluedyeontherightsideoftheneuralectoderm(figure3b). Itwasnotedthatwithtime,itbecamehardertosustainthemovementofthedye-markedpopulationsofcells,wherethedyebecamelighterandmuchmoredifficulttotrace.Also,someembryosbecamesickanddied.Inthefuture,itwouldbeagoodideatodyemoreembryosandpossIBLyeventakephotosmorefrequently(i.e.,twiceaday). Figure2.DyemarkedAxolotlembryosondayone.Theembryojustbelowthestarwasdyedontheleftandrightsideoftheblastoporelip.Thatpopulationofcellswouldbeexpectedtofollowthepathwaythroughtheleftandrightneuralectodermshownbelow(figure3). Figure3.(A)dye-markedembryoonday2showingabluelongitudealstipeontherightsideoftheembryoandaredlongitudalstripeontherightsideoftheembryo.Thedyedregionisontheleftandtherightsideoftheneuralectoderm.(B)day3dyemarkedneurulalyingonitsleftside.Thepopulationofcellscontainingthebluedyecanbeseenalongtherightneuralectodermoftheneurula. Acknowledgements IwouldliketothanktheFranklinandMarshallBIOLOGydepartmentforallowingmetoconducttheexperimentintheirlaboratoryfacilitiesandfortheavailABIlityoftheirlaboratorysupplies.AnotherspecialthankyoutomylabpartnersMatthewsBandaandJohnDeLongfortheirassistanceindyingtheembryos.IwouldespeciallyliketothankprofessorCebra-Thomas,mydevelopmentalbiologyprofessor,forherassistnaceinconductingtheexperimentandinterpretingtheresultsoftheexperiment.
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