Objective Inthisexperiment,aregionoftheneuralcrestwillbetransplantedfromonestage-fourteenembryotoanother(Figure1).Tofacilitatethevisualizationofthegraftsandobservethecellfatesofthetranplantedregion,bothalbinoandwildtypeembryoswillbeused.Tentransplantswillbeattemptedinwhicharegionofanalbinoembryoneuralcrestwillbetranplantedintotheregularlypigmentedwild-typeembryo.Insucessfultransplants,aregiondevoidofpigmentshoulddevelopatthetransplantsitealongthedorsalsideofmatureaxolotls. Introduction TrADItionally,amphibianshavebeenoneofthemostwidelystudiedorganismsinthefieldofdevelopmentalBIOLOGy.TwoamphibianspeciesoftenusedindevelopmentexperimentsaretheXenopuslaevisfrogandtheAmbystomamexicanum,orAxolotlsalamander.Axolotlsareparticularlyusefulforstudyoftheearlystagesofdevelopmentbecausetheeggsarelarge,easytoharvestyearround,andcanbearrestedatvariousstagesofdevelopmentbystoringat4°C(Gilbert,2000). Oneofthemostcommonlyusedtechniquesforobservingthefateofcellswithinearlygastrulasandneurulasistotransplantregions,eithertolocationswithinthesameembryo,orformoneembryotoanother.Intheearlytwentiethcentury,HildeMangoldtransplantedrgionsofthedorsallipofanamphibianembryoandobservedthestructuralanddevelopmentalimplicationsofthetransplants(Gilbert,2000).HildeMangold,alongwithHansSpemann,usedthesimilartransplantationtechniquestoidentifytheOrganizer--latertermedthedorsallip--alongthedorsalsideoftheembryo.Thisdiscoverywasaseminalworkindevelopmentalbiologybecauseitdemonsratedsomeofthefirstevidenceofregulativedevelopment,andlentsignificantsupporttotheargumentforepigenesisandagainsttetheoryofthehomonculus(Spemann,1929).Sincethen,severalothertechniqueshavebeendeveloped.Inthisexperiment,thetransplantwilloccurbetweenalbinoandwildtype(regularlypigmented)embryos.Transplantationbetweenpigmentedandnon-pigmentedembryoswillprovideaneasilyvisIBLeindicatorofthefateofthetransplanedcels. Inthisexperiment,theprotocolsdevelopedbyE.S.WilsonandD.L.LawrencehavbeenadaptedinordertotransplantregionsofanalbinoAxolotlembryoandapigmentedembryo(Wilson,2002,Lawrence,2000).Becausealbinoembryosarelessrobustandaremoresusceptibletobeingdestroyedinthecourseofthesurgery,thealbinoembryoswillserveastherecipientembryo. Protocol note:Whentheembryosarenotbeingmanipulated,keeptheminasolutionofHBStcontainingantibioticsinordertokeepthecellhydratedandtominimizethechanceofinfection. 1.ManuallyremovethejellycoatingsurroundingastagefourteenAxolotlembryousingbluntforeceps. 2.RinseanagarosePetridishwithanalliquotoftheHBStsolution. 3.PlaceapreparedembryointotheoperatingdishcontainingHBStwithanitbiotics. 4.Maintainsterilesurgicalconditionsthroughouttheprocedurebycleaningalltoolsin70%ethanolandfollowingsurgicalprocedure. 5.Removethemembranearoundtheembryobygentlytearingwithfineforeceps.Becarefulnottocrushembryo,andmakesuretokeeptheembryounderthesurfaceoftheHBStsolutionwhileremovingthemembrane.Ifdonecorrectly,theembryoshouldslideoutonesideofthemembrane. 6.Usinganeyebrowknife,createasmallventralpocketintheright-handsideoftheneuralcrestregionoftherecipient(regularlypigmented)embryo(Figure2a).Noremovalofcellsisnecessaryintherecipient. 7.Removeasectionoftheneuralfoldfromtherightsideofthedonor(albino)embryo.Usetheeyebrowknifetomakeathin,consistentincisioninthelightlypigmentedepidermisveryclosetothedistaledgeoftheneuralfold(Figure2a).Continuetocutuntiltheunpigmentedcellsbeneaththesurfaceareexposed. 8.Makeanincisionparalleltotheinitialincisionalongtheinneredgeoftherightneuralfold(Figure2b). 9.Extractthesectionwithtwoshortcutsinordertoharvesttherectangularcellsample(Figure2c).Iftheexcisionissucessful,thesampleshouldincludethewhitishendodermalcellsaswellasalayerofthetan-coloredectoderm. 10.Usingasurgicallooptomanipulatetheexcisedportion,transfertheneuralfoldsegmentfromthealbinodonortotheregularlypigmentedrecipient.Theimplantshouldfitsnuglywithintheincisiomadeinstep6. 11.Allowtheembryostoheal.ThentransferboththedonorandthehostembryosintoasteriledishcontaininglowerconcentrationHBStbygraduallyreducingtheconcentrationuntil20%HBStisacheived. 12.Allowdevelopmenttocontinueuntilfullgrownaxolotlsareobtained. 13.Observewhetherthegraftingprocedurewassucessful,andnoteanydifferenceinthedevelopmentofthegraftedareas,particularlythepigment.Photographtheresultingembryos. Results Tentransplantsweresucessfullycompletedusingportionsofalbinoembryosinsertedintoregularlypigmentedembryos(Figure3).Ofthesetentransplants,ninecontinuedtodeveloptwenty-fourhourspostsurgery.However,afterfortyeighthours,oncetheprocessofdilutingtheHBStsolutionwasbegun,developmentofallremainingembryosfailedduetodevelopmentalabnormalitiesunrelatedtothetransferregionoftheembryo(Figure4).Cellslosttheircohesivenessandfailedtodividewithintheexpectedpatterns,resultinginfataldelaminationandabnormalextensionsofmesoderm.Theseabnormailitesresultedintheterminationofdevelopment. Discussion Terminationofdevelopmentwascausedbytheabnormalcellgrowthofregionoftheembryosnotdirectlyaffectedbythetransplant.Thesegrowthsmayhavebeencausedbyinsufficientcadherinexpressionbetweencells(Gilbert,2000),giventhattheprotectiveandsomewhatshapesustainingmembranehadbeenremoved.Oncethemembranewasremovedfromtheembryo,theremaynothavebeensufficientstructuralconstrictionstomaintainproperformanddirectionofgrowth.FurThermore,theagarplatesusedasoperatingdishesinthisexperimenthaddepressionsinordertofacilitatethesurgery,andtoholdtheembryosstablepost-surgery.Postsurgery,however,theembryoslosttheirshapeandmesodermalandendodermalcellsextendedintothedepressions.Thechangesinshapemostlikelyweresufficienttodestroytheembryos. However,withtheexceptionofoneembryo,alltransplantsweresucessfuluptotwentyfourhoursaftersurgery(Figure5).Inthefailedembryo,thetransplantmayhavebeeninsertedintothetransplantincisionincorectly,thatis,thecellsthatwerefatedtobecomeectodermwerealignedwiththeendodermoftherecipientembryo.Graftstransplantedinthismannerwouldfailtohealbecausetheectodermalcellswouldexpressdifferentcadherinsthanwouldtheendodermalcells(Gilbert,2000).Experimentalerrorssuchasthismaybeavoidableifthetransplantwastakenfromapigmenteddonorandgraftedtoanalbinorecipientbecauseinpigmentedspecimens,theectodermiseasilydistinguishablefromtheendoderm,andinversionsofthegraftswouldnotoccur. Intheremainingnineembryos,however,thegraftsbegantoheal,indicatingthat,hadotherunrelatedproblemsnotarisen,thetransplantswouldhaveresultedinfullydevelopedAxolotlembryosdisplayingalbinoregionsontheirdorsalside.Inthefuture,dilutionsoftheHBStsolutionshouldbemademoregradually,andtheembryosshouldbetransferredtoapetridishwithoutdepressionsassoonafterthetransplantaspossible. Figures Figure1.Apigmentedstagefourteenembryo.Stagefourteenembryosarecharacterizedbytheclosingoftheneuraltube.Thename"keyholestage"hasbeenusedforstagefourteenduetotheuniqueshapeoftheneuralcrest.Pigmentedembryossuchasthisonewereusedasrecipientembryosinthetransplantprocedure. b) c) Figure2.Astep-by-stepschematicoftheincisionsnecessarytoexcisearegionoftheaxolotlembryo.a)Thisinitialincisionshouldbemadeonboththerecipientembryo(tobecomethesiteoftranplantation)andthedonorembryo.b)Thesecondparallelincisiontobemadealongsidetheinitialincisionofthedonorembryo.c)Thesetwosmallcutsshouldbemadeperpendiculartothefirsttwoincisionsinthedonorembryoinordertofreearegionoftheneuralfoldforexcisionandtransplantation. Figure3.Sucessfultransplantsofregions(circatwenty-fourhoursaftertransplant)neartheneuralcrestregionoftheaxolotlembryo.Thetransplantedregionorginatingfromanalbinoembryocanbeeasilyidentifiedintherecipientpigmentedembryoasaregiondevoidofpigment. Figure4.Developmentalabnormalitiesleadingtotheterminationofdevelopment.Thetransplantedpigment-freeregion(indicatedbytheblackarrow)hadbeguntohealbeforedevelopmentfailed.Howeverotherregionsoftheembryofailedtodevelopproperly(indicatedbythewhitearrow). Figure5.Afailedtransplantoftheneuralcrestregioninanaxolotlembryo.Thecellsofthegraftfailedtoadheretothesurroundingcellsintherecipientembryo,possiblybecauseofincorrectalignmentoftheectodermalandendodermalcellsofthedonorandrecipient.Developmentarrestedlessthanfortyeighthoursafterthesurgery.
