Developmental and phenotypic implications of transplantation of neural crest regions of albino Axolotl embryos相关交流-蚂蚁淘在线

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.

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.

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容