FromthevariousmicrosurgicalproceduresofSpemannandothers,severaldevelopmentalprincipleshaveemergedaboutamphibianembryos.Oneofthemostimportantdiscoverieswastheunderstandingthatthedevelopmentalfatesofvariousgroupsofcellsarenotrigidlydeterminedearlyindevelopment.Inmanycases,ifamulticellularfragmentofanearlyfroggastrulaistransplantedfromoneregionoftheembryotoanother,thecellswillbehaveasiftheywerecellsfromtheinsertedsite,producingstructuresthatwouldnormallydevelopinthatpartoftheembryo.Laterindevelopment,however,cellsoftransplantedfragmentsdevelopdifferently.Theytendtocontinuealongthesamedevelopmentalpathwaysthattheywouldhavefollowedintheiroriginallocation.Theydifferentiateasiftheyhadnotbeentransplanted,thusproducingananomalouspatchofmisplacedtissue.ExperimentsbyDuShaneinthe1930srevealedthattheneuralcrestelementsareprecursorsofpigmentcells.Todemonstratethatthepigmentcellsarederivedfromtheneuralcrest,weplantotransplantthecrestmaterialtoanotherregionofthefrog"sbody.Neuralcrestcellsformadorsallyplacedwedgebetweenthelateralwalloftheneuralfoldandtheectodermoneachsideoftheembryo,thustheremovalofthelateralneuralfoldremovesaportionoftheneuralcrest.

Ayoungaxolotlembryo.

Objective

Inthisexperimentwewilltransplantneuralcrestelementsfromoneembryointotheventralsurfaceofanotherembryoandobservehowthetransplantationeffectsthedevelopmentoftheembryo.Becausepigmentcellsdevelopfromtheneuralcrestregion,asuccessfultransplantationwouldresultinagroupofpigmentcellsarisingwheretheneuralcrestcellsaretransplantedto.Finallywewillobservewhetherthedonorembryocanrecoverfromthesurgeryandstillmaketheneuralcrestafterwehaveremovedthelateralneuraltube.

Anaxolotlembryo,anexampleofwhatwasusedinthislab.

Protocol

I.PreparationofMicrosurgeryTools

Followprotocolfoundonthegeneralprotocolwebpage.

II.Microsurgerypreparation:

1.Manuallydejellyembryoofstage14andkeepinHSBtcontainingantibioticsuntiluse.Formicrosurgery,alsoremovemembranearoundtheembryo.

2.Prepare2%agarose-coatedoperatingdish.RinsewithHBStandantibiotics.

3.Placeembryosintooperatingdishcontaining1XHBStandantibiotics.

4.Usecleantechniquewhileperformingmicrosurgery(i.e.dipalltools,Pipettes,andglassbridgesinto70%ethanolandthenintosterileHBStbeforeusing).

III.Transplantationoftheneuralcrest

1.Prepareasmallpocketintheventralsurfaceofthehostembryo.Onlyaslitisrequiredbycuttingwithaglassneedle.Thepocketcanbeenlargedtoreceivethetransplantbycarefullyremovingsomeofthemesodermcellswiththetipoftheglassneedle.

2.Usingafine-tippedneedle,removepartoftheneuralfoldontherightsideoftheembryo.Withasinglemotionfromtheanteriortotheposterior,usetheglassneedletomakeathintearinthepigmentedepidermisveryclosetotheouteredgeoftherightneuralfold.Now,tearcarefullythroughthewhitishmesodermalcellsthatlieunderneath.Whenthegrayish-tanendodermalcellsbecomevisIBLe,stopcutting.Followthefirstlongitudinalcutwithasecondparallelcutattheinneredgeoftherightneuralfold.Next,maketwotransversecuts,anteriortoposterior,tocompletearectangularareaofneuralfoldtissue.

3.Lifttheentirerectangularareaofneuraltissueawayfromtheembryowiththetipoftheglassneedle.Thepieceshouldincludeawhitishmesodermwiththeoverlyingpigmentedectoderm.

4.Transfertheneuralfoldtransplantonthetipoftheglassneedletotheventralpocketontherecipientembryo.Gentlyandfirmlypushthewholemassthroughthenarrowincisionandanchoritagainsttheyolk.Atightfittingtransplantisdesirable.

5.Quicklycovertheimplantwiththeflatsurfaceofapiecefromathin-gradecoverslip.Waitonehour.

6.Carefullyremovethecoverslip.Ifthetransplanthasnothealedproperly,placeitunderpressureagainforanotherhalfhour.Afterhealingiscomplete,transferboththedonorandthehostembryostoaglassdishcontainingspringwater.

7.Observethedifferentiationoftheimplantedtissue.Notewhethergraftwassuccessfullytransplanted,howtheembryorespondedtothegraft,andwhethertherehavebeenanydifferenceswhichhaveoccurredinthedevelopmentoftheembryo.

Results

Twentyembryosweresurgicallymanipulated.Ofthose20,eightweredeemedpossiblysuccessfultransplants.However,uponcheckingtheembryosafter48hours,itwasdeterminedthatnoneoftheembryosuccessfullyacceptedthegrafts.Whydidthishappen?Itwasmostlikelybecausewedidnotpossessthemanualdexteritywhichwasrequiredtoperformmicrosurgeryonthesmallembryos.Additionally,theglasscoverplateswhichwereplacedontopoftheembryospost-surgerycausedtheembryostobepulledapartafterweattemptedtoremovethem48hourspost-op.Wefeelthatthislabwouldbebettertoperformwithlaterstageembryosorbyapersonwiththenecessarydexteritytomanipulatethedelicateembryos.

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