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Axolotl Protocol: Transplantation of the Gill Organ Field
Objective:
Background:
Procedure:1.Obtainaxolotlembryos.Forthisexperiment,wewillbeusingwildtypeandalbinoaxolotlembryosatstage21intheirstagingseries.2.Preparetheaxolotlembryosasfollows:a.manuallydejellyembryoofappropriatestageandkeepinHEPES-bufferedModifiedSteinberg"ssolution(HBSt)containingantibioticsuntiluse.Formicrosurgery,alsoremovemembranearoundembryo.b.placeembryosintooperatingdishcontaining1xHBStandantibioticsc.usecleantechnique;diptools,Pipettes,andglassbridgesin70%EtOHandthenintosterileHBStbeforeusing3.Startmicrosurgeryonyourdonorembryo.Locatethegillorganfieldwhichisinthefirstanteriorthirdoftheembryo(towardhead)andrightabovethesmallcrestorindentionclosertotheventralside(Rugh,1962).Isolatethegillorganfield(ortheregionthatwillbecomethegillorganfield)bycuttingarounditanddetachingthatspecificpieceoftissuefromtheembryo.4.Starttransplantationonyourrecipientembryo.Cutasmallslitinaregionclosetothegillorganfieldontherecipient.Gentlyslipdonorgillorganfieldintoslitandbesuretosecurethegraftinplace.ItiscriticaltotrytogetthegraftasfarunderthecutaspossIBLetoensurethattherecipientwillbegintoincorporatethenewtissueintoitsdevelopmentprocess.
Figure1:
A.A21stagealbinoaxolotlembryowithatransplantedwildtypeaxolotlgraft(supposedlythegillfield).Thepicturewastakenimmediatelyaftermicrosurgery.
B.A21stagewildtypeaxolotlembryowithatransplantedalbinoaxolotlgraft.Thepicturewastakenimmediatelyaftermicrosurgery
5.Oncethesurgeryiscomplete,besuretotakepicturesofanormaldonorandrecipientandthenthenewlytransplantedgraftoftherecipient.Picturesshouldbetakenatvariousstagestoobservedevelopment(tobedeterminedatdiscretionofresearcher).6.TheembryosshouldbeleftinHBStatroomtemperaturetocontinuetodevelopment.Observebothdonorsandrecipientsastimecontinuestoexaminetheeffectofthetransplantedgraft.7.MakeOBSERVATIONSasdevelopmentcontinues...thisisthekeytoobtainingresults!8.Afterthefirstday,itisimportanttoreplacetheoperatingsolutionwith10%modifiedSteinberg"ssolution.Thegraftwilldevelopbetterinalowerconcentrationofcertainchemicals.Todothis,simplypipetteofftheoperatingsolutionandpourinthe10%modifiedSteinberg"ssolution.
Results/Discussion:
Figure2:Apictureofawildtypeaxolotlembryo2daysafterthealbinoaxolotlgrafthadbeentransplantedintoit.Thegraftislocatedbelowthegillfieldbutstillhasbeensuccessfullytransplantedandhasbecomeapartofthedevelopingembryo.
Weexpectedthatthetransplantedgraftwouldhavedevelopedasifitwereapartitsoriginalenvironment.Thisimpliesthatthegillwouldhavebeenspecifiedpriortotransplantation.Upontransplantation,inductiveeffectswouldallowthetransplantedgrafttoeventuallyformthegill.Therearetworeasonswhywemightnothaveexaminedanyofourexpectedresults.Thefirstisthepossibilitythatthegillorganfieldhadnotbeenspecifiedpriortotransplantation,whichdidnotallowittodevelopproperlywhenitwastransplantedintotherecipients.Thesecondreasonisduetotechnology.Thiswasaverycomplicatedexperimentthatrequiredskilledmicrosurgeryonembryos.Sincethiswasafirstattemptatmicrosurgery,ourtechniqueandtechnologymighthavebeeninsufficientwhichcausedpoorresultstobeobtained.
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