Introduction

Therearetwomodesofdevelopmentcommontomostspeciesintheanimalkingdom.Virtuallyallembryosundergobothmosaicandregulativedevelopmentatsomepointduringtheirgrowth.Regulativedevelopmentgenerallyoccursinearlygastrulationwhencellsareinducedtoformdifferentstructuresaccordingtothecell-cellsignalinginteractionsinaspecificareaoftheembryothatleadtotheconditionalspecificationofacell\"sfate.Acellundergoingregulativedevelopmentcanbetransplantedtoanotherpartoftheembryoandformwhateverstructurebelongsinthatareainsteadofthestructurethatitwouldhaveoriginallyformedbecauseitiscompetenttoreceivethedifferentsignalsfromthenewcellsaroundit.

Thesecondmodeofdevelopmentismosaicdevelopment.Mosaicdevelopmentresultsfromtheautonomousspecificationofacell\"sfate.Thesecells,insteadofdependingoncell-cellinteractions,aredeterminedbycytoplasmicfactorscontainedwithinthecellitself.Thesecellswillformagivenstructureeveniftheyaremovedtoanewlocationandareexposedtocell-cellinteractionsandsignalsthatdifferfromtheiroriginalposition.Mostorganismscontaintissueswhichmayundergooneorbothofthesedevelopmentalmechanismsatagiventime(Gilbert,2003).

Thedevelopmentalpatternoftheamphibianheartwillbestudiedbybisectingtheheartprimordialfieldandobservingthesubsequentdevelopmentoftheheart,ifpresent.Iftheheartweretodevelopusinganautonomousmechanism,thecellswoulddevelopnormallyevenifthetwosideswerenotinphysicalcontactwithoneanother.Thiswouldleadtohalfaheartgrowingoneithersideoftheincision.However,ifinsteadtheheartdevelopedusingaregulativemechanism,thenthecellsonthetwosideswouldcompensatefortheperceivedabsenceoftheothercells.Thiswouldleadtothedevelopmentoftwodistinct,andperhapsfunctional,hearts(Hamburger,1960).

Theheartmorphogeneticfieldwassplitusingtwodifferenttechniques.Firstthefieldwassplitusingaluminumfoil.Thisbisectionensuresthatanimpenetrablebarrierexistsbetweenthetwoprimordiaandcutsoffallsignalingbetweencellsonoppositesidesofthefoil.However,giventhatthismaterialiscompletelyunnaturaltotheembryo,thisimplantationtechniquecouldbeexcessivelystressfultotheembryos.ThusasecondtechniquewasmodeledaftertheHamburgerexperimentfortheformationoftwohearts,andinvolvedthebisectionoftheheartmorphogeneticfieldofusingatransplantofnon-hearttissuefromolderembryos.Thesetissueswouldserveasabarriertotheintracellularsignalsandalreadybespecifiedassomeothertissue.Thereforethegraftswouldbeimpervioustotheheartsignals.Sincethesetransplantswouldbeofaxolotltissueasopposedtosomeforeignsource,suchbisectionwouldbelessstressfulandwouldpresumablyhealfasterthanbisectionusingfoil.

Foiltechnique

1.Preparemicrotoolsforsurgery.

2.Prepare1%agarose-coatedoperatingdish;rinsewith100%HBStandantibiotics.

3.Autoclaveasmallpieceofaluminumfoil;thisfoilwillbeusedtobisectthemorphogeneticfieldsoftheheart.

4.Sortembryosbyage,stopdevelopmentofstage15axolotlembryobyplacingin4°C.Thedevelopingembryoissensitivetotemperatureandwilldevelopfasterathighertemperatures(Hamburger,1960).

5.ManuallydejellyembryoinSteinbergssolutionasdemonstratedinvideoonnextpage.Usefineforcepstobreakholeinthehardjellycoat.Maneuverembryoout.Becarefulnottopuncturetheembryo!

6.TransferembryosintowellofoperatingdishcontainingincreasingconcentrationsofHBStandantibiotics.RemovehalfthesolutionwithPipettemanandfillwith100%HBSt.Afteraminuteremovehalfthesolutionandfillwith100%HBSt.Allowembryotosit2minutesinhighsaltsolution.Thiswillcausethevitellinemembranetoswellandmakeiteasiertoseeandremoveasshowninthevideo.Demembranateembryoin100%HBStbycarefullygrabbingnearneuralcrestcellswithbothtweezersandpullingtoformaholeasdescribedinthedejellyingstepabove.

7.Dipalltoolsinto70%EtOHandthenintosterileHBStbeforeusing.

8.Holdrecipientembryoventralsideupwithhairloopandpickasmallslitdownthemidlineoftheembryoalongtheanterior-posterioraxiswitheyebrowmicroscalpelortungstenmicroscalpel.

9.Placefoilintheincisiondeeplyenoughsothatitdoesnotfalloutbutnotsodeeplythatitcutsthroughtheembryo.Observedevelopmentuntilheartforms.

10.Leaveoneembryowithonlytheincisionbutnofoil,andoneembryouncutascontrols.

11.WhilethehighCa2+concentrationoftheHBStenhanceshealingbysaturatingtheCa2+bindingsitesofcadherinmolecules,ahighsaltconcentrationalsocausesdevelopmentalabnormalities.Therefore,afterembryoshealfromsurgery,gentlyreplace100%HBStwith50%,then20%HBSt.Changesolutionconcentrationsafter12hrs.

12.Collectpicturesandmoviesdailyusingamicroscopeequippedwithacameraandrecordingcameras.

Grafttechnique

1.Followpreviousprotocolforpreparationofmicrotoolsandoperatingdishes,dejellying,anddemembranationofstage15recipientembryos(Figure1A,below)andstage20donorembryos(Figure1B).

2.Positiondonorembryoonlateralsideandrecipientembryoondorsalside(neuralcrest-sidedown)exposingventralsideinseparatewellsofoperatingdish.

3.HolddonorembryowithhairloopinstrumentanduseTungstenmicroscalpeltoremovesmallchunkoftissuefromlateral,gill-formingsurface.

4.Holdrecipientembryoventralsideupwithhairloopandpickasmallslitdownthemidlineoftheembryowitheyebrowmicroscalpelortungstenmicroscalpel(Figure2A).

5.Imbeddonorgill-formingpieceintheincisionontherecipientembryo.Patdownandensureitspositionwitheyebrowortungstenmicroscalpel.

6.Leaveonerecipientembryowithonlytheincisionbutnograft,andoneembryouncutascontrols.

7.Observethedevelopmentoftheembryosuntilheartforms.

8.Asdescribedpreviously,thehighCa2+concentrationoftheHBStenhanceshealingbysaturatingtheCa2+bindingsitesofcadherinmolecules,butahighsaltconcentrationalsocausesdevelopmentalabnormalities.Therefore,afterembryoshealfromsurgery,gentlyreplace100%HBStwith50%,then20%HBSt.Changesolutionconcentrationsafter12hrs.

9.Collectpicturesandmoviesdailyusingamicroscopeequippedwithastillcameraandrecordingcameras.

Figure1.Dejelliedtestembryos.(A)Stage15,earlyneurulaIIrecipientembryo.ClearvitellineenvelopeisswollenandvisIBLeaftersoakingin100%HBSt.(B)Dejellied,demembranated,stage20,lateneurulaIVdonorembryo.Donorgraftsweretakenfromthegill-formingregionofastage20,lateneurulaIV,shownbyarrow.

Figure2.Recipientembryos.(A)Stage16recipientembryowithincisionsdownmidlineofventralside.Actualincisionswerelargerthanthisphotographportrays.(B)Recipientembryowithgraftinincision.Darkdonortissuesitsinlighterareaofincision.

FoilProcedure:

Theresultsofthestudyindicatethatdevelopmentintheaxolotlisunderregulativecontrol.Figure1showsacontrolandbisectedaxolotlembryos.Theinsicisionofthisparticularembryoshowsthebisectionoftheheartmorphogeneticfield.Themoviesshowtheresultofthebisectionoftheheartmorphogeneticfield.Itisclearthatthebisectedembryohastwohearts.

Figure1.Thebisectedheartfieldofanaxolotlembryoafterthreedays(A).Thecontrolembryoafterthreedays(B)Bisectedembyo

GraftingProcedure:

Ofthealmostonehundredembryosusedinthegraftingprocedurealone,onlysixty-twosurvivedthedejellyingprocess.Twenty-oneembryoswerekilledduringthedemembranatingprocedures.TwentywereintheearlyneurulaIIorIIIstage(stages15or16)ofdevelopmentandappropriateforthisexperiment(Bordzilovskaya,210).Elevenembryoswerekilledduringthegraftingsurgery.Theseembryosgenerallyhadtoolargeanincisionmadedownthemidlineoftheventralside,whichresultedinthespillingoutofthelighttancoloredendo-andmesodermalcells(Figure3).Onlynineembryosweresuccessfullygraftedandsetasidewiththetwocontrolembryos

Forty-eighthoursaftersurgery,mostoftheembryoshadreachedstage27andsomeoftheembryosweremovingbythemselves(Figure4).Oneembryorespondedtotouchbywigglingtheanteriorhalfitsbodybackandforth.Fouroftheembryoshaddamageneartheanteriorend,consistingofanareawherethedarkectodermalcellsweremissingorbarelyattachedtothebodyandthelightercoloredendo-andmesodermalcellswerevisibleorhangingoutthebody(Figure5A).ClumpsofcellshadfallenoutoftheembryosandwerefloatingintheHBStsolution.Afterfivedays,sevenoftheninetestembryoshaddisintegratedintoanunorganizedmassofcells(Figure5B).Bydaysix,allembryos,includingthecontrols,haddied.

Figure3.Surgicallydamagedrecipientembryo.Theincisionwastoolarge,resultinginthelossoflighttancoloredendo-andmesodermalcells.Embryosdamagedinthiswaywereineligiblefortheexperiment.

Figure4.Post-operationaldevelopmentoftestembryos.(A)12hoursafteroperation,graftisstillclearlydiscernablefromhostembryotissue.(B)48hoursaftersurgery,embryoshadgrowntostage27.Thegraftispartiallycoveredwithhostectodermcellsandisbecominglessprominent.(C)By72post-surgery,embryoshaddevelopedtostage32andtheincisionwasbarelyvisible.

Figure5.(A)Injuredstage34embryofourdayspostsurgery.Embryoswerefoundwithinjuriesneartheanteriorhead-formingregionandoftenhadlostsomeendo-ormesodermalcellsaswell.(B)Embryossixdayspostsurgery.Embryoseventuallydisintegratedintomassesofcellswithnodiscernableorganization.

Theresultsofthefoilproceduresupportthehypthesisthattheheartmorphogeneticfieldsintheaxolotlareunderregulativecontrol.Morphogeneticfieldsareveryimportantinthattheyhelpdeterminehowdifferentorgansintheaxolotldevelop(fromtheheartandgillstothelimbs).Theresultsofthestudyconfirmthisideabyshowingthattwoheartsformedwhenanimpermeablebarrierwasplacedbetweentheheartprimordia.Themoviesshowthatthesameaxolotlhadtwohearts,oneoneachsideofthebody.Afterthebisectionthetwosidesofthemorphogeneticfieldwerenotindirectcommunication.Asaresultbothheartprimordiadevelopedintoacompleteheart.Iftherehadbeennobisection,theheartfieldwouldhavejustdevelopedintooneheart.

Noembryossurvivedtostage37withfullydevelopedheartsinthegraftingprocedure.Thereareanumberofpossiblereasonsforthedeathsoftheembryos.Beingcaughtinthewellsoragarofthesurgicaldishescouldhaveirreparablyinjuredtheembryos.Onlyonecasewasdocumentedinwhichanembryowasleftinawellandgrewthereuntilitgotstuck.Thisembryowaspartiallydamagedwhenitwasremovedfromthewell.Injuriestotheheadregionsmayhavebeenduetotheagar.Topreventfurtherpossibledamage,theembryosweretransferredtoagarlesspetridishesassoonastheyappearedsufficientlyhealedfromsurgery.Thisterminatedtheinstancesofembryosbeingdamagedbytheagar.

Iftheembryosdidnothealbeforetheyreachedstage29,theywouldbegintomoveandinjurethemselves,eventuallycausingthemselvesseveredamage.Healingcouldhavebeendisruptedifthegraftswerecomposedofneuraltissue.However,specialcarewastakentocutthegraftfromthenon-neural,gill-formingectodermregiononthelateralsideofthedonorembryo.Thus,whileitislikelythattheembryosdidnothealfromthesurgery,itwaspresumablynotduetotheuseofneuralcellsinthegraft.

Bythesecondpostoperationalday,someembryoswerebeginningtomoveindependentlyandtoreacttoexternalstimuli.Someweremovingandgettingcaughtindifferentareasofagar.Thiscouldhavedisruptedordamagedthegraftinsomeway.Whentheycouldnotheal,theembryosdiedandtheirbodiesdegeneratedtothepoolsofunorganizedcellsseeninFigure5B(J.Cebra-Thomas,personalcommunication,2004).

Thefailureoftheembryostodevelopfullyfunctionalheartsdidnotnegatethevalueofthisexperiment.Themethodologycanbeausefultoolforstudyingthetypesofregulationthatoccurindevelopingembryos.Iftheembryoshadbeenmovedtoagarlessdishesearlier,someoftheinjuriesthatmayhavecontributedtotheembryodeathcouldhavebeenavoided.Similarly,mistakesduetoinexperiencemanipulatingthemicrotoolsareverycommonandwouldbeavoidedifthisexperimentwererepeatedforpractice.Theincisionswouldbesmaller,neater,andwouldthereforehealfaster.Bothofthesechangeswouldincreasethesurvivalrateandallowtheembryostodevelopfarenoughtodisplayeitherregulativeorautonomousdevelopmentintheheart.