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Regulative Development in Axolotl Embryos; Splitting the Heart Field
Objective
ThisexperimentwillseektodemonstrateregulativedevelopmentinAmbystomamexicanumembryos.Specifically,wewillsplitthemorphogeneticfieldthatisresponsIBLeforheartformationusinggraftedtissuefromthegillarea,andattempttoformtwohearts–oneoneithersideofthegraftedtissue.
Background
Organismsthatuseregulativedevelopmentrelyoncell-cellinteractionstodeterminewhatdevelopswhere,ratherthanhavingfixedcellfates(asinmosaicdevelopment).Becauseofthis,embryosthatundergoregulativedevelopmentareabletocompensateformissingordamagedparts(Gilbert,2003).Thissortofcompensationisparticularlyseeninregionsknownasmorphogeneticfields.Theseregionsofcellsareeachcommittedtoformacertainorgan,butmadeupofindividualcellsthatarenotyetcommittedtoformaspecificpartofthatorgan.Damagetothesefieldscanbecompensatedforbytheothercellsinthefield(Gilbert2003).
Intheaxolotlsalamander(Ambystomamexicanum),theheartformsthroughthefusionoftworegionsofheartformingtissue--theheartprimordia(Hamburger,1973).Inorderfortheaxolotltohaveonefullyformedheart,thesetworegionsmustcommunicatewitheachothersothattheycan"decide"whenandhowtocometogether.Theoretically,ifthiscommunicationwereinterruptedbydamagetothetissue(orabarrierplacedbetweentheprimordiatissue),theheartprimordiawouldbeunabletofuseintoone.ThiscommunicationandsubsequentABIlitytocompensatefordamage,isaprimeexampleofregulativedevelopment.
Thisexperimentwilltakeadvantageofthemorphogeneticfieldoftheaxolotlheart.Thefactthattwoheartsformisnotonlyamazingtobehold,butdemonstratesthatthecells,whenpreventedfromcommunicatingwitheachother,willgoontoformtwoseparateorgans.Thisisadirectresultofregulativedevelopment.
Materials
Axolotlembryos(stage14-16)1%agarose-coatedoperatingdishes100%HEPES-bufferedModifiedSteinberg"sSolution(HBSt)withantibiotics(gentamycinat70mg/ml)50%HBSt20%HBStMicrosurgerytools(tungstenknivesandhairloops)
Procedure
1)Removethefirsttwojellycoatsfromseveralstage14-16embryos
a.Transferembryosintodishof20%HBStusingplasticpipet(thetipshouldbecutoffsothatyoucansuckuptheembryosasiftheywerethebubblesinbubbletea).b.Withfinestforceps,grabandPiercethemoreinteriorofthetwovisiblejellycoats.Thistakespracticeandpatience,asthejellyisveryslippery.
c.Oncepierced,firmlypullthejellycoatapart,takingcarenottosquashtheembryointheprocess.Theembryoshouldpopoutnicelyoncethejellycoatsareopenedupenough.
2)Removethevitellinemembrane
a.Prepare1%agarose-coatedoperatingdishi.HeatglasspipettipusingBunsenburneruntilmeltedintoalittleballatthetipii.Presshottiptoseveralplacesinthedishtoformlittleindentationsintheagarose.Thiswillhelpholdtheembryosinplacewhileoperating.
iii.Filldishwith100%HBStwithgentamycin
b.Placeembryosinoperatingdishandletsitforabout5minutes.Thiswillallowthevitellinemembranetopuffupwithwatersomewhat,makingiteasiertoremove.
c.Withplentyoflightfromthestereoscope,lookforvitellinemembranearoundembryo.Gentlytrytograbitwithfineforcepsandpulloff.Again,thistakespracticeandpatience.*Damagedembryosshouldbetransferredtoaseparatedishforuseastissuedonorsetc.*
3)Obtaingilltissuefromstage16donor
a.Placedonorembryoinoneoftheindentationsinthedishwithitsflanksidefacingup,ventralsidetowardyou,anddorsalaway.b.Intheloweranteriorcornerthereshouldbeaslightlylightercoloredregion(thepresumtivegilltissue);thisiswhatyouwanttocutout(Figure1);useatungstenknifetocutandahairlooptohelpholdtheembryoinplace.Thiscuttingtakessomegettingusedto;don"tbesurprisedifatfirstyouendupsquishingtheembryointomanylittleyolkycells.
4)Preparehost(~stage15)fortissuetransplant
a.Placehostembryoinoneoftheindentationswithventralsideup.b.Usingthetungstenknifetocutandhairlooptohelpholdtheembryoinplace.Makeanincisionthatisaboutathirdthelengthoftheembryo,buttowardstheanterior.Youjustwanttobreaktheexteriorandscrapealittlewayintothecellssotherewillbeenoughroomforthegraft.Thisalsotakesabitofpractice.
5)Puttissuetransplantintohost
a.Pickuptissuetransplantwithhairloopandtransferintoincisioninhost.b.Usetungstenknifeandhairlooptogentlybutfirmlypushtissueintoincision
6)Allowtohealovernightintheiroperatingdishes,thengraduallyreplacesolutiontoreduceconcentrationto20%HBSt.
7)Afterembryosheal,transfertofreshagarose-coateddish.
8)After3or4days,checkforheartpulsations,beingsuretonoteiftwoheatshaveformed,andiftheyhave,whetherornottheybeatinsynch.
9)Collectphotosandvideoofsuccessfulhosts.
ResultsSixaxolotlembryoswereinitiallypreparedsuccessfully,andofthose,atleastoneshowedadistinctheartbeatby6daysaftersurgery.Determiningwhetherornottheheartfieldhadbeenaffectedbythegraftwasdifficultduetotheopacityoftheaxolotlskin,andthepositioningoftheembryointheoperatingdish;onesidedown,suchthatonlyonesidecouldbeviewedatatimewithoutdamagingtheembryo. Evidencethattheheartfieldwasnotparticularlyaffectedincludesthefactthatthegraftedtissuewasatleastpartiallyrejected.Additionally,whenviewingthegillscloseup,theflowthroughthemseemedcompletelysynchronousbetweentheleftandrightgills,aswellasinsynchwiththevisibleheartbeat.Asforthestageofheartdevelopment,itappearstohavedevelopedatleastsomewhatnormallytoapointsomewherebetweenstagesAandBinFigure7.Specificallythebloodflowseemedtostarttowardtheventral-anteriorcorneroftheheart,flowupwardstowardthedorsalarea,andfinallydowntotheventral-posteriorregion(Figures5and6). Thereweresomeirregularitieshowever.InFigures3,4,5&6wecanseethattheheartregioniswellbehindthegills.Thisissomewhatfartherbackandmoretoonesidethanonewouldnormallyexpecttofindtheheart.Thisirregularityopensupthepossibilitythattheheartfieldwasatleastsomewhataffected,despiteappearingtohaveasynchronousheartbeat. | |||
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A | B | ||
Figure7.Twoearlystagesofamphibianheartdevelopment;heartdevelopmentinabovefigures3-6ispresumedtobebetweenthesetwostages.DiagramssimplifiedafterRugh,1951. DiscussionFromourobservationofasingledistinctheartbeat,itappearedthattheattempttodividetheheartfieldwasnotsuccessful.Additionallytheobserveddirectionofbloodflowandapparentsynchronousflowthroughtheleftandrightgillsseemedconsistentwithnormalamphibianheartdevelopment(Rugh,1951).However,theabilitytoobservetheheartbeatswashinderedbytheopacityoftheaxolotlskin,aswellasthepositionoftheembryointheoperatingdish,sotheseobservationsmaynotaccuratelyreflectthetrueconditionoftheheart.Inaddition,theheartappearedtobesomewhatmoreposteriorthanwouldnormallybeexpected.Thepresenceofthisirregularityleadsustobelievethattheheartfieldwasaffectedinsomeway.Ifthiswerethecase,theheatcouldactuallybesplit,orpartiallysplit,buthavetwoheartbeatsthathappentoappearsynchronous. Thefactthatthegraftedtissuewasobservederuptingfromthebelly/ventralregionoftheembryomaysuggestthatthegraft,whichwasintendedtosplittheheartregion,wasnotincorporatedintotheembryoenoughtosufficientlyblockcommunicationbetweenthetwohalvesoftheheartfield(Figures3,4,&5).Itshouldbenotedthatthereareotherexplanationsforthesedatathatmightalsobeevidenceforregulativedevelopment.Forinstance,thegilltissuecouldbetransformingintohearttissuethroughcell-cellinteractions.However,therelativelyadvanceddevelopmentalstageofthedonortissue,andthefactthatthephotosseemtoclearlyshowtissuerejection,donotsupportthisconclusion.Intheend,theseresultsareatbestinconclusiveinregardstoregulativedevelopmentintheformationoftheaxolotlheart. Itshouldbenotedhowever,thatpreviousexperimentsusingsmallpiecesofsterilefoilasabarrierratherthangilltissue,didyieldresultssupportingtheexistenceofregulativedevelopmentintheaxolotlheart.Specifically,twohearts,oneoneithersideofthefoilbarrier,wereseentodevelopandclearlybeatasynchronously(VélezandKrsmanovic,2004).Otherexamplesofthemanipulationofmorphogeneticfields,andthereforeevidenceofregulativedevelopment,havebeenshowninlimbregenerationstudies.Axolotlsareactuallyabletocompensatefordamagetothelimbmorphogeneticfieldsthroughouttheirlives,tothepointwhereentirelimbsmayberegeneratedfromtheremainingtissueofalimbstump(Gardineretal2002). Sincestudiesshowthatthereisampleevidenceforregulativedevelopmentinaxolotlsitwouldthereforeseemthattheourresultslikelyhavemoretodowithexperimentaltechniquethanabsenceofregulativedevelopmentinaxolotls.Whilethistissuegraftprocedurewasnotparticularlycomplex,andwasthemethodoriginallysuggestedinHamburger"sManualofExperimentalEmbryology,inretrospectitdoesnotseemwellsuitedtothosewhohavehadlittletonopracticedoingmicrosurgery.Additionally,itshouldbenotedthatwhiletheembryoswerelargelyattheappropriatestageatthebeginningoftheprocedure,astimewenton,thewarmthoftheroomallowedthemtodevelopmorequicklythanwehadanticipated,leADIngtofewersuitableembryos.Itisimportanttorememberthatthereisonlysomuchtimebeforetheheartprimordiafuse,andgraftsmustbedonebeforethishappensinordertobesucessful.Inthefutureitwouldbeadvisabletokeeptheembryoscoldwhenevertheyarenotactuallybeingworkedon,ratherthanlettinganumberofthemsitoutatroomtemperature.
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