Toobservetherolesulfateplaysinseaurchingastrulation,andtoreplicatethefindingsofKarpandSolursh,thatseaurchinembryosfailtogastrulatewithoutsulfate.Totestwhetherendodermdifferentiationcanoccurintheabsenseofthemovementsofgastrulation. Seaurchindevelopmentrequiresnotonlyinternallyproducedsignals,butalsomaterialsincorporatedfromtheenvironment.KarpandSolurshhavehypothesizedthatsecondarymesenchymecells,whichformthefilopodiaofthedevelopingarchenteron(primarygut)requiresulfate(toformsulfatedproteoglycanswhichactassomethinglikeanadhesive)inordertomigratealongtheacidmucopolysaccharideoftheextracellularmatrixwithintheblastocoelofadevelopingseaurchin(1974).Developingseaurchinembryoswereraisedineitherartificialseawaterorsulfate-freeseawater,fixed,andstainedforalkalinephosphotaseenzymeactivityandwithimmunofluorescentantibodiestovegetalarchenteroncells.Embryosraisedinasulfate-freeenvironment,unlikecontrols,didnotdeveloparchenterons.Stainingindicated,however,thatthesecellsdiddifferentiatetobecomegutcellsandhadinvaginated,butthecellscouldnotmigrateuptheblastocoelwall.ThisdatasupportsthefindingsandhypothesisofKarpandSolursh(1974). Introduction Seaurchindevelopmentprogressesinapredictableandeasilyobservableway(Gilbert,2000).Thevegetalplatethickensandprimarymesenchymecellsingressandformspicules,theurchinskeleton(Figure1).Thevegetalplatetheninvaginates,formingthearchenteron,orprimitivegut.Thearchenteronmigratesuptheseaurchin"sblastocoelwallwiththehelpofsecondarymesenchymecells(Figure1). Themigrationofthearchenterondependsnotonlyonsignalsandproteinsalreadypresentintheegg,butalsoonextracellularmaterialsthathavebeenincorporatedintotheorganism.KarpandSolurshhavehypothesizedthatsecondarymesenchymecells,whichformthefilopodiaofthedevelopingarchenteron(primarygut)requiresulfate(assomethinglikeanadhesive)inordertomigratealongtheextracellularmatrixwithintheblastocoelofadevelopingseaurchin(1974). Presumably,seaurchinembryosincorporatesulfatefromtheenvironmentintotheirextracellularmatrixes.Theextracellularmatrixcontainsacidmucopolysaccharide,whichwhenboundtosulfate,isroughinappearance(KarpandSolursh,1974).Thisroughnessisakintovelcro"sroughness,allowingsecondarymesenchymecellstopullthearchenteronupalongtheblastocoelcavity.Ifsulfateisnotpresent,ithasbeenobservedthatanarchenterondoesnotform(KarpandSolursh,1974).Fixedandstainedembryoswillindicateifthegutendodermcellshavedifferentiated(andhavesimplyfailedtomigrate). Figure1.Aschematicdrawingofagastrulatingseaurchinembryo.Notethesecondarymesenchymecells,intheformofafilopodia,attachingtotheblastocoelwalltopullthearchenteronupthroughtheblastocoeltoformthegutcavity. MaterialsandMethodsLytchinuspitusorStrongylocentrotuspurpuratus(fromthePacificcoast),andArbaciapunctulataorLytchinusvariegates(fromtheAtlanticandGulfofMexico)canbeusedforthisexperiment.Lytchinusvariegatuswasusedforourexperiment.SeaurchingametereleasewasinducedbyinjectionofKClandeggsandspermwereobtainedinseparatebeakersasdescribedinbasicprotocol"seaurchingametecollection". Eggswereobtainedinsulfatefree(SF)water,toensurethattheydidnotincorporatesulfate.AlleggswerethenwashedfourtimesinSFwater.Eggswerefertilizedasdescribedinthebasicprotocol"seaurchinfertilization".Eggswerecheckedforfertilizationunderthemicroscope.Abeakerwasfilledwithartificialseawater(ASW),andanotherbeakerwasfilledwithsulfate-freewater.FertilizedeggswerethenPipettedintoeachbeaker.Embryoswereincubatedatabout24°C(roomtemperature)overnight.Sinceembryoswerenotfullydevelopedbythenextday,theywereincubatedforanadditionalday.Ondaytwo,becauseyieldofhatchedblastulaswaslowandembryosweredilute,swimmingembryosfloatingatthetopofthewaterwerecentrifugedandconcentrated.Theseconcentratedembryoswerethenfixedasdescribedinthe"PreparationoffixedembryosforimmunocytochemistryandAPstaining".Embryoswerethendividedintotwogroupsandstainedwith5C7,amonoclonalantibodythatstainsendoderm,asdescribedinthe"ImmunofluorescentstainingofSeaUrchinembryos"orhistochemicallystainedasdescribedinthe"Histochemicalstainingofseaurchinembryosforalkalinephosphatase(AP)enzymeactivity". ResultsWhenobservedbeforefixing,embryosfromthecontrolgrouphadbecomeearlypluteuslarvaewithfullyformedarchenterons.Embryosfromthesulfate-freegroupwerestillspherical,andhadnoobservablearchenteronsaftertwodaysofincubation.Embryosstainedbothhistochemicallyandimmunofluorescentlyshowedsimilarpatterns.Alkalinephosphotase(AP)stainingrevealedactivitythroughoutthemidgutandhindgutofafullyformedarchenteroninacontrolembryo(Figure2A).However,APactivitywasonlyapparentatthevegetalplateinasulfate-freeembryo(Figure2B). A.B. Figure2.Fixedtwo-dayoldseaurchinembryosstainedforalkalinephosphotaseenzymeactivity.(A)Whitearrowpointstostaininginacontrolembryo,inpluteuslarvastage,whichrevealsenzymeactivitythroughoutafullyformedarchenteronorguttube.(B)Whitearrowpointstostaininginanembryoraisedinasulfate-freeenvironment,whichrevealsactivityonlyattheposteriororvegetalplateandnodiscernablearchenteronformation. Immunofluorescentstainingforvegetalplateandposteriorarchenteroncellsrevealedstainingthroughoutafullyformedarchenteroninapluteuslarvacontrol(Figure3A).Aringofimmunofluorescentstaininginanembryoraisedinasulfate-freeenviromentappearedonlyatthevegetalplate,withnodiscernablearchenteronformation(Figure3B). A.B. Figure3.Fixedtwo-dayoldseaurchinembryosstainedwith5C7,anantibodytovegetalplateandposteriorarchenteroncells.(A)Whitearrowindicatesstainingthroughoutafullyformedarchenteroninacontrolembryoinpluteuslarvastage.(B)Thewhitearrowpointstoaringofstainedvegetalplateandarchenteroncellsthathavenotformedadiscernablearchenteron. Discussion AshypothesizedandfoundbyKarpandSolursh(1974),embryosraisedinasulfate-freeenvironmentdoindeedfailtoformarchenterons.Supportingthefindingsthatsulfateactsonlyasanadhesivetoallowsecondarymesenchymecellstopullthearchenteronupthroughtheembryo,sulfate-freeembryocellshaddifferentiated.APactivityindicatedthatsulfate-freeraisedembryoshavegutcells.Thesecellssimplyhavenotmigratedtothecorrectlocation.Additionally,theimmunofluorescentstainingpatterninthesulfate-freeembryoappearedtobearing,suggestingthatthecellshadinvaginated.Thisfindingwouldfurthersupportthehypothesisthatsulfateisnecessaryfortheadhesionofsecondarymesenchymecellstotheblastocoelwalltoextendandformthearchenteron.Thecellsinvaginated,butlackedtheABIlitytomigrateandformanarchenteron.ThesedatasupportthefindingandhypothesisofKarpandSolursh(1974).Interstingly,embryosinthisexperimentdidnotferitilixeaswellanddevelopedmoreslowlythaneggsfromthesamedonorcollected,fertilized,andraisedinartificialseawater.Furtherexperimentsshouldbedonetodetermineifsulfate(orthelackthereof)affectsfertilizationanddevelopmentprocessesmoregenerally.Objective
Abstract