Objective

Toobservetherolesulfateplaysinseaurchingastrulation,andtoreplicatethefindingsofKarpandSolursh,thatseaurchinembryosfailtogastrulatewithoutsulfate.Totestwhetherendodermdifferentiationcanoccurintheabsenseofthemovementsofgastrulation.

Abstract

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.

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