ObjectiveThepurposeofthisexperimentistodeterminetheeffectsofcalcium-freeseawateronthedevelopmentofseaurchinembryos.Embryoswereplacedincalcium-freeseaafterfertilizationandattheinitiationofgastrulationIntroductionCalciumisnecessaryintwoprocessesshortlyfollowingfertilization.Oneprocess,theacrosomalreaction,isthefusionoftheacrosomalvesicleandthespermplasmamembrane,whichresultsintheextensionoftheacrosomalprocess.Theacrosomalreactionisinitiatedbyafucose-containingpolysaccharidewithintheeggjelly,whichbindstothespermandallowscalciumtoenterthespermhead.Thesecondmechanismisthecorticalgranulereaction.Uponfertilization,thecalciumionconcentrationoftheeggincreasesgreatly,whichcausesthecorticalgranulemembranestofusewiththeeggplasmamembrane,releasingtheircontents.Awaveofcortical,releasedfromtheendoplasmicreticulum,beginsatthepointoffertilizationandtravelscompletelyaroundtheegg.Thecalciumionsneededforthesetwomechanismsarenotaresultofaninfluxofcalcium,butcomefromwithintheeggitself.Thereforewithdrawingcalciumduringfertilizationwouldnotaltertheseprocesses.Laterindevelopment,calciummayplayaroleincelladhesion.Themajorcelladhesionmoleculesareknownascadherins.Cadherinsarecalcium-dependentadhesionmolecules.Cadherinsarecriticalinestablishingandmaintainingintercellularconnections,andappeartobecrucialtothespatialsegregationofcelltypesandtotheorganizationofanimalform.Calciumionsareneededfortheadhesionofthesamecadherinmoleculesbetweencells.

Theseaurchinembryoprovidesapowerfulmodelsystemforthestudyofmorphogenesisandcadherinactivity.InastudyperformedbyJeffreyMillerandDavidMcClay,threedevelopmentaleventswhereshowtobedirectlyrelatedtocadherinmolecules:1)theacquisitionofcellpolarityincleavage-stageblastomeres;2)theepithelial-mesenchymalconversionofepithelialcellstomesodermalderivatives;and3)theconvergentextensionmovementsinvolvedinconstructingthearchenteron.Theirdataprovidednewinsightintothefunctionofcadherinsduringvariousmorphogeneticeventsandprovidedbuildingblocksforfutureexperimentsexploringcadherinlocalizationandadhesiveactivityduringdevelopment.

Figure:Earlyseaurchinembryoperformingfirstcleavage

Procedure*Gametecollectionfromadultseaurchins*Fertilizationofgametes*Calcium-FreefromFertilization*UsingasterilePipette,transferfertilizedeggstoashallowfingerbowl.*Addcalcium-freeseawater,alloweggstosettle,thanremoveexcessliquidandreplacewithcalcium-freeseawater.*Alloweggstodevelopedandcapturedigitalimagesseveraltimesduringa24-hourperiod.*ComparetocontrolembryoswhichdevelopedinASW*Calcium-FreefromVegetalPlatestage*Usingsterilepipette,transferfertilizedeggstoashallowfingerbowl.*FillwithASWandallowdevelopmentfor7to10hours.Capturedigitalimageinordertodocumenttransfertocalcium-freeseawater.*RemoveexcessASWandreplacewithcalcium-freeseawater.RepeatseveraltimesinordertoremoveasmuchcalciumaspossIBLe.*Alloweggstocontinuedevelopmentforanother14-17hours.Capturedigitalimagesofembryos.*ComparewithcontrolembryoswhichdevelopedinASW*ControlEmbryos*Usingsterilepipette,transferfertilizedeggstoashallowfingerbowl.FillwithASW.*Allowgrowthforthesametimeastheexperimentalembryos.Captureimagesasametimeasexperimentalembryoforcomparison.

ResultsTheembryoswereobservedaftera24hourstimeperiod.Therewasstrongevidencetoshowthecalciumisamajorcontributorinthedevelopmentoftheseaurchinembryos.Thecontrolembryosdevelopednormallyandattimeofobservationwereperforminggastrulation(SeeFigure1).Theembryos,whichwereplacedincalcium-freeseawaterimmediatelyfollowingfertilization,begandevelopmentnormallyandweresimilartothecontrolembryosatthe7-hourstage(SeeFigure2and3).Butwhentheywereobservedatthe20-hourstagetheembryoshadnotcontinuedthroughgastrulation(SeeFigure4).Theembryosappearedtostopdevelopmentsoonafterthe7-hourstage.Theembryos,whichwereplacedincalcium-freeseawaterafter7hoursofdevelopment,showedverydifferentresults(SeeFigure5).Theembryoshaddiedandnolongermaintainedtheirshape.Itappearedasifthecellswerenolongeradheredtogether.Basedontheseresults,majoreventsduringdevelopmentaredependentonthepresenceofcalcium.Inthefuture,furtherdevelopmentscanbemadeinvolvingtheimportanceofcadherinmoleculesinembryogenesis.