TheEffectsofNiCl2onSpiculeFormationJessicaAnnBillet,FranklinandMarshall,Classof2000BackgroundandObjectiveSeaurchinsexhibitrADIalholoblasticcleavage,eventuallyformingablastula.Shortlyaftertheblastulahatchesfromthefertilizationmembrance,theembryobeginsgastrulation.Gastrulationbeginswhenthevegetalsideoftheblastulabeginstothickenandflatten.Thisflatsheetofcellsiscalledthevegetalplate.Inthecenterofthevegetalplateasmallgroupofcellsbeginstochange.Thesecellsextendandcontractlong,thinfilopodia.Thesecellsthenbreakofffromtheepitheliumandmigrateintotheblastocoel.Thesemigratingcellsareknownastheprimarymesenchymecells(PMCS).Eventuallythemigratingcellslocalizewithintheventrolateralregionoftheblastocoel.ItisinthisareathatthePMCsfusetogethertoformsyncytialcables.Syncytialcableswilleventuallyformtheaxisofthecalciumcarbonatespiculesofthelarvalskeleton.Thepurposeofthislabistoexplorethedevelopmentofspiculesinaseaurchin"slarvalskeleton.NiCl2interfereswithspiculeandskeletalformation,byintroducinghalfoftheembryosintoasolutionofNiCl2,wehopetoobservetheeffectsofblockingskeletonformation.WewillstainalloftheembryoswithanIg8immunoflourescentantibody.Ig8willstainor"tag"theprimarymesenchymecellsofthedevelopingembryo.Thislabwilltaketwoweekstoaccomplish.Inthefirstweek,wewillcollectandfertilizeseaurchingametes.OnehalfoftheembryosproducedwillthenbesubjectedtoaNiCl2solutionuntilFridaymorning.OnSaturday,theembryoswillbefixedinpreparationforstaininginweektwo.DuringlabnextweekwewillstainthelabswithIg8antibody,animmunofluorescentstain.WewillusethisstaintoidentifyPCMsanddevelopingspicules.**WewillbeusingHytechinusvariegatusfromtheFloridacoast.Thegametesandembryoscanbeleftatroomtemperatureinordertodevelop**ExperimentalProtocolA.FertilizationofUrchinsSeestandardprotocol.B.NiCl2Treatment1)Afterfertilizationhasoccured,transferhalfoftheembryosintoasolutionof10mMNiCl2.Transfertheremaininghalf,thecontrol,intoasolutionofASW.2)Thetwogroupsshouldbeleftatleast24hourstodevelop.Trackthedevelopmentthroughthemesenchymeblastulastage.3)Thegroupsshouldbepreparedforstainwhenthecontrolgroup(ASW)reachesgastrulationandhasformedthepluteuslarva.C.StainingSeestandardprotocol.ResultsandDiscussion Thetwosamplesbehavedquitedifferently.TheIg8stainingrevealedthatPMCsofthecontrolgrouphadinfactmigratedaftergastrulationtoformtheendoskeleton,orspicules,oftheurchin.Intheexperimentalgroup,noneoftheembryoshadundergonecompletegastrulation.TheIg8stainingshowedthatthePMCcellshadfailedtomigrateout,theembryoitselfhadfailedtodevelop.Ouroriginalhope,thatwecouldblockskeletonformationintheurchin,wassatisfiedbythisexperiment.WealsoobservedthatNiCl2disruptsnotonlyskeletonformation,buttheprocessofgastrulationaswell.  References: -InfluenceofNiCl2ontheSkeletonFormationinSeaUrchin:Armstrongetal.(1993)Development119:833-840;Hardinetal.(1992)Development116:671-685.-FertilizationAndEarlyDevelopmentOfTheSeaUrchin.Cebra-Thomas,Judy.Spring1998.-BasicsofSeaUrchinDevelopment.Cebra-Thomas,Judy.Spring1999.GilbertSF.editor.1997.DevelopmentalBIOLOGy.5thed.:SinauerAssociates,Inc.,Sunderland,Mass,918p.
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