Detectionofapoptosisduringplanarianregenerationbytheexpressionofapoptosis-relatedgenesandTUNELassayJungShanHwanga,ChiyokoKobayashib,KiyokazuAgatab,KazuhoIkeoa,TakashiGojoboria,*aCenterforInformationBiologyandDNADataBankofJapan,NationalInstituteofGenetics,Yata1111,Mishima,Shizuoka411-8540,JapanbLaboratoryforEvolutionaryRegenerationBiology,CenterforDevelopmentalBiology,RIKENKobe,Kobe,Hyogo650-0047,JapanReceived10July2003;receivedinrevisedform27August2003;accepted5February2004Availableonline20April2004AbstractApoptosisisatightlyorganizedcelldeathprocessthatplaysacrucialroleinmetazoandevelopment,butithasnotyetbeenrevealedwhetherapoptoticeventsareinvolvedintheprocessofregeneration.Here,wetriedtodetectapoptoticcellsduringplanarianregenerationusingtheTdT-mediateddUTPnick-endlabeling(TUNEL)assayaswellastheexpressionofapoptosis-relatedgenes.ThreenovelcDNAswereisolatedfromaplanariancDNAlibraryandshowntobecloselyrelatedtoothermetazoancaspasesattheaminoacidsequencelevel.OneofthesecDNAs,Caspase-likegene3(DjClg3),wasexpressedprimarilyinapoptoticcellsbydoubledetectionswiththeTUNELassay.WholemountinsitustudiesindicatedthatDjClg3wasexpressedinthecellsofthemesenchymalspaceandalsoaroundthepharynxoftheintactbody.Itsexpressionintheregeneratingheadpiecewasseenintheblastemaandlesssignificantlyinthebrain,whileintheregeneratingtailpiece,DjClg3expressionwasdetecteduniformlythroughouttheentireregion.Inparallelexperiments,weperformedinsituTUNELassaystolocalizetheregionswherecelldeathoccurredduringregenerationandcomparableresultstotheDjClg3expressionpatternswereobtained.Thisisthefirstreporttoshowthatplanarianshaveapoptosis-relatedgenesandtheresultssuggestthattheapoptoticmechanismprobablytakesplacetoalargeextentinnormalintactwormsaswellasduringtheirregeneration.Wehypothesizethatthepresenceofapoptosisinplanariansmayhavearoleincontrollingcellnumbers,eliminatingunnecessarytissuesorcellsandremodelingtheoldtissuesofregeneratingbodyparts.D2004ElsevierB.V.Allrightsreserved.Keywords:Caspase;Programme...
