Immunofluorescence localization of the crystallins in amphibian lens development, with special reference to the γ-crystallins☆

摘要

TheontogenyandlocalizationofthecrystallinsinthedevelopinglensofR.pipienshasbeeninvestigatedbymeansoftheimmunofluorescencetechnique.AntibodiestoadultR.pipienstotal-lensproteinsandadultR.pipiens?3-crystallinsa€”whoseantigen,tissue,anddevelopmentalspecificityhadbeendemonstrateda€”wereusedinboththedirectandindirect(ora€?sandwicha€?)immunofluorescencetechniques;bothmethodsgavecomparableresults.Theseantibody-dyeconjugateswereappliedtosectionsofembryonicR.pipiens;aseriesoflensdevelopmentalstageswasrecognizedanddefinedbyustoaidinidentificationofthosestagessubjectedtoimmunofluorescenceanalysis.Apositivereactionfortheanti-total-lens-proteinimmunofluorescencereagentwasfirstnotedatLensDevelopmentalStageVI,inanumberofprospectiveprimarylensfibercells,andaweak,irregularreactionwasalsonotedintheexternallayer.TheintensityofimmunofluorescencewiththisreagentincreasedinthefiberareaandexternallayeruntilatLensDevelopmentalStageXallcellsofthelenswerepositive;theprimaryandsecondarylensfibercellsandlensepitheliumexhibitedimmunofluorescenceinorderofdecreasingintensity.Thefirstpositivereactionwiththeanti-?3-crystallinimmunofluorescencereagentcouldalsobeobservedatLensDevelopmentalStageVI.However,theimmunofluorescencewaspresentonlyintheregionoftheprospectiveprimarylensfibercellsandwasabsentintheexternallayer.TheintensityoftheimmunofluorescenceincreasedinthefiberareauntilLensDevelopmentalStageX,atwhichtimeitwasgreaterintheprimaryfibercellsthanthatinthesecondaryfibercells.NoimmunofluorescencecouldbedetectedintheexternallayerorinthelensepitheliumwiththisreagentuntilStageX.Atthistime,afterthelensiswelldifferentiatedandhasassumedthebasicadultconfiguration,aweakimmunofluorescencereactionwasnotedinthelensepitheliuminasmallpercentageofcases.Atnotime,witheitherimmunofluorescencereagent,couldanimmunofluorescencereactionbeobservedoutsidethelenstissue.Suchresultsindicatethatthe?3-crystallins,previouslyimplicatedinbovineandregeneratingnewtlensfiberdifferentiation,arealsoindicativeoflensfiberdifferentiationinthenormal,developinglensoftheanuranamphibian,R.pipiens.Inaddition,therelativelylateappearanceinlensdevelopment(i.e.,afterformationofalensvesicle)ofanyimmunofluorescencereactionforthecrystallinssuggestthattheyarenotnecessaryforlensinductionandsubsequentlensplacodeformation.