Origin of the blastema in regenerating limbs of the newt Triturus viridescens: An autoradiographic study using tritiated thymidine to follow cell proliferation and migration☆

[摘要] ThepresentstudyhasutilizedautoradiographytodetectincorporationoftritiatedthymidinebythecellsoftheregeneratingTriturusviridescenslimbduringblastemaformationandtofollowthesubsequentmigrationofcellslabeledbytheisotope.NewinformationwasobtainedonsitesofDNAsynthesisduringblastemaformationandtheroleoftheinternaltissuesandepitheliumwasre-evaluatedbytracing,forthefirsttime,theactualfateoflabeledcellsduringnormalregeneration.Threeseriesofexperimentswereperformed.Inthefirst,regeneratinglimbs1–28dayspostamputationwerefixedthesamedaythattheanimalswereinjectedwithtritiatedthymidine.ThisexperimentrevealedthatDNAsynthesisbegins4–5daysafteramputationinthededifferentiatingmuscle,endomysium,epimysium,periosteum,nervesheaths,andlooseconnectivetissueofthestumpforadistance∼1mmproximaltotheamputationsurface.ThenumberofcellsintheinnertissuessynthesizingDNAincreasesrapidly10–20dayspostamputation.TheepidermiswhichmigratesoverthewoundsurfaceceasestosynthesizeDNAwithinabout2days.Epitheliumproximaltotheamputationsurface,however,reachesahighlevelofDNAsynthesis8daysafteramputation,anditscellsmigratedistallytoincreasethesizeoftheapicalcap10–15dayspostamputation.Duringblastemaformation,nomorethan2%ofthecellsintheapicalcapincorporatethymidine,theseratherfeebly.Aftertheblastemaisestablishedandtheregeneratehasbeguntoelongate,theapicalcapthinsoutanditscellsbegintosynthesizeDNAagain.Inthesecondexperimentalseries,limbsweretreatedwithtritiatedthymidineduringregeneration(5,10,and15dayspostamputation)andrepresentativelimbswerefixedatdailyintervalsaftertreatment.Thededifferentiatinginnercellsincorporatedalargeamountofthymidineonthedayofinjection,whereastheapicalepitheliumdidnot.Almostalltheblastemacellsthatappearedsubsequentlywerelabeled,anditcanbeconcludedthattheywerederivedfromthededifferentiatinginternaltissues.Inthethirdexperimentalseries,animalswereinjectedwithtritiatedthymidinebeforetheirlimbswereamputated.Epidermisistheonlylimbtissuethatlabelsunderthesecircumstances.Bloodcellsarelabeledintheirsitesoforigin,theliverandspleen.Afteramputationthelabeledepidermismigratesoverthewoundsurfaceandformsanapicalcapwhichremainswelllabeledthroughoutblastemaformation.TheisotopebecomesdilutedinthatareaofproliferatingproximalepitheliumshowninSeriesItobeactivelysynthesizingnewDNA.Bloodcellslabeledatthetimeofinjectionextravasateintothewoundedlimbtissues,butdisappearfromthelimb12–15dayspostamputationTheblastemacellswhichformintheselimbsthatcontainedlabeledbloodandapicalepitheliumarenotlabeled.Inthediscussion,theconceptthatthemorphologicalandphysiologicalchangeswhichtakeplaceindedifferentiatingtissuesmakepossibleaphaseofactivecellularproliferation,isemphasized.TheapicalepidermalcapdoesnotexhibittheDNAsynthesisoranyoftheotheressentialfeaturesofdedifferentiatingcellsand,whenthisepidermisislabeledbyappropriatetreatment,notransformationofepithelialcellsintoblastemacellscanbedemonstrated.Indeed,thecellsoftheapicalcapgiveevidenceofbeingmorehighlydifferentiatedthanepidermiselsewhere.

[学科分类] 生物科学（综合）