Cell-cell interaction by mouse limb cells during in vitro chondrogenesis: Analysis of the brachypod mutation☆

[摘要] Thispapercontainsobservationsandexperimentswhichcollectivelydemonstratearequirementforcell-cellinteractionsamonglimbbudmesenchymecellsduringchondrogenicdifferentiation.LimbbudcellsisolatedfrombrachypodismH(bpH)andwild-typemouseembyrosbetweenThielerstage16–17andmidstage21werecomparedwithrespecttotheirabilitiestoundergochondrogenicdifferentiationinhigh-densitymicromasscultures.NodulesformedbydissociatedDay12(stage20)bpHlimbbudcellshavebeenreportedpreviouslytobeabnormallyreducedinsizeandnumber,anddelayedinformation.Wecorroboratetheseresults,butfindthatbpHculturespreparedfromearlier-stagelimbbuds(betweenstages16–17andearlystage21)areprogressivelymorelikewild-typecultures.Stage16–17bpHculturesat72hractuallycontainnormalnumbersofandsizenodules,whilestage18bpHculturesareintermediatebetweenstages16–17andstage21innoduleformation.Ontheotherhand,wealsofindthattheinitialrateofaggregateformationisnormaleveninbpHculturespreparedfromstage20culturesinwhichnoduleformationisnotnormal.Preparationofculturescomposedprimarilyofearlystage21bpHlimbbudcellsmixedwithsmallquantities(e.g.,5%)ofstage16–17wild-typelimbbudcellsshowedsignificantincreasesincartilagenoduleformationovercontrolculturescomposedonlyofearlystage21bpHcells.Greaterproportionsofwild-typecellsobtainedfromembryosolderthanstages16–17wererequiredforthesamedegreeofnormalization,supportingthehypothesisthataspecificcelltype,whoseproportiondecreasesnormallyinthelimbbudovertime,isrequiredtoincreaseinvitrochondrogenesisbybpHcells.Additionally,culturescontainingstage23chicklimbcellsandearlystage21bpHcellsataratioof1:20containedwild-typelevelsofnodulespersquaremillimeterofculture.Thus,bpHcellsappeartorespondtochondrogenicinductivesignalsfromnormallimbmesenchymecells.InordertotestfortheabilityofbpHlimbbudmesenchymetoinducechondrogenesis,stage16–17bpHandwild-typelimbbudcells,whichformidenticalnumbersofaggregatesandnodulesinculture,wereeachmixedwithearlystage21bpHcellsatratiosof1:20,1:10,and1:3.Althoughlowproportionsofwild-typestage17cellssignificantlyincreasedthenumberofaggregatesandnodulesinthesemixedcultures,lowproportionsofbpHstage16–17cellsdidnot.Itis,therefore,concludedthattheprimarydefectofthebpHmutationislikelytoresideinthereducedabilityofaspecificmesenchymecellsubpopulationtoprovideaninductivestimulusforchondrogenesis.

[学科分类] 生物科学（综合）