Extended exposure to Sonic hedgehog is required for patterning the posterior digits of the vertebrate limb

摘要

Sonichedgehog(Shh)isakeysignalinestablishingdifferentdigitfatesalongtheanteriorndash;posterioraxisofthevertebratelimbbud.AlthoughtheanteriordigitsappeartobespecifiedbydifferentialconcentrationsofShhinatraditional,morphogen-likeresponse,recentstudieshavesuggestedthatposteriordigitsarespecifiedbyanextendedtimeofexposuretoShhratherthan,orinadditionto,athresholdconcentrationofShh.ThismodelfordigitpatterningdependsuponcontinuedShhsignalingintheposteriorlimbthroughmid-to-latebudstages.Wefindthatcyclopamine,apotentantagonistofShhsignaling,candown-regulatehedgehogtargetgenesintheposteriorlimbthroughoutthetimeemShh/emisexpressed,indicatingthatcontinuedactiveShhsignalingindeedtakesplace.TofurtherexploretherelativerolesoftimeandconcentrationofShhduringlimbdevelopment,wecarriedouttwoadditionalseriesofexperiments.Totesttheeffectoflimitingthetime,butnottheamountofShhproduced,wetreatedchickembryoswiththehedgehogantagonistcyclopamineatvariousstagesoflimbdevelopment.WefindthatshortexposurestoShhresultinspecificationofonlythemostanteriordigitsandthatmoreposteriordigitsarespecifiedsequentiallywithincreasingtimesofuninterruptedShhactivity.TotesttheeffectoflimitingthelevelofShhproduced,butnotthetimeofexposure,wegeneticallymodifiedShhproductioninmice.Aspreviouslyshown,reducingboththeconcentrationofShhproducedandthedurationofShhexposureresultsinalossofposteriordigits.WefindthatmaintainingalowlevelofShhproductionthroughoutthenormaltimeframeofZPAsignalingresultsinanearcompleterestorationoftheposterior-mostdigits.Thesedataareconsistentwith,andlendadditionalsupportto,themodelthatconcentrationofShhseenanddurationofexposurebothcontributetothedose-dependentspecificationofdigitidentities,butfortheposterior-mostdigitsthetemporalcomponentisthemorecriticalparameter./p/div