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神经系统特异表达的新基因NDNF功能及发育学研究
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ROLEOFGDNFINCOLONICENSDE；statisticalsigni?cance.A；respectively.Thesevalues；cimanyDlatnempoleveDFig.；4.；Fig.5.；identi?edintherateofENCC；GdnfIsChemoattractivetoE；Inadditiontoregulati
ROLEOFGDNFINCOLONICENSDEVELOPMENT1407
statisticalsigni?cance.AtE12,withcolorectalENCCcolonizationcom-plete,therateofcellproliferationdecreaseddramatically.ProliferationratesatthisstageforRCAS-GFP,-Gdnf,andCRNAiwere8.8,9.1,and8.3%,
respectively.Thesevaluesshowsimi-lartrendsasatE9,butwerenotstat-isticallysigni?cant.Nodifferencewass
cimanyD latnempoleveDFig.
identi?edintherateofENCCapopto-sis,whichwasdeterminedusingantibodiestocleavedcaspase-3andHNK-1.Veryfewapoptoticcrest-derivedcellswereidenti?edintheinjectedcolons,regardlessoftheretrovirusexpressed(notshown).
GdnfIsChemoattractivetoENCCsintheColon
Inadditiontoregulatingcellularpro-liferationanddifferentiation,modula-tionofGdnfexpressioncouldimpactENScolonizationdirectlybyaffectingENCCmigration.Totestthis,beadscoatedwithrecombinantGdnfproteinoranti-GdnfantibodywereembeddedintothehindgutmesenchymepriortoENCCarrival.After3daysin
Fig.4.Gdnfinducesprematureneuronaldif-ferentiationandpromotesENCCproliferation.Top:Thepercentageofneuronaldifferentia-tionwascalculatedinE9-andE12-injectedembryos.Intestinewasharvestedatthosestagesandimmunohistochemistryperformedoncross-sectionsthroughthemid-colorectumusingantibodiestoENCCs(p75NTRorHNK-1)andneurons(Hu).Left:MyentericganglionfromanE12RCAS-GFPhindgut.ThenumberofneuronsandENCCswascountedtodeter-minethepercentageofHu-immunoreactiveENCCs(arrows).Bottom:ENCCproliferationwasdeterminedusingBrdUincorporationintoHNK-1tENCCs.Left:MyentericganglionfromanRCAS-GFP-infectedhindgutwitharrowsmarkingBrdUtcells.ThepercentageofproliferatingENCCswascalculatedbydividingthetotalnumberofHNK-1tENCCsbythenumberofBrdUt/HNK-1tdouble-im-munoreactivecells.Thegraphsshowtheav-erageandstandarddeviationforeachgroup.Lineswithasteriskdenotestatisticalsignifi-cance(P&0.02).
Fig.5.GdnfischemoattractivetomigratingENCCsinthecolorectum.E5.5intestinewasisolatedandthecolonimplantedwithaPBSbead(A,D),Gdnfbead(B,E),oranti-Gdnfbead(C,F),thenculturedincollagengelfor3days.WholemountTuj1stainingshowsENCCmigrationcontinuingpastthecontrolbead(A),whileGdnf-andanti-Gdnf-coatedbeadsarrestedmigration(B,C).TheproximalendofthecolonismarkedwithanasteriskandthemigratorywavefrontwithanarrowinACC.Insetsshowmagnifiedviewsoftheareanearthebead.Longitudinalsections(proximalendtotheleft)werelabeledwithHuantibody.ENCCsmigratearoundthePBSbead(D).Incontrast,theGdnf-coatedbeadattractsen-tericneurons,causingthemtoclusteraroundthebead(E).Beadscoatedwithanti-Gdnfantibodystopmigration,butdonotleadtoneuronalclustering(F).
cimanyD latnempoleveD1408MWIZERWAETAL.
duringwhichtimeENCCsnormallycolonizethecolon,ENSdevelopmentwasvisualizedimmunohistochemi-callyonlongitudinalsections.AsshownbywholemountstainingwithTuj1antibody,PBSbeadsdidnotaffectENCCmigration(Fig.5A).ENCCswereabletomigratepastthebeadandintothedistalcolon.Incon-trast,bothGdnf-andanti-Gdnf-coatedbeadsarrestedmigration(Fig.5B,C).ENCCsstoppedintheimmedi-atevicinityofthesebeads,withonlyafewmigratingashortdistancebeyondthem.LongitudinalsectionsofgutsstainedwithHuantibodyrevealedmoredetail.ThePBSbeadhadnoimpactonmigrationandENCCswereabletomigratepastthebead(Fig.5D).WeagainobservedthatbothGdnfandanti-Gdnfbeadsarrestedmigration.OverexpressionofGdnfcausedENCCstoaccumulatearoundthebeadandalsotodifferentiate,asshownbythestrongHuexpressionaroundthebead(Fig.5E),preventingmigrationpastthebead.Incontrast,whileanti-Gdnfbeadsalsostoppedmigration,noENCCaccumulationoccurredonthebead(Fig.5F).
DISCUSSION
FormationoftheENSrequiresthemigration,proliferation,anddifferen-tiationofentericneuronalprecursorsoriginatingprimarilyfromthevagalneuralcrest.Beginningasasmallpop-ulationofprogenitorcells,theENSreliesoncontinuedcellularprolifera-tiontogenerateasuf?cientpoolofcellstocolonizetheentirelengthofthegut.Whileproliferationandmigrationarecritical,someENCCsmustexitthecellcycleanddifferentiateintoneuronstoformthecomplexfunctionalnetworkthatregulatesintestinalmotility.Thisbalancebetweenmigration,prolifera-tion,anddifferentiationisessentialandabnormalmigration,inadequateproliferation,orprematuredifferentia-tioncanallresultinintestinalagan-glionosis,asoccursinhumanHirsch-sprung’sdisease.
TodeterminetheeffectsofGdnfonENSdevelopment,wemodulateditsactivityinavianembryosusingretro-virus-mediatedgeneoverexpressionandretroviralvector-basedgenesilencing.WhiletheessentialroleofGdnfsignalinginENSdevelopment
hasbeenestablishedinhumans(AmielandLyonnet,2001),rodents(Mooreetal.,1996;Picheletal.,1996;Sanchezetal.,1996),andzebra?sh(Shepherdetal.,2001),mostofthesestudiesusedculturedneuralcrestCderivedcellsdevoidofthemesenchy-malenvironmentknowntobeimpor-tantduringENSdevelopment(Chala-zonitisetal.,1998;Hearnetal.,1998;Heuckerothetal.,1998;Taravirasetal.,1999;Worleyetal.,2000;Youngetal.,2001;Natarajanetal.,2002;Nganetal.,2008).OurapproachallowedustomodulateGdnfactivityinvivo,speci?callyinthepresumptivemesodermofthedistalgut,whichhasmajorclinicalimportanceinneuroin-testinaldiseases,suchasHirsch-sprung’sdiseaseandintestinalneuro-naldysplasia.WefoundthatlossofGdnfsignalingcausesasigni?cantdelayinENCCcolonizationofthecolorectum,leadingtodistalcolorectalaganglionosisandseverehypoganglio-nosisproximally.Thisphenotypeisconsistentwiththatseeninrodentmodels,wheretheextentofENSde?-ciencyvarieswithGdnfdosage.NullallelesofGdnfleadtototalintestinalaganglionosis(Mooreetal.,1996;Picheletal.,1996;Sanchezetal.,1996),whileheterozygousmiceexhibitsigni?canthypoganglionosis(Shenetal.,2002;Gianinoetal.,2003;Flynnetal.,2007),especiallyaffectingthedis-talgut(Shenetal.,2002).Asimilardose-responseisobservedwithRet,whereprogressivereductionsinRetexpressioncorrespondwithlongerseg-mentsofintestinalaganglionosis(Uesakaetal.,2008).We?ndthatthecauseofthehypoganglionosis/aganglio-nosisismultifactorial,consistentwiththeknownpleiotropiceffectsofGdnfonENCCproliferation,neuronaldifferen-tiation,andmigration.Gdnfinhibitionalsoresultedinashorterlengthofcolor-ectum,suggestingotherGdnf-mediatedeffectsinthegutthatneedtobeexploredfurther.Additionally,inhibitionofGdnfresultedinamarkedreductioninthesizeofthenerveofRemakandinthenumberof?bersextendingintothecolon.These?ndingssuggestthat,likethevagalcrestCderivedcells,sacral-derivedENCCsarealsodependentonGdnfsignaling,whichmayexplainwhytheterminalgutremainsaganglionicinHirschsprung’sdiseaseandRet-de?-cientmousemodels.
GdnfPromotesENCCProliferationintheColorectumatE9ButNotE12
InvivooverexpressionofGdnfenhancedENCCproliferationby29%,whileinhibitionofthegenereducedproliferationby18%.ThisabilityofGdnftopromotecellularproliferationhasbeendemonstratedpreviouslyinculturedENCCs(Chalazonitisetal.,1998;Hearnetal.,1998;Heuckerothetal.,1998;Taravirasetal.,1999;Wor-leyetal.,2000;Barlowetal.,2003;Nganetal.,2008).Theanti-mitogeniceffectofdownregulatingGdnfhasbeenshowninGdnfheterozygousmice,whichdemonstrateasigni?cantlyreducedrateofENCCproliferationcomparedtowild-typemice(Gianinoetal.,2003).Interestingly,themito-geniceffectofGdnfwasnotobservedinE12embryos.Asimilar?ndingwasreportedbyChalazonitisetal.(1998),whoshowedthatGdnfstimulatedtheproliferationofneuralcrestCderivedcellsisolatedfromE12intestine,butnotfromE14orE16gut.Inbothaviansandrodents,themajorproliferativeeffectofGdnfappearstobepresentwhileENCCsarestillmigratingalongthegut.ThustheeffectofGdnfonENCCdevelopmentchangeswithtime,highlightingthedynamicnatureofitsroleduringENSdevelopment.TherecentdemonstrationthatinactivationofGFRa1duringlategestationinmice(atE15,5,wellaftercolonizationofthecoloniscomplete)leadstoentericneu-ronalcelldeathspeci?callyinthecolonmaybeduetothefactthatexpressionofbothGdnfandGFRa1ismarkedlyreducedinthesmallintestineandup-regulatedinthecolonatthisstage(Uesakaetal.,2007),illustratingthedynamicspatialandtemporalroleofGdnfduringENSdevelopment.
ModulatingGdnfExpressionInVivoLeadstoPrematureNeuronalDifferentiation
OurresultsdemonstratethatbothoverexpressionandinhibitionofGdnfinduceprematureneuronaldifferen-tiation.RCAS-Gdnfledtoa20%increaseintheproportionofcolo-rectalENCCsthatdifferentiatedintoneurons,whiletherewasa40%increaseinRCAS-Gdnf-RNAi
ROLEOFGDNFINCOLONICENSDEVELOPMENT1409
cimanyD latnempoleveDembryos.Withbothviruses,thepro-portionofdifferentiatedENCCsintheE9colon(60%inRCAS-Gdnfand70%inRCAS-Gdnf-RNAi)didnotchangesigni?cantlybyE12(59%inRCAS-Gdnfand72%inRCAS-Gdnf-RNAi).Incontrast,theproportionofneuronaldifferentiationincontrolgutsincreasedfrom50to66%betweenE9andE12,respectively.Theseresultsdemonstratethatmodu-latingGdnfexpressioneitherupordownduringcolonizationofthecolo-nicENSinducesprematureneuronaldifferentiation.
Previousstudieshaveshowninduc-tionofneuronaldifferentiationbyadditionofGdnfinvitrotoculturedENCCs(Chalazonitisetal.,1998;Hearnetal.,1998;Taravirasetal.,1999;Nganetal.,2008),consistentwithourinvivo?ndingsthatGdnfoverexpressioninducesdifferentia-tion.However,the?ndingthatGdnfinhibitionalsoinducesENCCdiffer-entiationisinteresting.Flynnetal.(2007)analyzedneuronaldifferentia-tioninmiceheterozygousforGdnfanddidnot?ndastatisticaldiffer-encecomparedtowild-typemice,althoughtheirdatadoshowaconsist-enttrendtowardincreaseddifferen-tiationintheheterozygotes.Onepos-sibleexplanationforour?ndingsisthatsincethelossofGdnfexpressiondelaysmigration,ENCCsareleftwithaprolongedexposuretoasyetunidenti?edlocalfactorsinthecolonthatmaydriveneuronaldifferentia-tion.FurtherstudiesintothefactorsregulatingENCCdifferentiationinvivoarenecessary.Nevertheless,ouranalysisdemonstratesthatGdnflev-elsmustbetightlyregulatedinthedevelopinggutduringthearrivalofENCCs,asanychangeinthebaselinelevelofexpressionhasasigni?cantimpactonthetimingofneuronaldif-ferentiation,whichcanleadtodown-streameffectsonENCCmigration.Asexpected,theincreaseinneuronaldifferentiationwasassociatedwithhypoganglionosisanddistalaganglio-nosisinRCAS-Gdnf-RNAiguts.How-ever,RCAS-Gdnfintestineswerenor-mallycolonizedbyENCCsdespitetheirprematuredifferentiation.WehypothesizethatthisisduetothemarkedincreaseinENCCprolifera-tionobservedintheseembryos,whichmaycompensateforthepremature
differentiationandallowthegenera-tionofanadequatepoolofprecursorcellstocompleteENScolonizationofthedistalgut.
AlteringLocalGdnfLevelsintheColonArrestsENCCMigration
Gdnfhasbeenproposedtobechemo-attractivetoENCCsbasedoninvitrostudiesusingorgancultures(Youngetal.,2001;Natarajanetal.,2002).TheexpressionofGdnfinembryonicmouseintestineisconsistentwithapotentialroleasachemoattractantintheforegutandmidgut,sinceGdnfisinitiallyexpressedstronglyinthestomachandlaterinthececumatstageswhenENCCsarestillrostraltothatlevel(Natarajanetal.,2002).However,asimilarpatternofGdnfexpressionhasnotbeenobservedinthehindgut(Natarajanetal.,2002).Inavians,we?ndGdnfmRNAstronglyexpressedinthececaandclo-acaatE5,withrapidexpansionofexpressionthroughoutthelengthofthehindgutmesodermbyE7,whenENCCsarestillintheproximalcolon(NagyandGoldstein,2006).Thispat-ternofexpressionmakesitunclearwhetherGdnfhasasimilarchemoat-tractiveroleinthedistalgut.WefoundthatthepresenceofGdnf-coatedbeadsinthecolorectalmesen-chymewashighlychemoattractivetoENCCs,whichweredrawntothebeadandfailedtomigratefarbeyondit.Theseresultscon?rmthatcolo-rectalENCCsarechemoattractedtoGdnf.TheadditionaleffectofGdnftopromoteneuronaldifferentiationmayhavealsocontributedtothearrestofmigration.Interestingly,beadscoatedwithanantibodythatblocksGdnffunctionalsoresultedinagangliono-sisdistaltothebead,likelyviaadif-ferent,andmultifactorial,mecha-nism.LocalinhibitionofGdnfexpressionledto(1)anabsenceofthemigratorycue,(2)promotionofneuro-naldifferentiation,and(3)down-reg-ulationofENCCproliferation.The?ndingthateithertoomuchortoolit-tleGdnfexpressioninterfereswithENCCmigrationagaindemonstratesthecriticallevelsoftheproteinneces-sarytopromotenormaldevelopmentofthecolorectalENS.
EXPERIMENTALPROCEDURESAnimals
FertilizedWhiteLeghornchicken(Gallusgallus)andquail(Coturnixcoturnixjaponica)eggswereobtainedfromcommercialbreedersandmain-tainedat37??Cinahumidi?edincuba-tor.EmbryoswerestagedaccordingtoHamburgerandHamilton(HH)tables(HamburgerandHamilton,1992)orthenumberofembryonicdays(E).
RCASViruses
TotalRNAwasobtainedfromE5CE10chickgutsusingTrizolandcDNApre-pared.ThefollowingprimersweredesignedbasedonthechickGdnfsequence(NCBIAccessionno.XM_425018):forwardAGTAATGGGCAGAGCAGCTT,reverseTCAGACACATCCACACCTTT,withtheadditionofanupstreamKozaksequenceandNotI/ClaIrestrictionsites.Theampli-?edproductwasclonedintotherepli-cation-competentretroviralvectors,RCAS(A)andRCAS(B),andbothvec-torswereinjectedsimultaneouslytomaximizeGdnfexpression.
Retroviralvector-basedGdnfgenesilencingwasachievedusingthemethoddescribedbyDasetal.(2006).TwoRNAitargetsequenceswereselected(GTGATGCAGTTGAGACCACGTAandACTCTAATATGCCAGAGGATTA)andeachwasclonedintoamicroRNAoperoninthepRFPRNAiCshuttlevector(ARK-Genomics).TheoperonwasexcisedwithNotIandClaIanddirectionallyclonedintoRCAS.OneRNAitargetwasclonedintoRCAS(A)andtheotherintoRCAS(B)andbothinjectedinovosimultaneouslytoachievemaximalgenesilencing.
DF1cellstransfectedwiththeviralconstructweregrowntocon?uenceandthesupernatantharvested.Viralharvesting,concentration,andtiter-ingwereperformedasdescribed(Cepko,1991).ControlinfectionswereperformedusinganRCAS-green?uo-rescentprotein(GFP)vector.
InOvoViralInfection
EmbryoswereincubateduntilE2($HH12),windowed,andviewedunderaNikonSMZ800dissection
cimanyD latnempoleveD1410MWIZERWAETAL.
microscope.Approximately1mlofvi-rus($1?105cfu)wasinjectedintoeachsideoftheembryo,directedatthepresumptivegutmesoderm,basedonestablishedchickfatemaps(Mat-sushita,1995).Theeggswerethensealed,returnedtotheincubator,andharvestedatvarioustime-points,5C10daysafterinfection.
OrganCulture
TostudymigrationofENCCsalongtheintestine,E5(HH27)quailorE5.5(HH27)chickgutwasdissectedfromtheumbilicustothecloacaandem-beddedinathree-dimensionalserum-freecollagengelmatrix,preparedasdescribed(NagyandGoldstein,2006).ThecollagengelwassupplementedwithGdnf(10ng/R&DSystems,Minneapolis,MN)oranti-Gdnffunc-tion-blockingantibody(10mg/R&DSystems).After3days,theexplantswereremovedandprocessedforimmunohistochemistry.
Forbeadexperiments,heparin-acrylicbeads(70C150Sigma,St.Louis,MO)wererinsedinPBS,soakedinprotein(100ng/mlGdnfor100mg/mlanti-Gdnf)at37??Cfor1hrand4??Covernight.Theintes-tinewasdissectedfromE5.5(HH27)chick,fromumbilicustocloaca,andthehindgutincisedwithtungstenneedlestocreateaspaceinthemes-enchymeforthebead.Thebeadwasinsertedwithablunt-endglassneedleandthegutsembeddedinathree-dimensionalcollagengelmatrixfor3days(NagyandGoldstein,2006).
Immunohistochemistry
Gutswere?xedin4%formaldehyde,gelatin-embedded,frozen,andsec-tionedat12mm.PrimaryantibodiesincludedTuj1(1:1,000;Covance,Princeton,NJ),p75NTR(kindgiftofLouisReichardt)(WeskampandReichardt,1991),HuC/D(1:100;Mo-lecularProbes,Eugene,OR),HNK-1(1:100;Fisher,Pittsburgh,PA),cleavedcaspase-3(1:100;CellSignal-ing,Danvers,MA),and3C2(1:5;De-velopmentalStudiesHybridomaBank,IowaCity,IA).Detectionwasbyvisiblelightor?uorescence.Forvisiblelight,sectionswereincubatedwithantibodyfor45min,followedbybiotinylatedgoatanti-mouseIgMor
IgG(VectorLabs,Burlingame,CA)andavidin-biotinylatedperoxidasecomplex(VectastainEliteABCkit,VectorLabs).Endogenousperoxidaseactivitywasquenchedbyincubationfor10minwith3%hydrogenperoxide(Sigma)andprimaryantibodybind-ingsitesvisualizedwith4-chloro-1-naphthol(Sigma)ordiaminobenzi-dine(DAB).For?uorescence,second-aryantibodiesincludedAlexaFluor594and488goatanti-mouseIgG,AlexaFluor594and488goatanti-mouseIgM,andAlexaFluor594and488goatanti-rabbitIgG(MolecularProbes).CellnucleiwerestainedbyDAPI(VectorLabs).
Forwholemountimmuno?uores-cence,intestineswere?xedwith4%formaldehydefor3hr.AfterseveralwashesinPBS,specimenswereincu-batedovernightwithprimaryanti-bodies(Tuj1orp75NTR)followedbysecondaryantibodies(AlexaFluor594anti-mouseIgGorAlexaFluor488anti-rabbitIgG)for5hr.
Cellproliferationwasdeterminedbyincubatingthegutfor4hrin5mg/mlBrdU(Roche,Nutley,NJ)solution.DNAwasdenaturedbyincubatingslideswith2NHClinH2Oat37??Cfor30min.Afterneutralizationwith0.1Mboricacid(pH8.5)for30min,sec-tionswerestainedwith?uorescein-conjugatedanti-BrdUantibody(Roche).
InSituHybridization
Tissueswere?xedin4%formalde-hyde,dehydratedinmethanol,andstoredatà20??Cuntilreadyforproc-essing.RiboprobesynthesisandwholemountRNAinsituhybridiza-tionwasperformedaspreviouslydescribed(Jowett,1999).AntisenseGdnfRNAprobewasgeneratedbyamplifyingafragmentofGdnfsequencefromtotalRNApreparedfromE9chickgut(NagyandGold-stein,2006).
ACKNOWLEDGMENTS
WethankLouisReichardtforanti-chickp75NTRantibody.3C2antibodywasobtainedfromtheDevelopmentalStudiesHybridomaBank,developedundertheauspicesoftheNICHD,andmaintainedbytheUniversityofIowa.A.M.G.issupportedbyNIHR01DK080914.N.N.issupportedbya
BolyaiFellowshipoftheHungarianAcademyofSciences.
REFERENCES
AmielJ,LyonnetS.2001.Hirschsprungdisease,associatedsyndromes,andgenetics:areview.JMedGenet38:729C739.
BarlowA,deGraaffE,PachnisV.2003.EntericnervoussystemprogenitorsarecoordinatelycontrolledbytheGpro-tein-coupledreceptorEDNRBandthereceptortyrosinekinaseRET.Neuron40:905C916.
BarlowAJ,WallaceAS,ThaparN,BurnsAJ.2008.Criticalnumbersofneuralcrestcellsarerequiredinthepathwaysfromtheneuraltubetotheforeguttoensurecompleteentericnervoussystemformation.Development135:.BurnsAJ,LeDouarinNM.1998.Thesac-ralneuralcrestcontributesneuronsandgliatothepost-umbilicalgut:spa-tiotemporalanalysisofthedevelopmentoftheentericnervoussystem.Develop-ment125:.
CepkoCL.1991.Transductionofgenesusingretroviralvectors.In:AusubelFM,KingstonRE,MooreDD,SeidmanJG,SmithJA,StruhlK,editors.Cur-rentprotocolsinmolecularbiology.NewYork:JohnWileyandSons.p9:9.10.11C19.14.13.
ChalazonitisA,RothmanTP,ChenJ,GershonMD.1998.Age-dependentdif-ferencesintheeffectsofGDNFandNT-3onthedevelopmentofneuronsandgliafromneuralcrest-derivedpre-cursorsimmunoselectedfromthefetalratgut:expressionofGFRalpha-1invitroandinvivo.DevBiol204:385C406.DasRM,VanHaterenNJ,HowellGR,FarrellER,BangsFK,PorteousVC,ManningEM,McGrewMJ,OhyamaK,SaccoMA,HalleyPA,SangHM,StoreyKG,PlaczekM,TickleC,NairVK,Wil-sonSA.2006.ArobustsystemforRNAinterferenceinthechickenusingamodi?edmicroRNAoperon.DevBiol294:554C563.
deGraaffE,SrinivasS,KilkennyC,D’AgatiV,MankooBS,CostantiniF,PachnisV.2001.DifferentialactivitiesoftheRETtyrosinekinasereceptoriso-formsduringmammalianembryogene-sis.GenesDev15:.
DruckenbrodNR,EpsteinML.2007.Behaviorofentericneuralcrest-derivedcellsvarieswithrespecttothemigra-torywavefront.DevDyn236:84C92.DruckenbrodNR,EpsteinML.2009.Age-dependentchangesinthegutenviron-mentrestricttheinvasionofthehindgutbyentericneuralprogenitors.Development136:.
EmisonES,McCallionAS,KashukCS,BushRT,GriceE,LinS,PortnoyME,CutlerDJ,GreenED,ChakravartiA.2005.Acommonsex-dependentmuta-tioninaRETenhancerunderliesHirschsprungdiseaserisk.Nature434:857C863.
ROLEOFGDNFINCOLONICENSDEVELOPMENT1411
cimanyD latnempoleveDEnomotoH,ArakiT,JackmanA,Heucker-othRO,SniderWD,JohnsonEMJr,Mil-brandtJ.1998.GFRalpha1-de?cientmicehavede?citsintheentericnervoussystemandkidneys.Neuron21:317C324.FlynnB,BergnerAJ,TurnerKN,YoungHM,AndersonRB.2007.EffectofGdnfhaploinsuf?ciencyonrateofmigrationandnumberofentericneuralcrest-derivedcells.DevDyn236:134C141.GianinoS,GriderJR,CresswellJ,Eno-motoH,HeuckerothRO.2003.GDNFavailabilitydeterminesentericneuronnumberbycontrollingprecursorprolif-eration.Development130:.HamburgerV,HamiltonHL.1992.Ase-riesofnormalstagesinthedevelop-mentofthechickembryo.1951.DevDyn195:231C272.
HearnCJ,MurphyM,NewgreenD.1998.GDNFandET-3differentiallymodulatethenumbersofavianentericneuralcrestcellsandentericneuronsinvitro.DevBiol197:93C105.
HeuckerothRO,LampePA,JohnsonEM,MilbrandtJ.1998.NeurturinandGDNFpromoteproliferationandsur-vivalofentericneuronandglialpro-genitorsinvitro.DevBiol200:116C129.HottaR,AndersonRB,KobayashiK,New-greenDF,YoungHM.2009.Effectsoftis-sueage,presenceofneuronesandendothelin-3ontheabilityofentericneu-roneprecursorstocolonizerecipientgut:implicationsforcell-basedtherapies.NeurogastroenterolMotil22:331Ce86.JowettT.1999.Analysisofproteinandgeneexpression.MethodsCellBiol59:63C85.LandmanKA,SimpsonMJ,NewgreenDF.2007.Mathematicalandexperimen-talinsightsintothedevelopmentoftheentericnervoussystemandHirsch-sprung’sdisease.DevGrowthDiffer49:277C286.
LeDouarinN,TeilletM.1973.Themigrationofneuralcrestcellstothewallofthediges-tivetractinavianembryo.JEmbryolExpMorphol30:31C48.
MatsushitaS.1995.Fatemappingofthesplanchnopleuralmesodermofthe1.5-day-oldchickembryo.Roux’sArchDevBiol204:392C399.
MooreMW,KleinRD,FarinasI,SauerH,ArmaniniM,PhillipsH,ReichardtLF,RyanAM,Carver-MooreK,RosenthalA.1996.Renalandneuronalabnormal-itiesinmicelackingGDNF.Nature382:76C79.
NagyN,GoldsteinAM.2006.Endothelin-3regulatesneuralcrestcellprolifera-tionanddifferentiationinthehindgutentericnervoussystem.DevBiol293:203C217.
NatarajanD,Marcos-GutierrezC,Pach-nisV,deGraaffE.2002.RequirementofsignallingbyreceptortyrosinekinaseRETforthedirectedmigrationofen-tericnervoussystemprogenitorcellsduringmammalianembryogenesis.De-velopment129:.
NewgreenDF,SouthwellB,HartleyL,AllanIJ.1996.Migrationofentericneuralcrestcellsinrelationtogrowthofthegutinavianembryos.ActaAnat(Basel)157:105C115.
NganES,ShumCK,PoonHC,ShamMH,Garcia-BarceloMM,LuiVC,TamPK.2008.Prokineticin-1(Prok-1)workscoordinatelywithglialcellline-derivedneurotrophicfactor(GDNF)tomediateproliferationanddifferentiationofen-tericneuralcrestcells.BiochimBio-physActa.
PichelJG,ShenL,ShengHZ,GranholmAC,DragoJ,GrinbergA,LeeEJ,HuangSP,SaarmaM,HofferBJ,Sar-iolaH,WestphalH.1996.Defectsinen-tericinnervationandkidneydevelopmentinmicelackingGDNF.Nature382:73C76.
SanchezMP,Silos-SantiagoI,FrisenJ,HeB,LiraSA,BarbacidM.1996.RenalagenesisandtheabsenceofentericneuronsinmicelackingGDNF.Nature382:70C73.
SchuchardtA,D’AgatiV,Larsson-BlombergL,CostantiniF,PachnisV.1994.Defectsinthekidneyandentericnervoussystemofmicelackingthetyrosinekinaserecep-torRet.Nature367:380C383.
ShenL,PichelJG,MayeliT,SariolaH,LuB,WestphalH.2002.Gdnfhaploin-suf?ciencycausesHirschsprung-likein-testinalobstructionandearly-onsetlethalityinmice.AmJHumGenet70:435C447.
ShepherdIT,BeattieCE,RaibleDW.2001.Functionalanalysisofzebra?shGDNF.DevBiol231:420C435.
SimpsonMJ,ZhangDC,MarianiM,LandmanKA,NewgreenDF.2007.Cellproliferationdrivesneuralcrestcellinvasionoftheintestine.DevBiol302:553C568.
TaravirasS,Marcos-GutierrezCV,DurbecP,JaniH,GrigoriouM,SukumaranM,WangLC,HynesM,RaismanG,Pach-nisV.1999.SignallingbytheRETre-ceptortyrosinekinaseanditsroleinthedevelopmentofthemammalianen-tericnervoussystem.Development126:.
UesakaT,JainS,YonemuraS,UchiyamaY,MilbrandtJ,EnomotoH.2007.Con-ditionalablationofGFRalpha1inpost-migratoryentericneuronstriggersunconventionalneuronaldeathinthecolonandcausesaHirschsprung’sdis-easephenotype.Development134:.
UesakaT,NagashimadaM,YonemuraS,EnomotoH.2008.DiminishedRetexpressioncompromisesneuronalsur-vivalinthecolonandcausesintestinalaganglionosisinmice.JClinInvest118:.
WeskampG,ReichardtLF.1991.Evi-dencethatbiologicalactivityofNGFismediatedthroughanovelsubclassofhighaf?nityreceptors.Neuron6:649C663.
WorleyDS,PisanoJM,ChoiED,WalusL,HessionCA,CateRL,SanicolaM,BirrenSJ.2000.Developmentalregula-tionofGDNFresponseandreceptorexpressionintheentericnervoussys-tem.Development127:.
WuJJ,ChenJX,RothmanTP,GershonMD.1999.Inhibitionofinvitroentericneuronaldevelopmentbyendothelin-3:mediationbyendothelinBreceptors.Development126:.
YoungHM,HearnCJ,FarliePG,CantyAJ,ThomasPQ,NewgreenDF.2001.GDNFisachemoattractantforente-ricneuralcells.DevBiol229:503C516.YoungHM,BergnerAJ,AndersonRB,EnomotoH,MilbrandtJ,NewgreenDF,WhitingtonPM.2004.Dynamicsofneu-ralcrest-derivedcellmigrationintheembryonicmousegut.DevBiol270:455C473.
ZhangD,BrinasIM,BinderBJ,Land-manKA,NewgreenDF.2010.Neuralcrestregionalisationforentericnervoussystemformation:implicationsforHirschsprung’sdiseaseandstemcelltherapy.DevBiol339:280C294.
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　A. NGF、BDNF 和 GDNF 是神经营养素家族成员。 B. Trk 受体是神经营养素家族成员的高亲和力受体。 C. p75NTR 是神经营养素家族成员的低亲和力受体。 D. Trk... 　GDNF GFAP GFI1 GJA8 GLI1 GNB2L1 GPER GPI GPI GPRC5A GRB2 GREM1 Gremlin GRIA1 GRIA2 GRIA3 GRIK1 GRIK2 GRIK4 GRIK5 GRIN1 GRIN2A GRIN2B GRIN2... 　(3)经酶切后的载体和 GDNF 基因进行连接, 连接产物经筛选得到的载体主要有三种:单 个载体自连、 GDNF 基因与载体正向连接、 GDNF 基因与载体反向连接(如图 1 ... 　研究人员构建了含 GDNF 版权所有:中华资源库
基因的表达载体(如图 1 所示) ,并导入到大鼠神经干细胞中,用于干细胞基因治疗的研究。 请回答: ...