Molecular cloning and expression of a novel CYP26 gene (cyp2

GeneExpressionPatterns5(2005)733–739

http://doc.guandang.net/locate/modgep

MolecularcloningandexpressionofanovelCYP26gene(cyp26d1)

duringzebra shearlydevelopment

XingxingGua,b,FangXua,XiaolinWanga,c,XiangGaoa,QingshunZhaoa,*

a

ModelAnimalResearchCenterandStateKeyLaboratoryofPharmaceuticalBiotechnology,NanjingUniversity,Nanjing,Jiangsu210093,China

b

JiangsuProvinceKeyLaboratoryofNeuroregeneration,NantongUniversity,Nantong,Jiangsu226001,China

c

SchoolofLifeSciences,NanjingNormalUniversity,Nanjing,Jiangsu210097,China

Received8February2005;receivedinrevisedform14April2005;accepted14April2005

Abstract

ProperrestrictionofretinoidsignalingbyCyp26sisessentialfordevelopmentofvertebrateembryoswhileinappropriateretinoidsignalingcancauseteratogenesis.Here,wereportcloningandexpressionanalysisofanovelcyp26gene(cyp26d1)isolatedfromzebra sh.Thepredictedproteinencodedbycyp26d1consistsof554aminoacids.Itexhibits54%aminoacididentitywithhumanCyp26C1,50%withzebra shCyp26B1and38%withzebra shCyp26A1.Whole-mountinsituhybridizationshowsthatcyp26d1is rstexpressedinspherestage,thendisappearsat50%epibolyandresumesitsexpressionat75%epiboly.Duringsegmentationperiod,cyp26d1messageisfoundatpresumptivehindbrain.Doubleinsituhybridizationwithkrox20andcyp26d1revealsthatcyp26d1isexpressedinpresumptiverhombomere2–4(r2–r4)at2-somitestage.At3-somitestage,cyp26d1geneisexpressedinr6andpharyngealarch(pa)oneinadditiontoitsexpressionatr2andr4.At6-somitestage,cyp26d1messageispresentincontinuousbandsatr2–r6andinpa1.Thisexpressionpatternismaintainedfrom10-somitestagethrough21-somitestageexceptthattheexpressionlevelisgreatlyreducedatr2andr4.At21-somitestage,cyp26d1isalsofoundinagroupofcellsintelencephalonanddiencephalons.At25–31hpost-fertilization(hpf),thezebra shcyp26d1expressiondomainisextendedtoeyes,oticvesiclesandmidbraininadditiontoitsexpressioninhindbrain,telencephalon,diencephalons,andpharyngealarches.At35–48hpf,theexpressionofcyp26d1ismainlyrestrictedtooticvesicles,pharyngealarchesandpectoral nsandtheexpressionlevelisgreatlyreduced.

q2005ElsevierB.V.Allrightsreserved.

Keywords:Zebra sh;cyp26;cyp26a1;cyp26b1;cyp26d1;CytochromeP450;Retinoidsignaling;Development;Hindbrain;Rhombomere;Eye;Oticvesicle;Pharyngealarch

1.Results

Retinoicacid(RA)playscrucialrolesinvertebrateembryonicdevelopmentbysignalingthroughheterodimersofretinoicacidreceptors(RARs)andretinoid-Xreceptors(RXRs)(reviewsbyRossetal.,2000;Maden,2002).However,RAcanbeapotentteratogenifitisavailabletoembryosinthewrongplacesoratthewrongtimes(Shenefelt,1972).Therefore,inorderforvertebrateembryostodevelopnormally,amechanismmustbein

*Correspondingauthor.Address:ModelAnimalResearchCenter,NanjingUniversity,12XuefuRoad,PukouDistrict,Nanjing210061,China.Tel.:C862558641527;fax:C862558641500.

E-mailaddress:qingshun@http://doc.guandang.net(Q.Zhao).

1567-133X/$-seefrontmatterq2005ElsevierB.V.Allrightsreserved.doi:10.1016/j.modgep.2005.04.005

placetocreateaboundarytokeepRAactivityincheck.IthasbeendemonstratedthatvertebrateembryossynthesizeRAbyinitiallyconvertingtheprecursorvitaminAintoretinalusingretinoldehydrogenase,thenirreversiblyintoRAusingmainlyretinaldehydrogenases2(Raldh2)(Maden,2002;Rossetal.,2000).Ontheotherhand,theembryosmetabolizeRAinto4-OHRA,18-OHRA,4-oxo-RAor5,6-epoxyRAusingCyp26s(cytochromeP450RA)(Fujiietal.,1997;Taimietal.,2004,Whiteetal.,1996,2000).cyp26a1isthegenethatwas rstidenti edtobeexpressedinareasofvertebrateembryossensitivetoRAteratogenesisanditsgeneproductcanmetabolizeRAintoinactivepolarforms(Whiteetal.,1996;Fujiietal.,1997;Niederreitheretal.,2002).Inzebra sh,overexpressionofCyp26A1inembryoscansuppresstheexpressionofposteriorgeneswhileabrogationofCyp26A1activitycausesmoderateposteriorization,whicharesimilar

to

734X.Guetal./GeneExpressionPatterns5(2005)733–739

theresultsfromRAinactivationandRAtreatment,respectively(Kudohetal.,2002).InXenopuslaevis,injectionofcyp26a1mRNAintoembryosalsocausesphenotypesresemblingRAdeprivationandcanrescueembryosfromteratogenesiscausedbyRAtreatment(Hollemannetal.,1998).Inmice,knockoutofcyp26a1genecausesRAsignalingtoexpandanteriorlyintothehindbrainandposteriorlyintothetail(Abu-Abedetal.,2001;Sakaietal.,2001),whereasthecyp26a1nullphenotypescouldbesuppressed/Kanditshindbraindefectpartiallyrescuedwhencyp26a1Khomozygousmicewerebredintoaraldh2K/Cbackground(Niederreitheretal.,2002).Together,theseresultssupportthehypothesisthatcyp26functionstocreateboundariesinareasofRAsignaling(reviewinMaden,1999).Todate,threeCyp26s(includingCyp26A1,Cyp26B1,andCyp26C1)havebeenfoundinmammals(Whiteetal.,1997,2000;Taimietal.,2004),whileonlytwozebra shorthologs,Cyp26A1andCyp26B1,hadbeendiscovered(Whiteetal.,1996;Zhaoetal.,2005).Inthispaper,wereportanovelcyp26familymember(cyp26d1)isolatedfromzebra shanddescribeitsexpressionpatterninearlydevelopment.Additionally,wecompareindetailthedifferencesandsimilaritiesintheexpressionpatternofcyp26d1tothoseofcyp26a1andcyp26b1inzebra sh.

1.1.Isolationofazebra shcyp26d1cDNAandanalysisofitsgenomicstructure

EmployingtheRACE-nestedPCRstrategy,weclonedacDNAwiththelengthof1689basepairs(bp)anddepositedthesequenceintoGenBank(GenBankaccessionnumber:AY920470).ThecDNAcontainsafullcodingregion,whichispredictedtoencodea554aminoacidprotein(Fig.1A).Bydoingproteinalignmentanalysis,wefoundthatthepredictedproteinshareshighidentityandsimilaritywithmembersofCyp26family.Itexhibits54%identitywithhumanCyp26C1(Taimietal.,2004),51%identitywithmouseCyp26C1(Tahayatoetal.,2003),51%identitywithhumanCyp26B1(Whiteetal.,2000),51%identitywithmouseCyp26B1(MacLeanetal.,2001;MenkeandPage,2002),51%identitywithratCyp26B1(GenBankAccessionnumber:NM_181087)and50%identitywithzebra shCyp26B1(Zhaoetal.,2005;Fig.1A).IncomparisonwithmembersoftheCyp26A1family,thepredictedproteinshows40%identitywithhumanCyp26A1(Whiteetal.,1997),40%withmouseCyp26A1(Rayetal.,1997;Abu-Abedetal.,1998),39%withratCyp26A1(Wangetal.,20 …… 此处隐藏：21586字，全部文档内容请下载后查看。喜欢就下载吧 ……