New Early Zygotic Regulators Expressed in Endomesoderm of Sea Urchin Embryos Discovered by Differential Array Hybridization

Developmental Biology

Eric H. Davidson¹  Cristina Calestani¹  Jonathan P. Rast¹  Andrew Ransick¹  Takuya Minokawa¹ 

[1] Division of Biology 156-29, California Institute of Technology, Pasadena, California, 91125

关键词: endomesoderm;    macroarray;    subtractive hybridization;    sea urchin;    embryo;    specification;    zygotic;   

DOI  :  10.1006/dbio.2002.0607

学科分类：生物科学（综合）

来源: Academic Press

【 摘 要 】

GenesthatareupregulatedbyLiCltreatmentofseaurchinembryosand/ordownregulatedbyinjectionintotheeggofmRNAencodinganinternalfragmentofcadherin(Cad)weredetectedinadifferentialmacroarrayscreen.ThemethodwasthatrecentlydescribedbyJ.P.Rastetal.(2000,Dev.Biol.228,270–296).Almost105clonesfroma12-hcDNAlibrarywerescreened.MeasurementsoninternalstandardsshowedthatthescreeningprocedurewassufficientlysensitivetoafforddetectionofdifferentiallyexpressedmRNAsofthemostrareclass,thosepresentinonlyafewcopiesperaveragecell.TheinjectionofCadmRNA,whichspecificallyblocksnuclearizationofβ-catenin,resultedinmany-folddecreasesinthelevelsoftranscriptsofasuiteofmarkergenesexpressedzygoticallyduringendomesodermspecification.ThesemeasurementssubstantiatedtheuseofCadmRNAasthebasisforadifferentialscreenfordiscoveryofnewendomesodermalgenes.ByuseofthenewlydevelopedBioArraysoftwareforanalysisofmacroarrayscreens,1106clonesrepresentingdifferentiallyexpressedgenesandyieldingusefulsequencewererecovered.The367clonesthatgavesignificantBLASTXmatchestoknowncellularproteinsfellinto264nonredundantsequenceclasses.ThoseofparticularinterestforthisworkwereclonesencodingDNA-bindingtranscriptionfactors,signaltransductionpathwaycomponents,proteases,kinases,andphosphatases.QuantitativePCRanalysisof66suchselectedclonesrevealedthatthelargemajorityofthesecloneshadbeenselectedbecausetheyareupregulatedbyLiCltreatment,whichaffectstheexpressionofamuchgreaterdiversityandnumberofgenesthanareinvolvedinendomesodermspecification.SeventranscriptspecieswereidentifiedthatrespondedsharplytoinjectionofCadmRNA,andthatarenotrepresentedinmaternalmRNA.Sixofthoseencodetranscriptionfactors.Wefocusedonthreetranscriptionfactorgenesofthissetthatwerepreviouslyunknowninseaurchinembryos.Bywhole-mountinsituhybridization,thesegenesareexpressedinspecificdomainsoftheendomesodermalterritory.Theyare:(1)Speve,anevenskippedorthologueexpressedveryearlyinallvegetalblastomeresandthengraduallyshiftingtoveg1derivativesbythemesenchymeblastulastage;(2)Spgcm,anorthologueofthefruitflygeneglialcellsmissing,whichisfirstexpressedspecificallyandexclusivelyinpartoftheprospectivesecondarymesenchyme(mesodermal)domainatlate-cleavageblastulastage;and(3)Spfoxc,whichisfirstexpressedintheearlyblastulaonlyinthefoursmallmicromeres,andlateronlyexpressedinthatcoelomicpouchwhichgivesrisetothemesodermoftheventralsurfaceoftheadultrudiment.

【 授权许可】

Unknown