InterplaybetweenFGF,one-eyedpinhead,andT-boxtranscriptionfactorsduring - PDF document

InterplaybetweenFGF,one-eyedpinhead,andT-boxtranscriptionfactorsduringzebraÞshposteriordevelopmentKevinJ.P.GrifÞn*andDavidKimelmanDepartmentofBiochemistry&CenterforDevelopmentalBiology,Box357350,UniversityofWashington,Seattle,WA98195-7350,USAReceivedforpublication5May2003,revised10September2003,accepted10September2003 ThezebraÞshT-boxtranscriptionfactorsspadetail(spt)andthenotail(ntl)aretogetheressentialforposteriormesodermformation.Inadditiontobeingfunctionallyredundant,alsogeneticallyinteractwithzygoticmutantallelesofpinhead(Zoep),leadingtosynergisticmesodermaldefects.Herewehaveusedgeneticandpharmacologicalassaystoaddressthemechanismoftheseinteractions.Weshowthataretogetherrequiredupstreamofexpression,accountingfortheseverityofthemesodermaldefectsinembryos.SinceXenopusbrachyuryisproposedtoregulateexpression,andFGFsignalingisrequiredforexpression,weanalyzedtheinvolvementoftheFGFsignalingpathwayinthesegeneticinteractions.UsingaspeciÞcinhibitorofFGFRactivitytoindirectlyassaythestrengthofFGFsignalinginindividualembryos,wefoundthatmutantembryoswerebothhypersensitivetotheFGFRinhibitor.ThishypersensitivityisconsistentwiththepossibilitythatSptandNtlfunctionupstreamofFGFsignaling.Furthermore,weshowthatminorpharmacologicalorgeneticperturbationsinFGFsignalingaresufÞcienttodramaticallyenhancetheZoepmutantphenotype,providingaplausibleexplanationforwhygeneticallyinteractswith.Finally,weshowfunctionareessentialfortheformationofallposteriortissues,includingspinalcord.Takentogether,ourdataprovidestronginvivosupportfortheregulationofFGFsignalingbyT-boxtranscriptionfactors,andthecooperativeactivityofOepandFGFsignalingduringtheformationofposteriorstructures. Thedevelopmentofposteriorstructures(spinalcord,somites)invertebratesinvolvesthespatiallyandtemporallycontrolleddifferentiationofsmallpopulationsofmultipo-tentprogenitors,andisdependentuponFGFsignaling.InhibitionofFGFsignalingusingadominantnegativeFGFreceptor(FGFR)preventstheformationofposteriormeso-derm,andsuchembryosdevelopwithoutposteriorstruc-tures(Amayaetal.,1991;GrifÞnetal.,1995).AlthoughthesestudiesillustratetherequirementforFGFsignalinginthisprocess,itisnotclearfromthemwhetherFGFsignalingisrequiredfortheformation,maintenance,orbehaviorofposteriorprogenitors.Astudyofchickspinalcorddevel-opmentindicatesthatoneimportantroleofFGFsignalingistoinhibitprogenitordifferentiation,therebymaintainingapopulationofstemcell-likecells(Mathisetal.,2001).GeneticandmolecularinterventionshaveattemptedtoidentifyfactorsactingdownstreamofFGFinthemesoder-malprogenitorpopulation,andhaveshownthatatleastoneimportantroleofFGFsignalingistoregulateexpressionofcertainT-boxtranscriptionfactors(Amayaetal.,1993;Isaacsetal.,1994;GrifÞnetal.,1995,1998;Schulte-MerkerandSmith,1995;Ruvinskyetal.,1998).Inze-braÞsh,theT-boxtranscriptionfactorsspadetail/tbx16(spt)notail(ntl;theorthologofmurineextensivelycoexpressedinmesodermalprogenitors(Schulte-Merkeretal.,1992;GrifÞnetal.,1998;Amacheretal.,2002),andtheirexpressionislostwhenFGFsignal- *Correspondingauthor.Currentaddress:DepartmentofMolecular,Cell,andDevelopmentalBiology,Box951606,MolecularBiologyInsti-tute,UniversityofCalifornia,LosAngeles,CA90095-1606,USA.Fax:E-mailaddress:kjpg@ucla.edu(K.J.P.GrifÞn).
       DevelopmentalBiology264(2003)456Ð466www.elsevier.com/locate/ydbio0012-1606/$Ðseefrontmatter©2003ElsevierInc.Allrightsreserved. ingisblockedusingthedominantnegativeFGFR(Grifnetal.,1995,1998).aretogetheressentialfortheformationand/ormaintenanceofposteriormesodermalpro-genitors(Amacheretal.,2002).doublemutantsdonotformanyposteriormesodermalderivatives,andcloselyresembleembryosinwhichFGFRsignalinghasbeenin-hibited.Incontrast,singlemutantembryosdonotshowthisseverephenotype,indicatingthatthesefactorsarefunctionallyredundant(Kimmeletal.,1989;Halpernetal.,1993).mutantsdo,however,havemorecdefects,affectingeithertrunksomitogenesis,ortailandnotochordformation,respectively.InadditiontoT-boxtranscriptionfactorsactingdown-streamofFGFsignaling,evidencefromthattheymayalsoactupstreamofligandexpression(Isaacsetal.,1994;Schulte-MerkerandSmith,1995).IthasbeenproposedthatexpressionofXenopusbrachyury(Xbra)isregulatedbyanindirectautoregulatoryloopinvolvingFGFsignaling.Simply,FGFsignalingmaintainspressionandXbrainturnactivatesexpression.Con-sistentwiththis,aconsensusT-boxbindingsiteispresentinpromoter(Caseyetal.,1998),andcoexpressedwith(Isaacsetal.,1994).DespitetheproposedtightlinkagebetweenT-boxtranscriptionfactorfunctionandtheFGFpathway,thisrelationshiphasnotyetbeenadequatelytestedusingageneticapproach.Further-more,thereisgeneticevidencethatthesituationmaynotbesostraightforward.Forexample,thesimplestinterpretationoftheautoregulatorymodelpredictsthatexpressionshoulddependuponNtl/Brachyuryfunction.However,thisisnotthecaseeitherinzebrash(Schulte-Merkeretal.,1994a)ormouse(Schmidtetal.,1997),withtheexceptionofexpressioninnotochordprogenitors.ThisindicatesthateitheradditionalfactorsmaintainFGFsignalingintheabsenceofNtlfunc-tion,and/orthattheregulationofexpressionismorecomplexandinvolvesadditionalsignalingpathways.InadditiontotheFGFpathway,thefunctionofOne-EyedPinhead(Oep)isalsointricatelyassociatedwithSptandNtlfunction.Oep,anextracellularEGF-CFCfactor,ismaternallyaswellaszygoticallyexpressed.DuetothepresenceofmaternalmutantallelesmerelyattenuateOep-dependentsignalinganddirectlycauseonlyendodermandprechordalmesodermdefects.dramaticallyenhancesthemutantphenotypes(Schieretal,1997;GrifnandKimel-man,2002).Whereassinglemutantsformtrunksomitesandblood,doublemutantembryosfailtoformblood,andsomitesarealmostcompletelyabsent(Schieretal.,1997).Similarly,whereassinglemutantembryoshavereducedanddisorganizedtrunkparaxialmesoderm,doublemutantembryosformnoparaxialmeso-dermwhatsoeverandhaveanunexpectedmidlineprogen-itordefect(GrifnandKimelman,2002).Althoughthemechanismsofthesegeneticinteractionsareunclear,theyneverthelessprovideaglimpseintothegeneticcomplexityofposteriormesodermformation.HerewehaveaddressedtherelationshipbetweenFGFsignaling,andtheT-boxtranscriptionfactors.WeshowthataretogetherrequiredtomaintainexpressionofandproposethatthelossofSptfunctionissufcienttoaccountforthesynergisticmeso-dermaldecienciesobservedinembryos.UsingapharmacologicalinhibitortomanipulateFGFRsignalingindifferentmutantbackgrounds,weshowthatembryoshaveanapparentdecitinFGFsignaling,consis-tentwiththesefactorsactingupstreamofFGFligandex-pression.Inaddition,reductioninFGFRsignalinginmaycausetheirgeneticinteractionswithweshowthatandFGFsignalingactsynergisticallyinvivo,andaretogetherrequiredfortheformationofposteriorstructures.Thesedataprovideinsightsintothegeneticcom-plexityofposteriormesodermformation,andaresuggestiveofcriticalmolecularinteractionsamongtissuesinthetailInsituhybridizationandantibodystainingWholemountinsituhybridizationandantibodystainingwasperformedaspreviouslydescribed(Grifnetal.,1998).Digoxygenin-labeled(Boehringer)RNAprobeswerepre-paredaspreviouslydescribed:netal.,1998),(Weinbergetal.,1996),(LunandBrand,1998),and(Liaoetal.,1997).MF20antibodyrecog-nizesanepitopeinmyosin(Shimizuetal.,1985);MF20supernatantwasusedat1:50,andvisualizedwithHRP-conjugatedgoatanti-mouseI(Sigma)secondaryantibodies;HRPwasvisualizedusingDABandstandardreactioncon-ditions(Westereld,1995).FishmaintenanceandmutantallelesshwereABstrain.Allmutantstrainswerekeptasheterozygousadultsidentiedbyrandomcrossing,andweremaintainedbyout-crossingtoABsh.Double-mutantlineswereobtainedbyintercrossingheterozygousadults;doublyheterozygousF1progenywereidentiedbyrandomcrosses.Thefollowingallelecombinationswere,Zoep;ace,Zoep;acegenotypesyieldedsimilarphenotypesat24hpf;however,subsequentanalysiswasperformedonly;aceSU5402treatmentSU5402(Mohammadietal.,1997;Calbiochem)wasdissolvedinDMSOandstoredatC;priortoadditiontoembryos,stockSU5402wasdilutedinembryorearingK.J.P.GrifÞn,D.Kimelman/DevelopmentalBiology264(2003)456Ð466 medium(1oceansalts).EmbryosintheirchorionsweretreatedwithSU5402fromthemidorlategastrulationstageandculturedovernightinthepresenceofthedrug.Treat-mentswereperformedin24-wellplates,2025embryosperwellin0.5mlofmedium.Allexperimentswereper-formedatleasttwicewithmultipleconcentrationsofSU5402.Experimentswithmutantembryoswereper-formedasfollows.Embryoswerecollectedfromheterozy-gousadultsanddividedintopoolsof2025.Onepoolwasleftuntreated,toconrmthepresenceandproportionofhomozygousmutantembryos.NoeffectswereobservedbyexposuretoDMSOvehiclealone.Treatedembryoswerecollectedat24hpostfertilization,xedin4%paraformal-dehydeinPBS,andprocessedforimmunohistochemistryorinsituhybridization.MorphologicalcriteriawereusedtodeterminetheeffectsofSU5402onhomozygousmutantembryos.Inaddition,SU5402-treatedtantembryoswerealsogenotypedfollowingphotography.Individualembryosweredigestedwith0.5mg/mlprotein-aseKin0.1MTris,pH8.0,0.1%TritonX-100,andusedasatemplateforPCRamplicationusingprimersspanningthemutantbase.ThegenotypewasdeterminedbydirectsequencingofthePCRproduct.ZoepandntlaretogetherrequiredtomaintainexpressionofsptGeneticanalysishasshownthatthezebrashT-boxnotail(ntl),theorthologofmurineisrequiredfornotochordandtailmesodermformation,althoughitisexpressedtransientlybyallmesodermalpro-genitors(Halpernetal.,1993;Schulte-Merkeretal.,1994b).Zygoticoep(Zoep)isrequiredforendodermandprechordalmesodermformation,butposteriormesodermformationisremarkablynormal(Schieretal.,1997;Grits-manetal.,2000).Incontrast,embryosdoublymutantforhaveprofounddecienciesintrunkparaxialmesodermandblood,demonstratingthatthereisacombi-natorialrequirementforthesefactorsincertainmesodermaltissues(Schieretal.,1997).Wewereinterestedinunder-standingwhygeneticallyinteract.WeembryosformesodermalderivativesthatwerenotexaminedbySchieretal.,(1997).Weobservedthatmyosin-expressingcardiacmesodermwaseasilyde-tected(Fig.1E),aswerevascularendothelialprogenitors(Fig.1D),whichwereextremelydisorganizedandmayevenbesubstantiallyincreasedinnumber.Thusonlyacsubsetofmesodermaltissues(blood,paraxialme-soderm)aredefectiveinTrunkparaxialmesodermandblood,butnotvascularendothelium,arebothtissuesthatdependuponoftheT-boxtranscriptionfactorSpadetail(Spt;Kimmeletal.,1989;Thompsonetal.,1998;GrifnandKimelman,2002).WethereforecharacterizedexpressioninembryosderivedfromanintercrossofadultstoascertainifSptwasinvolvedinthemesodermaldefects(Fig.2).Priortotheonsetofgastrulation,expressionappearednormalinallembryosfromsuchacross,indicatingthattheinitiationofexpressionoccurrednormallyinembryos(datanotshown).However,aftertheonsetofgastrulation(6.5h,60%epiboly),Zoep,ntl,mutantembryoscouldbedistinguishedbasedonalterationsintheexpressionof.Insinglemutantembryos,expressionwasweakincellsadjacenttothenotochordprogenitors,aspreviouslyreported(Fig.2B;Grifnetal.,1998),whereasinmutantembryoswasundetect-ableinthemigratingprechordalplateprogenitors(Fig.2CandE;conrmedusingembryosderivedfromanintercross,datanotshown).mutantembryoswereablebyadditivechangesinexpression(Fig.2DandF).Beginningatmidgastrulation(8hpostfertilization,hpf),however,expressioninmutantembryosbegantodecline(datanotshown)and,bytheendofgas-trulation,wasbarelydetectable(10hpf;Fig.2H).Thisindicatesthataretogetherrequiredtomaintainexpressionduringtheformationoftrunkmesoderm.TodetermineifthiswassolelyaneffectofNodalsignalingweexpressioninembryoswithdoublemutantembryos(Fig.2I).At10hpf(budstage),continuedtobeexpressedathighlevelsinthetailbudofdoublemutantembryos.Similarresultswereob-tainedwithembryos(notshown).Thisdemon-stratedthatexpressionisnotexclusivelyregulatedbyNodalsignalingbutalsodependeduponNtlfunction.Fur-thermore,itdemonstratesthatthelossofexpressioninembryoswasnotsimplyduetodefectiveNodalsignaling.Similarresultswereobtainedwithbryos(notshown).SincethepresenceofSptproteincorre-lateswellwiththedistributionofmRNA(Amacheretal.,2002),thedeclineinmRNApresumablyrepresentsalate-onsetlossofSptfunction.Theearlydeclineinexpressioninembryosisthereforesufcienttoaccountforthedecitsinparaxialmesodermandblood.InhibitionofFGFRsignalingwithSU5402causesdevelopmentaldefectsT-boxtranscriptionfactorsareimplicatedinanautoregulatoryloopviaeFGFsignaling(Isaacsetal.,1994;Schulte-MerkerandSmith,1995;Caseyetal.,1998).IfalsoautoregulateviaFGFsignaling,thenmutantembryosmighthavedecreasedFGFRactivity,whichinturnmayplayanimportantroleinthegeneticinteractionwith.Totestthis,weneededasensitiveassaytocomparethelevelsofFGFRsignalingfoundinindividualwild-typeormutantembryos.Preliminaryexper-imentsusingwholemountantibodystainingtodetectphos-phorylatedMAPkinase(Shinyaetal.,2001),orwholemountinsituhybridizationtodetecttheFGF-regulatedK.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 (RaibleandBrand,2001;RoehlandNusslein-Volhard,2001),didnotrevealanymajorchangesbetweenwild-typeandembryosinjectedwithanpholino(datanotshown),butthesetechniquesmaynotbesensitiveenoughtodetectsmallchanges.Wethereforedevelopedanalternatemethodbasedonapharmacologicalchallenge,whichismorespecictoFGFRsignalingthanphosphorylatedMAPkinasestaining,andmoresensitivethaninsituhybridizationorantibodystaining.SU5402isaspecic,dose-dependentinhibitorofFGFRsignalingincellculture,butdoesnotappreciablyinhibitothertyrosinekinasereceptorsatdosesofupto100(Mohammadietal.,1997).WecharacterizedtheeffectsofincreasingconcentrationsofSU5402addedtoembryosatthemidorlategastrulationstageonthedevelopmentofwild-typezebrashembryos.SU5402induceddefectsincerebellumandposteriordevelopment,bothofwhichareknowntodependuponFGFsignaling(Grifnetal.,1998;Reifersetal.,1998).Anacerebellarphenotypewastypicallyobservedat8MSU5402(datanotshown),whereassig-canttailmesodermdefectsbegantobeobservedat15M,andtrunkmesodermdefectsathigherdosesstill(Fig.D).Defectswerealsoobservedinneuroectodermalderivativessuchastheretinaathigherconcentrations,butthesewerenotspecicallycharacterized(datanotshown).Embryostreatedwith30MSU5402formedonlyanteriortrunkparaxialmesoderm(Fig.3D)andaresimilartoem-bryosexpressingthedominantnegativeFGFreceptor(Grif-netal.,1995).Somevariabilityinthedose-responsewasobservedusingdifferentstrains(datanotshown).Sinceatleastsomeandmyosin-positivecellsweredetectedinembryostreatedwith30MSU5402,theposteriordefectsobservedatthisandlowerconcentrationsofSU5402areunlikelytobecausedbyinhibitionofmuscleterminaldifferentiation,butratherbyinhibitionofcriticaleventsearlierinthemesodermformationpathway.ThesedefectsareconsistentwithSU5402actingasaspecicinhibitorofFGFRsignalinginthezebrashembryo.Inaddition,thedose-responserelationshipweobservedisinaccordwiththedose-inhibitionrelationshipdenedinvitro,where50%inhibitionoccurredat15M(Mohammadietal.,1997).FGFRinhibitionselectivelyenhancestheace/fgf8mutantWewishedtousesensitivitytoSU5402treatmentasanindirectassayofFGFRsignalingactivityinmutantem-bryos.Totestthefeasibilityofthisapproach,weanalyzedtheeffectsofSU5402treatmentonembryoswithadedefectinFGFsignaling.Acerebellar(ace)isahypomorphicmutantalleleofaffectingthesplicingofexons2and3(Reifersetal.,1998).mutantembryosfailtodevelopthecerebellumbuthaverelativelynormalposteriordevel-opment(Fig.3E),eitherduetoresidualamountsofcor-rectlysplicedmRNA(Reifersetal.,1998;Draperetal.,2001),orfunctionalredundancywithotherFGFligands(Draperetal.,2003).Whateverthebasis,wereasonedthatthereductioninFGFsignalinginmutantembryosshouldmakeposteriordevelopmenthypersensitivetoSU5402whencomparedwithwild-typeorheterozygoussiblings.Fig.3showsrepresentativeembryosfromanin-tercrossofadultstreatedwiththe5MSU5402,adosethatonlyrarelycausesacerebellardefectsandnevercausesposteriordefectsinwild-typeembryos.Inthisex-periment,embryoswithnormalmidhindbrainformationhadrelativelynormalposteriordevelopment(Fig.3F),whereasembryosthatwereacerebellar(Fig.3G)alsohadsevereposteriormesodermaldefectsthatcouldbeassevereasthedefectsinwild-typeembryostreatedwith20SU5402(Fig.3CandD).Genotypingwasperformedonembryosfromonesuchexperiment(30),demonstratingthatinheritanceofthemutantallelesignicantlyin-creasedthephenotypicseverityduetominorFGFRinhibi-tion(0.001;Table1).ThisdemonstratedthatminorinhibitionofFGFRsignalingcouldbeusedtoinducepro-nouncedpatterningdefectsinembryosinwhichFGFRsignalingwasalreadycompromised.SptandntlmutantsarehypersensitivetoSU5402ThehypersensitivityofmutantembryostoSU5402demonstratedthatreductionsinFGFRsignalingcouldbephenotypicallyenhancedusingthispharmacologicalap- Fig.1.arenotrequiredfortheformationofcardiacmesodermorvascularendothelium.Anterior,left;dorsal,uppermost;genotypesasindicated(bottomleft)(Aexpression.Notethepresenceofexpressingcellsintheembryo,whicharedisorganizedandpos-siblymorenumerousthaninwild-typeoreithersinglemutantembryos.(E)Myosinstaining(brown)detectsthecardiacprimordium(arrow),aswellassomitictissueintheanteriortrunk,aspreviouslyreported.K.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 proach.WethereforeusedSU5402todetermineifmutantembryosmightbesimilarlysensitivetoSU5402.EmbryosfromintercrossesofadultswereexposedtoarangeoflowconcentrationsofSU5402,andassayedforthepresenceofparaxialmesodermusingmyosinstainingat24hpf(Fig.4).Approximately25%ofembryos(10of41)fromanintercrossofadultstreatedwith4MSU5402hadseveredefectsinposteriordevelopment,andmyosinwasonlydetectedintheanterior-mostregionofthetrunk(Fig.4B).Theremaining75%oftheembryoswerethesameaswild-typeembryostreatedwiththisdoseofSU5402(Fig.4C).Sinceembryoswiththetypicalappearanceofsinglemutants(Fig.4A)werenotobservedinthetreatedgroup,butwerepresentintheuntreatedcontrolsiblingembryos,theseverelyaffectedembryoswerelikelytobemutantembryosthathadbeenaffectedbytheFGFRinhibitor.Similarly,theformationofparaxialmesoderminmutantembryostreatedwith7MSU5402wasdramati-callydecreasedcomparedtountreatedcontrols(Fig.4DandE),whereasdefectsinparaxialmesodermformationinwild-typeorheterozygoussiblingembryoswereinfre-quentlyobserved(Fig.4F).(SU5402-treated-mutantem-bryoswereidentiableat24hpfbythecharacteristicpres-enceofmesenchymalcellsatthetipofthetail.)embryosweresignicantlymorelikelythanwild-typeorheterozygousembryostobeaffectedby7MSU5402(0.0001;Table1).ThehypersensitivityofthemutantembryostotheFGFRinhibitorsuggestedthatre- Fig.2.aretogetherrequiredtomaintainexpression.(Asevenhours(60%epiboly),dorsalview,animalpoleuppermost.(EandF)sevenhours(60%epiboly),vegetalview.(GI)Tenhours(budstage),posteriorviewofthetailbud,dorsalisup.(A)Wild-typeembryo.expressedinparaxialmesodermprogenitorsinvolutingatthemarginandthemigratingprechordalmesoderm(arrow),butisexcludedfromthenotochordprogenitors(N).(B)mutantembryo;isexpressedinparaxialandprechordalmesoderm(arrow),butisweakerinparaxialmesodermadjacenttothenotochordprogenitorsandtheborderofexpressionlacksthedenededgeobservedinwild-typeembryos.(C)mutantembryo;expressionisnormalincellsatthemargin,butisnotobservedinprechordalmesodermprogenitors.(D)doublemutantisexpressedatthemarginbutisweakeradjacenttothenotochordandisnotdetectedinprechordalmesodermprogenitors.(EandF)Vegetalview,dorsalsideuppermost,ofembryosinCandD,showingTable1 615/?334114Intheexperimentusingembryosderivedfromadults,embryoswereclassiedasifposteriordevelopmentwassimilartoeitherthemildorseveresyndromescharacterizedinexperimentsusingwild-typesh(Fig.3).embryoswereclassiedasaffectedifthepostanaltailwassignicantlydiminished,andtherewasasignicantreductioninmyosinstainingrelativetountreatedembryos.Intheseexperi-ments,untreatedembryoshadsubstantialamountsofmyosinDeterminedbygenotyping.BasedontesttodeterminedeviationfromexpectedifthemutantallelesdidnotinuencesensitivitytoSU5402. thedistributionofexpressioninmesodermatthemargin.(G)Expres-sionofinawild-typeembryo.Notetheintensestaininginthetailbud(arrow)andsegmentalplatecellseithersideofthenonexpressingnoto-chord.(H)doublemutantembryo;expressionisonlyobservedweaklyinasmallnumberofcellsinthetailbud(arrow).(I)mutantembryo.Notetheintenseexpressionofinthetailbud.K.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 ducedFGFRsignalingwasacommonfeatureofthemutantphenotypes,consistentwiththeexpressionofthesefactorsbeingregulated,atleastinpart,byindirectautoregulatoryloopsinvolvingFGFsignaling.ZoepandFGFsignalinginteractsynergisticallyinposteriordevelopmentIfthereductioninFGFRsignalinginwasimportantforthegeneticinteractionswithmildperturbationsinFGFsignalingshouldbesufcientbythemselvestocausesynergisticposteriordefectsinmutantembryos.WetestedthispharmacologicallyusingtheFGFRinhibitorSU5402,andgeneticallyusingthemutantallele.EmbryosobtainedfromweretreatedwithavarietyofdosesofSU5402andtheamountofparaxialmesodermassayedat24hpf(Fig.5).SincecyclopiawasneverinducedbySU5402atanydosetested(upto50M,datanotshown),cyclopiawasusedtohomozygousmutantembryos.Paraxialmeso-dermformationinmutantembryoswasverysensitivetoSU5402.At10MSU5402,trunkandtailparaxial Fig.3.EffectoftheFGFRinhibitorSU5402onposteriordevelopmentinwild-typeandmutantembryos.(ADandF)Wild-typeand(EandG)mutantembryos.Allembryosare2432h,anteriortotheleft.Embryosarestainedwithanantibodytodetectmyosin,exceptEG,whichareunstained.D)IncreasingtheconcentrationofSU5402ledtoadose-dependentlossinmusclestaining,beginningwiththetail.Onlytailmuscledefectswereobseat15M,whereastrunkandtailmuscledefectswereobservedat30M.(EG)Embryosfromobtainedfromadults.(E)Untreatedembryo,showingtypicallygoodposteriordevelopment.(F)Wild-typeembryofromparentstreatedwith5MSu5402;(G)mutantembryotreatedwith5MSU5402showingsevereposteriordefects. Fig.4.mutantembryosarehypersensitivetoFGFRinhibition.(AC)Embryosfromadults.(A)Myosinstaininginanuntreatedembryo.(B)Presumptivemutantembryotreatedwith4MSU5402;myosinstainingisdramaticallyreducedrelativeto(A)and(C)embryostreatedwith4MSU5402.(DF)Embryosobtainedfromadults.(D)Untreatedmutantembryoshavepatchymyosinstaininginthetrunkandsegmentedstaininginthetail.(E)mutantembryostreatedwith7MSU5402arealmostdevoidofmyosinstaining,whereas(F)embryostreatedwith7MSU5402appearsimilartountreatedwild-typeembryos.K.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 mesodermwasreduced(Fig.5E),andat15MSU5402musclewasonlydetectedintheanteriortrunk(Fig.5F).Incontrast,wild-typeandheterozygoussiblingembryoshadnormalposteriordevelopmentat10Mandonlytailde-fectswereobservedat15MSU5402(Fig.5BandC).AsanadditionaltestoftherelationshipbetweenandFGFsignaling,andtospecicallyaddresstheroleoftheFgf8ligand,weanalyzedthephenotypeofmutantembryos(Fig.6).At48h,mutantembryoshaveonlyminordefectsintrunkandtailmesodermformation(Fig.6BandC).mutantembryoswereeasilyidentiedbythecombinationofcyclopiaandabnormalmidhindbrainmorphology.Incontrasttoeithersinglemutant,embryoshadextremelypoorposteriordevelopmentthatappearedtoaf-fectmesodermalandectodermalderivatives(Fig.6D).Atearlierstages,extensivecelldeathwasapparentthroughoutthetailbudandposteriormesoderm(Fig.6N).Themutantphenotypewasalwaysmuchmoreseverethanmerelyadditivebutsomevariabilitywasobserved,proba-blyreectingvariabilityintheinheritanceofmaternalandpossiblyalsovariabilityintheextentofprocessingoftranscript.T-expressingcellswereonlyobservedintheanteriortrunk,andinseverelyaf-fectedembryosexpressionwasbarelydetectable(Fig.6H).Notochordwaspresent,butwasonlyobservedintheanteriortrunk.Expressionofthattherewereadditivedefectsineyeandmidhindbrain,butsynergisticdefectsinoticvesicle,nephricmesoderm,andspinalcord,whichwasseverelytruncatedposteriorly(Fig.6L).Takentogether,thesedatademonstratethatsynergisticallyinteractswiththeFGFsignalingpathway,andthataretogetheressentialfortheformationoftailbud-derivedtissues(somites,noto-chord,andspinalcord).Weareinterestedinunderstandingthemolecularandgeneticpathwaysunderlyingtheformationofposteriorme-soderm.Amongthefactorsknowntoberequiredforpos-teriordevelopmentinzebrashare:FGFsignaling(Grifetal.,1995),Nodalsignaling(Feldmanetal.,1998),andOep(Gritsmanetal.,1999),aswellasatleasttwomembersoftheT-boxtranscriptionfactorfamily,SptandNtl(Kim-meletal.,1989;Halpernetal.,1993;Schulte-Merkeretal.,1994b;Grifnetal.,1998).Primarily,therolesofthesefactorshavebeenestablishedusingeithergeneticanalysisormisexpressionanddominantnegativestudiesanalyzingtheimportanceofsinglefactorsorpathways.However,inthenormalcourseofdevelopment,cellsareexposedtomultiplesignalssimultaneouslyandcoexpressmultipletranscriptionfactorsinuencingcellfate,eachofwhichmayobscureoralterthefunctionofotherfactorswithwhichtheyareexpressed(Goeringetal.,2003).Complexgeneticnetworksarelikelythereforetobetheruleratherthantheexception.Ultimately,weneedtounderstandhowcombinationsoffactorsinteractintheformationofapar-ticulartissue.HerewehaveaddressedhowmesodermallyexpressedT-boxtranscriptionfactorsgeneticallyinteractwithZoep,andhowFGFsignalingisinvolved. Fig.5.ThemutantphenotypeisenhancedbyFGFRinhibition.Embryosat24hofdevelopment,anteriortoleft,hybridizedtodetect(brownstain).(A/?;(D.Defectsinwild-typetailsomiticmesodermarerstobservedat15M(C),asdescribedinFig.3.(D)mutantembryo.(E)mutantembryostreatedwith10Mshowaberrationsinthenumberof-positivecells.Unlikethedefectsobservedinwild-typeembryostreatedwith10MSU5402,lossofmuscledidnotoccurstrictlyfromposteriortoanterior,asindicatedbythegapinexpressionintheposteriortrunk.Musclestaininginthetailwascontinuousacrossthemidline,indicatingtheabsenceofposteriornotochord.(F)embryostreatedwith15stainingisonlydetectedintheanteriortrunk.K.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 OepandNtlarerequiredtomaintainsptexpressionSchieretal.(1997)observedthatdoublemutantembryoshadmesodermaldefectsthatwerenotobservedineithersinglemutant,notablytheneartotalabsenceofparax-ialmesodermandblood.Themolecularbasisforthisge-neticinteractionwasunknown,andwasnotclariedbythediscoverythatencodesanextracellularfactoressentialforNodalsignaling(Zhangetal.,1998;Gritsmanetal.,1999).HerewehaveshownthatareessentialtomaintainhighlevelsofexpressionoftheT-boxtranscrip-tionfactor.Thefailuretomaintainexpressioninthemutantbackgroundissufcienttoaccountforthesynergisticmesodermaldefectsinsomiticmesodermandbloodforthefollowingreasons.Sptplaysanimportantroleinbloodformation,isfunctionallyredundantwithintheformationofposteriormesodermalprogenitors,andincom-binationwithisessentialfortheformationofsomiticmesoderm.Furthermore,vascularendotheliumwasunaf-fectedbytheinteractionbetweenandisnotdependentuponZoepandSptfunction(Thompsonetal.,1998;GrifnandKimelman,2002).Wehavepreviouslyshownthataretogetherrequiredfortheformationofmyocardialcells(GrifnandKimelman,2002).Itisinterestingthereforethatmyocardialcellsarepresentinembryosat24hpf,despitethe Fig.6.geneticallyinteractswith.Allembryosareshowninlateralview,anteriortotheleft.(A,E,I,andM)wild-type;(B,F,andJ)mutants;(C,G,andK)mutants;(D,H,L,andN)doublemutants.(AD)Liveembryosat48h.(A)Wild-typeembryo.(B)embryoslackthecerebellumandhaveanenlargedtectum(arrow).(C)mutantembryosareobviouslycyclopic(arrow).Bothmutantshavewelldevelopedposteriormesodermandneuroectoderm.(D)doublemutantembryo;thephenotypewasadditiveinanteriorneuroectoderm.Posteriordevelopmentinembryoswasextremelypoor,andmesodermalderivatives(somitesandnotochord)weredifculttodistinguishmorphologically.(Eexpression(24h).(E)Wildtype,(F)and(G)mutantembryosshowstrongexpressionthroughoutthesomites.(H)Severelyaffectedisexpressedonlyinafewcellsintheanteriortrunk(arrow)andnotmoreposteriortothis.(Iexpression(24h).(I)Inwild-typeembryos,isexpressedintheretina,midhindbrainborder,oticvesicle,dorsalspinalcord,andpronephricmesoderm.(J)Inmutantembryos,expressionisabsentfromthemidhindbrainborderandisreducedintheretinaandoticvesicle.(K)Inmutantembryoswithastrongphenotype,expressionisabsentfromtheretina,andisreducedintheoticvesicle.(L)Severelyaffected-expressingcellsareonlydetectedintheanteriorspinalcord(arrow).(MandN)Midsomitogenesisliveembryos,anteriortoleft.Notethelargenumbersofopaquedeadordyingcellsinposteriortissuesoftheembryo(N),whicharenotapparentinthewild-typeembryos(M).K.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 factthattheylackand,indirectly,Sptfunction.Sinceexpressionisinitiallynormalinembryosonlyhavealate-onsetdefectinSptfunc-tion.Takingthisintoconsideration,wesuggestthatSptislikelytoplayanearlyroleincardiacmesodermformation.Incontrast,sinceSptisalsorequiredforbloodformation(Thompsonetal.,1998)andthisisdefectiveinembryos,theroleofSptinblooddevelopmentislikelytobecantlylater,aftergastrulation.SptandntlembryoshaveenhancedsensitivitytoreducedFGFRsignalingWehaveusedhypersensitivitytoSU5402,aspeciinhibitorofFGFRactivity(Mohammadietal.,1997),toindirectlyassaytheoverallstrengthofFGFRsignalingindifferentmutantbackgrounds.Wefoundthistobeanef-fectivetoolwithwhichtouncoverimpairmentsintheFGFsignalingpathwaythatalonedonotyieldasigniphenotype.Ingeneral,thisapproachcouldbeusedinmanyothercontexts,usinganyoftheincreasinglylargenumberofspecicinhibitorsofsignaltransductionpathways.Usingthisapproach,wehaveshownthatparaxialme-sodermformationinmutantembryosishyper-sensitivetoFGFRinhibition,suggestingthatreducedFGFRsignalingisanimportantfeatureofthesemutantpheno-types.AlikelyexplanationforreducedFGFRsignalinginmutantsisthatSptandNtlregulateexpressionofligands,assuggestedforBrachyuryinmesoderm(Isaacsetal.,1994;Schulte-MerkerandSmith,1995),andinthelimbbudsofmouseandchickembryos(Liuetal.,2003).InXenopus,Xbraexpressionismain-tainedbydirectregulationofexpressionbyXbra,whichinturnactivatesexpression(Isaacsetal.,1994;Schulte-MerkerandSmith,1995).AlthoughourdataareconsistentwithsimilarT-box/FGF-positivefeedbackloopstheirregulationislikelytobemuchmorecomplexandsubtlethanthesimplemodelfromsuggests.Forexample,withtheexceptionoftheexpressiondoesnotdependuponNtl/Brachyuryfunction(Schulte-Merkeretal.,1994a;Schmidtetal.,1997).WhileourworksupportstheexistenceoftheseFGFloops,theymustinvolvemultipledownstreamfactorsregulatingthefeedbackandarelikelytoinvolvemultipleFGFligandsand/orinteractionsacrosstissuelay-ers,suchasoccursinthechicklimbbud(Ohuchietal.,1997;Xuetal.,1998).PosteriormesodermrequiresOepandFGFsignaling,actingsynergisticallyUsingtheFGFRinhibitoraswellasageneticapproach,wehaveshownthatOepactssynergisticallywithFGFsignaling,specicallyFgf8,intheformationofposteriorwereespeciallyusefulforthisdemonstrationsincebothmutantallelescauseonlyhypo-morphicreductionsinactivitythroughtheirrespectivesig-nalingpathways,therebysensitizingtheembryotoreduc-tionsinfactorsactinginparallelordownstream.Inmutantembryos,thepresenceofmaternallyinheritedOeppermitssufcientOep-dependentsignalingtosupportpos-terioraxialandparaxialdevelopment.Similarly,asde-scribedabove,themutantalleleishypomorphic,andinadditionthereisfunctionalredundancybetweenFgf8andanothermesodermallyexpressedFGFligand(Draperetal.,2003).However,indoublemutantembryos,thecombinationofreducedOepandhypomorphicFgf8signal-ingcausedasynergisticposteriordefect.ThisinteractionandtheFGFpathwaysuggestsanattractiveexplanationforthegeneticinteractionsbetween(Fig.7).SincemutantembryosmayhavereducedFGFRactivity,thealterationsinthesignalingenvironmentinmutantembryosmayresemblethesignalingenvironmentinembryos,andmutantembryostreatedwiththeFGFRinhibitorSU5402.OurdataimplicateSptandNtlintheregulationofFGFsignaling(Fig.7),butamajorquestioniswhichsignalingpathwayOepisinvolvedwithinthesemutantscenarios?AlthoughOepisstronglyimplicatedinsignalingbycertainssuchastheNodalligandsCyclopsandSquint(Gritsmanetal.,1999)aswellasVg1andGDF-1(Chengetal.,2003),thereisalsoevidencefromthatEGF-CFCproteinsaredirectlyinvolvedwiththeFGFpathway.RecentworkhasimplicatedtheOep-relatedpro-teinFRL-1withFGFsignalingintwocontextsgentextension,viatheFGFR1(Yokotaetal.,2003),andinneuralinduction(Yabeetal.,2003),andwasoriginallyedasanatypicalFGFRligand(Kinoshitaetal.,1995).Consistentwiththelatter,neuralinductionbyFRL-1 Fig.7.SimpliedschemedepictingtherelationshipsbetweenOep,FGFsignaling,andtheT-boxtranscriptionfactorsSpadetailandNoTailduringposteriordevelopment.SpadetailandNoTailarebothupstreamanddownstreamofFGFsignaling,duetoputativepositivefeedbackloops.OepandFGFsignalingactcooperativelyduringposteriordevelopment,ren-mutantembryossensitivetoalterationsinFGFsignalingfromanyofthefollowingcauses:FGFRinhibition,hypomorphicFgf8function,ormutationsineithernotailK.J.P.Grifn,D.Kimelman/DevelopmentalBiology264(2003)456 involvesMAPkinasesignalingandrequiresactiveFGFRsignaling(Yabeetal.,2003).AlthoughOepisabletorescueFRL-1depletionphenotype,thereisstrongcircumstantialevidencethatOepmediatesTGFduringzebrashposteriordevelopment.InterferencewithtwotranscriptionaleffectorsofTGFnandKimelman,2002)and(Rojoetal.,2001),amutantalleleofincombinationwithmutantallelesphenocopiestheposteriormeso-dermaldefectsobservedin.Further-more,wewereunabletodetectanyeffectofOeponFGFRactivityinaoocyteassay,withorwithoutadditionofeFGFligand(unpublishedobservations).Thus,itislikelythattheinteractionsbetweenspt,ntl,andtheFGFpathway(Fig.7)inzebrashrepresentsynergybetweenandFGFsignaling,consistentwithavarietyofinvitromodelsofmesoderminduction(KimelmanandKirschner,1987;Greenetal.,1992;Kimelmanetal.,1992).However,withoutmoredenitiveproof,theroleofOepinthesecontextsremainscontroversial.Incomparisonwithmanyotheraspectsofearlydevel-opmentourunderstandingofhowthetailbudfunctionsisextremelyrudimentary,althoughsomestudieshavebeguntodemonstrateitscomplexityandtheimportantroleoftissueinteractionswithinthisstructure(Agathonetal.,2003).Inparticular,astudyinthechickclearlydemon-stratedthespecialpropertiesofasmallgroupofcellslocatedatthejuxtapositionofaxialandparaxialprogenitor(theaxial-paraxialhinge).Surgicalremovaloftheaxial-paraxialhingesecondarilycausedlossofchordoneuralhinge-derivedtissues(oor-plateandnotochord)and,sub-sequently,massiveapoptosisthroughoutthespinalcordandsomites(Charrieretal.,1999).Thephenotypeisremarkablysimilartothissyndromeofdefects.ItwillbeveryinterestingtodeterminethespecicrolesofOepandFgf8signalingpathwaysintissueinteractionsintheze-shtailbud.WewishtothankSteveWilson,DidierStainier,andDebbieYelon,formutantlinesandreagents;TomRehforagiftofSU5402;AlexSchier,MichaelBrand,BruceDraper,SharonAmacher,CharlesKimmel,andCarolynVivianoforcommentsand/orunpublishedresults;DavidRaibleandmembersoftheKimelmanlabfordiscussionandadvice;andKenLiuandLauraSwaimformaintainingtheshfacility.ThisworkwassupportedbyagrantfromNSF(0078303),andaPHSTrainingGranttoK.J.P.G.Agathon,A.,Thisse,C.,Thisse,B.,2003.Themolecularnatureoftheshtailorganizer.Nature424,448Amacher,S.L.,Draper,B.W.,Summers,B.R.,Kimmel,C.B.,2002.TheshT-boxgenesnotailandspadetailarerequiredfordevelop-mentoftrunkandtailmesodermandmedialoorplate.Development129,3311Amaya,E.,Musci,T.J.,Kirschner,M.W.,1991.ExpressionofadominantnegativemutantoftheFGFreceptordisruptsmesodermformationinXenopusembryos.Cell66,257Amaya,E.,Stein,P.A.,Musci,T.J.,Kirschner,M.W.,1993.FGFsignal-lingintheearlyspecicationofmesoderminXenopus.Development118,477Casey,E.S.,OReilly,M.A.,Conlon,F.L.,Smith,J.C.,1998.TheT-boxtranscriptionfactorBrachyuryregulatesexpressionofeFGFthroughbindingtoanon-palindromicresponseelement.Development125,Charrier,J.B.,Teillet,M.A.,Lapointe,F.,LeDouarin,N.M.,1999.De-ningsubregionsofHensensnodeessentialforcaudalwardmove-ment,midlinedevelopmentandcellsurvival.Development126,4771Cheng,S.K.,Olale,F.,Bennett,J.T.,Brivanlou,A.H.,Schier,A.F.,2003.EGF-CFCproteinsareessentialcoreceptorsfortheTGF-betasignalsVg1andGDF1.GenesDev.17,31Draper,B.W.,Morcos,P.A.,Kimmel,C.B.,2001.Inhibitionofzebrafgf8pre-mRNAsplicingwithmorpholinooligos:aquantiablemethodforgeneknockdown.Genesis30,154Draper,B.W.,Stock,D.W.,Kimmel,C.B.,2003.Zebratopromoteposteriormesodermaldevelopment.Development130,4639Feldman,B.,Gates,M.A.,Egan,E.S.,Dougan,S.T.,Rennebeck,G.,Sirotkin,H.I.,Schier,A.F.,Talbot,W.S.,1998.Zebrashorganizerdevelopmentandgerm-layerformationrequirenodal-relatedsignals.Nature395,181Goering,L.M.,Hoshijima,K.,Hug,B.,Bisgrove,B.,Kispert,A.,Grun-wald,D.J.,2003.AninteractingnetworkofT-boxgenesdirectsgeneexpressionandfateinthezebrashmesoderm.Proc.Natl.Acad.Sci.USA100,9410Green,J.B.A.,New,H.V.,Smith,J.C.,1992.ResponsesofembryonicXenopuscellstoactivinandFGFareseparatedbymultipledosethresholdsandcorrespondtodistinctaxesofthemesoderm.Cell71,n,K.,Patient,R.,Holder,N.,1995.AnalysisofFGFfunctioninnormalandnotailzebrashembryosrevealsseparatemechanismsforformationofthetrunkandthetail.Development121,2983n,K.J.,Amacher,S.L.,Kimmel,C.B.,Kimelman,D.,1998.Molec-ularidenticationofspadetail:regulationofzebrashtrunkandtailmesodermformationbyT-boxgenes.Development125,3379n,K.J.,Kimelman,D.,2002.One-EyedPinheadandSpadetailareessentialforheartandsomiteformation.Nat.CellBiol.4,821Gritsman,K.,Talbot,W.S.,Schier,A.F.,2000.Nodalsignalingpatternstheorganizer.Development127,921Gritsman,K.,Zhang,J.,Cheng,S.,Heckscher,E.,Talbot,W.S.,Schier,A.F.,1999.TheEGF-CFCproteinOne-eyedpinheadisessentialforNodalsignaling.Cell97,121Halpern,M.E.,Ho,R.K.,Walker,C.,Kimmel,C.B.,1993.Inductionofmusclepioneersandoorplateisdistinguishedbythezebrashnotailmutation.Cell75,99Isaacs,H.V.,Pownall,M.E.,Slack,J.M.,1994.eFGFregulatesXbraexpressionduringXenopusgastrulation.EMBOJ.13,4469Kimmel,C.B.,Kane,D.A.,Walker,C.,Warga,R.M.,Rothman,M.B.,1989.Amutationthatchangescellmovementandcellfateintheshembryo.Nature337,358Kimelman,D.,Kirschner,M.,1987.SynergisticinductionofmesodermbyFGFandTGFandtheidenticationofanmR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