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DevelopmentalCellVol9593603November2005Copyright2005byElsevierIncDOI10


DevelopmentalCellferenttissuesTogaininsightintothemechanismsbywhichSoxEproteinsmightberegulatedsuchthattheycandirectdevelopmentofdiversecelltypeswecar-riedoutayeasttwo-hybridscreenthatidentifiedSUMO-1

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1 DevelopmentalCell,Vol.9,593–603,November
DevelopmentalCell,Vol.9,593–603,November,2005,Copyright©2005byElsevierInc.DOI10.1016/j.devcel.2005.09.016SoxEFactorsFunctionEquivalentlyduringNeuralCrestandInnerEarDevelopmentandTheirActivityIsRegulatedbySUMOylationKimberlyM.TaylorandCaroleLaBonne*DepartmentofBiochemistry,MolecularBiology,andCellBiologyandRobertH.LurieComprehensiveCancerCenterNorthwesternUniversityEvanston,Illinois60208Sox9andthecloselyrelatedfactorSox10areessen-tialfortheformationofneuralcrestprecursorcells,andplaydivergentrolesintheprocessbywhichthesecellsaresubsequentlydirectedtoformspecific DevelopmentalCellferenttissues.TogaininsightintothemechanismsbywhichSoxEproteinsmightberegulatedsuchthattheycandirectdevelopmentofdiversecelltypes,wecar-riedoutayeasttwo-hybridscreenthatidentifiedSUMO-1andUBC9asSoxE-interactingproteins.WereportherethattheSUMOylationstateofSoxEproteinsplaysakeyroleinregulatingthefunctionofthesefac-torsduringneuralcrestandinnereardevelopment.Ourfindingssuggestanovelmechanismbywhichtheac-tivityofthesewidelydeployeddevelopmentalregula-toryproteinsmightbedirectedtospecificdevelop-mentalevents.Sox9andSox10HaveEquivalentEffectsonNeuralCrestFormationAlthoughSox9andSox10haverecentlyemergedaskeydeterminantsofbothneuralcrestandinnerearde-velopment,theextenttowhichSoxEfactorsplayfunc-tionallyequivalentrolesintheseprocesseshasbeenlessclear.Wethereforeexpressedthesefactorsinearlyembryosandcomparedtheirabilitytoinflu-enceneuralcrestandinnerearfates.Tocontrolfordosage-dependenteffects,epitope-taggedformsofbothSox9andSox10weregenerated.Theactivitiesofthetaggedanduntaggedformsofeachproteinwerecompared,andtheepitopetagswerefoundtohavenoeffectonfunction(datanotshown).Experimentsdi-rectlycomparingSox9andSox10activitywerecarriedoutusingtheepitope-taggedfactors,andproteinex-pressionlevelsweremonitoredviaWesternblots.mRNAencodingepitope-taggedSox9orSox10wasin-jectedintoonecelloftwo-cellstageandtheinjectedembryoswereallowedtodeveloptoneurulastages,whentheeffectsonneuralcrestprecur-sorcellscouldbeassayedbyinsituhybridization.-galactosidasemRNAwascoinjectedasalineagetracer,andtheuninjectedsideoftheembryoservedasacontrolfornormaldevelopment.WefoundthatbothSox9andSox10wereabletoincreasetheformationofneuralcrestprecursorcells,asevidencedbyexpandedexpressionofmarkerssuchFigures1A–1D),consistentwithpre-viousreports(Aokietal.,2003;Honoreetal.,2003Significantly,however,wealsonotedthatneuralcrestmarkersweresometimesinhibitedinSox9-andSox10-injectedembryos.Forexample,inanexperimentinwhichmostembryosshowedexpandedexpressionofneuralcrestmarkers,wealsonotedembryosinwhichexpressionofthesamemarkershadbeeninhibitedFigures1Eand1F)(Sox9-injected:75%increased,10%decreased,n=47;Sox10-injected:68%increased,8%decreased,n=38).Thissuggestedthatintheem-bryosshowingadecrease,Sox9orSox10activitymighthaveledtotheformationofsomeothercelltypeattheexpenseofneuralcrestprecursors.Wheninthesameexperiment5-foldhigherlevelsofSox9orSox10wereexpressed,thisinhibitionofneuralcrestprecursorformationwasnotedmorefrequentlythanatlowerdoses(Sox9-injected:45%increased,34%decreased,n=29;Sox10-injected:40%increased,45%decreased,n=40).Importantly,weobservednodifferencesintheabilitiesofSox9andSox10toexpandorinhibitthefor-mationofneuralcrestprecursorcells.Moreover,theobservationthatthedifferencesinthesephenotypeswerenotfullydosedependentsuggestedthepossi-bilitythattheactivityofSoxEproteinsmightbedynam-icallyregulated,perhapsbyposttranslationalmodifi-Sox9andSox10PromoteMelanocyteandGlialFormationandInhibitNeuronalDifferentiationWenextcomparedtheabilityofSox9andSox10todirectthe

2 formationofspecificneuralcrestderivative
formationofspecificneuralcrestderivatives.Inparticular,wewereinterestedincomparingtheef-fectsofthesetwoproteinsondevelopmentofmela-noblastsandglia,aspreviousstudieshavesuggestedthatSox10isanimportantdeterminantoftheselin-eages(Southard-Smithetal.,1998;Kelshetal.,2000;Britschetal.,2001;Stoltetal.,2002).Sox9andSox10wereexpressedatlevelsthatpredominantlyexpandtheneuralcrestprogenitorpool,andinjectedembryoswereharvestedattailbudstagesforinsituhybridiza-tionwithmarkersofdevelopingmelanoblastsoral-lowedtodeveloptostageswhenformationofdif-ferentiatedmelanocytescouldbedirectlyevaluated.Intheseexperiments,wefoundthatbothSox9andSox10potentlyinducedtheformationofmelanoblastsasevi-dencedbyscatteredectopiccellsontheembryonicflankthatexpressedmelanocytemarkerssuchas,andTrp2Figures1Gand1Handdatanotshown;Sox9-injected:51%ofembryos,n=129;Sox10-injected:53%ofembryos,n=103),aswellasbytheformationofsupernumerarymelanocytesatswimmingtadpolestages(seebelow,anddatanotshown).Atthestagesexaminedintheseexperiments,expressionmarksthreecellpopulations:melanoblasts,glia,andthedevelopingear.Inadditiontonumerousectopicmelanoblastsincaudalregions,wenoteddra-maticallyincreasedexpressionincranialre-gions.Suchstainingisconsistentwithanincreasednumberofcellsadoptingglialand/oroticfates.Todis-tinguishbetweenthesepossibilities,weexaminedex-pressionofatstage28,whenitservesasaglialmarker(Kelshetal.,2000;Gilmouretal.,2002).WefoundthatbothSox9-andSox10-injectedembryosshowedsignificantlyenhancedstaininginthecranialganglia(Figures1I,1J,1M,and1N)(Sox9-injected:69%increased,n=55;Sox10-injected:74%increased,n=46).Together,thesefindingsindicateSox9andSox10canbothdirecttheformationofatleasttwocelltypescommonlyassociatedwithSox10function,melanocytesandglia.RecentworkhasimplicatedSox10inthemainte-nanceofmultipotencyinneuralcreststemcellsandintheinhibitionofneuronaldifferentiation(Paratoreetal.,2002;Kimetal.,2003).Whilethesestudiesdidnotad-dresswhetherotherSoxEfactorshadsimilaractivities,morerecentworkhasdemonstratedthatforcedex-pressionofSox9blocksneuronaldifferentiationofneu-ralcrestcellsinavianembryos(CheungandBriscoe,).WethereforeaskedwhetherSox9orSox10couldinfluencetheadoptionofneuronalfatesinneuralcrestcells.Wefoundthatbothfactorsinhib-itedthedifferentiationof-expressingcellsin SoxESUMOylationduringNeuralCrestDevelopment Figure1.EffectsofSox9andSox10onNeuralCrestDevelopment(A–F)Insituhybridizationexaminingtheexpressionofneuralcrestmarkers(A,B,E,andF)and(CandD)inSox9-(A,C,andE)orSox10-(B,D,andF)injectedembryos.InjectionofeitherSox9orSox10frequentlyleadstoanincreaseinneuralcrestprecursorformation(A–D,whitearrowhead)butcouldalsoresultinlossofneuralcrestprecursorformation(EandF,whitearrowhead).(GandH)BothSox9andSox10inducetheformationofsupernumeraryandectopicmelanocytes(blackarrows).(I–P)Anincreasein-expressingglia(IversusJ;MversusN)andadecreasein-expressingneuronsinthecranialganglia(KversusL;OversusP)arefoundinSox9-(I–L)andSox10-(M–P)injectedembryos.Lightblueorredstainisthelineagetracerthecranialganglia,evenatdosesthatsignificantlyin-creasedformationof-expressinggliainsiblingembryos(Figures1K,1L,1O,and1P)(Sox9-injected:69%decreased,n=49;Sox10-injected:72%de-creased,n=42).Sox9CanRescueNeuralCrestFormationinSox10-DepletedEmbryosTheabovedatademonstratethatinoverexpressionas-says,Sox9andSox10arefunctionallyequivalentwithrespecttotheirabilitytomediateneuralcrestforma-tion.However,theseexperimentsdonotexcludethepossibilitythatthesefactorsperformequivalentlyintheseassayssecondarytoanabilitytopositivelycross-regulateeachother’sexpression.Forexamp

3 le,theabil-ityofSox9toinducepigmentcells
le,theabil-ityofSox9toinducepigmentcellsmightbeduetoitsabilitytoinduceexpressionofendogenousSox10,whichwouldtheninitiateaprogramofmelanocytedif-ferentiation.Toexplorethispossibility,wecomparedtheabilityofSox9andSox10torescueneuralcrestdevelopmentinembryosdepletedforSox10.Embryosinjectedattheeight-cellstagewithmorpholinostar-getingSox10showreducedorabsentexpressionofearlyneuralcrestmarkers(Figures2Aand2B;90%in-,n=59).SubsequentinjectionofmRNAencod-ingeitherSox9orSox10significantlyrescuedforma-tionofneuralcrestprogenitorsintheseembryosFigures2C–2F;Sox9:18%inhibited,n=40;Sox10:21%inhibited,n=52).ThesefindingsconfirmthatSox10doesnothavefunctionsduringneuralcrestpre-cursorformationthatcannotbecompensatedforbySox9.Importantly,whenrescuedembryoswereal-lowedtodeveloptotailbudstages,wefoundthatSox9wasstillabletoinduceformationofectopicmelano-cytesdespiteSox10depletion(Figures2Iand2J).ThisfindingdemonstratesthattheabilityofSox9todirectmelanocyteformationisnotsecondarytoanabilitytoSox9andSox10DirecttheFormationofEnlargedandEctopicOticVesiclesBecauseSoxEfactorshavealsobeenimplicatedinoticplacodeformation,wecomparedtheeffectsofSox9andSox10activityoninnereardevelopment.Thecon-sequencesofSoxEmisexpressiononthedevelopmentofthiscelltypehavenotbeenpreviouslyreported.WefoundthatinjectionofmRNAencodingeitherfactorfre-quentlyledtoexpandedexpressionofotocystmarkers,or“enlargedears”(Figures3Aand3B)(Sox9-injected:55%enlarged,n=94;Sox10-injected:61%enlarged,n=154).Moreover,in3%–5%ofcases,Sox9-andSox10-injectedembryosdevelopedsupernumeraryotocysts,suchthatbetweentwoandfourdistinctoticvesiclesformedonasinglesideoftheinjectedembryoFigures3C,3D,and3F).Toaskwhethertheappear-anceofenlargedorectopicoticvesiclescorrelatedwithanincreaseinsizeoftheoticplacodeatneuralplatestages,weexaminedtheexpressionofwhichisamongtheearliestmarkersofthisstructureHellerandBrandli,1999).WefoundthatbothSox9andSox10couldexpandthedomainofexpressioncorrespondingtothisplacode(Figures3Gand3H)(Sox9-injected:52%increased,n=50;Sox10-injected:48%increased,n=62).Similarly,bothfactorscouldmediateincreasedexpressioninthedevelopingearatstage25(datanotshown).Importantly,however,Sox9andSox10didnotdifferintheirabilitytoinduce DevelopmentalCellFigure2.Sox9andSox10RescueNeuralCrestDevelopmentinSox10-DepletedEm-EmbryosinjectedwithSox10morpholinos(MO)showreducedorabsentexpressionofearlyneuralcrestmarkerssuchasandB)andreducedoticvesiclesandcranialneuralcrest(GandH).ThesedefectscouldberescuedequallywellbySox9orSox10(C–FandI–L).Sox9retainsitsabilitytoinduceectopicmelanocytesinSox10-depletedem-bryos(J,blackarrows).Lightredstainisthelineagetracer-gal. theformationofenlargedorectopicears,providingfur-therevidencethatthesefactorsfunctionequivalently.UBC9andSUMO-1AreSoxE-InteractingFactorsTheabovefindingsfurtherhighlightthequestionofhowtheactivityofSoxEfamilyproteinsisregulatedduringtheformationofdiversecelltypessuchastheneuralcrestoroticplacode.Toaddressthis,wesoughttoidentifyinteractingproteinsthatmightmodifythefunctionoftheseproteinsduringdevelopment.Wecar-riedoutayeasttwo-hybridscreen,usingaSox10cDNAtruncatedbeforetheactivationdomainasbaitandagastrulastagecDNAlibraryasprey.ThisscreenidentifiedanumberofSoxE-interactingpro-teins,amongwhichwere7isolatesofthesmallubiq-uitin-likemodifierSUMO-1and27isolatesoftheE2SUMO-conjugatingenzymeUBC9,andthesefactorswerealsofoundtointeractwhentheSox9N-terminuswasusedasbait(Figure4Aanddatanotshown).To-gether,thesefindingssuggestedtheintriguingpossi-bilitythatSoxEactivitymightberegulatedposttransla-tionallyviaSUMOylation.Examinationofth

4 eSox9andSox10se-quencesshowedthattheyeac
eSox9andSox10se-quencesshowedthattheyeachpossesstwocon-servedSUMOylationconsensussites,KXE(Gill,2004ThefirstofthesesitesislocatedjustN-terminaltotheE1domain,whilethesecondsiteislocatedwithintheC-terminalactivationdomain(Figure4B).BecausenotallproteinscontainingthismotifareSUMOylatedinvivoHay,2005),weaskedwhetherSox9andSox10areSUMOylatedinembryos.WhenSox9orSox10wereexpressedinthepresenceorab-senceofepitope-taggedSUMO-1,Westernblotanaly-sisindicatedthatSox9andSox10couldeachbeSUMOylatedontwosites,andthateachmodificationleadstoadistinctanddistinguishableshiftinmobilityonSDS-PAGEgels(Figures4Cand5A).BlottingagainstthemyctagonSox9orSox10followingimmunoprecip-itationofSUMO-1(flag)confirmedthattheslowermo-bilitySoxEisoformsrepresentSUMOylatedproductsFigure3D),andSUMOmodificationofSox9hasalsorecentlybeenobservedinhumanembryonickidneycells(Komatsuetal.,2004).Wefoundthatatgastrulastages,Sox10isSUMOylatedatlysine44intheNter-minusaswellasatlysine333intheactivationdomain.InSox9,thepredominantsiteofSUMOylationislysine365intheCterminus,althoughtheproteincanalsobeSUMOylatedonlysine61.TheidentitiesofthesemodifiedresidueswereconfirmedbymutatingoneorbothreactivelysinesinthehypothesizedSUMOylationsitestoarginineinordertoblocktheirSUMOylation.ExperimentsinwhichSox9andSox10isoformscarry-ingtheselysinemutationswerecoexpressedwithSUMO-1inearlyembryosconfirmedthattheseweretheonlySUMOylationsitesintheseproteins(FiguresBand5C).Consistentwiththis,Sox9andSox10pro-teinslackingtheselysineresiduesnolongerinteractwithSUMO-1orUBC9inyeast(Figure4A).Interest-ingly,whenonlyasinglemodifiedlysineinSox10wasmutated,onlyoneofthetwoslowermigratingSox10specieswaslost(Figure5C).ThesefindingsindicatethatthemobilitydifferencesapparentbySDS-PAGEre-flectwhichsiteintheproteinhasbeenSUMOylated,ratherthanthenumberofSUMOmoietiesappended.SUMOylationModulatesSoxEFunctionduringNeuralCrestDevelopmentMorethanhalfofallidentifiedSUMOsubstratesaretranscriptionalregulatoryproteins,andSUMOylationcaneitherup-ordownregulatetheactivityofthesefac-tors(Girdwoodetal.,2004).Havingconfirmedbio-chemicallythatSoxEproteinsareSUMOylatedinembryos,wesoughttobetterunderstandhowSUMOylationmodulatesSoxEfunctionduringneuralcrestdevelopment.WeinitiallyfocusedthesestudiesonSox9,asthisproteindisplayedonlyonepredomi- SoxESUMOylationduringNeuralCrestDevelopment Figure3.Sox9andSox10InduceEnlargedandEctopicEarStruc-tures(A–F)Insituhybridizationshowingexpressionintheearsofstage28embryosinjectedwithSox9(AandC)orSox10(B,D,andF).Expressionofeitherofthesefactorsconsistentlyleadstotheformationofenlargedears(AandB,whitearrows)and/ortotheformationofoneormoreectopicears(C,D,andF,blackarrow-(GandH)Increasedexpressionof,whichmarkstheoticplac-ode(whitearrowhead)inSox9-(G)orSox10-(H)injectedembryosatstage13.LightblueorredstainisthelineagetracernantsiteofSUMOincorporation.Inadditiontoourloss-of-SUMOylationmutants,wegeneratedaformofSox9whichhadSUMO-1fusedin-frametoitsCter-minus(Figure6A).SuchfusionshavebeenfoundtomimictheconstitutivelySUMOylatedstateofaproteinandhaveprovenparticularlyusefulwhenthenativeSUMOylationsiteoccursneartheterminusofaproteinofinterest,asitisinSox9(Rossetal.,2002;Holmstrometal.,2003;Longetal.,2004).Inordertofacilitateinter-pretationoftheseexperiments,weensuredthatthefusedSUMOmoietywouldrepresenttheonlySUMO-ylationoftheproteinbyappendingittothedouble-lysinemutanttocreateSox9WefirstwishedtoascertainwhethertheSUMO-ylationmutantsaffectedSox9proteinstability,asSU-MOylationoftargetedlysinesinsomeproteinsregu-latedbyubiquitin-dependentproteolysisresultsinstabilizationofthosepr

5 oteins(Hay,2005).Wedidnotexpectthistobet
oteins(Hay,2005).WedidnotexpectthistobethecaseforSox9andSox10,how-ever,aswehavefoundtheseproteinstobeverystablewhenexpressedin,andhavebeenunabletodetecttheirubiquitination(datanotshown).Neverthe-less,todirectlyaskwhetherblockingSUMOylational-tersSox9stability,weexpressedwild-typeSox9andsuchthatequivalentproteinlevelsweredetectedatblastulastages,andthencomparedtheex-pressionlevelsoftheseproteinsovertime.Wede-tectednodifferenceinproteinstabilitybetweenwild-typeSox9andthedouble-lysinemutant(Figure5Danddatanotshown),makingitunlikelythatSUMOylationregulatesSoxEstabilityinthissystem.ToexaminetheeffectsofSox9SUMOylationonneuralcrestdevelopment,mRNAencodingwild-typeSox9,Sox9,orSox9/SUMO-1wasinjectedintoonecelloftwo-cellstageembryosandeffectsonneuralcrestprecursorcellswereassayedbyinsituhybridizationatneurulastages.Theseproteinswereepitope-taggedandwereexpressedatequivalentlevelsasdeterminedbyWesternblot.Whentheex-pressionofmarkerssuchaswereex-aminedintheseembryos,wefoundthatSox9andSox9/SUMO-1haddramaticallydifferentef-fectsonneuralcrestprecursorformation.Forexample,whenexpressedatlevelsatwhichthewild-typeproteinmediatesamodestincreaseinexpression,consistentlyinducedadramaticincreaseexpression(97%increased,n=81).Con-versely,Sox9/SUMO-1stronglyinhibitedexpression(97%inhibited,n=82)(Figure6B).Giventhatwild-typeSoxEproteinscanbothpositivelyandnegativelyinfluencetheformationofneuralcrestpro-genitors(Figure1),theseresultssuggesttheSUMOyla-tionstateoftheexpressedproteinmaybeanimportantdeterminantofthesedifferentoutcomes.andSox9/SUMO-1werealsofoundtodifferentiallyaffecttheformationofneuralcrestderivatives.Forexample,aswithwild-typeSox9,embryosinjectedwithSox9developednumer-ousscattered-expressingmelanoblastsontheinjectedsideoftheembryo(92%,n=121).Bycontrast,/SUMO-1wasunabletoinduceectopicmelanoblasts(0%,n=162)(Figure6C,blackarrows).Similarly,bothwild-typeSox9andSox9theformationofsupernumerarydifferentiatedmelano-cytesontheinjectedsideoftheembryo,while/SUMO-1doesnot(Figure6D).Alsolikewild-typeSox9,wefoundthatSox9couldres-cueneuralcrestdevelopmentinSox10-depletedem-bryos(Figure6E;Sox10MO:93%inhibited,n=28;:29%inhibited,n=34).Impor-tantly,however,Sox9/SUMO-1isnotinactiveintheseassays,asitstronglyinducedectopicSox10ex-pressionincranialregions(Figure6C,whitearrow-heads).Someofthisstainingwasconsistentwithen- DevelopmentalCellFigure4.Sox9andSox10AreSUMOylated(A)Growthofseriallydilutedculturesonse-lectivemediashowingthatbothSox9andSox10(activationdomaindeleted)interactwithUBC9andSUMO-1inayeasttwo-hybridassay.MutationoftheSUMOylationsitespreventsinteraction.(B)SchematicofSoxEproteindomains.(C)Westernblotoflysatespreparedfromembryosinjectedasnotedshowingmodifi-cationofSox9orSox10withSUMO-1.Ar-rowsmarkreducedmobilityformsresultingfromconjugationtooneormoreSUMOmoi-(D)LysatesfromembryosinjectedwithSox9orSox10alone,ortogetherwithSUMO-1,wereimmunoprecipitated(IP)withantibod-iesagainsttheepitopetaginSUMO-1(flag)andthenimmunoblotted(IB)usingantibod-iesagainsttheepitopetagontheSoxEfac-tors(myc),confirmingthatmoreslowlymi-gratingSoxEspeciesareSUMOylated.Directimmunoblottingofthecrudelysatewitheithermycorflagantibodiesservedasinputcontrol(bottompanels). largedotocysts,althoughstainingcorrespondingtoanincreaseincranialgliawasalsoapparent(FigureS3ToconfirmthatourfindingswithSox9SUMOylationmutantsweregenerallyapplicabletoSoxEfactors,wemadeananalogoussetofmutationsinSox10tocreateandSox10/SUMO-1.WefoundthattheeffectsofoverexpressingtheseSox10SUMO-ylationmutantscloselymimickedtheeffectsoftheirSox9counterparts(FigureS2SUMOylationof

6 SoxEProteinsIsImportantforInnerEarDevelo
SoxEProteinsIsImportantforInnerEarDevelopmentGivenourfindingsthatSoxEproteinscouldmediateoticplacodeformation,wecomparedtheabilityofFigure5.IdentificationofSoxESUMOyla-tionSites(A)Westernblotoflysatespreparedfromstage11embryosexpressingwild-typeorly-sinemutantSox9proteins.OnemajorandoneminorSUMO-modifiedformofSox9arenotedatthisstage(blackandredarrows).(B)MutationofthereactivelysineintheC-terminalSUMOylationsiteeliminatesthemajormodifiedspecies.(C)Westernblotoflysatespreparedfromembryosexpressingwild-typeorlysinemu-tantSox10proteins.TwoSUMO-modifiedformsofSox10ofdifferentmobilitiesarenoted(blackandredarrows).Eachsinglely-sinemutationeliminatesonlyasingleoneofthesespecies.(D)WesternblotshowingthatmutationofSUMOylationsitesdoesnotaffectSox9pro-teinaccumulation. orSox9/SUMO-1tomodulatede-velopmentofthistissue.Incontrasttoitseffectsonneuralcrestprecursorformation,wefoundthat/SUMO-1increasedthesizeoftheoticplacode,asevidencedbyanexpandeddomainofexpression(47%increased,n=44).Conversely,expressioninplacodalre-gions(61%decreased,n=55)(Figure6F).Consistentwiththis,whenweexaminedexpressionoftheoticvesicleattailbudstages,wefoundthatinhibiteditsexpression(50%decreased,n=51),whereasSox9/SUMO-1-injectedem-bryosfrequentlyshowedexpandedregionsofpression(55%increased,n=47)(Figure6G).These SoxESUMOylationduringNeuralCrestDevelopment Figure6.RegulationofSox9ActivitybySUMOylation(A)SchematicshowingexpressedSox9isoforms.(B)InsituhybridizationshowingneuralcrestexpressionofinSox9-(i),Sox9-(ii),andSox9/SUMO-1-(iii)injectedem-(C)Insituhybridizationshowingectopic-expressingmelanoblasts(blackarrows)instage28embryosinjectedwithwild-typeSox9or.Sox9/SUMO-1-injectedembryosneverdevelopectopicmelanoblasts(noteabsenceofthesecellsinregionofredarrows)butdoshowincreasedexpressionincranialregions(whitearrowheads;compareinjectedandcontrolsidesofsameembryos).(D)Closeupsoftheheadsoftheswimmingtadpolesinjectedwithwild-typeSox9,Sox9,orSox9/SUMO-1.Bothwild-typeSox9andSox9mediateformationofsupernumerarydifferentiatedmelanocytesontheinjectedsidesoftheembryos(redarrowheads),whileSox9/SUMO-1isunabletodoso.(E)Sox9canrescueearly(ii)andlate(iv)aspectsofneuralcrestformationinSox10MO-injectedembryos(whitearrowheads),andretainsitsabilitytoinduceectopicmelanocytes(iv,blackarrows).expressionintheoticplacodeofstage13embryospreviouslyinjectedwithSox9orSox9expression,whichmarkstheotocyst,instage28embryospreviouslyinjectedwithSox9orSox9(H)TablesummarizingtheeffectsofSox9andSox9/SUMO-1intheseassays.Inpanelsshowinginsituhybridization,lightredorbluestainisthelineagetracer DevelopmentalCellfindingsunderscoredthattheconstitutivelySUMO-ylatedformofSox9hasactivitiesthataredistinctfromthosepossessedbytheunmodifiedprotein.Moreover,ourresultsstronglysuggestthattheSUMOylationstateofSoxEfactorsplaysacentralroleindeterminingwhetherthesefactorswillmediateneuralcrestoroticplacodeformation,mostlikelybyregulatingtheirchoiceofpartnerinteractions.WorkinanumberofmodelorganismshasdetailedtheexpressiondomainsofindividualSoxEfactorsandtheembryonicconsequencesresultingfromthelossofthisexpression(reviewedinHongandSaint-Jeannet,2005Bycontrast,verylittleisknownaboutthecomparativeactivitiesofthesefactors.Sox9andSox10,togetherwithSox8,mostlikelyaroseviaduplicationofasingleancestralSoxEfactor,andthemostcommonlyem-ployedexplanationsfortheretentionofsuchduplicategenesduringvertebrateevolutionareneofunctionaliza-tionandsubfunctionalization.Neofunctionalizationas-sumesthattheduplicationeventfreesonecopyofthegenefromselectivepressuretomaintainessentialfunctions,allowingthiscopytoevolv

7 enewfunctions.Whileexamplesofneofunction
enewfunctions.Whileexamplesofneofunctionalizationhavebeenre-ported(McClintocketal.,2001),subfunctionalizationmaybeamorecommonexplanationforthehighreten-tionrateofduplicategenes.Undertheduplication-degeneration-complementation(DDC)modelofsub-functionalization,thereciprocallossofaspectsoftheancestralexpressionpatternineachoftheduplicatescouldaccountfortheselectivepressuretomaintainbothcopies(LynchandForce,2000areinitiallyexpressedinallneuralcrestprecursorsin,theirlaterexpres-sionisrestrictedtodistinctsubsetsofneuralcrestde-rivativesinallmodelorganismsexamined,consistentwitharoleforsubfunctionalizationinretainingtheseparalogssubsequenttotheirduplication.Whileneo-functionalizationsuggestssomedegreeoffunctionaldivergence,duplicategenesmaintainedasaresultofsubfunctionalizationarelikelytoretainsimilaractivities.ToaskwhetherthisisthecaseforSox9andSox10,wecomparedtheiractivitiesbyexpressingtheminearlyembryosandassayingtheireffectsonneuralcrestandinnereardevelopment.Wealsocomparedtheirabilitiestorescuemorpholino-mediateddepletionofSox10.Significantly,ourexperimentsdetectednodifferencesintheactivityofthesefactors.However,inthecourseofthiswork,weuncoveredseveralpre-viouslyunreportedactivitiespossessedbythesefac-tors.First,wefoundthatinadditiontoitspreviouslydescribedabilitytoinducetheformationofneuralcrestprogenitorcells,Sox9canalsoinducetheformationofbothmelanocytesandcranialglia(Figure1),celltypestypicallyassociatedwithSox10function.Moststrik-ingly,wefoundthatbothSox9andSox10werecapableofinducingtheformationofenlargedorectopicearsFigure3BecauseourfindingsstronglysuggestedthatthefunctionalactivityofSoxEparalogshasbeenlargelyconserved,wesoughttodeterminehowtheseproteinsmightberegulatedsuchthattheycandirecttheforma-tionofcelltypesasdistinctastheneuralcrestandoticplacode.TheidentificationofUBC9andSUMO-1asSoxE-interactingproteinssuggestedonemechanismbywhichtheactivityofthesefactorsmightberegu-latedposttranslationally,andweconfirmedbiochemi-callythatbothofthesefactorsundergoSUMOylationembryos(Figures4and5).WhileSox10canbemodifiedattwosites,Sox9ispredominantlymodi-fiedonasinglesiteintheCterminus(K365),andourexperimentstodatehavefailedtoidentifyfunctionalconsequencesofmutatingtheN-terminalSUMOac-ceptorsiteofeitherfactor(ourunpublisheddata).WhilewecannotruleoutthatSUMOylationatthissitemodi-fiesSoxEfunctioninamannernotdetectedbyouras-says,inlamprey,theprototypemodelforthebasalver-tebrate,allthreeidentifiedSoxEparalogshaveonlytheC-terminalSUMOylationconsensussite(D.McCauley,personalcommunication),emphasizingtheprimaryim-portanceofthissite.AlthoughSUMOylationwasfirstdescribedasapost-translationalmodificationalmost10yearsago,thecellularconsequencesofSUMOmodificationremainpoorlyunderstoodatthemolecularlevel,andappeartodiffersignificantlyfromsubstratetosubstrate.More-over,SUMOmodificationofdifferentsiteswithinthesameproteincanhavedifferentconsequencesforthatprotein’sactivity(Poukkaetal.,2000;Gill,2004;Hay,).AmongthereportedeffectsofSUMOylationontranscriptionfactorsarethemodulationofprotein-pro-teininteractions,protein-DNAinteractions,andproteinlocalization,aswellastheregulationofproteinstabilityviaantagonizationofubiquitination(Gill,2004;Hay,).TheconsequencesofSUMOylationfortranscrip-tionalactivityarealsodiverse.Inanumberofcases,SUMOmodificationoftranscriptionalactivatorsinhibitstheirpotencyasactivators(Girdwoodetal.,2003;Gill,);however,SUMOmodificationofotherproteinsleadstoanincreasedabilitytoactivatetranscriptionGostissaetal.,1999;Rodriguezetal.,1999;Goodsonetal.,2001;Hongetal.,2001TheconsequencesofSUMOmodificati

8 oncanalsobehighlycontextdependent.Forexa
oncanalsobehighlycontextdependent.Forexample,onsomepromoters,SUMOylationofSmad4resultsintranscrip-tionalrepression,whileonotherpromotersthismodifi-cationhasbeenfoundtoenhanceSmad4-dependenttranscriptionalactivation(Longetal.,2004).OncetheeffectsofSUMOylationarebetterunderstood,itmayprovetobethecasethat,likeotheraspectsoftran-scriptionalregulation,suchpromotercontext-depen-denteffectsaretheruleratherthantheexception.In-deed,givenourfindingsthatSoxEisoformsthatcannotbeSUMOylatedorthatmimicaconstitutivelySUMO-ylatedstatehavedistincteffectsonneuralcrestandoticplacodeformation,cellularcontextappearslikelytoplayanimportantroleindeterminingSoxEfunctioninthesetwocelltypes.NodirecttargetsofSoxEtranscriptionalregulationhavebeenidentifiedatearlystagesofneuralcrestde-velopment;however,anumberofpromotersareknowntobedirectlyregulatedbyoneormoreofthesefactorsduringneuralcrestdifferentiation.Duringmelanocytedevelopment,Sox10bindstheDct/Trp2promoterinco-operationwithMITF(Potterfetal.,2001;Ludwigetal., SoxESUMOylationduringNeuralCrestDevelopment2004;MurakamiandArnheiter,2005),whereasduringthedevelopmentofgliathisfactorregulatesboththePoandMBPpromoters(Peiranoetal.,2000;Stoltetal.,2002,2004).Similarly,Sox9bindstoandregulatestheCol2a1promoterduringchondrogenesis(Lefebvreetal.,1997;Tsudaetal.,2003).Inallcaseswheretheyhavebeenexaminedmolecularly,SoxEfactorshavebeenshowntofunctionastranscriptionalactivatorsontargetpromoters.Itwillthereforebeimportanttodeter-minehowoverexpressionofconstitutivelySUMOylatedformsofSox9orSox10leadstosuchadramaticdown-regulationofearlyneuralcrestmarkerssuchasSox10andSlug(Figure6FigureS2).AnimportantfirststepinthisdirectionwillbeidentificationofdirecttargetsofSoxEregulationduringthesestages.InsightintopotentialmechanismsviawhichSUMOmodificationmightregulateSoxEactivitymayalsode-rivefromrecentworkonMITF.Inthatstudy,SUMOmodificationofMITFwasfoundtohavenoeffectontheregulationofpromoterscontainingasingleMITFbindingsitebutsuppressedsynergisticactivationofpromoterswithmultiplebindingsites(MurakamiandArnheiter,2005).IfSUMOylationregulatesSoxE-medi-atedtranscriptionbyselectivelymodulatingcoopera-tiveinteractionamongfactorsconstitutingtranscrip-tionalcomplexes,thenourfindingssuggestthatdistincttypesofcomplexesmaybedeployedintissuessuchastheneuralcrestandtheinnerear,andfutureworkwillexplorethispossibility.ElucidatingtheprecisemechanismsthroughwhichSUMOylationmodifiesSoxEactivitymayhavebroadimplicationsforunder-standinghowdevelopmentalregulatoryfactorswithconservedactivitycanmediateverydifferentfunctionaloutcomeswhenexpressedindifferenttissues.ExperimentalProceduresDNAConstructsandEmbryoMethodsApartialcDNAwasisolatedfromanarrayedcDNAlibrary,andafull-lengthclonewasgeneratedby5RACE(BDClontech,MountainView,CA).wasisolatedfromstage17cDNAusinglowcopynumberPCRandahigh-fidelitypolymerase(Tgo;Roche,Indianapolis,IN).cDNAswereclonedintoapCS2variantthataddsfiveC-terminalmyctags(giftofR.Davis)andconfirmedbyse-quencing.TheSox9K61RandK365RmutationsandSox10K44RandK333RmutationsweregeneratedusingtheQuickChangemethod(Stratagene,LaJolla,CA;primersequencesavailableuponrequest).hSUMO-1wasinsertedinthevectorpCS2-FlagNandusedforinvivoassays.Sox9/SUMO-1andSox10SUMO-1werecreatedbyligatingSUMO-1in-frameC-terminaltothefull-lengthSox9orSox10mutantsusingPCRmethods,insertingaprolineandaglycinebetweenthetwose-quences.ThefusionproteinswereN-terminallyepitopetaggedbyinsertionintovectorpCS2-Myc(providedbyD.Turner).Allcon-structswereconfirmedbysequencing.Allresultsshownarerepre-sentativeofatleasttwoindependentexperiments.RNAforinj

9 ec-tionwasproducedinvitrofromlinearizedp
ec-tionwasproducedinvitrofromlinearizedplasmidtemplatesusingtheMessageMachinekit(Ambion,Austin,TX).mRNAconcentra-tionsinjectedwereintherangeof5–50pg.Collection,injection,andinsituhybridizationofembryoswereasdescribedBellmeyeretal.,2003probeisdirectedagainstthe3ofthemessageanddoesnotrecognizethecodingregioncon-structusedformisexpressionexperiments.ConstructsformakingPax8andDlx3probeswereprovidedbyA.BrandliandT.Moreno,respectively.YeastTwo-HybridAssaysSox10aminoacids1–333insertedinthevectorpEG202wasusedasbaittoscreenagastrulastageyeasttwo-hybridlibraryinthepJG4-5vector(giftofS.Sokol)essentiallyasdescribed(Itohetal.,2000).Positiveinteractorswererecoveredfromyeast,shuttledthroughbacteria,andretestedinyeastforstringencyofinteractionbygrowthonselectivemediumandbyassaying-galactivity.Clonesthatretestedweresequenced,andanumberofthesewereidentifiedaseitherUBC-9orSUMO-1(GenBankaccessionnumbersBC046273andZ97073,respectively).BaitsconsistingofSox9aminoacids2–305,Sox9aminoacids2–305,andSox10aminoacids1–333werealsoconstructedinpEG202,andinteractionswithbothSUMO-1andUBC9werecomparedbyplatingserialdilutionsofthetransformantsonselec-tivemedium.WesternBlotsandSUMOylationAssaysWild-typeormutantSox9orSox10proteinswereexpressedinthepresenceorabsenceofSUMO-1,andembryoswerecollectedatgastrulastagesunlessotherwisenoted.ForWesternblots,em-bryoswerelysedinRIPAbuffer(50mMTrisHCl[pH7.4],150mMNaCl,1mMEDTA,1%TritonX-100,1%sodiumdeoxycholate,0.1%SDS)supplementedwithphenylmethylsulfonylfluoride,aprotinin,leupeptin,N-ethylmaleimide,cytochalasinB,and1,10-phenanthroline.SampleswereresolvedonSDS-PAGEandproteinsweredetectedusingantibodiesagainsttheepitopetags(Myc:9E10;SantaCruzBiotechnology,SantaCruz,CA;Flag:M2;Sigma-Aldrich,St.Louis,MO).Forloadingcontrols,blotswerestrippedandreprobedforactin(-actin;Sigma-Aldrich).Secondaryanti-bodieswerehorseradishperoxidasecoupledanddetectedbychemiluminescence(Pierce,Rockford,IL).MorpholinoOligonucleotideRescueExperimentsASox10morpholinoantisenseoligonucleotidedesignedagainstthe5UTRandcodingregionofSox10(5CATCACTCATGGTGCC-3,Sox10MO)wasobtainedfromGeneTools,LLC(Philomath,OR).TodepleteSox10,10ngofSox10MOwasinjectedintoasingleblastomereattheeight-cellstage.Forrescues,mRNAencodingepitope-taggedformsofeitherSox9,Sox10,orSox9wassubsequentlyinjected,togetherwithmRNAencodinglineagetracer-gal.Injectedembryoswerecul-turedtotheindicatedstage,andharvestedforinsituhybridization.SupplementalDataSupplementalDataincludethreefiguresandcanbefoundwiththisarticleonlineathttp://www.developmentalcell.com/cgi/content/TheauthorsgratefullyacknowledgeM.Walker,V.Rao,andJ.Lind-grenfortechnicalassistance,A.VernonforassistancewithWest-ernblotsandSUMOassays,andA.Smithforassistancewithyeastassays.WethankL.Hicke,R.Cornell,andmembersofthelabora-toryforhelpfuldiscussions,andS.Sokol,A.Brandli,T.Moreno,C.Kinter,andL.Hickeforprovidingreagents.K.M.T.issupportedbyapredoctoralfellowshipfromtheNIDCD.ThisworkwassupportedbyaBasilO’ConnerawardtoC.L.,aswellasbygrantsfromtheAmericanCancerSocietyandtheNationalInstitutesofHealth.C.L.isaScholaroftheGMCancerResearchFoundation.Received:July13,2005Revised:September2,2005Accepted:September26,2005Published:October31,2005Aoki,Y.,Saint-Germain,N.,Gyda,M.,Magner-Fink,E.,Lee,Y.,Credidio,C.,andSaint-Jeannet,J.(2003).Sox10regulatesthede-velopmentofneuralcrest-derivedmelanocytesin.Dev.,19–33.Bellmeyer,A.,Krase,J.,Lindgren,J.,andLaBonne,C.(2003).Theprotooncogenec-Mycisanessentialregulatorofneuralcrestfor-mationin.Dev.Cell,827–839. DevelopmentalCellBondurand,N.,Pingault,V.,Goerich,D.E.,Lemort,N.,Sock,E.,

10 Caignec,C.L.,Wegner,M.,andGoossens,M.(20
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