Together, theseresultssuggestthatWntsignalingactsinstructivelytodirect expansion oftheoticplacodeatexpenseepidermis,andresultingtissueexpressesexclusivelydorsalotocystm the signalsthatdivide *Author forcorrespondence (e-mail:[email protected]) School ofMedicine,KyotoUniversity, Kyoto,Japan. epidermal, fateandpromotesdorsalcellidentitiesintheotocyst. normally activatedinasubsetof Phillips etal.,2001;2004;WrightandMansour, 2002; Liuetal.,2003;Lombardo1998;Maroon mammals (Ladheretal.,2000;Ladher2005;Leger andBrand, and crucialroleinthisinductionfish,amphibians,birds and members ofthefibroblastgrowth factor (Fgf)family playacentral induction processindifferent speciesisstilluncertain,itclearthat and whereastherelative contribution ofthesetwo tissuestothe hindbrain andcranialparaxialmesoderminoticplacodeinduction, Studies indifferent specieshave suggestedrolesforboththe et al.,2002;Riley andPhillips,2003;Torres andGiraldez,1998). (Barald andKelley, 2004;Brown etal.,2003;Groves, 2005;Kiernan ectoderm, theoticplacode,thatarisesnext totheposterior hindbrain embryonic induction.Itderives fromasimplepatchofthickened particular organs. how they areorchestratedintheinductionanddevelopment of different signalingstrategies, muchstillremainstobelearnedabout (Levin, 2002).Despitethegreatprogressinunderstandingthese Tsakonas etal.,1999;Lai,2004)orgapjunctionalcommunication by cell-cellinteractionssuchaslateralinhibition(Artavanis- grained patterningatthecellularlevel canalsoemerge inacellfield an essentiallyall-or-none response(Bier, 2000;Shilo,2003).Fine- other cases,inducingsignalsactover very shortdistancestoproduce Lander etal.,2002;Mizutani2005;Stamataki2005).In gradient (Briscoeetal.,1999;DeRobertisandKuroda, 2004; differentiating accordingtotheirpositionrelative tothemorphogen responds toagradientofinducingsignals,withdifferent celltypes establish suchinductive interactions.Insomecases,afieldofcells territories orcelltypes.Embryosuseavariety ofstrategies to homogeneous fieldofcellsundergoes patterningtogive distinct One ofthecentralquestionsindevelopmental biologyishow a INTRODUCTION KEY WORDS:Mouse,OticPlacode,Wnt factor The oticplacode,theanlagenofinnerear, developsfromanectodermalfieldcharacterizedbyexpressionofthetranscripti Takahiro Ohyama and epidermis Wnt signalsmediateafatedecisionbetweenoticplacode Development 133,865-875doi:10.1242/dev.02271 Accepted 3January 2006 1 3rd Street, Los AngelesCA90057,USA. McGill University, Montreal, Canada. epidermal markersattheexpenseofoticplacode.Conversely, conditionalactivationof Gonda DepartmentofCellandMolecular Biology, HouseEarInstitute, 2100West The innerearisanincreasinglywell-characterizedexample of Pax2 . Previousfatemappingstudiessuggestthatthese 1 , OthmanA.Mohamed Pax2 3 Department ofPharmacology, Graduate 2 + Department ofObstetricsandGynecology, field intoplacodalandepidermalterritoriesareunknown.We reportthatWntsignalingis Pax2 + cells, andthatconditionalinactivationof 2 , MakotoM.Taketo Pax2 + Pax2 possible thatWntsignalingisnotnecessaryfortheinduction of embryos wereusuallyofreducedsize(Phillipsetal.,2004). It is Wnt8 suggest thatoticplacodeinductioncanproceedintheabsence of (Ladher etal.,2000).However, morerecentstudiesinzebrafish members hasbeenproposedtoparticipateinoticplacodeinduction epidermis? using Pax2-Cre micealsoshows thatmany that cellslyingwithinthe otic placodeortheinnerear. Fate-mapping studiesinchicken show that areinitiallyinducedbyFgfsignalingwillnotcontribute tothe Pax2 that thecanonicalWntsignalingpathway isactivated inasubsetof placodal tissue,ratherthanepidermis.Inthepresentstudy, weshow placode byinstructingtheseprecursorcellstodifferentiate into of activation ofthecanonicalWntsignalingpathway bystabilization expense oftheoticplacode andotocyst. Conversely, constitutive ␤ on afieldof mediates aplacode-epidermisfate decisionbyactinginstructively at theexpense ofepidermis. OurresultssuggestthatWntsignaling several linesofevidence suggestthatmany to besynonymous withtheinductionofoticplacode. However, markers (suchasthetranscriptionfactors 2003). Fgfsignalinginducestheexpression ofavariety ofmolecular Wnt signalingpathway in field of light oftheseobservations, whatarethemechanismsbywhicha possibly toepibranchialplacodes(OhyamaandGroves, 2004b).In presumptive placodalregion ultimately give risetoepidermisand placodes (Streit,2002).Geneticfate mappingof other thantheotocyst, suchastheepidermisorepibranchial Wright andMansour, 2003). morphologically distinct(Alvarez etal.,2003;Ladher2005; presumptive placodalectodermbeforetheplacodebecomes cells willgiverisetobothoticplacodetissueandepidermis,but -catenin geneleadstoanexpansion ofcranialepidermisatthe Activation oftheWntsignalingpathway byWnt8family The inductionof ␤ -catenin in + + 3 expression inthehindbrain,althoughotocysts ofsuch cells duringearlydevelopment. Disruption ofthecanonical precursor cells,but insteaddeterminesthesizeof otic , DanielDufort Pax2 Pax2 Pax2 + ␤ + - precursor cellsissub-divided intooticplacodeand aei nteecellscausesanexpansionof catenin inthese cells toanoticplacodal,ratherthan Pax2 + precursors todirect themtoanoticplacodefate. Pax2 + cells causesanexpansion oftheoticplacode ␤ in cranialectodermiscommonlythought 2 - Pax2 catenin in Pax2 and Andrew K.Groves + domain cangive risetostructures cells byconditionaldeletionofthe RESEARCH ARTICLE Pax2 + Pax2 cells causesan Pax2 Pax2 + Pax2 ectodermal cells + and cells inthe 1, + * ectoderm Pax8 arkers. on ) in 865

DEVELOPMENT cryosectioning. embryos werefixed with4%paraformaldehyde andprocessedfor For theimmunohistochemical detectionof reporter micewas performedbyastandardprotocol(Mohamedetal.,2004). Detailed protocolsareavailable uponrequest.X-galstainingoftheTCF/Lef Color reactionwas performedwithaDAB development kit(Vector). horseradish peroxidase-conjugated secondary antibody(MolecularProbes). activated caspase3 was usedfortheprimaryantibody, togetherwitha blocked withPBScontaining0.1%Triton-X100 and20%goatserum.Anti- Photoshop foreaseofviewing. some imagestheblueDAPI stainingwas converted tomagentainAdobe detection togetherwithDAPI (MolecularProbes)fornuclearstaining.In antibodies fortheappropriatesubtype(MolecularProbes)wereused for tubulin (TuJ1; Covance). Alexa 488-or594-conjugatedsecondary Phospho-Histone H3(Upstate),activated caspase3(R&DSystems)and catenin and fluorescent protein(MolecularProbes), obtained asfollows: Pax2 (Zymed), containing 0.1%Triton-X100 and10%goatserum.Primaryantibodieswere 2004a). Theslideswerewashed withPBSat50°Candblocked inPBS, sections werecollectedasdescribedpreviously (OhyamaandGroves, stock number003465(Schwenketal.,1995);conditional the JacksonLaboratory:CMV-Cre mice[BALB/c-Tg(CMV-cre)1Cgn/J], Mohamed etal.,2004).Thefollowing mouselineswerepurchasedfrom 2000). Tg(ACTB-Bgeo/GFP)21Lbe/J) stocknumber004178(Novak etal., number 00310(Soriano,1999);Z/EGCrereportermice(B6.Cg- catenin ( following matingprocedure.First,micecarryingafulldeletionof described inthetext. 3-10 ofbothmutantandcontrolembryoswereexamined forallmarkers mice asdescribedpreviously (OhyamaandGroves, 2004b).Inallcases, performed bycrossingPax2-Cre micewitheitherR26RorZ/EGreporter this studywere et al.,2005).Detailedprotocolsareavailable uponrequest.Probesusedin protocol ofSternandcolleagues(Stern,1998)asdescribedpreviously (Kil Whole-mount insituhybridizationwas performedbymodificationsofthe Whole-mount insituhybridization allele (B6.129- (Catnb reporter miceandtheconditionallyactive alleleof Regional ResourceCenters(Stocknumber010569-UNC).TCF/LefWnt Groves, 2004b)andwillbedistributed bytheMutantMouse and Pax2-Cre transgenicmiceweregeneratedinourlaboratory(Ohyama Genetically modifiedmice MATERIALS ANDMETHODS 866 embedded in7.5%gelatinand15%sucrosesolution,10 Embryos werefixed in4%paraformaldehyde,washed inPBSand Immunostaining anddetectionof (provided byDorisWu), catenin crossed withPax2-Cre micetogenerateaPax2-Cre; catenin in maintained bybreedingheterozygotes. 01;R26RCrereportermice(129S- 2001); line was thencrossedtoahomozygous a CMV-Cre line(Schwenketal.,1995)andthe crossed withPax2-Cre mice.Geneticmarkingof pGEM-T Easy(Promega) forproductionofinsituprobes. GTCTGTGCACATTTGTG-CCTG). ThePCRproductwas subclonedinto with PCRprimers(forward; AGTGCCTGGTAACACCACCAC; reverse, fragment (0.6kb)ofmouse Thomas Lufkin)andrat by MeinradBusslinger), Wilkinson), For whole-mountdetectionofapoptoticcells,embryoswere fixed and A conditionaldeletionof lox(ex3) floxed/del RESEARCH ARTICLE ␤ -catenin Pax2 ␥ Fgf3 -catenin (BDTransduction Laboratories),E-cadherin(Sigma), ) have beendescribedelsewhere (Haradaetal.,1999; ; Pax2-Cre embryos(CKO) foranalysis.To activate Foxi2 Ctnnb1tm2Kem + (provided bySuziMansour), del cells andtheirdescendants,Catnb ) weregeneratedbycrossing (Ohyama andGroves, 2004a), Shh Pax2 Krox20 Wnt8a /J), stocknumber004152(Braultetal., ␤ (provided byHenkRoelink).Apartialexon 6 ctnnin -catenin (provided byGreg Dressler), , was amplifiedfrommousegenomicDNA Hoxb1 ␤ ␤ -galactosidase ␤ -galactosidase (Promega), green Pax2 Gt(ROSA)26Sor -catenin ␤ and ␤ -catenin (Zymed,Sigma), -catenin Dlx5 + Epha4 ␤ cells was generatedbythe -galactosidase, dissected ␤ floxed Lfng and -catenin ␤ ␤ del -catenin -catenin (provided byDavid Hmx3 , line togenerate lox(ex3) Pax2 Tbx1 mice werethen tm1Sor Pax8 floxed ␤ -catenin , + (provided by del del Gbx2 mice were ␮ ␤ cells was mice with /J), stock (provided line. This m frozen line was -catenin , Msx1 floxed ␤ III ␤ ␣ ␤ ␤ - - - - with themostmedialpartof activity was presentinthepresumptive oticectoderm,overlapping ectodermal 2004b). Thesemicedrive Crerecombinaseexpression intheentire al., 2001)withPax2-Cre transgenicmice(OhyamaandGroves, pronephric tubules (Fig.2A). mice alsoexpress Cre inthemid-hindbrainboundaryand Pax2 the Pax2 domainadjacenttotheneuraltube,but notinmorelateral Pax2 showed that at E8.5(Fig.1C).Doubleimmunostainingfor We observed strong reporteractivity inpresumptive oticectoderm ectoderm alreadyexpressed activity was detectedinpresumptive oticectoderm,althoughthis mice betweenembryonicday(E)8.0and9.5.AtE8.0,noreporter the reporter gene(Mohamedetal.,2004).We examined expression of sites arecoupledtoaminimalpromoterandthe reporter transgenicmouselineinwhichsixTCF/LefDNA-binding were displayedgraphicallyasthenumberofpH3 medial-lateral lengthofectodermfrom These resultssuggestthatthemostlateralpopulationof expressed inrhombomere4betweenE8.0andE8.5(Fig.1A,B). candidate forthesourceofWntsignalingis the oticvesicle beingeitherweakornegative. Onepossible region oftheoticvesicle, withactivity intheposterolateralregion of E9.5, reporteractivity isstillobserved mainlyintheanterior-medial reporter activity inectodermlateraltothethickened placode.By posterolateral regions (Fig.1D).Atthistime,wedidnot observe regions oftheoticplacode,withexception ofthemost morphologically distinctatE8.75,reporteractivity isseeninmost embryos werenormalizedper150 placode was countedineachsectionandthecountsfromcActcontrol compared withpH3 a 150 activity inasubsetofPax2 A TCF/Lefreporter mousereveals Wntsignaling RESULTS digital cameraandanAxiophot2microscope.ThenumberofpH3 reveal cellnuclei.ImagesofsectionswerecapturedwithaZeissAxiocam to phospho-histoneH3(pH3)and Serial sectionsofnormalandmutantembryoswerestainedwithantibodies Normalization ofproliferating cellsinnormalandmutantmice Pax2 We usedconditionalgenetargeting toinactivate the expenseofoticplacode precursors causesanexpansion ofepidermisat Conditional inactivationof cells toanoticplacodal,ratherthanepidermal,cellfate. Wnt pathway was activated in binding sites(LoganandNusse,2004).To determineifthecanonical family proteinsandactivates transcriptionfromTCF/LefDNA- allows itstranslocationtothenucleus,whereitbinds toLeforTCF In thecanonicalWntsignalingpathway, stabilizationof embryos usingAdobePhotoshopsoftware. ThenumberofpH3 apical surface ofthethickened placodewas measuredincActandcontrol otic placodewas greatlyenlarged comparedwithcontrols,thelengthof epidermis, wehypothesizedthatWntsignalingmightdirect As thislateralpopulationofcellsislikely togive risetocranial exhibit littleornoactivation ofthecanonicalWntsignalingpathway. In thecaseofconditionallyactive ␤ + + -galactosidase reporterinthepresumptive oticregion ofthese ␮ cells bycrossingaconditional allele of m lengthofoticplacodefromeachcontrolsectionwas countedand cells (Fig.1C).Astheinvaginating oticplacodebecomes Pax2 domain adjacenttothehindbrain. Inaddition,these ␤ + -galactosidase isexpressed inamedialsubsetof cell countsineachsectiontaken fromanequivalent Pax2 Pax2 ␤ ␮ -catenin andthenstainedwithDAPI to Pax2 Pax2 m lengthofplacode. ␤ is initiallyexpressed inabroad + -catenin CKO embryos.Cellcounts (Fig. 1A).ByE8.25,reporter ␤ ␤ precursors + expression domain(Fig.1B). -catenin inPax2 -catenin allele(cAct)wherethe precursor cells,weuseda + Development 133(5) cells persection. ␤ ␤ -galactosidase and -catenin (Braultet Wnt8a ␤ ␤ -galactosidase -catenin inall Pax2 , whichis + ␤ cells inthe -catenin + + cells in + Pax2 cells +

DEVELOPMENT shows theextentofPax2domain,andarrowhead showsPax2 presumptive oticplacode.ThelowerpanelsshowasimilarembryoimmunostainedforPax2(red) and whole-mount E8.5TCF/Lefreporter embryostainedfor onset of and TCF/Lefreporter the otocyst,andblackarrows indicate ␤ cells. ( Wnt signalsinoticplacodedevelopment high degree ofpenetrance.We verified thatthehindbrainsof were consistentlyobserved inallembryosexamined, suggestinga 20% ofitsnormalsizeinCKO embryos(Fig.2A). Bothphenotypes The sizeoftheoticvesicle was significantlyreducedtolessthan that seeninWnt1-Cre/ hindbrain region ofCKO miceisentirelymissing,asimilarresultto (CKO) embryossurvived until atleastE10.5.AtE10.5,themid- (Haegel etal.,1995),Pax2-Cre/ and Groves, 2004b). expression becomesrestrictedtotheotocyst by E9.0-9.5(Ohyama of awideregion ofcranialectoderm,even thoughendogenous mice thereforedrive Cre-mediatedrecombinationinthedescendants restricted totheventromedial region oftheotocyst atE9.5.Pax2-Cre becomes localizedtotheinvaginating oticplacodeatE8.75andis rhombomere 6atE8.5(OhyamaandGroves, 2004b).Itthen region ofcranialectodermextending fromrhombomere3to size oftheoticplacode,weexamined expression of determine whetherthesmallotocyst was duetoareductioninthe (rhombomere 4;seeFig.S1A in thesupplementarymaterial).To (rhombomeres 5and6) expression asamarker ofrhombomeres3and5(Fig.2B) catenin CKO micewerepatternednormallyby examining Fig. 1.DetectionofWntsignalinginPax2 CKO embryoscomparedwithcontrols (Fig.2B).To test whetherthe Dlx5 -galactosidase In contrastto D at E8.5.Allthreemarkers werestronglydownregulated in ) Sectionsthrough theoticcupandotocystofE8.75E9.5reporter embryos.Thewhitearrowhead andblackbarsindicate Pax2 expression atE8.25butnotE8.0(arrowheads). – regions ofplacodalepithelium.Theblackarrowhead indicates ␤ -catenin-null mice,whichdieduringgastrulation ␤ -galactosidase activityatE8.0andE8.25.Significantreporter expression isobservedinpresumptive oticectodermafterthe ␤ , Epha4 -catenin mutantmice(Braultetal.,2001). (rhombomeres 3and5) ␤ -catenin conditionalknockout ␤ -galactosidase + ectoderm withTCF/Lefreporter mice. + Pax2 ␤ migrating neuralcrest cells.n,neuraltube.Scalebars:100 -galactosidase activity, withsectionsthrough theanteriorandposteriorregions ofthe , Pax8 Krox20 Wnt8a Hoxb1 Pax2 Fgf3 and ␤ + - , expression isobservedinrhombomere 4atbothE8.0andE8.25.( ␤ -galactosidase the sizeofoticplacode,whichis and relative tocontrols, asrevealed bythedownregulation of otic placodeinCKO embryosisalreadygreatlyreducedinsize cells intheoticregion of CKO embryosafterE8.5.Bythistime,the CKO embryosbetween E8.5andE9.0.We onlyobserved apoptotic (pH3) andactivated caspase3asamarker ofapoptosisinnormal and survival withantibodiestotheM-phasemarker phospho-histone H3 tissue, weexamined expression of loss ofotictissuewas accompaniedbyanexpansion ofepidermal excluding theoticplacode(OhyamaandGroves, 2004a). factor expressed incranialectodermimmediatelysurrounding,but gives anestimationofthesizeboth correspondingly reduceddomainof survival of size oftheoticplacodemightbeduetoadecreaseinproliferation or placode tissue.However, itisalsopossiblethatthereductionin Cre/ significantly expanded region of Our resultssuggestedtheexpansion of the oticplacode cell proliferation and survival atlaterstagesin Conditional inactivationof 2B). ␤ ␤ Dlx5 -galactosidase -catenin conditionalmutantsoccurredattheexpense ofotic , andtheexpansion of – ( Pax2 A ectoderm. Thearrow indicatesmigratingneuralcrest , B ) Comparisonof + + cells. We assayedcell proliferationandcell vestibuloacoustic ganglioncellsmigratingfrom ␤ -galactosidase (green) protein. Thebracket Pax2 Foxi2 Foxi2 Foxi2 RESEARCH ARTICLE ␮ ␤ Foxi2 expression, m. -catenin altersboth Foxi2 (Fig. 2B).After E8.5,the Foxi2 + , aforkheadtranscription Foxi2 – epidermal cells,anda – otic placodecells(Fig. . CKO embryoshada + Wnt8a domain andalsoof ectoderm inPax2- expression Pax2 Foxi2 C ) A , Pax8 thus 867

DEVELOPMENT complexes inCKO embryos.Despitethefact thatPax2-Cre mice cadherin-mediated celladhesion, weanalyzedE-cadherinadhesion number ofpH3 the reductioninsizeofoticplacode. 868 with theepidermalmarker, 3A) andbyco-localizationofactivated caspase-3-expressing cells than thickened placodalepithelium,onthebasisofmorphology(Fig. majority ofapoptoticcellsoccurredinunthickened epidermis,rather otocyst tissuein 2005; NelsonandNusse,2004). To determinewhether thelossof mediated adhesioncomplexes (Bienz,2005;HarrisandPeifer, pathway, but alsooneofthemaincomponents ofcadherin- ␤ otocyst, butnotinearlyPax2 Cadherin-mediated celladhesionisaltered inthe phenotype. Pax2 switch fromplacodetoepidermis(asshown bytheexpression of size oftheoticplacodeinCKO embryos,itismore likely thatafate is possiblethatdecreasedproliferationmaycontribute tothesmall staining orBrdUincorporation(datanotshown). Thus,althoughit significant differences inthenumberofcellsSphasebyPCNA presumptive oticplacode(Fig.3C,D),although wecouldnotdetect unthickened, downregulation ofplacodemarkers andpredominantlyin death isobserved inCKO embryos,itoccursaftertheinitial -Catenin isnotonlyakey mediatorofthecanonicalWntsignaling Between E8.5andE9.0,wenoticedathreefoldreductioninthe , Pax8 RESEARCH ARTICLE , Dlx5 Foxi2 + ␤ and + cells incranialectodermatthelevel of the -catenin CKO embryos was duetoafailure in epidermis andassuchisunlikely toaccountfor Foxi2 ) isalsoresponsiblefortheobserved Foxi2 . Thus,althoughincreasedcell + precursors localized tothe cellmembrane.As The of epithelialcellsstronglyexpressing that thesmalloticcupremainingatthistimeconsistedofamixture a resultofdeleting E8.5 isunlikely tobeduechangesincelladhesionweobserve as concomitant expansion ofepidermalmarkers seeninCKO miceat 4A). Theseresultssuggestthatthelossofoticmarkers andthe the levels ofeitherE-cadherin, junction complexes. We didnotobserve significantdifferences in hours isrequiredtoclearall limiting amounts(Hincketal.,1994)andsuggeststhatatleast12 preferentially associateswithadherensjunctionswhenpresentin delete catenin 2004b), andbelieve thisisresponsibleforthe residualnumberof recombination inPax2-Cre transgenicmice(OhyamaandGroves, compared withcontrols,but normallevels of contained significantlylower levels ofE-cadherinand remaining otocyst cellsinwhich consist entirelyofeither of aseriessmallcysts fusedtooneanother. Thecysts tendedto lower panels).ByE10.5, theresidualotocyst inCKO mice consists noted thatasmallnumberof expressing no E8.75 (Fig.4A).Thisprobablyreflectsthefact that membrane-localized When weexamined ␤ -catenin + ␤ -catenin fromE8.0,westillobserved low levels of otic cellsinE9.0CKO embryos.Thesmallnumber of ␤ – reporter signal(green) isdetectedinthe lateral (top)anddorsal(bottom)aspects.Cre-loxP images ofthe analysis ofPax2-Cre mice(left)andwhole-mount Pax2 patch (dashedoval).InE8.5CKOembryosthe Foxi2 CKO embryos(asterisks)atE8.5.Theepidermalmarker (arrowheads) andinthemid-hindbrainboundaryof downregulated inpresumptive oticectoderm smaller thancontrols. ( the sizeofoticvesicle(broken line)issignificantly mid-hindbrain region isentirely missing(asterisk)and in hybridization ofotic,epidermalandhindbrainmarkers expense oftheoticplacode. images. control embryos.Scalebars:100 remains strongly expressed intherimofotic cupin expression isgreatly diminishedinCKOembryos,but CKO isthesamelevelasthatincontrol. adjacent tor5.Theposteriorendoftheoticcupin control. Asmalloticcup(dashedoval)isformed marks rhombomeres (r)3and5intheCKO ectodermal domainisexpanded.AtE8.75, (arrowheads). InE10.5 (blue) isdetectedinvirtuallytheentire oticvesicle staining isshowninmagenta.ByE10.5,reporter signal ectoderm (brackets)lateraltotheoticplacode.DAPI detected bothinthethickenedoticplacodeand ectoderm from E8.5.ByE8.75,reporter signalis Fig. 2.Conditionalinactivationof Pax2 -catenin proteinatall(Fig.4A).We have previously cysts containedbothE-cadherin and ␤ -catenin CKOembryos. ␤ / + Pax8 indicates thesizeofoticplacodeasawhite -catenin. cells promotes epidermalfatesatthe ␤ ␤ -catenin proteininotic-level ectodermat -catenin CKO embryosatE9.0,wenoticed -expressing domainissmallerandthe ␤ -catenin ␤ -catenin CKOembryoatE10.5from ␥ ␤ -catenin or -catenin proteinfrommembrane + Pax2 ␥ ␤ B ␤ or -catenin hasbeen reportedto -catenin proteinwas absent ) Whole-mountinsitu -catenin CKOembryos,the ␤ ␤ -catenin proteinandsome + Pax2 -catenin ␣ cells fail toundergo -catenin atE8.75(Fig. ( Development 133(5) A ␮ and ␥ ) Cre-loxP reporter m influorescent -catenin (Fig.4A, – ␤ Pax8 cells (Fig.4B). -catenin in Dlx5 Pax2 are Krox20 ␤ ␣ ␣ + -catenin -catenin -catenin Foxi2 ␤ + -

DEVELOPMENT Wnt signalsinoticplacodedevelopment experiment byconditionally activating astabilizedformof from oticplacodetoepidermis, weperformedacomplementary To confirmthatlossof canonicalWntsignalingcausesafate change placode attheexpenseofepidermis precursor cellscauses anexpansionoftheotic number ofNeurod1 integrity incellslacking al., 1995),webelieve thatitisresponsibleformaintainingepithelial localize toadherensjunctionsintheabsenceof Conditional activationof supplementary material). geniculate andpetrosalplacodesafterE9.0(seeFig.S2in the to theCKO otocyst. We alsoobserved areductioninthesizeof proceed inthevery smallnumbersof suggesting thatbothotocyst formationandneurogenesiscould from thesmallremainingotocysts ofCKO embryos(Fig.4C), in normally targets residues whosephosphorylation bytheAPC/axin/GSK3complex (Harada etal.,1999).Thiseliminates allserineandthreonine which selectively deletesexon 3inthepresenceofCrerecombinase Pax2 + precursors. We madeuseofaconditional ␤ -catenin fordegradation. Asaresult,the mutant + and TuJ1 ␤ -catenin. We alsoobserved asmall + ␤ ␤ -catenin -catenin inPax2 ␤ -catenin – neurons delaminating ␤ – -catenin (Haegel et cells thatgave rise ␤ -catenin allele + ␤ -catenin in all transgenic micetoconstitutively activate thecanonicalWntpathway fashion (Haradaetal.,1999).We crossedthesemicewithPax2-Cre Pax8 laterally andventrally intothecranialectoderm,asassayedby supplementary material).ByE8.75,theoticplacodeexpanded Fgf3, Epha4 patterning oftheposteriorhindbrainwas normalbasedon mutant embryosformedatthelevel ofrhombomeres 4and5, proliferation, as wecoulddetectnosignificant increaseinpH3 of placodalepitheliumappeared nottobedueanincreaseincell expression of (Fig. 5B).Thisepitheliumwas verified tobeoticincharacterbyits ventral pharynxandoccasionally invaginating inectopic locations lateral surface oftheembryo, extending ventrally tothelevel ofthe region ofthickened placodalepitheliumextended over much ofthe invaginating placodalepithelium.ByE9.5,thegreatlyexpanded embryos atE8.75showed highlevels of form of loss of otic placodeincActembryoswas accompaniedbyaconcomitant The oticplacodeofconditionallyactivated (cAct) and Pax2 Foxi2 ␤ Pax2 -catenin becomesstabilizedandactsinadominantactive + precursor cells. + Dlx5 epidermal cells(Fig.5A).ImmunostainingofcAct expression (Fig.5A).Significantly, expansion ofthe and , Gbx2 Hoxb 100 (see Materialsandmethods).Scalebars: the lengthofapicalsurfaceplacode the CKOoticcup.Numbersare normalizedto E8.75 showsareduction individingcells pH3-positive cellspersectionintheoticcupat and DAPI(blue).( Foxi2 capase 3cellscanbeseeninthethickened, CKO embryosbutare notseenincontrols. No Fig. 3.Apoptosisandproliferation in (pH3) atE8.75co-stainedwith arrowheads) markedbyphospho-HistoneH3 rectangles inA).( per sectionintheoticregion (indicatedas number ofactivatedcaspase3-expressing cells unthickened, Foxi2 (magenta) andprocessed withaprobe to with activatedcaspase3(green), DAPI parts oftheoticregion are shownstained pouches. Sectionsofanteriorandposterior show thefirst(1p)andsecond(2p)branchial the contralateraloticplacode.Broken lines (arrowhead). Bracketsindicatethepositionof (A) region oftheoticectoderminCKO caspase 3cellsdetectedmainlyintheanterior embryos. Increased numbersofactivated anti activatedcaspase3staininginE8.5 CKOembryos. catenin and ␮ – . Apoptoticcellsare observedin expression (seeFig.S1Binthe m. otic placode(brackets).( Msx1 RESEARCH ARTICLE Foxi2 (Fig. 6A).Thehugeexpansion C D ) Dividingcells(green, + ) Average numberofthe ectoderm (arrowheads) in ( A ␤ ) Whole-mount -catenin inthe B ␤ ) Average -catenin (red) ␤ -catenin Krox20 Dlx5 ␤ 869 - , ,

DEVELOPMENT Fig. 4.Cadherin-mediatedadhesionin 870 signaling pathway in Together, theseresultssuggestthatactivation ofthecanonicalWnt staining intheepitheliumbetweenE8.75andE9.5(Fig.5B,C). marker ofthefutureendolymphaticduct, embryos. Two markers ofthedorsalotocyst, battery ofregional markers oftheotocyst incontrolandcAct activity was duetoWntsignaling,weanalyzedtheexpression ofa 2005; Riccomagnoetal.,Wu et al., 1998).To testwhetherthis neural tubemayregulate dorsalcellfates intheotocyst (Boketal., A numberofstudiessuggestthatsignalingactivity fromthedorsal Wnt signalingpromotes dorsalotocystcellfates epidermis. into oticplacodecellsandprevents themfromdifferentiating into ␣ embryos (Fig.6B).ByE9.5, markers suchas to theventral midline(Fig.6A).Expressionofotherdorsalotocyst deeply invaginated partoftheplacode,whichtendedtolayclosest (Fig. 6A). throughout thethickened placodalepitheliuminE9.5cAct embryos immunolabeling ofE10.5embryoswith otocysts. BothE-cadherinand Neurod1 such aslunatic fringe, placode (Fig.6B).Markers ofneurogenesisintheventral otocyst expression was expressed inanexpanded region oftheinvaginating We saw virtuallyno to ventromedial anddorsomedial regions oftheotocyst, respectively. white arrow) andnegative(dashedline)cells. cochleovestibular; p,petrosal andn,nodoseganglia.Scalebars:100 -catenin ( RESEARCH ARTICLE -positive ganglioncanbeseendelaminatedfrom thesmallCKOotocyst(arrowhead). Thisganglionisamixture of ␣ Dlx5 -cat) or and Hmx3 ␥ Msx1 -catenin ( Pax2 Pax2 Neurod1 did notappeartobeexpanded incAct were expressed atlower levels inthemost + expression incActembryos and ␣ precursors forcesthemtodifferentiate ␥ Pax2 -catenin are expressed inthe -cat) atE8.75andE9.0.Bracketsindicate (Fig. 6C)and and ␤ Pax8 -catenin andeitherE-cadherinor ␤ -catenin CKOembryos. ␤ -Catenin-negative cellsare still Msx1 are normallyrestricted Dlx5 Ngn1 , wereexpressed and (not shown) Gbx2 ␤ -catenin-negative cysts.( , anda Pax8 ␮ ( A m. ) Doubleimmunolabelingof ␤ ␣ -catenin-negative regions ofectoderminCKOembryos.( provide evidence thatWntsignalingacts instructively on refinement intodifferent tissuetypes.Inthepresentstudy, we by thespecificationofabroadcellulardomain,followed by Development ofaparticularregion oftheembryofrequentlyoccurs DISCUSSION within theoticepithelium. an oticplacodefate, Wntsignalingalsopromotesdorsalcellfates activity andmorelateralectoderm displayinglittleornoreporter cells closesttotheneuraltube displayinghighlevels ofreporter Wnt signals as candidates to pattern Pax2 Wnt signalsascandidatestopattern placode cellsearlyintheirdevelopment. decision, Wntsignalingalsospecifiesadorsalidentityin otic also show thatinadditiontomediatingtheplacode-epidermisfate Pax2 We monitoredactivation of the canonicalWntsignalingpathway in precursor cells of Wntsignaling, precursors todirectthemanoticplacodefate, andinthe absence results suggestthatinadditiontodirecting was alsoentirelyabsentinmutantembryos.Taken together, these were absentincActembryos,andtheposteroventral marker subset of (Mohamed etal.,2004).We observed activation ofthereporterina -catenin. Dashedovalsindicatetheoutlineof ␤ III tubulin(TuJ1, green) positive.t,trigeminal;g,geniculate;cv, + precursor cellsusingaTCF/Lef- Pax2 C ) Neurogenesis in + cells incranialectodermfromE8.25 onwards, with Pax2 ␤ + -catenin ( precursors differentiate asepidermis.We ␤ -catenin CKOembryosatE10.5.A ␤ -cat) andE-cadherin(E-cad), lacZ Pax2 ␤ ␤ -catenin-negative Development 133(5) -catenin-positive (red, transgenic reporter + precursor cellsto B ) Double + Pax2 Tbx1 +

DEVELOPMENT placodal epithelium.Numbersare normalizedtothelengthofapicalsurfaceplacode(seeMaterialsandmethods).Sca accumulated placodal epitheliumfailstocloseincActembryos,formingagrossly enlargedplacodeextendingventrallytothelevelof A.K.G., unpublished),suggestingthatvery low levels ofnuclear at laterstagesintheinnerear(T.O., O.A.M.,M.M.T., D.D.and only beenabletoreproduciblydetectnuclear and cAct embryos.Theectodermlateraltotheoticcupisthickenedfrom E8.75incActembryos markers in marker are upregulated inectodermlateraltotheoticcup(arrows) atE8.75,whiletheepidermal Wnt signalsinoticplacodedevelopment considered circumstantial. case fora family membersandloss-of-function datafor However, intheabsenceofgeneexpression dataforotherWnt expressed thelowest levels of and otocyst –regions thatliefarthest fromrhombomere4– 1993). We notethatthemostposteriorregions oftheoticplacode replaced by E8.0 andE8.5(Fig.1A,B),afterwhichtimeitdisappears,to be show that in rhombomeres5and6zebrafish(Lekven etal.,2001).We and Dodd,1993;Kiletal.,2005;Niederreither2000), and in rhombomere4mouseandchick(Bouilletetal.,1996;Hume our study?Wnt8homologuesareexpressed fromavery earlystage Fuchs, 1999;Dorsky etal.,2002). binding sites,but notinmicewithonlythreesites(DasGupta and presumptive placodeinourreportermicewhich carrysixTCF/Lef- This mayexplain whyreporteractivity can beobserved inthe catenin drive TCF/Lefreporterexpression attheoticplacodestage. be involved intheinitial induction ofthe ectoderm (Fig.1A,B),suggestingthatWntsignalingisunlikely to after theonsetof Significantly, expression oftheTCF/LefreporteratE8.25occurs tube –anddeclinedinmorelateralventral ectoderm. signaling emanatedfromthedorsalmidline–mostlikely theneural activity. Thispatternofactivation suggestedthatthe source ofWnt cAct embryos.( placodal domain.Markersofthemid-hindbrainboundary(asterisk)are alsoexpandedin Fig. 5.Conditionalactivationof the expenseofepidermis. What isthesourceandidentityofWntsignalsobserved in ␤ -catenin protein (red) isexpressed strongly intheplacodalepithelium.ByE9.5, Foxi2 ␤ Wnt8a Wnt8a -catenin cActembryos.Expression oftheearlyoticmarkers ␤ is downregulated atE9.5.Thebroken lineindicatestheoutlineof Wnt1 -catenin andectopicinvaginatingregions (asterisk).( B ) Expansionoftheplacodaldomainwithoutanincrease incelldivision signaling sourceinrhombomere 4muststillbe is expressed inmouserhombomere4between and Pax2 Wnt3a expression inthepresumptive placodal ( A ) Whole-mountinsituhybridizationofoticandepidermal in thedorsalhindbrain(Parr etal., ␤ ␤ -galactosidase reporter(Fig.1). -catenin in Pax2 Pax2 ␤ -catenin expression Wnt8a cell field.We have + cells expandstheoticplacodeat in mice,the C ) TheaveragenumberofpH3-positivecellsisnotsignificantlyincreased incAct ␤ - Pax2, Pax8 previously shown that placode–isalsoreducedcomparedwithcontrols.We have otic forced activation of territory thatwould normallyformtheoticplacode.Conversely, catenin CKO embryos, adjacent totheoticplacode(OhyamaandGroves, 2004a).In greatly reduced,while embryos, expression oftheearlyoticmarkers signaling differentiate asepidermis.InE8.5 that inductionof gene expression studies (OhyamaandGroves, 2004b;Streit,2002) Our presentresultsnow reinforcethenotionfromfate mappingand have tendedto view otic placodeinduction–including thosefromourown laboratory– otic placodetissue,while ear development involve theinductionofabroadfield Fate-mapping experiments suggestthattheearliestevents ininner between oticplacodeandepidermis Wnt signalingmediatesacellfatedecision pathway intheinitial mutations of present study, weprovide evidence fromloss-andgain-of-function placode, andotherswhichwilldifferentiate asepidermis.Inthe some ofwhichwillmaintain Dlx5 expansion ofthickened placodalepithelium,andexpansion of in support theideathatWntsignaling ismediatingacellfate decision corresponding lossofepidermal itself. Thisexpansion ofoticplacodeterritoryisaccompaniedbya Pax2 and otherearmarkers, suchas and Foxi2 + cells betweenoticplacodeandepidermis. Moststudiesof Dlx5 – ␤ ctnnthatactivation ofthecanonicalWntsignaling -catenin Pax2 ␤ Pax2 -catenin intheinitial Pax2 in cranialectoderm andformationoftheotic Pax2 Foxi2 Foxi2 as anexclusive marker oftheoticplacode. Pax2 + field causescellstodifferentiate into – whichbyE8.5islocalizedtothe Pax2 labels aregion ofepidermalcells expression expands tooccupy the + Foxi2 cells thatarenotexposed toWnt Gbx2 RESEARCH ARTICLE expression andformtheotic expression. Ourresultsthus and Pax2 pharynx, with Msx1 + field causesagreat le bars:100 Pax8 ␤ , aswell -catenin CKO and Pax2 Dlx5 + ␮ Pax8 m. cells, Pax2 871 ␤ is - ,

DEVELOPMENT almost exclusively restrictedto expression ofplacodalandepidermalmarkers seenatE8.5,andare conditional deletionmutantsalsooccurafterchangesin the Similarly, thechangesincellsurvival weobserve in after placodalandepidermaldefectsarealreadyapparentatE8.5. late stagesofotocyst formationinCKO embryos,theseoccurwell However, althoughweobserve somechangesincelladhesionat markers. downregulated atE9.5,while with bars. invaginated partoftheplacode,closesttoventralmidline(asterisks).Borders ofthe embryos atE9.5. of theoticplacode. Asoticvesicle invagination isdriven inpartby by E9.0leadstosecondarymorphological defectsintheremnants 872 deleting junctions, andsothephenotypesweobserve byactivating or pool ofbothplacodalandepidermalprogenitorcells. should moreproperlybethoughtofasinitiallymarkinga‘pre-otic’ placode areactuallyexperimentally separableevents, andthat Fig. 6.Activationof malformed otocystandopenrim.Anterior(A)posterior(P)orientationisindicatedwitharrows. embryos, clearanceof results isaWntsignaling-mediated changeincellfate. IntheCKO of theoticplacodeatE8.5inboth ourgain-andloss-of-function that themostparsimoniousexplanation forthechangesinsize ␤ -Catenin alsofunctionsasanimportantcomponentofadherens RESEARCH ARTICLE Neurod1 ␤ Msx1 -catenin mightbeexplained bychangesincelladhesion. expression isjuststartingtoappearinthecontrol endolymphaticbudatE9.5(arrowhead). ( Dlx5, Gbx2 and ␤ Lfng -catenin promotes dorsalotocystcellfates. ␤ -catenin proteinfromadherens junctions (anteroventral markers)and and Pax8 Msx1 is expanded.Adorsalmarker, Foxi2 are upregulated intheplacodalepithelium,although + epidermis. Thus,wefeel Tbx1 ␤ (posteroventral marker)are notexpressed incAct embryos.Broken linesrepresent the -catenin Hmx3 Pax2 ( , isnotexpanded.( A ) Whole-mountinsituhybridizationofdorsalotocystmarkerscAct inner earwhereby Ladher andcolleaguesproposedaschemeforinductionofthechick Wnt andFgfsignalinginoticplacodeinduction otic vesicle. placode ofcActembryosfrominvaginating toformanenlarged likely thatphysicalconstraintsprevent thegreatlyexpanded 1995; Moro-Balbasetal.,2000;Visconti andHilfer, 2002),itis contacts withtheneuraltube(Brown etal.,1998;Gerchman inactivating associated withanullmutation of attempted toovercome theseproblems,andproblemsoflethality complete lossofWntfunction. Inthepresentstudywehave possible, however, thatsuch failed toblockoticplacode induction(Phillipsetal.,2004).Itis overexpression ofthesecretedWntantagonistdickkopf 1( Wnt signalinginzebrafishoticplacodeformationastudywhere 2000). Bycontrast,Riley andcolleagueshave questionedtheroleof Pax2 and othermarkers inunspecifiedectoderm(Ladheret al., Dlx5 ␤ C -catenin onlyin ) Expression ofneurogenic andnon-neurogenic and Dlx5 Fgf19 Gbx2 and Msx1 expressions incontrol embryosare indicated and Dkk1 B Pax2 are downregulated inthemostdeeply Wnt8c ) Pax2 -expressing cells. experiments donotrepresent a expression incActis act synergistically toinduce ␤ -catenin, byconditionally Development 133(5) Dkk1 )

DEVELOPMENT (magenta) of-function (cAct)phenotypes.Control: Wnt-TCF/Lefisactivated otic placode.E8.0: shown asgraph.( panels asabroken line.Thestrength oftheWnt-mediated response is panels represent atransversesectionatthelevelshown inthetop top panelsrepresent theoticplacode.A,anterior;P, posterior. Lower expanding theoticplacodeatexpenseofepidermis.Ovalsin responsive genesinawideregion ofthesurfaceectoderm,thus fates (Riccomagnoetal.,2002;Riccomagno2005). Shh activateexpression ofventralotocystgenesandrepress dorsalcell placode invaginates,signalsfrom theventralmidline(orange)suchas dorsal oticmarkerssuchas signaling inthe Wnt signalsinoticplacodedevelopment pathway, orelsebysynergizing with Fgf-induced transcriptionin ectoderm toFgfsignaling,either atsomestepinthesignaling cell statebyenhancing,sensitizing orperpetuatingtheresponseof proposing thatWntsignalingis required tostabilizetheoticplacode express epidermal-specificgenessuchas fates. the oticplacode-epidermisfatedecisionanddorsalotocystcell Fig. 7.Proposed modelfortherole ofWntsignalinginmediating to delineatethesizeofoticplacode(darkblue). Pax2 signaling from rhombomere (r)4directs themedial-mostregion ofthe signaling from underlyingtissuesand/orthehindbrain.E8.5:Wnt of theoticplacode.IncAct,stabilized absence ofWntsignaling,epidermis(gray)isexpandedattheexpense not receive Wntsignalsdifferentiate asepidermis (gray).InCKO,inthe We reconcileourresultswiththoseofLadherand Riley by + ectoderm toanoticplacodefateandactivatesexpression of ( A ) Summaryofthe in Pax2 Pax2 B + Pax2 ) Amodeloftheinductionandpolarization surface ectodermbyWnt(blue)from hindbrain(HB) + ectoderm are directed toanepidermalfateand in thesurfaceectoderm(SE)isinducedbyFgf ␤ Dlx5 -catenin loss-of-function(CKO)andgain- . Cellsthatdonotreceive Wnt ␤ -catenin activatesWnt- Foxi2 . E8.75 Pax2 + : Astheotic + cells thatdo threshold ofWntsignaling, receives asourceofWntsignalingfromthemidline.Above acertain We suggestthatsubsequentWntsignalingnotonlyinstructs in whichFgfsignalingfirstestablishes afieldof‘pre-otic’ identity intheotocyst are regulated byWnt-independentsignals. Wnt signaling(Fig.6B),suggesting thatsomeaspectsofdorsal expressed genessuchas observation ofRiccomagnoandcolleaguesthatsomedorsally in thesupplementarymaterial).Ourresultsalsoconfirm the downregulate expressed incActembryos,andassuchisagoodcandidateto midline (Fig.6A,centerpanel).We have confirmedthat By contrast, placode closesttotheventral midline(Fig.6A,leftandrightpanels). these genesareexpressed attheirlowest levels inregions ofthe of Wnt signalinginatleastsomecases:forexample, whileexpression from theventral midlinesuchasShharecapableofcounteracting the expense ofventral markers (Fig.6).We alsoshow thatsignals cAct embryoscausesalarge expansion ofdorsalotocyst markers at We findthatactivation ofthecanonicalWntsignalingpathway in for correctdorsoventral otocyst polarity(Riccomagnoetal.,2005). fates, andthatabalanceofopposingWntShhsignalsisrequired signaling fromthedorsalneuraltubeactstospecifyotocyst Riccomagno andcolleagueshave shown Wnt1and/orWnt3a fates intheotocyst (Riccomagnoetal.,2002).Morerecently, midline suchassonichedgehog(Shh)arerequiredtospecifyventral beginning tobeunderstood–forexample, signalsfromtheventral specify thedorsoventral, mediolateralandanteroposterioraxes are et al.,2000;Fekete, 1996;Fekete andWu, 2002).Thesignalsthat shortly afteritsformation(reviewed byBrigandeetal.,2000;Cantos developing earbecomespatternedinallthreeembryonicaxes The work ofFekete, Wu andothershasclearlyshown thatthe dorsoventral polarityoftheinnerear The role ofWntsignalinginspecifying (Gutknecht andFritzsch,1990). 1996)] leadstomultipleexpanded andinterconnected otic vesicles (Phillips etal.,2004),whereasexposure of of Wntsignalingwith morpholino knockdown of concomitant lossofepidermalmarkers. Insupportofourmodel, catenin cActembryoscausesanexpansion ofearlyoticmarkers, and Foxi2 as the absenceofWntsignalingcauseslossearlyoticmarkers such al., 1999;Begbie andGraham,2001).In signals, intoepibranchialplacodes(Begbie etal.,2002;Begbie et differentiate intoepidermisor, inthepresenceofadditionalinducing placode anditsderivatives. Below thisthreshold,cellseither Fig. 7A.Aninitialfieldof et al.,2005).Asimplemodelsummarizingourresultsisshown in al., 2002;HolnthoneretIsrasena2004;Mansukhani the level ofregulation ofGSK3(Dailey etal.,2005;Hashimoto crosstalk betweentheWntandFgfpathways exist, particularlyat the nucleus.Indeed,recentobservations suggest that potentialfor in thesecells.As theplacodeinvaginates, themost ventral regions of precursors toanoticplacodefate, but alsospecifiesdorsaloticidentity inhibiting GSK-3 lithium chloride[whichactivates canonicalWntsignalingby In Fig.7B,wedescribearevised modelofoticplacodeinduction, Our resultsconfirmaspectsofthesepreviously publishedstudies. Dlx5 Pax8 . Conversely, activation oftheWntsignalingpathway in and and Gbx2 Msx1 Dlx5 Msx1 , andanexpansion ofepidermalmarkers suchas expression seemedunaffected byproximity tothe ␤ is seenthroughouttheplacodeofcActembryos, and (Klein andMelton,1996;Stambolicetal., Dlx5 Dkk1 Hmx3 Pax2 Wnt8 Pax2 in ventral cActotictissue(seeFig.S3 results ingreatlyreducedotocysts appear toberefractorychangesin + cells –inducedbyFgfsignaling mRNA inzebrafish,orinhibition + RESEARCH ARTICLE cells differentiate intotheotic ␤ -catenin CKO embryos, Xenopus Shh embryos to Pax2 was still + Pax2 cells. 873 ␤ + -

DEVELOPMENT Lufkin forcDNAprobes. Theconditionallyactivealleleof Meinrad Busslinger, HenkRoelink,SuziMansourandThomas DavidWilkinson, for excellenttechnicalsupport.We are gratefultoDoris Wu, Greg Dressler, Welly MakmuraandSheriJuntillaforcolonymanagement;JuemeiWang We thankRebeccaFerreira andherstaff foranimalmaintenance;JuanLlamas, colleagues have shown thatectopicactivation of correct development ofthedorsalstructures.For example, Fekete and in thisregard thatWntsignalingmayberequiredcontinuouslyforthe cells willcontribute tothedorsalotocyst. Itisinterestingtospeculate express dorsalfates, anduponclosureoftheoticpit, thesemostlateral – even thoseatthemostlateraledgeofoticplacode–continueto ventralizing signalssuchasShh.Cellsthatdonotreceive Shhsignals otic epitheliumisbroughtclosetotheventral midline,exposing itto 874 Artavanis-Tsakonas, S.,Rand,M.D.andLake,R.J. Alvarez, Y., Alonso,M.T., Vendrell, V., Zelarayan,L.C.,Chamero, P., Theil,T., References Bier, E. Bienz, M. Begbie, J.,Ballivet,M.andGraham,A. Begbie, J.andGraham,A. Barald, K.F. andKelley, M.W. http://dev.biologists.org/cgi/content/full/133/5/865/DC1 Supplementary materialforthisarticleisavailableat Supplementary material A.K.G. the HouseEarInstituteandbyNIHgrantsDC004876DC004675to made availabletousbyDrKhashayarshaKhazaie.Thisworkwassupported otic placodewithrespecttoadjacentectodermindifferent species. and rangeofWntsignalingactstospecifytheparticularsize derivative attheexpense oftheother. Itispossiblethatthestrength by modulatingWntsignaling,weareabletoincreasethesizeofone expressing cellsintooticplacodeandepidermalderivatives, andthat study, weshow thatWntsignalingsubdivides afieldof Johnston andGallant,2002;Kenyon etal.,2003).Inthepresent and Raff, 1999;DayandLawrence, 2000;Jacobsonetal.,1997; inductive interactionswithcelldivision andcelldeath(e.g.Conlon the cochlea(Stevens etal.,2003). ventral otocyst canultimatelygive risetovestibular haircellswithin Bouillet, P., Oulad-Abdelghani,M.,Ward, S.J.,Bronner, S.,Chambon,P. and Bok, J.,Bronner-Fraser, M.andWu, D.K. Begbie, J.,Brunet,J.F., Rubenstein,J.L.andGraham,A. Briscoe, J.,Sussel,L.,Serup,P., Hartigan-O’Connor, D.,Jessell,T. M., Brigande, J.V., A.E.,Gao,X.,Iten,L.E.andFekete,D.M. Kiernan, Brault, V., Moore, R.,Kutsch,S., Ishibashi,M.,Rowitch,D.H.,McMahon,A. 130 Requirements forFGF3andFGF10duringinnerearformation. Bosl, M.R.,Kato,S.,Maconochie,M.,Riethmacher, D.etal. cell fatecontrol andsignalintegrationindevelopment. development. 511. of sensoryneurons bytheneurogenic placodes. in innereardevelopment. Curr. Biol. the epibranchialplacodes. placodes andthehindbrain. Mech. Dev. expressed duringearlyembryogenesis andisectopicallyinducedbyretinoic acid. Dolle, P. Development dorsoventral butnotanteroposterior axialspecificationoftheinnerear. 398 specification ofneuronal identitybygradedSonic hedgehogsignalling. Rubenstein, J.L.andEricson, boundaries playarole? formationintheinnerear:docompartment Molecular geneticsofpattern 1264. malformation andfailure ofcraniofacialdevelopment. catenin genebyWnt1-Cre-mediated deletionresults indramaticbrain P., Sommer, L.,Boussadia,O.andKemler, R. The processesthatspecifythesizeofamatureorgan coordinate , 6329-6338. , 622-627. (2000). DrawinglinesintheDrosophila wing:initiationofwingvein RESEARCH ARTICLE (2005). beta-Catenin:apivotbetweencelladhesionandWntsignalling. (1996). AnewmousememberoftheWntgenefamily, mWnt-8, is 15 58 , R64-R67. 132 Curr. Opin.Genet.Dev. , 141-152. , 2115-2124. Proc. Natl.Acad.Sci.USA Development (2001). Integrationbetweentheepibranchial Development Science (2004). From placodetopolarization:newtunes (1999). HomeoboxgeneNkx2.2and 294 10 131 , 595-598. 126 , 393-398. (2002). Earlystepsintheproduction (2005). Roleofthehindbrainin , 4119-4130. , 895-902. (2001). Inactivationofthebeta- Mol. CellNeurosci. 97 , 11700-11706. (1999). Notchsignaling: Development Science ␤ - (1999). Inductionof catenin waskindly ␤ -catenin inthe Development 284 (2003). , 770-776. 128 21 (2000). Nature , 502- , 1253- Pax2 - Ladher, R.K.,Anakwe,K. U.,Gurney, A.L.,Schoenwolf,G. C.andFrancis- Klein, P. S.andMelton,D. A. Kil, S.H.,Streit, A.,Brown, S.T., Agrawal,N.,Collazo,A.,Zile,M.H. and Kiernan, A.E.,Steel,K.P.Kiernan, andFekete, D.M. Gutknecht, D.andFritzsch,B. Brown, S.T., Martin,K.andGroves, A.K. Johnston, L.A.andGallant,P. Jacobson, M.D.,Weil, M. and Raff, M.C. Israsena, N.,Hu,M.,Fu,W., Kan,L.andKessler, J.A. Harada, N.,Tamai, Y., Ishikawa,T., Sauer, B.,Takaku, K.,Oshima,M.and Haegel, H.,Larue,L.,Ohsugi,M.,Fedorov, L.,Herrenknecht, K.andKemler, Groves, A.K. Fekete, D.M.andWu, D.K. Dorsky, R.I.,Sheldahl,L.C.andMoon,T. De Robertis,E.M.andKuroda, H. Day, S.J.andLawrence, P. A. Dailey, L.,Ambrosetti, D.,Mansukhani,A.andBasilico,C. Conlon, I.andRaff, M. Brown, J.W., Beck-Jefferson, E.andHilfer, S.R. Kenyon, K.L.,Ranade,S.S.,Curtiss,J.,Mlodzik,M.andPignoni,F. Holnthoner, W., Pillinger, M.,Groger, M.,Wolff, K.,Ashton,A.W., Hinck, L.,Nathke,I.S.,Papkoff, J.andNelson,W. J. Hashimoto, M.,Sagara,Y., Langford, D.,Everall,I.P., Mallory, M.,Everson, Harris, T. J.andPeifer, M. Cantos, R.,Cole,L.K.,Acampora,D.,Simeone,A.andWu, D.K. Gerchman, E.,Hilfer, S.R.andBrown, J. W. Fekete, D.M. DasGupta, R.andFuchs,E. Hume, C.R.andDodd,J. lithium ondevelopment. development. West, P. H. deficient quail. oftheinnerearrevealedinduction andpatterning byastudyofvitamin-A- Groves, A.K. Academic Press. Organogenesis inner ear. In animal development. or neuronal differentiation ofneuralstemcells. FGF2 signalingdetermineswhetherbeta-cateninexertseffects onproliferation Xenopus press). Ear induction inXenopusembryos. in theDrosophila head. Coordinating proliferation andtissuespecificationtopromote regional identity Drosophila. induction. 369. adhesion moleculeintheformationofavianinnerear. 1147-1160. mutation ofthebeta-cateningene. Taketo, M. Development R. 235-237. ear. matrix intheformationofinnerear. Neurobiol. throughout zebrafishdevelopment. catenin-dependent reporter isexpressed inspatiallyrestricted domains size andshape. Development transcription complexesduringhairfollicledevelopmentanddifferentiation. Factor Rev. Mechanisms underlyingdifferential responses toFGFsignaling. 244. between adhesionandtranscription. 11713. ofthemammaliancochlea. Patterning role inprimitivestreak formationandhindbrainorganization. endothelial cells. Fibroblast growth factor-2 inducesLef/Tcf-dependent transcriptioninhuman Albanese, C.,Neumeister, P., Pestell,R.G.andPetzelbauer, P. complex assembly. cadherin/catenin complexformation:novelprotein interactionsandpathwaysof for neuroprotection. regulates signalingviatheglycogensynthasekinase-3betapathway. Implications A., Digicaylioglu,M.andMasliah,E. (1995). Lackofbeta-cateninaffects mousedevelopmentatgastrulation. (ed. A.N.PopperM.W. KelleyandD.K.Wu). NewYork: Springer-Verlag (in Curr. Opin.Neurobiol. into multipleoticvesiclesconnectedbytubes. Curr. Top. Dev. Biol. 6 16 BioEssays (2000). Identification ofsynergisticsignalsinitiatinginnerear (2005). Theinductionoftheoticplacode.In , 533-541. Mouse Development:Patterning,Morphogenesisand (1996). Cellfatespecificationintheinnerear. 121 126 , 233-247. Science (2005). Distinctroles forhindbrainandparaxialmesoderminthe Dev. Biol. (ed. J.RossantandP. L.Tam), pp.539-566.SanDiego,CA: (1999). Intestinalpolyposisinmicewithadominantstable Development J. Biol.Chem. , 3529-3537. , 4557-4568. J. CellBiol. J. Biol.Chem. Cell 24 290 (1999). Sizecontrol inanimaldevelopment. Dev. Cell , 54-64. Proc. Natl.Acad.Sci.USA (1993). Cwnt-8C:anovelWntgenewithpotential 285 (2005). Decisions,decisions:beta-cateninchooses 88 , 1965-1967. 12 (1999). Multipleroles foractivatedLEF/TCF (2002). Revisitingcellfatespecificationintheinner , 347-354. , 252-271. (1996). Amolecularmechanismforthe effect of , 35-42. (2000). Measuringdimensions:theregulation of 127 125 (1990). Lithiumcantransformearplacodesof (2002). Control ofgrowth andorgansizein 57 277 Annu. Rev. CellDev. Biol. 5 , 115-149. , 2977-2987. , 1327-1340. , 403-414. (2004). Dorsal-ventral patterning andneural (2004). Dorsal-ventralpatterning 277 , 45847-45853. EMBO J. Dev. Biol. Trends CellBiol. Proc. Natl.Acad.Sci.USA , 32985-32991. (2002). Fibroblast growth factor1 Dev. Dyn. (1997). Programmed celldeath in (2003). Molecularbasisofinnerear 18 (1995). Involvementofextracellular (2002). AtransgenicLef1/beta- (2002). Developmentofthemouse 241 , 5931-5942. Dev. Biol. (1998). Arole forneuralcell , 229-237. 202 93 15 Development 133(5) (1994). Dynamicsof , 8455-8459. , 421-432. (2004). Thepresence of Naturwissenschaften , 234-237. Development ofthe 20 268 Dev. Dyn. Curr. Opin. , 285-308. (2005). , 220-231. Development Cytokine Growth 97 (2002). Cell (2000). , 11707- 213 (2003). 96 , 359- , 235- 119 77 , ,

DEVELOPMENT Logan, C.Y. andNusse,R. Liu, D.,Chu,H.,Maves,L.,Yan, Y. L.,Morcos, P. A.,Postlethwait, J.H.and Lombardo, A.,Isaacs,H.V. andSlack,J.M. Novak, A.,Guo,C.,Yang, W., Nagy, A.andLobe,C.G. Niederreither, K.,Vermot, J.,Schuhbaur, B.,Chambon,P. andDolle,P. Levin, M. Lekven, A.C.,Thorpe,C.J.,Waxman, J.S.andMoon,R.T. Maroon, H.,Walshe, J.,Mahmood,R.,Kiefer, P., Dickson,C.andMason,I. Mansukhani, A.,Ambrosetti,L.andBasilico,C. D.,Holmes,G.,Cornivelli, Leger, S.andBrand,M. Lander, A.D.,Nie,Q.andWan, F. Y. Nelson, W. J.andNusse,R. Mohamed, O.A.,Clarke,H.J.andDufort,D. Mizutani, C.M.,Nie,Q.,Wan, F. Y., Zhang,Y. T., P., Vilmos, Sousa-Neves,R., Ladher, R.K.,Wright, T. J.,Moon,A.M.,Mansour, S.L.andSchoenwolf,G. Wnt signalsinoticplacodedevelopment Moro-Balbas, J.A.,Gato,Alonso,M.I.,Martin,P. anddelaMano,A. Ohyama, T. andGroves, A.K. Lai, E.C. and disease. 130 transcription factorsare required foroticplacodespecification. Westerfield, M. 192. junctional communicationinembryonicpatterning. FGF-3 in Cre-mediated excision. reporter mouselinethatexpresses enhancedgreen fluorescent protein upon Development inthemouseembryo. Retinoic acidsynthesisandhindbrainpatterning mesoderm andneurectoderm patterning. wnt8 encodestwoproteins onabicistronic transcriptandisrequired for 108. placode induction,maintenanceandinnerearpatterning. cadherin pathways. and vesicle. (2002). Fgf3andFgf8are required togetherforformationoftheoticplacode signaling andosteoblastdifferentiation. (2005). Sox2inductionbyFGFandFGFR2activatingmutationsinhibitsWnt diffusion? gradient intheDrosophila embryo. Bier, E.,Marsh,J.L.andLander, A.D. Development C. invagination inchickembryos. (2000). Basallaminaheparansulphateproteoglycan isinvolvedinoticplacode Dev. Dyn. marks theprospective siteofprimitivestreak formation in themouseembryo. early craniofacialdevelopment. 603-613. (2005). FGF8initiatesinnerearinductioninchickandmouse. , 2213-2224. (2004). Notchsignaling:control ofcellcommunicationandfate. (2002). Isolationandcommunity:areview oftherole ofgap- Xenopus 231 Dev. Cell Development Annu. Rev. CellDev. Biol. 127 131 , 416-424. (2003). Fgf3andFgf8dependentindependent , 75-85. , 965-973. development. 2 Science , 785-796. Genesis (2002). Fgf8andFgf3are required forzebrafishear 129 (2004). TheWntsignalingpathwayindevelopment (2004). ConvergenceofWnt,beta-catenin,and 303 , 2099-2108. (2004a). Expression ofmouseFoxiclassgenesin Anat. Embryol. (Berl.) Anat. Embryol. Dev. Dyn. 28 , 1483-1487. Int. J.Dev. Biol. , 147-155. Dev. Cell (2002). Domorphogengradientsariseby 20 J. CellBiol. , 781-810. (2005). FormationoftheBMPactivity 231 Dev. Cell (1998). Expression andfunctionsof 8 , 640-646. , 915-924. (2004). Beta-cateninsignaling 42 , 1101-1107. 1 168 , 103-114. 202 J. Membr. Biol. , 1065-1076. , 333-343. (2000). Z/EG,adouble Mech. Dev. (2001). Zebrafish Development Genes Dev. 185 119 , 177- (2000). , 91- 19 , Wu, D.K.,Nunes,F. D.andChoo, Phillips, B.T., Storch, E.M.,Lekven,A.C.andRiley, B. Ohyama, T. andGroves, A.K. Riccomagno, M.M.,Takada, S.andEpstein,D.J. Riccomagno, M.M.,Martinu,L.,Mulheisen,Wu, D.K.andEpstein,J. Phillips, B.T., Bolding,K.andRiley, B. Parr, B.A.,Shea,M.J.,Vassileva, G.andMcMahon,A.P. Stern, C.D. Stern, Schwenk, F., Baron, U.andRajewsky, K. Riley, B.andPhillips,T. Shilo, B.Z. Soriano, P. Wright, T. J.andMansour, S.L. R.P.Visconti, andHilfer, S.R. Torres, M.andGiraldez,F. Stevens, C.B.,Davies,A.L.,Battista,S.,Lewis,J.H.andFekete,D.M. Stambolic, V., Ruel,L.andWoodgett, J.R. Stamataki, D.,Ulloa,F., Tsoni, S.V., Mynett,A.andBriscoe,J. Streit, A. 125 organs versusnon-sensorystructures ofthechickeninnerear. otic placodeinduction. for FgfbutnotWntinoticplacodeinduction. 365. redundant functionsrequired foroticplacodeinduction. modification ofaPax2bacterialartificialchromosome. interactions inoticdevelopment. supporting roles ofShh. regulation ofinnerearmorphogenesisisbalancedbytheopposingand germ cells. for theubiquitousdeletionofloxP-flankedgenesegmentsincludingin hedgehog. (2002). SpecificationofthemammaliancochleaisdependentonSonic limb buds. genes exhibitdiscrete domainsofexpression intheearlyembryonicCNSand strain. Mech. Dev. Curr. Top. Dev. Biol. hybridization andimmunohistochemistryinwholemountsofavianembryos. Biol. synthase kinase-3activityandmimicswinglesssignallinginintactcells. tube. gradient ofGliactivitymediatesgradedSonicHedgehogsignalingintheneural pathway duringdevelopment. rhombencephalon andinhibitssubsequentinvagination. prevents associationoftheoticprimordium withtheposterior placode. organ identityinthechickeninnerear. (2003). Forced activationofWntsignalingaltersmorphogenesisandsensory , 11-20. 6 Genes Dev. , 1664-1668. Nat. Genet. (2002). Extensivecellmovementsaccompanyformationoftheotic Dev. Biol. (1999). GeneralizedlacZexpression withtheROSA26Cre reporter (2003). SignalingbytheDrosophila epidermalgrowth factorreceptor (1998). Detectionofmultiplegeneproducts simultaneouslybyinsitu Development Nucleic AcidsRes. Genes Dev. 71 , 5-21. 19 249 21 36 , 626-641. , 70-71. , 223-243. , 237-254. 16 Development Genes Dev. 119 , 2365-2378. (1998). Thedevelopmentofthevertebrateinnerear. (2003). Ringinginthenewear:resolution ofcell , 247-261. 23 (2002). Perturbationofextracellularmatrix (2004b). GenerationofPax2-Cre miceby Exp. CellRes. (2003). Fgf3andFgf10are required formouse , 5080-5081. Dev. Biol. 19 (1998). Axialspecificationforsensory 130 , 1612-1623. Dev. Biol. , 3379-3390. (1995). Acre-transgenic mousestrain (2001). Zebrafishfgf3andfgf8encode RESEARCH ARTICLE 261 (1996). Lithiuminhibitsglycogen 284 Development , 289-312. , 140-149. 261 (2005). Wnt-dependent , 149-164. Genesis Dev. Biol. Dev. Dyn. (2004). Adirect role (1993). MouseWnt 131 Development 38 , 923-931. (2005). A , 195-199. 235 223 , 351- , 48-58. Curr. 875

DEVELOPMENT