in thehindbrain,compromisinginductionandmaintenanceofoticfate. depletion inwildtype.Thus,excessRAexpandsoticcompetence,whereasthelossofimpairsexpression double mutants,andinactivationof maintenance. We showthatrhombomere5 otic vesiclessimilartoRA-signaling-depletedembryos,suggestingasignalfromrhombomere5-6mayalsoberequiredforf both Fgf8 have beenimplicatedtohave overlapping functions;lossof Whitfield etal.,2002;Brown etal.,2003).Inzebrafish, Fgf3and (reviewed inFritzschetal.,1997;Torres andGiráldez,1998; species, withonlyhindbrain-derived Fgf3playingaconserved role members fromvarious sourcesregulate oticinductionindifferent (Fgfs) intheinductionofoticplacode.Various Fgffamily 1997; Whitfield etal.,2002;BaraldandKelley, 2004). (Noden andvan deWater, 1986;Coulyetal.,1993;Fritzsch membranous labyrinthandneuronsofthestatoacousticganglion otocyst gives risetoallstructures of theinnerearincluding epithelial structurewithsharplydefined borders.Subsequently, the cavitates toformtheoticvesicle, alsoknown astheotocyst, an hindbrain. Dependingonthespecies,placodeinvaginates or thickening, theoticplacode,visibleoneithersideofdeveloping and vestibular functions.Itdevelops fromatransientectodermal The vertebrate innerearisthesensoryorgan thatprovides auditory INTRODUCTION Accepted 30April 2007 2 1 KEY WORDS:Competence, never achieveanormalsize,suggestingthatanadditionalfactorisrequiredtomaintainoticfate. induction inRA-depletedembryos.Oticisrescuedby producefewoticcells,andthesecellsfailtoformavesicle,indicatingthatFgf8istheprimaryfactorresponsible signaling competence, butinsteadresultsindelayedonsetof expressed throughoutthepreplacodaldomain.Bycontrast,lossofRAsignalingdoesnotaffect provides competencetoadoptanoticfate.Subsequently, have originsindifferent tissues.ExcessRAleadstoectopic enlarged orreducedoticvesicles,respectively. Hereweelucidatethemechanismsthatunderliethesechangesandshow Retinoic acid(RA)haspleiotropicfunctionsduringembryogenesis.Inzebrafish,increasingorblockingRAsignalingresultsin Stefan Hans Changes inretinoicacidsignalingalteroticpatterning (2007)doi:10.1242/dev.000448 Development 134,2449-2458 *Author forcorrespondence (e-mail:[email protected]) Technology, Dresden, Germany. signaling cascaderequiredforoticinductioninthisspecies(Ladher Fgf8 hasbeenshown toplayacrucialroleupstreamoftheFgf (Wright andMansour, 2003;Alvarez etal.,2003).Furthermore, and Fgf10resultsinthecompleteablationofoticdevelopment Fgf10 isexpressed inthemesodermbeneathit,andlossofbothFgf3 expressed inthehindbrainabutting thepreoticdomain,whereas Fgf3is induction (WrightandMansour, 2003;Alvarez etal.,2003): 2002). Inmouse,Fgf3andFgf10actasredundantsignalsduring otic tissue (Phillipsetal.,2001;Maroon2002;LégerandBrand, Zebrafish Biotechnology CenterandofRegenerative Therapies,Universityof Institute ofNeuroscience, UniversityofOregon, Eugene,OR97403,USA. Previous studiessupporttheprimacy offibroblast growth factors fgf3 and fgf8 1,2 and MonteWesterfield together resultsinnearortotalablationofotic dlx3b , Danio rerio wnt8b , in wnt8b fgf3 1,* fgf3 , fgf8 mutants bymorpholinoinjectionresultsinsmalloticvesicles,similartoRA expression isabsentinbothRA-signaling-depletedembryosand fgf3 , foxi1 expression andimpairedoticinduction. pax8 foxi1 ne a,Mrhln,Oi nuto,Oticplacode,Retinoicacid, Innerear, Morpholino,Oticinduction, , , theexpressionofwhichdependsuponFoxi1andFgf,isalso fgf8 expression throughouttheentirepreplacodaldomain.Foxi1 expressed with 2003; Nissenetal.,2003).Thehomeoboxgene, highly variable earphenotypes(Thisseetal.,2005;Solomon expression leadstosevere defects in oticplacodeformationand the preoticdomainatlategastrulastages,anddisruptionof The expression of Fgf signalinginzebrafish (Hansetal.,2004;Solomon2004). Foxi1 andDlx3btranscriptionfactors tobecompetentrespond effectors downstream ofFgf-signalingandthatcellsneedtoexpress 1994; Lombardoetal.,1998). al., 1995;Ladheret2000;2005;SongandSlack, Fgf2 andFgf3,have beenimplicatedinoticinduction (Mahmoodet et al.,2005).Inchick,Fgf3,Fgf8andFgf19and,inamphibians, to acompleteloss ofr5-r7accompaniedbyan expansion ofr3-r4, Dupé andLumsden,2001).Complete absenceofRAsignalingleads hindbrain attheexpense ofposteriorhindbrain(Dupéetal.,1999; compromised RAsignalingleads toanexpansion ofanterior the development oftheposteriorhindbrain,particularlyr5-r7,and RA isrequiredinaconcentration- andtime-dependentmannerfor Gavalas and Krumlauf,2000;Romand,2003).Inamnioteembryos, the hindbrainisknown toregulate oticdevelopment (reviewed by crucial roleinhindbrainpatterningandrhombomere(r)identity, and hematopoietic system(reviewed by Maden,2002).RAplaysa show defectsinthecirculatorysystem,limbs,trunkand proper embryonicdevelopment. Embryosdeficient inRAsignaling 2004; Hansetal.,2007). activated Fgfsignalingpathway (Hansetal.,2004;Solomon otic inductioneven inthepresenceofafully functionalorover- et al.,2003).LossofbothDlx3bandFoxi1 ablatesallindicationsof Ellies etal.,1997;Kudoh etal.,2001;SolomonandFritz,2002;Liu and olfactory tissues(Akimenko etal.,1994;Ekker etal.,1992; knockdown of corresponding tocellsofthefutureneuralplateborder, and overexpression inRA-depletedembryos,althoughoticvesicles Recently, studieshave shown thatPax2a andPax8 arethemain Retinoic acid(RA),aderivative ofvitaminA,isrequiredfor dlx3b dlx4b foxi1 and in lategastrulastageembryosastripe is restrictedtobilateraldomains,including dlx4b foxi1 fgf8 together causesasevere lossofotic fgf3;tcf2 expression orotic mutants depletedofRA RESEARCH ARTICLE double mutantsform fgf3 fgf3;tcf2 and dlx3b forotic wnt8b , isco- 2449 foxi1 ate y

DEVELOPMENT fates, duetocompromised signaling resultsinlower inductionandimpairedmaintenanceofotic throughout thepreplacodaldomain.Bycontrast,reduced RA to anincreaseinoticcompetencedueexpanded expression of expression inthehindbrain.Instead,thislow doseofexcess RAleads show thatRAaffects oticinductionwithout expanding used a50-foldlower doseofRAthanthatprevious studies,and more orlessRAsignalingareproducedbydifferent mechanisms.We RA (Phillipsetal.,2001). and/or Fgf8bymorpholinoinjectionblockstheeffects ofexogenous expression surroundingtheanteriorneuralplate.Inactivation ofFgf3 expanded intotheanteriorneuralplate,inducingpreotic MATERIALS ANDMETHODS phenotype canberescuedbyan increaseinFgf-signaling. RA-deprived embryosandthattheinduction but notthemaintenance hindbrain. We further show that of RA,hindbrainexpression domainsof 2001). Inzebrafish embryostreatedwithteratogenicdoses(1 ectopic oticvesicles inanFgf-dependentmanner(Phillips etal., than reduced,RAresultsintheformationofsupernumeraryand In zebrafish, theoppositeresulthasbeenreported;excess, rather expansion ofFgf3expression (Whiteetal.,2000;Kil2005). expansion ofposteriorhindbrainand,inparticular, withthecaudal vesicles caudaltothemainotocyst, whichcorrelateswiththe embryos, however, leadstotheformationofsupernumeraryotic moderate lossofRAsignaling,suchasinweakquailorratVAD placode (Perz-Edwards etal.,2001;Whitfield etal.,2002).A expression inthepresumptive r4and fortheinductionofotic suggested, but notyetshown, thatRAisrequiredfornormal knockdown ofRAbypharmacologicaltreatmentandithasbeen The sameoticphenotypecanbeobserved inzebrafish after abnormally distantfromthehindbrain(Niederreitheretal.,1999). and notproperlyrestricted,otocysts arehypoplastic and expressed inthepresumptive r5-r6 inwild-typeembryos,isweak Aldh1a2 function studies.InembryoswithnoRAsignaling,suchasmouse because hindbrainpatterningisdisturbedinRAgain-andloss-of- mostly secondaryconsequencesduetochangesinFgfsignaling, al., 1992;Kudoh etal.,2002). expense ofpresumptive fore-andmid-brainstructures(Marshallet r3 tor4-r5identityandexpansion ofposteriorhindbrainatthe posteriorizes theanteriorneuralplatewithtransformationofr2- Kimmel, 2005).Treatment ofvertebrate embryoswithexcess RA something inadditiontoAldh1a2(Grandeletal.,2002;Maves and by pharmacologicaltreatment,suggestingthatRAisproduced expansion ofr3andr4isobserved afterknockdown ofRAsignaling Kimmel, 2005).However, alossofr5-r7accompaniedbyan embryos (Begemann etal.,2001;Grandel2002;Maves and similar toweakvitaminAdeficiency syndrome(VAD) inamniote only alossofr7accompaniedbyslightexpansion ofr5andr6 according tostandard criteria(Kimmeletal.,1995) orbyhourspost produced usingstandardprocedures (Westerfield, 2000)andstaged Embryos wereobtainedfromtheUniversity ofOregon zebrafish facility, Animals 2000). Mutationsin in theearlyembryo(Niederreitheretal.,1999;Niederreither dehydrogenase thatisresponsibleforthemajorityofRAproduction Raldh2 as observed inmouseembryosmutantfor 2450 Here, weshow thattheoppositeoticphenotypesgeneratedby Otic defectsgeneratedbychangesinRAsignalingareconsidered RESEARCH ARTICLE – MouseGenomeInformatics).Aldh1a2isanaldehyde mutants, expression ofFgf3,whichisspecifically aldh1a2 in zebrafish arelessprofoundandshow fgf3 fgf8 and is theprimaryinducingfactor in wnt8b fgf3 Aldh1a2 and expression inthe fgf8 (also known as fgf3 are greatly and pax8 foxi1 ␮ fgf3 fgf8 M) et al.,1998); as Hopkins, 2001;Hansetal.,2007),andwerefertothehomozygousmutants described previously (Brandetal.,1996;Herzog2004;Sunand of medium asfollows: DEAB, 10 embryo treatments,dilutionsofthesechemicalsweremadein in DMSOand1mMall-transretinoicacid(RA;Sigma)DMSO.For stored at–80°C:100mM4-(Diethylamino)-benzaldehyde(DEAB;Sigma) pharmacological treatments,thefollowing stocksolutionsweremadeand (Liu etal.,2003;SolomonandFritz,2002;Kim2002).For The Morpholinos (MOs)andpharmacologicaltreatments al., 2001); cDNA probesthatdetectthefollowing geneswereused: In situhybridization conventions (http://zfin.org/zf_info/nomen.html) areused. Approved geneandproteinnamesthatfollow thezebrafish nomenclature Genes andmarkers acerebellar fertilization at28°C(hpf).Thewild-typelineusedwas AB.Thelines expression incontrolembryos,theotic placode was reducedinsize At the12-somitestage,when the placodewas outlinedby but increased upto40%inthepresenceof10nMRA(Fig.1D-F). size was reducedbyupto 50% inRA-signaling-depletedembryos, throughout theentireepithelium ofcontroloticvesicles, oticvesicle expression oftheoticmarker semicircular canalprotrusions (Fig. 1A-C).Assessedat22hpfby enlarged oticvesicles containingthreeotolithsandmultiple excess RAataconcentrationof10nMledtotheformation small otolithandimpairedsemicircularcanalprotrusions,whereas of RAsignalingledtostronglyreducedoticvesicles withonlyone al., 1988).Comparedwiththemorphologyofcontrolsat50hpf, loss (DEAB), apotentretinaldehydedehydrogenaseinhibitor(Russo et used thepharmacologicalinhibitor4-(Diethylamino)-benzaldehyde placodal andpreplacodalstages.To interferewithRAsignaling,we analyzed theexpression ofseveral oticmarkers atthevesicle, 2001). To studytherole ofRAsignalinginoticdevelopment, we have remainedobscure(Perz-Edwards etal.,2001;Phillips leads tooppositeoticphenotypes,but theunderlying mechanisms It hasbeenshown previously thatexcess ordeficient RAsignaling induction Excess orlossofRAsignalinginterferes withotic RESULTS et al.,1998); al., 1993;Jowett andYan, 1996;WhitlockandWesterfield, 2000). hybridization was performedessentially aspreviously described(Thisseet (Kelly etal.,1995).Probe synthesis andsingle-ordouble-colorinsitu hypomorphic alleleof or lossof mutants werescoredeitherbytheirmorphologicalphenotypeand/orPCR conducted inthedark. incubated incorrespondingdilutionsofDMSO.Allincubationswere rinses withembryomedium.For controltreatments,siblingembryoswere stage weretreatedwith1 previously (Kudoh etal.,2002);dechorionatedembryosat40%epiboly Treatments withateratogenicdoseofRAwerecarriedoutasdescribed chorions andtransferredintoPetridishescontainingthetreatmentsolution. gastrulation (30%epibolyor4.7hpf)embryoswereremoved fromtheir maintained for30minutesinaheatingblock. fresh 37-39°Cembryomediumina1.5mltube(20embryospertube)and Heat-shock-treatment embryosweretransferred,stillintheirchorions,into fgf8 tcf2 dlx3b , ) andthetransgenicline fgf3 pou1f1 -MO, dlx3b ti282a and foxi1 pou1f1 tcf2 foxi1 (Ekker etal.,1992); expression (Herzogetal.,2004;SunandHopkins,2001). (a stronghypomorphicalleleof (Solomon etal.,2003); mutants, respectively. Homozygousmutantsanddouble (Nica etal.,2004); -MO and fgf3 ); ␮ M RAfor80minutesfollowed bythorough tcf2 Tg(hsp:fgf8) wnt8b ␮ starmaker hi2169 M; andRA,10,201000nM.Priorto fgf3 -MO have beenpreviously described (a nullorstronghypomorphicallele stm (Phillips etal.,2001); otx2 (Söllner etal.,2003);and (to misexpress ( stm (Li etal.,1994); Development 134(13) ,wihwas expressed ), which fgf8 cldna fgf8 ); pax8 fgf8 (Kollmar et ) have been lia t24149 (Reifers (Pfeffer cldna wnt8b (a

DEVELOPMENT unaffected by20nMRAbut arecompletelyabolished inthe In comparisontocontrols,expressions of altered theexpression of earlyanteroposterior-axis-specific genes. showed thatteratogenicdoses,but not 20nM,ofRAsignificantly material). Subsequentmarker analysisoftheanteroposterioraxis compared themtocontrols(see Fig.S1inthesupplementary orateratogenicdoseofRAand embryos witheither20nMRA doses ofRA(Phillipsetal.,2001;Kudoh etal.,2002),wetreated the anteriorneuralplateinsamemannerastypicalteratogenic 2B-D). To demonstrate thatourRAtreatmentdidnotposteriorize patches ofectopicoticcellsinembryostreatedwith20nMRA(Fig. limit ofthehead,weobserved onlyrandomlydistributed small doses (1 al., whoreportedthat20-30%ofembryostreatedwithteratogenic previously (Phillipsetal.,2001).However, incontrasttoPhillipset anterior neuralplate(Fig.2A),consistentwithresultspublished of RAto20nMamplifies the entire anteriorneuralplatecanbedetected(Fig.1J-L).Anincrease domain isenlarged andweak expression of whereas, inRA-treatedembryos,thepreotic depleted embryos,thepreoticexpression of signaling arealreadyevident atpreoticstages.InRA-signaling- the oppositephenotypesgeneratedbyexcess ordeficient RA the oticanlagenpriortoformationofplacode,showed that increase (Fig.1G-I).Useof in RA-signaling-depletedembryos,whereasexcess RA ledtoasize Retinoic acidandoticfate 35 the top.(D-L)Dorsalviewswithanteriortowards thetop.Scalebar: views ofliveoticvesicleswithanteriortotheleftanddorsaltowards stage) stagesafterlabelingwith evident atplacodal(G-I,12-somitestage)andpreplacodal (J-L,5-somite ( morphology at50hpf(A-C)or (DEAB) oraftertheapplicationof10nMRA,asassessedbothby reduced orincreased insize,respectively, afterdepletionofRAsignaling phenotypes. Fig. 1.LossandexcessofRAsignalinggenerateopposite G-L ␮ Reduction orincrease inthenumberofpreotic cellsisalready ) m. ␮ M) ofRAproduceectopicoticvesicles attheanterior ( A-F ) Compared tocontrol embryos,oticvesiclesare pax8 stm cldna pax8 expression at22hpf(D-F). expression, whichisinitiatedin (G-I) or expression surroundingthe pax8 pax8 six3a (J-L). (A-C)Lateral pax8 pax8 surrounding the and is reduced, expression otx2 was 35 of thesepatches.Dorsalviewswithanteriortowards thetop.Scalebar: vesicles. (C,D)HighermagnificationsofBshowtheepithelialstructure patches ofcellsthatexpress theoticmarker with Fig.1J,L).( pax8 material). Inembryostreatedwith20nMRA,expression of with ateratogenicdoseofRA(seeFig.S1G-Iinthesupplementary misexpressed throughouttheentireneuralplateofembryos treated expanded inembryostreatedwith20nMRA,but itwas strongly et al.,1996)inthecaudalhindbrainofcontrolembryoswas mildly material). ExpressionoftheRA-controlledgene presence ofateratogenicdose(seeFig.S1A-Finthesupplementary the endogenousear. induction andtotheformationofectopicoticcellsoutside 20nMRAleadstowidespread ectopic otic Applicationof Fig. 2. RA signaling,whereas inembryostreatedwith20 nMRA, r3-r4, weconsistentlyobserved enlarged RA signalingledtoalossofr5-r7accompaniedbyanexpansion of depleted ofRAsignaling(Fig.3A,B).Becausethecompleteloss of control embryos,but itsexpression was severely reducedinembryos 95% epiboly, (Phillips etal.,2001;Maroon2002;LégerandBrand,2002). At implicated tohave overlapping functionsinoticplacodeinduction otic inductionandcompetence.Inzebrafish, Fgf3and Fgf8 have been possibilities, weexamined theexpression ofthe factors thatregulate respond totheinductive signals,orboth.To distinguishamongthese underlying theinductionofoticfates orinthecompetenceofcellsto development. Thiscouldbeduetochangesinthemechanisms the oticvesicle, whereasincreasedRAresultsinenhanced otic Our resultsindicatethatlossofRAsignalingimpairstheformation signaling reduces Excess RAinduces aspects ofembryonicdevelopment. occurs withlow dosesofRAthatdonotapparentlyaffect other be tracedbacktooticinductionandthatectopic opposite phenotypesgeneratedbygainorlossofRAsignalingcan supplementary material).Together, ourresultsshow thatthe treated withateratogenicdoseofRA(seeFig.S1J-Linthe whereas itwas misexpressed intheanteriorneuralplateofembryos the rostralhindbrainwas indistinguishablefromcontrolembryos, to controlembryos, the innerear(Hansetal.,2004;Solomon etal.,2004).Incomparison proposed thatFoxi1 and Dlx3bprovide competenceforcellstoform 20 nMRA(Fig.3C,F,I anddatanotshown). We andothersrecently fgf3 with controlembryos(Fig.3D,E,G,H). Bycontrast,expression ofboth the level of domains inDEAB-treatedembryosatthethree-somitestage,although ␮ and m forA;50 expression encompassingtheentire preplacodal domain(compare fgf8 fgf3 was completelyunaffected inembryostreatedwith10or fgf3 B-D expression appearedtostillbereducedincomparison ␮ m forB;10 ) Embryostreated with20nMRAdisplayectopic was expressed stronglyinther4primordiumof foxi1 ( A ) Applicationof20nMRAleadstoectopic fgf3 foxi1 expression was unchanged intheabsenceof ␮ m forC,D. expression , whereas lossofRA RESEARCH ARTICLE stm fgf3 and formenlarged and hoxb1b fgf8 (Alexandre expression foxi1 gbx1 2451 was in

DEVELOPMENT compensated forbyexpanded hindbrain-specific domainsof early These resultsshow thatthecompletelossofRAsignalingimpairs complete lossorectopicactivation of RAsignaling(Fig.3M-O). L). Bycontrast,weobserved nochangein misexpressed inastripesurroundingtheanteriorneuralplate(Fig.3J- views withanteriortowards thetop.Scalebar:90 domain isunaffected byloss(N)orexcess(O)ofRAsignaling.Dorsal preplacodal domain(L).( expression withintheentire application of20nMRAleadstoectopic from control embryos(J)afterRA-signalingdepletion(K),whereas the nM RA-treated (I)embryos.( 20 embryos depletedofRAsignaling(H)compared withcontrol (G)or ( reduced inanenlargedr4primordium atthethree-somite stage(E). gastrulation inembryosdepletedofRAsignaling(DEAB,B)andremains RA (C,F),r4-specific ( Fig. 3.LossandexcessofRAsignalingaffect different tissues. 2452 causes anexpansion of fgf8 1992; Kudoh etal.,2002).Subsequently, otherplacode-derived in embryostreatedwithateratogenic doseofRA(Marshalletal., in theseembryos,similartofate changesinthedeveloping neuralplate suggested thepossibilitythat preplacodaldomainisposteriorized Our observation that ectopic RAsignaling Lens developmentisdisturbed inthepresence of preplacodal ectoderm. G-I A-F ) Anenlargedr4-specific ) Incomparisontocontrols (A,D)ortoembryostreated with20nM expression atlaterstages.ExcessRAsignaling,ontheotherhand, fgf3 RESEARCH ARTICLE expression inthedeveloping hindbrain,whichcannotbe fgf3 foxi1 M-O expression isdelayedbytheendof foxi1 fgf8 is misexpressed inRA-treated embryos ) Expression of J-L expression domainisalsopresent in ) expression throughouttheentire foxi1 expression isindistinguishable dlx3b dlx3b ␮ in thepreplacodal expression afterthe m. fgf3 and not shown). Bycontrast,expression of expression was unaffected inthepresence of10or20nMRA(data anterior pituitaryplacodes(Duttaetal.,2005),andwefoundthat its pitx3 expression domains(datanotshown). Atearly segmentation stages, treatment, specifically inthelensplacodewithout affecting other 2005), corroboratedourresults,showing expression changes,afterRA the pituitaryplacodeat24hpfinwild-typeembryos(Duttaet al., expressed intheventral andposteriordiencephalon,inthelens expression inthedeveloping hindbrain,wehypothesized thatthe because lossofRAsignalingled toasignificant reductionin In zebrafish, Fgf3and Fgf8arenecessaryforoticinductionand, primarily duetoFgf8signaling Residual oticinductionin RA-depletedembryosis leads toalossofthelens. amounts ofRAimpairlensplacodeformation,whichsubsequently control embryos(Fig.4G-I).Together, ourresultsshow thatincreasing embryos treatedwith10or20nMRA,respectively, incomparisonto marker (Thisseetal.,2001),was downregulated oralmostabsentin connexin 44.1 or 20nMRAshowed severe downregulation ornoexpression of changed. Incomparisontocontrolsat24hpf,embryostreatedwith10 which isexpressed onlyintheocularlens(Casonetal., 2001),was top. Scalebar:30 and dorsaltowards thetop.(D-I)Dorsalviewswithanteriortowards the pax6b evident atpreplacodal stages(five-somitestage,5S),asindicatedby wild-type embryos.( completely lostaftertheapplicationof10or20nMRA,compared with and ( Fig. 4.Increased RAsignalingcompromises lensspecification. a lens(Fig.4A-C).Consistentwiththis,expression of with 20nMRAcompletelyfailed toshow any morphologicalsignof with 10nMRAdeveloped amuchsmallerlensandembryostreated (data notshown). Comparedwithcontrolsat50hpf,embryostreated lens, allofthesestructuresformedinembryostreatedwith20nMRA lens, trigeminal,laterallineandepibranchialplacodes.Exceptforthe molecular markers fortheformationofanteriorpituitary, olfactory, structures mightbeaffected. To examine thispossibility, wetested A-F sesdbt ymrhlg t5 p (DICmicroscopy, A-C) ) Assessedbothbymorphologyat50hpf cx44.1 expression defines anequivalence domainforthelensand labeling. (A-C)Lateralviewsofliveeyeswithanteriortotheleft expression at24hpf(D-F),thelensisreduced insizeor , respectively (Fig.4D-F).Labelingfor ␮ m forA-C;50 G-I ) Compromised lensspecificationisalready ␮ m forD-F;35 pax6b Development 134(13) , aspecific lensplacode ␮ m forG-I. pitx3 connexin 44.1 , whichis fgf3 ,

DEVELOPMENT otic specification in pax8 hsp:fgf8 induction dependsprimarilyonFgf8. hypothesis that,intheabsenceofRAsignaling,residualotic no effect onoticvesicle size(datanotshown), supportingour hindbrain. Furthermore,lossofRAsignalingin consistent withthedelayedandreducedexpression of combined lossof alone ledtoreduced already evident atpreoticstages.Lossof Analysis of fgf8 signaling orin comparison tocontrols,oticvesicles intheabsence ofRA 10 presence ofsomeresidualoticcellsin remaining oticinductionisdependentprimarilyon Retinoic acidandoticfate Dorsal viewswith anteriortowards thetop. Scalebar:35 late gastrulationstages(F)leadsto theformationofmuchlargeroticvesiclesincomparisontotheirnon-transgenic siblings of Fig. 6.Misexpression of comparison tonon-transgenicembryos (A).( maintained. ( fgf8 only oneotolith(Fig.5A-C).Bycontrast,lossofRAsignalingin vesicle (Fig.5D).Nevertheless, the oticmarker n =25 outof25 fgf8 fgf8 mutants producedadifferent andhighlypenetrantphenotype mutants, RA-depletedorcontrolembryos(Fig.5E-H). (Fig. 5I-L).Ourobservation ofsomeindicationsresidual at theendofgastrulation(D),ascan heat-shockedtransgenic mutants) depletedofRAsignalingcomparedtountreated embryos depletedofRAsignaling (H)donotshowanincrease inoticcellsbutratherresemble non-transgenicRA-depletedsibli ( A pax8 , fgf8 B Preotic ) fgf8 fgf8 expression showed thatthisphenotypewas mutants arereducedinsizeandtypicallyform fgf8 pax8 mutants); embryosnever formedanotic and RAsignalingcausedasevere lossof fgf8 pax8 mutants depletedofRAsignalingis expression inthepreoticregion, but at theendofgastrulationrescues oticinductioninRA-depletedembryos,buttherescue isnot expression isexpandedintransgenic fgf8 C , D fgf8 mutants ( ) Reducedpreotic fgf3 or RAsignaling stm ␮ revealed the mutants had n m. =10 outof fgf3 fgf8 in the pax8 . In hsp:fgf8 hsp:fgf8 expression inRA-depleted embryos (C)canberescued bythemisexpression In embryosdepletedofRAsignaling,oticvesicles were only slightly formation ofenlarged oticvesicles was impaired(Fig.6C,D,G,H). stages inembryosdepletedofRAsignaling,but thesubsequent expression domainaftermisexpression of al., 2007).Bycontrast,wealsoobserved anexpanded pathway andmuchlarger oticvesicles formed(Fig.6E,F)(Hanset Subsequently, many morecellsembarked ontheoticdevelopment non-transgenic siblings(Fig.6A,B)(Hansetal.,2007). Misexpression of temperature-inducible allows ustoexpress activate ectopicFgfsignaling,weusedastabletransgeniclinethat we hypothesizecouldberescuedbyectopicFgfsignaling.To fgf3 suggests thatreducedoticinductioncausedbythedelayedonsetof to initiateoticfate isnotimpairedinRA-depletedembryos.This Our resultsindicatethatcompetenceprovided byFoxi1 andDlx3b maintained inRA-depletedembryos Otic inductioncanberescued butisnot pax8 thatof induction andcausedexpanded marker expression, suchas embryos (B)aftermisexpression of embryos withnormalRAsignaling (B).( expression isresponsiblefortheobserved oticphenotype,which , pax2a 35 top. (D-L)Dorsalviewswithanteriortowards thetop.Scalebar: live oticvesicleswithanteriortotheleftanddorsaltowards the 5S), asdetectedbylabelingwith evident asearlythepreplacodal stages(five-somitestage, size reduction in ( embryos, inwhichmore oticcellsare present. (H) compared to only residual oticcellsin (A). ( mutants depletedofRAsignaling(D),incomparisontocontrols (B) andin microscopy), oticvesiclesare reduced afterthedepletionofRA signaling. Fig. 5.Fgf8mediatesoticinductionintheabsenceofRA ␮ E-H m. and ) Labelingwith fgf8 sox9a fgf8 ( A-D fgf8 mutants (C),andare completelylostin ) Assessedbymorphologyat50hpf(DIC at lategastrulationstagesledtoectopicotic , withinthepreoticregion incomparisonto fgf8 fgf8 uniformly underthecontrolofzebrafish hsp70 mutants depletedofRAsignalingis mutant (G),RA-depleted(F)orcontrol (E) stm fgf8 promoter (Hansetal.,2007). at 22hpfreveals thepresence of fgf8 mutants depletedofRAsignaling RESEARCH ARTICLE pax8 at theendofgastrulationin E , F ). Misexpression of . (A-D)Lateralviewsof fgf8 (E).( at lategastrulation G , H I-L ) Transgenic ) Oticvesicle ngs (G). fgf8 fgf8 2453 pax8 at

DEVELOPMENT towards thetop.Scalebar:35 comparable toRA-depletedembryos(L).(A-G)Lateralviewswithanteriortheleftanddorsaltowards thetop.(H-O)Dorsalv size reduction isnotassevere asinembryosdepletedofRAsignaling(L).LossWnt8bfunction reduced earsize(labeledwith midbrain-hindbrain boundaryandinr3,butnotr5.( dorsal halfoftheotocystat22hpfislostinembryosdepletedRAsignaling.( single mutants,combinedloss of detected inthemidbrain-hindbrainboundary, inr3andr5(H),whereas Semicircular canal formation,however, whichwas severely double mutants),similartoRA-depleted embryos(Fig.7A-E). vesicles thatformedonly oneotolith( Hopkins, 2001).Incomparison toeitherwildtypeor maintenance ofoticfate is compromised(Fig.7A,D)(Sunand 2001), oticvesicles arereduced insizeatlaterstagesindicatingthat is unaffected atearlystagesinthesemutants(SunandHopkins, into anr4identity(Hernandezetal.,2004).Althoughoticinduction region ofthedeveloping hindbrainislostandpartiallytransformed Hernandez etal.,2004).Instrong expression domainandofr5coincide(SunHopkins,2001; manner atearlysegmentation stages.Theanteriorbordersofthe posterior hindbrainandanteriorspinalcordinanRA-dependent The homeoboxgene of hypothesized thatthesourcemightbelocatedwithinr5-r7.Analysis to theexpansion ofr3-r4accompaniedbylossr5-r7,we required tomaintainoticfate and,becauselossofRAsignaling led Our resultssuggestedthatanFgf-independentmechanismis Wnt8b isrequired tomaintainoticfate maintained intheabsenceofRAsignaling. gastrulation stages.However, oticfate cannotbeproperly embryos, aftertheectopicactivation ofFgfsignalingatlate region canundergo ectopic oticinduction,similartocontrol demonstrate that,intheabsenceofRAsignaling,cellspreotic comparison tocontrolembryos.Taken together, ourresults shown), but, ingeneral,oticspecification was always reducedin of and posteriorlytotheendogenousoticvesicle aftermisexpression frequently observed smallectopicoticvesicles locatedanteriorly they werestillmuchsmallerthaninuntreatedcontrols. We also stages thaninnon-transgenicsiblingsdepletedofRAsignaling,and bigger afterectopicactivation ofFgfsignalingatlategastrulation fgf3 reduced in Fig. 7.Rhombomere 5-specificWnt8bfunctionisrequired forthemaintenanceofoticfate. 2454 fgf8 fgf3;tcf2 and at lategastrulationstagesinRA-depletedembryos(datanot RESEARCH ARTICLE tcf2 fgf3 double mutantsconfirmed thishypothesis(Fig.7A-E). together results inoticvesiclesthatformonlyoneotolith,asisobservedRA-depletedembryos(B).( (C) and tcf2 tcf2 (D) singlemutantscompared towild-typeembryos(A),butnotasmuchinRA-depleted(B).( (previously stm ␮ m forA-EandH-O;20 at 22hpf),similartothesizeobservedin tcf2 tcf2 vhnf1 n and mutant alleles,ther5-r6 =10 outof10 ) isexpressed inthe fgf3 resulted inotic K-N ␮ m forF,G. ) InactivationofWnt8binwild-typeembryosbymorpholinoinjection(MO,N)leadstoa tcf2 fgf3;tcf2 or fgf3 tcf2 tcf2 fgf3 affected in impaired inRA-signaling-depletedembryos,was lessseverely Wnt8b functionin morpholino injectionintowildtype.Furthermore,wecompromised to maintainoticfate, wecompromisedWnt8bfunctionusing expression specifically inr5(Fig.7H-J).To testwhether both somitogenesis onwards (Kelly etal.,1995).Compared tocontrols, candidate becauseitisexpressed stronglyinr1,r3andr5 frommid- suggesting thatWntsignalingmaybeaffected. Wnt8bisalikely was completelylostinRA-depletedembryosat22hpf(Fig.7F,G), later intheoticvesicle forexpression of Wnt signalingisrequiredearlyforthemaintenanceofoticfate and the zebrafish oticvesicle. Recentstudiesinmouse have shown that (Romand, 2003),RAsignalingisinvolved inpatternformationof signaling-depleted embryos,whichhave defectsin wnt8b found thatexpression of of adorsalfate (Ohyamaetal.,2006;Riccomagno2005).We depleted embryos. mimics thesizereductionof the oticvesicle observed inRA- expression andlossofWnt8b functiontogether compromised Fgf3 derived factor Wnt8bfromr5maintainsoticfate, andthat embryos. Together, ourresultsdemonstrate thatthehindbrain- might partlycompensateforthe lossof depleted embryos(datanotshown), andexpression of the expansion ofr3-r4,oticvesicles werelocated closertor3inRA- reduction inoticvesicle size(datanotshown). However, becauseof RA signalingandWnt8bfunctiontogetherresultedinaslightfurther in RA-depletedembryos( an additive effect, producingsmalleroticvesicles similartovesicles N). However, compromisedWnt8bfunctionin not asprofoundinembryosdepletedofRAsignaling(Fig. 7K- to theformationofsmalleroticvesicles, but thesizereductionwas expression (Fig.7K-O).LossofFgf3orWnt8bfunctionaloneled mutants (I)andRA-depletedembryos(J)show H-J mutants (M),compared tountreated wildtype(K);however, the tcf2 ) Incontrol embryosat22hpf, together mimicstheoticvesicle sizereductionofRA- mutants andRA-depletedembryosshowed alossof fgf3;tcf2 double mutants,indicatingthat,similartomouse fgf3 fgf3 ( A-D dlx3b mutants totestwhetherlossof n mutants ( =14 outof18)(Fig.7L,O).Depletion ) Assessedbymorphology, oticvesiclesare in thedorsalportionofoticvesicle O wnt8b F produces reduced oticvesicles ) , G dlx5 ) Expression of expression canbe wnt8b Development 134(13) and fortheacquisition wnt8b fgf3 iews withanterior from r5inthese mutants showed fgf3 in the E ) Lossofboth dlx3b wnt8b wnt8b and fgf3 in the wnt8b wnt8b in r3 acts and

DEVELOPMENT provided bythegraft,because ectopic oticinductionmightalsobenefit fromectopic Fgfsignaling inducing signalsbecauseofectopicactivation of the competenceofanteriorpreplacodalcellstorespondotic- RA producedbythegraftedventral andlateralgermringincreases (Begemann etal.,2001;Grandel2002).Thus,weproposethat ventral andlateralgermringbut notdorsallyintheembryonicshield genes, and,duringzebrafish gastrulation, activity ofaldehydedehydrogenases,whichareencodedbyAldh vesicles (Woo and Fraser, 1997).RAisgeneratedbytheenzymatic into theprospective forebrainregion never inducesectopicotic of ectopichindbraintissue,thedirecttransplantation shield) doesnot.Althoughgermringgraftsalsoleadtotheinduction whereas thetransplantationofdorsalgermringtissue(theembryonic prospective forebrainregion can induce ectopicoticvesicles, 1997). Transplantation ofventral andlateralgermringtissueintothe explanation forthefindings ofWoo andFraser(Woo andFraser, foxi1 throughouttheentirepreplacodaldomainprovides aconsistent et al.,2006). required toobtainthesameeffect onwild-typeembryos(Hernandez sufficient toposteriorizecyp26a1 mutants,whereas200nMRAis with thisinterpretation,arecentstudyhasshown that5nMRAis al., 2004;Kudoh etal.,2002;Hernandez2006). Consistent neural platebut notinthepreplacodaldomain(Dobbs-McAuliffe et enzymes oftheCyp26classthatareexpressed within the anterior difference ispresumablyduetothepresenceofRA-degrading whereas ateratogenicdoseofRAaffected bothtissues.This the preplacodaldomain,but notwithintheanteriorneuralplate, changes aftertheapplicationof20nMRAwereobserved onlyin anterior endinRA-treated severely reducedtoasmall,residualdomainofexpression atthe expression observed in20nMRA-treatedwild-typeembryoswas et al.,2003;Nissen2003),wefoundthattheenlarged showing thatpreotic domain ofpreotic sufficient toproduceectopicexpression of do notinterferewiththepatterningofanteriorneuralplateare Our resultsdemonstratethatmuchlower concentrations ofRAthat enlarge thepreoticexpression domainof completely transformforebrainandmidbrainintohindbrain Kudoh etal.,2002).Furthermore,teratogenicdosesofRA presumptive fore-andmid-brainstructures(Marshalletal.,1992; expansion oftheposteriorhindbrainatexpense ofthe which leadstotransformationofr2-r3intor4-r5andthe of theanteriorneuralplateinaconcentration-dependentmanner, Treatment ofvertebrate embryoswithRAcausesaposteriorization competence endogenous RAisnotrequired forotic cells torespond tootic-inducingsignals,but Ectopic RAsignalingincreases thecompetenceof DISCUSSION Retinoic acidandoticfate effect ontheexpression of show thatlossofRA signalingduringzebrafish gastrulationhasno al., 2004;Reiferset1998;Cao2004). highest levels onthedorsalside(Fürthauer etal.,1997;Fürthauer expressed inadorsoventral gradientinthezebrafish germring,with expression mightratherreveal aregulatory mechanismemployed events ofotic induction.Theobserved effect ofexcess RAon hence, weconcludethatRAsignaling isnotrequiredfortheearly RA-depleted embryosisnotdue toaneffect onFoxi1 level and, Our observation thatexcess RAleadstoectopicexpression of Although RAissufficient toinduce pax8 pax8 expression. Consistentwithrecentreports expression dependsonFoxi1 (Solomon foxi1 foxi1 fgf3 . Thus,impairedoticinduction in mutants (Hansetal.,2007).Fate , fgf8 foxi1 pax8 aldh1a2 , foxi1 fgf17b expression, ourdataalso (Phillips etal.,2001). , whichexpands the foxi1 is expressed inthe and . Theobserved fgf24 are all foxi1 pax8 but nothing isknown about itsexpression laterin development, prior expressed duringearlystages ofoticinduction(Phillipsetal.,2004), downstream ofaminimal promoterandfourLef-bindingsites,isnot TOPdGFP It hasbeenreportedthat,in zebrafish, theWntreportergene is unknown whether Wntsignalingplaysthesameroleinzebrafish. directing preoticcellstoanotic fate (Ohyamaetal.,2006).Sofar, it that Wntsignalingmediatesaplacode-epidermisfate decision by expansion ofoticfate attheexpense ofepidermal fate, suggesting canonical WntsignalinginpreoticPax2-positive cellsleadstothe (Ohyama etal.,2006).Conversely, constitutive activation of of oticfate, andoticvesicles aresignificantly reducedinsize positive cellsleadstoanexpansion ofepidermalfate attheexpense disruption ofthecanonicalWntsignalingpathway inpreotic Pax2- induction but priortotheformationofplacode. Furthermore, preotic region inasubsetofPax2-positive cellsafterFgf-dependent signaling was detectedusingaTcf/Lefreporterconstruct within the maintain oticfate (Ohyamaetal.,2006).Inthelatterstudy, Wnt that WntsignalingisrequiredafterFgf-dependentinduction to 2001; LégerandBrand,2002),arecentstudyinmouseshows (Niederreither etal.,1999;Whitfield etal.,2002;Phillips Fgf3 hasbeenproposedfromwork inbothmammalsandzebrafish expression. Reducedoticinductionduetoweaker expression of compromised maintenanceofoticfate duetolossof otic inductionduetodecreased RA signalingformedsmalloticvesicles becauseofcompromised changes inhindbrainpatterning.We foundthatembryosdepletedof caused bylossofRAsignalingareasecondaryconsequence Our analysisfurtherdemonstratesthatdefectsinoticspecification Wnt signalingduringoticinduction inducing r4fromthepreoticregion. embryos, ther3primordiumisgreatlyexpanded, displacing the impaired oticinduction,because,inRA-signaling-depleted positioning ofinducingandinducedtissuemightalsocontribute to developing hindbrainafterthelossofRAsignaling.Incorrectspatial respond totheendogenousFgf3andFgf8emanatingfrom after exposure toahighuniformdoseofFgf8but areunableto control embryos.Thus,cellsarestillcompetenttoadoptoticfate activation ofFgfsignalingatlategastrulationstages,similarto preotic region canundergo ectopicoticinductionafter here, weshow thateven intheabsenceofRAsignaling,cells segmentation stages(Hansetal.,2007).Usingthesameapproach zebrafish occursbetweentheendofgastrulationandearly hsp70 uniformly underthecontrolofzebrafish temperature-inducible experiments withastabletransgeniclinethatexpresses stage (Phillipsetal.,2001;LégerandBrand,2002).Basedon morpholino injectionseverely reduces and not becompensatedforbyanexpanded hindbraindomainof RA-signaling-depleted embryosdelayedoticinductionandcould zebrafish. Impaired during therelatively shorttime-window foroticinductionin tissues. however, whether al., 2001;Market2004;Romand,2003).Itiscurrentlyunknown, been implicatedinthedevelopment ofallthesetissues(Drageret (Thisseetal.,2005),andRAsignalinghas septum oftheoticvesicle expressed intheretina,pharyngealpouchesanddorsal during otheraspectsofdevelopment. Inzebrafish at48hpf, Our resultsfurtherindicatethatearly fgf8 promoter, werecentlyproposedthatoticinductionin expression atlaterstages.Consistently, depletionof , atransgeneconsistingof GFP-codingsequence foxi1 fgf3 expression dependsonRAsignalinginthese expression inthedeveloping hindbrainof fgf3 RESEARCH ARTICLE pax8 expression andbecauseof fgf3 expression atthetailbud expression iscrucial foxi1 fgf3 wnt8b 2455 fgf8 fgf3 by is

DEVELOPMENT addition to wnt3a maintenance afterFgf-dependentinduction.Expressionof is very likely thatseveral Wntmoleculesareinvolved in oticfate difficult toaddresslaterWnt8afunctionduringoticdevelopment. It compromising oticinduction(Phillipsetal.,2004)andmakingit fgf8 al. (Ohyamaetal.,2006).However, properexpression of (Phillips etal.,2004),supportingthemodelproposedbyOhyama function bymorpholinoinjectionresultsingreatlyreducedotocysts anlagen (Kelly etal.,1995;Lekven etal.,2001).LossofWnt8a epiboly (8hpf),ORF2canbedetectedinr5-r6adjacenttotheotic complete open-readingframes,ORF1andORF2,and,at75% zebrafish, catenin orbytheexpression ofaconditionallyactivated formof Wnt signalingpathway inmousebyconditionalknockout of autonomous disruptionorconstitutive activation ofthecanonical formation oftheoticregion (ourunpublishedresults).Cell- that gene foundthatexpression oftheendogenousWntreporter previously induction canberescuedbut notmaintained.Furthermore,wehave for Wntsignaling,wefoundthat,inRA-depletedembryos,otic to theformationofoticplacode.Consistentwithasimilarrole 2456 later stages. also contribute tofewer ectopicoticcellsinRA-treatedembryosat organs werepresumablyintactunderourexperiments, whichmight Furthermore, pathways to generateotherplacode-derived sensory is essentialforthemaintenance ofoticfates afterinduction. hindbrain boundarypresumably donotreceive Wntsignaling,which neural platefate, excess RAsignaling(20nM)didnotapparentlychangeanterior (Wilson andHouart,2004).Becauseour conditionstogenerate early neuralplateanteriortothefuturemidbrain-hindbrainboundary cells. Inwild-typeembryos,however, Wntsignalingisabsentinthe these RA-treatedembryos,maintainingthefate oftheectopic otic domains. Wntsignalingispresumablyalsoshiftedanteriorly in leads totheanteriorexpansion ofhindbrain posteriorized aftertreatmentwithateratogenicdoseofRA,which cells. Phillipsetal.showed thattheanteriorneuralplateisstrongly we observed onlyrandomlydistributed smallpatchesofectopicotic morphologically visibleoticvesicles attheanteriorlimitofhead, reported that20-30%ofRA-treatedembryosproduceectopic (Phillips etal.,2001).However, incontrasttoPhillipsetal.,who neural plateborder, similartotheresultspublishedbyPhillipsetal. led tothemassive ectopicexpression of embryos atlaterstages.Ourtreatmentofwith20nMRA responsible forthereducednumberofoticcellsinRA-treated otic fate maintenance. will benecessarytoaddresstheroleofparticularWntmoleculesin we have shown for (Lekven etal.,2003;Buckles2004;Lecaudey etal.,2006),as and defects of results insmalleroticvesicles thatmimicthesizeandpatterning mutants andthatinactivation ofWnt8bbymorpholinoinjection signaling isstronglydeficient intheposteriorhindbrainof expression inr5isthesource,consistentwithfindings thatWnt8b et al.,2006).Ourdata,ontheotherhand,indicatethat propose that catenin withinpreoticPax2-positive cellsledOhyamaetal.to Absent orinappropriateamountsofWntsignalingmightalsobe wnt3a axin2 axin2 sdsubdinthehindbrainoftheseembryos,thus is disturbed and RESEARCH ARTICLE is severely reducedinthecaudalhindbrainof mafb wnt10b wnt8a wnt8a is absentduringtheinitialstagesofoticinductionbut is expressed withinthepreoticregion priortothe Wnt8a mutants (M.Brand,personalcommunication).In pax8 and encodes abicistronicmessageencodingtwo wnt8b has beenreportedinthezebrafish hindbrainin from r4isthesourceofWntsignaling(Ohyama -positive cells anteriortothefuturemidbrain- wnt8b . Conditionalandcombinatorialknockout , and,interestingly, expression of pax8 fgf3 around theanterior and fgf8 tcf2 expression fgf3 mutants wnt8b wnt1 mafb wnt1 and ␤ ␤ - - , gene replacementinthemousehasshown that (Eberhard etal.,2000).Pax2 ishighlyrelatedtoPax5 andPax8, and via interactionwithco-repressorsoftheGrouchoproteinfamily Pax6 canbeconverted fromtranscriptionalactivators torepressors and Watanabe, 2005).Otherstudieshave shown that both Pax2 and the di-mesencephalicboundary(Schwarz etal.,2000;Nakamura specification ofmammalianeye territoriesandinformationof the paired-domain proteinsPax2 andPax6 hasbeenimplicatedin ectopic expression of posteriorization ofthepreplacodaldomain,but ratherduetothe lens tissueisnotduetomisexpression of et al.,2006).However, itisalsolikely thatthereductionorlossof pax6b 20,000 zebrafish genesshowed thatonlyseven genes,including with thisinterpretation,microarrayanalysiscovering approximately treated embryosmightbeinvolved inlensfate repression.Consistent region ofwild-typeembryosandexpanded Foxi1 expression inRA- promoting oticfate inresponsetoFgfsignaling,Foxi1 inthepreotic fate inprospective non-lensdomains(Bailey etal.,2006).By territory andthatsubsequentdevelopment requiresrepressionoflens tissue, implyingthatlensfate isadefault stateofthepreplacodal shown thattheentirepreplacodaldomainisinitially specified aslens sensory organs, except forthelens.Interestingly, itwas recently observe any expansion oftheotictissueatexpense ofother foxi1 We foundthattheapplicationof20nMRAledtoexpression of Ectopic RAsignalingandlensplacodeinduction Bailey, A.P., Bhattacharyya,S.,Bronner-Fraser, M.andStreit, A. Alvarez, Y., Alonso,M.T., Vendrell, V., Zelarayan,L.C.,Chamero, P., Theil,T., Alexandre, D.,Clarke,J. Oxtoby, E.,Yan, Y. L.,Jowett, T. andHolder, N. Akimenko, M.-A.,Ekker, M.,Wegner, J.,Lin,W. andWesterfield, M. References http://dev.biologists.org/cgi/content/full/134/13/2449/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Feodor Lynen fellowship oftheAlexandervonHumboldtfoundation. supported byNIHgrantsDC04186andHD22486.S.H.isarecipient ofa of the manuscript; andValdimarsson Gunnar materials.for This work was Michael Brandforsharingunpublishedresults; LisaMavesforcriticalreading We wishtothankSandraBrown andLauren Clanceyfortechnicalassistance; Bouchard, M.,Pfeffer, P. andBüsslinger, M. Begemann, G.,Schilling,T. F., Rauch,G.J.,Geisler, R.andIngham,P. W. Barald, K.F. andKelley, M.W. among thesefactors. other fates willhelpclarifythefunctionalsimilaritiesanddifferences of Foxi1 andPax2 orPax8 inoticinductionandtherepressionof al., 2004;Mackereth etal.,2005).Furtherinvestigation oftheroles system andthezebrafish innerear(Bouchardetal.,2002;Hans for Pax2 andPax8 duringdevelopment ofthemouseurogenital 2000). Combinedandredundantgenefunctionhasalsobeenshown biochemically interchangeable(Pfeffer etal.,1998;Bouchard substitute for promotes olfactoryidentity. specification istheground stateofallsensoryplacodes,from whichFGF homeobox codeforthehead. Combinatorial expression of three zebrafishgenesrelated to 130 Requirements forFGF3and FGF10 duringinnerearformation. Bösel, M.R.,Shigeaki,K.,Maconochie,M.,Riethmacher, D. et al. phenotype. mandibular arch neuralcrest andphenocopiesaretinoic acid-induced (1996). Ectopicexpression of Hoxa-1inthezebrafishaltersfateof 3094. thehindbrain. mesodermal signalsthatpattern (2001). Thezebrafishnecklessmutation reveals arequirement forraldh2in wrinkles inthedevelopmentofinner ear. , 6329-6338. within theentirepreplacodaldomain.However, wedidnot , aresignificantly repressedafteroverexpression ofFoxi1 (Yan Development Pax2 , indicatingthatPax2, Pax5 andPax8 proteinsare 122 pax8 Dev. Cell , 735-746. J. Neurosci. 20) From placodetopolarization: new (2004). . Reciprocalinhibitionbetweenthe 11 , 505-517. 14 (2000). Functionalequivalence of , 3475-3486. Development Development Development 134(13) Pax5 131 Distal-less can functionally foxi1 Development , 4119-4130. 128 (2006). Lens , 3081- or toa : partofa (2003). (1994).

DEVELOPMENT Hernandez, R.E.,Putzke,A.P.,Hernandez, Myers,J.P., Margaretha, L.andMoens,C.B. R.E.,Rikhof,H.A.,Bachmann,andMoens,C.B. Hernandez, Hans, S.,Christison,J.,Liu,D.andWesterfield, M. Hans, S.,Liu,D.andWesterfield, M. Grandel, H.,Lun,K.,Rauch,G.J.,Rhinn,M.,Piotrowski, T., Houart,C., Gavalas, A.andKrumlauf,R. Fürthauer, M.,Van Celst,J.,Thisse,C.andB. Fürthauer, M.,Thisse,C.andB. Fritzsch, B.F., Barald,K.F. andLomax,M.I. Brown, S.T., Martin,K.andGroves, A.K. Brand, M.,Heisenberg,C.P., Jiang,Y. J.,Beuchle,D.,Lun,K.,Furutani-Seiki, Bouchard, M.,Souabni,A.,Mandler, M.,Neubüser, A.andBusslinger, M. Herzog, W., Sonntag,C.,vonderHardt, S.,Roehl,H.H.,Varga, Z. M.and Ellies, D.,Stock,Hatch,G.,Giroux, G.,Weiss, K. and Ekker, M.E. Ekker, M.E.,Akimenko,M.-A.,BreMiller, R.andWesterfield, M. Eberhard, D.,Jiménez,G.,Heavey, B.andBusslinger, M. Retinoic acidandoticfate Dutta, S.,Dietrich,J.E.,Aspöck,G.,Burdine, R.D.,Schier, S.,Westerfield, Dupé, V., Ghyselinck,N.B.,Wendling, O.,Chambon,P. andMark,M. Dupé, V. andLumsden,A. Drager, U.C.,Li,H.,Wagner, E.andMcCaffery, P. Dobbs-McAuliffe, B.,Zhao,Q.andLinney, E. Buckles, G.R.,Thorpe,C.J.,Ramel,M.andLekven,A. Couly, G.F., Coltey, P. M.andLeDouarin,N. Cason, N.,White,T. W., Cheng,S.,Goodenough,D.A.andValdimarsson, G. Cao, Y., Zhao,J.,Sun,Z.,Postlethwait,J.andMeng,A. for hindbraindevelopment. (2006). Cyp26enzymesgeneratetheretinoic acidresponse necessary pattern hindbrain developmentinzebrafish. andlocalFGFsignalstodirectintegrates globalRApatterning posterior 5. induction requires competenceprovided byFoxi1andDlx3b. patterning. transcription factors. synergistically inoticspecification,downstream oftheFoxi1andDlx3b induce apectoralfinbud. theanterior-posteriorsegmentation stagestopattern axisoftheCNSandto Retinoic acidsignallinginthezebrafishembryoisnecessaryduringpre- Sordino, P., Kuchler, A.M.,Schulte-Merker, S.,Geisler, R.etal. 2853-2864. controls ofthezebrafishembryo. thedorsoventralpatterning 4264. dorsoventral patterning ofthezebrafishgastrula. dorsoventral patterning Popper andR.Fay),pp.80-145.NewYork: Springer-Verlag. vertebrate ear. In expression ofzebrafish patterns Relationship betweenthegenomicorganizationandoverlappingembryonic of theGroucho family. Transcriptional repression byPax5(BSAP)through interactionwithcorepressors Mech. Dev. regulate retinoic acidproduction anddegradationinthezebrafishembryo. induction. midbrain andhindbrain. Mutations inzebrafishaffecting theformationofboundarybetween M., Granato,Haffter, P., Hammerschmidt,M.,Kane,D.etal. 2970. (2002). NephriclineagespecificationbyPax2andPax8. 127 the transcriptionfactorsPax2andPax5inmousedevelopment. adenohypophysis. required forearlyspecificationandsubsequent survivalofthezebrafish Hammerschmidt, M. embryos. Regional expression ofthree homeoboxtranscriptsinthe innerearofzebrafish anterior pituitaryplacode. M. andVarga, Z.M. 126 caudal hindbrain,pharyngealarches andotocystinthemouse. Key roles ofretinoic acidreceptors ofthe alphaandbetainthepatterning responses toretinoic acidsignalling. 579-587. synthesis andbreakdown inthedevelopingmouseretina. 429. Mech. Dev. Combinatorial Wntcontrol ofzebrafishmidbrain-hindbrainboundaryformation. in highervertebrates:astudyquail-chickchimeras. connexin44.1: azebrafishlensgapjunctionprotein. (2001). Molecularcloning,expression analysis,andfunctionalcharacterizationof Biol. fgf17b , 3707-3713. , 5051-5059. 271 , anovelmemberofFgffamily, zebrafishembryos. helpspatterning , 130-143. Neuron Curr. Top. Dev. Biol Curr. Opin.Genet.Dev. 121 121 , 339-350. , 437-447. Development of the Auditory System Development oftheAuditory 9 Development , 27-35. Development (2005). pitx3definesanequivalencedomainforlensand (2004). Fgf3signalingfrom theventraldiencephalonis EMBO J. Development (2001). Hindbrain patterning involvesgraded Hindbrainpatterning (2001). Development Development Development . (2000). Retinoidsignallingandhindbrain 57 19 dlx 131 , 115-149. , 2292-2303. 131 10 genes. , 3681-3692. Development Development (2004). Pax8andPax2afunction , 380-386. 123 , 5091-5102. (1997). Arole forFGF-8inthe 129 132 1 , 179-190. (2003). Molecularbasisofinnerear Genomics 34 , 2851-2865. , 1579-1590. (1997). Earlyembryologyofthe , 177-187. (2004). Feedbackmechanisms (1993). Thetripleoriginofskull Development 128 131 (2001). Retinoicacid Dev. Dyn. (2007). Fgf-dependentotic 45 Development (2004). Fgfsignalling , 2199-2208. , 4511-4520. (ed. E.W. Rubel,A.N. Genes Dev. , 580-590. Prog. BrainRes. (2000). BMC Dev. Biol. Development (2004). 221 Development 124 Development , 238-247. (2004). vhnf1 (1992). , 4253- 16 117 (2004). (2002). (1996). , 2958- (1997). , 409- (1999). 131 Dev. 131 7 , , , Lekven, A.C.,Buckles,G.R.,Kostakis,N.andMoon,R.T. Lekven, A.C.,Thorpe,C.J.,Waxman, J.S.andMoon,R.T. Li, Y., Allende,M.L.,Finkelstein,R.andWeinberg, E.S. Ladher, R.K.,Wright, T. J.,Moon,A.M.,Mansour, S.L.andSchoenwolf,G. Ladher, R.K.,Anakwe,K.U.,Gurney, A.L.,Schoenwolf,G.C.andFrancis- Léger, S.andBrand,M. Lecaudey, V., Ulloa,E.,Anselme,I.,Stedman,A.,Schneider-Maunoury, S. Kil, S.H.,Streit, A.,Brown, S.T., Agrawal,N.,Collazo,A.,Zile,M.H.and Kelly, G.M.,Erezyilmaz, D.F. andMoon,R.T. Kudoh, T., Wilson,S.W. andDawid,I.B. Kudoh, T., Tsang, M.,Hukriede,N.A.,Chen,X.,Dedekian,Clarke,C.J., Niederreither, K.,Vermot, J.,Schuhbaur, B.,Chambon,P. andDollé,P. Kim, S.H.,Shin,J.,Park,H.C.,Yeo, S.Y., Hong,S.K.,Han,S.,Rhee,M.,Kim, Jowett, T. andYan, Y. L. Niederreither, K.,Subbarayan,V., Dollé,P. and Chambon,P. Nica, G.,Herzog,W., Sonntag,C.andHammerschmidt,M. Nakamura, H.andWatanabe, Y. Kollmar, R.,Nakamura,S.K.,Kappler, J.A.andHudspeth, Kimmel, C.B.,Ballard, W. W., Kimmel,S.R.,Ullmann,B.andSchilling,T. F. Mackereth, M.D.,Kwak,S.J.,Fritz,A.andRiley, B. Maden, M. Lombardo, A.,Isaacs,H.V. andSlack,J.M. Liu, D.,Chu,H.,Maves,L.,Yan, Y.-L., Morcos, P. A.,Postlethwait.P. and Mark, M.,Ghyselinck,N.B.andChambon,P. Mahmood, R.,Kiefer, P., Guthrie,S.,Dickson,C.andMason,I. Maves, L.andKimmel,C.B. Marshall, H.,Nonchev, S.,Sham,M.H.,Muchamore, I.,Lumsden, A.and Maroon, H.,Walshe, J.,Mahmood,R.,Kiefer, P., Dickson,C.andMason,I. two zebrafishorthodenticle-related genesintheembryonicbrain. Dev. Biol. wnt10b functionredundantly atthezebrafishmidbrain-hindbrainboundary. mesoderm andneurectoderm patterning. wnt8 encodestwoproteins onabicistronic transcript andisrequired for 108. placode induction, maintenance and inner ear patterning. placode induction,maintenanceandinnerearpatterning. C. development. West, P. H. 4346. retinoic acidinposteriorizingtheneuralectoderm. study ofthezebrafish and Pujades,C. 603-613. deficient quail. oftheinnerearrevealedinduction andpatterning byastudyofvitamin-A- Groves, A.K. pathways. wnt8b share acommonactivitybutare involvedindistinctdevelopmental zebrafish embryos. development. Embryonic retinoic acidsynthesisisessentialforearlymousepostimplantation Endocrinol. expression screen inzebrafishembryogenesis. Kiang, A.,Schultz,S.,Epstein,J.Toyama, R.etal. Sci. USA Expression andphylogenyofclaudinsinvertebrateprimordia. 49 regionalization ofthemesencephalonandmetencephalon. 48 pit1 gastrulation inzebrafish. neuroectoderm byFrizzled8a-mediatedWnt8bsignallingduringlate patterning C. H.,Chitnis,A.B.andHuh,T. L. 310. (1995). Stagesofembryonicdevelopmentthezebrafish. system. genes inthemaintenanceofoticplacode. is required foroticplacodeinductionandplaysaredundant role withPax2 FGF-3 inXenopusdevelopment. 130 transcription factorsare required foroticplacodespecification. Westerfield, M. the developmentofbranchialarches. Development Multiple roles forFGF-3during cranialneuraldevelopmentinthechicken. zebrafish posteriorhindbrainbyretinoic acid. transformation ofrhombomeres 2/3intoa4/5identity. Krumlauf, R. and vesicle. (2002). Fgf3andFgf8are required togetherforformationoftheoticplacode , 229-244. (2005). FGF8initiatesinnerearinductioninchickandmouse. , 231-235. , 2213-2224. mutants lackthree pituitarycelltypesanddevelopsevere dwarfism. Nat. Rev. Neurosci. 98 15 (2002). Retinoidsignalinginthedevelopmentofcentralnervous Development , 10196-10201. 18 (2000). Identificationofsynergisticsignalsinitiatinginnerear Development , 172-187. 121 Science Nat. Genet. (1992). RetinoicacidaltershindbrainHoxcodeandinduces , 1196-1209. (2005). Distinctroles forhindbrainandparaxialmesoderminthe Dev. Biol. (2006). Role of the hindbrain in patterning theoticvesicle:A (2006). Roleofthehindbraininpatterning (2003). Fgf3andFgf8dependentindependent , 1399-1410. Trends Genet vhnf1 290 (2002). Fgf8andFgf3are required forzebrafishear (1996). Doublefluorescent insituhybridizationto 121 285 Development 129 , 1965-1967. 21 17 (2005). Dynamic and sequential patterning ofthe (2005). Dynamicandsequentialpatterning mutant. , 1787-1799. , 252-271. , 444-448. , 2099-2108. , 843-853. . Int. J.Dev. Biol 12 (2005). Isthmusorganizerand (2002). Specificationofananterior , 387-389. Dev. Biol. Curr. Opin.Genet.Dev. 129 (2002). Distinctroles forFgf,Wntand RESEARCH ARTICLE Dev. Cell , 4443-4455. (1998). Expression andfunctionsof Dev. Biol. Genome Res. Development (2004). Retinoicacidsignallingin 303 . (1995). Zebrafishwnt8and 42 , 134-143. Development , 1101-1107. 1 , 103-114. 285 Nature (2005). Zebrafishpax8 (2001). Agene (1994). Expression of Mech. Dev , 593-605. Dev. Dyn. (2003). Wnt1and 11 132 (2001). Zebrafish (2004). Zebrafish 14 Proc. Natl.Acad. (1999). , 1979-1987. 360 Development Int. J.Dev. Biol. , 591-598. , 371-382. Genes Dev. 129 (1995). Mech. Dev (2001). , 737-741. , 4335- . 203 119 Mol. (2000). , 253- 2457 , 91- 19 . ,

DEVELOPMENT Phillips, B.T., Storch, M.K.,Lekven,A.C.andRiley, B. Reifers, F., Bohli,H.,Walsh, E.C.,Crossley, P. H.andStainier, D.Y. R. Russo, J.E.,Hauguitz,D.andHilton, Riccomagno, M.M.,Takada, S.andEpstein,D.J. Phillips, B.T., Bolding,K.andRiley, B. Perz-Edwards, A.,Hardison, N.L.andLinney, E. Schwarz, M.,Cecconi,F., Bernier, G.,Andrejewski, N.,Kammandel,B., Romand, R. Ohyama, T., Mohamed,O.A.,Taketo, M.M.,Dufort,D.andGroves, A.K. Noden, D.M.andvandeWater, T. R. Nissen, R.M.,Yan, J.,Amsterdam, A.,Hopkins,N.andBurgess,S.M. Solomon, K.S.andFritz,A. Söllner, C.,Burghammer, M.,Busch-Nentwich,E.,Berger, J.,Schwarz,H., 2458 Pfeffer, P. L.,Gerster, T., Lun,K.,Brand,M.andBusslinger, M. for FgfbutnotWntinoticplacodeinduction. 365. redundant functionsrequired foroticplacodeinduction. Pharmacol. aldehyde dehydrogenase by4-(diethylamino)-benzaldehyde. Top. Dev. Biol. supporting roles ofShh. regulation ofinnerearmorphogenesisisbalancedbytheopposingand 125 maintenance ofmidbrain-hindbrainboundaryandsomitogenesis. Fgf8 sensory placodeformation. 3063-3074. dependency of Characterization ofthree novelmembersofthezebrafish gene expression intransgenicreporter zebrafish. Development territories byreciprocal transcriptionalrepression ofPax2andPax6. Wagner, M.andGruss,P. epidermis. (2006). Wntsignalsmediateafatedecisionbetweenoticplacodeand R. vandeWater andE.W. Rubel),pp.15-46.Amsterdam: Elsevier. and interactions.In Zebrafish integrity ofearandjaw. Development inthemouseembryo. Retinoic acidsynthesisandhindbrainpatterning by starmakerinotolithbiomineralization. Riekel, C.andNicolson,T. , 2381-2395. is mutatedinzebrafish RESEARCH ARTICLE foxi one (2003). Theroles ofretinoic acidduringinnereardevelopment. Development 37 127 127 , 1639-1642. 57 Pax5 , 261-291. , 75-85. , 4325-4334. modulates cellularresponses toFgfsignalingrequired forthe The BiologyofChangeinOtolaryngology and Development Genes Dev. 133 Pax8 (2000). Spatialspecificationofmammalianeye Development (2002). Concertedactionoftwo acerebellar (2003). Control ofcrystalsizeandlatticeformation , 865-875. on the 19 Pax2. 1 (1986). Thedevelopingear:tissueorgins 130 , 1612-1623. ( (1988). Inhibitionofmousecytosolic ace Science (2001). Zebrafish 129 , 2543-2554. mutantsandisrequired for ) , 3127-3136. ( Development noi Dev. Biol. function. ) 302 (2001). Retinoicacidmediated (2005). Wnt-dependent , 282-286. Dev. Biol. Pax2/5/8 229 (2004). Adirect role fgf3 131 Development Biochem. dlx (ed. R.J.Ruben,T. , 89-101. and , 923-931. paralogs in (1998). Development 235 family: fgf8 , 351- (1998). (2003). encode 125 Curr. , Yan, J.,Xu,L.,Crawford, G.,Wang, Z.andBurgess,S.M. Wright, T. J.andMansour, S.L. Woo, K.andFraser, S.E. Wilson, S.W. andHouart,C. Whitlock, K.E.andWesterfield, M. Song, J.andSlack,M. Whitfield, T. T., Riley, B.B.,Chiang,M.-Y. andPhillips,B. Thisse, C.,B.,Schilling,T. andPostlethwait,J.H. Thisse, B.,Fauny, J.-D.,Hamadbachir, A.andThisse,C. Thisse, B.,Pflumio,S.,Fürthauer, M.,Loppin,B.,Heyer, V., Degrave,A., Sun, Z.andHopkins,N. Solomon, K.S.,Kwak,S.-J.andFritz,A. Solomon, K.S.,Kudoh,T., Dawid,I.G.andFritz,A. White, J.C.,Highland,M.,Kaiser, M.andClagett-Dame, Westerfield, M. Torres, M.andGiráldez,F. chromosomes andstablyremodels chromatin structure. forkhead transcriptionfactorFoxI1remains boundtocondensedmitotic otic placodeinduction. nonaxial signals. forebrain. Development zebrafish formbyconvergenceofcellularfieldsattheedgeneuralplate. of thezebrafishinnerear. the zebrafish pronephros, andhindbrain. associated gene,regulates regional specificationofthezebrafishgut, development. fibroblast growth factor(FGF-2)mRNAandprotein inearlyXenopus otic placodeinductionandformation. shortening ofthecaudalhindbrainratembryo. deficiency results inthedose-dependentacquisitionofanteriorcharacterand Mech. Dev. 168. mutant embryos. http://zfin.org/. Submission, ZDB-PUB-051025-1, throughput expression analysisofZF-modelsconsortiumclones.ZFINDirect Data ZDB-PUB-010810-1, http://zfin.org/. of thezebrafishgenomeduringembryogenesis.ZFINDirect DataSubmission, Woehl, R.,Lux,A.,Steffan, T., Charbonnier, X.Q.etal. 940. mediates oticplacodeformationandjawdevelopment Zebrafish (Daniorerio). Dev. Cell 71 snail 127 Mech. Dev , 5-21. (2000). Science Development , 3645-3653. gene anditsexpression inwild-type, 6 , 167-181. The Zebrafish Book. A Guide for the Laboratory useof The ZebrafishBook.AGuidefortheLaboratory Eugene: UniversityofOregon Press. Development (2001). vhnf1,theMODY5andfamilialGCKD- (1994). Spatialandtemporalexpression ofbasic (1997). Specificationofthezebrafishnervoussystemby 277 . Dev. Dyn. 48 (1998). Thedevelopmentofthevertebrateinnerear. Genes Dev. , 254-257. , 141-151. (2004). Earlystepsinthedevelopmentof 119 (2003). , 1203-1215. 223 (2000). Theolfactoryplacodesofthe 130 Dev. Dyn. Fgf3 15 , 427-458. (2004). Geneticinteractionsunderlying , 3379-3390. , 3217-3229. and 230 Fgf10 Development 134(13) , 419-433. (2003). Zebrafish Dev. Biol. . Development are required formouse spadetail Mol. Cell.Biol. (2005). High (1993). Structure of (2002). Development (2006). The (2001). Expression (2000). Vitamin A (2000). Vitamin 220 and , 263-284. no tail 130 26 foxil , 155- , 929-

DEVELOPMENT