Hiroshi Kikuta Germany Institute ofStemCellResearch, IngolstaedterLandstrasse1,D-85764 Neuherberg, *Present address: GSF-NationalResearch CenterforEnvironment and Health, London, UK. Developmental Neurobiology, NewHunt’s House,King’s CollegeLondon,SE19RT segregating thetelencephalicandeyefieldsisisolatedfromdiencephalicpatterning,mediatedbyRx3. expression oftheWntantagonistTlcandwinged-helixtranscriptionfactorFoxg1.Theseresultssuggestthatprocess is cellautonomous.Transfating oftheeyefieldinabsenceRx3functioncorrelateswithasubstantialposteriorexpansio Houart etal.,2002;van deWater etal.,2001),andthelackof the expense ofthetelencephalonandeyes (Heisenberg etal.,2001; function ofAxin1,leadstoanenlargement ofthediencephalonat in theposteriordiencephalon(Kelly etal.,1995),orbythelossof activity, forinstancebyoverexpression ofWnt8bnormallyproduced and eyes (Houartetal.,2002).Conversely, increasedcanonicalWnt abrogation ofTlcfunctionimpairstheformationtelencephalon mimics ectopicANBactivity intelencephalicinduction,and Frizzled RelatedProtein(sFRP)Tlc.Ectopicexpression ofTlc et al.,2002).Inzebrafish,oneoftheseantagonistsisthesecreted and Dempewolf, 2002),aswellpotentWnt antagonists(Houart The ANBexpresses thesecretedfactors Fgf3andFgf8(Eagleson et al.,2002;Houart1998;ShimamuraandRubenstein,1997). ANR), controlsdevelopment ofanteriorforebrainidentities(Houart organizer, locatedattheanteriormargin oftheneuralplate(ANBor patterning (Foley andStern,2001;Wilson andHouart,2004).One gastrulation, localorganizers refineandmaintainforebrain initially establishedisincompletelyunderstood. hypothalamus andthecaudaldiencephalon.How theirdomainsare anteriorly locatedtelencephalonandretinae,theventral The vertebrate forebrainisprefiguredatembryonicstagesbythe INTRODUCTION KEY WORDS:Zebrafish,Telencephalon, Eyefield,Forebrain, Rx3 ckh direct celltracingdemonstratesthatretinalprecursorsacquireatelencephalicfateinembryoshomozygousforthe Rx3 biasescellspecificationchoicestowardstheretinalfatewithinapopulationofbipotentialprecursorsanteriorfor subdivided remainslargelyunknown.We demonstratethatatlategastrulationthePaired-likehomeodomaintranscriptionfactor anterior fields(telencephalonandeye)fromthediencephalon.Howtelencephalicretinalprimordiaaresubsequently Anteroposterior patterningofthevertebrateforebrainduringgastrulationinvolvesgradedWntsignaling,whichsegregates Christian Stigloher the zebrafishforebrainterritoryiscontrolledbyRx3 Segregation oftelencephalicandeye-fieldidentitiesinside Development 133,2925-2935(2006)doi:10.1242/dev.02450 Accepted 22May 2006 ‡ † 1 Bergen, Thormoehlensgt.55,N-5008Norway. Germany Neuherberg, Germany. Institute ofDevelopmentalGenetics,IngolstaedterLandstrasse1,D-85764 Research CenterforEnvironment andHealth,DepartmentZebrafishNeurogenetics, University-Munich, Trogerstrasse 4b,D-81675,Munich,GermanyandGSF-National Author forcorrespondence (e-mail:[email protected]) Present address: MischterlichandPartners,Postfach330609,80066Munich, Zebrafish Neurogenetics JuniorResearch Group, InstituteofVirology, Technical Following thespecificationofforebrainidentityduring ne2611 , characterizedbyanenlargedtelencephalonandalackofeyes.ChimeraanalysesfurtherindicatethatthisfunctionRx3 2 2 , ThomasS.Becker Sars Centre forMarineMolecularBiology, Universityof 1 , JovicaNinkovic 2 , CorinneHouart 3 MRC Centre for 1, *, MaryLaplante 3 and Laure Bally-Cuif 2 , Andrea Geling out ofthesinglemouse expressed attheopenneuralplatestage(Chuangetal.,1999).Knock- pattern isobserved forzebrafish telencephalic field(Bailey etal.,2004;Chuang1999).Asimilar intense intheeye fieldandisnon-overlapping withtheadjacent proteins. Atlategastrulation,expression ofthemouse and Jamrich,2000). development (Bailey etal.,2004;Graw, 2003;Hanson, 2003; Mathers expression ofPax6, Six3andRx1-Rx3,shown tobecrucialforeye specification choices,however, remainsunknown. cellular andmolecularlevels, andwhether itindeedcontrolscell diencephalic size(Esteve etal.,2004).How olSfrp1actsatthe complementarily enlarged telencephalon,withoutmodifying oligonucleotides producesembryoswithreducedeyes anda abrogation ofolSfrp1functionusingmorpholinoantisense expressed intheanteriormostregion oftheneuralplateinMedaka: forebrain areunknown. OnecandidatemightbeolSfrp1,asFRP separation ofthetelencephalonandeye fieldwithin theanterior early responsetoWntactivity. Thefactors controllingthelater suggesting thatthesetwo domainsareinitiallydefinedasoneintheir simultaneously thepresumptive telencephalonandeye field, development duringgastrulation(Wilson andHouart,2004). posterior localsourceandanteriorinhibitors,patternsforebrain canonical Wntactivity, determinedbytheantagonismbetweena et al.,2003).Theseresultssuggestamodelwherethelevel of zebrafish andmouse(Dorsky etal.,2003;Kim2000; Lagutin forebrain development atthebenefitofmoreposterioridentitiesin abrogation oftheWntinhibitorsTcf3orSix3abolishesanterior telencephalon canbecorrectedbyincreasedlevels ofTlc.Similarly, the tissue (ChuangandRaymond,2001). Theseobservations suggestthat rx2 2003; Mathersetal.,1997).Conversely, ectopicexpression of function, abolishestheformation ofeye structures(Casarosaetal., function ofzebrafish andMedaka progenitors (Bailey etal., 2004),incontrastwiththeproposedlater The specificationoftheeye fieldiscorrelatedwithsustained Manipulations ofWntoritsantagonistsatanearlystageaffect Rx by mRNA injection inzebrafishtriggersanexpansion ofretinal genes areinvolved inthespecificationormaintenanceof retinal 1,† 1,‡ , BirgitTannhäuser Rx Rx genes encodepaired-like homeodomain gene, andinhibitionof rx3 rx3 RESEARCH ARTICLE , theearliestandonly in retinalevagination; innull 1 , StefanieTopp rx3 Xenopus -null allele Rx ebrain: rx rx1 gene is n of 2925 Xrx1 gene 1 and ,

DEVELOPMENT Sat coding sequence.ThisTtoCtransitionleadstheintroductionofanew and RNA was injectedattheconcentration of50or100ng/ mMessage mMachineKit,following therecommendedprocedure.Capped by PCRfromreverse-transcribed RNA fromhomozygous screened torecover specific locusratewas 1/670againstthe 1996), except thatanincrosswas conductedintheF1generation.The CNS defects.Thescreensetupfollowed thatofHaffter etal.(Haffter etal., 3 where theforward primerwas designedcontainingtheATG-start site. exons toincludeputative splicesitemutations.Anexception was exon 1, embryos. Primersweredesignedtobindintronicsequencesflankingthe exons comparinghomozygouswild-typewithmutant by directsequencingofPCRproducts(Sequiserve) ofeachthethree carriers. al., 2003)mutantswereobtainedbypairwisematingofheterozygousadult The Molecular identificationof according toKimmeletal.(Kimmelal.,1995). Embryos ofABwild-typeorENU-treatedfishwereraisedandstaged Fish strains mutants forRx3[ 2926 (http://www.compbio.dundee.ac.uk/~www-jpred/). Theinput was the secondary structurethathasbeen analyzedindetail related The JPREDalgorithm(Cuff etal.,1998)was usedtofindanearly Bioinformatic analysis and subsequentlysubclonedintothepCS2+Vector. (Deutsches RessourcenzentrumfürGenomforschungGmbH,www.rzpd.de) chk/rx3 RNA andBACinjections MATERIALS ANDMETHODS forebrain patterning. choices betweeneye fieldandtelencephalonduringanterior results identifyRx3asakey determinantcontrolling specification telencephalic expansion arealsoapparentinthismutant.These at theone-cellstage. and subcloningintopCS2+: embryos, followed bydirectcloningusingtheTOPO cloning kit(Invitrogen) allele of telencephalon andalackofeyes. We reportthat the Rxfamily controllingretinalmorphogenesis. had notbeennoted,Rx3was proposedtobeanunusualmemberof al., 2001;Winkler etal.,2000).Becauseforebrainpatterningdefects fails toevaginate (Kennedy etal.,2004;Loosli2003; anterior genessuchas express (QIAGEN) andinjectedataconcentrationof35ng/ from RZPD,amplifiedandpurified withtheLarge-Construct Kit reanalyzed thepublishedallele one-cell stage. Ј ; The onlysignificantalterationwas found atnucleotideposition382ofthe rx3_exon2_ reverse, 5 rx3_exon1_ reverse, 5 rx3_exon3_ reverse, 5 rx3_exon3_forward, 5 rx3_exon2_forward, 5 chk/rx3 rx3_cDNA_reverse, 5 rx3_exon1_forward, 5 The BAC CHORB736A01233Q containingthe rx3_cDNA_forward, 5 We describehereazebrafishmutant, I endonucleaserestrictionsite. rx3 RESEARCH ARTICLE locus of chk tlc cDNA (IMAGp998G108961Q) was obtainedfrom RZPD and rx3 ne2611 at lategastrulationandacquireatelencephalicfate. We chk , andthatretinalprecursorsin chk ne2611 fish wererecovered inasmallscalescreenfocusingon ne2611 chokh ne2611 /rx3 six3 Ј Ј Ј Ј Ј Ј Ј -TATTATTGCTGTATTAGTTTGAACAGAA-3 Ј -TCTCATCTACCACGTCTTCCCTATA-3 -ATAAGCTCCTCAACTACATCTTTAACTT-3 -AGACCACTGATTTTGAAGATACAAA-3 -AAGTTAGAAGTTAGGATAAAGTTGTCAA- -TGCACTTTCTCACATATTTCTCACTG-3 -GCACGAGGTTCAATGAGGC-3 -AAATCGTTCAATGAGGCTTGTT-3 . capped RNA was synthesizedusingthe Ambion ( mutants was analyzedforputative mutations ckh or ne2611 pax6 ) and ckh are expressed, but theopticvesicle s399 eyeless golden and demonstratethat ne2611 locus and442genomeswere ( el chokh chk ), respectively], early rx3 ␮ ne2611 , withanexpanded l intoembryosatthe ne2611 ␮ locus was obtained ( ne2611 l intotheembryos chk /rx3 ne2611 s399 Ј ectopically ; ) (Loosliet was cloned is anull Ј ; and tlc mutant Ј and Ј Ј . ; Ј Ј ; ; ventricle iswideopen(Fig.1A,B)( lies far posteriorcomparedwithwildtype(wt)andthediencephalic the constrictionseparatingtelencephalonfromdiencephalon In subsequently fixed ataround30hpf. fluorescent light,anddonorrecipientembryosweredocumented The appropriatelocalizationoftransplantedcellswas checked under cells was conductedinahomotypicmannerattheanimalpoledomestage. embryos maintainedinthedarkatallstages.Transplantation ofaround10 as previously described(HoandKane,1990),withrecipientdonor Probes) inwater (Ambion)attheone-cellstage.Celltransplantationswere emx2 somites stagewithmolecularmarkers ( size ofthenasalplacodesandepiphysis werenotaffected. only asmalllensisvisible(Fig.1A for anti-fluoresceinimmunocytochemistry. subsequently fixed overnight in4%paraformaldehydeat4°Candprocessed Hrm CameraandtheAxiovision Software Package (Zeiss),and the FITC-channelonaZeissAxioplan2MicroscopewithAxiocam with apinhole.Irradiatedembryoswereimagedatthe24hpfstage,using dye was activated inafew cellsusingaUV-beam (DAPI-channel) focused were allowed todevelop furtherinthedark.Atearlytailbud stage,the (5 mg/ml;MolecularProbes),was injected intoone-cellembryos,which A solutionofDMNB-CagedFluoresceindextran andbiotin,lysinefixable Uncaging experiments fixable (fluoro-ruby)Tetramethylrhodamine Dextran (10,000 detection ofthebiotintracer. embryos. Recipientembryoswereprocessedat30hpfforimmunochemical and isochronicallyontotheanimalpoleofshield-stagewild-typeor Probes) attheone-cellstage.Thirtyto40cellsweretransplantedisotopically Wild-type donorembryoswereinjectedwithbiotin-dextran (Molecular Cell transplantationsbetween (http://kinemage.biochem.duke.edu/software/mage.php). and structureanalysisusingtheMAGE software package version 6.36 primary outputofthealgorithm(1FJL.pdb)was usedforfurthersequence zebrafish assemblyversion 4(Zv4)usingtheENSEMBLserver. The protein sequenceofRx3(ENSDARP00000022866) fromtheSangerCentre forebrain domainin Selective enlargementoftheanteriormost RESULTS of the CAATATTACAAG) mapstochromosome10,37.390 basepairsupstream (Ellingsen etal.,2005).Thissequence(TAAAAAAAAATTTGGGGT- insertion was identifiedbylinker-mediated PCRasdescribedpreviously detection screenforitsexpression intheretina.The3 CLGY469 Isolation andmappingofthe stereomicroscope oraZeissAxioplanphotomicroscope. Embryos werescoredandphotographedunderaZeissSV11 secondary antibody(JacksonImmunoResearchLaboratories)(1/200). ImmunoResearch Laboratories)orCy3-conjugatedgoatanti-mouse using FITC-conjugatedgoatanti-rabbitsecondaryantibody(Jackson biotechnology) was usedina1/500to1/1000 dilution.They wererevealed in afinaldilutionof1/200.Purifiedrabbitanti-GFPantibody(ams The anti-Phospho-HistoneH3antibody(UpstateBiotechnology)was used carried outaccordingtostandardprotocols(Hammerschmidtetal.,1996). Probe synthesis,insituhybridizationandimmunohistochemistrywere In situhybridizationandimmunohistochemistry compared therelative sizes ofthedifferent forebrain andmidbrain this phenotypereflectedbroader APpatterningdefects,we anteroposterior (AP)andmediolateral axes. To determinewhether CLGY469 Telencephalic expansion in ne2611 , rx3 pax6.1 locus (SangerCentrezv5release). was recovered in aretrovirus-mediated large-scale enhancer homozygous embryosat36hourspost-fertilization(hpf), transgenic donorembryoswereinjectedwith1.5%lysine- ) (Fig.1C,D;datanotshown), andoccursalongthe ne2611 CLGY469 ne2611 ne2611 Ј n ,B >100, 100%ofcases);theeye, Ј , arrowheads). Bycontrast,the mutant embryos and wildtype insertion was confirmedatthe15- emx3 – Development 133(15) Ј sequence flankingthe previously M r , Molecular emx1- ne2611 ,

DEVELOPMENT Rx3 activitysegregates telencephalonandeyefield pretectum andmidbrain).We used domains (telencephalon,hypothalamus,prethalamus,thalamus, not shown; seealso schemeinFig.1H,I). to reveal the anteriorandposteriorhypothalamus(Fig.1E,F;data prethalamus andposteriorhypothalamusat15somites, prethalamus (Fig. 1G,redbars, suffer fromsubstantially elongatedneuraltissueanteriortothe variations inembryo length.We confirmed that hindbrain, limitedby the midbrain-hindbrainboundary, andthesizeofanterior krox20 , served asareference to correctfor P <0.01, lhx5 n =10 embryosmeasured for and her5 arx expression identified as markers ofthe ne2611 embryos nkx2.1b ath5 – somitogenesis stages(see telencephalon of domain ofmutantembryos. We foundthat the enlarged whether cellswithretinalidentitywerepresentinthetelencephalic S1E-H inthesupplementarymaterial).Thispromptedustoanalyze resolve this issue,weprobed non-specification orthenon-maintenance ofeye precursors,and,to domain absentin (Fig. 1G,bluebars, expression ofseveral regional markers, suchas supplementary material)].We noted,however, aperturbed and anormalmediolateralpatterning(seeFig.S1C,Din the dlx2a the dorsalandventral telencephalicmarkers telencephalon maintainsagrosslynormaldorsoventral polarity[see of cellshaving maintainedmoleculareye identity. We foundthatthe phenotypes intelencephalicorganization, aswellforthepresence 1H,I). n between wildtypeand embryos measuredforeachgenotype)(sizeofthe lhx5 prethalamus itself,aswellstructureslocatedposteriortothe each genotype),andfoundthatthisphenotypeislocal,asthe or missing(seealsoFig.S1I,Jinthesupplementarymaterial). the telencephalon.Later, theanteriorhypothalamusappearsreduced (hence thelighterred color)bytheanteroposterior enlargementof hypothalamus appearselongated,butmightbesimplystretched hypothalamus are unchangedinthemutants.Theanterior arx only, prethalamus; red: somites. Thegenesusedaslandmarksare colorcoded(yellow: forebrain territoriesinwildtypeversus ( sample independentStudent’s length betweenwildtypeand genes indicatedinE.Barsindicates.e.m.Thedifference inA/C anterior hindbrain(domainC),andthedomainsare definedwiththe siblings (F).(G)Measurements are normalizedtothesizeof (domain B)at15somitesin (domain A)versusprethalamus, thalamus,pretectum andmidbrain ( massive anteroposterior enlargementofthe wild-type siblings(C);scalebar:0.02mm.Themutantsdisplaya telencephalic marker due totheabsenceofeyes.( arrows) ishypothalamic,itdisplacedtowards thelensin both genotypes.Theadjacentdomainof expression of ne2611 ne2611 (arrowheads) andanexpandeddiencephalicventricle(arrows) in dorsal viewsofthesameembryosshowingabsenceretina telencephalon, delimitedbyadashedline,in parasagittal andlateralfocus,respectively. (A,B)Notetheenlarged ne2611 views are lateral,anteriorleft.( Fig. 1.Enlargedtelencephalonandlackofretina in H,I E-G =10 embryosmeasuredforeachgenotype)(schematizedinFig. The absenceofexpression ofeye markers couldresultfromthe We next analyzed + ) Schematicrepresentation ofthesizedifferent anterior nkx2.1 Relativeanteroposterior sizesoftheanteriormostforebrain ) / arx (see Fig.S1A,Binthesupplementarymaterial),respectively, and , albeitsmallerthaninwild-typesiblings.Insetsshow . (A’,B’) Notethemaintenanceofalens(arrowhead) in (B,B’) andwild-typesiblings(sib;A,A’) at36hpf;viewswith domain, isunchangedcomparedwithwild-typesiblings , posteriorhypothalamus).Prethalamus andposterior vax2 pitx3 , notshown). ne2611 ne2611 , molecularlyidentifyingthelens(arrowheads) in emx3 nkx2.1 P ne2611 ne2611 =0.75, i.e.nosignificantchange, ) or otx2 never expressed retinalmarkers atpost- at the15-somitestagein ne2611 C,D ne2611 ne2611 t only, anteriorhypothalamus;orange: emx2 A-B -test, in Fig.2A,B,and : Compared expression ofthe ) embryos forpotentialpatterning P =0.15, i.e.nosignificantchange, Ј ) Compared morphologyof (E) compared withwild-type RESEARCH ARTICLE (scattered in P mutants forthe expression of is statisticallysignificant(two- ne2611 values are giveninthetext). pitx3 emx3 ne2611 emx3 expression (small siblings at15 atoh7 – domain. ne2611 flh (Fig. 3D-F)and . Insetsare ne2611 (telencephalic ne2611 arx previously ne2611 ) (seeFig. domain (D) and arx 2927 n . =10 All

DEVELOPMENT ckh incomplete eye-field identity( By contrast, but thattheirexpression was lostduringsomitogenesis(notshown). ne2611 rx3 (Chuang etal.,1999;ChuangandRaymond,2001).We foundthat rx3 the earliesteye-field markers. Inorderofappearance, weselected 2928 dorsal viewsofflat-mountedembryos), andof gray dotsinB.( Lateral views;t,optictectum;telencephalon (arrowhead) delimitedby is absentintheenlargedtelencephalon of labeling thewild-typeretina (A,bluearrow) butnotthetelencephalon, ne2611 Fig. 2.Theeyefieldispartiallyspecifiedbutnotmaintained in in normally detected at3-somitesinwildtype(K,inset), isneverinitiated whole embryos),isinitiatednormally in however, that development (Kennedy etal., 2004;Looslietal.,2003).We found, et al.,2001;van deWater etal.,2001)and ( Reduced orabsenteyes characterizethezebrafishmutants ne2611 of allretinalmarkers. ne2611 leading toforebraintruncations,whiletheexisting hdl et al.,2004;Loosli2003;Rojas-Munoz2005).Inaddition, hdl ne2611 w29 and (at 70%epiboly),followed by ; and tcf7l1a and RESEARCH ARTICLE , but thistransientphaseisfollowed bythelossofexpression mbl mutants comparedwiththeirwild-typesiblings(Fig.2C-H), mutants. rx1 (K). Lateralviews ofwholeembryos. is anullalleleof ) (Kimetal.,2000), embryos displayvarious degrees ofbrainposteriorization ckh expression followed anormalspatiotemporalpatternin ne2611 C-H rx2 hu499 l iw,atro et ( All views,anteriorleft. ) Expression oftheearliest eye-field marker was never expressed (Fig. 2I-K). Thus,an were describedasnotaffecting telencephalic is allelicto masterblind rx3 ckh rx3/chokh rx1 + , (23 embryoslackingeyes in78 ne2611 rx1 and ne2611 A,B + chokh rx2 but ( ) Expression of mbl rx1 . ( (at the3-somitestage) I-K rx2 (F-H; lateralviewsof ( ; sibling embryos(B). ckh axin1 ) Expression of ckh – ) isspecifiedin ; rx3 alleles ) (Heisenberg ) (Kennedy otx2 headless rx3 ckh , (C-E; rx2 s399 , , prominent frombud stage onwards (Fig.4E-G). foxg1 their expression withthat of ( defects werenotedfollowing injectionof (10% ofcases, embryos withheadtruncationsat24hpfinadose-dependentmanner Rx3 telencephalic development. staining atthetail-bud stage(100%of ckh domain attail-bud stage(Fig.4E,F).Theearliestphenotype inboth and eye field(Houartetal.,2002)tobecomeadjacentthe forebrain atlategastrulation,andisexcluded fromthehypothalamus rescued, and RZPD;79%ofthemutantembryoswereatleastpartially CHORB736A01233Q, whichcontainsthe defects of shown). We alsocouldrescueboththeretinalandtelencephalic normal eyes fromsuchcrosses,hadanenlarged telencephalon(not embryos froma similar phenotype, confirmed bygenotyping,have expanded of eye phenotypecausedbythe type embryos.Ectopicexpression ofwild-type support thisinterpretation,weoverexpressed This suggeststhat embryos froma using theJPREDalgorithmwithstructureof position 128oftheRx3protein(T382N,Fig.3B,C).Comparison (Fig. 3A),leadingtoaSerineProlinesubstitutionataminoacid revealed aTtoCtransitionwithinexon 2innucleotideposition382 experiments). Sequencingofthe and and expanded telencephaloninmorethan50embryostestedbyPCR creates a DNA sequencedfromeightindependentembryos.Themutation Zebrafish eye fieldatgastrulation Rx3 controlsofthetelencephalonand patterning 3M,N). Theseresultssuggestthat ne2611 that mutantembryosdisplayanenlarged telencephalonidenticalto ckh conclude that of embryosfromacrossbetween segregated withthe al., 2003;Rojas-Munozet2005).Theabove T382Nmutation in thestudiesofexisting helix1 and2oftheRx3homeodomain. homeodomain predictsthissubstitutiontoacoileddomainseparating during thesestagesinboth development, weexamined expression oftelencephalicmarkers of Houart, 2004).To determine whetherthetelencephalicphenotype al., 1999),whichabut thetelencephalicprimordium (Wilson and restricted tothepresumptive eye fieldandhypothalamus(Chuanget bf1 ckh As mentionedabove, structuralconsiderationspredictthatthe tlc We werepuzzledthatnotelencephalicdefectshadbeenreported ckh ne2611 ne2611 Sat ne2611 ) alsolabelthepresumptive telencephalon,andwecompared s399 is oneoftheearliestmarkers ofthepresumptive anterior expression appearedidenticalinwild typeand 1 digestion(notshown). Inaddition,wefoundthat alleyeless reflects anearlyroleofRx3inanteriorneuralplate (100% ofcases, , andreveals apreviously undiscovered roleofRx3in n , whichtruncatestheRx3homeodomain(Looslietal.,2003). Sat protein isdysfunctional,andwefoundthetelencephalic and =64; Fig.3G-K).Finally, are-analysisof rx3 1 restrictionsitethatalsosegregated withlossofeyes ne2611 expression isinitiatedatlategastrulationandfirst ckh ne2611 ne2611/+ n ne2611/+ =56, Fig.3L,N).Bycontrast,nomorphological s399 ne2611 n ne2611 =82; Fig.4A-D).Ectopic mutants was theposteriorexpansion of is analleleof embryos following injectionofBAC ckh ϫ n ϫ =23; Fig.3D-F, seealsoFig.4D).We ckh might representanullalleleof ckh tlc ne2611 ckh alleles (Kennedy etal.,2004;Loosli telencephalon andeye phenotypesin ne2611 s399 expression inatime-course analysis. s399 ne2611 rx3 /+ /+ mutation tobeassevere asthose and intercross, but noembryoswith ckh ckh cDNA from intercross intwo independent ckh s399 , henceforthreferredtoas ckh is anullalleleof tlc rx3 ne2611 Development 133(15) s399 expression, rx3 rx3 heterozygotes have a rx3 locus (Sangercentre . ne2611 ne2611 tlc Drosophila mutant embryos, mRNA produced ne2611 emx3 ckh expression was RNA inwild- mRNA (Fig. s399 n and >50; 24% rx3 ckh embryos revealed rx3 Paired . foxg1 ne2611 . To rx3 tlc

DEVELOPMENT ectopic in Rx3 activitysegregates telencephalonandeyefield ckh the one-somitestageonwards; mutants atbud stage(Fig.4H),but was expanded posteriorlyfrom Accordingly, overexpression of result fromafailure to be repressedwithinthe largely overlaps Double stainingsfurtherdemonstrated that becoming visiblyectopicafew hoursafterwards (notshown). injections into wild-type embryos,reducedexpression ofearly ne2611 embryos untilthe3-somitestage(Fig.4K-M),only ckh (Fig. 4G,J),suggestingthatthese posteriorexpansions rx3 expression atthestages whenthey become emx3 rx3 , but not expression was unaffected in tlc and rx3 ne2611 foxg1 rx3 , bymRNA expression domain. emx3 positive domain, followed bythedownregulation of by theearlydownregulation of the anteriorneuralplate.This functionisnormallymanifested Rx3 atitsonsetofexpression inlimitingtelencephalicextent in conclude thattelencephalicexpansion in not shown). (58% ofcases, telencephalic markers suchas Given that . n rx3 =18; seeFig.S2inthesupplementarymaterial;data expression startsat late gastrulation,we expression ofwild-type ne2611 ne2611 telencephalic marker aristaless-rx domain.( red box,homeodomain;lightgrayotp- (asterisk inC).Darkgraybox,octapeptide; exchange withintheRx3homeodomain ( development in79%ofmutantembryos. embryos. BACinjectionrestored retinal BAC injectioncompared withnon-injected (K) Percentage ofembryoslackingeyesafter small lensinHindicatedbythewhitearrow). with uninjectedwildtype(G)or note therestoration oftheretina compared IJ Representative injected (I,J) locus andare observedat24hpf. BAC CHORB736A01233Qcontainingthe cross were injectedattheone-cellstagewith (C) inwildtype(+)and ( (left) reveals aTtoCtransition(arrows). ne2611 (right twobars)followinginjectionof supplementary material)orheadtruncation (left twobars)(seeFig.S2inthe embryos showingreduction of rx3 mm). (lateral views,anteriorleft;scalebar:0.02 ( expansion similartothatof ( Fig. 3. rx3 compare withwildtype,inset)whereas of (lateral views,anteriorleft).Ectopicexpression the one-cellstageandobservedat24hpf embryoswere injectedat mRNA. Wild-type L B,C G-K A , Sequencingtracedataof ) M rx3 ne2611 ne2611 Rx3protein sequences(B)andstructures ) Phenotypestriggered byectopic ) Embryosfrom a ) ckh tlc causes headtruncations(L,arrow; ne2611 (E) and mutation leadstoaSerineProline mutant (right)andawild-typesibling mRNA (asindicated). has noeffect (M).( and s399 tlc RESEARCH ARTICLE embryos displayatelencephalic hesx1 expression withinthe is anewnullalleleof ckh s399 ( anf emx3 D-F ne2611/+ ckh (F) embryosat48hpf ne2611 rx3 ) atthetail-bud stage ) Expression ofthe reflects aroleof versus in wild-type(D), ne2611 ne2611 N rx3 Percentage of ) tlc mutants. The ϫ ne2611 cDNA from a expression ne2611/+ rx3 foxg1 mutants; mutants. ne2611 rx3 rx3 2929 (H; rx3 or rx3 and . -

DEVELOPMENT are non-genotypedembryosfrom crosses between labeled by downregulated from thepresumptive eyeandhypothalamusfields, wild-type andmutantembryosat100%epiboly, when siblings (A,C).( plate. We analyzedcelldeathprofilesin ectopic specificationofsuchprecursorswithintheanteriorneural in telencephalic precursorsmightaccountforexpansion Rx3. Several non-exclusive mechanismsaffecting early We next addressedtheprocessesunderlyingthisearlyfunctionof telencephalic precursors of eye-fieldcellswhencompared with Rx3 functionaccountsforthehigherproliferation 2930 ckh forebrain. Fig. 4.Rx3controlsofthepresumptive earlypatterning anterior or flatmounts(E-M).( heterozygotes. Allviewsare dorsalanteriorleftinwhole embryos(A-D) affected in mutantsbefore the3-somite stage. ( bud stage(H)butisvisibleattheone-somite stage(J,bluearrows). arrows). ( stage theectopic positive domainat100%epiboly; arrows inE,absentF).Attail-bud ckh telencephalon attail-budstageandismassivelyexpandedinboth K-M ckh ne2611 ne2611 ) Therelative expression of : theirreducedcelldeath,increasedproliferation,oran RESEARCH ARTICLE H-J (B) and (B,G,J), rx3 Expression ofthemarkersindicated(colorcoded)in ) Expansionof (see thefewremaining E-G ckh ckh tlc ) Nodifference in expression overlapsthe s399 s399 A-D (D) ortheirwild-typesiblings(A,C,F,I); E,H,K-M (D) mutantscompared withtheirwild-type ) foxg1 tlc expression identifiesthepresumptive emx3 across theeyefieldisabsent attail- tlc tlc and expression isobservedbetween -positive cellsinthe rx3 ckh rx3 are notnoticeably ne2611 domain (G,blue ckh embryos between ne2611 tlc expression is /+ rx3 - 3 and1,respectively) andthe corresponding territoriesin presumptive telencephalicandeyefieldsinwild-typeembryos(domains plate of phase markerPhosphohistone-H3(red nuclei)intheanteriorneural Corresponding domainsin 3 inFig.5)andtheeye fieldby of presumptive telencephalicidentityimposesalow proliferation inside theeye fieldduringgastrulation,and/orthattheacquisition This observation suggestseitherthatRx3promotes proliferation embryos andofthetelencephalon+eyefieldin blue) servesasamarkerofthepresumptive telencephaloninwild-type panel) attail-budstage(dorsalviews,anteriorleft). the presumptive telencephalon, in significantly decreased inthe presumptive eyefield,butisunaltered in ( ckh PH3-positive cellsindomains1-4 (see C,D)inwild-type(red) versus siblings (domains4and2,respectively) attail-budstage.( versus telencephalicprecursors. Fig. 5.Rx3accountsforthehigherproliferation ofeyefield telencephalon of these stages( significant difference betweenmutantandwild-type siblingsat immunostaining ofcleaved caspase3.We didnotdetectany 90% epibolyand3somitesusingacridineorange telencephalic anlagewas identifiedbyitsexpression of immunocytochemistry. Inwild-typeembryosattail-bud stage,the telencephalon andeye fieldusinganti-phosphohistoneH3(PH3) is notaffected (comparedomains3and4; whereas proliferationwithinthemoreanterior with wild-typesiblings( values are giveninthetext). embryos attail-budstage(two-sample independentStudent’s phase withinthe domain revealed significantlydecreasednumbersofcellsinM (domain 2inFig.5).CountsofPH3-positive cellswithineach positive, C , D We next monitoredproliferationinthepresumptive ne2611 Schematicrepresentation oftheembryosinA,Btolocalize the ) ckh (gray) embryos.Barsindicatestandard errors. Proliferation is rx3 ne2611 negative (domain4inFig.5),versus n =19), althoughwedidobserve apoptosisinthe mutants (rightpanel)andtheirwild-typesiblings(left rx3 ckh -positive domainin ne2611 mutants at28hpf(notshown). P ckh <0.02; comparedomains1and2), ne2611 ckh ( A , ne2611 B ) ImmunodetectionoftheM- mutants weredefinedas rx3 compared withwild-type ckh P (domain 1inFig.5). =0.86; Fig.5A,B,E-G). Development 133(15) ckh ne2611 rx3 ne2611 tlc -negative domain expression (ISH, when compared . ckh E rx3 ) Numberof tlc ne2611 t (domain -test, positive P tlc

DEVELOPMENT Table 1).( (21% ofcases);(E)retina and telencephalon(31%ofcases)(seealso Retinaonly(45%ofcases);(D)retina andanteriorhypothalamus (C) (overlay offluorescence and Nomarskiopticsviews,anteriorleft). ( clone immediatelyfollowinguncaging(B)(dorsalviews,anteriorup). at theearlytail-budstage,andphotomicrograph ofthefluorescent compared withthenotochord, polsterandenveloping layer(insetinA) in mutants. specification extends posteriorlyattheexpense ofeye identityinthe early foxg1 telencephalic expansion. Bycontrast,theco-expression of for thelackofeyes of Decreased proliferationofretinalprecursorsmightpartiallyaccount anterior forebrain precursors Rx3 attributeseyeversustelencephalicidentityto role alreadywithintheanteriorneuralplate. evagination (Looslietal.,2003).We show thatRx3mayexert this precursors hadbeensuspectedatthestageofopticvesicle rate. AroleforRx3incontrollingpositively proliferationofretinal Rx3 activitysegregates telencephalonandeyefield at 24hpfbymorphologicalinspectionunderfluorescenceand location oftheprogeny oftheselabeledcellswas thendetermined field precursorcellsattheearlytail-bud stage(Fig.6A,B);the stage embryos,andwas activated inasmallnumber(~10)ofeye- identity intheabsenceofRx3. Fig. 6.Cellsofthepresumptive eyefieldacquire atelencephalic telencephalon (100% ofcases,seeTable1). fluorescence onlyinG-I,anterior left).Allclonescontributetothe 24 hpfstage(overlayoffluorescence andNomarskiopticsviewsinF, C-E To testthishypothesis,wetracedthefate ofeye-field precursors ckh ) Fateoftheuncagedprogeny inwild-typeembryosat24hpf and mutant ne2611 ckh F-I ne2611 ) Fateoftheuncagedprogeny in mutants. Cagedfluoresceinwas injectedintoone-cell embryos (Fig.4G,J)suggeststhattelencephalic rx3 RNAs inthepresumptive eye-field territoryof ckh mutants, but cannotbethedirectcauseof ( A , B ) Locationofuncagedcells ckh ne2611 embryos atthe tlc or turn onexpression ofthetransgene(80% ofcases, at thespherestage,werepeatedly observed thatsomeofthesecells exclusively ofwild-typecells(Fig. 7A).Bycontrast, evaginated retina(36%ofcases, telencephalon of detected expression ofretinalmarkers in the expanded precursors lackingRx3tendtojointhetelencephalon.We never 6E; compared with34%ofcasescontributing tothetelencephalon(Fig. except thetelencephalonin45+21=66%ofcases(Fig.6D).This and clonescontributing totheretinaandotherforebrainderivatives 1): pureretinalcloneswererecovered in45%ofcases(Fig.6C), 6A mostlyrevealed retinalprecursorsinwild-typeembryos(Table however, we foundthatuncagingatthepositionindicatedinFig. telencephalic fieldsareintermingled(Woo andFraser, 1995); Nomarski optics(Fig.6C-I).Attheearlytail-bud stage,theeye and telencephalon in shield-stage cells (25-40)arehomotopicallytransplantedtotheanimalpoleofa The sameistrueforzebrafishRx3:whenlarge numbersofwild-type is responsibleforretinalevagination inacell-autonomousmanner. 2000) proposedthatMedakaEyeless,theorthologofzebrafishRx3, Using transplantationexperiments, Winkler etal.(Winkler etal., telencephalic identityiscellautonomous The role ofRx3incontrolling retinal versus function. cells mayacquireatelencephalicfate intheabsenceofRx3 not shown), supportingtheinterpretationthatpresumptive eye-field al., 2005),werecovered atransgenicline, following thetechnologydescribedbyEllingsenetal.(Ellingsen sensitive transgenicretinalmarker. Inanenhancerdetectionscreen identity oftheprogeny ofthesetransplantedcells,wemadeusea adopt telencephalicidentity. type cellsaresubjectedtoanon-autonomouscellfate changeand when inaminorityinsideRx3-depletedenvironment, thewild- in amutantenvironment but keep theireye-field identity, orthat suggests eitherthatthewild-typecellsaretopologicallymisplaced evagination takes place(0%ofcases, the animalpoleofa with alow number ofcells.Whenafew wild-typecellstaken from marker forretinalspecification inourtransplantationexperiments therefore used evagination andisanearlymarker ofretinalspecification.We Fig. 7Cfora5-somiteembryo),andthusprecedesretinal CLGY469 (see Materialsandmethods).YFPexpression isobserved in As mappingindicates,thislineislikely todetectan expresses YFPintheretinabut notinthetelencephalon(Fig.7D). and isochronicallytransplanted intoa ckh type cellsaretransplantedintothepresumptive eye fieldofa cells (4-5).Indeed,weobserved thatwhenalow numberofwild- from morphogenesiscontrolbytransplantingasmallnumberof earlier functionofRx3was alsocellautonomous,weuncoupledit of eye fieldversus telencephalicidentity. To determinewhetherthis shown). transplanted intoawild-typehostnever contribute totheretina(not activated Fig. 6F-I).Becauseextensive labeledcloneswererecovered inall To discriminatebetween thesetwo possibilitiesandtoassessthe Our resultssupportanearlierroleofRx3inspecificationchoices ne2611 n =29). Bycontrast,alllabeledclonescontributed tothe host, theseintegrate intotheanteriorforebrainandno ckh transgenic embryosfromthetail-bud stageonwards (see ckh ne2611 CLGY469 ne2611 ckh ckh CLGY469 mutants (Table 1),weconcludethatretinal ne2611 ne2611 embryo, they oftengive risetoanormally embryos (100%ofcases, (see expression asaselective andsensitive transgenic donorwerehomotopically otx2 n =11), whichisalways composed RESEARCH ARTICLE on Fig.2B,or ckh n =14; Fig.7B).Thisfinding ne2611 non-transgenic host CLGY469 n atoh7 =5; Fig.7F),in n ckh rx3 =10; Table 1, ne2611 and enhancer , which vax2 2931 cells ,

DEVELOPMENT the anteriorforebrain ofa oa 63 100 1 10 9 90 10 100 3 1 34 the expense ofatelencephalicfate. Thisfunctionisperformedina 31 the determinantbiasingcellsofeye fieldtowards aretinalfate at 9 Our resultsdemonstratethat,atlategastrulation,zebrafishRx3is DISCUSSION autonomously encodedbyRx3expression. 21 field identity, andrepressionoftelencephalonfate, iscell- 6 66 7G; 86%ofcases, that contributed tootherstructures(usuallythetelencephalon;Fig. to YFPexpression incellsthatpopulatedtheretina,but notincells 45 similar graftsintonon-transgenicwild-typehostsusuallygave rise transplantation, werealreadydeterminedtoexpress thetransgene: 13 that thetransplantedcells,althoughYFP-negative atthetimeof conditions wherenomorphologicalretinaisvisible.We canruleout 100 29 Ret, retina; Hyp,hypothalamus;Tel, telencephalon;Tec, tectum. labeled clonescontributetothestructures indicated. Five independentexperimentswere conducted.Labeledcloneswere recovered inallembryos.Thevalueslistedare thenumber(or *Ventral structures oftherostral CNS(opticnerveandhypothalamus). Total % % Sum 5210102204740302203532003212124341220131110110Experiment Table 1.Fateofcellclonesuncaged,asindicatedinFig.6A,attheearlytail-budstagewild-typeand 2932 Fig. 7.Rx3controls eyefieldversustelencephalicfateinacell-autonomousmanner. white arrow). retina (red arrowheads) andtelencephalon(whitearrowhead; G,inset), but onlyretinal cellsexpress thetransgene(G,green a is rescued insometransplanted cells(red, inset),indicatingrescue oftheeye-field fate.Inawild-typehost,transplantedc transgenic a lownumberofcellsistransplanted, retinal evaginationdoesnotoccur. ( evaginate (arrow). Theyare entirely composedofwild-typecells,whereas transplantedcellsdistributerandomlyinawild-type pole ofa their descendants.Itisabsentin RESEARCH ARTICLE ckh CLGY469 ne2611 (F) orwild-type(G)host.Inamutant host(F),retinal evaginationdoesnotoccur(asinB),but expression ofthetransgene n embryos before (C;dorsal view) andafter(D;lateralretinal evagination.YFPexpression isrestricted toeye-fieldcells =7). We concludethatthemaintenanceofeye- n ckh ne2611 ckh eiaol e+eta N*RtTlTel Ret+Tel Ret+ventralCNS* Retina only ne2611 or wild-type(inset)host.(A)Whenalargenumberofcellsare transplanted,retinal structures are rescued and mutants (seetext).( e / e e±tesTel±others Tel±others Ret w/oTel Wild type E-G ) Transplantation ofafewwild-type cellstransgenicfor C,D molecule, Rx3. epithelium, areaccomplishedatleastinpartbytheuseofsame retina intoanopticcupandthespecificationofpigmentedretinal maintenance ofretinalprecursors,themorphogeneticshaping between several crucialstepsofretinaldevelopment, namelythe epithelium (Rojas-Munozetal.,2005),they alsosuggest thatthelink Winkler etal.,2000)andformationofthepigmentedretinal (Kennedy etal.,2004;Loosli2003;2001; demonstrated rolesofRx3incontrollingretinalevagination field duringanteriorforebrainpatterning.Given thepreviously shed lightontheprocessessegregating thetelencephalon andeye molecular componentoftheanteriorforebrain-patterningcascade, tlc cell-autonomous manner, andisaccompaniedbytherepressionof ) Expression ofYFP(revealed byanti-GFPimmunocytochemistry)in and foxg1 expression byRx3.Thesefindings,whichidentifya ( A,B ) Transplantation ofwild-typecells(brown) into percentage) ofuncagedembryoswhere n ckh ells contributetothe CLGY469 chokh ne2611 Development 133(15) e Tel+Tec Tel host(inset).(B)When rrows, asopposedto ne2611 mutant siblings into theanimal (green) and

DEVELOPMENT our re-analysisof further decisive argument towards thisinterpretationisprovided by controls theearlydevelopment ofbothtelencephalonandeyes. A BAC supporttheinterpretationthatRx3,directlyorindirectly, and areconcomitantlyrescuedbyinjectionofan ckh stages onwards, respectively, inamannerindistinguishablefrom expanded in thespecificationofearlyanteriorprogenitors. (Bailey etal.,2004)lendssupporttoaprimaryancestral roleofRx genes duringbraindevelopment invertebrates andinvertebrates However, theevolutionary conservation ofanearlyexpression ofRx dependent effect ofRx3inzebrafish(Rojas-Munozetal.,2005). relate totherecentdemonstrationofageneticbackground- all canberevealed intheMedakacontext. Thisobservation might and MedakaRx3/elmighthave similarregulatory capacities,but not rescue the cause headtruncations(Looslietal.,2001).Rx3/elcan,however, 2001) (thispaper),overexpression ofRx3/elinMedakadoesnot Winkler etal.,2000).Unlike zebrafishRx3(ChuangandRaymond, morphogenesis defects,isamorepuzzlingcase(Looslietal.,2001; where lossofRx3/elseemstobeonlyassociatedwith with anearlyroleofRxincontrollingretinalspecification.Medaka, downregulation ofretinalmarkers inthesemutantsiscompatible be difficult toappreciateexperimentally; however, theprecocious mouse harborsasingle anterior forebraintruncations(Mathersetal.,1997).Given thatthe Rx presumptive telencephalon(Chuangetal.,1999).Thephenotypeof rx2 zebrafish at lategastrulation.Thisfindingisinkeeping withtheonsetof an earlyroleforRx3inlimitingextension ofthetelencephalicfield telencephalic andeye phenotypesin results solelyfromthelossofRx3function.Ourobservations that al., 2005),makingitcrucialtoascertainthatthe defects (Kennedy etal.,2004;Loosli2003; Rojas-Munozet Previous analysesof loss ofRx3function The complete Rx3 activitysegregates telencephalonandeyefield , andinadomainadjacenttobut non-overlapping withthe ne2611 mutant miceiscomplex andincludesbothlackofeyes and (Fig. 3F, Fig.4D).Theseobservations leadustopropose rx3 ckh tlc expression duringgastrulation,earlierthan and phenotype (Looslietal.,2003).Thus,zebrafishRx3 ckh ne2611 emx3 s399 ckh Rx , andthefindingthat expression fromthetail-bud andsomite mutant allelesdidnotreporttelencephalic gene, successive functionsforRxmight phenotype results from the ckh ne2611 ckh always co-segregate s399 ne2611 mutants display rx3 -containing phenotype rx1 and ckh 2003). Whethertheimpairedproliferationofeye fieldin progenitors inectopicexpression experiments (Casarosaetal., which hasbeenshown toincreasetheclonalproliferationofretinal This findingisinlinewithprevious analysesof promotes proliferationofitsexpressing cellsatlategastrulation. suggesting thatRx3,inadditiontoimpartingcellidentity, also proliferation ofeye-field precursorsatthetail-bud stage(Fig.5), and identity isaltered,asrevealed bytheco-expression of first, theirproliferationisreduced(Fig.5B,E);andsecond, expressing retinal precursors,althoughatleastpartiallyspecified(e.g. independent rolesofRx3.Contrarytothishypothesis,wefoundthat have beensubsequentbut unrelatedphenomenareflectingtwo The expanded telencephalonandlackofeyes of telencephalic identity Rx3 functionmaintainsretinal versus factors actinatimelyregulated cascade. identities isageneralpropertyofRxfactors, and,ifso,whetherthese determine whetherthedistinctionbetweeneye andtelencephalic be importanttosupportthesedatabyloss-of-functionanalyses foxg1 observed atalatestage(neuralkeel), whilenoalterationin fate switchimposedbyinjectionof and Raymond,2001).Notablyhowever, thetelencephalictoretinal postulated topromoteretinalversus telencephalicidentity(Chuang retinal versus telencephalicspecificationduringgastrulation. by postulatinganearlyroleofRx3inpermittingthemaintenance telencephalic fate (Fig.6).Together, theseresultsarebestinterpreted telencephalon insteadoftheretinaandprobablyacquirea further demonstratesthatthesecells,inmutants,populatethe embryos. Directtracingofeye-field cellsfromthetail-bud stage stage (Fig.4G,J),combinationsnever observed inwild-type and retinalidentity. remains toberesolved. Similarly, Xrx1promotesbothproliferation cells orreflectsanindependentroleofRx3inproliferationcontrol late gastrulationin An additionalphenotypeof Based onoverexpression studies,zebrafishRx1andRx2were ne2611 rx3 expression couldbedetectedattheneuralplatestage. It will within theeye fieldin mutants isaconsequenceofthealteredidentitythese rx3 (red) represses thelatterdomain,Rx3activity anterior forebrain. Within star) isset-upinfront ofthediencephalon,isolating boundary(blackarrowpanel), aposteriorpatterning with telencephalon andeyes.Atthetail-budstage(middle anterior domain(purple)includingthepresumptive diencephalon (green), whereas ‘lowWnt’definesan and Houart,2004).‘HighWnt’definesthepresumptive antagonists (purplegradient)locatedattheANB(Wilson the posteriordiencephalon(green gradient)andWnt activities ofaposteriorWntsource locatedatthelevelof that theforebrainasawholebyopposite ispatterned arrows. Duringgastrulation(leftpanel),aprevalent view is patterning. and eyeanlageduringzebrafishforebrain Fig. 8.Modelforthesegregation ofthetelencephalic eyes andanenlarged telencephalon(rightpanel). fate (bottompanel),leadingat24 hpf totheabsenceof absence ofRx3,retinal precursors acquire atelencephalic retinal andtelencephalicidentities (toppanel).Inthe at theexpenseofatelencephalicfate, thereby segregating (blue bar)andattributesretinal fatetoitsexpressing cells and ckh rx1 ne2611 RNAs, Fig.2C-D,H),areaffected alreadyat Patterning boundariesarePatterning indicatedbyblack tlc mutants. Thisisnoticeableintwo ways: expression (bluegradient) and ckh ckh ne2611 ne2611 RESEARCH ARTICLE rx1 at thetail-bud orone-somite mutants isthedecreased or rx2 ckh mRNA was only Xenopus mutants might tlc foxg1 or emx3 Xrx1, foxg1 2933 or

DEVELOPMENT and permit ustodeterminewhethertheregulation ofexpression of unresolved (ChuangandRaymond,2001),ourresultsdonot factors displaytranscriptionalrepressororactivator functionsis that thesegenesrespondtotheearlyfunctionofRx3.WhetherRx the normaltimingofopticvesicle evagination, stronglysuggesting process. First,theearliestalterationingeneexpression in possible involvement ofWntsignalinginthisearlyRx3-dependent and Houart,2004),remainsunknown. Several findingssuggesta reminiscent ofglobalforebrainpatterningatanearlierstage(Wilson process involves gradedpositionalinformation,inamanner also necessitateinstructive information. defined bydefault bytheabsenceofRx3expression, orwhetherthey to bedemonstrated.Itisalsounknown whethertheseprecursorsare at thatstagewould alsobecapableofacquiringaretinalfate remains choices inbipotentialprecursors.Whethertelencephalicprecursors adopt atelencephalicfate, demonstratingthatRx3biasescellfate telencephalic identities.Without Rx3function,retinalprecursors process specifictotheanteriorforebrainandsegregating eye versus field (Fig.8,asterisk),demarcatingtheposteriorlimitofapatterning the patterningfieldisestablishedbetweendiencephalonandeye global APforebrainpatterningduringgastrulation,aboundaryof segregation ofthetelencephalonandeye field(Fig.8).Following These resultspermitforthefirsttimeproposalofamodel possible exception oftheanteriorhypothalamus(see Fig. 1E-I). retinal fieldsbut sparesmostotherdiencephalicdomains,withthe cell identitychoicesbetweenthepresumptive telencephalicand A majorfindingofourstudyisthat,atlategastrulation,Rx3controls forebrain intotelencephalonandeyefield A modelforthesubdivisionofanterior maintenance process. might beanintegral partoftheRx3-encodedspecification distinguish themfromtelencephalicprecursors.Thisphenomenon cells withspecificcellsurface recognitionpropertiesthat field identitytoitsexpressing cells,Rx3mayalsoendow these telencephalon (notshown). Thus,inadditiontoattributing aneye- animal poleofawild-typehostpreferentiallypopulatethe Rx3: identify here,however, two new andpossiblyimportanttargets of preventing astraightforward interpretationoftheseresults.We both genesalsolabelthetelencephalicprimordiumatthatstage, unaffected in in of Rx3include in ourunderstandingofanteriorforebrainpatterning.Known targets Rx3-mediated retinalspecificationcascadewillbeanimportantstep (Carl etal.,2002),andidentifyingtheupstreamregulators ofthe The initiationof telencephalon development ofthepresumptive eyefieldversus Molecular cascadeofRx3functioninthe 2934 belongs, like Tlc,tothesFRPfamily of Wnt-bindingfactors (Esteve recently proposedtoalsoparticipate inanteriorforebrainpatterning, interaction ofRx3withWntactivity. Finally, olSfrp1,whichwas overexpression phenotypemightresultfroma(premature)positive exaggerated Wntsignaling (Kimetal.,2000).Thus,theRx3 to headtruncations(Fig.3),and asimilarphenotypeiscausedby gradient). Second,overexpression of is theectopicmaintenanceof Finally, weobserved that Whether thespecificRx3-mediatedanteriorforebrainpatterning ckh foxg1 tlc (Kennedy etal.,2004). RESEARCH ARTICLE and by Rx3isdirect. foxg1 ckh rx2 ne2611 rx3 . Theirexpression ismodifiedin and expression doesnotdependonPax6 orSix3 mab21l2 at the3-somitestage(notshown); however, tlc ckh six3 , whichareabsentordownregulated expression (Fig.4A-G;Fig.8,blue ne2611 or rx3 pax6.1 cells transplantedintothe by mRNA injection leads expression appeared ckh ne2611 ckh prior to ne2611 tlc during this work, to H. Baier and J. Wittbrodt forthe during thiswork,toH.BaierandJ.Wittbrodt We are gratefultoK.Imai,R.Koesterandotherlabmembersforcriticaladvice anterior forebraincell-specificationdefectsof et al.,2004).Thus,whetherTlcactivity isinstrumental inthe Bailey, T. J.,El-Hodiri,H.,Zhang,L.,Shah, R.,Mathers,E.H.andJamrich,M. References http://dev.biologists.org/cgi/content/full/133/15/2925/DC1 Supplementary materialforthisarticleisavailableat Supplementary material University ofBergen. (FUGE) intheResearch CouncilofNorway, andbyadditionalfundingfrom the core grant,bytheNationalProgramme inFunctionalGenomicsNorway 2003-503466. Work inT.S.B.’s laboratoryisfurthersupported byaSarsCentre and C.S.,L.B.-C.T.S.B. byEU6thFrameworkcontractnumberLSHC-CT- probes. C.S.andL.B.-C.are supportedbyaVWStiftungjuniorgroup grant, help withENUmutagenesis,andtonumerous colleaguesforthegiftof might accountforthe issues toaddress.Another, non-exclusive, interestingcandidatethat field/diencephalon borderatlategastrulation,remainimportant an appropriatesourceofWntispositionedattheeye bars). However, theearlyexpression domainof Casarosa, S.,Amato,M.A.,Andreazzoli, M.,Gestri, G.,Barsacchi,G.and Carl, M.,Loosli,F. andWittbrodt, J. or absenceofRx3. context-dependent activities, whichmightberelatedtothepresence versus eye specificationwould becomplex andislikely toimply and C.H.,unpublished).Thus,Foxg1 involvement intelencephalic includes asmallportionoftheventral retinalfield(LisaWinstanley is notcompletelyrestrictedtothetelencephalicprimordiumbut also Chuang, J.,Mathers,P. andRaymond,P. Chuang, J.C.andRaymond,P. A. Heisenberg, C.P., Houart, C.,Take-Uchi, M.,Rauch,G.J.,Young, N., Hanson, I.M. Hammerschmidt, M.,Pelegri,F., Mullins,M.C.,Kane,D.A.,vanEeden,F. J., Haffter, P., Granato,M.,Brand,Mullins,M.C.,Hammerschmidt, Graw, J. Foley, C.D. A.C.andStern, Esteve, P., Lopez-Rios,J.andBovolenta,P. Ellingsen, S.,Laplante,M.A.,König,M.,Kikuta,H.,Furmanek,T., Hoivik,E. Eagleson, G.W. andDempewolf,R.D. Cuff, J.A.,Clamp,M.E.,Siddiqui,A.S.,Finlay, M.andBarton,G.J. 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