required fortheinductionactivityofisthmicorganizerinMHB. Fgf8 our resultsindicatethat ad .Johnson L. Randy Accepted 15November 2006 † 4 2 1 Fgf8 expression islocatedanteriortothatof Meyers etal.,1998;Reifers1998).Thedomainof (Chietal.,2003; MHB leadstothelossoftectumandcerebellum 1999; Nakamuraetal.,1986).Conversely, inactivation of (Crossley andMartin,1995;Crossley etal.,1996; Martinezetal., ectopic tectum(dorsalpartofthemidbrain)andcerebellum regarding theinductionof effects ofMHBgraftsinchickembryos Wurst andBally-Cuif,2001).BeadscoatedwithFGF8mimicthe and (LiuJoyner, 2001b;Nakamuraetal.,2005; ofthemidbrain forthedevelopment arerequired Cuif, 2001). (Liu andJoyner, 2001b;Nakamuraetal.,2005;Wurst andBally- planar signalsforregulating thedevelopment ofthemid/hindbrain anteroposterior axis.Theisthmicorganizer isthoughttosecrete ofthecentralnervous system(CNS)alongthe regionalization the bestmodelstostudymolecularmechanismof (MHB).Theisthmicorganizer serves asoneof hindbrain boundary - the isthmicorganizer intheisthmus,aconstrictionat by The development ofthemidbrainandcerebellumarecontrolled INTRODUCTION KEY WORDS: embryos showedthatmanygeneswerelostbyE9.5.IntheMHBof and itsexpressionbecomesprogressivelyrestrictedtotheisthmusatE9.0.AnalysisofgeneinMHBmutan remains tobeelucidated.Hereweshowthatthe the midbrain-hindbrainboundary(MHB).However, transcriptionalregulationofthesesecretedfactorsduringdevelopment Secreted factorsFGF8andWNT1areessentialeitherfortheinductiveactivityofisthmusorganizerorregionalizat Chao Guo development inmice isthmic organizerduringtectumandcerebellum Lmx1b Development 134,317-325(2007)doi:10.1242/dev.02745 these moleculardefects,thedevelopmentoftectumandcerebellumwasseverelyimpairedin expression intheMHBwasfurtherconfirmedby Teeatoscnrbtdeulyt thiswork *These authorscontributedequallyto School ofMedicinePainCenter, Washington University, StLouis,MO63110,USA. Medicine, Shanghai200025,China. for BiologicalSciences,ChineseAcademyofShanghai200031,China. Biology, UniversityofTexas, MDAndersonCancerCenter, Houston,TX77030,USA. transcription factors at the4-somitestage,wascompletelyabsent,whereas the 4-somitestage.Bycontrast, Author forcorrespondence (e-mail:[email protected]) ofAnesthesiology, Psychiatry, MolecularBiologyandPharmacology,Departments Department ofNeurology, RuijinHospital,ShanghaiJiaotongUniversitySchoolof Institute ofNeuroscience andKeyLaboratoryofNeurobiology, ShanghaiInstitute w ertdfcos G8adWT,emanatedfrom the FGF8andWNT1, Two secretedfactors, , , Wnt1 Wnt1 and severalisthmus-relatedtranscriptionfactorsintheMHB,isacrucialcomponentofcross-regulatorynetwork 1, does nothave inductionactivity but isessentialfor Lmx1b *, Hai-Yan Qiu is essentialfor , Isthmicorganizer, En1 3 , Zhou-FengChen Lmx1b and 3 Department ofBiochemistryandMolecular Pax2 2, plays anessentialroleinthedevelopmentoftectumandcerebellumbyregulatingexpression *, Ying Huang Otx2 were alsodownregulatedpriortothe4-somitestage,whereas and Fgf8 Fgf8 Pax6 , Tectum, Cerebellum 4 in theMHB.Unlike and Yu-Qiang Ding expression wasnotaffected in Fgf8 1 Wnt1 LIM , HaixuChen Fgf8 -Cre-mediated region-specificconditionalknockoutof Wnt1 and in the Wnt1 was downregulatedbeforethe4-somitestage.Moreover, 3 , Rong-QiangYang Lmx1b 1,† Wnt1 al., 1999).Theexpression of Li andJoyner, 2001;Milletetal.,1999;Rhinn1998; that of of the (Crossley andMartin,1995;LiuJoyner, 2001b).Theexpression Cuif, 2001).Within theMHB, et al.,2005;Simeone,2000;Simeone2002;Wurst andBally- (Joyner, 1996;Joyner etal.,2000;LiuandJoyner, 2001b;Nakamura andposteriorepiblast,respectively anterior in theCNSandmark development. expressed intheMHBarealsoinvolved inmid/hindbrain the inductionactivity oftheisthmicorganizer. Bradley, 1990).Therefore,FGF8isthekey moleculeformediating development (McMahonetal.,1992;McMahonand mid/hindbrain known functioninneuronaldifferentiation, 2003; Dingetal., 2004;Smidtetal.,2000).In additiontoitswell horn (Asbreuketal.,2002; Chengetal.,2003;Ding serotonergic neurons,andmedullaryspinaldorsal hindbrain is essentialforthedifferentiation ofmidbraindopaminergic neurons, al., 1998;Morelloet2001). DuringCNSdevelopment, nail-patellasyndrome (Chenet inheriteddiseasecalled dominantly development (Chenetal.,1998).Itsmutation inhumancausesa orthologue ofchicken controlof involved intranscriptional the MHB(Ye etal.,2001).However, whetherthereareothergenes of is foundtobenecessaryandsufficient fortheinduction Thomas andCapecchi,1990;Wurst et al.,1994).Bycontrast, of initiation for theinductionof to setupthepositionofisthmicorganizer, but arenotrequired organizer isunclear. nadto to In addition The LIMhomeodomaintranscriptionfactor Lmx1b En1/En2 –/– En2 embryos, theexpressionof is expressedintheanteriorembryoasearlyE7.5 Lmx1b and expression inthe Fgf8 Otx2 isrequiredforthemaintenancebut notforthe –/– Pax5 Fgf8 and or embryos. Therequirementofspecific Fgf8 1 , Sheng-DiChen Wnt1 Gbx2 is laterthan Lmx1 and or Wnt1 En1 r mn h earliestgenesexpressed are amongthe expression (LiuandJoyner, 2001a; Wnt1 , whichisrequiredforthelimbbud Gbx2 Otx2 Lmx1b , xrsin(Broccolietal.,1999; expression Pax2 , transcriptionfactors thatare RESEARCH ARTICLE Fgf8 and Fgf8 downregulation occurredat Fgf8 –/– and mice. Taken together, Gbx2 , whichnormallyoccurs expression intheisthmic expression intheMHB 2 Lmx1b Wnt1 , Lmx1b act antagonistically Lmx1b is earlier, whereas . Asaresultof is expressed in is amouse Lmx1b ion of Fgf8 Lmx1b t Pax2 317 in

DEVELOPMENT MATERIALS ANDMETHODS organizer duringmid/hindbraindevelopment. initiation andmaintenanceoftheinductionactivity oftheisthmic Wnt1 expression andforthemaintenanceofseveral othergenes,including that genes.Ourresultsdemonstrate on otheristhmicorganizer-related organizer. Inaddition,weexamined theeffect of of functionof We focusedonthe analyze theroleof et al.,2005). structures andthelossof Wnt1 heterozygous a mixed geneticbackground.To inactivate Kimmel etal.,2000;Zhao2007).Allmouselinesweremaintainedon (Chenetal.,1998;Danielian genotyped aspreviously described band andthelittermates(e.g. obtained, were fixed again with4%PFA for1hour andpermeabilizedinasolution of treated withproteinase K(20 in methanol,thenrehydratedPBS containing0.1%Tween 20(PBT) and withsomemodifications. Embryoswerefixed in4%PFA andstored 2003) Whole-mount TUNELwas performedasdescribedpreviously (Chietal., Whole-mount TUNEL andsectionedat12 embedded inparaffin wax saline (PBS;pH7.4).For NisslstainingwithCresylViolet, thebrainswere perfused with4%paraformaldehyde(PFA) in0.01Mphosphatebuffered 0pupswere (P) E18.5embryosandpostnatalday (Ding etal.,2004). andimmunocytochemistry wereperformedasdescribed Nissl staining hybridization assays Histological analysis,immunohistochemistryandinsitu Lmx1b Genotyping andmaintenanceofanimals of (Adams etal.,2000;Matsunaga2002),whereasknockdown tectum andcerebelluminduces Lmx1b the developing MHBinchickandzebrafish, andmisexpression of 318 Fgf8 embryos wereused. experiments, atleastthreemutantembryosandanequalnumberofcontrol Ineachsetof (Kaufman,1998). according tocriteriadescribedpreviously differ, theactualdevelopmental stageoftheembryowas furtherascertained embryonic day(E)0.5].Becausethematingtimeamongmicemay age ofembryoswas determinedaccording totheplugdate[consideredbe Lmx1b cre expression of 4% PFA inPBS,anddigoxigenin-labeled ribroprobeswereusedtodetect proliferating cells. were employed tolabel Rabbit anti-Ki67 antibodies(Novocastra) used. IgG(Jackson)were IgG(Jackson)andFITC-labeleddonkey anti-goat rabbit (Sigma)withBRN3b(SantaCruz),Cy3-labeleddonkey anti- calbindin after cryoprotectionwith15%sucroseinPBS.For doublestainingof immunostaning, thePFA-fixed brainsweredirectlysectionedonacryostat double insituhybridizationof and (Schwarz etal.,1999) al., 2003), ioiei and visualizedindarkbluewithsubstratesofBCIPandNBT. digoxigenin TR/Napthol AS/MX(Sigma),whereastheotherprobeswerelabeled with situ probewas labeledwithfluoresceinandvisualizedinredFast Red Whole-mount insituhybridizationwas performedonembryosfixed in In thepresentstudy, weused aloss-of-functionapproachto Fgf8 , Lmx1b - (Wassarman etal.,1997), Lmx1b , cre +/flox En1 mutant, RESEARCH ARTICLE , theessentialfactor fortheinductionactivity oftheisthmic in theMHBofchickembryocausesexpansion ofthe , Lmx1b in thezebrafish resultsinthelossofisthmicandcerebellar +/– Otx2 eeoe omly hywr sda oto mro.The normally;they wereusedascontrolembryos. ) developed , En2 offspring werematedwith Lmx1b expression isnecessaryfortheinitiationof Lmx1b -/flox (Martinez-Barbera etal.,2001;Suda2001), Lmx1b , Pax2 (hereafter referredtoas Lmx1b (Ding etal.,2004), flox/flox mice with and Wnt1 ␮ , and nmdhnbandvlpetinmice. in mid/hindbraindevelopment Lmx1b Gbx2. g/ml inPBT)for2-8 minutes.Theembryos Gbx2 and Lmx1b Wnt1 DneinadMMhn 96.For (Danielian andMcMahon,1996). Wnt1 Wnt1 (Li andJoyner, 2001), with wnt1 Thus, - cre En1 Cemc weregeneratedand -Cre mice Lmx1b Wnt1 Cemice,andtheir -Cre Lmx1b in regulating theexpression Fgf8 expression aswell(O’Hara / Lmx1b Lmx1b Lmx1b , ␮ En2 m inthesagittalplane.For or with flox/flox and in theMHB,wecrossed (Li andJoyner, 2001), +/flox w is essentialforthe CKO) progeny were Lmx1b Wnt1 Fgf8 mice. Thedesired , or Hoxa2 ,the Lmx1b expression expression Lmx1b (Chi et –/flox Wnt1 Fgf8 Pax2 or in - the expression (G,H,red arrows) overlappedwith there wasa sequentially forthedetectionof isthmus,asshown forthesameembryoprocessed arrows) inthe ( MHB atE8.5(B,arrow) andintheisthmusatE9.5(C,arrow). E7.5 intheanteriorembryo(A,arrow), appeared atthe prospective mount insituhybridization.Expression of localization intheMHB. Fig. 1.Expression of the MHB.( To characterizedynamicexpression of Lmx1b RESULTS BCIP. inalkalinephosphatasebuffer containingNBTand signals werevisualized antibodies(Roche),andfinally the phosphatase-labeled anti-digoxigenin (Roche)at37°Cfor1hour, thenwithsheepalkaline digoxigenin-dUTP embryos wereincubatedwith1 0.1% sodiumcitrateandTriton X-100for5minutesonice.The Wnt1 the MHB, wecompared shown). To furtherdefine thelocationof the isthmusbetweenE9.0andE10.5(Fig.1C,Gdatanot restrictedto presumptive MHB(Fig.1B),andbecameprogressively the Fgf8 oancvrdtemjrt of thesetwodomains.( domain covered themajority (arrowheads), whereas the non-overlapping patterns 4-somite stage.Expression of Fig. 1A).ByE8.5, detected intheanteriorembryoearlyhead-foldstage(E7.5; of ThemRNA transcript . atdifferent stagesof performed mount insituhybridizationwas covering thecaudalhalfof whereas Fgf8 D-F Lmx1b Fgf8 Expression of ) + hwda neootro o-vrapn xrsinpattern, showed ananteroposteriornon-overlapping expression and oani h H tte4-somitestage(Fig.1D-F).This the domain intheMHBat + Lmx1b domain (bluearrow) intheisthmus. is expressed intheisthmicorganizer + I Fgf8 ) Doubleinsituhybridizationfor Lmx1b domain (red arrow) overlaps withtheanteriorportionof at the4-somitestage.Theexpression of expression overlapped withboth + Wnt1 oanlctdpseirt the domain locatedposteriorto Lmx1b Lmx1b (D), ( Lmx1b A-C Wnt1 Fgf8 ϫ Wnt1 in thedevelopingembryoandits ) D ufr(Promega) containing40 TDT buffer Lmx1b Lmx1b expression was observed inthe E and (E) expression domainwiththoseof and + n oto h ertr fthe of and mostoftheterritory and expression revealed bywhole- Fgf8 Lmx1b Lmx1b Lmx1b Lmx1b Wnt1 Lmx1b showed anteroposterior Wnt1 Development 134(2) was firstdetectedat F intheMHBat (F) in theMHB,whole- RNAs. Notethat and expression (H,blue Wnt1 G Lmx1b expression inthe Lmx1b Fgf8 , H Fgf8 ) Lmx1b + domain in . Notethat expression and Wnt1 was first Wnt1 and ␮ M ,

DEVELOPMENT Lmx1b reduced cerebellum fusedwiththesuperiorcolliculus(SC)in sections ofP0brains.Theinferior colliculus (IC)wasmissingandthe PT, pretectal region; Th,thalamus.Scalebars:700 plexus;Hy, hypothalamus; Purkinje cellsinthe cerebellum). cp,choroids BRN3b (markingICandlaminated SC)andforcalbindin(marking and thetectumwere much smallerinthe cerebellum (Cb)wasmissing,andtheremaining part ofthecerebellum ofthe mid/hindbrain region ofP0mousebrains.Themedialportion connected withthesuperiorcolliculusinmutant(Fig.2F).In colliculus inthecontrolbrain(Fig.2C,E),becamedirectly 2E,F). Thecerebellum,whichislocatedcaudaltotheinferior calbindin, markers forthetectumandcerebellum,respectively (Fig. and MouseGenomeInformatics)(Xiangetal.,1996) (POU4F2 – 2D), whichwas furthershown bydoubleimmunostaining ofBRN3b appeared tofusewiththemuchreducedsuperiorcolliculus(Fig. colliculus) was missing(Fig.2B,D).Thereducedcerebellum the cerebellumwas notvisibleandthecaudaltectum(inferior of localized totheisthmicorganizer andoverlaps withtheexpression compared withthewild-type control (A).( adlybyn the caudally beyond Fgf8 Wnt1 insituhybridizationfor double whole-mount was alsoobserved atE9.0by overlapping expression characteristic Lmx1b of thetectumandcerebellum. Fig. 2. the caudal aitlbanscin.I the sagittal brainsections.In wild-type (Fig.2A,B).Thiswas corroboratedbyexamination of withthe cerebellum weresignificantly reducedinsizecompared alonethatthetectumand apparent frommacroscopicexamination To testthehypothesesthat and cerebellum development Lmx1b induction activity oftheisthmicorganizer. Lmx1b the thedevelopment ofthetectumandcerebellumin analyzed induction activity oftheisthmicorganizer, wesystematically Wnt1 + or –/– domain (Fig.1I).Thisconfirmed that Lmx1b –/– is vitalforisthmicorganizeractivity and brain. ( Fgf8 gene deletionleadstodefectivetectum mutant. AtE18.5andP0,intheabsenceof Lmx1b Fgf8 . The deletion results inamarkedreduction inthesize E , F suggestingthat , + ) Doubleimmunostainingofthebrain sectionsfor domain overlapped withtheanteriorpartof Lmx1b Wnt1 + + domain (redarrows inFig.1G,H),and oanoelpe u loextended domain overlapped but also Lmx1b Lmx1b ( A , B Lmx1b –/– ) Dorsalviewofthe mutant, themedialportionof in theMHBregulates the Lmx1b C , D may beimportantforthe ) ParasagittalNissl-stained –/– Lmx1b ␮ ri B as (B) brain Lmx1b m. Lmx1b expression is with either it was Lmx1b the expression was detectedfromthe4-somitestagetoE12.5in Fgf8 of inFig.4B),suggesting specific regulation was normal(arrowhead By contrast, expression of type control(arrows inFig.3I,J).Intheventral tegmentum, inthemutantembryoascomparedwithwild- a caudalextension marker forthemidbrain;seebelow) expression domaindidnotshow the severe abnormalitiesinthetectumandcerebellum, that inthecontrolembryoatthisstage(Fig.3G,H).Despitethese presumptive cerebellumwas dramaticallyreducedcomparedwith Math1 diin h eerlcortex andpretetcalregion in addition, thecerebral the thatany changeinthegene expression profile in 2003), itispossible attheMHBregion (Chi etal., loss ofexpression ofother genes inactivation of En1 Lmx1b Fgf8 that a lal bevdi the was clearlyobserved in smaller inthemutantembryo(Fig.3A,B).Lossofthisconstriction the midbrainandhindbrainwas notapparentandrhombomere1was observed atE10.5.We foundthatthenormalconstrictionbetween different embryonicstages.Morphologicaldefects werefirst eetbei the detectable in (see below). control (Fig.2C,D),presumablyasaresultofthereducedtectum wild-type seemedtoextend morecaudallycomparedwiththe mice xrsinwsasn nthe expression was absentin presumptive MHBatthe4-somitestage(Fig.4A).Strikingly, the at whichmorphologicaldefectsintheMHBwerefirst observed in E10.5,thestage during mid/hindbraindevelopment atstagespriorto betweentheexpression of relationship Bally-Cuif, 2001).Therefore,wesoughttodissectthepossible hindbrain (LiuandJoyner, 2001b;Nakamuraetal.,2005;Wurst and ofthemidbrainand essential forthedevelopment is organizer, whichisemanatedfromtheisthmic The secretedfactor FGF8, expression Lmx1b cerebellum. defectsinthedevelopment ofthe tectumand causes severe 3N). Taken together, thesefindings indicatethatdeletionof (Fig. 3P),althoughthecerebellarvermis was notseendorsally(Fig. atE15.5 tegmentum, remainedunchangedinthemutantembryo Genome Informatics),amarker fortherednucleusin (Smidt etal.,2000).However, expression of 3L,R anddatanotshown), whichisconsistentwithprevious findings midbrain) weredownregulated atE12.5andwerelostE15.5(Fig. inthe tyrosine hydroxylase(markers fordopaminergic neurons Lmx1b alsoaffected bythedeletionof was et al.,2002) genes(Joyner, 1996;Joyner etal.,2000;Simeone organizer-related examined whethertheexpression of We next To define theearlieststageatwhichhistologicalchangesis In thewild-typeembryo, Fgf8 Lmx1b Lmx1b Lmx1b , expression intheisthmicorganizer. expression intheMHBdisappearedaroundE12.5.No En2 –/– ( expression by is requiredfortheinitiation of Atoh1 is required forthemaintenanceof is required fortheinitiationof mro(i.4 n aantson.Tu,weconclude embryo (Fig.4Danddatanotshown). Thus, –/– –/– and expressed intheMHBisrequiredforinitiationof Fgf8 mromay beduetothelossof embryo embryo. Fgf8 Nurr1 – MouseGenomeInformatics)expression inthe Lmx1b Pax2 xrsini te ein ftemtn embryo expression inotherregions ofthemutant in theMHBcausesdownregulation andeventual ( Nr4a2 Lmx1b –/– expression mutant, Nisslstainingwas performedat Lmx1b Fgf8 Lmx1b MouseGenomeInformatics)and – in theMHB.Incontrolembryo, expression was first observed inthe –/– –/– RESEARCH ARTICLE embryo atE12.5(Fig.3F),and mutant atthisstage(Fig.4B). Lmx1b Brn3a Fgf8 Wnt1 Fgf8 and thatof and otheristhmic ( expression, and Lmx1b Pou4f1 Fgf8 expression. We Lmx1b Wnt1 –/– – Mouse Otx2 . Since mutant Lmx1b Fgf8 Fgf8 Fgf8 , 319 (a

DEVELOPMENT and r1of Ki67, showingareduced numberofproliferating cells inthetectum bars: A-D,1000 wild-typeembryo (Q).Aq,aqueduct;Cb,cerebellum. Scale the with (TH) immunostaininginthetegmentum ofthemutant(R)ascompared sections ofembryoatE15.5,showing thelossoftyrosine hydroxylase compared withthecontrol brain(A,arrow). ( and lessconspicuousisthmusinthemutantbrain(B,arrow), as sections ofembryosatE10.5,showingthereduced rhombomere 1(r1) embryos ascompared with control brains(M,O).( expression inthepresumptive red nucleus(P, arrow) inthemutant the lossofcerebellar vermis (N,arrow) andnormal control (K).( ventral midbrainofthemutantembryo(L)ascompared withthe embryos atE12.5,showingreduction of with control brains(E,G).( presumptive cerebellum (H,arrow) inthemutantembryo,ascompared arrowhead) andreduced loss oftheconstrictionbetweenmidbrainandhindbrain(F, (C) atE10.5.( not showacaudalextensionin rlfrto nthe proliferation in Fig. 3.Earlymorphologicalchangesandreduced cell Fgf8 areexpressed intheMHBpriorto first focusedonthosegenesthat 320 compared withwild-type(I)atE12.5.( (Fig. 4H,L,P).Becausethereductionin byE9.5 werehighlyexpressed inthewild-type embryo (Fig.4E,I,M).Theexpression was lostaltogether they whereas 4F,J,N), was drasticallyreducedintheMHBat3-somitestage(Fig. . In RESEARCH ARTICLE Lmx1b Lmx1b M-P E-H ␮ –/– ) Transverse sectionsofembryosat E15.5,showing –/– ) SagittalsectionsofembryosatE12.5,showingthe m; E-L,700 embryo (D)ascompared withthatinthecontrol mro,theexpression of embryos, Lmx1b Math1 I , J ) Expression domainof –/– ␮ Lmx1b ;M-R,500 m; embryo. xrsin(,,arw)inthe (E,F, arrows) expression –/– K , L Nurr1 uatebys(J)as mutant embryos ( ) Transverse sectionsof A , B C ␮ ) Nissl-stainedsagittal , m. D expression inthe Wnt1 ) Immunostainingof Q Wnt1 , Otx2 R ) Transverse , Brn3a En1 , En1 (arrow) did and and Pax2 Pax2 TUNEL gene downregulation was prominent. A smallbut similarnumberof correct positioningoftheisthmicorganizer, thelossof Lmx1b in hindbrain (arrow inFig.5C), which the embryo,in somite stage(Fig.5B).However, unlike thewild-type Otx2 However, wefoundnochangeineitherthelevel orthelocationof (Simeone, 2000;Simeoneetal.,2002;Wassarman etal.,1997). trbtdsml oicesdcl et,a hsocre afterthese toincreasedcelldeath,asthis occurred simply attributed the Lmx1b determine whetherthiswas causedbyabnormalcell death in in midbrain/cerebellumdevelopment was downregulated. To of Lmx1b embryo, thereductionshouldhave resultedfromtheinactivation of expression occurredbeforetheabsenceof between the3-and13-somitestages,when Gbx2 Fig. 5C,D),suggestingspecific in expression isnotaffected bythedeletionof death was detectedintheMHB(seebelow). Taken together, increasedcell until the13-somitestage(Fig.5K-N),atwhichpoint no changesinthedistancebetweentwo expression domainsup rhombomere 3)(Chietal.,2003)inthemutantembryos.We found we examined expression of Lmx1b the samelevel of Abnormal celldeathin midbrain. MHB isnotchangedin in themutantembryo(Fig.5P),suggestingthatpositionof pretectal marker in Fig.5J)remainedunchanged,andtheexpression pattern ofthe distance betweenthetwo endsofthemidbrain(double-arrowed line E9.5 (Fig.5H,J)ascomparedwiththewild-type5G,I).The The above resultsshowed thatin stage, TUNEL 7-somitestage (Fig.5R).However, atthe13-somite increased the slightly at number 7-somitestage;this the type embryosbefore Wnt1 that expression maydependon appear tobecausallyrelated not shown). Thus,theexpression of downregulated but maintainedatalow level untilatleastE9.5(data expression, whichisnormallyexpressed laterthan hindbrain ofbothwild-typeand that 2000; Simeoneetal.,2002;Wurst andBally-Cuif,2001).We found positioning oftheisthmicorganizer (Joyner etal.,2000;Simeone, and anteriorhindbrain,respectively, andbotharerequiredforproper In thenormalembryo, expression Effects of maintenance intheMHB. Fgf8 Lmx1b Lmx1b Lmx1b Gbx2 Lmx1b , expression inthe in therostralhindbrain.Since –/– –/– –/– ahrta of , ratherthan En1 was absentandtheexpression ofmany other genesinvolved + –/– mro,btnti idtp mro Fg ST.Thus, embryos, but notinwild-type embryos(Fig.5S,T). –/– el eefudi h MHBofboththemutantandwild- cells werefoundinthe embryos, weperformedwhole-mountTUNELstaining expression was normalin embryo maycauseacaudalshiftofthemidbrain deletion-induced changesingene expression cannotbe Gbx2 , expression, althoughnotrequiredfortheinitiationof mro tte4-somitestage(Fig.5D).Bycontrast, the embryos at embryos doesnotresultinacaudalexpansion ofthe Lmx1b En2 + Pax6 cells weremarkedly increased intheMHBof transcript was foundattherostralendof and Gbx2 Otx2 deletion on WlhradGus 91 was notaffected (Walther andGruss,1991) Lmx1b Lmx1b Pax2 Fgf8 expression was detectedinthecaudal Fgf8 and Gbx2 . Furthermore,wefoundthat –/– expression, isnecessaryfortheir –/– Lmx1b Otx2 Gbx2 expression. Nevertheless, itisclear embryos atthe7-somitestageand Lmx1b Lmx1b Lmx1b embryos. To furtherconfirm this, xrsinwsbrl detectable expression was barely Fgf8 Lmx1b Gbx2 Wnt1 are expressed inthemidbrain and Otx2 –/– –/– –/– -dependent expression of expression, whereas and embryos –/– embryos (arrowheads in Lmx1b mro,theexpression embryos, Lmx1b , Hoxa2 En1 Fgf8 Development 134(2) mro eoete4- the embryos before and Otx2 and , andlossof deletion-induced in the are requiredfor Gbx2 (a marker for Fgf8 Pax2 Gbx2 , was also Lmx1b did not in the Gbx2 Otx2 En2 En2 –/–

DEVELOPMENT Lmx1b increased celldeathin geneexpression. Ontheotherhand,because changes in Lmx1b rhombomere 1inthe celldeathandlower cellproliferationinthetectumand abnormal also reducedcellproliferation.Therefore,itislikely thatthe embryos(Fig.3C,D),suggestingthat in wild-type and to theexistence ofnon-overlapping expression domainsof incomplete deletionof absence of expression intheMHBat3-somitestage,but anearlycomplete was lower inthe Ki67-labelled proliferatingcellsinthetectumandrhombomere1 ofthesechangesingeneexpression. consequence developmental genes,itislikely thattheabnormalcelldeathisa Fgf8 inactivation usinga organizer isessential,we performedregion-specific gene whetherspecific expression of confirm essential forthedevelopment oftheisthmic organizer. To further As describedabove, wefoundthat results inasimilarphenotype Wnt1 size ofthetectumandcerebellum. of of expression oftheisthmus-related genescausedbytheinactivation Lmx1b MHB (seeDiscussion).Theexpression of greatly reduced at the4-somitestage(datanotshown) andwas lost In additiontoincreasedcelldeath,wefoundthatthenumberof Lmx1b Wnt1 xrsinadtedownregulation ofseveral further expression andthe -Cre-mediated deletion of w at the6-somitestagemaybeascribed tothedownregulation is vitalforisthmicorganizeractivity CKO embryo,therewas aslightreduction in n otemta dependence oftheirexpression inthe , andtothemutual in theMHB(Fig.1).Thenearly completedeletionof Lmx1b Lmx1b expression atthe6-somitestage(Fig.6A,B).The Wnt1- Lmx1b Lmx1b Lmx1b –/– Cre conditionalknockout method.Inthis embryos atE10.5,ascomparedwiththat –/– –/– before the6-somitestagemaybedue embryo leadstothereductionin mro curdatrtels of occurredaftertheloss embryos Lmx1b Lmx1b expression intheMHBis Lmx1b Fgf8 in theMHBwas Lmx1b in theMHB in theisthmic deletion Lmx1b Lmx1b in the absenceof complete tectum andcerebellumareduetothespecific actionof (Fig. 7).We thusconclude thatthedevelopmental defectsinthe exhibited adrasticreductioninthesizeoftectumandcerebellum embryo, wefoundthat isthmus atE9.0.Amongmany genesthatareexpressed intheearly restrictedtothe progressively embryo asearlyE7.5,andbecomes presentstudyrevealed that The DISCUSSION MHB. described above. Consistentwiththis, loehratE9.5(Fig.6C,D).If altogether activity (e.g.thelossofinferior colliculusandgreatreduction in phenotype thatischaracteristic ofthelossisthmicorganizer genes inthemutantmice.Infact, downregulation ofseveral otheristhmicorganizer-related as bythe further exemplified bytheabsenceofexpression of inactivation of conditional tectum andcerebellumweredrasticallyreducedinsize, the complete deletionof whereas thatof completely lost,andthatof 6E,F), eesnilformid/hindbraindevelopment. In be essential deletion of expression atthe4-somitestagemaybeattributed totheincomplete expression inadosage-dependentmanner, theremaining in asimilarphenotype.Thefunction of are similartothosefoundinconventional ealsoexamined theexpression ofotherdevelopmental genes We the MHBof Fig. 4.Analysisof arrow) inthe at the4-somitestage(B,arrow) andintheisthmusatE9.5(D, theabsenceof hybridization showing ( compared withthatinthewild-typeembryo(A,arrowhead). as embryo(B,arrowhead) wasnormal ofthemutant other areas control (I,K).( arrow), ascompared withthatinthe expression atE9.5(L, in theMHBat3-somitestage(J,arrow) andthelossof ( the roof platewasmaintainedinthemutantbrainatE9.5(H). that inthewild-typebrain(E,G).Note expression intheisthmusatE9.5(H,arrow) ascompared with MHB atthe3-somitestage(F, arrow) andthelossof changes in E-H I-L Lmx1b Pax2 ) ) Lmx1b Lmx1b Lmx1b (Fig. 6G,H), w K embryos.AtE9.5,expression of CKO Otx2 Pax2 deletion causedamarkedreduction in deletion resulted inreduced Lmx1b M-P Lmx1b in the Fgf8 xrsin(ros otoefudfor found tothose (arrows) expression (Fig. 6O,P)was unchanged.Allthesepatterns Lmx1b ) Lateralviewofembryosshowingsimilar Lmx1b Fgf8 –/– –/– En1 expression. Lmx1b Lmx1b embryo. Notethat embryos. , En2 after the6-somitestageresultsin Wnt1 (Fig. 6I,J)and steeris ee besides is theearliestgene, Lmx1b by w was downregulated (Fig.6K,L), Lmx1b , CKO embryo,whereasnearly RESEARCH ARTICLE En1 Wnt1 ( A-D Lmx1b is expressed intheanterior Fgf8 and Lmx1b Lmx1b -Cre intheMHBresulted Whole-mountinsitu ) mutant brainsdisplaya Wnt1 Gbx2 Fgf8 Lmx1b Pax2 Wnt1 expression intheMHB can regulate Lmx1b w expression in in theMHBwas expression inthe (Fig. 6M,N)was K micealso CKO expression in expression in –/– En1 Lmx1b Fgf8, Wnt1 –/– embryos as Wnt1 expression En1 mice, the Otx2 as well En1 (I-K). in the (Fig. Fgf8 Fgf8 321 ,to

DEVELOPMENT absent intheMHBof Lmx1b of organizer. Inthepresentstudy, weshowed thatexpression fortheinductionactivity oftheisthmic shown toberesponsible only partiallycharacterized.Thegrowth factor FGF8hasbeen 2001). Themolecularbasisoftheisthmicorganizer patterningis and Joyner, 2001b;Nakamuraetal.,2005;Wurst andBally-Cuif, responsible forpatterningofthemidbrainandcerebellum(Liu It iswellestablishedthatthereanisthmicorganizer intheMHB isthmic organizer Lmx1b intheMHB. developmentally importanttranscriptionfactors through regulation oftheexpression of controllingthedevelopment oftheisthmicorganizer development, the cerebellum). 322 that of thetectumandcerebellum(Matsunagaetal.,2002),suggesting embryos inducesectopicexpression of previous studyhasshown thatmisexpression of sufficient fortheinitiationof previous studies,weconcludethat with expression intheisthmicorganizer ofmouseembryos.Together first evidence that of Conversely, knockdown of Lmx1b abletoinduce ectopic is Joyner, 2001a), FGF8-coated beadstomouse midbrainsliceculture(Liuand oraddition of in thechickembryo(Matsunaga etal.,2002), amongthedifferent species. Furthermore, itisinterestingto notethatmisexpression of maybedifferent machinery isthmic organizer development, althoughtheintrinsicmolecular between Thus, suchcross-regulation development oftheisthmicorganizer. fgf8 Lmx1b RESEARCH ARTICLE expression (O’Haraetal.,2005).Ourresultsprovide the precedes thatof and Lmx1b is required fortheinitiationofFgf8in Fgf8 is sufficient fortheinitiationof and Lmx1b pert rs-euaeec te uigthe cross-regulate eachotherduring appear to Lmx1b Fgf8 Lmx1b Fgf8 is thereforeessentialformid/hindbrain may comeinto play onlyatthe4-somite is requiredfortheinitiationof lmx1b Fgf8 –/– (Fig. 1),and mro (Fig.4).Importantly, a embryos in zebrafish resultsintheloss expression intheMHBduring Fgf8 Lmx1b Lmx1b Fgf8 Fgf8 and causesexpansion , expression. Thus, Fgf8 is necessaryand Wnt1 Lmx1b expression was expression. and other in chick Fgf8 Fgf8 development oftheisthmic organizer. Fgf8 Taken together, et al.,1992). independently of Wnt1 the 1990; McMahonetal.,1992).In development ofthemidbrainandhindbrain(McMahonBradley, the isthmicorganizer andmicelacking The secretedfactor expression Lmx1b proper activity oftheisthmicorganizer. between andapositive feedbackregulation either directlyorindirectly, precedes stage when independent but Wnt1 expression by it ispossiblethatthe ta. 98,and et al.,1998), 2002), andthatknockdown of Wnt1 findings thatmisexpression of Wnt1 This resultindicatesthat downregulated intheMHBandprematurelylostbyE9.5(Fig.4). Fgf8 unlikely that theabsenceof the chickembryo(Ye etal.,2001),itis although ectopticallyexpressed development oftheMHBpriorto4-somite stage.Furthermore, maintaining regulated by wnt1 expression inthe expression andforthemaintenance of , becausetheinitiationof occurred priortothe4-somitestage,ourdatarevealed an expression (O’Haraetal.,2005).Whether expression ectopically(Adamsetal.,2000;Matsunaga expression intheMHB.Thisisconsistentwithprevious is required formaintainingWnt1 Fgf8 Fgf8 Fgf8 Wnt1 arrowed line)markedby (O). Notethattherostral-caudal distance(double- in themutantembryos(P)ascompared withwildtype Lmx1b number ofTUNEL ie between line) (double-arrowed showing nochangesinthedistance ( embryos (H,J)ascompared withwild-typecontrols (G,I). ( (C,E). arrowhead), ascompared withcontrols caudal hindbrain(D,arrowhead) andoticvesicle(F, arrow). Notethat atE9.5(F, intheisthmus arrow) andlostaltogether detectable inthemutantbrainat4-somitestage(D, atE7.75(B),butwasbarely in mutantembryos of TUNEL However, there wasasignificantincrease inthenumber downregulation ofgeneexpression wasobserved. embryo atthe7-somitestage(R),whenmarked Fig. 5.Analysisof Lmx1b expression andneuronal deathintheMHBof controls (K,M).( with embryos atthe13-somitestage(N),ascompared inthisdistancethemutant stage (L)andareduction type embryo(S). at the13-somitestage(T),ascompared withthewild- Lmx1b Lmx1b K-N G-J , and Lmx1b Fgf8 Lmx1b Wnt1 expression initiates.Because Expression of ) expression intheMHB(Chietal.,2003;Reifers obei iuhbiiainfor hybridization ) Doubleinsitu Wnt1 Fgf8 –/– Lmx1b –/– is absentintheMHBof in theMHB.However, asdownregulation of Lmx1b is unclear. Because + (Danielian andMcMahon,1996; McMahon embryo. ( embryos. -dependent el arw nteMBo h uatembryo intheMHBofmutant (arrow) cells is likely toregulate expression of is necessaryfortheproperdevelopment of Lmx1b activity mediatesthemaintenanceof Otx2 may exist afterwards toensurethe O Lmx1b –/– , + Gbx2 P lmx1b Wnt1 + Otx2 Q-T embryo isduetodownregulation of el arw intheMHBmutant (arrow) cells Lmx1b ) Expression of Gbx2 is requiredforthemaintenanceof and ( A-F ) There wasaslightincrease inthe Wnt1 expression wasnormalinthe is essentialfortheinitiationof was notchangedinmutant Pax6 Hoxa2 Lmx1b can induce ) , in zebrafish leadstotheloss Gbx2 Otx2 Fgf8 in chickembryosinduces was maintainedinthe expression profile inthe + Wnt1 , Wnt1 –/– expression wasnormal domains atthe5-somite Fgf8 Hoxa2 Development 134(2) Pax6 expression occurs embryos, Lmx1b Otx2 Fgf8 xrsinduring expression Lmx1b Wnt1 show abnormal was notchanged is requiredfor and and expression in expression –/– Pax6 is directly Wnt1 Hoxa2 embryo, Fgf8 Fgf8 Wnt1 was - ,

DEVELOPMENT Pax2 recombination method.( nteMBa h -oiesae(,arw ythe in theMHBat6-somitestage(B,arrow) by in theMHBof Gbx2 expression of midbrain, paralleledbyposteriorized caudal expansion ofthe contrast, (Fig.4).By detectable atthe4-somitestageandabsentthereafter the4-somitestage,but becamebarely expression was normalbefore (O). conditional knockoutembryo(P)ascompared withthecontrol embryo distance ofthemidbrain(double-arrowed line)were normalinthe per odfeetal euaeteepeso of appears todifferentially regulate theexpression 5).Thisisinterestingbecause the wild-typecontrol(Figs3, precedes al., 2002;Wurst andBally-Cuif,2001). Expressionof Barbera etal.,2001;Rhinn1998;Simeone,2000;Simeone et initiation of forthe notrequired are positioning oftheisthmicorganizer, and It iswellestablishedthat Is Lmx1b ( Cre conditionalknockoutanditseffect ongeneexpression. Fig. 6.Region-specificdeletionof (C,E,G,I) are shownforcomparison.( E9.5. Normalexpression (arrows)ofeachgeneinthecontrol patterns compared withthatinthecontrol (arrow, K).( control embryo(M).( but normalintheoticvesicle(N,arrowhead) ascompared withthe Gbx2 dependent regulation of geneexpression intheMHB.Moreover, the MHB.Theseresultsalsoargue forthespecificity of areknown toantagonize eachotherinsettingthepositionof which A , B Lmx1b Whole-mount insituhybridizationshowinginactivationof ) (H) and –/– of the is vitalforisthmicorganizeractivity ielc h neirhindbrain anddisplayabnormal anterior mice lackthe Otx2 Gbx2 Lmx1b Fgf8 required forthepositioningofMHB? En1 Otx2 expression in , but after (J) waslostintheisthmusof or n1Lmx1b Wnt1 w , O CKO embryoswaslostintheisthmus(N,arrow) Wnt1 Wnt1 , P C-J ) Otx2 Otx2 Otx2 expression (Joyner etal.,2000;Martinez- xrsin(ros of (arrows) ) Expression and Lmxb w expression andtherostral-caudal and (Fig. 1).In CKO embryoatE9.5(arrow, L)as Fgf8 –/– K Gbx2 Lmx1b , L mro a iia tothatin embryos was similar (Broccoli etal., 1999;Liand ) Decreased expression of are requiredforthecorrect Lmx1b in theMHBby M Lmx1b , N ) Theexpression of Wnt1 Fgf8 –/– w Gbx2 CKO embryosat embryos, -Cre (D), Wnt1 and Wnt1 Lmx1b Lmx1b Lmx1b Lmx1b Otx2 Gbx2 En2 (F), - - , 700 pretectal region; SC,superiorcolliculus;Th,thalamus.Scalebars:C,D, plexus;Hy, hypothalamus;IC,inferiorcolliculus;PT, 2E,F). cp,choroids asdescribedfortheconventionalknockoutmice(seeFig. patterns cerebellar Purkinjecells,respectively, showingthesame staining immunostaining ofBRN3bandcalbindin,markingthetectum ntecniinlkoku ie(seeFig.2C,D).( in theconditionalknockoutmice seentothosefound stained sectionofP0brains.Similardefectswere conventional tectum andcerebellum (Cb)wasobservedtothatdescribedforthe brains. Asimilarreduction inthesizeanddisplacedlocationof that caudal extension ofthemidbrain(Fig.5).Thisraisespossibility to rather than intheMHB, to beduetheincreasedcelldeath embryoatthe13-somitestageismost likely mutant distance inthe Otx2 Hoxa2 in the such acaudalshiftofthemidbrainwhen Joyner, 2001;Wassarman etal.,1997).However, wedidnotfind CKO mice. developmentin Fig. 7.Impaired tectumandcerebellum 2001). We have shown thattheexpression of 2001b; Nakamuraetal.,2005;Wurst andBally-Cuif,2001;Ye etal., PAX2, EN1andEN2areexpressed intheMHB(LiuandJoyner, genesencodingtranscriptionfactors The isthmicorganizer-related of theisthmicorganizer maintenance looprequired forthedevelopment Lmx1b Otx2 putative expanded domainfor be requiredcell-autonomouslyinthe in theabsenceof caudalshiftofthemidbrain compensatory mechanismthatprevents xrsini h omleby,i sulkl htthis activity inthemutant downregulation iscausedby thelossofFGF8 itisunlikely that expression inthenormal embryo, initiationof transcription factors occurred atatimepriortothe ofthese (Fig.4).Becausethedownregulation their expression of Pax2 ␮ Lmx1b expression intheMHB. m; E,F, 250 and Lmx1b + domains inthemutantembryo(Fig.5).Thereductionthis and , acrucialcomponentinpositive Pax6 ( A Lmx1b En1 , –/– deletion intheMHBmayactivate anunknown B ) Dorsalviewofthemid/hindbrainregion ofintactP0 embryo, asindicatedbythenormalexpression of and unchangeddistancebetweenthe ␮ Fg ) n that (Fig. 1),and Gbx2 m. –/– ie(seeFig.2A,B).( mice activity. Itisalso possiblethat Lmx1b RESEARCH ARTICLE C Gbx2 , deletion downregulated D ) ParasagittalNissl- Lmx1b E expression was lost , F ) Double precedes that Lmx1b Otx2 Lmx1b + Fgf8 may 323 and w

DEVELOPMENT Wnt1 organizer development. loopforisthmic is acrucialcomponentofthepositive maintenance that Wurst etal.,1994;Ye etal.,2001).Ourstudyindicates Joyner etal.,2000;McMahon1992;Meyers etal.,1998; expression. Becausethedownregulation of that isrequiredfortheinitiationof initiation ofexpression ofthesegenes,withtheexception of genes. isthmicorganizer expression andthemaintenanceofotherkey organizer activity, key componentofthegeneticpathway underlying theisthmic isthmic organizer isestablishedintheMHB.We propose that,asa begins whenthe MHB. Thesecondstepoftheregionalization example, wehave foundthat remainstobefullyelucidated.For loop positive maintenance may inturnregulate expression of initiation of be sufficient toexplain thelossof hn,L,Ce,C . u,P,Tn . i,M,Jhsn .adM,Q. R.andMa, M.,Johnson, P., M.,Qiu, Tan, C.L.,Luo, L.,Chen, Cheng, C.V., K.C.,Pepicelli, H.,Oberg, D.,Kokubo, Y., Ovchinnikov, H.,Lun, Chen, R.D. J.A.andRiddle, J.M.,Golden, K.A.,Maida, Adams, References ‘Pujing Project’ ofShanghai, China(06PJ14116). and Technology ofChina(2006CB806600and2006CB943900), andthe ‘973’ Program andKeyStateResearch Program from theMinistryofScience the NationalNaturalScienceFoundationofChina(30525014,30628016), F. Zhang fortechnicalassistance.Thisproject wassupportedbygrantsfrom A. Groves andW. L.Ye forhelpwithwhole-mountTUNELstaining;andMrsY. Simeone, S.Aizawa,G.R.MartinandP. Gruss forproviding insituprobes; Drs We thankDrA.P. McMahonforproviding theWnt1-Cre mice;DrsA. in theearlyembryo,forwhich Robertson, 1998).Thefirst stepisthepositioningoffutureMHB (Adamsetal.,2000;Beddingtonand developing mid/hindbrain Lmx1b normally expressed earlierthan atE9.5(C.G.andY.-Q.D., unpublished).Since embryos Pax2 Bally-Cuif, 2001).Thisisbasedontheevidence that thelossof proposed (Joyner etal.,2000;Nakamura2005;Wurst and Pax 2003). stages, because may contribute totheexpression ofthesetranscriptionfactors atlater embryo. Thisdoesnotexclude thepossibilitythat 324 hr a ocua hf fthe there was nocaudalshiftof h,C . atnz . us,W n atn G. R. W. andMartin, S.,Wurst, C.L.,Martinez, Chi, W. E.andWurst, V., Boncinelli, Broccoli, J. M.P. andBurbach, A.,Smidt, C.F., C.H.J.,Vogelaar, Hellemons, Asbreuk, this processis edntn .S .adRbrsn E.J. R.S.P. andRobertson, Beddington, cerebellum. organizer signalFGF8 isrequired forcellsurvivalintheprospective midbrainand neurotransmitter phenotype. (2003). Lmx1b,Pet-1,andNkx2.2coordinately specifyserotonergic Nat. Genet. mutant micesuggestaninvolvementof LMX1Binhumannailpatellasyndrome. R.L. B.andJohnson, L.,Lee, Can, expression positionstheisthmic organizer. organizer. Trends Genet. systems. suggest functionalcooperativityinthedevelopmentofforebrain motorcontrol P. In summary, therearetwo stepsintheregionalization ofthe loopinvolving A positive maintenance (2002). CNSExpressionof pattern genes duringthedevelopment oftheisthmicorganizer hasbeen , Fgf8 and RESEARCH ARTICLE a eur osbefeedbackregulation by may requirepossible Mol. Cell.Neurosci. Development Pax2 Development 19 or Fgf8 14 , 51-55. Lmx1b Wnt1 , 277-284. Fgf8 expression, but alsofortheinitiationof either directlyorviaanunknown pathway, which Lmx1b Lmx1b function affects themaintenancebut notthe idpnetbecauseinthe -independent 127 is requiredfortheirmaintenance(Chietal., 130 maintains Wnt1expression within theisthmic Lmx1b , 1857-1867. 21 J. Neurosci. , 2633-2644. is responsiblefortheinitiationof , 410-420. Otx2 Lmx1b is requirednotonlyformaintaining Lmx1b (1998). LimbandkidneydefectsinLmx1b En1 Otx2 Fgf8 and (1999). Thecaudallimitof 23 (1998). Anterior patterning inmouse. (1998). Anteriorpatterning Lmx1b , theimmediatemaintenanceof Nature Fgf8 , 9961-9967. and coexpression withPtxgenes + expression islostin Gbx2 expression (Chietal.,2003; domain, whichmarksthe , 401 h opeiyofthis . Thecomplexity Lmx1b Fgf8 Wnt1 (2003). Theisthmic are two majorplayers; , 164-168. , and Wnt1 Lmx1b may controlthe (2000). The Fgf8 En1 Pax2 and . –/– Otx2 normally Lmx1b may not embryo En Lmx1b En1 Pax2 Fgf8 Fgf8 En1 and –/– is , onr .L,Lu .adMle,S. A.andMillet, A.L.,Liu, Joyner, ig .Q,Yn . ai,A,Za,Z . ono,R .adCe,Z.F. R.L.andChen, Z.Q.,Johnson, A.,Zhao, J.,Kania, Y. Q.,Yin, Ding, i,A n onr A.L. A.andJoyner, Liu, A.L. Joyner, iml .A,Trbl,D . lnut . us,W,Loi,C.A.and W., Loomis, V., Wurst, D.H.,Blanquet, R.A.,Turnbull, Kimmel, M.H. Kaufman, aila,P . ucn,D,Rwth .H,Mcal S.K.andMcMahon, D.H.,Michael, D.,Rowitch, P. S.,Muccino, Danielian, ig .Q,Mrln,U,Ya,W,Yn . emn . rco,J.,Deneris, L.,Ericson, J.,Wegman, W., Yin, U.,Yuan, Y. Q.,Marklund, Ding, i,A n onr A.L. A.andJoyner, Liu, i .Y .adJye,A.L. J.Y. H.andJoyner, Li, ilt . apel . pti,D . oo,K,Hri,E n onr A.L. E.andJoyner, K.,Harris, D.J.,Losos, K.,Epstein, S.,Campbell, Millet, G.R. M.andMartin, E.N.,Lewandoski, Meyers, G.R. P.Crossley, H.andMartin, atnz . rsly .H,Cbs . uesen .L n atn G. R. J.L.andMartin, I.,Rubenstein, P. S.,Crossley, H.,Cobos, Martinez, A.P. P. S.andMcMahon, Danielian, oel,R,Zo,G,Dee,S . avy .J,Nnmy,Y,Tonr P. Y., S., Thorner, S.J.,Ninomiya, S.D.,Harvey, G.,Dreyer, R.,Zhou, Morello, G.R. S.andMartin, P.Crossley, H.,Martinez, aaua . aaia . asng,E n ao T. E.andSato, T., Matsunaga, H.,Katahira, Nakamura, H. S.andFujisawa, H.H.,Takagi, K.E.,Igawa, H.,Nakano, Nakamura, J.A. A.andMcMahon, A. L.,Bradley, A.P., Joyner, McMahon, D., P. E.,Acampora, P., M.,Boyl, Puelles, J.P., Signore, Martinez-Barbera, cao,A .adBaly A. A.P. andBradley, McMahon, ’aa .P,Bc,E,Br,L . og .L,Kslr .S n ide R.D. D.S.andRiddle, L.L.,Kessler, F. L.K.,Wong, P.,O’Hara, Beck,E.,Barr, H. T. andNakamura, E.,Katahira, Matsunaga, efr,F,Bhi . as,E . rsly .H,Sane,D .adBad M. D.Y. P. andBrand, E.C.,Crossley, H.,Stainier, H.,Walsh, F., Bohli, Reifers, hn,M,Deih . hwo,W,Bhigr .R,L er M.andAng, R.R.,LeMeur, W., Behringer, A.,Shawlot, M.,Dierich, Rhinn, development. midbrain andcerebellum. (2004). 191. themousemid/hindbrainregion.of FGF8inpatterning neuronshorn ofthespinalcord. ectodermal ridgeformation. A.L. Joyner, Press. 741. position andmaintainamid-hindbrainorganizer. signalling pathwayinvertebratemidbraindevelopment. tamoxifen-inducible formofCre recombinase. A. P. development ofserotonergic neurons. R.L.andChen,Z.F. E., Johnson, 4991. and notinductionofmid-hindbraingeneexpression. organizer. (1999). Arole forGbx2inrepression ofOtx2andpositioningthemid/hindbrain 141. series generatedbyCre- andFlp-mediatedrecombination. 595. induced byFGF8inthechickembryo. in thedevelopingembryo. of polypeptidesandisexpressed inregions thatdirect outgrowth andpatterning deletion ofengrailed-expressing cellsby9.5dayspostcoitum. midbrain-hindbrain phenotypeofWnt-1-/Wnt-1-miceresults from stepwise Development isthmocerebellar developmentviaarepressive effect onOtx2expression. (1999). FGF8inducesformationofanectopicisthmicorganizerand renal diseaseinnailpatellasyndrome. glomerular basementmembranecollagenexpression byLMX1Bcontributesto B.etal. A.,Zabel, W., Winterpacht, J.H.,Cole, Miner, 49 organizer formidbrainandhindbraindevelopment. chick chimeras. (1986). 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