*These authorscontributedequallytothis work USA. Molecular BiologyandPharmacology, Washington University, StLouis,MO63110, and expressed intheventral telencephalon,suchas of basalgangliacells(Muzioetal.,2002).Conversely, or maintainacorticalfate andinsteadadopt,atleastinpart,thefate expressed inthedorsaltelencephalon,precursorcells fail toadopt For example, inembryosmutantfor Rubenstein, 2000;Campbell,2003;Zakietal.,Hébert,2005). required tospecifyprecursorcellsasdorsalorventral (Wilson and cognition andemotions.Homeoboxtranscriptionfactors are which have multiplefunctionsrelatedto motor coordination, cognitive andperceptualfunctions;theventral basalganglia, two areas:thedorsalcerebralcortex, whichistheseatofourhighest derived fromtheembryonictelencephalon,arecomposedlargely of generated duringdevelopment? Theadultcerebralhemispheres, organized intodistinctfunctionalareas.How aretheseareas The vertebrate braincomprisesamultitudeofneural celltypes INTRODUCTION KEY WORDS:Basalganglia,Ganglioniceminence,FGF, SHH,GLI3,Telencephalon, Mouse Patterning, indicating thatFGFsactdownstreamof generate ventralprecursors.Moreover, the as does mimicking thephenotypeobtainedpreviouslywithalossofSHHsignalling.Yet, inthe results inthelossofdifferentiated ventromedialcells;andsecond,inthe ventral cells,however, hasrecentlybeenquestionedbecauselossofthe (SHH)isrequiredtogenerateventralcelltypesthroughoutthecentralnervoussystem.Itsroleindirectlyspe Grigoriy Gutin independently ofSHH FGF signallinggeneratesventraltelencephaliccells Development 133,2937-2946(2006)doi:10.1242/dev.02465 Accepted 30May 2006 † 1 Medicine, 1410PelhamParkwaySouth, Bronx, NY10461,USA. Biological Sciences,Stanford University, Stanford, CA94305,USA. ua .McConnell K. Susan required forventraltelencephalondevelopment.First,simultaneousdeletionof isalsomutant.Consequently, anotherventraldeterminantmustexist.Here,geneticevidenceestablishesthatFGFsare Nkx2.1 a lossofventral celltypesthatnormallyexpress the forebrain.Intelencephalon,lossofSHHsignallingleads to induction ofventral celltypesthroughouttheneuraltube,including unclear (Hébert,2005). of thesegenestoventral ordorsaldomains,many pointsremain made inunderstandinghow morphogensmightrestrictexpression and Campbell,2001;Yun etal.,2003).Althoughprogresshasbeen Horton etal.,1999;SusselCorbin2000;Toresson (Anderson etal.,1997;SzucsikCasarosa1999; Informatics) areessentialforproperspecificationofventral fates Author forcorrespondence (e-mail:[email protected]) Departments ofNeuroscience andMolecularGenetics,AlbertEinsteinCollegeof Sonic hedgehog(SHH)isasecretedfactor thatisrequired forthe Dlx2 , attheexpense ofdorsalcelltypesthatexpress Gli1 , andthebHLHgene , thetranscriptionofwhichdependsonSHHactivity, suggestingthatFGFsignallingactsindependentlyofSHHto 1, *, MarieFernandes 3 and JeanM.Hébert Mash1 Pax6 ( Shh Ascl1 1, and *, LauraPalazzolo Fgfr1 and – MouseGenome Emx2 Gli3 1,† Dlx2 ; 2 Fgfr2 Department of 3 Gsh2 Department of , two genes to generateventraltelencephaliccelltypes. , Gsh2 Pax6 phenotype, unlikethe , Nkx2.1 and and 1 , HunKiPaek Fgf14 both theridgeandrostralmidlineexpress several FGFgenes, forms therostralmidlineonceneuraltubehasclosed.Inmice, growth factors (FGFs). 2002). Candidatesfortheventralizing signalarethe fibroblast pattern thisaxisofthetelencephalon(Aotoetal.,2002;Rallu normal dorsoventral patterning,implyingthatotherfactors can Remarkably, embryosmutantforboth 1999; Aotoetal.,2002;Kuschel etal.,2003;Rallu al.,2002). Shh process. Thezinc-fingertranscriptionfactor gene cells toadoptaventral fate, itisnotstrictlynecessaryforthis features (Jessell,2000;BriscoeandEricson,2001). thought toactdirectlyonneuralprecursorcellsinduceventral Rubenstein, 1997;Kohtz etal.,1998).Inthe spinalcord,SHHis Wilson, 1995;HauptmannandGerster, 1996;Shimamuraand telencephalon ofzebrafishandmice(Ericsonetal.,1995;Barth can induceexpression of pathway orbyknocking down further decreasedbyinhibition oftheintracellularsignalling fgf8 specific populationsofventral neuronsisdiminishedorabsentin the ventral telencephalon toform.Theexpression ofmarkers for (Gunhaga etal.,2003).Inzebrafish,FGFsignallingisrequired for dorsal features,suchasexpression oftheneocorticalmarker activation oftheWntpathway is required forinducingexpression of telencephalon. Inchickembryos,FGFsignalling,following postulated tospecifybothdorsalandventral celltypesinthe 1992; Petersetal.,1993;Hébert2003).FGFshave been cells, Three FGFreceptorgenesarewidelyexpressed inneuralprecursor et al.,1998;Xu1999;Crossley etal.,2001;Wang etal.,2000). Fuccillo etal.,2004).Likewise, ectopicexpression ofthe Emx2 reduced orabsent (Shinya etal.,2001;Walshe andMason,2003). In morpholinos, ventral precursor cellsexpressing The anteriorneuralridgeattheendofplate Although SHHsignallingisessentialfortelencephalicprecursor Shh and dorsalizesthetelencephalon(Grove etal.,1998;Theil mutants (Shanmugaligametal., 2000).WhenFGFsignallingis Fgfr1 Fgfr1 , (Ericson etal.,1995;Chiang1996;Ohkubo2002; Fgf15 gene haslittleeffect onventraldevelopmentifthe Shh ; , Fgfr2 Fgfr2 Fgfr1 , 1 , KaiYu Fgf17 phenotype, isnotrescuedbylossof double mutant,ventralprecursorcellsarelost, and and Fgfr1 and Fgfr3 Fgfr3 2 , DavidM.Ornitz ; Fgf18 Fgfr2 (Orr-Urtreger etal.,1991;Peters specifically inthetelencephalon Dlx2 mutant, (McWhirter etal.,1997;Maruoka fgf8 RESEARCH ARTICLE and Shh and Shh and Nkx2.1 fgf3 remains expressed, 2 Gli3 , expression using Gli3 in thedorsal exhibit grossly Gli3 or antagonizes nk2.1b , further Shh cifying Emx1 Fgf8 2937 gene Gli3 are ,

DEVELOPMENT are specifiedbut fail todifferentiate, whereasinthe phenotypes. Inthe Foxg1 ages indicatedintheexpected ratioswithnosignsofnecrosis.Atotal19 heterozygotes wereusedinthecross.Mutantembryosobtainedat dilution ofpolyclonalrabbitserum (generousgiftfromS.A.Anderson). performed aspreviously described(Marinetal.,2000)with a1:2500 embryo was obtainedandanalysed).ImmunohistochemistryforNPYwas the E16.5 mutant andthreecontrolembryoswereanalyzedforeachprobe(except for instead ofNBT/BCIPtoreveal expression patterns.Aminimumofthree (Henrique etal.,1995)withtheexception thatBMpurple(Roche) was used digoxigenin-labelled probes,theprocedurewas performedasdescribed 1994). For theinsituhybridizationofwhole-mountmouse embryosusing al., 2003). expression (Ralluetal.,2002),the although the occurs despiteevidence ofactive SHHsignalling.Furthermore, and cellsallalongthedorsoventral axisadoptadorsalfate. This mutant, precursorcellswithventral characteristicsfail todevelop crossed to of thesegeneswereusedtogeneratetelencephalicspecificknockouts when 1994; Yamagushi etal.,1994;Arman1998),thereforefloxed alleles or two FGFRgenesexpressed inthetelencephalon(Fig.1A).Alossof crosses weredesignedtogeneratemicemutantforallthreecombinationsof To studytheroleofFGFsignallinginpatterningventral telencephalon, Generation ofmutantembryos normal, mutants thatretainonlyonefunctionalcopy of examined forpatterningdefects.Although development. Allcombinationsoftwo receptorsaredeletedand disrupted specificallyinthetelencephalonatearlieststagesofits conditional geneticapproachinthemouse,FGFsignallingis dorsoventral axisofthetelencephalonisexamined. Usinga with othersignallingmolecules,suchasSHH,isunclear. telencephalic cellsremainsunknown. Moreover, how FGFsinteract mammalian telencephalonforspecifyingeitherdorsalorventral et al.,2003).WhetherFGFsignallingisrequiredintheembryonic ventral markers indorsaltelencephalicexplants inculture(Kuschel Moreover, inmice,FGF8-soaked beadscaninduceexpression of Nkx2.1 chick embryos,applicationofsolubleFGFR4leadstoaloss 2938 these embryonic ages,Cre-mediateddeletionof intraperitoneal injection withBrdUandwereeuthanized 1hourlater. At Female micepregnant withE9.5andE10.5embryosreceive an BrdU incorporationandTUNELassays hybridized to whole mount.For theradioactive insitu,frozensectionswerepreparedand radioactive RNA probesonsectionsandtheotherDIGlabelledin In situhybridizationwas carriedoutaccordingtotwo protocols:oneusing RNA issituhybridizationandimmunohistochemistry and 31 Laboratories. carrying the three embryosofeachgenotypewereusedforexperiment. Mice the 1996). As were usedascontrols.The MATERIALS ANDMETHODS ventral cells. placing FGFsignallingdownstream ofthesegenesingenerating Fgfr2 In thisstudy, theroleofFGF signallinginpatterningthe Foxg1 cre/+ Foxg1 -expressing telencephaliccells(Marklundetal.,2004). RESEARCH ARTICLE in thewholeembryoleadstolethalityatgastrulation(Dengetal., Fgfr1 cre ;Fgfr1 Fgfr1 Fgfr1;Fgfr2 Foxg1 Fgfr3 allele donotexhibit thephenotypesdescribedinthisstudyand Gli3 cre/+ 35 ; lox/+ Fgfr3 S-labelled probes,aspreviously described(Frantzetal., Shh lox/lox cre ;Fgfr1 -null ( homozygous nullmiceareviablebut breedpoorly, mice, aspreviously described (Hébertetal.,2003;Yu et , Fgfr1 Fgfr1 ;Fgfr3 phenotype canberescuedbylossof and double mutantinFig.4forwhichonlyoneviable lox/lox extra lox/+ ; Fgfr3 Fgfr1 null/null Fgfr3 ;Fgfr2 ;Fgfr3 , three ; mutant, ventromedial precursorcells allele usedisanull(Dengetal., Fgfr2 ) allelewereobtainedfromJackson lox/lox null/+ Fgfr1 Foxg1 mutants exhibit severe ventral and embryos wereusedandatleast ; Fgfr1 Fgfr2 cre/+ ;Fgfr2 Fgfr1 lox/+ Fgfr2 Fgfr1 phenotype cannot, ;Fgfr2 lox/lox ; appear grossly Fgfr3 Fgfr1 and lox/+ ;Fgfr3 Fgfr2 carrying double ; Fgfr2 null/null Fgfr1 Gli3 is (arrowhead). having asmallertelencephalon(arrow), losesthefrontonasal process and Fgfr3 mutants. The designed toobtainthethree possiblecombinationsofFGFRdouble Allthree FGFRdoublemutantsare generated. Fig. 1. and TUNELcellcounts. corresponding to the MGEareawerecountedasdescribed above forBrdU anti-rat secondaryandcounterstained withHoechst33342.Cellsinsectors BrdU (1:1,000,OxfordBiotechnology), incubatedwithTexas Red-coupled HCl, neutralizedwith100mMNaBorate, blocked, incubatedwithratanti- incubated 1hourwithFITC-coupled secondary, post-fixed, treatedwith 2N overnight at 4°C withKi67antibody(1:100,Novocastra monoclonal), antigen retrieval, blocked (5%goatserum,0.1%Triton X-100), incubated fixed (4%paraformaldehyde),boiledin10mMsodiumcitrate forKi67 were collectedatE11.5andembeddedinOCT. Cryosections(10 Female micecarryingE10.5embryoswereinjectedwithBrdU.Embryos Cell cycleexitassay three separateembryosarecountedineachcase. within thissegment arethencounted.Atleasttwo segments fromeachof points toestablishthe200-cellsegment. TUNEL-orBrdU-positive cells ventrally. A100Syto11-positive cellsarecountedoneithersideofthese through thepointsweretelencephalichemispheresmeetdorsally and defined asanareaencompassing200cellsandobtainedbydrawing aline dorsaltotheventral midline.Midlinesegments are segments are45° ventral tothedorsalmidlineandventral to countdorsalcellsareat45° cells inthesegment (segments contain~200totalcells).Segments used the outeredgeofneuraltissueanddividing bythetotalnumberof these cellsinaradialsegment spanningfromtheventricular surface to BrdU orTUNEL-positive cellsisdeterminedbycountingthenumberof were counterstainedwithSyto11(MolecularProbes).Thefractionof position alongtheanteroposterioraxisoftelencephalon.Sections control andmutantswerematchedsothatthey correspondtothesame specifications (Roche,Catalogue#2156792).Coronalsectionsfor 2003), orforTUNELanalysisaccordingtothemanufacturer’s used foreitherBrdUstaining,aspreviously described(Hébertetal., Embryos werecollectedandfrozeninOCT. Freshfrozensectionswere complete (Hébertetal.,2003)andprecursorcellsacquireventral fates. Fgfr3 Fgfr1 allele isanull.Theembryoshomozygousmutantfor are alsoheterozygous mutantfor ; Fgfr2 Fgfr1 double mutant( and Fgfr2 mutant allelesare floxed,whereas the C ) embryos.Themutant,aswell Fgfr1 . WholeE12.5control ( Development 133(15) ( Fgfr2 A ) Crosses ␮ m) were and B )

DEVELOPMENT contributes totheobserved phenotypes. Discussion), itisunlikely thatlossofonecopy of obtained inothersystemswhich FGF signallinginventral telencephalicdevelopment have been as phenotypesanalogoustotheonesdescribedhereimplicating and asmallertelencephaloninthe were viabletoatleastE14.5.Exceptforthelackofanasalprocess crosses depictedinFig.1A.Allthreedoublemutantembryos combinations ofFGFRdoublemutantsaregeneratedusingthe olfactory bulb defect(Hébertetal.,2003).Inthisstudy, all shown), whereaslossof own doesnotleadtosignificantpatterningdefects (datanot et al.,1994;Arman1998).Lossof embryo knockouts ofthesegenes(Dengetal.,1994;Yamaguchi bypass theearlyembryoniclethalityassociatedwithwhole- were deletedinthetelencephalonusingaCre/ neighbouring ectodermalormesodermaltissues. telencephalic precursorsbyaffecting thedevelopment of disruption ofligandgeneexpression canhave indirecteffects on Crossley etal.,2001;Hébert2003).Inaddition,the 1997; Maruokaetal.,1998;Xu1999;Wang etal.,2000; 1991; Petersetal.,1992;1993;McWhirter Fgfr3 three receptorsexpressed inthetelencephalon, receptors ratherthanligandsweretargeted becausethereareonly FGF signallingwas disruptedgenetically. GenesencodingFGF To testtheroleofFGFsinpatterningventral telencephalon, telencephalon are viabletoatleastE14.5 Mutants thatlackanytwoFGFRgenesinthe RESULTS FGF signallingventralizesthetelencephalon observing wholeE12.5embryos. (Fig. 1C),nogrossmorphologicaldefectcouldbeidentifiedupon Foxg1 revealed between is betweenE9.5andE12.5,(2)asnogeneticinteraction defect heterozygosity onitsown doesnotleadtoany patterning loss ofFGFRgenesdescribedbelow. However, (1)as this locuscouldintheorycontribute tothedefectsobserved with The lossofonecopy of , comparedwithatleastfive ligands(Orr-Urtreger etal., cre/+ ;Fgfr1 +/– ;Fgfr2 Foxg1 –/– Fgfr1 Foxg1 ;Fgfr3 and FGFRgeneseven inthe in thetelencephalonleadstoan resulting frominsertionof –/– Fgfr1 mutant (Fig.2B,F,J), and(3) Foxg1 Fgfr3 ; Fgfr2 is wildtype(see loxP Fgfr1 or Fgfr1 double mutant Fgfr2 approach to , Fgfr2 and on their Foxg1 Foxg1 Fgfr2 cre and at in thismutant,furtheranalysesfocusedonthe the telencephalon.Owingtolackofanobvious patterningdefect copy of Nkx2.1 Fgfr1 addition tobeingdeficientin grossly normalmorphologyatE12.5(Fig.2B,F,J). Thismutant,in Fgfr1 Nkx2.1 In the cells requires FGFsignalling Generation ofventraltelencephalicprecursor Nkx2.1 Emx2 or survival, isdisruptedinthe than specificationofventral precursors,suchascelldifferentiation without lossofventral precursorcells,suggeststhataprocess other mutant (Tole etal.,2006).Lossofthenormalventral morphology, is missing.Thisphenotypealsoobserved inthe ganglionic eminencesnormallydifferentiate. Inaddition,theseptum No sulcusformsbetweenareaswherethemedialandlateral morphology oftheventral telencephaloninthismutant isabnormal. of dorsalandventral precursorshasoccurrednormally. However, the (Fig. 2C,G,Karrowheads; Fig.4K),suggestingthat thespecification by RNA insituhybridizationanalysisusingprobesfor The telencephalonofE12.5doublemutantembryoswas examined dorsoventral patterning One copyof below). Dlx2 in thetelencephalon(Fig.2H,L;Fig.4L;7L).Theremaining this mutant,ahighlevel of morphology disrupted,but normaldorsoventral patterningislost.In ehptaai s najcn etos expression ofthe hypothalamicas,inadjacentsections, be Nkx2.1 dorsoventral axis(Fig.2D;Fig.7I),whereasthedomainsof ventral midlineinsteadofendingapproximatelymidway down the By contrast,inthe ; and Fgfr1 Fgfr2 (Fig. 1A).Despitethis,normalpatternsof (dorsal markers), and and (ventromedial marker). The expression areobserved (Fig.2B,F,J), indicatingthatone Fgfr1 Nkx2.1 Mash1 Mash1 ; mutants. Fgfr3 is largely sufficient topatternthedorsoventral axisof Fgfr1 expression neartheventral midlineislikely to expression matchthosefoundincontrolembryos expression aregreatlyreducedorcompletelylost mutant, thedorsoventral bordersof loss of extends totheventralmidline(D)and bythe are lost,indicatedbyexpression of normal. ( morphology ofthetelencephalonare grossly and mutant, dorsoventralpatterning radioactive probes. ( analysis ofE12.5coronal brainsectionsusing double mutants. Fig. 2.Ventral celltypesare lostinFGFR flattened inappearance.( of theventraltelencephalonisabnormal and specification ofprecursor cells, themorphology (arrowheads), indicatingproper dorsoventral Dlx2 (G)andNkx2.1(K)are normal although theborders ofexpression ofPax6(C), telencephalon. ( ventral andventromedial areas ofthe Nkx2.1 embryo, expression of Fgfr1 Fgfr1 is largelysufficient fornormal Dlx2 Pax6 –/– Dlx2 ; ; Fgfr2 (I) are usedtoidentifythedorsal, Fgfr2 Fgfr2 and C and , G Fgfr1 (H) and , –/– K Mash1 RESEARCH ARTICLE Emx2 Fgfr2 and ) Inthe mutant, notonlyisventral mutant, ventralprecursor cells B , ; F Fgfr3 RNA insituhybridization , Fgfr3 J Nkx2.1 A expression extends tothe ; ) Inthe Fgfr3 , (ventral markers), and E Pax6 Fgfr1 , I ) Inthecontrol D , lacksonecopy of mutant (addressed , (L) expression. –/– (A), H mutant displayed Fgfr2 Fgfr1 , ; Pax6 L Fgfr3 )In the Dlx2 Fgfr1 –/– Pax6 Pax6 ; , ; –/– Fgfr3 Fgfr3 Pax6 Dlx2 (E) and mutant, , that single Dlx2, Dlx2 2939 –/– and and and ,

DEVELOPMENT Fgfr1 changes incellsurvival orproliferation, telencephalic precursorcells. Fgfr1 S1 inthesupplementarymaterial).Thisphenotypedemonstratesthat expression ofthehypothalamicmarker telencephalic marker 2940 observed inany sectorbetweencontroland percentage ofcellsincorporatingBrdUorstainingwithTUNELis components (seeMaterialsandmethods).Nodifference inthe defined sectorsthatincludedorsal,ventral anddorsomedial neurogenesis. Sectionsoftelencephalonswerepartitionedinto floxed allelesinthemutantiscompleteandpriortostartof incorporation assaysatE10.5,astagewhenrecombinationofthe Fgfr1 P in thedorsalmidlineofmutant(21.3±2.6%versus 13.5±1.5%, between controland embryos. However, statisticallysignificantdifferences areobserved specified inthe precursor cells. versus 48.8±2.1%, telencephalon ofthismutantcomparedwithcontrol(42.9±1.2% Namely, slightlylessproliferation isobserved intheventral telencephalic, but notdiencephalic,expression of role forFGFsignallinginspecifyingventral precursor cells, ventral diencephalon)whereitisalsoexpressed. Consistentwitha basis withthetelencephalonasopposedtohypothalamus(or age, expression of readily detectableincontrolembryosatthisage.Inaddition, in theE9.25-9.75mutant.Unlike otherventral markers, analyzed theexpression oftheventral telencephalicmarker the E9.25-E9.75 In the requires FGFsignalling Differentiation ofventraltelencephaliccells mark differentiating ventral cellswas examined. Expressionof the telencephalonisimpaired.Thereforeexpression of genesthat precursor cellssuggeststhatdifferentiation ofventral celltypesin between theganglioniceminenceswithoutalossofventral <0.0001). Thereducedlevels ofventral proliferationinthe To assesswhethertheobserved phenotypesmightbedueto Another possibilityisthatventral precursorcellsfail tobe ; ; Fgfr1 Fgfr2 Fgfr2 RESEARCH ARTICLE and ; double mutantcouldinpartexplain thelossofventral Fgfr3 Fgfr2 mutants wereanalyzedusingTUNELandBrdU Fgfr1 Fgfr1 P mutant, thelossofseptumandsulcus Nkx2.1 are requiredtogethertogenerateventral =0.02) andanincreaseinapoptosisisobserved Foxg1 ; Fgfr2 Fgfr1 ; Fgfr2 is reducedorabsentinthisarea,whereas can beassociatedonamorphological ; mutant. To addressthispoint,wehave Fgfr2 double mutant(Fig.3C-F). mutant embryos(Fig.3A,B). Foxd1 is increased(seeFig. Fgfr1 Fgfr1 Nkx2.1 ; Fgfr3 ; Fgfr3 Nkx2.1 is lostin mutant Nkx2.1 Lhx6 and is , expression of zone (VZ)andsubventricular zone(SVZ),was examined. AtE12.5, normally markthegerminative layersoftheMGE,ventricular medial areaofthe the earlylossofMGE-derived neurons(Fig.4B,E). E18.5 (seeFig.S2inthesupplementarymaterial),consistentwith Anderson etal.,2001),aremissinginthe Nkx2.1 marker forasubclassofmigratoryinterneuronsderived from produced atearlierages(Fig.4H).However, expression ofNPY, a might beexpected, asneuronsderived fromtheLGEarestill Tag1 However, inthe expense ofthedorsalneuronalmarker, interneurons, mutant (Fig.4C,F,I), expression ofaventral marker forGABAergic early lossofalldifferentiated ventral celltypesinthe presence ofventral anddorsalneuronsatE16.5.Consistentwiththe was affected inolderFGFRmutantembryos,weexamined the given thelossofventral precursorcells(Fig.2D,H,L). expression lost,but sois cell types.Inthe promote thedifferentiation, ratherthanthesurvival, ofventromedial described above, thissuggeststhatFGFsignallingisrequiredto is unaffected (Fig.4H).Together withtheTUNELandBrdUresults which marksdifferentiating cellsofthelateralganglionic eminence, the mutant(Fig.4B,Eandnotshown), whereasexpression of the medialganglioniceminenceandseptalareas,isalmostabsentin Lhx7 compared withcontrols(0.8%versus 0.2%, percentage ofTUNEL-labelledcellsintheventral medial area generated. Althoughmutantsexhibited asignificantly higher examined atE11.5,astagewhenneuronshave justbegun tobe neurogenesis, theratesofcellcycle exit andcelldeathwere neurons. of SVZprecursors,whichdoesnotreadilyexplain the lossof This suggeststhatthereisalossofVZprecursorcellsattheexpense reductions inthelevels of Prox1 Prox1 To addressthenatureofdisruptedneurogenesisinventral In ordertodeterminewhetherdifferentiation ofventral celltypes To furtheraddressthecauseunderlyingdisrupted expression aresimilartothoseincontrols(Fig.4M,N,P,Q), as and marks theSVZ.Inmutant,however, elevated levels of expression arefoundintheVZ,coincidentwithapparent -positive precursorsintheMGE(Marinetal.,2000; Shh , whichisnormallyfoundinthedifferentiating field of in the rates ofcelldeathinthedorsalmidline(B)are observed incorporation intheventraltelencephalon(A)andhigher mutant telencephalons.SlightlylowerratesofBrdU incorporation andTUNELanalysesofE10.5control and border. OS,opticstalk. indicate theputativetelencephalic-diencephalicventral absent asearlyE9.25inthemutant.Arrowheads perturbed inthe Fig. 3.Cellfate,proliferation, anddeathare anterior domainof ( C-F Gad67 Hes5 Fgfr1 Fgfr1 ) Whole-mountinsituhybridization.Themore Fgfr1;Fgfr3 Fgfr1 and , islostintheventral telencephalonatthe ; Fgfr2 ; Fgfr3 –/– Ebf1 Lhx2 ; Hes5 Fgfr2 mutant, notonlyis mutant, thedomainsof (Fig. 4C,F,I), aswould beexpected, mutant, theexpression ofgenesthat Fgfr1 Nkx2.1 normally markstheVZ,whereas –/– and mutant compared withcontrols. –/– Lhx2 ; expression intheforebrain is Fgfr2 expression (Fig.5A-C). Development 133(15) Fgfr1;Fgfr3 –/– P Tag1 <0.03; Fig.5F),itis mutant. Lhx6 (Fig. 4O,R,S). , Lhx7 Fgfr1 Gad67 ( A mutant at , B and ) BrdU ; Fgfr2 Ebf1 Shh and ,

DEVELOPMENT expressed inthe differentiating cellsofthelateralganglioniceminence(G),remains (B,C,E,F). eminence (A,D);however, thisexpression islostinthemutants normally expressed indifferentiating cellsofthe medialganglionic of not the Fgfr1 E12.5 coronal brainsectionsusingradioactiveprobes. FGF signallingventralizesthetelencephalon been cycling adaybefore.Thefraction ofdouble-labelled (Scholzen andGerdes,2000), BrdU,whichmarkscellsthathad labelled insectionsforKi67, whichmarksallcycling cells hours priortocollectingE11.5 brains.Thesewerethendouble rate ofcellcycle exit, pregnant femaleswereinjectedwithBrdU24 low, canfullyaccountforthedramaticlossofneurons. To assessthe questionable whetherthisincreased level ofcelldeath,whichisstill ventral precursors (J),remains expressed inthe Fig. 4.Neurogenesis isperturbedinthe bars: 0.5mminA-L;1M-S. boundaries of Fgfr1 expressing areas (arrows) ofthe E16.5 coronal brainsections. Tag1 –/– –/– ; ; Fgfr1 Fgfr2 expression (O,R,S),whereas inthe Fgfr2 Ebf1 –/– –/– , ontheotherhand,whichisnormallyexpressed in –/– Gad67 ; Fgfr2 mutant (I).Mash1,whichisnormallyexpressed in Fgfr1 mutants. –/– and –/– (L) mutant.( ; Fgfr3 Tag1 ( A-L Gad67 –/– ) RNAinsituhybridizationanalysisof expression are normal(M,N,P,Q). Scale Fgfr1 mutant (H),butnotthe M-S expression islostin –/– ; Fgfr2 ) RNAinsituhybridizationof Fgfr1 Fgfr1 –/– Fgfr1 –/– mutant attheexpense –/– ; Fgfr3 ; –/– Fgfr3 ; Shh Fgfr3 –/– Foxg1 –/– mutant, the and –/– and - (K) but Lhx7 are rescue the is devoid of Fgfr1 Dlx2 expression extends totheventral-most areaofthetelencephalonand mutant thatcarriesjustonecopy of Gli3 that carryoneortwo mutantallelesof the mutant, the ( abolished inthetelencephalonusingatissue-specificknockout of the phenotypeobserved inmicewhichSHHsignallingis The lossofventral precursorcellsinthe mutant Shh observed inthe is likely tobeinlarge partresponsibleforthelackofneurons compared with25%incontrols( (BrdU+Ki67) cellsover totalBrdU-labelledcellsis66%inmutants signalling actsdownstream of Gli3 Foxg1 hypothalamus, asrevealed inserialsectionsbyreducedorabsent expression inthemutants islikely tocoincidewiththeanteroventral little ornodifference betweencontroland to berequiredforinducingventral celltypes. gene inthemesendodermunderlyingtelencephalonisstilllikely ventral celltypes. signalling actseitherthroughorindependentlyofSHHtogenerate matching phenotypes,thequestionarisesastowhetherFGF telencephalic expression of Fgfr2 embryos (Fig.6A),indicatingthatFGFsignallingthrough to maintain downstream ofSHH.Consistentwiththeseresults, and, consequently, thatFGFsignallingactsindependentlyor SHH requires it isnotsufficient togenerateventral celltypes.Thisindicatesthat expressed, but thatitisalsoactive inthe and mutantatE10.5(Fig.6B,C),suggestingthatnotonlyisSHH expressed in dependent onSHHactivity (Baietal.,2002),was examined. mutant, theexpression of mesendoderm. To addresswhetherSHHisactively signallingin the of ventral celltypesinthe (Aoto etal.,2002;Rallu2002).Can mutants thatlackbothcopiesof lost. However, grosslynormaldorsoventral patterningis restored in in the types arelostanddorsalonesexpand, andtheopposite isobserved the telencephalon(Ralluetal.,2002).In Shh phenotype Loss ofGli3doesnotrescue theFgfr1;Fgfr2 inhibited (Kuschel etal.,2003). telencephalic explants inculture,even whenSHHsignallingis and FGF8-soaked beadscaninduceventral geneexpression indorsal This questionwas addressedbyanalyzing To addressthisquestion,theexpression of Fgfr1 and does notrescuethelossofventral celltypes.Inthe to rescuethe ; and expression islargely orcompletelylostasitisinthe Gli3 Fgfr2 expression (notshown). Thefailure ofonemutantcopy of is notrequiredtomaintain Gli3 Pax6 ; Fgfr2 Shh Gli1 mutant –ventral celltypesexpand anddorsalones are Fgf8 double mutant(Fig.7E-M).Theventromedial areathat Foxg1 act antagonisticallytopatternthedorsoventral axisof Fgfr1 Pax6 expression expands totheventral midline,while phenotype (Ralluetal.,2002), indicatesthatFGF Fgfr1;Fgfr3 mutant atE10.5,atimewhichexpression ofthis expression (Ohkuboetal.,2002;Aoto2002) remain expressed inthe Fgfr1 Smo -positive telencephaliccellsinboththecontrol and and ) gene(Fuccilloetal.,2004).Inthe ; Fgfr2 Fgfr2 Emx2 Fgfr1 Nkx2.1 mutant. Gli1 to generateventral telencephaliccells phenotype, incontrasttoitsability to Gli3 ; expression but positive for P Shh Fgfr2 , agenewhosetranscriptionis <0.0001; Fig.5D,E).Thischange and RESEARCH ARTICLE and and oneorbothcopiesof Gli3 Shh mutant? Fgfr1 Fgfr1 Shh Gsh2 . Inthesemutants,lossof Gli3 . Shh expression inventral ; Gli3 Shh ; Fgfr2 Shh Fgfr1 Fgfr2 are lost.Given these Fgfr1 also rescuetheloss mutant, ventral cell expression shows , was examined in Fgfr1 Pax6 ; mutant andthat Shh mutant mimics ; Fgfr2 Fgfr2 Fgfr1 is required and ; Fgfr1 Fgfr2 mutants mutant Gli1 ; Emx2 Fgfr2 2941 Dlx2 Gli3 Smo and is

DEVELOPMENT not rescuethelossofventral cellsobserved inthe supplementary material),suggestingthatcompletelossof and telencephalon (Fig.7D).Nevertheless, in rather aflatteningandsignificantreductioninthesizeof not exhibit ahighfrequency ofexencephaly (lessthan20%),but et al.,2002),the which earlier thanE8.75usingthe parallel toSHH signalling. In thisprocess,FGFscouldbe acting upstream,downstream orin signalling aresimilarlyrequired forgeneratingventral precursors. deleted(Stormetal.,2006). Therefore,bothSHHandFGF is The samephenotypeisalsoobtained inembryoswhich ( over totalBrdU-labelled cellsispresent inthemutantcompared withthecontrol, indicatinganabnormally lowrateofcellcy and mutantE11.5brainsdoubled-labelledforKi67BrdU (injected24hrspriortosacrifice).( (A). Thisexpansioncoincideswithanapparent decrease intheexpression oftheVZmarkers coronal sectionsthrough theventromedial area. Unlikethecontrol, present inthe Wnt3a for thecorticalhemtoform(Grove etal.,1998),expression of mutant andthatFGFsignallinginfact actsdownstream of Fig. 5.The 2942 Fgfr1 promoting theirdifferentiation. Thelossofventral precursorsinthe signalling isrequiredbothforgeneratingventral precursorsandfor loss ofdifferentiated ventromedial cells(Figs 2-4).Hence,FGF ventral precursorcells,whereaslossof mouse, simultaneouslossof their differentiation. Usingaconditionalgeneticapproachinthe in thetelencephalonforbothgenerationofventral precursorsand The resultsofthisstudydemonstratethatFGFsignallingisrequired DISCUSSION mutants (Fig.7Q-Sandnotshown). Fgfr1 (Fig. 7A-D).Unlike the this casedevelopment ofthetelencephalonisgreatlycompromised the lossofventral precursorcellsisalsonotrescued.However, in F ) indicatesahigherrateofcelldeathinthemutantatE11.5. Consistent withprevious reportsindicatingthat In the Emx2 ; –/– Fgfr2 Smo RESEARCH ARTICLE ;Fgfr2 and Fgfr1 , but not Fgfr1 is conditionallydeletedinthetelencephalon startingno mutant mimicsthephenotypeobtainedinembryos Wnt2b ; –/– Fgfr2 –/– Fgfr1 ;Gli3 Fgfr1 Dlx2 ; Fgfr3 , markers forthehem,areabsentin mutant thatcarriestwo mutantcopiesof –/– ; , areexpressed (Fig.7N-P;seeFig.S3inthe –/– Fgfr2 Shh –/– ;Fgfr2 mutant (Fig.7T-U andnotshown), but SVZ isexpandedandundergoesmore celldeathandlesscycleexit. ; Gli3 Foxg1 ; Gli3 Fgfr1 –/– double homozygousmutant(Rallu Cre triple homozygousmutantdoes and and allele (Fuccilloetal.,2004). Fgfr1 Foxg1 Fgfr1 Fgfr2 and -positive areas, –/– leads toalossof ;Fgfr2 Fgfr3 Gli3 Fgfr1 is required –/– leads toa Gli3 Gli3 ;Gli3 ; Fgfr2 Gli3 Pax6 Fgf8 does Prox1 . +/– , expression inthemutantexpandsfrom theSVZtoventricularsurface et al.,2002),ismaintainedinthe downstream of S3 inthesupplementarymaterial), placingFGFsignalling 2002), itdoesnotdosointhe rescues thelackofventral precursorsinthe and dependsonFGFsignalling.Andfinally, althoughlossof indicating thatSHHactivity isnotsufficient togenerateventral cells Second, Fgf8 the of Previous evidence supportsamodelinwhichFGFsactdownstream FGFs actdownstream ofSHH maintenance of of SHH(Aotoetal.,2002;Rallu2002).Second,the indicating thatanotherfactor cangenerateventral cellsindependent ventral cellscanstilldevelop if both SHHandGLI3arelost, 1995; Chiangetal.,1996;Ohkubo2002;Fuccillo2004), required toinduceventral telencephaliccelltypes(Ericson etal., proliferation or survival oncethey have alreadybeenspecified. specifies thesecellsorwhether itisrequiredtopromotetheir downstream ofSHH togenerateventral telencephaliccells,directly downstream of generating ventral telencephaliccelltypeshasbeenlacking. genetic evidence inmammalsdemonstratingaroleforFGFs with morpholinosagainst 2003). Andfinally, zebrafishembryosthatlack when SHHsignallingisinhibitedwithcyclopamine (Kuschel etal., when placedonculturedexplants ofdorsaltelencephalon, even expression ofventral markers andrepressexpression ofdorsalones Ohkubo etal.,2002).Third,FGF8-soaked beadscaninduce a lossof Mason, 2003)andchickembryostreatedwithsolubleFGFR4show types (Shanmugalingametal.,2000;Shinya etal.,2001; Walshe and The questionremainsastowhether FGFsignalling,acting Three resultsdescribedinthisstudyestablishthatFGFsfact act Shh Fgfr1 , whereby to generateventral celltypes(Fig.8).First,althoughSHH is Nkx2.1 Gli1 ; Fgfr2 expression, whichisdependentonSHHactivity (Bai Shh Hes5 Shh Shh expression (Marklundetal.,2004).However, direct Fgf8 mutant (Fig.6).Thisisalsothecasewithlossof E expression ismaintained(Kawauchi etal., 2005). and and ) Alargerpercentage ofKi67+BrdU labelledcells and expression requires Gli3 Gli3 Lhx2 Fgf8 . First, . (B,C). Scalebar:0.02mm.( ( A-C Fgfr1 and ) RNAinsituhybridizationofE12.5 Shh ; Fgf3 Fgfr1 Fgfr2 expression isnotaffected in show lossofventral cell ; Shh Shh Fgfr2 mutant (Fig.7;seeFig. Development 133(15) cle exit.TUNELanalysis mutant (Ralluetal., (Aoto etal.,2002; Fgf8 mutant (Fig.6), or aretreated D ) Control Gli3

DEVELOPMENT Foxg1 through theanteriorprosencephalon. Intelencephalicareas expressing ( ventral mesendodermunderlyingthetelencephalon(arrowheads). the ventralareas ofthemutant(C,arrows). situ hybridizationanalysisofE10.5control and FGF signallingventralizesthetelencephalon telencephalic cellsthatnormallyexpress Second, even atearlystagesoftelencephalondevelopment, ventral ectopically induceventral telencephaliccells(Kuschel etal.,2003). induce orspecifyventral cells.First,FGF8-soaked beadscan Several linesofevidence suggestthatFGFsignallingdoesinfact precursors inthe Fig. 6.SHHsignallingisinsufficient togenerateventral embryos indicatesthat how canFGFsgenerateventral cellsif 2002; Ralluetal.,2002),presumablyowing toFGFsignalling.But In SHH antagonizesGLI3torelieve repression ofFGF mutant asearlyE9(Stormetal.,2006). proliferation andincreasedapoptosisareobserved inthe in the fact, reducedproliferationintheventral telencephalonisobserved promotes theproliferationandsurvival ofventral precursorcells.In This doesnotexclude thepossibilitythatFGFsignalling also consistent witharoleforFGFsignallinginspecifyingventral cells. the of unchecked repressionby by The likely answeristhatFGFgeneexpression isnormallyrepressed expression ofFGFgenes,inparticular FGF geneexpression, Fgf8 Furthermore, inthe (Theil etal.,1999;Aoto al., 2002;Kuschel etal.,2003). expression onlyindirectlybyrepressing expands (Aotoetal.,2002), suggestingthat B , C Shh Gli3 RNA insituhybridizationanalysisofserialE10coronal sections ) Fgfr1 is nolongerrepressedby (B), expression of ; Fgfr1 Gli3 (Fig. 8).Inthe ; Fgfr2 double mutants,ventral cellsaregenerated(Aotoetal., ; Fgfr2 or Fgfr1 mutant atE10.5(Fig.3A)andbothreduced Shh Fgf8 Shh Gli1 Fgf8 –/– Shh ; Gli3 ; Fgfr2 remains expressed inthemutant , whichrequires SHHactivity, islocalisedto mutants (Fig.3)(Stormetal.,2006), Gli3 mutant, expression expands inthe double mutant, Gli3 –/– , but inthe mutant. . Consistentwith Fgf8 Fgf8 Shh Gli3 expression islostbecause Nkx2.1 ( Shh A Fgfr1 is requiredtomaintain (Ohkubo etal.,2002)? ) Whole-mountRNAin (Fig. 8). Fgf8 ; Shh Gli3 –/– are notpresentin ; Fgfr2 expression also promotes Gli3 double mutant, Gli3 –/– repressing mutant mutant Fgf8 Fgf8 unclear. Interestingly, lossof signalling occurinotherpartsofthedeveloping CNSremains signalling inthespinalcord.In al., 2003).Itispossiblethatinthe Wnt signallingdorsalizestelencephalicprecursorcells(Gunhagaet evidence hassuggestedthatFGFsignallingincombination with loss of that atleastinthespinalcord,but perhapsalsointhetelencephalon, that generateventral celltypesinthespinalcord.Itshouldbenoted 1998; Xuetal.,1999),FGFsbecomeexcellent candidatesforfactors cord (Crossley andMartin,1995;Borjaetal.,1996;Maruoka genes areexpressed withinorimmediatelyadjacenttothespinal Wijgerde etal.,2002).Given theresultspresentedhereandthatFGF the spinalcord(LitingtungandChiang,2000;Perssonetal.,2002; patterning, indicatingthatotheryettobeidentifiedfactors pattern mutants, ventral neurongenerationisrescuedasdorsoventral copy of deficient forbothcopiesof precursor cells, Of thethreeFGFreceptorgenesexpressed intelencephalic telencephalon FGFR1 isthelikelyreceptor forFGF8intheearly telencephalon. al., 1998)accountsforthedorsalizationofremaining expressed beyond thecorticalhemarea(suchasWnt7a)(Grove et remaining FGFsignallingthroughFGFR3alongwithaWntthatis the goes beyond inactivating GLI3. 2002), suggestingthatSHHhasaroleinpatterningneuraltissue (Litingtung andChiang,2000;Perssonetal.,2002;Wijgerde etal., secondarily through FGFR2. the earlytelencephalonFGF8 acts primarilythroughFGFR1and loss of the similaritiesinphenotypes obtainedtodatewithreductionsor FGF8 actsnotonlythroughFGFR1, but alsothroughFGFR2.Given similar tothe et al.,2006).However, inthelattercase thephenotypeismore and thoseinwhich Fgf8 et al.,2001;Walshe and Mason,2003).Mouseembryosinwhich expression islostorreduced(Shanmugalingametal.,2000;Shinya ventral, midlineandcommissuraldefectsareobserved when phenotypes tothoseobtainedwithlossof commissural defects(Dodeetal.,2003). to Kallmannsyndrome,whichincludesolfactory bulb and species. Inhumans,reductionorlossofexpression ofthisgeneleads indicating that and differentiated ventromedial celltypes(Tole etal.,2006), (Hébert etal.,2003),alossofmidlineglia,cerebralcommissures telencephalon, lossof Fgfr3 of theFGFsignallingload.Conversely, althoughlossof that FGFR1producedfromonealleleissufficient totransmitmost The key rolesplayedby significant roleinthepatterningprocessesdescribedthisstudy. J.M.H., unpublished),suggestingthat are the development. The Whether similarinteractionsbetween The natureofthesignalsthatdorsalizetelencephaliccellsin Reduction orlossof Fgfr1;Fgfr2;Gli3 is hypomorphiccanlackolfactory bulbs (Meyers etal.,1998) on theirown have littleornopatterningdefectinthe Fgf8 Fgfr2 Gli3 Fgfr1 and withthedifferent FGFRmutants,itislikely thatin does notcompletelyrescuethelossof and Fgfr1 exhibit grosslynormalpatterning(Fig.2),indicating Fgfr1 Fgfr1 Fgfr2 Fgfr1 ; Fgfr2 Fgf8 plays adominantrole.Telencephalons thatare Fgfr1 ; on itsown isnecessaryfortelencephalic Fgfr3 triple mutantremainunclear. Previous and is nulllackventral precursorcells(Storm phenotype describedhere,suggesting that Fgfr1 Fgf8 Fgfr2 alone resultsinanolfactory bulb defect Fgfr1 phenotypes (Fig.2;G.G.,S.K.M.and Gli3 are likely tobeconserved across expression leadstocomparable and ; Fgfr3 Shh RESEARCH ARTICLE Fgfr1;Fgfr2;Gli3 can alsorescuelossofSHH Fgfr3 ; Gli3 phenotypes aresimilar, as Fgfr3 but thatretainonlyone Gli3 or Fgfr1 Smo , does notplaya Shh Shh . Inzebrafish, ; Gli3 , andFGF mutant, the phenotype Fgfr2 double 2943 fgf8 or

DEVELOPMENT in thisstudynorolewas uncovered for 2005), but itsexpression orroleinthemouseisunknown. Although zebrafish inpromotingventral forebraindevelopment (Miyake etal., characterized mayalsobeexpressed. Ofnote, which thetelencephalicexpression patterninmicehasnotyetbeen neuroepithelium (e.g.Donoetal.,1998),andotherFGFgenes for believed tobewidelyexpressed throughoutthetelencephalic with FGFR3. phenotype, itremainsunclear which ligandsarelikely tointeract have notyet revealed asimilaroligodendrocyte differentiation characterized postnatally, andthatmutationsinFGFligand genes et al.,2003).Given thattheexpression ofFGFligandsispoorly oligodendrocyte terminal differentiation thatoccurspostnatally(Oh cell types,thisgeneisrequired forregulating theonsetof lateral telencephalon(Masonetal.,1994), 1998; Xuetal.,1999;Wang etal.,2000), similar patternto 2944 variants ofthe potentially beexplained bythepresenceofalternatively spliced (Ornitz etal.,1996;Chellaiah1999).Thisdiscrepancy could assays, andthatFGFR3isthehighestaffinity receptorforthisligand has littleornoaffinity forFGFR1incellmitogenicityandbinding whether differential expression ofunidentifiedco-factors. Nevertheless, Fgf14 mutant. Fgfr2 This issurprising,given previous studiessuggestingthatFGF8 Fgf8 , in theearlytelencephalonawaits analysisofthetripleFGFR Fgf15 RESEARCH ARTICLE is nottheonlyFgfgeneexpressed intheearlytelencephalon. Fgfr3 , Fgf17 in any way compensatesforlossof Fgfr3 Fgf8 and transcript inthedifferent celltypesorthe (McWhirter etal.,1997;Maruoka Fgf18 are expressed inanoverlapping or Fgfr3 Fgf7 in generatingventral Fgf19 Fgf1 is expressed inthe plays arolein and Fgfr1 Fgf2 and/or are Fgfr1 telencephalon isalsolikely toberequiredformidlineformation. The midline (HayhurstandMcConnell,2003).FGFsignallinginthe not onlythelossofventral cellsbut alsolossof therostralanddorsal expression, whichisnormallypresentonlyventrally, canresultin andmice,itremainsamysteryhow theabsenceof SHH pathway areknown tocauseholoprosencephaly inboth in humans(Muenke andBeachy, 2001).Althoughmutationsinthe hemispheres, isthemostcommondevelopmental forebraindefect Holoprosencephaly, theincompleteseparationoftelencephalic midline A modelfortheformationoftelencephalic commissures (Tole et al.,2006);andthe indusium griseumandmidlinezipperglia,allthreecerebral 2001). can inducestructuresresembling adorsalmidline(Crossley etal., regulating expression appearsto regulate dorsalmidlineformationby al., 2000;Walshe and Mason, 2003).Furthermore,thelevel of and severe commissuralaxonpathway defects(Shanmugalingamet zebrafish, lossofFGFactivity alsoleadstodisruptionofthemidline formation oftherostrodorsalmidline. not shown) (Stormetal.,2003),indicatingthatFGFsregulate and awiderdomainof mutants have ahigherrateofapoptosis(Fig.3B)(Stormetal.,2006) FGF signalling are likely tointeractinregulating formationofthe 1998; Theiletal., 1999;Kuschel etal.,2003).Therefore and WNTgenesforforming thedorsalmidline(Grove etal., Consistent witharoleforFGFsignallinginmidlineformation, in ; Fgfr3 Gli3 Bmp4 Fgfr1 the telencephalonisseverely compromised inthe Fgfr1 cortical hemappearsnormalforallgenotypesexcept express mutant (T,U). Scalebar:0.5mminE-U. Foxg1 brain sections.Inthe ( other genotypes(A-C).t,telencephalon;d,diencephalon. phenotype. Fig. 7.Lossof Fgfr1 expression inneighbouringsections(notshown).Inthe coincides withareas withdecreased orabsent to behypothalamicratherthantelencephalicasit remaining expression of the expenseofventralmarkerssuchas extends totheventromedial area ofthetelencephalonat the is alsorequiredforpromotingthe expression ofBMP E-U mutant lacksrostralmidlineglialcelltypes,the Fgfr1 ) RNAinsituhybridizationanalysisofE12.5coronal –/– –/– –/– expression (Stormetal., 2003);andectopicFGF8 -positive telencephalicarea inanexencephalic ; ; ; Pax6 Fgfr2 Fgfr2 Fgfr2 –/– ; Fgfr2 Bmp4 –/– –/– –/– but not ( A-D ; ; ; Gli3 Gli3 Gli3 Gli3 –/– ) E12.5wholeembryos.Developmentof expression inthedorsalmidline(data mutant, –/– –/– –/– does notrescue the Dlx2 Fgfr1 (Q-U). Broken linesindicatethe mutant, mutant (D)compared withthe Dlx2 (N-P). –/– Pax6 ; Fgfr2 in bothmutants(L,M)islikely Foxg1 Wnt3a and Development 133(15) Fgfr1 –/– ; Gli3+/– -positive areas Emx2 Dlx2 expression inthe ; Fgfr2 Fgfr1 expression (E-M). The mutant, asin Foxg1 –/– and Gli3 ; Fgfr2 Fgf8 Fgf8 Shh and –/–

DEVELOPMENT Casarosa, S.,Fode,C.andGuillemot,F. Campbell, K. http://dev.biologists.org/cgi/content/full/133/15/2937/DC1 Supplementary materialforthisarticleisavailableat Supplementary material NIMH 1R21MH075779,1R01MH70596(J.M.H.). by theJamesS.McDonnellFoundationforBrainTumor Research, andby Start-Up Award, bytheAlexandrineandAlexanderL. SinsheimerFoundation, NIMH MH65261(S.K.M.),byaHoward HughesMedicalInstituteJuniorFaculty Anderson andCelineZimmerforplasmids.Thisworkwassupportedinpartby Briscoe, J.andEricson, Borja, A.Z.M.,Murphy, C. andZeller, R. Barth, K.A.andWilson,S.W. Bai, C.B.,Auerbach,W., Lee,J.S.,Stephen,D.andJoyner, A.L. Arman, E.,Haffner-Krausz, R.,Chen,Y., Heath,J. K.andLonai,P. ( Juha PartanenandJanetRossant( We thankGord Fishellandmembersofhislaboratory forinsightfuldiscussions; signalling. midline development indirectlybyregulating GLI3andFGF presented here,pointtoamodelwherebySHHpromotesventral and dorsal midline(Fig.8).Theseprevious reports,alongwiththeresults FGF signallingventralizesthetelencephalon Anderson, S.A.,Qiu,M.,Bulfone,Eisenstat,D.D.,Meneses,J., References and thatFGF8regulates expression ofWNTs andBMPsforformationofthedorsalmidline, cell types.Previous evidenceindicatesthatGLI3isrequired for are thennecessarytogenerateventralandatleastsomerostral midline thus relieving repression ofFgfgeneexpression (seeDiscussion).FGFs required fortheseprocesses onlyindirectly byantagonizingGLI3and types, butalsorostral anddorsalmidlineones.SHHislikelytobe developing telencephalon,isrequired togeneratenotonlyventralcell in thetelencephalon. Fig. 8.Modelforthegenerationofventralandmidlinecelltypes Aoto, K.,Nishimura,T., Eto,K.andMotoyama,J. C.,Jennings,K.andRubenstein,J.L.R. Anderson, S.A.,Marin,O.,Horn, indirectly byregulating GLI3andFGFsignalling. Hence, SHHislikelytopromote ventralandmidlinedevelopment Fgfr3 in theventraltelencephalon. Curr. Opin.Neurobiol. neural tube. analysis duringneuraltubedevelopment. associated FGF-2protein isoform:itsembryonicdistributionandfunctional 1768. neuronal differentiation intheembryonicforebrain. influenced bysonichedgehog/vertebratehedgehog-1anddemarcates azoneof Shh pathway. but notGli1,isrequired forinitialShhsignalingandectopicactivationofthe Sci. USA for FGFsignalinginpregastrulation mammaliandevelopment. Targeted disruptionoffibroblast growth factor(FGF) 2suggestsarole limb bud. regulates striatalneurons.of lateborn genes Pedersen, R.andRubenstein,J.L. eminence. (2001). Distinctcorticalmigrationsfrom themedialandlateralganglionic ) formice;andSoniaGarel, AlexJoyner, JohnRubenstein,Stewart Dlx-1 95 Fgf8 Dev. Biol. Development , 5082-5087. (2003). Dorsal-ventral patterning inthemammaliantelencephalon. (2003). Dorsal-ventralpatterning and Curr. Opin.Neurobiol. Development expression andapoptosisin the developingneuraltube,face,and Dlx-2 251 13 disrupt thestriatalsubventricularzoneanddifferentiation , 320-332. 128 (2001). Specificationofneuronal fatesintheventral , 50-56. SHH, whichisexpressed ventraltothe Bmp4 129 , 353-363. Neuron Development (1995). Expression ofzebrafish , 4753-4761. Fgfr1 expression inadose-dependentmanner. 11 19 , 43-49. ), andChu-XiaDengPhilipLeder , 27-37. (1997). Mutationsofthehomeobox (1999). Mash1regulates neurogenesis (1996). AltFGF-2,anovelER- Dev. Biol. 126 , 525-534. (2002). MouseGLI3 1802 Development , 680-692. nk2. 2 Proc. Natl.Acad. 121 (2002). Gli2, (1998). is , 1755- Marin, O.,Anderson,S.A.andRubenstein, J.L.R. Litingtung, Y. and Chiang,C. Marklund, M.,Sjodal, M.,Beehler, B.C.,Jessell, T. M.,Edlund, T. and Henrique, D.,Adam,J.,Myat,A.,Chitnis,Lewis,J.andIsh-Horowicz, D. Hébert, J.M.,Partanen,J.,Rossant,andMcConnell,S.K. Kuschel, S.,Ruther, U.and Theil,T. Horton, S.,Meredith, A.,Richardson, J.A.andJohnson,E. Kohtz, J.D.,Baker, D.P., Corte,G.andFishell, Kawauchi, S.,Shou,J.,Santos,R.,Hébert,J.M.,McConnell,S.K.,Mason, I. Jessell, T. M. Crossley, P. H.andMartin,G.R. Hébert, J.M. Dode, C.,Levilliers,J.,Dupont,J.M.,DePaepe,A.,LeDu,N.,Soussi- Chiang, C.,Litingtung,Y., Lee,E.,Young, K.E.,Corden, J.L.,Westphal, H. Crossley, P. H.,Martinez,S.,Ohkubo,Y. andRubenstein, J.R. Corbin, J.G.,Gaiano,N.,Machold,R.P., Langston,A.andFishell,G. Chellaiah, A.,Yuan, W., D.M. Chellaiah,M.andOrnitz, Hayhurst, M.andMcConnell,S.K. Dono, R.,Texido, G.,Dussel,R.,Ehmke,H.andZeller, R. Deng, C.,Wynshaw-Boris,A.,Zhou,F., Kuo,A.andLeder, P. Deng, C.,Wynshaw-Boris,A.,Shen,M.M.,Daugherty, D.M.and C.,Ornitz, Hauptmann, G.andGerster, T. Gunhaga, L.,Marklund,M.,Sjodal,Hsieh,J.C.,Jessell,T. M.andEdlund, Grove, E.A.,Tole, S.,Limon,J.,Yip, L.andRagsdale,C.W. Ericson, J.,Muhr, J.,Placzek,M.,Lints,T., Jessell,T. M.andEdlund,T. Fuccillo, M.,Rallu,McMahon,A.P. andFishell,G. Frantz, G.D.,Weimann, J.M.,Levin,M.E.andMcConnell,S.K. molecular specificationofstriatalinterneurons. 979-985. mediated byanantagonisticinteraction betweenShhandGli3. telencephalon. Bmp/Wnt andFgfsignalingunderliesthe ventralizationofthe and transcriptionalcodes. Development signaling through FGFR1isrequired forolfactorybulbmorphogenesis. Nature (1995). Expression ofaDelta homologueinprospective neurons inthechick. telencephalon development. Sonic Hedgehog. the mammaliantelencephalonismediatedbychangesinresponsiveness to bHLH factorMASH1. coordination ofneuronal differentiation eventsinventralforebrain requires the development. cell production andmaintanence ofneurogenesis duringolfactoryepithelium and Calof,A.L. in thedevelopingembryo. of polypeptidesandisexpressed inregions thatdirect outgrowth andpatterning development. The Gsh2homeodomaingenecontrols multipleaspectsoftelencephalic Curr. Opin.Neurol. dominant Kallmannsyndrome. Baverel, F. etal. Yanicostas, N.,Coimbra,R.S.,Delmaghani,Compain-Nouaille, 921. growth factorreceptor 3isanegativeregulator ofbonegrowth. Neuroscience prosencephalon duringdevelopmentofthetelencephalicandopticvesicles. Coordinate expression ofFgf8,Otx2,Bmp4,andShhintherostral lacking Sonichedgehoggenefunction. and Beachy, P. A. 34794. Multiple regions determineligandbindingspecificity. ligand bindingdomainsinchimericfibroblast growth factorreceptor molecules. hedgehog. gene intheembryoniczebrafishbrainisaltered byoverexpression ofsonic mice. cerebral cortexdevelopmentandbloodpressure regulation inFGF-2-deficient and axialorganization. Leder, P. FGF signaling. T. 2325. genes andiscompromised inGli3-deficientmice. the embryoniccerebral cortexisdefinedbytheexpression ofmultipleWnt Development requirement forhedgehogsignalinginventraltelencephalic patterning. cerebellum. and common signal for ventral patterning withintheneuraltube. common signalforventralpatterning Sonic hedgehoginducesthedifferentiation ofventralforebrain neurons: a (2003). SpecificationofdorsaltelencephaliccharacterbysequentialWntand Otx2 EMBO J. 29 (1994). MurineFGFR-1isrequired forearlypostimplantationgrowth define layersandregions indevelopingcerebral cortexand , 787-790. Development (2000). Neuronal specification inthespinalcord: inductivesignals (2005). Unravelingthemolecularpathwaysthatregulate early J. Neurosci. 108 130 131 Development Development Nat. Neurosci. 17 Dev. Biol. (2005). , 4213-4225. Development (2003). Loss-of-functionmutationsinFGFR1causeautosomal , 183-206. , 1101-1111. , 5031-5040. (1996). Cyclopia and defective axial patterning inmice (1996). Cyclopiaanddefectiveaxialpatterning 16 Mol. Cell.Neurosci. , 135-141. Genes Dev. 14 Fgf8 260 122 Nat. Rev. Genet. , 5725-5740. Development 132 127 Curr. Top. Dev. Biol. , 484-495. 6 , 1769-1780. (2000). Specificationofventralneuron typesis defines aneurogenic domain required forstem , 701-707. (1996). Complexexpression ofthezp-50pou Nat. Genet. 125 (1995). Themouse , 5211-5223. , 5007-5020. 8 (2003). Mousemodelsofholoprosencephaly. , 5079-5089. (2003). Adisruptedbalancebetween , 3045-3057. Nature 121 14 RESEARCH ARTICLE 1 33 , 355-369. , 20-29. , 439-451. , 463-465. 383 J. Neurosci. 69 Development (1998). Regionalizationwithin , 407-413. Fgf8 , 15-35. (2000). Originand J. Biol.Chem. (2004). Temporal gene encodesafamily (1999). Mapping (1998). Impaired 20 (1998). Thehemof Cell , 6063-6076. Gli3 (2003). FGF (1996). Fibroblast (1999). Correct 125 Nat. Neurosci. (2001). Cell 81 mutant (1994). 274 , 2315- , 747-756. 84 , 34785- (2000). , 911- (1995). 2945 Otx1 3 ,

DEVELOPMENT Scholzen, T. andGerdes, J. Rallu, M.,Machold,R.,Gaiano,N.,Corbin,J.G.,McMahon,A.P. andFishell, D.,Werner,Peters, K.,Ornitz, S.andWilliams,L. Persson, M.,Stamataki,D.,teWelscher, P., Andersson,E.,Bose,J.,Ruther, Shanmugalingam, S.,Houart,C.,Picker, A.,Reifers,F., Macdonald,R.,Barth, Peters, K.G.,Werner, S.,Chen,G.andWilliams,L.T. Orr-Urtreger, A.,Givol,D.,Yayon, A.,Yarden, Y. andLonai,P. D.M.,Xu,J.,Colvin,J.S.,McEwen,G.,MacArthur,Ornitz, C.A.,Coulier, 2946 Ohkubo, Y., Chiang,C.andRubenstein,J.L. Oh, L.Y. S.,Denninger, A.,Colvin,J.S.,Vyas, A.,Tole, D.M.and S.,Ornitz, Maruoka, Y., Ohbayashi,N.,Hoshikawa,M.,Itoh,Hogan,B.M.and Meyers, E.N.,Lewandoski,M.andMartin,G.R. McWhirter, J.R.,Goulding,M.,Weiner, J.,Chun,J.andMurre, C. Mason, I.J.,Fuller-Pace, F., Smith,R.andDickson,C. Miyake, A.,Nakayama,Y., Konishi,M.andItoh,N. Muenke, M.andBeachy, P. A. Muzio, L.,DiBenedetto,B.,Stoykova,A.,Boncinelli,E.,Gruss,P. and unknown. 4974. mutants lackingbothGli3andHedgehogsignaling. G. 423-430. oftheFGFreceptorpattern 3geneduringmouseorganogenesis. limb formationandorganogenesisinthemouse. genes are differentially expressed inepithelialandmesenchymaltissuesduring cord requires Gli3transcriptionalrepressor activity. U., Ericson,J.andBriscoe, A., Griffin, K.,Brand,M.andWilson,S.W. and Developmental expression oftwomurinefibroblast growth factorreceptors, growth factorfamily. F., Gao, G.andGoldfarb,M. 111 regulate morphogenesisofthetelencephalicandopticvesicles. synergistic actionsofBMP4,SHHandFGF8intherostral prosencephalon the developingtelencephalon. Gunhaga, L. Development forebrain commissureofthetelencephalon. formationandpatterning onset ofoligodendrocyte terminaldifferentiation. Bansal, R. Fgf8 Furuta, Y. Development is adownstream targetofthechimerichomeodomainoncoprotein E2A-Pbx1. novel fibroblast growth factorgeneexpressed inthedevelopingnervoussystem interactions. roles inmyogenesis,forebrain regionalisation andepithelial-mesenchymal (keratinocyte growth factor)expression duringmousedevelopmentsuggests 141. series generatedbyCre- andFlp-mediatedrecombination. Hh signalingisrequired forzebrafishdevelopment. Emx2( Mallamaci, A. and D.Valle), pp.6203-6230.NewYork: McGraw-Hill. Molecular BasesofInheritedDisease (2002). Dorsoventral patterning isestablishedinthetelencephalonof (2002). Dorsoventralpatterning , 1-17. bek , Fgf17 –/– RESEARCH ARTICLE . ) Pax6(Sey/Sey)double-mutantmice. Development J. CellPhysiol. (1998). Comparisonoftheexpression ofthree highlyrelated genes, (2003). Fibroblast Growth Factorreceptor 3signalingregulates the and Mech. Dev. (2004). Retinoicacidsignallingspecifiesintermediatecharacterin 127 124 (2002). Conversionofcerebral cortexintobasalgangliain Fgf18 , 2549-2561. , 3221-3232. J. Biol.Chem. , inthemouseembryo. 113 45 182 (2000). TheKi-67protein: from theknownand , 1419-1434. , 15-30. , 311-322. (2001). Holoprosencephaly. In Development (1996). Receptorspecificityofthefibroblast (2002). Dorsal-ventral patterning ofthespinal (2002). Dorsal-ventralpatterning 271 (ed. C.R.Scriver, A.L.Beaudet,W. S.Sly , 15292-15297. 131 Nat. Neurosci. (2002). Coordinate regulation and Mech. Dev. (2000). , 4323-4332. Development J. Neurosci. (1998). An Genes Dev. (1993). Uniqueexpression Dev. Biol. Development (2005). Ace (1994). FGF-7 (1992). Two FGFreceptor 74 / Nat. Genet. Fgf8 5 , 175-177. The Metabolicand Fgf19 , 737-745. 288 Fgf8 23 16 114 Neuroscience is required for (1991). , 883-894. , 2865-2878. , 259-275. Dev. Biol. 129 mutant allelic , 233-243. regulated by (1997). A , 4963- 18 , 136- 155 flg , Wang, D.M. Q.,McEwen, D.G.andOrnitz, Wijgerde, M.,McMahon,J.A.,Rule,M.andA.P. Walshe, J.andMason, I. Toresson, H.andCampbell,K. Xu, J.,Lawshe,A.,MacArthur, D.M. C.A.andOrnitz, Wilson, S.W. andRubenstein,J.L.R. Sussel, L.,Marin,O.,Kimura,S.andRubenstein,J.L. Tole, S.,Gutin,G.,Bhatnagar, L.,Remedios,R.andHébert,J.M. Szucsik, J.C.,Witte,D.P., Li,H.,Pixley, S.K.,Small,K.M.andPotter, S. Storm, E.,Garel, S.,Borello, U.,Hébert,J.M.,Martinez,S.,McConnell,S.K., Yun, K.,Garel, S.,Fischman,S.andRubenstein,J.L.R. Zaki, P. A.,Quinn,J.C.andPrice,D. Yamaguchi, T. P., Harpal,K.,Henkemeyer, M.andRossant,J. Shimamura, K.andRubenstein,J.L.R. Storm, E.,Rubenstein,J.L.andMartin,G.R. Shinya, M.,Koshida,S.,Sawada,A.,Kuroiwa, A.andTakeda, H. Theil, T., Alvarez-Bolado, G.,Walter, A.andRuther, U. Yu, K.,Xu,J.,Liu,Z.,Sosic,D.,Shao,Olson,E.N.,Towler, D.A.and factor 14. developmental expressionsplicedformsoffibroblast ofalternatively growth 4349. and Fgf8duringzebrafishforebrain development. progenitor domainsinthepresumptive mammalianspinal cord. requirement forhedgehogsignalingnormalspecification ofallventral 4780. striatum andolfactorybulbofGsh2mutantmice. the cerebrum. Development ofmidlinecelltypesandcommissuralaxontractsrequires Dev. structure, mapping,activityandexpression offibroblast growth factor17. ofthetelencephalon. patterning 2849-2864. into thestriatum. within thebasaltelencephalon:evidenceforatransformationofpallidum homeobox genefunctionresults inaventraltodorsalmolecularrespecification 1844. developing forebrain. determines whethercellsurvivalispositivelyornegativelyregulated inthe Development for Emxgeneexpression duringdorsaltelencephalondevelopment. Gsh-2 homeoboxgene. (1997). Altered forebrain andhindbraindevelopmentinmicemutantforthe Fgf8 Martin, G.R.andRubenstein,J.L. development. axons through thebasalganglia. required forhistogenesisofthestriatumandolfactorybulb andthegrowth of the lateralganglioniceminenceby growth. essential role forFGFsignalingintheregulation ofosteoblast functionandbone D.M. Ornitz, telencephalon inzebrafishembryos. signalling through MAPKcascadeisrequired fordevelopmentofthesubpallial early regionalization oftheforebrain. gastrulation. required forembryonicgrowth duringmouse andmesodermalpatterning 83 in regulatingcenters. telencephalicpatterning , 165-178. Development Mech. Dev. Genes Dev. 126 Curr. Opin.Genet.Dev Dev. Biol. (2003). ConditionalinactivationofFGFreceptor 2reveals an , 3561-3571. Development Proc. Natl.Acad.Sci.USA 90 130 Dev. Biol. 289 , 283-287. (2003). UniqueandcombinatorialfunctionsofFgf3 8 , 3032-3044. , 3063-3074. , 141-151. (2001). Arole forGsh1inthedeveloping 126 Neuron J. Comp.Neurol. 191 , 3359-3370. Development . Development , 230-242. 13 (2000). Inductionanddorsoventral (2003). Mousemodelsoftelencephalic Gsh1 (1997). Inductiveinteractionsdirect (2006). Dosagedependentfunctionsof , 423-437. 28 , 641-651. and (2000). Subcellularand (2003). DosageofFgf8 100 Gsh2 Development 128 461 Development Development 124 Development 133(15) , 1757-1762. , 4153-4164. , 151-165. homeobox genesis (1999). LossofNkx2.1 , 2709-2718. (1999). Genomic (1999). Gli3isrequired (2003). Patterning of (2003). Patterning (2002). Adirect (1994). Genes Dev. 128 130 133 (2006). (2001). Fgf , 4769- , 4337- , 1831- Fgfr-1 Fgfr1 Mech. 16 in is ,

DEVELOPMENT