telencephalic patterningcenters death. Inaddition,alterationsintheexpressionof subdivisions. modifications intherelativestrengthofFGFsignalingcanhaveprofoundeffects ontherelativesizeandnatureoftelencephal regulate neocorticalpatterning.Thesedatasuggestthat ua .McConnell K. Susan *These authorscontributedequallytothis work Stanford University, Stanford, CA94305-5020, USA. and Universidad Miguel Hernández, Spain. and UniversidadMiguelHernández, Ohkubo etal.,2002;Shimagori2004)ormolecules that (Shimamura etal.,1995;Kim2001;Anderson2002; molecules suchasNogginandSFRPthatrestrictligandavailability Patterning centersarealsoregulated bytheexpression ofsecreted other’s expression (Ohkubo etal.,2002;Shimogori2004). and thereisevidence thatFGF8andBMP4reciprocallyrepresseach maintain Garel andRubenstein,2004).For instance,SHHis required to Crossley etal.,2001). not beenfirmlyestablished,expresses al., 2002).Theventral patterningcenter, thefunctionofwhichhas (Grove etal.,1998;Galceran2000;Lee1999;Hebert genes andcontrolsthedevelopment ofdorsocaudalstructures dorsal patterningcenterexpresses anestedsetofBMP andWNT et al.,1998;BachlerandNeubuser, 2001;Gimenoetal.,2003).The a nestedsetofFGFgenes: Garel andRubenstein,2004).Therostralpatterningcenterexpresses midline (Crossley etal.,2001;Grove andFukuchi-Shimogori,2003; telencephalon, atleastthreepatterningcentersextend fromthe signals thatregulate regional identityandgrowth. Intheembryonic juxtaposed indeveloping tissuestoprovide qualitatively distinct embryonic morphogenesis.Multiplepatterningcentersare Secreted moleculesproducedbypatterningcentersregulate INTRODUCTION KEY WORDS: Bmp4 dorsal andventralstructures.Furthermore,nonlineareffects of We provideevidencethatthehypoplasiaresultsfromdecreased Mouse embryosbearinghypomorphicandconditionalnull Elaine E.Storm Dose-dependent functionsof Development 133,1831-1844(2006)doi:10.1242/dev.02324 Accepted 29March 2006 § ‡ † 1 of Medicine,Bronx, NY10461,USA. USA. Psychiatry, SanFrancisco,CA94143-2611, UniversityofCalifornia, San Francisco,CA94143-2611,USA USA. Author forcorrespondence (e-mail:[email protected]) Present address: INSERM,U368,EcoleNormaleSupérieure, Paris,France Present address: DepartmentofPsychiatry, SanFrancisco, University ofCalifornia, Department ofAnatomy, SanFrancisco,CA94143-2711, UniversityofCalifornia, There iscrossregulation betweenpatterningcenters(reviewed by 3 2 Departments ofNeuroscience andMolecularGenetics,AlbertEinsteinCollege Nina Ireland LaboratoryofDevelopmentalNeurobiology, Departmentof and Fgf8 Msx1 Fgf8 expression (Ohkuboetal.,2002;Aoto2002), , correlatewithaholoprosencephalyphenotypeandthenonlinearexpressionoftranscriptionfactorsthat , Forebrain,Mouse Patterning, 1, * ,† , SoniaGarel 5 , GailR.Martin Fgf8 4 Instituto deNeurociencias deAlicanteCSIC , Fgf18 5 Department ofBiologicalSciences, Shh , 2, Fgf17 * ,‡ (Shimamura etal.,1995; , UgoBorello 2 and JohnL.R.Rubenstein and Fgf15 Bmp4 (Maruoka , Fgf8 2, Wnt8b *, JeanM.Hebert Fgf8 functions tocoordinatemultiplepatterningcenters,andthat Fgf8 mutations havesmallandabnormallypatternedtelencephalons. Fgf8 , Foxg1 Nkx2.1 can bedeletedbyCre-mediatedrecombination), (Sun etal.,1999),whereastelencephalic conditional Fgf8 ( al., 1998).Micelacking Fgf8 Storm etal.,2003;Huffman etal.,2004).Thesestudiesusedfour telencephalic development (Meyers etal.,1998;Garel2003; facilitated theanalysisofmultiplefunctions telencephalon (Shinya etal.,2001;Walshe andMason,2003). in regulating Sansom etal.,2005).FGFsignalinginzebrafishisalsoimplicated Garel etal.,2003;StormWalshe andMason,2003; Shimogori andGrove, 2001;Fukuchi-ShimogoriandGrove, 2003; and otherregulatory genes(Crossley etal.,2001;Fukuchi- rostral telencephalonbymodulatingtheexpression of increase ordecreaseFGFsignalinginfluencethepatterningof al., 2000;Stormet2003).Furthermore,manipulationsthat in asmalltelencephalon(Meyers etal.,1998;Shanmugalingam et 2005; Martynogaetal.,2005). Papalopulu, 2000;Hanashimaetal.,2004;Mucioand Mallamaci, fates (Xuanetal.,1995;Dou1999;Hardcastleand proliferation andrepressesdifferentiation anddorsaltelencephalic (Dou etal.,2000;Seoane2004)andtherebypromotes winged helixtranscriptionfactor thatrepressesTGF (Shimamura andRubenstein,1997;Ye etal.,1998).FOXG1 isa patterning center)positively regulates theexpression of mouse anteriorneuralridge(theanlageofthetelencephalicrostral Sagi, 2004). signaling (Minowada etal.,1999;Lin 2002;KimandBar- function intracellularly, suchassproutyorSEFproteins,torepress including the frontal neocortex; (2) rostral expansion of the types ofdefects:(1)hypoplasia ofrostraltelencephalicstructures Previously, wereportedthatthesemutantsexhibit threegeneral telencephalon patterning(Storm etal.,2003;Garel2003). normal expression), Fgf8 gene doseontheexpressionofasubsetgenes,including An allelicseriesofmutationsatthemouse Fgf8 Previous studiessupportamodelinwhich expression, reducedcellproliferationandincreased Neo/Neo alleles: and flox/flox 2,§ in regulating hypomorphic mutationsinbothmouseandzebrafishresult Shh mutants survive untilbirthandexhibit gradeddefectsin 3 Fgf8 recombined using , SalvadorMartinez are associatedwithabnormaldevelopmentof Shh (wild type), expression anddevelopment oftheventral Fgf8 Fgf8 Null (exons 2and3aredeleted)(Meyers et Fgf8 ( Fgf8 flox Foxg1-Cre RESEARCH ARTICLE Null/Null (exons 2and3arepresentbut 4 , ) dieduringgastrulation Fgf8 ), Fgf8 expression inthe Fgf8 Fgf8 Fgf8 Null/Neo Fgf8 Emx2 ␤ Neo locus has TelKO signaling gene in (~40% , and Foxg1 , ic 1831 Otx2 nulls

DEVELOPMENT Fgf8 expression andapoptosis. MATERIALS ANDMETHODS phylogenetic andintra-individual diversity. likely tocontribute totheir telencephalic subdivisions thatare signaling have profoundeffects ontherelative sizeandnatureof modifications intherelative strengthofFGF/BMP/WNT/SHH gradients oftranscriptionfactor expression demonstratesthat of regulatory interactionsbetweenpatterningcentersthat control transcription factors thatregulate neocorticalpatterning.Thenexus holoprosencephaly phenotypeandalterationsintheexpression of intact in growth. Fgf8 midline development; hereweconcentrateontheeffect ofreducing focused ontheeffects ofreducing stage embryos.Furthermore,Stormetal.(Stormal.,2003) dose-dependent effects onneuralplateandearly post-neurulation to produce of following neuraltubeclosure.Becauseprosencephalic expression and didnotexamine primaryphenotypesintheneuralplateorjust Fgf8 to eitherdecreases( interactions betweentherostralanddorsalpatterningcentersleading properties [e.g. expression oftranscriptionfactors thatregulate neocorticalregional 1832 histochemistry (counter-stained withDAPI). ThenumberofPH3 Every other sectionwas stainedusingeitherPH3or TUNEL E9.0 embryos(~14-17somites)were sectionedinthehorizontalplane. Cell proliferation andapoptosisanalyses: analysis was performedon10-16 microscope andimageswereacquiredusingaSpotCCDcamera.TUNEL and fluorescentimmunohistochemicalstainingwereanalyzedunderaLeica 1/400 (Upstate)wereusedasprimaryantibodies.Hoechstcounterstaining described previously (Yun etal.,2001).Rabbitanti-phosphohistone-3(PH3) Immunohistochemistry was performedon10-16 embryos werefixed overnight in4%paraformaldehyde (PFA) inPBSat4°C. For immunohistochemistry, TUNELassaysandinsituhybridization, Immunohistochemistry, TUNELandinsituhybridization background. All Mice andgenotyping embryos ascontrols did notshow any discernablephenotypeandwereusedwithwild-type Fgf8 considered asembryonicday0.5(E0.5).Heterozygous al., 2003).For stagingofembryos,noononthedayvaginal plugwas performed asdescribedpreviously (HebertandMcConnell,1999;Stormet effects of turn affect patterningofbothdorsalandventral structures.Nonlinear Fgf8 contribute totelencephalichypoplasia.We alsodemonstratethat expression, areducedmitoticindex, andincreasedapoptosis stage toelucidatethemechanismsunderlying essential toinvestigate themutantphenotypesshortlyafterthis Shimamura andRubenstein,1997;Crossley etal.,2001),itis previously (Depew et al.,2002). hybridization was performedonwhole-mountembryosasdescribed Kit following themanufacturer’s recommendations (Intergen). Insitu The previous studiesconcentratedonthephenotype ofthe We reportourfindingthatspecificationoftheprosencephalonis Fgf8 Fgf8 Null/Neo TelKO Null/Neo dose ontelencephalicpatterningcenters,regionalization and regulates theexpression of RESEARCH ARTICLE mutant allelesweremaintainedonamixed 129/CD1Swissgenetic begins atneuralplatestages(Crossley andMartin,1995; and Fgf8 Fgf8 embryos. phenotypes. Therefore,herewereportstudiesof Fgf8 Fgf8 Fgf8 flox/Null Emx2 mutants; however, amajorreductionin Null/+ dose on Null/Neo Fgf8 ; Foxg1-Cre Fgf8 and and flox/flox Null/Neo mutant telencephalonbeginning atE10.5 Fgf8 Nr2f1 and Bmp4 ( Fgf8 ␮ Neo/+ Foxg1-Cre ) orincreases( m cryostatsectionsusingtheApoptag Bmp4 ( elKO l Te COUP-TF1 expression correlatewitha mice werecrossedtoproduce Fgf8 ) embryos.PCRgenotypingwas , Wnt8b ; Fgf8 dose ontelencephalic ␮ Null/+ m cryostatsectionsas )]; and(3)complex Fgf8 and Fgf8 mice werecrossed TelKO Fgf8 Shh Null/+ , whichin ) in TelKO embryos Foxg1 Bmp4 + Fgf8 and cell In contrast,expression of strong rostrally, andmayhave expanded caudally(Fig.2C,C Crossley etal.,2001;Chi etal.,2003). evidence FGF8can repress caudally intotherhombencephalon (Fig.2A,A expanded bothrostrally intheprosencephalon(Fig.2B,B Six3 factors thatareimportantforprosencephalicdevelopment, 2001). Thus,weanalyzedtheexpression ofthreetranscription the 4-somitestage(Crossley andMartin,1995;Crossley etal., Fgf8 appeared widerandflatter(Fig.2). and morphologicaldefectsintheanteriorneuralplate,which complete atthisstage.Ineachcase,weobserved subtlemolecular somite stage,becauseCre-mediatedrecombinationmaynotyet be biases betweengenotypes,but couldaffect theabsolutenumbers. appeared independentofgenotype,andshouldnothave ledtosystematic than oneTUNELreactionproduct.However, thesecomplications TUNEL staining;(3)uncertaintyaboutwhetherasinglecellcanhave more different levels ofPH3staining;(2)thesmallsizedotsgeneratedby Precise quantificationofpositive cellnumberswas complicated by:(1) left andtotherightofmidline,cellswerecountedonbothsides. (i.e. row EinFigs4and6).Inagiven section,box2was drawn bothtothe Figs 4and6);sectionsthatweredorsaltotheopticstalknotanalyzed labeled cellsinsectionsfromventral telencephalicregions (rows A-Din the anlageofbasalganglia,septumandrostralcortex). We counted the edgeofbox1towards theopticstalkregion (probably encompassing and approximatedtherostroventral telencephalon,extending laterallyfrom ~45 telencephalon (box1andbox2,respectively inFigs3and5).Box1was regions oftheforebrain:rostralmidlineandrostroventral reduction of Fgf8 rostroventral structuresshowed the mostprofoundalterations.Both prepared fromE14.5wild-type, Comparative analysisofCresylViolet-stained horizontalsections mutants defects in Rostral andventraltelencephalicmorphological RESULTS nuclei andTUNEL stage defectsinlateneuralplate Molecular patterning earlier development ofthesemutants. mechanisms underlyingthesemorphologicaldefects,westudiedthe they appearedsmallerandthinnerthannormal.To elucidatethe amygdaloid structureswerepresent(Fig.1andnotshown), although relatively morepreserved, as choroidal, hippocampaland mutants. Caudalanddorsaltelencephalicstructuresappearedtobe et al.,2005).We didnot examine (Xuan etal.,1995;Simeone2002;Lagutin2003;Gestri somite stage(Fig.2)andotherneuralplatestages(datanotshown) more severe in and olfactory bulbs (Fig.1,andnotshown); thesephenotypeswere and lacked identifiableseptalandpreopticnuclei,anopticchiasm observed in Fgf8 There was somevariation intheseverity ofthephenotypes Reducing revealed thedysmorphologiesinmutantforebrains(Fig.1). differed: the ␮ Null/Neo Null/Neo and expression begins intheanteriorneuralplateatapproximately m wideandspannedtherostralmidline.Box2was ~170 Fgf8 Foxg1 Fgf8 and mutants, whereasnoclearphenotypicvariation was Fgf8 Fgf8 Null/Neo Fgf8 Fgf8 dose causedprogressive prosencephalichypoplasia. , inthe Fgf8 Foxg1 Null/Neo + Null/Neo TelKO TelKO cells intheneuroepitheliumwas countedintwo TelKO mutants: expansionof Fgf8 mutants. Therostralmidlineofthemutants midline was thicker thanthatin mutants hadasingleganglioniceminence mutants. Inallmutants,rostraland expression Foxg1 Otx2 Null/Neo hypomorphic and Fgf8 expression (Martinezet al.,1999; Fgf8 ( Otx2 mutant embryosatthe9-to10- Bf1 Null/Neo ) was reduced intheneural TelKO expression appearedtohave Six3 mutants atthe9-to10- and Development 133(9) expression remained Ј Fgf8 ), consistentwith Fgf8 Otx2 TelKO Fgf8 TelKO embryos ␮ Ј ,D,D and m wide Ј Otx2, ) and TelKO Ј ).

DEVELOPMENT Foxg1 intact in Defects inboth telencephalon expanded expression of Reduced expression of Regulation oftelencephalicpatterning (Garel etal.,2003; Stormetal.,2003). in Fig.3F,G), whichwas maintainedatlaterstages (seeFig.7) shown) andamore profoundrostralexpansion atE9.5(arrowheads contrast, olfactory placodeappeared unaffected (Fig.3C,C plate; arches (Fig.2E,E reduced in (arrowhead; Fig.3B,B Six3 the telencephalicvesicles was reduced(Fig.3A,A Six3 E9.0. Atthisage,theevagination ofthe opticvesicles (asmarked by discern in expression) was roughlynormalinthemutantsbut thesizeof expression intherostralmidlinewas reducedinbothmutants , someaspectsoftelencephalicmolecularspecificationappear Foxg1 Fgf8 Emx2 Fgf8 Fgf8 Null/Neo expression was even morereducedinthebranchial expression showed asubtlerostralshiftatE9.0(not Fgf8 TelKO Null/Neo Ј ,F,F mutants. Null/Neo embryos, whereas Ј ). Thus,basedontheexpression of Ј mutants andwas even moredifficult to ,B Љ and .Expressionof ). Foxg1 Fgf8 Emx2 TelKO and in theearly Foxg1 mutants wereobvious by Six3 Foxg1 expression inthe Ј ,C and Љ Ј ,D,D ,A was greatly Љ ,B,B Ј Six3 ,D Љ Ј ). By ,B and Љ ). rostroventral telencephalon(box2;Fig.3E,E no differences weredetectedbetweenthegenotypes.In Previously, weprovided evidence thatthemutationsin midline development. changes intheexpression ofregulators ofdorsal Fgf8 and mutant (Fig.3E,E embryos hadroughlyatwofold highermitoticindex thaneither mutants atE9.0.Intherostralmidline(box1,Fig.3E,E of theM-phasecellcycle marker phosphohistone-3(PH3)in telencephalon (Hanashimaetal.,2002),weassessedtheexpression Because theinactivation of rostroventral telencephalon Reduced cellproliferation intheearly differentiation (Fig.5). transcription factor genes implicated inregulating dorsalmidline we examined theexpression atE9.5ofsignalingmoleculeand embryos (Stormetal.,2003).To confirmandextend thesefindings, Bmp4 Fgf8 Null/Neo and thelevel ofapoptosisinthedorsalmidlineE10.5 TelKO and embryos have distincteffects ontheexpression of A,A were definedbytheirlocationandmorphology. eminence; OB,olfactorybulb;S,septum. ganglionic eminence;MGE,medial GE*, mutantganglioniceminenceregion; LGE,lateral A, amygdalaregion; A*,mutantamygdalaregion; E Fig. 1. telencephalon null( wild-type (A-E), telencephalon. rostral andmidlineregions ofthe hypoplastic andhavemorphologicaldefectsin Љ Ј ) brainswere stainedwithCresyl Structures Violet. ,E Fgf8 Ј Љ ,A and Fig.4;Table 1). Љ Fgf8 are mostdorsalandE,E TelKO Bmp4 Foxg1 hypomorphic ( mutants exhibitdistinct Fgf8 ( A-E expression, whichmarksthe reduces cellproliferationinthe Null/Neo RESEARCH ARTICLE Љ TelKO ) HorizontalsectionsofE14.5 (A ) mutantsare Null/Neo Ј Ј -E ,E Ј Ј ,E ) and Љ , Fig.4),wild-type Љ are mostventral. ) and Fgf8 Ј Fgf8 ,E TelKO Љ , Fig.4) Null/Neo (A 1833 Fgf8 Љ -

DEVELOPMENT E,E expression isnotdetected in themutantpharyngealregion (arrows in Foxg1 expression isintensifiedin caudal regions (arrowheads inC-D Whole-mount insituhybridizationshowing of themidbrain/hindbrainboundary (arrowheads inA,A towards thetop.Inneural plateofthemutants, anterior islocatedtotheright;on frontal views,anteriorislocated bythenumberofsomites(s).Onlateralviewsembryos, left corner embryos. Thedevelopmentalstageofembryosisindicatedinthelower signaling (Furutaetal.,1997;ShimamuraandRubenstein, transcription factor positively regulated intheforebrainbyBMP either mutant(Fig.5A,A rostrodorsal midlineatthisage,was notdetectedinthisregion of 1834 Fig. 2. Analysis of neural plate patterning in Fig. 2.Analysisofneuralplatepatterning slightly expandedrostrally (arrowheads inB,B expression ( Ј ). expression isslightlyreduced (arrowheads inF,F RESEARCH ARTICLE C-D Ј ) and Foxg1 Ј ,A expression ( Љ ). Expressionof E-F Ј Otx2 ) in9-to10-somite Ј ) andcaudallyatthelevel Fgf8 expression ( Msx1 Otx2 Ј Null/Neo ). Ј ); Foxg1 , ahomeobox expression is Six3 Ј embryos. A-B ); and Ј ), Six3 Fgf8 Fgf8 interactions inthedorsalmidline. expression. Thesechangesreflectthecomplexity ofregulatory shown insquare parentheses foreachmutantincomparisonwithwildtype.Values Fig. 6A expression was almosteliminatedin reduced orabsentfromtherostrodorsalmidline(Fig.5D). present throughoutmuchofthetelencephalicvesicle, but was Kawakami etal.,2004).Incontrolembryos, implicated inregulating regulated byFGF10andWNT/ zinc-finger transcriptionfactor gene,whichinthelimbbud is shown). 5B,B pattern resembledthatof Fgf8 apoptosis in Storm etal.,2003;Chi2003),thereforeweexamined (Trumpp etal.,1999;Crossley etal.,2001;Abu-Issa etal.,2002; Alterations intheexpression of ofapoptosisintherostralpatterns midline midline ofthe UE idtp ±. 2.5±1.3 below 0.05are statisticallysignificant. 38±20 [0.001] 5.5±1.2[0.37] 26±5.7 13±3.7 [0.003] 38±18 [0.005] 5±1.2 1.7±0.6[0.39] 1.6±0.9[0.005] 12±4.6 [0.016] 2 1.5±0.7[0.37] 1.4±1.2 Box 2 2 2 3 marker (phosphohistone3;PH3)andtheaveragenumberofTUNEL 2 TelKO The datashowtheaveragenumber±s.d.ofcellularnucleilabeledwithM-phase Null/Neo 3 TUNEL TelKO Wildtype Box1 TUNEL Null/Neo TUNEL Wildtype PH3 ofembryos PH3 Genotype PH3 assayed Marker Number rostroventral telencephalon Table 1.PH3andTUNELanalysisintherostral midlineand individual cellcanhavemore thanoneTUNEL (Box 2)(seePH3andTUNELdatainFigs3-6). We donotknowwhether ornotan (apoptosis marker)intherostral midline(Box1)andintherostroventral telencephalon of Feledy etal.,1999),was alsonotdetectedintherostrodorsalmidline approx. threefoldfewer TUNEL telencephalon atthisage(box1,Fig.5E,6A-E).However, cells arenormallyabundant intherostralmidlineof Thus reductioninthedoseof persisted but alsoencompassedthedorsalmidline(Fig.5D variance intheaveragenumberofpositive cells/embryo.Resultsof by thenumberofboxescounted.Thestandard deviation(inparentheses) reflects the the rightofmidline)inthree orfoursectionsoftwothree embryosanddividing total numberoflabeledcellsinBox1orBox2 (whichwasdrawntoboththeleftand description ofhowtheboxeswere drawn.Averages were determinedbycountingthe 5D to thoseobserved inmutantsatE10.5(Stormetal.,2003) andwere expansion of telencephalon inboth extended rostrallyandencompassedmuchofthehypoplastic expression (whichisinparamediandorsallongitudinaldomains) dorsal telencephalon.Thusweobserved thattelencephalic led toarostralexpansion oftheWNTexpression domaininthe (Shimogori etal.,2004),wefoundthatreductionsin E10.5 (Stormetal.,2003). We thenexamined theexpression of Consistent withtheresultsofinuteroelectroporationexperiments Fgf8 Ј ). Incontrast,in TelKO TelKO Ј Null/Neo B Null/Neo Љ Љ -E ), similartoprevious observations of mutants appearedsimilartowild-typecontrols(Fig.5E mutants exhibited distinctpatternsof Љ ; Table 1).Thesedistincteffects onapoptosisaresimilar Fgf8 and mutants but was maintainedin Fgf8 Wnt8b mutants atE9.0usingtheTUNELassay. TUNEL Null/Neo Fgf8 Fgf8 Fgf8 in bothmutants,whereas TelKO mutants (Fig.5E TelKO Emx2 mutants (Fig.5C,C Fgf8 embryos, mutants exhibitdistinct Fgf8 + expression (Belletal.,2003; (Fig. 3F,F + cells werevisibleintherostral Fgf8 ␤ signal. SeeMaterialsandmethodsfora are associatedwithcelldeath -catenin signaling,andis Fgf8 Sp8 resulted intherostral Sp8 Ј , Fig.6A Ј Development 133(9) Null/Neo Fgf8 ,F expression notonly Sp8 , abuttonhead-like Bmp4 Ј ,C Љ ,G,G Љ TelKO Fgf8 ). Itsexpression expression was T Msx1 Fgf8 + -test analysesare mutants (Fig. Ј profiles -E expression at Ј ,G brains (Fig. Ј Null/Neo ), whereas gene dose Љ and and not Wnt8b Sp8 Sp8 and Љ ). Љ + ,

DEVELOPMENT al. (Chietal.,2003) forapoptosisinthemesencephalon of Fgf8 observed intheopticstalk, hypothalamusandmesencephalonof wild-type embryos(Table 1). hypothalamus (Fig.6A,B,C).Large numbersofTUNEL cells werepresentincaudalregions oftheopticvesicles andinthe other partsof telencephalon (box2,Fig.5E,E al., 2004;RamosetPark etal.,2005).Intherostroventral transcription factor thatisapositive regulator ofapoptosis(Liuet Regulation oftelencephalicpatterning positively correlatedwiththeexpression of Fgf8 Changes inthenumbersofTUNEL Null/Neo TelKO embryos hadapprox.tenfoldmoreTUNEL mutant embryos(Fig.6A Fgf8 mutant brains.Inwild-typeembryos, apoptotic Ј E Љ , Fig.6),boththe Ј -E + Ј cells werealsoobserved in and notshown) [see Chiet Msx1 (Fig. 5B,B Fgf8 + + Null/Neo cells than cells were Ј ,B Fgf8 Љ and ), a reduction of these regions. consistent withthelower levels ofCrerecombinaseexpression in TUNEL regions (Crossley etal.,2001;Treier etal.,2001).Scattered hypothalamus correlatewithearlyexpression of Thehighlevels ofcelldeathintheopticstalkand mutants]. (Fig. 6A undoubtedly contribute tothetelencephalic hypoplasiaobserved in signals (proliferationand apoptosis). Thesemodifications rostrodorsal patterningsignals,and inthecellularresponsestothese Fgf8 Thus, atE9.0-E10.0,asthetelencephalic vesicles areforming,a Null/Neo + Љ cells werevisibleintheopticstalksof -E mutants inthehypothalamusandmesencephalon, Ј Fgf8 Note, thepanelsshowingPH3labelingare180 turned for proliferation inthetelencephalon(Xuanetal.,1995). one frontal views.The panel showslateralviewsoftheembryos,andlower (E9.5 embryos; reduced in expression domaininthetelencephalonisseverely The reduction inPH3 reduction in Fgf8 indicate theregions inwhichthenumbersofPH3 expression andthenumberofPH3 other; thiswasdonetofacilitatecomparisonof hybridizations, suchthattherostral regions faceeach with respect tothepanelsshowingfrontal viewsofinsitu mutants (arrowheads inF-F telencephalon expandsrostrally in in B-B rostroventral telencephalonappearssmaller(arrowheads E9 embryosshowing mutant embryos. Fig. 3. stalk; Tel, telencephalon. rostroventral telencephalon(box2)(seeTable 1).Os,optic were countedintherostral midline(box1)andinthe of mitoticcellsinwild-type( horizontal sectionsthrough theforebrain labelsthenuclei phosphohistone3 (PH3)immunofluorescence on in bothmutants(openarrowheads). Anti- Foxg1 reduced intheforebrain of ), but thesebrainsshowed lessapoptosisthanthe Fgf8 TelKO TelKO Љ ) and expression intheolfactoryplacodesisstilldetected dose leadstoanalterationinthe expression of Foxg1 ( embryos (solidarrowheads inC-D E Љ Fgf8 ) embryos(additionalsectionsare inFig.4). Emx2 Foxg1 expression andproliferation are F-G Null/Neo expression inthecaudodorsal Љ expression (D,D Whole-mount insituhybridizationon ) expression. Foreachprobe, thetop + Six3 embryos andalmostabsentin -labeled cellscorrelates withthe Six3 RESEARCH ARTICLE ( A-B Љ expression domaininthe ). Bycontrast,the E ), Fgf8 Љ Fgf8 ), Foxg1 Fgf8 + Ј Null/Neo ,D Null/Neo cells. Theboxes Љ ). Null/Neo Fgf8 ( Foxg1 C-D ( and Љ E Fgf8 ). Bycontrast, Ј TelKO Љ ) and ) and and Foxg1 is required Fgf8 Foxg1 + in these Fgf8 mutants Emx2 cells TelKO 1835 TelKO o

DEVELOPMENT 1836 RESEARCH ARTICLE and havescattered TUNEL the embryo inC whole mountwithprobes for Sp8 optic stalk,andhypothalamus.ThenumberofTUNEL midline isindicatedbyanarrow (B-B in bothmutants(arrows A-A concentration ofTUNEL expression withthenumberofTUNEL face eachother;thiswasdonetofacilitatethecomparisonof showing frontal viewsofinsituhybridization,suchthattherostral regions the regions inwhichTUNEL and particularlyinthehypothalamus. evidence forcelldeathinthetelencephalon(whitearrowhead), opticstalk, Fgf8 implicated indorsalmidline/paramedialdevelopmentE9-9.5 Fig. 5.Expressionsignalsandtranscriptionfactors ofpatterning arrowhead inE),theopticstalksandhypothalamus.In embryos, apoptoticcellsare detectedinthetelencephalicmidline(open embryos ( mutant, butispresent inthemidlineof andevenmore sointhe mutant the rostral midlinehasfewerTUNEL panels showingTUNELlabelingare180 turned midline correlates withtheexpression of e,telencephalon. Tel, Abbreviations: HT: hypothalamus,OP, olfactoryplacode;Os,opticstalk; 1) androstroventral telencephalon(box2)ofembryos (seeTable 1). olfactory placodes. forebrain labelsapoptoticcellsinwild-type, to midlineinD-D readily observed). Fgf8 ( Null/Neo D-D Null/Neo Љ E-E ). and Bmp4 Љ Љ is tiltedbackward, sotherostral expansioncanbemore ) (seeadditionalcelldeathanalysisinFig.6).Inwild-type mutant. Fgf8 Љ ). TUNELanalysisonhorizontalsectionsthrough theE9.0 Sp8 expression isabsentintheprosencephalic dorsalmidline Wnt8b TelKO expression isgreatly reduced inthe Msx1 + cells intherostral midline(openarrowhead, E + embryos. expression isexpandedrostrally inthe + cells inthetelencephalon(whitearrowhead), Љ cells were countedintherostral midline(box ). expression isalsonotdetectableinthe Bmp4 embryos. A-A telencephalon. the sameasinFig.3.Os,opticstalk;Tel, the mostdorsal.Note,sectionsB,B ( labels thenucleiofmitoticcellsinwild-type on horizontalsectionsthrough theforebrain phosphohistone3 (PH3)immunofluorescence Fgf8 Fig. 4.Analysisofproliferation inthe A-E Msx1 Fgf8 ), + mutants atE9.0. Љ ( Fgf8 TelKO ); thisexpression isnotdetectablein A-A cells, E expression inprosencephalic dorsal Frontal viewsofembryoshybridizedin + Fgf8 midline cells.Theboxesdemarcate Msx1 Null/Neo Љ mutant (C-C TelKO ), Fgf8 Ј Fgf8 Msx1 Љ , althoughitdoesexhibit are themostventral;E-E o embryos haveahigher at E9.0(B-B ( TelKO with respect tothepanels Null/Neo A ( Ј B-B -E Development 133(9) mutant (arrow points Ј + ) and Љ Љ Anti- Fgf8 cells intherostral ) (notethatthe and ), Wnt8b Fgf8 Љ Null/Neo ). Notethatthe Fgf8 Fgf8 Msx1 Null/Neo TelKO TelKO ( Fgf8 Ј C-C embryos, ,B Љ ( Љ A are Null/Neo ) and Љ Ј Љ are -E ), Љ )

DEVELOPMENT al., 2000;Zhouet2001;Bishop2002;O’Learyand expressed incaudal-to-rostralgradientsthetelencephalon (Liuet Shimogori andGrove, 2003),transcriptionfactor genesthatare (Crossley etal.,2001;Garel2003;Storm Fukuchi- Emx2 Previous reportssuggestedthatFGF8canrepresstheexpression of transcription factorsinthetelencephalon expression ofthe Fgf8 telencephalic regionalization. the these mutants.We next turnedourattentiontotheeffect ofreducing Regulation oftelencephalicpatterning Fgf8 mutants showshiftsinthegraded and dose ontheexpression oftranscriptionfactors thatcontrol COUP-TFI Emx2 ( Nr2f1 and – MouseGenomeInformatics) COUP-TF1 hypomorphic 7A,A Fgf8 two mutants.Theexpression of E11.5 telencephalicvesicle asthedoseof We foundthatthelevels of Shimogori, 2003;Hamasakietal.,2004;GarelandRubenstein,2004). Nakamgawa, 2002;MuzioandMallamaci,2003;Grove andFukuchi- their ventrodorsal expression differs. and not expand in to-ventral gradient,whereas In contrast, COUP-TF1 Null/Neo Ј ,A Љ ). Thesedataareconsistentwithobservations madeinmildly mutant embryos,but itsrostralanddorsalexpression did Fgf8 COUP-TF1 Fgf8 (arrowhead inA).Inboth is detectedinthecaudomedialtelencephalon Lateral viewsare presented. InC-C regions (arrowhead inB).In domain isrestricted tocaudolateraltelencephalic In wild-typeembryos,the Fgf8 with thedifference in a differential effect on position in (arrowhead inB COUP-TF1 telencephalic vesiclesof C,C (restricted todorsalmidline tissues;arrowhead in expression isthesameasin wild-typeembryos wild-type embryos,the showsanadditionaldorsalview.lower leftcorner In ( on dissectedE11.5rostral neuraltubeshowing mutant embryos. COUP-TF1 expanded laterallyandrostrally (arrowhead inA embryos, the Fig. 7.Rostrocaudal gradientsof (arrowhead inC A-A expression sharesimilarcaudorostralgradients, mutants ( TelKO Ј ). Bycontrast, Љ mutants. Indeed,in Os, opticstalk;Tel, telencephalon. ( apoptotic cellsinwild-type( sections through theE9.0forebrain labels mutants atE9.0. Fig. 6.Analysisofcelldeathinthe telencephalon (D the junctionofdorsalopticstalkwith the mostventral;E-E telencephalic primordium (A arrowheads indicatetheapoptoticcellsin arrowheads indicatetherostral midline;white sections B,B ), A COUP-TFI Ј -E mutants (Fig.7B,B -high-expression domainexpandsrostrally Ј expression was affected differently inthe Fgf8 ) and expression are modifiedin Emx2 Fgf8 Emx2 COUP-TF1 TelKO Ј Љ ) whereas itremains inacaudolateral ). Os,opticstalk; Tel, telencephalon. Fgf8 Ј Neo/Neo ( ,B B-B Bmp4 expression wereincreasedinthe -high-expression domainis COUP-TF1 Whole-mount insituhybridization Љ mutants (arrowhead inB RESEARCH ARTICLE are thesameasinFig.5.Black TelKO Љ Bmp4 Emx2 Ј ) and COUP-TFI ,D Emx2 ) (Gareletal.,2003). TUNEL analysisonhorizontal Fgf8 Fgf8 expression expandsinthe Љ Fgf8 COUP-TF1 Љ ,E embryos ( are themostdorsal.Note, Ј Fgf8 Bmp4 expression inthetwo TelKO Fgf8 is expressed inadorsal- ,E has aventral-to-dorsal -high-expression domain Null/Neo Ј Null/Neo Љ ,B ). HT, hypothalamus; Null/Neo expression correlates mutant embryos A-E Љ spread rostrallyin Ј ). Although ,B Љ was reduced(Fig. ( C-C embryos, A a boxinthe Emx2 -high-expression and Ј ,B ), Љ -E embryos, the Fgf8 Љ Љ ,C ) expression. Љ Fgf8 ). A-A Fgf8 Љ and ) andat Null/Neo Fgf8 Љ TelKO ). Such Bmp4 Љ Emx2 Ј Emx2 1837 ,A are Љ ).

DEVELOPMENT rostrally itsexpression appearedtobereduced(Fig.7C contrast, inthe As boththe rostroventral telencephalon Reduced expression of be mediatedbythedivergent effects onlevels of therefore examined hypothalamic expression of in theexpression ofboth Fgf8 intertwined withthatof areas) was greatlyreducedin expression inthesubpallium(preopticandanteriorentopeduncular region in mutants, thecaudalexpression of Rubenstein, 2004),suggestingthat Fukuchi-Shimogori, 2003;MuzioandMallamaci,Garel gradient (reviewed byO’LearyandNakamgawa, 2002;Grove and 1838 of thetwo in thedorsaltelencephalicvesicles (Fig.7C where itshowed acaudal-to-rostralgradient(Fig. 7C). In telencephalon atE11.5was restrictedtodorsalparamediantissues, examined theexpression of separate domains(Crossley etal.,2001). the chiasmaticregion intotheopticstalks,followed bysplittinginto pattern of During thispatterningphaseofthesubpallium,expression and subsequentlywithexpression of with theexpression of markers thatarecharacteristicoftheventral neuraltube,beginning neurulation, therostroventral region ofthetelencephalonexpresses cortical (pallial)structures(Cobos-Silleroetal.,2001).During subcortical (subpallial)structuresarerostraltotheprimordiaof other aspectsofrostraltelencephalicpatterningweredisrupted. expression) intotherostraltelencephalon,weinvestigated whether of caudalmolecularproperties(i.e. early duringtelencephalicregionalization (Fig.8B,B midline of Previous work showed that repressed bydorsalpatterningsignalssuchasBMPsand/orWNTs. telencephalic primordium. alterations ofrostrocaudalandmediolateralpatterninginthe The expression of Fate mapsoftheanteriorneuralplateshow thattheprimordiaof Our resultsrevealed thatdifferent dosesof expression (Ohkuboetal.,2002),andinzebrafish,areduction RESEARCH ARTICLE Fgf8 Fgf8 Fgf8 Fgf8 Fgf8 Fgf8 genotypes on TelKO is highlydynamic,includingitsextension through Null/Neo Null/Neo TelKO Shh Shh mutants (Stormetal.,2003).We therefore Nkx2.1 and mutants atE10.5,but isincreasedinthis Fgf8 mutant, expression in in therostroventral telencephalonisclosely Bmp4 Fgf8 Bmp4 Fgf3 . SHHfunctionisrequiredtomaintain Emx2 Bmp4 Shh ( Shh Titf1 Fgf8 TelKO Bmp4 expression isreducedintherostral (Walshe andMason,2003).We Bmp4 in and and COUP-TF1 Null/Neo – MouseGenomeInformatics) and expression inthewild-type Fgf8 mutants showed anexpansion Shh Fgf8 was morebroadlyexpressed COUP-TF1 Fgf8 appeared normal,whereas Љ Nkx2.1 Emx2 mutants atE11.5. ). Thedifferential effects brains. Althoughafew (Crossley etal.,2001). Null/Neo Fgf8 results indecreased Bmp4 expression maybe and mutants atE9.5, produce distinct expression may in the expression. COUP-TFI Fgf8 Ј ). Ј Null/Neo ). By Shh in the (Fig. 9E,F)(Eisenstatetal.,1999).Theexpression of expressed inlateprogenitorsandsubsetsofpostmitotic neurons Dlx2 expressed inmostofthesubcorticaltelencephaloncontrol mice; ( required forsubcorticaldevelopment) was greatlyreduced tissues intherostrodorsaltelencephalon.In expressing least onesubcorticalregion ismaintained.Thereasmall region Fgf8 Fgf8 clearly reduced,andthe scattered clustersof The pallium atE12.5 anddifferentiationPatterning defectsofthe mutants (Fig.9E both medial partoftherostroventral telencephaloncontinuedtoexpress molecular featureswereeliminatedinthesemutants.Indeed,the embryos atE10(Fig.8A,A zone oftelencephalic expression inthe expression (Susseletal.,1999).Therefore,weexamined wider andmoreintense. base oftheopticstalksandalonglaminaterminaliswas both disrupted (arrow inFig.8B,B telencephalon, morphogenesisoftheAEP/MGEwas severely thickened neuroepitheliumthatproduced E12.5 embryos(Fig.9).Both the subpalliumusinginsituhybridizationoncoronalsectionsfrom E10, thetelencephalicexpression of eminence (MGE;Fig.9). septum, lateralganglioniceminence(LGE)andmedial exhibited alossofrostralsubcorticalstructures,including the Given thereductionin subpallium atE12.5 anddifferentiationPatterning defectsofthe characteristics. FGF8 functionisrequiredforinductionofbasaltelencephalic in thetelencephalon(Fig.8A,A Fgf8 Nkx2.1 Consistent withthelossofsubcorticalmorphologyobserved at Fgf8 TelKO Null/Neo Dlx2 Null/Neo Fgf8 is expressed primarilyinprogenitors,whereas Null/Neo mutants, weexamined thepatterninganddifferentiation in function isrequiredfortheinductionoftelencephalic Null/Neo Dlx2 and ) orlost( mutants, of of bothgenesisreduced inthetelencephalicdomains the telencephalonof ganglionic eminence. telencephalon. HT, hypothalamus;MGE,medial Arrows inB,B induction inthetelencephalon(Sussel etal.,1999). of assessed becauseprevious work demonstratedthatloss Nkx2.1 dissected E10rostral neuraltubeusingprobes for expression. progressive reduction oftelencephalic Fig. 8. and Dlx5 Љ in therostroventral telencephalonofthe Fgf8 Nkx2.1 mutant suggeststhatsubpallialneurogenesisinat ). Fgf8 Fgf8 Nkx2.1 Fgf8 Null/Neo Shh Fgf8 ( Nkx2.1 (Fig. 9E A , Null/Neo Fgf8 function inthetelencephalonprevents A TelKO + Null/Neo Ј Nkx2.1 TelKO Whole-mount insituhybridizationon , Ј A and cells werevisibleintherostroventral expression thatremainedin indicate Shhexpression inthe basal TelKO Љ Ј Ј ) and ). Furthermore, mutants generateddifferent typesof ) revealed thatnotallsubcortical expression inthesubpalliumwas ) atE12.5(Fig.9C-D Ј and Fgf8 Fgf8 ,F and Ј mutant lacked ,A Ј expression in ). Thesehomeoboxgenesare Shh TelKO Fgf8 Null/Neo Љ Fgf8 ). Theseresultssuggestthat Fgf8 Nkx2.1 ( B embryos. TelKO , TelKO Tbr1 B TelKO Ј ) showsthatexpression and Fgf8 Development 133(9) mutants have Shh and mutants atE10.In -expressing neurons mutant wasnot Nkx2.1 Fgf8 Shh Null/Neo expression atthe Fgf8 Shh Dlx2 expression in TelKO Љ ). Thesmall Nkx2.1 Null/Neo (which are Fgf8 expression mutants, a Fgf8 and mutants Dlx5 Nkx2.1 Null/Neo Shh Dlx5 TelKO Shh and is

DEVELOPMENT cortical progenitors( Fgf8 not MGEprogenitors(Susseletal.,1999;Stoykova etal.,2000).In 2000; Yun etal.,2001); itisalsoexpressed atlow levels inLGE,but dorsal-high ventral gradientincorticalprogenitors(Toresson etal., (Fig. 9anddatanotshown). in neurons (Hevner etal.,2001). T-box transcriptionfactor expressed inpostmitoticpallial(cortical) resembled choroidplexus tissues(Fig.9B Unlike controls,however, both telencephalon, consistentwith thelossofsubpallialstructures. ventrodorsal gradientthat nearlyextended throughouttheentire Pax6 extended throughoutnearly theentiretelencephalon(Fig.9A,A Regulation oftelencephalicpatterning Fgf8 at thedorsalmidlinewas apparent(Fig.9B (Fig. 9A,A showed reduced We observed aventral expansion ofcorticalmolecularproperties Fgf8 Null/Neo TelKO was expressed in mutant micebasedontheexpression ofgenesthatmark mutants producedathin, Ј ,A mutants, Љ ). Pax6 Pax6 Pax6 expression inrostraltelencephalic regions Fgf8 ) andpostmitoticcells( expression intheprogenitorzone Pax6 Null/Neo Fgf8 is normallyexpressed inalow Null/Neo and Tbr1 Љ , arrow). Fgf8 -negative midlinethat and Ј TelKO , arrow), whereas Fgf8 Tbr1 Tbr1 mutants ina TelKO ) atE12.5 encodes a mutants Ј ). forebrain in analysis ofthepatterningrostralneuralplateandearly patterning andmorphogenesis.We have performedanindepth profound andcomplex rolesinmostaspectsoftelencephalic The expression of DISCUSSION identity (Figs2,3).However, atleastsome key regulators ofprosencephalic( in anterior neuralplateandearly tubeshowed thatareduction dorsal andventral patterning centers.Molecularanalysisofthe proliferation, apoptosisandcross regulation betweentherostral, (conditional null)mousemutants,focusingonregional specification, these domains(Fig.9B,B structures suchasthelateralandventral palliumareproducedin of therostraltelencephalon.Thissuggeststhatventral cortical domain ofexpression extended intomorphologically ‘ventral’ parts in theanteriorneural ridgeofboth Fgf8 Tbr1 expression doesnotprevent theinductionormaintenanceof continued tobeexpressed inboth Fgf8 Fgf8 the approximate pallial-subpallialboundaryinthe ( subpallium. ganglionic eminence;S,septum;SP*,unspecified LGE, lateralganglioniceminence;MGE,medial ( Fgf8 and Fig. 9. of thedorsalmidlinetissuesin on coronal sectionsfrom E12.5wildtype, properties inthetelencephalon. F-F B-B Љ Љ Fgf8 TelKO TelKO Fgf8 ) expression. Horizontalarrows inA Null/Neo ), Shh Fgf8 TelKO mutants (verticalarrows inB mutant. Alsonotethedivergentmorphology at therostrallimitoftelencephalonhas ( Ј C-C (severely hypomorphic)and mutants loseventralmolecular ,B embryos showing Љ Љ ). ), Nkx2.1 Fgf8 Six3 RESEARCH ARTICLE Null/Neo ( D-D ) ortelencephalic( Fgf8 Љ Pax6 ), Fgf8 and Dlx2 Fgf8 In situhybridization expression persists Fgf8 ( Ј A-A ,B uat,andits mutants, ( Null/Neo E-E Ј Љ ). CX,cortex; and E Љ TelKO Fgf8 ), Љ ) and Tbr1 and Fgf8 Null/Neo Љ mutants; indicate Foxg1 Dlx5 1839 TelKO )

DEVELOPMENT in that alterationsin et al.,1999;Dou2000;Seoane2004).Thuswesuspect Martynoga etal.,2005)throughrepressingSMADsignaling(Dou Hardcastle andPapalopulu, 2000;Hanashimaetal.,2002; Foxg1 expression (ShimamuraandRubenstein,1997;Ye etal.,1998). have suggestedthatFGF8isapositive regulator of winged-helix transcriptionfactor that thereductionismediatedbydiminishedexpression ofthe due, atleastinpart,toreducedcellproliferation.We hypothesize (Figs 3,4;Table 1),suggestingthatthetelencephalichypoplasiais Fgf8 proliferation andcelldeath.Themitoticindex of theE9.0 telencephalon sizemaybeaconsequenceofalterationsin was ~75%and~50%ofwild-type,respectively (Fig.7).Reduced Fgf8 telencephalon byregulating Evidence thatFGF8controls thesizeof not present. al., 2005);theseareregions where signaling, particularlyincaudodorsalpartsofthecortex (Muzioet Emx2 embryonic tissues,throughadditionalpathways. For instance, each genealsocontributes topatterningoftheneocortex, andother Fgf8 2004; Muzioetal.,2005).Therefore,itisourview thatalthough regional fate ofprogenitorsinthecaudal cortex (Hamasakietal., evidence that thereby contributes tocorticalpatterning),thereisvery strong Emx2 Whereas for 2004). et al.,2003;Fukuchi-ShimogoriandGrove, 2003;Shimogorietal., embryonic brain(Martinezetal.,1999;Crossley etal.,2001;Garel expression atlaterdevelopmental stagesandindifferent partsofthe ability ofFGF8tofunctionasarepressor (Figs 2,3,5,7).Theseobservations areconsistent withtheknown results inthemolecularcaudalizationofanteriorprosencephalon thus furtherstudiesinwhichall 1840 dimension ofthe mutants isnearlynormal(Gareletal.,2003),therostral-caudal of thetelencephalon.Whereastelencephalicsizein (Fig. 3); in theprosencephalonappearedtobereducedmutantsatE9.0 A progressive reductionof the telencephalon). Therostralexpansion of for thespecificationofanteriorprosencephalon(including region areneededtoestablishdefinitively whether proliferation. causing thetelencephalichypoplasia thandecreasedcell of thiseffect suggests thatapoptosismayplayagreaterrolein compared tocontrolembryos(Figs 5,6;Table 1);the magnitude increased signalintherostroventral telencephalonofbothmutants death was detected.TheTUNEL assayshowed roughlya10-fold proliferation. (Gestri etal.,2005),andthereforemayalsoregulate cell There iscurrentlysomecontroversy abouttherespective roles In additiontodecreasedcellproliferation, anincreaseincell Fgf8 Fgf8 Fgf8 Null/Neo Null/Neo and promotes telencephaliccellproliferation(Xuanetal.,1995; expression inprogenitorcellsoftherostralcortex (and has recentlybeenshown topositively regulate WNT RESEARCH ARTICLE mutants. Inaddition,thedomainofstrong and mutants thatweobserved shows thatreduced Six3 Fgf8 Emx2 and and Emx2 Emx2 is alsoimplicatedinrepressingSMADsignaling clearly hasacentralroleinregulating thelevel of Fgf8 Fgf8 contribute toregulating eachother’s expression, Foxg1 Fgf8 in contributing topatterningoftheneocortex. has anautonomousfunctioninspecifyingthe TelKO TelKO Null/Neo contribute tothereducedtelencephalicsize rostroventral telencephalonwas reduced mutants leadstoprogressive hypoplasia and Fgf8 Fgf8 Fgf8 Foxg1 Fgf8 gene doseinthe expression isblocked inthis Foxg1 TelKO Emx2 (Fig. 3).Previous reports expression isvery low or telencephalon atE11.5 Emx2 , expression Otx2 , Otx2 Fgf8 Six3 and Fgf8 Fgf8 expression and is required Fgf8 Wnt8b Neo/Neo Wnt3a Neo/Neo Foxg1 dose in , 2002). regulated TCFbinding sitesinthe Fgf8 midline phenotypes. in al., 2002).Ofcourse,othergeneswhoseexpression ismisregulated midline development (Liuetal.,2004;RamosHebert Fgf8 1997). We foundthat neuroepithelium byBMPsignaling(ShimamuraandRubenstein, neural tube(Bachetal.,2003)thatcanbeinducedinthe apoptotic homeoboxgeneexpressed inthedorsalmidlineof expansion of and Grove, 2003; Shimogorietal.,2004).Thecorrelationinthe the extracellular concentrations ofFGFligands(Fukuchi-Shimogori modulated usingexpression ofasecretedformFGFR3toreduce electroporation studiesinwhich thelevel ofFGFsignalingwas one dayearlier, atE9.5,thetelencephalicmidlineof expression (Stormetal.,2003).Inthepresentstudywefoundthat at E10.5;bothphenotypeswerepositively correlatedwith on midlinecelldeathand expressed inthe to caudalpartsofthetelencephalon,whereasitwas broadly the rostralcortex ofthe (Garel etal.,2003);however, itsexpression remainedrepressedin expanded rostrallyin mutants containedareducednumberofTUNEL mutants, andincreasedcelldeathinthemidlineof reduced celldeathinthetelencephalicmidlineof development oftherostrodorsalmidline.Previously, wereported Altering thedoseof development centerscontrolpatterning dorsalmidline Interactions betweentherostral anddorsal For example, theexpression of mutants, theexpression ofothergeneswas alteredingradedmanner. alterations in create aspectrumofdorsalmidlinedefectsthatarecorrelatedwith (Huffman etal.,2004).Thus,different levels of defects weresuggestedbythefailure ofthecorpuscallosumtoform (mild hypomorph)mutantsappearedgrosslynormal,althoughsubtle (Hebert etal.,2002).Incontrast,thedorsalmidlineof known rolesofBMPsignalinginchoroidplexus development was thinandappearedchoroidplexus-like, consistentwiththe responded inanon-linearfashion in TF1 that thismaybecausedbyBMP4-mediatedrepressionof wild-type, whereastherostrodorsalmidlineof cortex ( holoprosencephalic midlinewithmolecularfeaturestypicalof there werechangesintheexpression of we didnotdetect resembled thatofwild-typeembryos(Figs5,6;Table 1).Although By E11.5, expression patternwas positively correlatedwithmidlineapoptosis. E12.5 (Fig.9).Whereasthe with distinctpatternsofhistogenesisattherostrodorsalmidlineon findings in rostral regions ofthetelencephalon(Figs3,5),consistentwithprior Like These divergent effects ofthe Whereas Fgf8 . Null/Neo Null/Neo Tbr1 Bmp4 mutants, suchas Bmp4 mutants andmaintainedin Bmp4 and + Bmp4 Fgf8 Wnt8b ) (Fig.9),thedorsalmidlineof , COUP-TF1 Fgf8 expression inthe Bmp4 , Neo/Neo Fgf8 Msx1 and and TelKO Fgf8 Neo/Neo expression ineithermutantatE9.5(Fig.5), Fgf8 Msx1 Fgf8 Msx1 and Emx2 mutants (Gareletal.,2003)andin Sp8 mutant (Fig.7). Bmp4 Null/Neo expression respondednon-linearlyin TelKO COUP-TF1 , mayalsocontribute tothesedorsal Fgf8 expression, genesthatregulate dorsal resulted indistincteffects onthe mutants. expression was notdetectablein Fgf8 may reflectthepresenceofWNT- / Msx1 Wnt8b Fgf8 mutant (Fig.7).We hypothesize and Null/Neo Null/Neo Emx2 Fgf8 Null/Neo expression werecorrelated Fgf8 Fgf8 and expression andcelldeath COUP-TF1 mutant hadathickened, and Neo/Neo enhancer (Theiletal., Msx1. Msx1 TelKO Development 133(9) Null/Neo Emx2 mutant was restricted Fgf8 embryos; thusits Fgf8 Fgf8 + Fgf8 Fgf8 mutants (Fig.7) cells relative to TelKO expanded into and TelKO TelKO elKO l Te Fgf8 Fgf8 Fgf8 expression expression mutations Fgf8 is apro- mutants mutants COUP- mutant Null/Neo Null/Neo Neo/Neo Bmp4 TelKO

DEVELOPMENT lamina terminalis;M,mesencephalon;MGE,medialganglioniceminence;OC,opticchiasm;S,septum. function mightcontributetotheirphenotypes.CP, commissuralplate;Cx,cortex;HT, hypothalamus;LGE,lateralganglionic emi since plays acentralrole inmediatinginteractionsbetweenthetelencephalicsignalingcenters(Aotoetal.,2002;Kuschel al., 1998;Kuschelet2003)andanexpansionof telencephalon (Tole etal.,2000;Rallu2002). more caudalregions oftheneuraltube,expression ofventralmolecularfeatures expandsintodorsal structures withinthe signaling centers.GLI3represses SHH-mediatedeffects onventralizationthroughout thenervoussystem (reviewed byRuizetal. general role inventralneuralspecification.Notdescribedthisschemaistherole thatGLI3playsinregulating thebalance et al.,2004).FGF8signalingisrequired forinductionof that EMX2positivelyregulatesrepresses BMP/WNTsignalingthatinturn interconnecting FGF, BMP, WNTandSHHsignalingthrough theEMX2,FOXG1andNKX2.1transcriptionfactors.Notethatthere isev Fgf8 centersasmarkedby expressionview ofthetelencephalonshowingpatterning genesindicated,cross regulation b signaling through phosphorylationofthelinkerdomainSMAD(Kretzschmar etal.,1997;Pera2003).( that shown: FOXG1alsorepresses expression ofWNT genes.Dottedlinesindicatethattheinteractioniseitherindirect orpotential a singlealleleof have notobserved achangein et al.,1999;MuzioandMalamacci, 2005).However, todate,we loss in is plausiblefortwo reasons:(1)heterozygosityof disrupted byinsertionof locus (sincetheseembryoscarrya Fgf8 Regulation oftelencephalicpatterning reduced mutants might also beattributable to differences inthe timingof phenotype largely reflectsalossof genes contribute totheopposingeffects ofthe this effect. Itwillalsobeimportanttodeterminewhethersprouty FGF signaling(reviewed byKimand Bar-Sagi, 2004),contribute to whether sproutygenes,whichencodeFGF-inducedrepressors of 10) (Stormetal.,2003).Ongoingstudiesareaimedattesting theFOXG1-mediated repressionofBMP4signaling(Fig. repress Fig. 10.Summaryofproposedcenters. interactionsbetweenpatterning al., 2003);and(2) Fgf8 Differences inthe phenotypes ofthe wepostulatedthatincreasesinFGF8signaling Previously In principlesomeoftheincreasein Foxg1 TelKO and TelKO Shh Bmp4 Fgf8 Bmp4 expression isessentiallyeliminatedfrom thetelencephalonin mutations on mutants maybecausedbyheterozygosityatthe expression ismaintainedinpartbymTOR(Hentgesetal.,1999;Hentges2001),andthattheMAPKcascadeblocksBMP expression observed in expression. / Wnt3a Foxg1-Cre Foxg1 -expressing centers,andthepositiveinteractionsbetween Bmp4 –/– Cre Fgf8 , thereforeweconcludethatthe mutants ectopicallyexpress ) (HebertandMcConnell,1999).This / Msx1 Bmp4 Null/Neo Fgf8 expression. expression inembryosbearing Fgf8 Bmp4 Foxg1 mutants constitutively have expression. Fgf8 Null/Neo expression observed in Null/Neo allele thathasbeen mutants (Stormet Foxg1 Gli3 Fgf8 Fgf8 and Nkx2.1 mutants exhibitareduction inBMPandWNTexpression atthedorsalmidline(Grove et expression (Aotoetal.,2002;Kuschel2003),leadingtothemodelthatGLI3 Bmp4 rescued the Null/Neo Fgf8 Fgf8 Foxg1 expression inthetelencephalon;wehypothesizethatFGFsignalinghasa (Dou TelKO TelKO and Fgf8 to thephenotypeof expression duringgastrulationinnon-neuraltissuescouldcontribute (Shimamura andRubenstein,1997).Inprinciple,reduced rostral neuralplateexpression of negatively regulate increase inBMP expression. Third,BMP signalingislikely to initially lowers BMP expression, but furtherreductionsleadto an expression (andprobably signaling).Reducingthelevel of Second, telencephalic structures)(Galceran etal.,2000;Lee2000). development ofthehippocampalcomplex (caudodorsal (reviewed byWilson andHouart,2004)isrequiredfor because WNTsignalingisknown tocaudalizetheprosencephalon 2004). Thishasimportantimplicationsforforebrainregionalization lack represses interactions withthedorsalpatterningcenter(Fig.10).First, expressing rostralpatterningcenterhascomplex regulatory mechanism. found evidence thatatelencephalicphenotypearisesbythis reduced Fgf8 Thus, several linesofevidence supporttheview thatthe expression (Shinozakietal.,2004;Muzio2005;Shimogori ( Fgf8 A mutants, itisnotclearwhetheralterationsin ) Postulatedsignalingcascadedownstream ofFGF8.Not Fgf8 Fgf8 Wnt3a Fgf8 expression primarilyintheforebrain,beginning after expression inalltissues,whereas dose hasamorecomplex relationshipwithBMP and and Fgf8 Shh Wnt8b -expressing domains.( Fgf8 expression (Ohkuboetal.,2002;Shimogori Null/Neo expression (Fig.5)(Shimogorietal., Foxg1 B RESEARCH ARTICLE mutants, althoughwehave not ) Schemaofafrontolateral is initiated(~3somitestage) C Postulatedpathways ) 2003). However, betweenforebrain Fgf8 ly indirect. Note Gli3 Gli3 nence; LT, , 2002).Asin etween the TelKO mutant expression or idence mutants Fgf8 1841 Fgf8 Fgf8 Fgf8 -

DEVELOPMENT subpallial structuresseenin and ventral structuresinthesubpallialtelencephalon.Theresidual zone, expression of based ontheirexpression oflow levels of they mayhave alateralganglioniceminence/striatalphenotype histological identityofthesestructures,althoughwesuspectthat to progressive reductionsof Reductions in telencephalon (Fig.8A,A normal domainsof The rostralpatterningcenterisalsoessentialforestablishingthe specification development: evidencethatFGF8initiatesventral centerscontrolpatterning subpallial Interactions betweentherostral andventral Msx1 Foxg1 steady-state isfurtherregulated bytheBMP-mediatedrepressionof dorsal midline(Douetal.,1999;MuzioandMallamaci,2005).This is requiredtorestrict between FGFandBMP/WNTexpression andsignaling,since Foxg1 et al.,2004).We suggestthatFGF8-mediatedpositive regulation of 1842 Dlx5 et al.,2001). Fgf10 FGF genemaycompensatefor in thediencephalon expression. Interestingly, Comparison between unpublished results). Fernandes, K.Yu, D.Ornitz,S.K.McConnellandJ.M.H., forebrain phenotypesinFGFRconditionalmutants(G.Gutin, M. function. Thisinterpretationisconsistentwitharecentanalysis of establishing ventral fates intheneuraltubeupstreamof receptor tyrosinekinasesignalingmayplayageneralrole in is inducedinthetelencephalon(Susseletal.,1999),suggesting that Once dominant negative EPHreceptortyrosinekinase(Xuetal.,1996). forebrain structures(hypothalamus)following expression ofa Indeed, sucharoleforFGF8mightexplain theloss ofventral embryonic centralnervous system(von OhlenandDoe,2000). FGF signalinghasageneralroleforinducing controlled byFGFsignaling(Serlsetal.,2005).Itispossiblethat telencephalon. Likewise, intheendoderm, Nkx2.1 that FGF8signalingisessentialforinducingventral molecular (e.g. provide theinitialstepintelencephalicventralization. Ourdatashow telencephalon supportsthehypothesisthatFGFsignalingmay In zebrafish, zebrafish induction of analogous totheroleofreceptortyrosinekinasesignalingin zebrafish, reduced expression ofeither Fgf8 and Neubuser, 2001;Gimenoetal.,2003);interactionsbetween Fgf18 forebrain development. ByContrast,inmouse Mason, 2003),whereasinmouse, The failure of and theseotherFGFgenesremains tobedemonstrated.In Nkx2.1 (Fig. 9E (Furuta etal.,1997;Shimamura1997). expression intheventral hypothalamusplaysthisrole(Treier expression overlap with (Furuta etal.,1997;Ohkubo2002)andinductionof ) andhistologicalpropertieswithintherostral-most RESEARCH ARTICLE expression playsakey roleinmaintainingthebalance expression hasbeenestablished,theexpression of vnd Fgf8 Fgf8 Ј ,F Ј ( ); ongoingstudiesareaimedatelucidatingthe Nkx function iscompensatedby dose inthe Nkx2.1 Nkx2.1 Dlx2 Fgf8 Bmp4 homologue) expression inthe Shh and mutant embryos,suggestingthatanother Fgf8 and Shh , Ј Fgf8 induction inthe ,A and Wnt3a Fgf8 Dlx5 Fgf8 Fgf8 Shh , Љ Null/Neo ,B,B Nkx2.1 Nkx2.1 Fgf3 Null/Neo function inmouseand , andlackof in thisregion. Itispossiblethat expression intherostroventral (Maruoka etal.,1998;Bachler and Ј , Fig.9C,C embryos expressed has notbeenassociatedwith , expression aremaintained Dlx2 Wnt8b and Fgf8 Pax6 Fgf8 Nkx2.1 and Nkx2.1 or Fgf3 Fgf15, Fgf17 Fgf8 expression tothe in theventricular Nkx2.1 TelKO Dlx5 Fgf3 Ј ,C TelKO (Walshe and induction is expression, Drosophila Љ mutants led expression ,D,D results in and Dlx2 mutant Foxg1 Ј Tbr1 ,D Shh Shh and and Ј ). Crossley, P. H.andMartin,G.R. Cobos-Sillero, I.,Shimamura, K.,Rubenstein,J.L.R.,Martinez,S.and Bishop, K.M.,Rubenstein,J.L.andO’Leary, D. Bell, S.M.,Schreiner, C.M.,Waclaw, R.R.,Campbell,K.,Potter, S.and Bachler, M.andNeubuser, A. Bach, A.,Lallemand,Y., Nicola,M.A.,Ramos,C.,Mathis,L.,Maufras, Chi, C.L.,Martinez,S.,Wurst, W. andMartin,G.R. Crossley, P. H.,Martinez,S.,Ohkubo,Y. andRubenstein,J.L.R. phenotypes inthesetissuesthe 2003; Martinez-Moralesetal.,2005).We have alsoobserved zebrafish retinalanddiencephalicdevelopment (Walshe andMason, et al.,2003;Wiellette andSive, 2004),thesegeneshave rolesin zebrafish midbrain/hindbrainorganizer (Walshe etal.,2002;Jaszai Fgf8 (Shanmugalingam etal.,2000),ashasbeenalsonotedinmouse telencephalic midline,resultingincommissuraldefects reductions inFGFdosezebrafishaffect development ofthe zebrafish subpallialdevelopment (Shinya etal.,2001).Furthermore, consistent withthedemonstratedroleofFGF/MAPKsignalingin reduced expression ofsubpallialgenes(e.g. Aoto, K.,Nishimura,T., Eto,K.andMotoyama,J. Anderson, R.M.,Lawrence, A.R.,Stottmann,R.W., Bachiller, D.and Abu-Issa, R.,Smyth,G.,Smoak,I.,Yamamura, K.andMeyers,E.N. References MH065670). Ireland, LarryL.HillblomFoundation(NINDSNS34661-01A1andNIMH and bygrantstoG.R.M.from NICHD(HD25331)andtoJ.L.R.R.from Nina to U.B.from NationalAllianceforResearch OnSchizophrenia andDepression; Lymphoma Society; toU.B.andS.G.from HumanFrontiers ScienceProgram; This workwassupportedbyfellowshipstoE.E.Sfrom TheLeukemiaand brain subdivisions duringevolution andindiseasestates. provide afundamentalmechanismtomodifytherelative sizesof controlling therelative strengthofagiven patterningcentermay (Fukuchi-Shimogori andGrove, 2001;Gareletal.,2003).Therefore, reduces theratiooffrontaltosensoryregions ofthe neocortex given patterningcenter. For instance,areductioninFGF8signaling relative sizeofstructureswhosemorphogenesisiscontrolledbya Modulation ofthecross-regulation hasthepotentialto controlthe essential roleinpatterningtheearlytelencephalon(Fig.10). (BMP; WNT)andventral (SHH)patterningcentersplaysan Furthermore, cross-regulation betweentherostral(FGF),dorsal telencephalon show aprofoundsensitivity to The growth, regional specificationandmorphogenesisofthe Concluding remarks detailed analysisremainstobeperformed. vesicles. prosencephalon centers forthetelencephalicandoptic definepatterning in thedevelopingembryo. of polypeptidesandisexpressed inregions thatdirect outgrowth andpatterning chimeras. Puelles, L. cerebellum. organizer signalFGF8isrequired forcellsurvivalintheprospective midbrainand and theirreceptors duringmidfacial development. limb bud. regulates Fgf8expression andapoptosisinthedeveloping neuraltube,face,and neocortex. Emx1, Emx2,andPax6inregulating thespecificationofareas inthedeveloping Proc. Natl.Acad.Sci.USA Scott, W. J. Development and Robert,B. Evidence thatcoordinate expression of forebrain developmentinthemouse. Klingensmith, J. mouse. Fgf8 isrequired forpharyngealarch andcardiovascular developmentinthe In additiontothewell-describedfunctionsof mutants (Huffman etal.,2004)(datanotshown). Development Neuroscience Dev. Biol. Dev. Biol. J. Neurosci. (2001). Fatemapoftheavianforebrain atstage8withquail-chick Development (2003). Sp8iscrucialforlimboutgrowth andneuropore closure. 130 (2003). Msx1isrequired fordorsaldiencephalonpatterning. , 4025-4036. (2002). Chordin andnogginpromote organizingcenters of 239 251 129 108 22 , 46-67. , 320-332. , 7627-7638. , 4613-4625. 130 100 , 183-206. Development (2001). Expression ofmembers oftheFgffamily , 2633-2644. , 12195-12200. (1995). ThemouseFgf8geneencodes a family Development Fgf8 Fgf8, Otx2,Bmp4, 121 , 439-451. mutants (unpublished),but a Mech. Dev. (2002). MouseGLI3 129 (2002). Distinctactionsof dlx2 (2003). Theisthmic Development 133(9) , 4975-4987. Fgf3 ); thesedefectsare and Fgf8 100 and Shh , 313-316. gene dose. (2001). in therostral Fgf8 (2002). at the

DEVELOPMENT Kim, A.S.,Lowenstein, D.H.andPleasure, S. J. Kawakami, Y., Esteban, C.R.,Matsui,T., Rodriguez-Leon,J.,Kato, S.and R. Huffman, K.J.,Garel, S.andRubenstein,J.L. Hevner, R.F., Shi,L.,Justice,N.,Hsueh,Y.-P., Sheng,M.,Smiga,S.,Bulfone, Hentges, K.E.,Sirry, B.,Gingeras,A.C.,Sarbassov, D.,Sonenberg,N., Jaszai, J.,Reifers,F., Picker, A., Langenberg,T. andBrand,M. Hentges, K.,Thompson,K.andPeterson,A. Hebert, J.M.,Mishina,Y. andMcConnell,S.K. Hebert, J.M.andMcConnell,S.K. Hardcastle, Z.andPapalopulu,N. Dou, C.,Lee,J.,Liu,B.,F., Massague,J.,Xuan,S.andLai,E. Hanashima, C.,Li,S.Shen,L.,Lai,E.andFishell,G. Feledy, J.A.,Beanan,M.J.,Sandoval,Goodrich,S.,Lim,H., Eisenstat, D.D.,Liu,J.K.,Mione,M.,Zhong,W., Yu, G.,Anderson,S.A., Dou, C.L.,Li,S.andLai,E. Hanashima, C.,Shen,L.,Li,S.C.andLai,E. Fukuchi-Shimogori, T. andGrove, E.A. Depew, M.J.,Lufkin,T. andRubenstein,J.L. Regulation oftelencephalicpatterning Hamasaki, T., Leingartner, A.,Ringstedt, T. andO’Leary, D. Grove, E.A.,Tole, S.,Limon,J.,Yip, L.andRagsdale,C.W. Grove, E.A.andFukuchi-Shimogori,T. Fukuchi-Shimogori, T. andGrove, E.A. Furuta, Y., Piston,D.W. andHogan,B.L. Gimeno, L.,Brulet,P. andMartinez,S. Gestri, G.,Carl,M.,Appolloni,I.,Wilson,S.W., Barsacchi,G.and Garel, S.,Huffman, K.J.andRubenstein,L.R. Garel, S.andRubenstein,J.L.R. Galceran, J.,Miyashita-Lin,E.M.,Devaney, E.,Rubenstein,J.L.and embryos. transcription factors,regulate Fgf8expression andlimboutgrowth invertebrate Belmonte, J.C. activity. to-midbrain transformationintheabsence ofmidbrain-hindbrainorganizer development ofintra-neocorticalprojections. differentiation ofthepreplate andlayer6. A., Goffinet, A.M.andRubenstein, J.L.R. Natl. Acad.Sci.USA proliferation duringembryonicdevelopmentinthemouse. andpatterning Sabatini, D.andPeterson,A.S. Development required fortheexpansionandregionalization ofthetelencephalicprimordium. 1041. required the dorsaltelencephalicmidline. locallytopattern head structures. locus mediatesloxPrecombination inthetelencephalonandotherdeveloping 1303-1314. the cellcycleinhibitorp27(XIC1)andimpartinganeuralfate. suppresses earlycorticalcellfate. Smad partners. interferes withtransforminggrowth factorbetasignalingbyassociatingwith independent mechanisms. proliferation anddifferentiation ofneocorticalprogenitor cellsthrough Dlx3 andMsx1. oftheanteriorneuralplateinXenopusbyhomeodomainfactors patterning Matsuo-Takasaki, M.,Sato,S.M.andSargent,T. D. Comp. Neurol. DLX-5 expression definedistinctstagesofbasalforebrain differentiation. Ghattas, I.,Puelles,L.andRubenstein,J.R. growth ofthecerebral andpatterning hemispheres. subdivisions byDlxgenes. neocortex bydirect specificationofcorticalprogenitors. regulates sizesandpositioningoftheprimarysensorymotorareas in secreted signalingmoleculeFGF8. 2325. genes andiscompromised inGli3-deficientmice. the embryoniccerebral cortexisdefinedbytheexpression ofmultipleWnt area map. regulating FGFpositionalsignaling. in developingmousebrain. 2401-2413. promoting cellproliferation andinhibitingBmp4expression. Andreazzoli, M. 130 ina neocortical patterning Cambridge: MITPress. The CognitiveNeurosciences gyrus granulecellsisregulated byLEF1. Grosschedl, R. 2212. (BMPs) asregulators ofdorsalforebrain development. ,1903-1914. Development Development Annu. Rev. Neurosci. 126 414 Mol. Cell.Biol. (2000). Hippocampusdevelopmentandgenerationofdentate Dev. Biol. Dev. Biol. 20) Sp8andSp9,twocloselyrelated buttonhead-like (2004). , 1601-1609. 20) Six3functionsinanteriorneuralplate specificationby (2005). , 217-237. 98 130 , 13796-13801. 131 , 6611-6623. 212 222 Science (1999). Dualrole ofbrainfactor-1 inregulating J. Neurosci. Fgf8 , 4763-4774. Gene Expr. Patterns (3rd edn)(ed.M.S.Gazzaniga),pp.69-84. , 455-464. , 296-306. 20 26 hypomorphic mousemutant. (2004). Patterning ofthecerebral(2004). Patterning cortex.In , 6201-6211. Science Science (2000). Distincteffects ofXBF-1inregulating 298 , 355-380. (2001). FRAP/mTORisrequired for (1999). Targeting ofcre totheFoxg1(BF-1) Nat. Neurosci. , 381-385. 22 20) StudyofFgf15geneexpression (2003). Development (2001). Neocortex patterning bythe (2001). Neocortexpatterning (2003). Generatingthecerebral cortical theneocortexby (2003). Emx2patterns , 6526-6536. 303 (1997). Bonemorphogeneticproteins Neuron 294 (2002). Brainfactor-1 controls the , 56-59. J. Neurosci. (1999). Theflat-topgeneis , 1071-1074. (2001). Tbr1regulates (2002). Specificationofjaw 3 (2002). BMPsignalingis , 473-481. (2001). Wntreceptors and Development (1999). DLX-1,DLX-2and (2004). Fgf8regulates the (2003). Acaudalshiftin 6 29 , 825-831. Cereb. Cortex , 353-366. 127 Development Neuron (1999). Inhibitory (2004). Foxg1 24 , 469-482. Neuron , 8917-8923. Development (1998). Thehemof Development Development 125 (2003). Isthmus- 43 (2004). EMX2 , 359-372. (2000). BF-1 9 35 , 2315- , 543-550. 124 , 1029- , 2203- J. 132 Proc. 127 , , Lagutin, O.V., Zhu,C.C.,Kobayashi,D.,Topczewski, J.,Shimamura, K., Lin, W., Furthauer, M.,Thisse,B.,C.,Jing,N.andAng,S.L. Muzio, L.,Soria,J.M.,Pannese,Piccolo,S.andMallamaci,A. Martynoga, B.,Morrison,H.,Price,D.J.andMason,O. Ruiz, I.,Altaba,A.,Palma,V. andDahmane, N. Pera, E.M.,Ikeda,A.,Eivers,andDeRobertis,M. Park, K.,Kim,Rho,S.B.,Choi,D.,Oh,H.,J.,Lee,H. O’Leary, D.andNakagawa,Y. Maruoka,Y., Ohbayashi,N.,Hoshikawa,M.,Itoh, Hogan,B.L.and Lee, S.M.,Tole, S.,Grove, E.andMcMahon,A.P. Kuschel, S.,Ruther, U.andTheil,T. Kim, H.J.andBar-Sagi, D. Sansom, S.N.,Hebert, J.M.,Thammongkol,U.,Smith, J.,Nisbet,G.,Surani, Ohkubo, Y., Chiang,C.andRubenstein,J.L.R. Martinez-Morales, J.R.,DelBene,F., Nica,G.,Hammerschmidt,M., Martinez, S.,Crossley, P. H.,Cobos,I.,Rubenstein,J.L.andMartin,G.R. Liu, Q.,Dwyer, N.D.andO’Leary, D. Kretzschmar, M.,Doody, J.andMassague, Minowada, G.,Jarvis,L.A.,Chi,C.L.,Neubuser, A.,Sun,X.,Hacohen,N., Ramos, C.,Fernandez-Llebrez,P., Bach,A.,Robert,B.andSoriano,E. Rallu, M.,Machold,R.,Gaiano,N.,Corbin,J.G.,McMahon,A.P. andFishell, Muzio, L.andMallamaci,A. Muzio, L.andMallamaci,A. Meyers, E.N.,Lewandoski,M.andMartin,G.R. Liu, Y., Helms,A.W. andJohnson,J.E. Neurobiol. and extrinsicmechanismscontrolling arealization oftheneocortex. is required fordevelopmentofthemammalianhippocampus. essential forvertebrateforebrain development. (2003). Six3repression ofWntsignalingintheanteriorneuroectoderm is Puelles, L.,Russell,H.R.,McKinnon,P. J.,Solnica-Krezel, L.andOliver, G. telencephalon. Bmp/Wnt andFgfsignalingunderliestheventralizationofGli3mutant Cortex specifically promotes expansionofoccipitalcortexandhippocampus. mutually stimulatingloopinvolvingEmx2andcanonicalWntsignalling of dorsaltelencephalicprecursor proliferation andapoptosis. required forspecificationofventraltelencephalonandregion-specific regulation Fgf8 andSpry2duringembryogenesis. Cloning ofthemouseSefgeneandcomparativeanalysisitsexpression with 457-467. developing story. Mech. Dev. Wnt inhibitorsare expressed ingradientsthedevelopingtelencephalon. Neurosci. and thegrowth ofthebrain. 230 Msx1 disruptionleadstodiencephalon defects andhydrocephalus. Genes Dev. IGF, FGF, andanti-BMPsignalsviaSmad1phosphorylationinneuralinduction. inhibits tumorgrowth byinducing apoptosis. and Lee,J. 111 regulate morphogenesisofthe telencephalicandopticvesicles. and synergisticactionsofBMP4,SHHFGF8intherostral prosencephalon Fgf8, Fgf17andFgf18,inthemouseembryo. Furuta, Y. 113-127. Msx3 indorsalneuraltubedevelopment. differential displayPCR. TFI, CHL1,andtwonovelgenesindevelopingneocortexidentifiedby 389 signaling pathwaysconvergeontheTGF-betafamilymediatorSmad1. M. A.,McConnell, S.K.andLivesey, F. J. is coordinated byanFGFsignalingcenter. Bovolenta, P. andWittbrodt, J. Development isthmocerebellar developmentviaarepressive effect onOtx2expression. (1999). FGF8inducesformationofanectopicisthmicorganizerand Development induced byFGFsignalingandcancausechondrodysplasia whenoverexpressed. Krasnow, M.A.andMartin,G.R. 141. series generatedbyCre- andFlp-mediatedrecombination. 4974. mutants lackingbothGli3andHedgehog signaling. G. neuronogenesis andhippocampalmorphogenesistothedorsomedialpallium. 647. regionalization andarealization ofthecerebral cortex. (2002). Dorsoventral patterning isestablishedinthetelencephalonof (2002). Dorsoventralpatterning , 446-460. , 1-17. , 618-622. 15 , 2021-2028. 25 12 (1998). Comparisonoftheexpression ofthree highlyrelated genes, 103 15 (2005). HomeoboxMsx1interactswithp53tumorsuppressor and , 4435-4441. , 14-25. 126 126 , 3023-3028. Dev. Biol. , 167-172. Nat. Rev. Mol.CellBiol. , 1189-2000. , 4465-4475. J. Neurosci. 260 (2004). ModulationofsignallingbySprouty: a (2005). Foxg1confinesCajal-Retzius (2003). Emx1,emx2andpax6inspecification, , 484-495. Nat. Rev. Neurosci. (2002). Patterning centers,regulatory(2002). Patterning genes (2005). Differentiation ofthevertebrateretina (2003). Adisruptedbalancebetween 20 (1999). Vertebrate Sprouty genesare , 682-690. Mech. Dev. (2004). DistinctactivitiesofMsx1and (2000). Differential expression ofCOUP- 5 Development Dev. Cell RESEARCH ARTICLE , 441-450. (2005). Genomiccharacterisation of Cancer Res. Mech. Dev. (1997). OpposingBMPandEGF Genes Dev. (2002). Hedgehog-Glisignalling 3 (2002). Coordinate regulation , 24-33. (1998). AnFgf8mutantallelic 113 8 (2000). AlocalWnt-3asignal Development , 565-574. Cereb. Cortex , 163-168. 131 (2003). Integrationof 65 74 Nat. Genet. 17 , 1017-1028. (2005). Foxg1is , 749-757. , 175-177. Dev. Biol. , 368-379. Development Neuroscience 129 Dev. Dyn. Curr. Opin. (2005). A 13 Cereb. (2002). , 4963- Nature 283 18 , 641- (2004). , 136- 1843 , 127 J. ,

DEVELOPMENT Shinozaki, K.,Yoshida, M.,Nakamura,Aizawa,S.andSuda,Y. Theil, T., Aydin, S.,Koch,Grotewold, L.andRuther, U. Sussel, L.,Kimura,S.andRubenstein,J.L.R. Simeone, A.,Puelles,E.andAcampora,D. Shinya, M.,Koshida,S.,Sawada,A.,Kuroiwa, A.andTakeda, H. Shimogori, T., Banuchi,V., Ng,H.Y., Strauss,J.B.andGrove, E.A. Shimamura, K.andRubenstein,J.L.R. Serls, A.E.,Doherty, S.,Parvatiyar, P., Wells, J.M.andDeutsch,G.H. Stoykova, A.,Treichel, D.,Hallonet,M.andGruss,P. Shimamura, K.,Hartigan,D.J.,Martinez,S.,Puelles,L.andRubenstein,J. Shanmugalingam, S.,Houart,C.,Picker, A.,Reifers,F., Macdonald,R.,Barth, Sun, X.,Meyers,E.N.,Lewandoski,M.andMartin,G.R. Seoane, J.,Le,H.V., Shen,L.,Anderson,S.A.andMassague,J. Storm, E.E.,Rubenstein,J.L.andMartin,G.R. 1844 Dev. Emx1 andEmx2cooperateininitialphaseofarchipallium development. thecerebralpattern cortex. Embryonic signalingcentersexpressing BMP, WNTandFGFproteins interactto Development telencephalon: evidenceforatransformationofthepallidumintostriatum. gene functionresults inaventraltodorsalrespecification withinthebasal embryo. disruption ofFgf8causesfailure ofcellmigrationinthegastrulatingmouse telencephalon inzebrafishembryos. signalling through MAPKcascadeisrequired fordevelopmentofthesubpallial and glioblastomacellproliferation. Integration ofSmadandforkheadpathwaysinthecontrol ofneuroepithelial the dorsoventral patterning ofthemammaliantelencephalon. the dorsoventralpatterning developing forebrain. determines whethercellsurvivalispositivelyornegativelyregulated inthe Genet. Dev. tube. R. early regionalization ofthemouseforebrain. Development forebrain commissureofthetelencephalon. formationandpatterning A., Griffin, K.,Brand,M.andWilson,S.W. liver andlung. Different thresholds offibroblast growththeventralforegut factorspattern into 8042-8050. 3961. a Fgf-regulated gradient-basedneocorticalprotomap. 19) Longitudinalorganizationoftheanteriorneuralplateand (1995). 121 Development RESEARCH ARTICLE , 475-489. Genes Dev. 12 126 127 Development , 409-415. , 3359-3370. , 2549-2561. 121 13 Proc. Natl.Acad.Sci.USA , 1834-1846. , 3923-3933. Development 132 , 35-47. Cell Development 117 (1997). InductiveInteractionsdirect 131 , 211-223. (2002). TheOtxfamily. Development , 5639-5647. (1999). Lossof (2000). Ace/Fgf8isrequired for 100 (2003). DosageofFgf8 128 , 1757-1762. Development , 4153-4164. (2000). Pax6modulates 124 (1999). Targeted (2002). Wntand Nkx2.1 J. Neurosci. , 2709-2718. (2004). Curr. Opin. (2001). Fgf 132 homeobox (2004). (2004). , 3947- Mech. (2005). 20 , Walshe, J.,Maroon, H.,McGonnell,I.M.,Dickson,C.andMason, Trumpp, A.,Depew, M.J.,Rubenstein,J.L.,Bishop,andMartin,G.R. Treier, M.,O’Connell,S.,Gleiberman,A.,Price,J.,Szeto,D.P., Burgess,R., Toresson, H.,Potter, S.andCampbell,K. Tole, S.,Ragsdale,C.W. andGrove, E.A. Ye, W., Shimamura,K.,Rubenstein,J.L.R.,Hynes,M.andRosenthal,A. Xuan, S.,Baptista,C.A.,Balas,G.,Tao, W., Soares, V. C.andLai,E. Xu, Q.,Alldus,G.,Macdonald,R.,Wilkinson,D.G.andHolder, N. Wilson, S.W. andHouart,C. Wiellette, E.L.andSive,H. Walshe. J.andMason.I. von Ohlen,T. andDoe,C.Q. Yun, K.,Potter, S.andRubenstein,J.L.R. Zhou, C.,Tsai, S.Y. andTsai, M.J. FGF8. Establishment ofhindbrainsegmentalidentityrequires signalingbyFGF3and Fgf8 duringzebrafishforebrain development. Development ventral identityinthetelencephalon:opposingroles forPax6andGsh2. cell survival and patterning ofthefirstbranchialarch.cell survivalandpatterning Cre-mediated geneinactivationdemonstratesthatFGF8isrequired for (1999). 386. signaling isrequired forpituitaryglanddevelopment. Chuang, P. T., McMahon,A.P. and Rosenfeld,M.G. 254-265. telencephalon isdisruptedinthemousemutantextra-toes(J). telencephalon. Bmp signallingcooperativelyregulate gradedEmx2expression inthedorsal dopaminergic andserotonin neurons intheanteriorneural plate. (1998). IntersectionsoftheFGF8andShhsignalscreate inductivecentersfor cerebral hemispheres. helixtranscriptionfactorBF-1isessentialforthedevelopmentof Winged Nature Function oftheEph-relatedzebrafishforebrain. kinasertk1inpatterning forebrain. formationinzebrafish. hindbrain pattern ventral columns. signaling pathwayssubdividesthedrosophila neuroectoderm intothree dorsal- regionalization oftheneocortex. 766. telencephalon. complementary rolesofthemammalian indorsoventralpatterning Curr. Biol. 381 Dev. Cell. , 319-322. 127 Development Development 12 Dev. Biol. , 4361-4371. , 1117-1123. 6 , 167-181. Neuron (2003). UniqueandcombinatorialfunctionsofFgf3 224 20) Earlyrequirement forfgf8functionduring (2004). (2004). Earlystepsinthedevelopmentof (2000). Convergenceofdorsal,dpp,andegfr 129 128 14 , 362-372. , 1141-1152. Genes Dev. , 3045-3054. , 193-205. (2001). COUP-TFI:anintrinsicfactorforearly Dev. Dyn. (2000). Dorsoventral patterning ofthe (2000). Dorsoventralpatterning (2001). (2000). Geneticcontrol ofdorsal- 15 Development , 2054-2059. Gsh2 229 Genes Dev. Development Development 133(9) 393-399. , (2001). Hedgehog and 130 Pax6 Dev. Biol. , 4337-4349. 13 Cell play , 3136-3148. 128 (1996). 93 (1995). (2002). , 377- 217 , 755- ,

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