Zinc finger protein too few controls the development of monoaminergic neurons

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1 Zin finger protein too few ontrols the evelopment of monominergi neurons Gil Levkowitz 1, Jörg Zeller 1, Howr I. Sirotkin 2, Dorothy Frenh 3, Srh Shilh 3, Hisshi Hshimoto 4,Mshiko Hii 5, Willim S. Tlot 2 n Arnon Rosenthl 1 1 Rint Neurosiene Corportion, 3155 Porter Drive, Plo Alto, Cliforni 94304, USA 2 Deprtment of Developmentl Biology, Stnfor University Shool of Meiine, 279 Cmpus Drive, Stnfor, Cliforni 94305, USA 3 Deprtment of Pthology, Genenteh In., 1 DNA Wy, South Sn Frniso, Cliforni 94080, USA 4 Division of Moleulr Onology, Biomeil Reserh Center, Osk University, 2-2 Ym-ok, Suit, Osk , Jpn 5 Lortory for Verterte Axis Formtion, RIKEN Center for Developmentl Biology, Mintojim-minmimhi, Chuoh-ku Koe, Hyogo , Jpn Corresponene shoul e resse to G.L. (gillev@rintneuro.om) Pulishe online 25 Novemer 2002; oi: /nn979 The mehnism ontrolling the evelopment of opminergi (DA) n serotonergi (5HT) neurons in vertertes is not well unerstoo. Here we hrterize zerfish mutnt too few (tof) tht evelops hinrin 5HT n norrenergi neurons, ut oes not evelop hypothlmi DA n 5HT neurons. tof enoes forerin-speifi zin finger trnsription repressor tht is homologous to the mmmlin Fezl (forerin emryoni zin finger like protein). Mosi n o-stining nlyses showe tht fezl ws not expresse in DA or 5HT neurons n inste ontrolle evelopment of these neurons non-ell-utonomously. Both the eh1-relte repressor motif n the seon zin finger omin were neessry for tof funtion. Our results inite tht tof/fezl is key omponent in regulting the evelopment of monominergi neurons in the verterte rin. Dopminergi (DA) n serotonergi (5HT) neurons resie in restrite lotions in the verterte rin n re responsile for rewr-ssoite ehviorl, enorine n ognitive funtion s well s posturl reflexes 1. Culture explnt stuies in rt n hik emryos suggest tht the ientities of these neurons re regulte, in prt, y the floor plte erive growth ftor Soni hegehog (Shh), y firolst growth ftor-8 (FGF8), whih is erive from the mi-hinrin ounry n nterior neurl rige, n y n FGF4-like signl 2. In ition, gene ltion stuies in the mouse implite the orphn steroi reeptor Nurr1 (refs. 3,4) n the homeoomin proteins Lmx1 (ref. 5) n En1/En2 (ref. 6) s possile meitors of DA neuronl ifferentition. Finlly, mutgenesis stuies in zerfish 7 n Drosophil melnogster 8,9 ientify the trnsription elongtion inhiitor Spt5 n the zin finger genes egle n hukeein s key regultors of DA n/or 5HT ell fte. Severl geneti n ell ulture stuies hve resse the mehnism ontrolling the temporl n sptil expression of FGF8 n Shh. FGF8 expression is regulte y the trnsription tivtor Px2, the trnsription repressors Otx2, Gx2, Engrile 2 n Grg, n the serete protein Wnt1 (ref. 10 n referenes therein). Likewise, Shh is regulte in prt y the trnsription ftor HNF3β (ref. 11). Mny of these trnsription ftors influene one nother s expression; hene, their ext position in the regultory se ontrolling the evelopment of DA n 5HT neurons is not fully unerstoo. Both FGF8 n Shh ontrol the evelopment of ell types other thn DA n 5HT, n re not suffiient to inue DA n 5HT in ll etopi rin lotions 2. Thus, it is oneivle tht itionl regultory ses ting in onjuntion with or inepenently of FGF8 n Shh re require to fully efine the evelopment of these neurons. To eluite suh regultory ses, we etermine the moleulr nture n funtion of the gene responsile for the loss of DA n 5HT neurons, ut not other neuronl ell types, in the zerfish mutnt tof. RESULTS too few mutnts show efiits in DA n 5HT neurons We previously sreene for zerfish mutnts tht show efiits in DA neurons 12. The tof mutnt hs reue numer of tyrosine hyroxylse positive (TH + ), opmine β-hyroxylse negtive DA neurons (2 6 DA neurons in the mutnt versus in wil type) n ompletely lks 5HT neurons in the hypothlmus (Fig. 1 ). Otherwise, the size, morphology, ntomy, fertility, espe, seeking of prey n feeing responses of tof were inistinguishle from wil type (WT). Moreover, DA neurons in the olftory ul n retin, 5HT neurons in the hinrin, TH + norrenergi neurons in lous oeruleus, the rh-ssoite ells n the symptheti neurons of the gut ll ppere to e norml (Fig. 1 n t not shown). We next exmine the expression of the ptterning genes lx-2, nk-2.2, rx-3, lx-6, lim-3 n th-5 to etermine whether the loss 28 nture neurosiene volume 6 no 1 jnury 2003

2 e g i Fig. 1. Impirment of forerin DA n 5HT neurons in the tof mutnt emryos. ( ) Emryos (48 h fter fertiliztion, ventrl view) were sujete to whole-mount stining either with ntioies irete ginst tyrosine hyroxylse (TH;, ) or serotonin (5HT;, ). (e h) In situ hyriiztion with ntisense Nk-2.2 (e, f) n Dlx-2 (g, h) RNA proes (24 h fter fertiliztion, lterl view). (i, j) Emryos (48 h fter fertiliztion) were proe with n ntisense Px6 RNA, followe y immunohistohemistry with n nti-th ntioy (lterl view; eyes were remove). e, eye; Hy DA, hypothlmi opminergi; A, rhssoite norrenergi neurons; Tel, telenephlon; Dien, ienephlon; MB, mi rin; Te, tetum; Pr, pretetum; Vt, ventrl thlmus. Sle r, 100 µm ( ); 500 µm (e, f); 400 µm (g, h); 50 µm (i, j). of DA n 5HT neurons might e ue to impirment in ell pttern. The expression pttern of lx-2 (refs. 13,14) in two longituinl omins in the telenephlon n rostrl ienephlon, of nk-2.2 in the mi-ienephli ounry 15 n of the other ptterning genes were ll norml (Fig. 1e h n t not shown). More importntly, the numer of px-6/zf- positive neurons tht intermingle with hypothlmi DA neurons 16 ws omprle in WT n tof emryos (Fig. 1i n j). Finlly, the expression pttern of FGF8 n Shh ppere to e norml (t not shown). Although the first DA n 5HT neurons ppere t 22 hours fter fertiliztion, efiits in DA n 5HT neurons ppere only few hours lter (24 48 hours fter fertiliztion; Fig. 4 ), suggesting f h j f h Fig. 2. Chromosoml linkge, moleulr hrteriztion n phenotypi resue of tof. () Geneti mp of tof. The tof muttion ws mppe to linkge group 11 (LG 11). We lulte the istnes etween tof n eh geneti mrker y SSLP or SSCP nlysis (Methos) on 200 iniviul mutnt emryos oring to the rte of reomintion etween mrkers n tof. Geneti istnes in entimorgn (M) units re inite. () Autoriogrphy of n SSCP polyrylmie gel showing the geneti linkge etween tof n the fez-like (fezl) gene. Shown is n SSCP of pools (24 emryos) of mutnt emryos (tof) n their respetive silings (WT + het.). The lotions of ns representing WT n mutnt lleles re inite. () Shemti representtion of tof/fezl s gene n protein omin strutures. Amino i numers re inite. (, e) Resue of the too few phenotype. Ventrl view of too few mutnt emryos, whih were injete with 10 pg of either PAC DNA onstrut ontining the fezl gene (e) or PAC vetor lone (). 48 h fter injetion, emryos were fixe n stine with n nti-th ntioy. (f i) Injetion of ntisense tof/fezl DNA. Emryos (t 1 4 ells stge) were injete with either 1.5 ng of fezl ntisense oligonuleotie (h, i) or wter (f, g). 40 h fter injetion, emryos were fixe n stine with ntioies irete to either TH or 5HT. RD, repressor omin; ZF, zin finger; e, eye; Hy DA, hypothlmi opminergi; Dien, ienephlon. Sle r, 100 µm. tht the tof muttion reues the numer of progenitors tht will ifferentite into forerin DA n 5HT neurons. too few isrupts the zin finger ontining gene, fezl Using polymorphi mirostellite mrkers 17, we mppe the tof muttion to linkge group 11 (LG 11) etween the mirostellite mrkers Z25289A n Z4190 (Fig. 2). We then g i e nture neurosiene volume 6 no 1 jnury

3 e teste linkge etween tof n expresse sequene tgs (ESTs) n genes previously mppe to the tof region 18 y soring polymorphisms in tof mpping ross. One gene from LG 11 forerin emryoni zin finger like (fezl) ws tightly linke to the muttion (0 reominnts, n = 200) (Fig. 2). fezl is speifilly expresse in the emryoni telenephlon n ventrl ienephlon 19,20 efore n uring the emergene of hypothlmi DA n 5HT neurons (Fig. 4). Injetion of tof e f g i j h Fig. 3. Gin-of-funtion nlysis of WT n mutnt tof/fezl RNA. ( ) Emryos (1 16 ell stge) were injete with 5 pg of the ifferent forms of in vitro synthesize tof/fezl RNA, s inite. Control emryos were mok-injete (). Injete emryos (t 70 80% epioly) were sujete to in situ hyriiztion with n ntisense Fox2 RNA proe. Shown is orsl view (nterior t the top, posterior t the ottom). (e) The omin strutures n tivities of WT n mutnt forms of tof/fezl re shemtilly shown. A Cys-to-Ser sustitution in the tof mutnt is inite. The perentge of injete emryos in whih the expression of Fox2 ws represse is inite on the right. RD, repressor omin; EnR, engrile repressor; ZF, zin finger. Sle r, 100 µm. emryos (t the one-ell stge) with P1 rtifiil hromosome (PAC) DNA onstrut ontining the full-length fezl gene resue the neuronl efiienies of tof (Fig. 2e; 32%, n = 273). Hene, fezl is strong nite for the too few lous. In situ hyriiztion of fezl RNA showe norml expression pttern in tof (Fig. 4 ). Sequening the fezl gene from mutnt n WT silings, however, revele T-to-A hnge in the mutnt llele tht shoul result in Cys-to-Ser hnge t mino i position 287 (Fig. 2). Fezl ontins n mino (N)-terminl trnsription repressor motif tht is followe y six C2H2-type zin finger repets. The ientifie muttion in tof sustitutes the seon Cys of zin finger 2 n is likely to isrupt the DNA ining ility of this zin finger 21. To test whether the tof phenotype is use y the loss of Fezl funtion, we loke tof/fezl gene tivity y injetion of morpholino-moifie ntisense oligonuleoties 22. More thn hlf of WT emryos injete with 1.5 ng ntisense fezl oligonuleotie showe fewer DA neurons in the hypothlmus (55%; n = 320; Fig. 2i). Consistent with the more severe 5HT phenotype in tof, injetion of fezl ntisense le to omplete loss of 5HT neurons (84%; n = 85; Fig. 2h). Other groups of DA n 5HT s well s TH + norrenergi neurons were not ffete y the ntisense injetion (Fig. 2 n t not shown). Thus, the inhiition of Fezl tivity y ntisense oligonuleotie mirrore Fig. 4. tof/fezl t non-ell-utonomously. ( ) Expression omins of fezl n DA neurons. WT (, ) n tof (, ) emryos were fixe t 22 (, ) n 50 (, ) hours fter fertiliztion n were proe with n ntisense Fezl RNA followe y n nti-th ntioy. Wek expression of Fezl ws etete in the retin of 50-hour emryos. Arrows mrk the proximl lotion of Fezl n DA neurons, respetively. (g j) Mosi nlysis of WT/tof himers. tof or WT emryos were trnsplnte with ells erive from WT or tof emryos, respetively. Donor emryos were injete with iotinylte linege trer. Chimeri emryos were fixe t 48 h fter fertiliztion n were sujete to in situ hyriiztion with DIG-lele TH proe (purple). The linege trer mrking the onor-erive ells ws etete y peroxise-onjugte streptviin (rown). Shown re tof emryos in whih WTerive ells gve rise to telenephli (g) or ienephli (h) tissue in tof emryo. (i, j) tof-erive ells tht were trnsplnte into WT emryo ololize with hypothlmi DA neurons. Emryo in (i) ws sujete to oronl setioning, whih is presente in (j). For referene, un-trnsplnte WT (e) n tof (f) were stine with TH proe. DA, opminergi neurons; e, eye; Hy, hypothlmus; Tel, telenephlon; Dien, ienephlon; ret; retin. Sle r, 35 µm (, ), 100 µm ( h), 50 µm (i), 10 µm (j). 30 nture neurosiene volume 6 no 1 jnury 2003

4 the tof neuronl efiit, initing tht the Cys-to-Ser muttion intivte tof n le to the loss of oth DA n 5HT neurons. Struturl n funtionl nlysis of tof To further etermine the funtion of tof n eluite its mehnism of tion, we injete ifferent forms of fezl RNA into zerfish emryos (1 16 ell stge). Injetion of the tof mutnt RNA i not le to ny phenotypi normlities. In ontrst, injetion of wiltype fezl RNA le to morphologil efets, inluing short xis n isproportionl he n rin strutures (t not shown). This isturne in erly ptterning ws reflete y iminishe expression of the forkhe trnsription ftor Fox2/HNF3β, whih is mrker of enoerm n xil mesenoerm uring gstrultion n is one of the key genes regulting the expression of Shh 11,23 (Fig. 3). In ontrst, WT emryos injete with fezl RNA form ontining the sequene hnge ientifie in too few mutnts (enote C287S) ppere to e ientil to mokinjete ontrols, initing tht the Cys-to-Ser sustitution intivtes the Fezl protein (Fig. 3). Consistent with Fezl ting s trnsriptionl repressor, the ility of Fezl to repress Fox2/HNF3β ws mintine when its puttive repressor omin ws reple with tht of Engrile (Fig. 3). The suppressor tivity ws lost when the Phe resiue in the repressor omin ws mutte to Glu n fter eletion of its six zin fingers (Fig. 3e n t not shown). We i not etet ny etopi DA or 5HT neurons in these experiments, supporting the ie tht Fezl my not e limiting ftor in the WT emryo (t not shown). Tle 1. Mosi nlysis of too few. Linege Numer of emryos with trer/lone 1 2 DA 4 5 DA 6 9 DA DA lotion: neurons neurons neurons neurons WT tof* Telenephlon n = 191 Dienephlon 10 Other rin regions tof WT* Telenephlon n = 131 Dienephlon Other rin regions *Mutnt emryos were re from tof prents; neurons were etete in WT emryos tht were sore with DIG-lele TH RNA proe. Fig. 5. Resue of tof 5HT efiit y non-utonomous telenephli ue. too few emryos were trnsplnte with ells erive from WT emryos, whih were injete with iotinylte linege trer (rown stining). Chimeri emryos were fixe t 48 h n were sujete to whole-mount stining with n ntioy irete ginst serotonin (5HT, lk stining). The resue of hypothlmi 5HT ells ws oserve in 12% of the emryos with WT telenephli lones (n = 94), ut not in emryos with other rin lones (n = 51). Shown re WT (), nonresue () n resue () WT/tof himeri emryos. () Suggeste moel for the evelopment of DA n 5HT neurons in the zerfish forerin. Dien, ienephlon; e, eye; Hy, hypothlmus; Tel, telenephlon; Hin, hinrin. Sle r, 100 µm. tof ontrols DA n 5HT ells non-utonomously Doule-stining of fezl n DA neurons strting from the erliest stge t whih DA neurons re etete (22 hours fter fertiliztion) showe tht fezl expression is onfine to the telenephlon n ventrl ienephlon n tht DA n 5HT neurons never express fezl (Fig. 4 ). Furthermore, DA neurons, whih normlly evelop frther wy from fezl expressing ells, were the most suseptile to the too few muttion (Fig. 4 n ). Tken together, these finings suggest tht tof might ontrol the evelopment of monominergi neurons in non-ell-utonomous mnner. To iretly exmine this possiility, we generte himeri emryos in whih WT-erive ells were trnsplnte into tof emryos or tof-erive ells were trnsplnte into WT emryos. We then nlyze DA n 5HT neurons in the mosi fish. Wiltype lones tht were onfine to the telenephlon, ut not to other rin regions, prtilly resue the tof DA n 5HT efiit (Figs. 4g n 5; Tle 1), wheres WT hypothlmi lones i not evelop DA neurons when trnsplnte into the mutnt fish (Fig. 4h; Tle 1). Moreover, mutnt hypothlmi tof lones tht were trnsplnte into WT emryos ololize with DA neurons n showe norml numer of these neurons, wheres WT emryos ontining forerin tof lones h reue numer of DA neurons (Fig. 4i n j; Tle 1). Tken together, our finings ientify the trnsription suppressor tof/fezl s key omponent of the regultory se tht ontrols the evelopment of ienephli monominergi neurons in vertertes in non-ell-utonomous fshion. DISCUSSION It hs een suggeste tht neurl evelopment in the verterte nervous system is ontrolle y regionl n ell speifi trnsription ftors 24. Here we provie eviene tht tof/fezl is one of the trnsription ftors ontrolling the evelopment of DA n 5HT neurons in the verterte forerin. The expression of tof/fezl is restrite to the prospetive nterior neuroetoerm n to the evelopmentl winow in whih DA n 5HT neurons re speifie 19. fezl represents fmily of genes tht re onserve in vertertes 25, n the murine fezl ortholog is lso expresse in the emryoni forerin (M.H., unpu. t). A mjor question rising from the present work onerns the iohemil mehnism y whih the Fezl protein exerts its funtion. The onserve struturl motifs tht re present in Fezl inlue puttive Gruho-TLE-ining repressor omin followe y six onseutive C2H2 Kruppel-type zin finger omins 19,25. Similr to other C2H2- ontining trnsription ftors, some of those zin fingers my mei- nture neurosiene volume 6 no 1 jnury

5 te the ining of Fezl to promoter elements of its trget genes, wheres others my serve s protein protein interting interfes 26. Notly, the tof muttion reple ysteine with serine in the seon zin finger (ZF2) of the Fezl protein (Fig. 2). Consistent with the ruil role of this Cys resiue in ining zin tom to the protein, we showe tht the mutte tof/fezl hs no isernle iologil funtion. Similrly, we showe tht point muttion in Fezl s Engrile-repressor-like motif interfere with Fezl s funtion. Our finings lso rise new question: Wht re the tof/fezl trget genes? Although Fox2/HNF3β is suppresse fter overexpression of tof/fezl, it is unlikely to e iret trget euse it hs norml expression pttern in the tof mutnt (t not shown). tof seems to e n intrellulr trnsriptionl repressor. DA n 5HT neurons, however, o not express tof n inste evelop wy from the fezl expression omin. Moreover, our mosi nlysis inite tht WT ell lones resiing in the telenephlon resue the DA (Fig. 4) n 5HT (Fig. 5) neuronl efiit in the tof hypothlmus n tht WT neurons trnsplnte into the tof ienephlons i not give rise to DA neurons. These finings inite tht fezl ts non-ell-utonomously. Given the likelihoo tht Fezl is trnsription suppressor, we fvor the possiility tht the evelopment of DA n 5HT neurons is ontrolle y non-utonomous telenephli ue tht is suppresse in the sene of tof/fezl (Fig. 5). The ftes of multiple groups of DA n 5HT neurons re speifie y Shh n epen on FGF8 signls for their evelopment 2. Aitionl signls suh s FGF4, however, re require to speify 5HT neurons in preise rin lotion. The extrellulr signls tht meite the tivity of tof/fezl remin to e etermine. Although hypothlmi DA n 5HT neurons re ner soure of Shh n FGF8 in the zerfish ienephlon, they evelop normlly in the sene of Shh or FGF8 signls 27,28. We lso foun tht the zerfish hypothlmi DA n 5HT neurons evelop normlly in the sene of the Wnt signling omponent TCF/LEF (heless) (t not shown), suggesting either reunny in these signling systems or tht tof/fezl meite its tivity y extrellulr signls other thn Shh, FGF8 or Wnt. A high onentrtion of ntisense oligo trgeting the Fezl gene h more severe evelopmentl effets thn those reporte here (ref. 20 n t not shown). These finings suggest tht fezl/tof not only regultes the evelopment of DA n 5HT neurons, ut my lso regulte the evelopment of other neuronl ell types in onentrtion-epenent mnner 29. In sum, we hve ientifie tof/fezl s key omponent in new regultory se of trnsription n non-utonomous ues tht is essentil for the evelopment of monominergi neurons in the verterte rin in vivo. METHODS Fish stoks. Fish reeing n mintining were performe s previously esrie 12. Experiments were one in orne with Genenteh In. protool review ommittee. Plsmis n proes. C287S n WT forms of Fezl were mplifie y PCR from RNA tht ws isolte from too few n WT siling, respetively. Nuleotie sequening onfirme the orret sequene of oth DNAs. The F39E Fezl onstrut ws generte y PCR-se mutgenesis. To onstrut EnR-Fezl, the repressor omin of Drosophil Engrile ( resiues 1 226) n the roxy (C)-terminl region ( ) of Fezl were mplifie y PCR. All fezl vrints were susequently su-lone into the pcs2+ expression vetor. The lx2, nk2.2, fox2, th n px6 proes were previously esrie 12 15,23. Immunohistohemistry n in situ hyriiztion. Whole-mount stining with rit nti-tyrosine hyroxylse (TH) ntioy (Chemion, Temeul, Cliforni) n polylonl 5HT ntioy (DiSorin, Stillwter, Minnesot) n in situ hyriiztion were one s previously esrie 12. Mpping n loning. AB/EK femle fish rrying the tof muttion were out-rosse to WT WIK mle fish, n F1 progeny were rise to ulthoo. Emryos from tof heterozygous ross were immunostine with n nti-th ntioy, n mutnt n WT emryos were sore. SSLP (simple sequene length polymorphism) n SSCP (singlestrne onformtion polymorphism) nlyses were one on genomi DNA tht ws isolte from WT n mutnt pools of 24 iniviuls. Primer pirs for SSLP mrkers were otine from Reserh Genetis, In. (Huntsville, Alm). For SSCP nlysis of LG 11-mppe 18 genes n ESTs, we performe PCR of p DNA frgments, erive from the 3 untrnslte regions of these sequenes, with 32 P-lele oligonuleoties. Amplifie prouts were enture in 95% formmie, snp-oole on ie n run overnight t room temperture through non-enturing 0.5 MDE gel (BMA, Rokln, Msshusetts). A P1 rtifiil hromosome (PAC) lone ontining the full-length fezl gene ws isolte from zerfish PAC lirry (Inyte Genomis, St. Louis, Missouri), whih ws sreene y riolele fezl proe. The genomi fezl lone ws sequene, n the intron-exon ounries were etermine y ligning the gene sequene with the fezl DNA. Miroinjetion n mosi nlysis. Cppe RNA ws synthesize with mmessage mmachine kit (Amion, Austin, Texs) from linerize pcs2+ plsmis. The sequene of morpholino-moifie ntisense oligonuleotie (Gene Tools, Corvllis, Oregon) trgete to the fezl gene (GenBnk AY078361) ws s previously esrie 20. RNA n DNA were miroinjete to 1 8 ell stge emryos, n injete emryos were llowe to evelop t 28.5 C. WT/tof himeri emryos were generte s esrie 30 n were sujete to in situ hyriiztion with igoxigenin (DIG)-lele tyrosine hyroxylse (TH) proe. The iotinylte linege trer (Moleulr Proes, Eugene, Oregon) ws etete y peroxise-onjugte Streptviin. Aknowlegments We thnk T. Ye, S. Neuhuss, C. Liew, C. Ton, A. Jensen, G. Wilhelm Otto, T. Look n M. Austen for shring their unpulishe mpping informtion n R. Geisler n E. Golings for proviing the ove ontt informtion; M. Ekker, A. Shier, U. Strhle, P. Rymons n S. Wilson for proes; S. Guo, K. Poulsen n L. Prker for tehnil vie; T. Hirno for support n omments on this work, n memers of the Rosenthl l for stimulting isussions. G.L. ws supporte y long-term postotorl fellowship from the Europen Moleulr Biology Orgniztion. W.S.T. ws supporte y Ntionl Institutes of Helth grnt RR Competing interests sttement The uthors eline to provie informtion out ompeting finnil interests. RECEIVED 23 SEPTEMBER; ACCEPTED 24 OCTOBER Knel, E.R., Shwrtz, J.H. & Jessell, T.M. Priniples of Neurl Siene 1135 (Elsevier, New York, 1991). 2. Ye, W., Shimmur, K., Ruenstein, J.L., Hynes, M.A. & Rosenthl, A. FGF n Shh signls ontrol opminergi n serotonergi ell fte in the nterior neurl plte. Cell 93, (1998). 3. Zetterstrom, R.H. et l. Dopmine neuron genesis in Nurr1-efiient mie. Siene 276, (1997). 4. Sueo-Crens, O. et l. Nurr1 is essentil for the inution of the opminergi phenotype n the survivl of ventrl mesenephli lte opminergi preursor neurons. Pro. Ntl. A. Si. USA 95, (1998). 5. Smit, M.P. et l. A seon inepenent pthwy for evelopment of mesenephli opminergi neurons requires Lmx1. Nt. 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