LETTERS NTURE Vol pril 2009 a cat-4 Tyr GTPCH TH DOP bas-1 D D VMT DT b Mol 1 Mol 2 D B E C F D nuron class 1 D nuron class 2 D nuron class 1 D

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1 Vol pril 2009 oi: /natur07929 Gn rgulatory logic of opamin nuron iffrntiation LETTERS Nuria Flams 1 & Olivr Hobrt 1 Dopamin signalling rgulats a varity of complx bhaviours, an fcts in opamin nuron function or survival rsult in svr human pathologis, such as Parkinson s isas 1. Th common nominator of all opamin nurons is th xprssion of opamin pathway gns, which co for a st of phylogntically consrv protins involv in opamin synthsis an transport. Gn rgulatory mchanisms that rsult in th irct activation of opamin pathway gns an thrby ultimatly trmin th intity of opamin nurons ar poorly unrstoo in all systms stui so far 2. Hr w show that a simpl cis-rgulatory lmnt, th opamin (D) motif, controls th xprssion of all opamin pathway gns in all opaminrgic cll typs in Canorhabitis lgans. Th D motif is activat by th ETS transcription factor ST-1. Loss of ast-1 rsults in th failur of all istinct opaminrgic nuronal subtyps to trminally iffrntiat. Ectopic xprssion of ast-1 is sufficint to activat th opamin pathway in som cllular contxts. Vrtbrat opamin pathway gns also contain phylogntically consrv D motifs that can b activat by th mous ETS transcription factor Etv1 (also known as ER81), an a spcific class of opamin nurons fails to iffrntiat in mic lacking Etv1. Morovr, ctopic Etv1 xprssion inucs opaminrgic fat markr xprssion in nuronal primary culturs. Mous Etv1 can also functionally substitut for ast-1 in C. lgans. Our stuis rval a simpl an apparntly consrv rgulatory logic of opamin nuron trminal iffrntiation an may provi nw ntry points into th iagnosis or thrapy of conitions in which opamin nurons ar fctiv. Nrvous systms gnrally harbour istinct populations of opamin (D) nurons that riv from iffrnt prcursor clls. Dspit thir ivrs origin, all D nurons shar th xprssion of a cor st of fiv gns that co for nzyms an transportrs which synthsiz, packag an r-uptak opamin ( opamin pathway gns ; Fig. 1a). Th rgulatory logic of th trminal iffrntiation of D nurons, manifst by th inuction of th D pathway gns, can, in thory, b scrib by two istinct mols. In mol 1, ach opamin pathway gn is inpnntly activat by a istinct st of rgulatory factors an, as a rflction of thir istinct vlopmntal history, ach D nuron subtyp uss a istinct st of rgulatory molculs(fig. 1b). In mol 2, ach opamin pathway gn is rgulat by th sam rgulatory factor(s) an thos factor(s) ar th sam in ach D nuron subtyp (Fig. 1b). Ths two mols mak spcific prictions about th cis-rgulatory architctur of opamin pathway gns. In mol 1, ach opamin pathway gn is controll by istinct cisrgulatory motifs an iffrnt motifs ar activ in iniviual D nuron subtyps. In mol 2, thr is a singl motif for all pathway gns that is us in all iffrnt D nuron subtyps (Fig. 1b). To tst ths mols, w ma us of th D systm of th nmato C. lgans, which contains four istinct, linally unrlat classs of D nuron subtyps that xprss th sam st of highly consrv opamin pathway gns (Fig. 1c) 3. W systmatically issct th cis-rgulatory rgions of all D pathways gns in th contxt of gfp rportrs xprss in transgnic worms (Fig. 1ch an Supplmntary Fig. 1). Th cis-rgulatory analysis of two gns xclusivly xprss in th D nurons, th opamin transportr gn (opamin transportr) an th tyrosin hyroxylas gn (tyrosin hyroxylas), rval th xistnc of a small cis-rgulatory moul (CRM) in ach promotr that is rquir an sufficint to riv xprssion in all D nurons (Fig. 1, ). Dopamin pathway gns xprss in both D an srotonrgic (5-HT) nurons (, vsicular monoamin transportr, cat-4, GTP cyclohyrolas, bas-1, aromatic amino aci carboxylas) contain sparabl CRMs for xprssion in D an 5-HT nurons (Fig. 1fh). Th D-spcific CRM of th locus contains a small squnc motif that is consrv in thr othr Canorhabitis spcis (Fig. 1 an Supplmntary Fig. 5); mutational analysis monstrats that this motif is rquir for xprssion in all D nurons (Fig. 1). This motif was also sufficint to riv xprssion in all D nurons, ithr whn tst in isolation or whn appn to th CRM of anothr nuron-spcific gn (Supplmntary Fig. 2a). Bioinformatics analysis prict th bining of six iffrnt typs of transcription factors to this consrv motif (Supplmntary Fig. 2b). Point mutations that spcifically abolish th prict bining of som factors whil kping othrs intact rval that th only prict motif that can b ma rsponsibl for cis-rgulatory motif activity in th D nurons is a prict ETS transcription factor bining sit (EBS) fin by an invariant GGW cor squnc (Supplmntary Fig. 2b). Th D-xprss CRMs of all othr opamin pathway gns also contain prict EBSs, an mutational analysis corroborat thir rquirmnt for th corrct xprssion in all D nurons of C. lgans hrmaphroits (Fig. 1fh an Supplmntary Fig. 3) an in th thr aitional D nuron pairs prsnt in th mal (Supplmntary Fig. 4). ll th functionally charactriz EBSs ar consrv in othr Canorhabitis spcis; thy can occur in ithr orintation an at iffrnt istancs from th transcriptional start (Supplmntary Fig. 5). Th wight matrix gnrat with all ths squncs fins a consnsus EBS squnc motif that w trm th D motif (Fig. 1i an Supplmntary Fig. 5). By analysing th xprssion of th D markr ::gfp in mutants that lack ach of th tn C. lgans ETS family mmbrs (Supplmntary Fig. 6), w foun that all ts family mutants show wiltyp ::gfp xprssion xcpt for animals lacking th axon string fct-1 (ast-1) gn, prviously intifi as a gn controlling axon outgrowth in th vntral nrv cor 4. Morovr, w foun that a mutant alll, ot417, which w rtriv from an unbias forwar gntic scrn for mutants in which D fat is inappropriatly xcut 5, is an alll of ast-1 (Fig. 2a). Th xprssion of all fiv opamin pathway gns was strongly affct if not compltly lost in ast-1 mutants (Fig. 2b, c, Supplmntary Tabl 1 an Supplmntary 1 Howar Hughs Mical Institut, Dpartmnt of Biochmistry an Molcular Biophysics, Columbia Univrsity Mical Cntr, Nw York, Nw York 10032, US Macmillan Publishrs Limit. ll rights rsrv 885

2 LETTERS NTURE Vol pril 2009 a cat-4 Tyr GTPCH TH DOP bas-1 D D VMT DT b Mol 1 Mol 2 D B E C F D nuron class 1 D nuron class 2 D nuron class 1 D nuron class 2 c DE PDE...GCTTTCCGGC = D motif = GFP DE PDE 5HT Lins p3mut#1 2/3 mut#1 prom8 prom9 g 32 1 cat-4 ::gfp DE PDE 5HT Lins mut#1 n.. mut#1,#2,#3 prom8 / prom8 mut#1 D motif i Figur 1 Charactrization of th D motif in C. lgans. a, Schmatic rprsntation of a D nuron synaps. D, aromatic L-amino aci carboxylas; DOP, 3,4-ihyroxy-L-phnylalanin; Tyr, tyrosin. b, Schmatic rprsntation of two iffrnt mols for D trminal iffrntiation. S txt for xplanations. Capital lttrs rprsnt hypothtical trans-acting factors. c, Pictur of an ault worm xprssing GFP unr th control of th full-lngth promotr, lablling all C. lgans D nurons. Similarly, is also xclusivly xprss in D nurons (ata not shown), as C. lgans contains no arnrgic or norarnrgic nurons., promotr analysis. Schmatic rprsntation of th locus with its upstram rgion. Exons ar rprsnt as r blocks; th upstram gn is shown in gry. t th bottom is a rprsntation of clon an injct constructs, an th xprssion pattrn in th D an 5-HT nurons of transgnic worms. Thick black lins Fig. 7). Two othr D trminal iffrntiation markrs, th ion channls asic-1 (rf. 6) an trp-4 (rf. 7), also fail to b xprss in th D nurons of ast-1 mutants (Supplmntary Fig. 8). Both gns contain phylogntically consrv D motifs in thir rgulatory rgions. ast-1 thrfor appars to affct D fat broaly, which is furthr corroborat by th axon pathfining fcts of D nurons w obsrv in ast-1 mutants (Supplmntary Fig. 9). Loss of D markr gn xprssion is not a rflction of arly linag spcification fcts an/or absnc of th nurons, as assss by analysis of aitional fat markrs (Supplmntary Fig. 8). ast-1 is xprss in svral nurons 4, incluing all D nurons (Fig. 2), an acts cll-autonomously in D nurons, as th ast-1 mutant phnotyp can b rscu by xprssion of ast-1 spcifically in th D nurons (Fig. 2). ast-1 xprssion prsists in D nurons throughout postmbryonic stags, inicating that ast-1 is rquir not only to initiat D trminal cll fat but also to maintain D nuron intity, a notion w confirm through tmporally controll aition an rmoval of ast-1 gn activity (Fig. 2f an Supplmntary Fig. 10). 886 DE PDE 5HT Lins mut#1 / / / 2/3 / / / / 2/3 mut#2&#3 f h DE PDE 5HT Lins mut#2 / / mut#3 mut#2#3 / / prom8 prom9 prom9 mut#1 mut#1 mut#2 mut#1&#2 DE PDE 5HT Lins * * / / 2/3 / / bas-1 symboliz th promotr pic plac in front of GFP (grn box). R cross rprsnts a mutat EBS. 1 inicats.10% pntrant xprssion in mor than half of th transgnic lins xamin; 1/2 also mans.10% pntrant xprssion, but th pntranc is lowr than in th corrsponing full-lngth construct; 2 inicats,10% pntrant xprssion in mor than half of th transgnic lins xamin; n.., not trmin. h, nalysis of th rgulatory rgions of all othr opamin pathway gns. 1* mans immr GFP xprssion than th corrsponing wil-typ construct. S Supplmntary Figs 1 an 3 for all primary ata an th natur of th mutations. i, Th squnc alignmnt of all functional EBSs fins a position wight matrix (PWM) that is rprsnt by a squnc logo. Th consrv cor in all squncs constituts th D motif. S Supplmntary Fig. 5 for squncs us to fin th D motif Macmillan Publishrs Limit. ll rights rsrv To arss whthr ast-1 function is not only ncssary for propr D nuron iffrntiation but also sufficint, w ctopically inuc ast-1 xprssion throughout all cll an tissu typs at iffrnt stags of vlopmnt (Fig. 3). Ectopic inuction uring mbryognsis l to a substantial ctopic xprssion of both ::gfp (Fig. 3a) an ::gfp (ata not shown). Th morphology, location an pannuronal fat markr xprssion of ths clls inicats that th ffcts of ast-1 ar confin to th nrvous systm, in which som (20 clls;,10% of th mbryonic nrvous systm) but clarly not all clls can b inuc to ctopically xprss both /DT an /TH. Ectopic ast-1 was maximally ffctiv whn xprss aroun th tim of nuronal iffrntiation (Supplmntary Fig. 11). Morovr, ctopic ast-1 xprssion unr control of th D- an 5-HT-spcific bas-1 promotr inuc /DT xprssion in 5-HT nurons (Fig. 3) monstrating that ast-1 acts autonomously to control D nuron spcification an that 5-HT nurons provi th appropriat cllular contxt to allow ast-1 to inuc D nuron spcification. Th rlat ETS omain transcription factor LIN-1 was not abl to inuc ctopic

3 NTURE Vol pril 2009 LETTERS a b ::gfp D clls ast-1 gk463 ast-1() DE D nuron prouction whn xprss unr similar conition (ata not shown), monstrating th spcificity of ST-1 function. To assss whthr ETS transcription factor(s) hav a similar function in vrtbrat D nuron spcification, w analys thir xprssion in th D aras of th brain (Supplmntary Tabl 2). Distinct ETS factors appar to b xprss in istinct typs of D nurons an w focus hr on th ETS factor Etv1, which is xprss in th D nurons of th olfactory bulb (Supplmntary Fig. 12) 8,9. Mic lacking Etv1 (rf. 10) isplay a notabl ruction in th numbr of tyrosin hyroxylas (TH)-positiv clls in thir olfactory bulb compar to wil-typ siblings, whras othr priglomrular intrnuron subtyps wr not affct or wr lss svrly ruc (Fig. 4ac an Supplmntary Fig. 13). This phnotyp was not paralll by incras cll ath, by a ruction in th ovrall nsity of clls in th glomrular layr, by a ruction in ovrall nuron numbr or by prolifration fcts (Fig. 4f an Supplmntary Fig. 14). Morovr, th intity of D prognitor clls in th latral ganglionic minnc, which alray xprss Etv1 (rf. 11), appar unaffct in Etv1 mutants (Supplmntary Fig. 15). Thrfor, Etv1 may affct a lat stag in olfactory D nuron iffrntiation. Lik ast-1, Etv1 appars not only rquir for D nuron iffrntiation but also sufficint, bcaus ctopic xprssion of Etv1 in olfactory bulb primary cll cultur incrass th numbr of clls xprssing th D markr TH (Fig. 4g). Etv1 is also abl to activat irctly th cis-rgulatory rgion of th mous TH locus in a htrologous contxt (Fig. 4h). This activation pns on th prsnc of two phylogntically consrv D motifs (Fig. 4h). Lik in C. lgans, phylogntically consrv D motifs can also b foun in th 59 upstram rgulatory rgion of all four othr mous opamin pathway gns (Supplmntary Fig. 16). nothr inicator for a consrv function of mous Etv1 an worm ST-1 is that mous Etv1 is abl to rscu th ast-1 mutant phnotyp whn xprss in transgnic worms (Fig. 2) min hat shock at L1 stag Tim rh300 G>R h1 >V ot417 M>I ETS omain h92 ast-1(gk463) c ::gfp ::gfp ::rfp cat-4::rfp bas-1::rfp tph-1::gfp WT gk463 WT gk463 WT gk463 WT gk463 WT gk463 WT gk463 DE 5HT clls PDE DE f NSM DF Othrs ast-1(rh300); ::gfp ast-1::yfp ::mchrry Mrg Bfor hat shock i ::gfp k 4 h aftr hat shock No array ast-1 (h1) Ex[ prom ::ast-1] Ex[ prom ::tv1] ::gfp s DE In conclusion, w hav scrib hr a surprisingly simpl rgulatory logic for D spcification. Our cis-rgulatory analysis in worms rvals that all opamin pathway gns ar co-rgulat through a similar cis-rgulatory motif an trans-acting factor, an this rgulatory logic applis to D nurons of istinct linag origin. Our analysis monstrats that th ETS factor ast-1 is a trminal slctor gn for D cll fat, akin to othr trminal slctor gns that control th trminal intity of othr nuron typs Trminal slctor gns ar transcription factors that irctly rgulat th nut-an-bolts iffrntiation gn battris that trmin th spcific proprtis of a 3 ays aftr hat shock Figur 2 ast-1 is rquir to inuc an maintain D nuron iffrntiation. a, Schmatic rprsntation of th ast-1 locus an mutant allls availabl for this gn. b, Rprsntativ xampl of loss of D fat markr in ast-1 mutants. S Supplmntary Figs 7 an 8 for othr xampls an Supplmntary Tabl 1 for quantification of ata. Scal bar, 10 mm. c, Summary of ast-1 null mutant phnotyp. 1, fat markr xprss; 2, fat markr not xprss. Owing to arly larval lthality, only th mbryonically gnrat D ha nurons, but not th postmbryonically gnrat PDE nurons, coul b scor for vlopmntal fcts in ast-1 null mutants. Markrs that wr xprss in both D an 5-HT nurons wr assay with an rfp rportr in a transgnic backgroun in which 5-HT nurons wr labll with gfp (Is[tph-1::gfp]) to allow scoring of loss of xprssion spcifically in th D nurons. S Supplmntary Fig. 7 for primary ata. WT, wil typ., Exprssion of an ast-1::yfp rportr gn 4 in D nurons. D nurons ar labll with ::mchrry. Scal bar, 5 mm., Rscu of th ast-1 mutant phnotyp. W us a hypomorphic alll, h1, in which xprssion is unaffct (Supplntary Tabl 1), to riv ast-1 or a mous homologu, Etv1, unr control of th D-spcific promotr an assay xprssion of ::gfp (otis199). Scal bar, 8 mm. f, Dvlopmntally stag ast-1(rh300) animals, containing th hat-shockinucibl ast-1 array otis198 an th D fat markr ::gfp (otis199), wr grown unr non-inucibl conitions to th first larval stag (rsulting in an absnc of ::gfp xprssion in 100% of animals); ast-1 was thn inuc by hat shock at th L1 stag. Of th 40 animals foun to turn on xprssion of ::gfp 4 h aftr hat shock, 17 lost xprssion aftr 3 ays an all lost xprssion aftr 5 ays. Data with a tmpratur-snsitiv alll of ast-1 corroborat sustain ast-1 activity (Supplmntary Fig. 10). Scal bar, 10 mm. nuron by bining to simpl cis-rgulatory motifs shar by mmbrs of th trminal gn battris, trm trminal slctor motifs (or, in th cas of th D nurons, th D motif) 15. s xmplifi by ST-1, trminal slctor gns ar continuously xprss throughout th lif of a nuron to nsur that th trminal iffrntiation stat is proprly maintain. Th rgulatory logic of D nuron spcification sms to b phylogntically consrv. Vrtbrat opamin pathway gns also contain D motifs that ar rquir for th activation by a transacting factor that is homologous to th C. lgans trans-acting factor. Loss of th trans-acting factor ithr in worms or in mic las to a loss 2009 Macmillan Publishrs Limit. ll rights rsrv of th D phnotyp. Both Etv1 an ast-1 ar continuously xprss throughout th postmitotic lif of D nurons, an our analysis in worms inicats that ths factors also maintain th trminal intity of D nurons. Th function of vrtbrat ETS protins in D spcification may hav bn istribut ovr svral iffrnt ETS omain transcription factors, as Etv1 is not xprss in othr D nuron populations in th brain an as it os not affct th gnration of ths othr typs of D nurons (ata not shown). Thos othr aras xprss a rlat ETS factor, Etv5, which may fulfil a rol similar to that of Etv1 in olfactory D nurons; in support of this notion, Etv5 can also transactivat th TH promotr in a htrologous assay systm (Supplmntary Fig. 17). Th logic of istributing an ancstral gn function, obsrv in an invrtbrat spcis, ovr svral vrtbrat 887

4 LETTERS NTURE Vol pril 2009 hat shock hat shock a b c ::gfp ::gfp ::gfp, rgf-1::s-r Ex[unc-119 prom ::ast-1] Ex[bas-1 ::ast-1] s s DE PDE ::gfp Figur 3 Ectopic ast-1 xprssion can inuc D cll fat. a, Rprsntativ control mbryo aftr th thrfol stag. ::gfp xprssion starts at lat thrfol stag an can b tct in th six mbryonically gnrat D nurons. b, c, Rprsntativ pictur of an mbryo hat shock 4 h aftr th two-cll stag an analys 10 h aftr th hat shock. ::gfp is ctopically xprss in many clls of th mbryo. Scal bar, 20 mm. In th prsnc of a pan-nuronal markr (rgf-1::rfp; paralogus of th ancstral invrtbrat orthologu has bn not for othr transcription factors as wll 16 an sms an important componnt of riving nuronal ivrsification procsss in mor complx brains. Tuj1/DPI TH g GFP&TH()/TH() DE s ST-1 an Etv1 both act as trminal slctor gns for D trminal iffrntiation, but thir prsnc is not strictly sufficint to activat D gns bcaus both ST-1 an Etv1 ar also xprss in nurons a Olfactory bulb primary cultur * Mous Etv1 / Mous Etv1 / b GFP GFPEtv1 Mous ctgcgacagtggatgcaattagat Rat ctgtgacagtggatgcaattagat Dog ctgcggc-ggggatgcaattagat *** * * * ************** 789 Mous TH promotr Figur 4 Mous Etv1 is ncssary for th olfactory bulb D nuron spcification a, b, Coronal sction TH immunostaining of a wil-typ (a) an Etv1 mutant (b) nwborn pup (P0) olfactory bulb. Scal bar, 150 mm. c, Quantification of TH-positiv clls in wil-typ an Etv1 mutants at P0. Etv1 mutants show a significant ruction in th numbr of TH-positiv clls alray at this stag (n 5 3, ***P ).,, Coronal sction Tuj1 immunostaining (grn) an DPI staining (blu) of a wil-typ () an Etv1 mutant () P0 glomrular layr to labl nurons an cll nucli, rspctivly. Scal bar, 40 mm. f, Quantification of DPI nucli in wil-typ an Etv1 mutants at P0. Glomrular layr cll nsity is similar btwn 888 h P0 PDE ::gfp c), th clls ctopically xprssing D fat can b intifi as nurons.,, Ectopic xprssion of ast-1 unr th control of th ctormal promotr unc-119 an th D/5HT-nuron-spcific promotr bas-1 las to ctopic xprssion of ::gfp in aitional nurons compar to wil-typ worms (r arrowhas). Similar ffcts wr obsrv in multipl lins ( lins for bas-1 rivr; 2/3 lins for unc-119 rivr). Scal bar, 100 mm. Olfactory bulb 327 D motif Mous gtgga--gaggatgcgc Rat gtgga--gaggatgcgc Human aggggtgggggatgta- ** * ***** 182 D motif Macmillan Publishrs Limit. ll rights rsrv c TH clls DPI nucli 10 5 f 120 Etv1 / Etv1 / * ** P<0.05 P<0.005 * *** *** NS * * Luminscnc wil-typ an Etv1 mutants (n 5 3, P ). NS, not significant. g, Ovrxprssion of Etv1 can inuc D iffrntiation. Dissociat P0 olfactory bulbs wr transfct with gfp an vctor control or gfp an Etv1 xprssion vctor, plat an cultur for 4 ays. *P, h, nalysis of th activation of TH promotr by Etv1 in COS clls. Th ott lin inicats th lvl of lucifras activation of th mpty lucifras vctor obsrv on Etv1 transfction. D motif nots th phylogntically consrv match to th VGGWRNV consnsus. In th top panl, astrisks not consrv nuclotis. n 5 3 inpnnt xprimnts for ach construct. ll rror bars rprsnt s..m.

5 NTURE Vol pril 2009 LETTERS othr than D nurons 4,17. Our ctopic xprssion xprimnts also show that ability of ST-1 to inuc ctopic D fat is rstrict to som cllular an tmporal contxts. Classic mastr rgulators, such as fly Eylss or mous MyoD (also known as Myo1), also show similar contxt-pnncis in thir mo of action 18,19. ST-1 an Etv1 function may b activly inhibit in clls rfractory to ST-1/ Etv1 activity. ltrnativly, ST-1/Etv1 function may rquir aitional, cll-typ-spcific factors for appropriat function in D nurons. Such combinatorial coing mchanisms ar a common thm in nuron typ spcification 20, an our intification of a consrv rol of ETS factors as a cntral componnt of such a co is th first important stp in coing th rgulatory logic of D nuron spcification. It will b intrsting to s whthr th aitional spcificity trminants of ETS factors ar also consrv from worms to vrtbrats. METHODS SUMMRY Transgnic an mutant C. lgans strains. Rportr gn constructs wr gnrat by subcloning into th ppd95.75 backbon vctor, which contains th gfp coing squnc an th unc untranslat rgion. Mutagnsis an ltions wr prform using th Quickchang II L sit-irct mutagnsis kit (Stratagn). Rportr constructs wr injct into otis181(is[at- 1::mChrry;ttx-3::mChrry]) to facilitat th intification of th D clls. DN was injct at 50 ng ml 21 using rol-6 as an injction markr. For vry construct, 30 or mor animals wr scor from at last 2 iffrnt transgnic lins. Ets mutant strains wr obtain from th knockout consortia in Oklahoma, Vancouvr an Tokyo. For th hat-shock xprimnts, a transgnic strain of th following gnotyp was gnrat: OH7546 [otis198(hsp16-2::ast-1;hsp16-2::nls-mchrry;ttx-3::s-r),vtis1(::gfp;rol6)]. Hat-shock inuction conitions, as wll as a list of transgnic an mutants strains, ar provi in th Mthos. nalysis of vrtbrat Etv1. Stanar histological protocols wr us to analys wil-typ an Etv1 mutant mous sampls. For ctopic Etv1 xprssion, olfactory bulbs wr issct from wil-typ mic at postnatal ay zro to on, issociat an lctroporat using th maxa Nuclofctor Systm an th mous nuron kit, following th manufacturr s protocol. For th TH promotr analysis, COS clls wr transfct with lipofctamin (Invitrogn) an lucifras activity was masur using th lucifras assay kit (Stratagn) an th b-galactosias nzym assay systm (Promga). S Mthos for full scription of histology, cll cultur an quantification mthos. Full Mthos an any associat rfrncs ar availabl in th onlin vrsion of th papr at Rciv 8 Fbruary; accpt 26 Fbruary Publish onlin 15 March Ivrsn, S. D. & Ivrsn, L. L. Dopamin: 50 yars in prspctiv. Trns Nurosci. 30, (2007). 2. bliovich,. & Hammon, R. Mibrain opamin nuron iffrntiation: factors an fats. Dv. Biol. 304, (2007). 3. Nass, R. & Blakly, R. D. Th Canorhabitis lgans opaminrgic systm: opportunitis for insights into opamin transport an nurognration. nnu. Rv. Pharmacol. Toxicol. 43, (2003). 4. Schmi, C., Schwarz, V. & Huttr, H. ST-1, a novl ETS-box transcription factor, controls axon guianc an pharynx vlopmnt in C. lgans. Dv. Biol. 293, (2006). 5. Doitsiou, M., Flams, N., L,. C., Boyanov,. & Hobrt, O. utomat scrning for mutants affcting opaminrgic-nuron spcification in C. lgans. Natur Mthos 5, (2008). 6. Voglis, G. & Tavrnarakis, N. synaptic DEG/ENaC ion channl miats larning in C. lgans by facilitating opamin signalling. EMBO J. 27, (2008). 7. Li, W., Fng, Z., Strnbrg, P. W. & u,. Z.. C. lgans strtch rcptor nuron rval by a mchanosnsitiv TRP channl homologu. Natur 440, (2006). 8. lln, Z. J. II, Waclaw, R. R., Colbrt, M. C. & Campbll, K. Molcular intity of olfactory bulb intrnurons: transcriptional cos of priglomrular nuron subtyps. J. Mol. Histol. 38, (2007). 9. Saino-Saito, S. t al. ER81 an CaMKIV intify anatomically an phnotypically fin substs of mous olfactory bulb intrnurons. J. Comp. Nurol. 502, (2007). 10. rbr, S., Lal, D. R., Lin, J. H., Frank, E. & Jssll, T. M. ETS gn Er81 controls th formation of functional connctions btwn group Ia snsory affrnts an motor nurons. Cll 101, (2000). 11. Flams, N. t al. Dlination of multipl subpallial prognitor omains by th combinatorial xprssion of transcriptional cos. J. Nurosci. 27, (2007). 12. Wnick,. S. & Hobrt, O. Gnomic cis-rgulatory architctur an trans-acting rgulators of a singl intrnuron-spcific gn battry in C. lgans. Dv. Cll 6, (2004). 13. Duggan,., Ma, C. & Chalfi, M. Rgulation of touch rcptor iffrntiation by th Canorhabitis lgans mc-3 an unc-86 gns. Dvlopmnt 125, (1998). 14. Etchbrgr, J. F. t al. Th molcular signatur an cis-rgulatory architctur of a C. lgans gustatory nuron. Gns Dv. 21, (2007). 15. Hobrt, O. Rgulatory logic of nuronal ivrsity: trminal slctor gns an slctor motifs. Proc. Natl ca. Sci. US 105, (2008). 16. Lichtnckrt, R. & Richrt, H. Insights into th urbilatrian brain: consrv gntic pattrning mchanisms in insct an vrtbrat brain vlopmnt. Hrity 94, (2005). 17. Brown, T.. & McKnight, S. L. Spcificitis of protinprotin an protindn intraction of GBP alpha an two nwly fin ts-rlat protins. Gns Dv. 6, (1992). 18. Halr, G., Callarts, P. & Ghring, W. J. Inuction of ctopic ys by targt xprssion of th ylss gn in Drosophila. Scinc 267, (1995). 19. Wintraub, H. t al. ctivation of muscl-spcific gns in pigmnt, nrv, fat, livr, an fibroblast cll lins by forc xprssion of MyoD. Proc. Natl ca. Sci. US 86, (1989). 20. Shirasaki, R. & Pfaff, S. L. Transcriptional cos an th control of nuronal intity. nnu. Rv. Nurosci. 25, (2002). Supplmntary Information is link to th onlin vrsion of th papr at cknowlgmnts W thank Q. Chn for injction assistanc, M. Doitsiou an J. Chn for proviing th ot417 alll, E. Savnr for initiating som of th promotr analysis,. Kruyr for tchnical hlp, th CGC for strains, th C. lgans Gn Knockout Consortia, l by S. Mitani at Tokyo Womn s Mical Univrsity School of Micin an by D. Morman at th Univrsity of British Columbia, for knockout allls, H. Huttr, N. Tavrnarakis an T. Jssll for strains an ragnts, svral mmbrs of Columbia Univrsity community for sharing thir quipmnt, Hobrt laboratory mmbrs, spcially M. Doitsiou, for iscussion, an. bliovich, R. J. Johnston an I. Grnwal for commnts on th manuscript. This work was fun by th National Instituts of Halth (R01NS ; R01NS ), th Howar Hughs Mical Institut an a EMBO long trm fllowship an Mari Curi Outgoing Intrnational fllowship to N.F. uthor Information Rprints an prmissions information is availabl at Corrsponnc an rqusts for matrials shoul b arss to N.F. (nf2171@columbia.u) or O.H. (or38@columbia.u) Macmillan Publishrs Limit. ll rights rsrv 889

6 oi: /natur07929 METHODS Strains an transgns. For th ETS family analysis th following mutants wr cross into vtis1(::gfp) strain to chck for a D phnotyp: tag-97(ok284); C502.4(tm440); C52B9.2(tm413); ts-4(ok165); C241.2(tm801); ts-5(tm866, tm1734); lin-1(1777) ast-1(h1, rh300, h92, gk463). F19F10.1(tm456); F19F10.5(tm436) wr cross into otis181. Forfurthr ast-1 analysis th following strains wr cross into ast-1(gk463or h92): nis118 (::gfp), zis13 (tph-1::gfp), oyis59 (osm-6::gfp), 43 [lin15(n765);ex(gcy-36::gfp], TQ886 [trp-4::yfp1pha- 1(1);pha-1(ts);him-5]. Th ::mchrry, bas-1 ::mchrry, cat-4 ::mchrry an asic-1::gfp rportrs wr injct into th ast-1 balanc strain ast-1(gk463) bli-2(768) unc-4(120)/min1[mis14 py-10(128)] II at 50 ng ml 21. ST-1 xprssion was analys injcting ast-1::yfp 4 at 50 ng ml 21 into otis181. In rgar to our rportr gn analysis, w not that w hav prviously rport that, in som cass, PCR proucts ar xprss in mor clls than th quivalnt clon prouct 21. Som of th rportr gn constructs that gav no xprssion in th D clls wr injct both as subclon plasmis an as PCR proucts (using vctor primrs). No iffrnc in xprssion was foun in any of thm xcpt for th singl-copy 31 bp EBS motif from (Supplmntary Fig. 2) that gav no xprssion injct as a clon prouct an PDE xprssion whn injct as PCR fragmnt. Hat-shock xprimnts. Strain OH7546 [otis198(hsp16-2::ast-1;hsp16-2::nlsmchrry;ttx-3::s-r),vtis1(::gfp;rol6)] was us for th hat-shock xprimnts. For th rscu xprimnt shown in Fig. 2f (postmbryonic hat shock), first larval stag worms wr hat shock at 37 uc thr tims for 30 min with 1 h incubation at 20 uc btwn ach hat shock to lt worms rcovr. ftr hat shocks, worms wr kpt at 15 uc an scor at th inicat tims. For th ctopic D fat inuction xprimnts (Fig. 3), two cll mbryos wr rlas from hrmaphroit mothrs by sctioning thm in half, mount in slis, incubat at 20 uc, hat shock at 37 uc for 20 min at iffrnt stags of vlopmnt an analys th following morning (approximatly 24 h aftr first clavag). hsp16-2 promotr activity was monitor by mchrry xprssion from hsp16-2::nls-mchrry construct prsnt on th sam array. Evn though r fluorscnc was first tct 2 h aftr th hat shock, immunotction of protin prouction from th sam promotr has bn rport as arly as 30 min aftr hat shock 22. Histology. P0 mic wr ansthtiz in ic an transcarially prfus with 4% paraformalhy (PF). P10 mic wr ansthtiz with an ovros of ktamin HCl (Ktast) an xylazin HCl (mtch) an transcarially prfus with 4% PF. Brains wr rmov, postfix for 2 h at 4 uc, cryoprotct in 30% sucros in PBS, mb in Tissu-Tk OCT compoun an stor frozn at 280 uc. 20-mm coronal sctions wr cut on a cryostat. Th following antibois wr us: shp anti-tyrosin hyroxylas (1/1,000, Pl-Frz), rabbit anti-er81 (1/32,000, provi by T. Jssll), rabbit anticalbinin (1/5,000, Swant), rabbit anti-calrtinin (1/2,000, Chmicon), rabbit anti-caspas 3 clav (1/200, Cll Signaling), mous anti-b-tubulin (Tuj1) (1/500 Covanc), rabbit anti-phospho-histon H3 (1/500 Upstat). Etv1 mutant analysis was prform using a confocal microscop (Lica TCS- SP5). For P0 tyrosin hyroxylas, P10 tyrosin hyroxylas, P10 calrtinin an P10 calbinin olfactory bulb quantification, ight iffrnt fils corrsponing to two iffrnt lvls wr scor in ach animal. For P0 clav caspas 3 an phosphohiston 3 quantification, two whol sctions of olfactory bulb wr scor for ach animal. For cll nsity quantification 4,6-iamiino-2-phnylinol (DPI) nucli wr scor in a fil of th glomrular layr for ach animal. Olfactory bulb volum was stimat by th Cavaliri mtho 23.Inbrif,thwhol xtnt of th olfactory bulb was cut in 20-mm coronal sctions; on vry twnty sctions was analys using a gri of 0.04 mm 2. Data wr statistically analys using NOV tsts. In situ hybriization was prform as scrib bfor 11. CD-1 mous mbryos wr analys using th following probs: Dlx2 (prob provi by J. Rubnstin); Ehf (imag clon an ); Elf3 (MTF387 from th Gray t al. library 24 ); Elf5 (imag clon ); Elk1 (imag clon ); Elk3 (imag clon ); Elk4 (imag clon ); Etv1 (prob provi by T. Jssll); Erg (imag clon an ); Ets1 (imag clon ); Ets2 (imag clon an ); Etv3 (imag clon ); Etv4 (antiboy provi by T. Jssll); Etv5 (imag clon an ); Etv6 (prob provi by S. H. Orkin); Fli1 (imag clon an ); ngn2 (also known as Nurog2; prob provi by F. Guillmot), Sfpi1 (imag clon an ); Spib (prob provi by M. C. Simon); Spi-C (also known as Spic; MTF416 from th Gray t al. library 24 ). Olfactory bulb primary culturs. Olfactory bulb an cortx wr issct sparatly from 1012 pups to obtain nough numbr of clls for th lctroporation. Clls wr issociat an qual amounts of olfactory bulb an cortx clls wr mix to obtain approximatly clls pr ml. Clls wr lctroporat using th maxa Nuclofctor systm an th mous nuron kit, following th manufacturr s protocol. In brif, for ach raction clls wr lctropotat with 0.7 mg of pmaxgfp, 2 mg of PCDN3.1, 0.7 mg of pmaxgfp or 2 mg of Etv1/PCDN3.1, rspctivly. Clls wr immiatly plat in 8-wll glass chambr slis an cultur for 4 ays as scrib prviously 25. Clls wr thn fix with 4% PF an immunostain against with GFP an TH antisra. Two inpnnt sts of xprimnts wr prform an in ach: two wlls wr scor for th GFP-xprssing clls an two for th GFP1Etv1-xprssing clls. In un-transfct culturs, th fraction of TH-positiv clls was 1/3,000 plat clls. For ach wll w scor th total numbr of TH-positiv clls an th numbr of co-labll TH/GFP-positiv clls (ranking from 100 to 200 TH clls an 20 to 50 TH/GFP clls pr wll). Th prcntag of TH transfct clls vrsus TH not transfct clls was calculat. Lucifras assays. COS clls wr plat in M6 plats an incubat ovrnight (12 h); at approximatly 70% conflunc clls wr transfct with lipofctamin (Invitrogn). Each wll was transfct with 2.7 mg of total DN (200 ng of b- galactosias, 500 ng of PGL3 lucifras, 2 mg of Etv1/PCDN3.1(2) or PCDN3.1(2)).Twnty-fourhoursaftrtransfction,lucifrasanb-galactosias activitywas masurusinglucifrasassay kit (Stratagn), b-galactosias nzym assay systm (Promga) an 20/20 n Luminomtr (Turnr BioSystms) an Biora Mol 680 Microplat rar. For ach xprimnt ach valu rprsnts th man lucifras activity in thr iffrnt wlls an ach construct was analys in thr inpnnt xprimnts. Statistical analysis was prform applying th t-tst. D motif mutagnsis was prform using th Quickchang II L sitirct mutagnsis kit (Stratagn). In both cass th cor GGT of th D motif was mutat to GT. 21. Etchbrgr, J. F. & Hobrt, O. Vctor-fr DN constructs improv transgn xprssion in C. lgans. Natur Mthos 5, 3 (2008). 22. Fukushig, T. & Kraus, M. Th myognic potncy of HLH-1 rvals wi-spra vlopmntal plasticity in arly C. lgans mbryos. Dvlopmnt 132, (2005). 23. Rosn, G. D. & Harry, J. D. Brain volum stimation from srial sction masurmnts: a comparison of mthoologis. J. Nurosci. Mthos 35, (1990). 24. Gray, P.. t al. Mous brain organization rval through irct gnom-scal TF xprssion analysis. Scinc 306, (2004). 25. Cobos, I., Borllo, U. & Rubnstin, J. L. Dlx transcription factors promot migration through rprssion of axon an nrit growth. Nuron 54, (2007) Macmillan Publishrs Limit. ll rights rsrv