LETTER. Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice

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1 oi:1.138/ntur1923 Trns-synpti Tnurin signlling in nuromusulr synps orgniztion n trgt hoi Timothy J. Mos 1, Wizh Hong 1, Vrhn S. Dni 1, Vinnzo Fvloro 1 & Liqun Luo 1 Synps ssmly rquirs trns-synpti signls twn th prn postsynps 1, ut our unrstning o th ssntil orgniztionl moluls involv in this pross rmins inomplt 2. Tnurin protins r onsrv, pirml growth tor (EGF)- rpt-ontining trnsmmrn protins with lrg xtrllulr omins 3.HrwshowthttwoDrosophil Tnurins, n Tn-, r rquir or nuromusulr synps orgniztion n trgt sltion. Tn- is prsynpti whrs is mostly postsynpti; nuronl Tn- n musl orm omplx in vivo. Pr- or postsynpti Tnurin prturtions us svr synps loss n impir mny ts o orgniztion trns-synptilly n ll utonomously. Ths inlu ts in tiv zon pposition, rls sits, mmrn n vsil orgniztion, n synpti trnsmission. Morovr, th prsynpti mirotuul n postsynpti sptrin ytoskltons r svrly isrupt, suggsting mhnism whry Tnurins orgniz th ytosklton, whih in turn ts othr spts o synps vlopmnt. Supporting this, physilly intrts with -Sptrin. Gnti nlyss o tnurin n nuroligin rvl tht thy hv irntil rols tht synrgiz to promot synps ssmly. Finlly, t lvt nognous lvls, rgults trgt sltion twn spii motor nurons n musls. Our stuy intiis th Tnurins s ky i-irtionl trns-synpti signl involv in gnrl synps orgniztion, n monstrts tht protins suh s ths n lso rgult trgt sltion. Vrtrt tnurins r nrih in th vloping rin 4,, loliz to synpss in ultur 6, n pttrn visul onntions 7. Both Drosophil Tnurins, n Tn-, untion in oltory synpti prtnr mthing 8 n wr urthr intii in nuromusulr juntion (NMJ) t srns 9,1, with lso ting motor xon guin 11. W xmin thir rols n th unrlying mhnisms involv in synps vlopmnt. Both n Tn- wr nrih t th lrvl NMJ (Fig. 1 n Supplmntry Fig. 1). Tn- ws tt t nuronl mmrns: this stining ws unttl yon kgroun in null mutnts (Supplmntry Fig. 1) n rly ttl tr nuronl RNA intrrn (RNAi; Supplmntry Fig. 1), initing tht Tn- is prominntly prsynpti. Prtil o-loliztion ws osrv twn Tn- n th pritiv zon mrkr Fsilin 2 (r. 12) s wll s th tiv zon mrkr Bruhpilot 13 (Fig. 1, ), suggsting tht Tn- is loliz to th juntion twn th pritiv zon n th tiv zon. ppr strongly postsynpti n surroun h outon (Fig. 1 n Supplmntry Fig. 1, ). Musl-spii tn-m RNAi limint th postsynpti stining, ut unovr wk prsynpti stining (Supplmntry Fig. 1) tht uiquitous tn-m RNAi limint (Supplmntry Fig. 1). Thus, th signl ws spii n, lthough prtly prsynpti, nrih postsynptilly. Consistntly, musl ololiz xtnsivly with Dlg (Fig. 1) n ompltly with -Sptrin (Fig. 1) n is thus proly oinint with ll postsynpti mmrns. Th loliztion o Tn- n suggst thir trns-synpti intrtion. To xmin this, w o-xprss My-tgg Tn- in nrvs using th Q systm 14 n hmgglutinin (HA)-tgg in musls using GAL4. Musl ws l to oimmunopripitt nrv Tn- rom lrvl synptosoms (Fig. 1), suggsting tht th Tnurins orm htrophili trns-synpti rptor pir t th NMJ. To trmin Tnurin untion t th NMJ, w xmin th null lll n lrv with nuron or musl RNAi o n/or tn-m. Following suh prturtions, outon numr n siz wr ltr: th quntity ws ru y % (Fig. 2, g n Supplmntry Fig. 2) Tn- IP: HA () Tn- My: N + + HA: M + + Tn- Tn- Fs2 Fs2 HRP Tn- Tn- Brp Dlg α-sptrin IB: My (Tn-) IB: HA () IB: Brp (Input) Tn- HRP Brp HRP Dlg HRP α-sptrin HRP Figur 1 Tnurins r nrih t n intrt ross Drosophil nuromusulr synpss., Rprsnttiv singl onol stions o synpti outons stin with ntiois ginst Tn- (r) or (grn), horsrish proxis (HRP) to mrk th nuronl mmrn (lu), n synpti mrkr s init., Tn- is ssoit with prsynpti mmrns n lrgly with th surrouning postsynps ().,, Tn shows limit o-loliztion with th pritiv zon mrkr Fsilin 2 (), n Bruhpilot (Brp), n tiv zon mrkr ().,, o-lolizs with, n xtns yon, Dlg () n ompltly o-lolizs with musl -Sptrin ()., Immunolots (IB) o lrvl synptosoms xprssing nuronl Flg My-tgg Tn- (N) n musl Flg HA-tgg (M) n immunopripitt (IP) using ntiois to HA. Tn- is tt in th pullown, initing tht nrv Tn- n musl intrt ross th NMJ. This is not sn in ontrol lns. Owing to low xprssion, nithr trnsgn prout is ttl in input lysts, whih r nrih in Brp. Sl r, mm. 1 Dprtmnt o Biology, Howr Hughs Mil Institut, Stnor Univrsity, Stnor, Cliorni 943, USA. Ths uthors ontriut qully to this work. 12 APRIL 212 VOL 484 NATURE Mmilln Pulishrs Limit. All rights rsrv

2 RESEARCH LETTER g Bouton numr Brp DGluRIII HRP h l o tn-m M-IR m p + Tn- N j n q k r 1 7 s t u 1 mv 1 mv 1 s 1 ms i Prntg unppos AZs Prntg o T-rs Prntg o ontrol tn-m+ M-IR tn-m+ N-IR QC + Tn- M + Tn- N Norml Misshpn Apposit ontrtil tissu Dth Doul EPSP mp. mepsp mp. mepsp rq. Figur 2 Tnurins t th strutur n untion o th nuromusulr synps., Rprsnttiv NMJs stin with ntiois to HRP. Muslspii tn-m RNAi (M-IR) n lots o rs outon numr. Nuronl (Tn- N) ut not musl (Tn- M) rstortion o xprssion rsus this phnotyp. Ths ts rsml mutnts n r nhn in oul mutnts. g, Quntiition o outon numr. h j, Rprsnttiv NMJs stin with ntiois to Brp (grn), th glutmt rptor suunit DGluRIII (r) n HRP (lu). In ontrol lrv (h), Brp n DGluRIII punt proprly ppos. tnurin prturtions (i, j) isrupt this tiv zon (yllow rrowh) n glutmt rptor pposition (whit rrowh). k, Quntiition o unppos tiv zon/glutmt rptor pirs. For h quntiition, n $ 8 lrv, 16 NMJs. l q, Trnsmission ltron mirosopy o tiv zon T-rs (strisks) in ontrol lrv (l) n n th inin o lrg outons mrkly inrs (Supplmntry Fig. 2k). Both hngs init impir synpti morphognsis. Th rution in outon numr ws proly umultiv through vlopmnt, s it ws visil in irst instr mutnts n prsist (Supplmntry Fig. 2k). In th mutnt, outon morphognsis ws rsu y rstoring Tn- xprssion in nurons, ut not musls (Fig. 2, g n Supplmntry Fig. 2). Nuronl ovrxprssion oul not sustitut or th lk o Tn-, rvling thir nonquivln (Supplmntry Fig. 2, l). Nuronl knokown o Tn- or rsult in wr synpti outons (Supplmntry Fig. 2 h, l), initing tht oth hv prsynpti untion, lthough prsynpti Tn- hs mor prominnt rol (Supplmntry Fig. 2l). Morovr, knoking own postsynpti in th mutnt i not nhn th phnotyp (Fig. 2g). Thus, prsynpti Tn- (n, to lssr xtnt, ) n postsynpti r rquir or synps vlopmnt. tnurin prturtion lso us ts in th pposition twn prsynpti tiv zons (rls sits) n postsynpti glutmt rptor lustrs (Fig. 2h n Supplmntry Fig. 3): up to % o th tiv zons/rptor lustrs lk thir prtnr ompr to 1.8% in ontrols (Fig. 2h k). Unr ltron mirosopy, tiv zons mutnts (m q) showing oul T-rs (m), th T-rs (n), misshpn T-rs (o), mmrn ruling (p, wv rrows) n T-rs ing ontrtil tissu (q). Som imgs show multipl ts. r, Distriution o T-r ts s prntg o th totl T-rs. N-IR, nuronl RNAi. For h gnotyp, n $ 3 lrv, 4 outons. s, t, Rprsnttiv vok EPSP (s) n mepsp (t) trs rom ontrol n mutnt gnotyps. u, Quntiition o mn EPSP mplitu (lk), mepsp mplitu (r), mepsp rquny (lu) n quntl ontnt (QC, whit) xprss s prntg o th ontrol vrg. For ll gnotyps, n $ 7 lrv, 8 musls. Error rs rprsnt s..m. Sl rs, 1 mm ( ), mm (h j), 1 nm (l q). P,.1, P,.1, P,.,, not signiint. Sttistil omprisons r with ontrol unlss not. r mrk y ltron-ns mmrns n singl prsynpti spiliztions ll T-rs (Fig. 2l), whih nl synps ssmly, vsil rls n C 21 -hnnl lustring 16. Tnurin isruption us ts (Fig. 2m r n Supplmntry Fig. 3) in T-r ultrstrutur (Fig. 2m o), mmrn orgniztion, n pposition to ontrtil tissu (Fig. 2p, q). Tnurin prturtion lso impir postsynpti nsitis whil inrsing mmrn ruling (Supplmntry Tl 1), urthr initing orgniztionl iiny. Ths phnotyps rsml mutnts with hsion n T-r iognsis ts 17,18, suggsting rol or Tnurins in synpti hsion n stility. Synpti vsil popultions similrly rquir Tnurins or lustring t th outon primtr n propr nsity (Supplmntry Fig. 4). As ths ts r not synonymous with tiv zon isruption 19, Tnurins r lso rquir or synpti vsil orgniztion. Synpss lking tnurin wr lso untionlly impir. Th mn mplitu o vok xittory postsynpti potntils (EPSPs) in lrv ws rs y 28% in th mutnt (Fig. 2s, u). Spontnous minitur EPSPs show 2% rs in mplitu, 46% rs in rquny (Fig. 2t, u), n n ltr mplitu istriution ompr with ontrol (Supplmntry Fig. ). Ths ts rsult in 2% rution in quntl ontnt (Fig. 2u), whih oul prtly u to N AT U R E V O L A P R I L Mmilln Pulishrs Limit. All rights rsrv

3 RESEARCH wr outons n rls sits. Howvr, rls proility my lso ru, s suggst y n inrs pir puls rtio in mutnts (Supplmntry Fig., ). Th y kintis o rsponss wr str in mutnts, suggsting itionl postsynpti ts on glutmt rptors n/or intrinsi mmrn proprtis (Supplmntry Fig., ). Furthr, FM1-43 y loing rvl mrkly tiv vsil yling in mutnts (Supplmntry Fig., h). Consistnt with physiologil impirmnt, tnurin-prtur lrv xhiit prooun loomotor ts (Supplmntry Fig. i). In summry, Tnurins r rquir or multipl spts o NMJ orgniztion n untion. As potntil mhnism or synpti isorgniztion ollowing tnurin prturtion, w xmin th pr- n postsynpti ytoskltons. In th prsynpti trminl, orgniz mirotuuls ontin Futsh ( mirotuul-ining protin)-positiv loops, whrs isorgniz mirotuuls possss puntt, ununl Futsh 2. Eh lssiition normlly rprsnt,1% (otn istl) o outons (Fig. 3, n Supplmntry Fig. 6). Upon tnurin prturtion, mny mor outons h ununl Futsh (Fig. 3, n Supplmntry Fig. 6) whrs thos with loop mirotuuls wr rs y 62 9% (Fig. 3). Thror, propr mirotuul orgniztion rquirs pr- n postsynpti Tnurins. In ontrst to mil tiv zon/glutmt rptor pposition ts, most outons isply mirotuul orgniztionl ts. tnurin prturtion lso svrly isrupt th postsynpti sptrin ytosklton, with whih ololiz (Fig. 1). Postsynpti -Sptrin normlly surrouns th outon (Fig. 3). Prturing nuronl or musl Tnurins mrkly ru postsynpti -Sptrin without ting Dlg (Fig. 3 h n Supplmntry Fig. 7). Postsynpti -Sptrin 21, Auin 22 n Wsp wr similrly t (Supplmntry Fig. 8). In musl, -Sptrin is oinint with n ssntil or th intgrity o th mmrnous susynpti rtiulum (SSR) 21,23. Consistnt with this, tnurin isruption ru SSR with up to 7% (Supplmntry Fig. 9 g) n inrs th rquny o ghost outons, whih r ilurs o postsynpti mmrn orgniztion 23 (Supplmntry Fig. 9 ). Thus, Tnurins r involv in th orgniztion o th pr- n postsynpti ytoskltons n postsynpti mmrns. Furthr, nognous -Sptrin o-immunopripitt with musl-xprss, Flg-tgg (Fig. 3i), suggsting tht physilly links th synpti mmrn to th ytosklton. Bus th most svr ts ollowing tnurin prturtion wr ytoskltl, w propos tht Tnurins primrily orgniz th prsynpti mirotuul n postsynpti sptrin-s ytoskltons (Fig. 3j), whih thn orgniz itionl synpti spts 2,21.Howvr, suh solitry rol nnot ully xplin th osrv phnotyps. Th rution in outon numr ssoit with ytoskltl isruption is milr thn tht ollowing tnurin isruption 2,21,24. Also, lthough tiv zon ynmis r t y ytoskltl prturtion 21,ts in pposition r not 21,2. Morovr, th T-r struturl ts mor losly rsml synps hsion n tiv zon ormtion ts 17,18. Thus, Tnurins my rgult rls sit orgniztion n synpti hsion inpnnt o th ytosklton (Fig. 3j). Our t lso init tht Tnurins t i-irtionlly ross th synpti lt. Tn- ts prominntly in nurons, s vin y loliztion, phnotyps us y nuronl (ut not musl) knokown, n mutnt rsu y nuronl (ut not musl) xprssion (Figs 2 n 3 n Supplmntry Figs 2 4, 6, 7 n 9). Yt, in ition to th prsynpti phnotyps, mny othrs wr postsynpti, inluing ru musl sptrin, SSR, n mmrn pposition (Fig. 3 n Supplmntry Figs 7 9). Similrly, lthough is prsnt oth pr- n postsynptilly, musl knokown rsult in prsynpti ts, inluing mirotuul n vsil isorgniztion, ru tiv zon pposition, n T-r ts (Figs 2 n 3 n Supplmntry Figs 3, 4, 6 n 7). Thus, Tnurins untion in i-irtionl trns-synpti signlling to orgniz nuromusulr synpss. This my involv ownstrm pthwys or simply stlish n orgniztionl rmwork y th rptors thmslvs. Morovr, s th rsults o singl isruptions o nuronl or musl tn-m wr similrly svr n not nhn y omintion (Figs 2g n 3, h n Supplmntry Fig. 2k), oth Tn- n proly Futsh HRP α-sptrin Dlg HRP i (Flg) 2 kd 3 kd Input IP: Flg + + IB: α-sptrin IB: Flg Figur 3 Tnurin prturtion rsults in mrk ytoskltl isorgniztion., Rprsnttiv NMJs stin with ntiois to Futsh (grn) n HRP (mgnt). Arrowhs init loop orgniztion. Arrows init ununl Futsh., Quntiition o th prntg o totl outons with loop or ununl mirotuuls. g, Rprsnttiv NMJs stin with ntiois to -Sptrin (grn), Dlg (r) n HRP (lu). Following tnurin prturtion, -Sptrin stining is lrgly lost. Axonl -Sptrin is unt y musl tnurin RNAi (). h, Quntiition o -Sptrin (grn) g j Prsynpti motor nuron MT loop Loops (%) h α-sptrin (A.U.) Vsil orgniztion Bouton numr 1 1 Ativ zon Nlg1 Nrx Tn- GluRII + + Tn- M + Tn- N Sptrin ytosklton Mmrn Orgniztion Postsynpti musl n Dlg (r) luorsn. A.U., ritrry units. For ll gnotyps, n $ 6 lrv, 12 NMJs. i, Immunolots (IB) showing tht -Sptrin is tt in th Flg immunopripitts (IP) o lrv xprssing musl Flg HA-tgg ut not in ontrol lrv. Owing to low xprssion, Flg HA- is only ttl tr nrihmnt y immunopripittion. j, Mol showing th rols o Tnurins, Nurxin n Nuroligin t th NMJ. Arrow siz rprsnts th rltiv ontriution o h pthwy to th llulr pross s inrr rom mutnt phnotypi svrity. Sl rs, mm. P,.1,, not signiint Ununl (%) Dlg (A.U.) 12 APRIL 212 VOL 484 NATURE Mmilln Pulishrs Limit. All rights rsrv

4 RESEARCH LETTER untion in th sm pthwy. Our ining tht Tn- n o-immunopripitt rom irnt lls in vitro 8 n ross th NMJ in vivo (Fig. 1) urthr suggsts signl vi trns-synpti omplx. Tnurin untion, howvr, my not solly trnssynpti. In som ss (vsil nsity, SSR with), ll-utonomous knokown rsult in strongr phnotyps thn knoking own in synpti prtnrs (Supplmntry Figs 3, 4, 9 n Supplmntry Tl 1). This suggsts itionl ll-utonomous rols in ition to trns-synpti Tnurin signlling. Signlling involving th trnsmmrn protins Nurxin n Nuroligin lso mits synps vlopmnt 26.InDrosophil, Nurxin (nrx) n Nuroligin 1() muttions us phnotyps similr to tnurin prturtion: rutions in outon numr, tiv zon orgniztion, trnsmission, n SSR with 27,28. n nrx muttions o not nhn h othr, suggsting tht thy untion in th sm pthwy 28. Consistntly 27,28, w oun tht nrx n mutnts xhiit lrgly similr phnotyps (t not shown). To invstigt th rltionship twn th tnurin gns n nrx n, w ous on th null mutnt. Both Nlg1 tgg with nhn grn luorsnt protin (Nlg1 GFP) n nognous oupi similr postsynpti sp (Supplmntry Fig. 1). tnurin n loss-o-untion muttions lso isply similr outon numr rutions (Fig. 2, g), vsil isorgniztion (Supplmntry Fig. 4), n ghost outon rqunis (Supplmntry Fig. 9). Othr phnotyps show notl irns in svrity. In mutnts, thr ws 29% ilur o tiv zon/glutmt rptor pposition (Fig. 2k n Supplmntry Fig. 1), ompr to % or th strongst tnurin prturtion. Th ytosklton o mutnts, howvr, ws only milly impir ompr to tht sn with tnurin prturtions (Fig. 3, h n Supplmntry Figs 6 n 7). To xmin urthr th intrply o tnurin n, w nlys oul mutnts. Both singl mutnts wr vil, spit thir synpti ts. Doul mutnts, howvr, wr lrvl lthl. W otin rr sprs, whih show 72% rution in outons, ompr to % rs in singl mutnts (Fig. 2). Ativ zon pposition in oul mutnts ws nhn synrgistilly ovr ithr singl mutnt (Fig. 2k n Supplmntry Fig. 1). Cytoskltl ts in th oul mutnt rsml th mutnt (Fig. 3 n Supplmntry Figs 6 n 7). Ths t suggst tht tnurin gns n nrx n t in prtilly ovrlpping pthwys, ooprting to orgniz synpss proprly, with Tnurins ontriuting mor to ytoskltl orgniztion n Nurxin n Nuroligin to tiv zon pposition (Fig. 3j). In th ompnying mnusript 8, w show tht lthough th sl Tnurins r roly xprss in th Drosophil ntnnl lo, lvt xprssion in slt glomruli mits oltory nuron prtnr mthing. At th NMJ, this sl lvl mits synps orgniztion. Anlogous to th ntnnl lo, w oun lvt tn-m xprssion t musls 3 n 8 using th tn-m-gal4 nhnr trp (Fig. 4). W onirm this or nognous tn-m, n trmin tht it ws ontriut y lvt xprssion in oth nrvs n musls (Fig.4 g).in,tn-m-gal4 ws highly xprss in slt motor nurons, inluing MN3-I, whih innrvts musl 3 (r. 29; Supplmntry Fig. 11). This lvt lrvl xprssion lso vri long th ntrior postrior xis (Supplmntry Fig. 12), n ws spii or, s Tn- xprssion i not ir within or twn sgmnts (t not shown). To tst whthr lvt xprssion in musl 3 n MN3-I ts nuromusulr onntivity, w xprss tn-m RNAi using tn-m-gal4. Wil-typ musl 3 ws lmost lwys innrvt (Fig. 4h). Howvr, tr tn-m knokown, musl 3 innrvtion il in 11% o hmisgmnts (Fig. 4i, j). This rquir on oth sis o th synps, s th trgting phnotyp prsist ollowing nuronl or musl RNAi supprssion using tissu-spii GAL8 trnsgns (Fig. 4j). RNAi i not show this phnotyp (Fig. 4j), onsistnt with homophili trgt sltion vi. Th phnotyp GFP Phlloiin Dlg HRP Dlg Phlloiin Dlg HRP tn-m-gal4>mcd8gfp h k m7 m4 m3 m6 Synptotgmin 1 Musl Nrv (tn-m N-IR) () i m7 m4 Musl 3 Musl 4 ws spii to musl 3, s innrvtion onto th immitly proximl or istl musl ws unhng (Fig. 4j). Th low pntrn is proly u to runnt trgt sltion mhnisms 3. Whr innrvtion i our, th trminl isply similrly svr phnotyps to othr NMJs (not shown). Thus, in ition to gnrlly miting synpti orgniztion, lso ontriuts to orrt trgt sltion t spii NMJ. To trmin whthr ovrxprssion oul ltr onntivity, w xprss in musl 6 (ut not th jnt musl 7), n th motor nurons innrvting oth musls using H94-GAL4. Normlly, 6% o th outons t musls 6/7 r prsnt on musl 6 with 4% on musl 7 (Fig. 4k, m). ovrxprssion us shit whry 81% o outons synps onto musl 6 n only 19% onto musl 7 (Fig. 4l, m). This shit lso rquir oth nuronl n musl, s nuronl or musl GAL8 rogt it (Fig. 4m). Th t ws spii us Tn- ovrxprssion i not ltr this synpti ln (Fig. 4m), nor ws it sonry to ltr outon m3 m6 H94-GAL4 >tn-m j Prntg trgting rrors tn-m-gal4>tn-m IR l m 1 Prntg o totl outons g Signl (A.U.) Signl (A.U.) + tn-m IR + lv-g8 + Mh-G tn-m MN 4 Syt1 MN 3 Musl 4 Musl 3 + IR + tn-m IR + lv-g8 + Mh-G8 + IR + tn-m, lv-g8 + tn-m, Mh-G8 Musl 3 Musl 4 Musl 2 + tn-m IR + lv-g8 + Mh-G8 Figur 4 High-lvl xprssion rgults musl trgt sltion., Rprsnttiv imgs o hmisgmnt A3 stin with ntiois to Dlg (lu), phlloiin (r), n xprssing GFP vi tn-m-gal4 (grn). High-lvl xprssion is osrv in musls 3 n 8 n slly in ll musls.,, Musl 3 () n 4() NMJs show irntil (r) ut similr Synptotgmin 1 (Syt1; grn) xprssion (rom tn-m musl knokown niml)., Quntiition o prsynpti (r) n Syt1 (grn) luorsn t musl 3 n 4 NMJs. MN, motor nuron.,,nmjstmusls3()n4 () show irntil (r) ut similr Syt1 (grn) xprssion in musls (rom tn-m nrv knokown). g, Quntiition o postsynpti (r) n Syt1 (grn) luorsn t musl 3 n 4 NMJs. h, i, Rprsnttiv imgs stin with phlloiin (lu) n ntiois to HRP (grn) n Dlg (r) to visuliz motor nurons n musls in ontrol (h)or tn-m- GAL4.tn-m RNAi lrv (i). m, musl. j, Quntiition o th hmisgmnt prntg with il musl 3 (r), 4 (lk) or 2 (lu) innrvtion. IR, intrring RNA. k, l, Rprsnttiv imgs o th musl 6/7 NMJ lll with ntiois to Dlg (grn) n HRP (mgnt). Th hrtristi wil-typ rrngmnt o outons (k) is shit towrs musl 6 whn is ovrxprss in tht musl n th innrvting motor nurons (l). m, Quntiition o th totl outon prntg on musls 6 (lu) n 7 (r). All gnotyps ontin H94-GAL4, itionl trnsgns r init (or tils, s Mthos). Th -mit shit is olish y nuronl or musl GAL8 trnsgns. Sl rs, 1 mm(), mm( i), 1 mm(k, l). In ll ss, n $ 12 lrv. P,.1, P,.1,, not signiint. + + IR m6 m7 2 4 N AT U R E V O L A P R I L Mmilln Pulishrs Limit. All rights rsrv

5 RESEARCH numr, whih ws unhng (t not shown). Thror, lvt on oth sis o th NMJ n is trgt hoi. This, omin with vin tht n mit homophili intrtion in vitro 8, supports trns-synpti homophili ttrtion mol t th NMJ s in th oltory systm. W intii two-tir mhnism or Tnurin untion in synps vlopmnt t th Drosophil NMJ. At th sl lvl, Tnurins r xprss t ll synpss n ngg in htro- n homophili i-irtionl trns-synpti signlling to orgniz synpss proprly (Fig. 3j). Supporting this, Tnurins n mit homo- n htrophili intrtions in vitro 8 n htrophili intrtions in vivo (Fig. 1). At th synps, Tnurins orgniz th ytosklton, intrt with -Sptrin, n nl propr hsion n rls sit ormtion. Furthr, lvt xprssion rgults trgt sltion in spii motor nurons n musls vi homophili mthing n untions with itionl moluls 3 to mit pris nuromusulr onntivity. Tnurin-mit trgt sltion t th NMJ is nlogous to its rol in oltory synpti prtnr mthing 8. As Tnurins r xprss roly throughout th ntnnl lo, it rmins n ttrtiv possiility tht thy lso rgult synps orgniztion in th ntrl nrvous systm. METHODS SUMMARY Dtils o Drosophil stoks, immunostining, ltron mirosopy, untionl ssys, onstrution o pitop-tgg Tnurin onstruts, immunopripittion, imging n sttistil nlysis n oun in Mthos. Full Mthos n ny ssoit rrns r vill in th onlin vrsion o th ppr t Riv 14 Jun 211; pt 7 Frury 212. Pulish onlin 18 Mrh Willims, M. E., Wit, J. & Ghosh, A. Molulr mhnisms o synpti spiiity in vloping nurl iruits. Nuron 68, 9 18 (21). 2. Gigtzoglou, N., Ly, C. V. & Blln, H. J. Cll hsion, th kon o th synps: vrtrt n invrtrt prsptivs. Col Spring Hr. Prspt. Biol. 1, 379 (29). 3. Young, T. R. & Lmy, C. A. Tnurins: importnt rgultors o nurl iruitry. Int. J. Biohm. Cll Biol. 41, (29). 4. Knzlmnn, D., Chiqut-Ehrismnn, R., Lhmn, N. T. & Tukr, R. P. Tnurin- 1 is xprss in intronnt rgions o th vloping rin n is pross in vivo. BMC Dv. Biol. 8, 3 (28).. Li, H., Bishop, K. M. & O Lry, D. D. Potntil trgt gns o EMX2 inlu Oz/ Tn-M n othr gn milis with implitions or ortil pttrning. Mol. Cll. Nurosi. 33, (26). 6. Silv, J. P. t l. Ltrophilin 1 n its nognous lign Lsso/tnurin-2 orm high-inity trnssynpti rptor pir with signling pilitis. Pro. Ntl A. Si. USA 18, (211). 7. Lmy, C. A. t l. Tn_m3 rgults y-spii pttrning in th mmmlin visul pthwy n is rquir or inoulr vision. PLoS Biol., 241 (27) CrossR. 8. Hong, W., Mos, T. J. & Luo, L. Tnurins instrut synpti prtnr mthing in n oltory mp. Ntur (this issu). 9. Lil, F. L. t l. Gnom-wi P-lmnt srn or Drosophil synptognsis mutnts. J. Nuroiol. 66, (26). 1. Kurusu, M. t l. A srn o ll-sur moluls intiis luin-rih rpt protinssky mitors osynptitrgt sltion. Nuron 9, (28). 11. Zhng, L. t l. Drosophil n Filmin t motor nuron growth on guin. PLoS ONE 6, 2296 (211). 12. Son, M. t l. Synpti vlopmnt is ontroll in th pritiv zons o Drosophil synpss. Dvlopmnt 127, (2). 13. Wgh, D. A. t l. Bruhpilot, protin with homology to ELKS/CAST, is rquir or struturl intgrity n untion o synpti tiv zons in Drosophil. Nuron 49, (26). 14. Pottr, C. J., Tsi, B., Russlr, E. V., Ling, L. & Luo, L. Th Q systm: rprssil inry systm or trnsgn xprssion, ling tring, n mosi nlysis. Cll 141, (21).. Mrrus, S. B., Portmn, S. L., Alln, M. J., Mot, K. G. & DiAntonio, A. Dirntil loliztion o glutmt rptor suunits t th Drosophil nuromusulr juntion. J. Nurosi. 24, (24). 16. Wihmnn, C. & Sigrist, S. J. Th tiv zon T-r plstiity moul? J. Nurognt. 24, (21). 17. Arl, H. t l. wishul thinking nos BMP typ II rptor tht rgults synpti growth in Drosophil. Nuron 33, 4 8 (22). 18. Owl, D. t l. A Sy-1 homologu rgults pr- n postsynpti mturtion in Drosophil. J. Cll Biol. 188, 6 79 (21). 19. Kittl, R. J. t l. Bruhpilot promots tiv zon ssmly, C 21 hnnl lustring, n vsil rls. Sin 312, 1 4 (26). 2. Roos, J., Humml, T., Ng, N., Klmt, C. & Dvis, G. W. Drosophil Futsh rgults synpti mirotuul orgniztion n is nssry or synpti growth. Nuron 26, (2). 21. Pilg, J., Fttr, R. D. & Dvis, G. W. A postsynpti sptrin sol ins tiv zon siz, sping, n iy t th Drosophil nuromusulr juntion. J. Cll Biol. 17, (26). 22. Pilg, J., Bult, V., Zuhro, J. B., Fttr, R. D. & Dvis, G. W. Hts/Auin ontrols synpti lortion n limintion. Nuron 69, (211). 23. Mos, T. J. & Shwrz, T. L. Th nulr import o Frizzl2-C y Importins-11 n 2 promots postsynpti vlopmnt. Ntur Nurosi. 13, (21). 24. Fthrston, D. E., Dvis, W. S., Duruil, R. R. & Broi, K. Drosophil - n -sptrin muttions isrupt prsynpti nurotrnsmittr rls. J. Nurosi. 21, (21). 2. Mih, C., Pur, H. U., H, X. & Shwrz, T. L. Prsynpti lol signling y nonil winglss pthwy rgults vlopmnt o th Drosophil nuromusulr juntion. J. Nurosi. 28, (28). 26. Crig, A. M. & Kng, Y. Nurxin nuroligin signling in synps vlopmnt. Curr. Opin. Nuroiol. 17, 43 2 (27). 27. Li, J., Ashly, J., Bunik, V. & Bht, M. A. Cruilrol odrosophil Nurxinin propr tiv zon pposition to postsynpti nsitis, synpti growth, n synpti trnsmission. Nuron, (27). 28. Bnovi, D. t l. Drosophil Nuroligin 1 promots growth n postsynpti irntition t glutmtrgi nuromusulr juntions. Nuron 66, (21). 29. Lngr, M., Roy, S., Prokop, A., VijyRghvn, K. & Bt, M. vn-skipp trmins th orsl growth o motor xons in Drosophil. Nuron 22, 43 2 (1999). 3. Sns, J. R. & Ymgt, M. Mny pths to synpti spiiity. Annu. Rv. Cll Dv. Biol. 2, (29). Supplmntry Inormtion is link to th onlin vrsion o th ppr t Aknowlgmnts W thnk H. Arl, V. Bunik, A. DiAntonio, R. Duruil, D. Fthrston, N. Rist, T. Shwrz, S. Stowrs, D. Vn Vtor, R. Wis, th Bloomington Stok Cntr n th Dvlopmntl Stuis Hyriom Bnk or ly stoks, ntiois n rgnts; J. Prrino n D. Luginuhl or thnil ssistn; K. Shn, K. Zinn, D. Bnovi, D. Brns, Y. Chou, C. A. Frnk, X. Go, S. Hippnmyr, K. Miymihi, K. Sillr, B. Tsi, X. Yu n S. Zosimus or ritiqus. Support y Ntionl Instituts o Hlth (NIH) grnt (R1 DC-982 to L.L.), n Epilpsy, Nontology n Dvlopmntl Biology Trining Grnts (NIH T n HD7249 to T.J.M.). L.L. is n invstigtor o th Howr Hughs Mil Institut. Author Contriutions T.J.M. sign n prorm ll xprimnts (prt rom ltrophysiology). W.H. hrtriz n provi nw rgnts, n ssist in som xprimnts. V.S.D. n T.J.M. sign n V.S.D. prorm ltrophysiology xprimnts with ssistn rom T.J.M. V.F. provi nw rgnts. L.L. suprvis th projt. T.J.M. wrot th mnusript with k rom ll uthors. Author Inormtion Rprints n prmissions inormtion is vill t Th uthors lr no ompting innil intrsts. Rrs r wlom to ommnt on th onlin vrsion o this rtil t Corrsponn n rqusts or mtrils shoul rss to T.J.M. (tmos@stnor.u). 1 2 A P R I L VO L N AT U R E Mmilln Pulishrs Limit. All rights rsrv

6 RESEARCH LETTER METHODS Drosophil stoks. All Drosophil strins n ontrols wr ris t 29 uc to mximiz GAL4 xprssion. All mutnts n trnsgns wr mintin ovr GFP lnr hromosoms to nl lrvl sltion. Mh-GAL4 or M2- GAL4 (r. 31) ws us to riv xprssion in ll somti musls. Nrv2-GAL4 (r. 32) n lv-gal4 (r. 33) wr us to riv xprssion in ll nurons. H94- GAL4 ws us to riv xprssion in musls 6, 13 n 4 n thir orrsponing motor nurons 34. ughtrlss-gal4 ws us to riv xprssion uiquitously 3. Synj-QF 36 ws us to riv xprssion in ll nrvs. NP668-GAL4 (tn-m-gal4) ws us to riv xprssion in th pttrn o nognous xprssion 8. Th D(X) ltion ws us s null mutnt 8. For mutnts, th I96 n x2.3 llls wr us in trns 28 n oul mutnt lrv with n muttions wr otin using optimiz rring onitions 37. Bus o th rly lthlity o th tn-m mutnt 11, n to ssss inpnntly, tissuspii RNAi ws us to xmin tnurin prturtion using th ollowing RNAi trnsgni strins: or tn-m, UAS-tn-m RNAi-V1173 n or (r. 8), UAS- RNAi-V Th ollowing trnsgni strins wr us: UAS-Dr2 (r. 38), UAS-Fs2 (r. 34), UAS-mCD8GFP (r. 39), UAS-Nlg1-GFP (r. 28), UAS-Tn- (r. 8), P{GS}9267 or tn-m ovrxprssion 8. In ll ss, th iy o RNAi trnsgns, ovrxprssion trnsgns n th ltion mutnt wr ssss n vrii y th ltrtion o ntioy stining (loss, rution or inrs) using tissu-spii GAL4 rivrs. For ll ss, N-IR inits nuronl RNAi, M-IR inits musl RNAi, U-IR inits uiquitous RNAi. For rsuing mutnts, Tn- N inits nuronl ovrxprssion with lv-gal4 o UAS- n Tn- M inits musl ovrxprssion o UAS- with M2-GAL4. Immunostining. Wnring thir instr lrv wr pross s prviously sri 23. Th ollowing primry ntiois wr us: mous ntioy to (ma2, 1:) 4, guin pig ntioy to Tn- (1:1) 41, mous ntioy to Brp (man82, 1:2) 42, rit ntioy to Synptotgmin 1 (1:4,) 43, mous ntioy to Cystin String Protin (ma6d6, 1:1) 44, mous ntioy to Dlg (ma4f3, 1:) 4, rit ntioy to Dlg (1:4,) 46, mous ntioy to -Sptrin (ma3a9, 1:) 47, mous ntioy to Fsilin 2 (ma1d4, 1:2) 48, rit ntioy to Fsilin 2 (1:,) 46, mous ntioy to Futsh (ma22c1, 1:) 2, rit ntioy to DGluRIII (1:2,), rt ntioy to Elv (ma7e8a1, 1:2) 49, mous ntioy to Evn-skipp (ma3c1, 1:1), rit ntioy to -Sptrin (1:1,) 1, mous ntioy to Hts (1:) 22, guin pig ntioy to Wsp (1:1,) 2. Alx488-, Alx46- or Alx647-onjugt sonry ntiois wr us t 1:2 (Invitrogn). Txs-R-onjugt Phlloiin ws us t 1:3. FITC-, Cy3- or Cy-onjugt ntiois to HRP wr us t 1:1 (Jkson ImmunoRsrh). Eltron mirosopy. Wnring thir instr lrv wr pross n stion s sri 23. Stions wr img on JEM-14 (JEOL) trnsmission ltron mirosop t 33, to 32, mgniition. Eltrophysiology. Lrv wr isst in HL3 slin 3 ontining mm C 21 n 4 mm Mg 21. Thy wr thn trnsrr to slin ontining.6 mm C 21 n rorings onut y impling lrvl musl 6 in oy wll sgmnts A3 n A4 using shrp intrllulr ltros (1 2 MV), rit rom orosilit glss pillris n ill with 3 M KCl solution. For vok EPSPs, svr nrv unls wr stimult using sution ltro onnt to linr stimulus isolt (A39, Worl Prision Instrumnts). Dt, quir using Multilmp 7B mpliirs (Molulr Dvis), wr low-pss iltr t 3 khz n igitiz t 1 khz. Rorings wr quir n nlys using Igor Pro sotwr (Wvmtris) n ustom-writtn progrms. All rorings in whih th rsting mmrn potntil ws highr thn 26 mv n/or whos rsting potntil, input rsistn or ss rsistn hng y mor thn 2% uring th urtion o t quisition wr xlu rom nlysis. All rorings n t nlyss wr prorm lin to th gnotyp. Quntl ontnt ws orrt or nonlinr summtion 4. Lrvl loomotion. Crwling ssys wr onut s sri. FM1-43 y loing. FM1-43 (Invitrogn) y loing ws onut s sri 6 with th ollowing moiitions: loing ws onut in 1. mm C 21,9mMK 1 slin or 1 min ollow y six 2-min wshs in mm C 21 slin. Imging ws onut on Ziss LSM 1 Mt Conol (Crl Ziss) with 34, PlnApo NA 1. wtr immrsion lns (Crl Ziss). Constrution o pitop-tgg tnurin trnsgns. Th tn-m n oing squns lking th stop oon wr lon into pentr-d/topo (Invitrogn) rom pentr-tn-m n pentr- 8. pentr-tn-m(-stop) n pentr-(-stop) wr romin into th stintion vtors puastttbgtw-tfhah n pquastttb-gtw-tfmh, rsptivly, using LR Clons II (Invitrogn). puastttb-gtw-tfhah is puast-gtwy-ttb s vtor with C-trminl TEV rognition sit n 33Flg, 33HA n 13His tgs. pquastttb-gtw-tfmh is pquast-gtwy-ttb 8 s vtor with C-trminl TEV rognition sit n 33Flg, 63My n 13His tgs. Th rsulting onstruts wr vrii y rstrition igst n squning n intgrt into th ttp24 or 86F lning sits on th son n thir hromosoms 7. Trnsgni lis wr vrii y immunopripittion on wstrn lot n ovrxprssion xprimnts. Immunopripittion, wstrn lots n SDS PAGE nlysis. For n Tn-, QUAS-Tn--Flg-My ws xprss in nrvs using Synj-QF n UAS- -Flg-HA in musls using mh-gal4. Lrvl synptosoms wr prpr rom lrvl oy wll illts s sri 8. For n -Sptrin, ontrol lrv onsist o M2-GAL4 without UAS--Flg-HA whrs xprimntl lis omin th two. Immunopripittion ws onut s sri using M2-nti-Flg-onjugt gros (Sigm) or Ai-Prp Protin A s (Bio-R) n rt ntiois to HA (Roh) 23. Protins wr sprt on NuPAGE 3 8% Tris-Att Gls (Invitrogn) n trnsrr to nitrollulos. Primry ntiois wr ppli ovrnight t 4 uc n sonry ntiois t 21 uc or 1 h. Th ollowing primry ntiois wr us: mous ntioy to -Sptrin (ma3a9, 1:2,), mous ntioy to Brp (man82, 1:1), mous ntioy to Flg (M2, 1:,, Sigm-Alrih), mous ntioy to My (3E1, 1:1,, Snt Cruz Biothnology), rt ntioy to HA (3F1, 1:1,, Roh). HRP-onjugt sonry ntiois (Jkson ImmunoRsrh) wr us t 1:1,. Blots wr vlop using th SuprSignl Wst Fmto Mximum Snsitivity Sustrt (ThrmoSintii). Imging nlysis. Lrv wr img with Ziss LSM 1 Mt lsr-snning onol mirosop (Crl Ziss) using ithr NA or NA ojtiv. NMJ imgs wr tkn s onol z-stks with th uppr n lowr ouns in y HRP stining unlss othrwis not. For ll mtris, outons wr ssss in sgmnt A3 t musl 6/7 n musl 4 on oth th lt n right sis. Fluorsn intnsity msurmnts wr tkn rom trminls on musl 4. All phnotyps, howvr, wr osrv t ll synpss rgrlss o musl ir or sgmnt. For mmrn orgniztion, vsil istriution n Tnurin ololiztion, NMJ imgs wr tkn s singl optil stions t th pris ntr o th outon s trmin y HRP stining. Imgs wr pross with th LSM sotwr n Ao Photoshop CS4. Bouton numr, tiv zon/ glutmt rptor pposition, luorsnt intnsity n mirotuul orgniztion wr quntii s prviously sri 23. Trgting rrors or h lrv wr quntii s th prntg o hmisgmnts rom A1 to A7 in singl niml with ilur o trgt innrvtion. Thr ws no irn in trgting rrors s on oy wll sgmnt. Exprimnts using H94-GAL4 wr onut s sri 34, n thir ts onirm using Fsilin 2 ovrxprssion (ontrol 8.1% o outons on musl 6, 41.9% on musl 7; Fs 2 ovrxprssion 73.% on musl 6, 27.% on musl 7; n 8nimlsorh,P,.1) 34. In ltron mirogrphs, prmtrs wr quntii s prviously sri using ImgJ (NIH) 23. T-r ts wr lssii into on o iv tgoris: norml (no isrnil t), oul (two T-rs wr osrv in th sm, ontinuous tiv zon), th (whr th T-r ws lrly visil ut ws not xpliitly onnt to th mmrn ssoit with th nrst PSD), pposit ontrtil tissu (whr th T-r ws not ppos to th SSR, ut rthr, th ontrtil tissu o th musl), misshpn (whr n ltron-ns T-r ws visil ut i not onorm to th T shp. Otn, th T-rs wr X shp). For Fig. 2r, h t is xprss s prntg o th totl numr o T-rs osrv in prtiulr gnotyp. For grint lultion, singl optil stions wr tkn through th ntr o th NMJ on musl 3 or musl 4, s trmin y HRP immunortivity. Th GFP signl (tn-m-gal4) or ntioy signl ws thn msur using ImgJ (NIH). For h lrv, msurmnts wr tkn on th right n lt sis o h init sgmnt. Th luorsn or h sgmnt ws xprss s prntg o th luorsn rom sgmnt A1 in th sm niml, on th sm si o th lrv. For ll lrv, sgmnt A1 rprsnt th mximl luorsn. Sttistil nlysis. Sttistil nlysis us GrphP Prism (Grphp Sotwr). In ll ss involving mor thn two smpls, signiin ws lult using ANOVA ollow y Dunntt post-ho tst to th ontrol smpl n Bonrroni post-ho tst mong ll smpls. For two-smpl ss, n unpir Stunt s t-tst ws us to ssss signiin, unlss othrwis init. In ll ss, oth mthos provi similr signiin msurmnts. In ll igurs, signiin is with rspt to ontrol gnotyps unlss othrwis not. 31. Lilly, B. t l. Rquirmnt o MADS omin trnsription tor D-MEF2 or musl ormtion in Drosophil. Sin 267, (199). 32. Sun, B., Xu, P. & Slvtrr, P. M. Dynmi visuliztion o nrvous systm in liv Drosophil. Pro. Ntl A. Si. USA 96, (1999). 33. Luo, L., Lio, Y. J., Jn, L. Y. & Jn, Y. N. Distint morphognti untions o similr smll GTPss: Drosophil Dr1 is involv in xonl outgrowth n myolst usion. Gns Dv. 8, (1994). 34. Dvis, G. W. & Goomn, C. S. Synps-spii ontrol o synpti iy t th trminls o singl nuron. Ntur 392, (1998). 212 Mmilln Pulishrs Limit. All rights rsrv

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