The transmembrane semaphorin Sema6A controls cerebellar granule cell migration

Transcription

1 Th trnsmmrn smphorin Sm6A ontrols rllr grnul ll migrtion Gérlin Krjn 1, Jki Doln 2,Céil Humitr 3, Sylvi Shnir-Munoury 3, Hjim Fujisw 4, Kvin J Mithll 2 & Alin Chéotl 1 Th trnsmmrn smphorin protin Sm6A is roly xprss in th vloping nrvous systm. Sm6A rpls svrl lsss o vloping xons in vitro n ontriuts to thlmoortil xon guin in vivo. Hr w show tht uring rllum vlopmnt, Sm6A is sltivly xprss y postmitoti grnul lls uring thir tngntil migrtion in th p xtrnl grnul ll lyr, ut not uring thir ril migrtion. In Sm6A-iint mi, mny grnul lls rmin topi in th molulr lyr whr thy irntit n r ontt y mossy irs. Th nlysis o topi grnul ll morphology in Sm6 2/2 mi, n o grnul ll migrtion n nurit outgrowth in rllr xplnts, suggsts tht Sm6A ontrols th initition o grnul ll ril migrtion, proly through moultion o nulr n/or som trnslotion. Finlly, th nlysis o mous himrs suggsts tht this untion o Sm6A is primrily non-ll-utonomous. In th vrtrt ntrl nrvous systm (CNS), rllr grnul ll prursors gnrt in th mryoni rhomi lips migrt tngntilly ovr th rllr plt to orm th xtrnl grnulr lyr (EGL) 1. In ronts, this sonry grmintiv zon prous grnul lls only tr irth 2. In th pr prt o th EGL, postmitoti grnul lls migrt tngntilly in ll irtions 2,3 n unrgo sris o prooun morphologil hngs 4. Thy irst xtn pross horizontlly, whih is soon ollow y son on xtning in th opposit irtion, or thir pross vlops prpniulr to th sur. This prs th inwr ril migrtion o grnul ll ois, long Brgmnn gli irs 5 to th intrnl grnulr lyr (, Fig. 1). Th horizontl prosss om th grnul ll xons known s prlll irs. Th ril migrtion o grnul lls hs n xtnsivly stui (s r. 5 or rviw), ut muh lss is known out th moluls tht ontrol thir tngntil migrtion or th swith rom tngntil to ril migrtion 5. Smphorins r srt or mmrn-oun protins tht ontrol xon guin n ll migrtion 6. Mny, i not ll, trnsmmrn smphorins r xprss in th vloping CNS, ut littl is known o thir untions in vivo. Clss 6 smphorins ompris our protins, Sm6A Sm6D, tht r losly rlt to invrtrt trnsmmrn smphorins 6. Clss 6 smphorins n ollps vrious lsss o xons 7 9 ; this pross sms to mit y rptors o th plxin-a mily 9,10. Clss 6 smphorins n th rlt inst lss 1 smphorins 6,11 r lso pl o iirtionl signling, pprntly ting s rptors in rtin ontxts 10,12. Rntly, mous lin iint in Sm6A ws otin using gn-trpping strtgy 13,14. Sm6A-iint mi hv normlitis in svrl xonl trts, inluing thlmoortil projtions 13. Hr w show tht in th vloping rllum, Sm6A my ontrol th initition o grnul ll ril migrtion, proly y inluning nulus or som trnslotion in non-ll-utonomous mnnr. RESULTS Sm6A is trnsintly xprss in th pr EGL Sm6A mssngr RNAs (mrnas) wr tt in th EGL o th E15 E17 mryoni mous rllum s prviously sri (t not shown n r. 15). In th postntl rllr ortx, Sm6A trnsripts wr oun in th pr EGL n in th (Fig. 1). To loliz Sm6A protin, w us monolonl n polylonl ntiois irt ginst toomins o th humn or mous Sm6A (mino is ). On wstrn lots o ll xtrts ollt rom COS7 lls trnst with plsmis noing ull-lngth Sm6A (s Mthos), ths ntiois rogniz n t th xpt molulr wight (120 kd) ut i not rogniz othr lss 6 smphorins (t not shown). To onirm th spiiity o Sm6A ntiois, w ll stions rom postntl y (P) 10 Sm6 / mi 13. Ths mutnt mi rry gn-trp insrtion in th Sm6A gn tht rsults in th usion o portion o th xtrllulr rgion o Sm6A to th rportr gn TM--go (s Mthos). Suh usion protins r squstr intrllulrly, proly in th noplsmi rtiulum, rlily prouing phnotypi null llls 14. In th rllum o Sm6 / mi, Sm6A immunostining irs rom tht in wil-typ mi. Sullulr Sm6A-positiv ots wr osrv in th rllr ortx (Fig. 1), most likly orrsponing to vsils o th noplsmi 1 Cntr Ntionl l Rhrh Sintiiqu UMR7102, Univrsité Pris 6, Cs 12, 9 Qui Sint-Brnr, Pris, Frn. 2 Smurit Institut o Gntis, Trinity Collg Dulin, Dulin 2, Irln. 3 Cntr Ntionl l Rhrh Sintiiqu UMR7622, Univrsité Pris 6, Btimnt C, Cs 24, 9 Qui Sint-Brnr, Pris, Frn. 4 Th 21st Cntury Cntr o Exlln Progrm, Division o Biologil Sin, Ngoy Univrsity Grut Shool o Sin, Chikus-ku, Ngoy , Jpn. Corrsponn shoul rss to A.C. (hotl@inoiogn.r). Riv 11 July; pt 31 August; pulish onlin 2 Otor 2005; oi: /nn VOLUME 8 [ NUMBER 11 [ NOVEMBER 2005 NATURE NEUROSCIENCE

2 Sm6 EGL 1 2 β-gl Sm6A EGL PGL rtiulum. Conol mirosopy show tht Sm6A-positiv ots oxprss -gltosis (Fig. 1,) s xpt or usion protin. From irth to, Sm6A immunortivity ws osrv in th pr EGL s shown on stions oul stin with th Purkinj ll mrkr linin D28k 16 (Fig. 1 h n t not shown). At, BrU-positiv prolirting grnul ll prursors in th uppr EGL wr Sm6A ngtiv (Fig. 1i). Also, rilly migrting grnul lls in th molulr lyr n postmigrtory grnul lls in th wr not immunortiv to Sm6A, lthough thy xprss Sm6A mrna, initing pris trnsltionl rgultion o Sm6A xprssion. At P21, whn grnul ll prolirtion n migrtion h stopp 17, Sm6A immunortivity ws no longr tt in th molulr lyr or (Fig. 1j). Arrst grnul ll migrtion in Sm6A-iint mi To trmin th untion o Sm6A in grnul ll vlopmnt, w nlyz mi gntilly nginr to iint in Sm6A 13. Sm6 / mi r vil n rtil n o not show ny sustntil hviorl ts (t not shown). Th gross ntomy o th rllum rom Sm6 / mi ws inistinguishl rom tht o th wil typ (t not shown). In stions rom wil-typ mi n Sm6 +/ stin with rsyl violt, th ult rllr ortx h its typil rgulr orgniztion (Fig. 2,). In Sm6A-iint mi (Fig. 2,), Purkinj lls n molulr lyr intrnurons show norml istriution n nsity, ut th siz o th ws ru y 10%. Morovr, in ll oli, numrous lls with smll, ns nulus wr oun in th molulr lyr, somtims lustr t th rllr sur (Fig. 2). To Sm6A+CBP+Hohst h Sm6A+CBP+Hohst i Sm6A+BrU j P0 g P21 Sm6A+Hohst Sm6A+CBP Sm6A+Hohst Figur 1 Sm6A is xprss in tngntilly migrting grnul lls. () Phss o grnul ll vlopmnt. (1) Grnul ll prursors prolirt in th outr portion o th xtrnl grnul ll lyr (EGL). (2) Postmitoti grnul lls in th pr EGL xtn two horizontl prosss n migrt tngntilly. Thy nxt sn thir pross prpniulr (3) n migrt rilly through th molulr lyr n Purkinj ll lyr (gry irls) long ril gli (4) to sttl in th intrnl grnul ll lyr or (5). () At, Sm6 mrnas r xprss in th pr EGL n in th. () Crllum stion rom Sm6A / mous. In ontrst with th wil-typ (s h), Sm6A immuno-positiv ots r osrv throughout th EGL, molulr lyr n. (,) 1-mm onol img o th rllr ortx o Sm6 / mous, oul ll with ntiois to Sm6A (in ) n nti-gltosis (in ) ntiois, showing tht Sm6A n -gl r o-xprss, most likly in smll intrytoplsmi vsils. () At P0, Sm6A xprssion is rstrit to th pr EGL (rrowhs) ov CBP-positiv Purkinj lls (rrows). (g,h) rllum stion immunostin with ntioy to Sm6A n CBP with Hohst ountrstining. Sm6A is still rstrit to th pr EGL. (i) Sm6A immunortiv grnul lls (strisks) r immitly jnt to th uppr EGL ontining prolirting BrU-ll grnul ll prursors (rrowhs). (j) At P21, Sm6A is not xprss in th rllr ortx. Sl rs, 50 mm (,,,j), 500 mm (g), 35 mm (,,h,i). trmin whthr thy orrspon to grnul lls, stions rom ult rllum wr ll with ntioy to th grnul ll spii 6 GABA A rptor suunit (6) 18. In ll ss, Sm6 +/ mi wr intil to wil-typ mi. In th wil typ, 6 isxprssonlyy grnul lls tht hv rh th (Fig. 2,g n r. 18). In ontrst, in Sm6 / mi, lrg proportion o 6-positiv nurons ws oun ithr within th molulr lyr or ggrgt in lustrs unr th pil sur (Fig. 2,h). Ths topi lls lso xprss C 2+ /lmoulin-pnnt protin kins IV (CMKIV), nothr mrkr o mtur postmigrtory grnul lls 19 (t not shown; isuss low). Quntiition o CMKIV-positiv grnul lls init tht out 40% rmin in th molulr lyr. Th sm rsults wr otin using Sm6A knokout mi 1 yr o g (t not shown), initing tht th migrtion is not simply ly n tht th topi grnul lls surviv. Th irntition o Purkinj lls th postsynpti prtnr o grnul lls s visuliz with ntiois to CBP or zrin II (r. 20) or with BDA lling (s Mthos), i not sm to t (Fig. 2g,h n t not shown). Th numr o molulr-lyr intrnurons, s trmin y Hohst stining n prvlumin immunostining, ws lso omprl to tht in th wil typ (Fig. 2g,h n t not shown). To urthr stuy th grnul ll migrtion t in Sm6Aiint mi, w prorm puls lling o migrting grnul lls with BrU (s Mthos). At 108 h tr singl BrU injtion, out 90% o BrU ll lls wr oun in th in wil-typ mi (Supplmntry Fig. 1 onlin), whrs 40% o th BrU-ll lls wr still in th molulr lyr in Sm6A / mi (Supplmntry NATURE NEUROSCIENCE VOLUME 8 [ NUMBER 11 [ NOVEMBER

3 Figur 2 Anorml grnul ll migrtion in Sm6A-iint mi. ( ) Crsyl violt stining o rllum stions rom ult wil-typ (,) or Sm6 / (,) mi. In th wil-typ (wt) rllum, th orr twn th molulr n Purkinj ll lyrs is shrp (rrow in ). In ontrst, in Sm6 / mi this orr is irrgulr n uzzy (rrow in ; s lso ). Morovr, ll nsity is highr in th molulr lyr () o Sm6 /. Th nsity o molulr lyr intrnurons (rrowh in n ) is similr, ut thr r mny lls with smll n ns nuli (rrows in ) isprs in th molulr lyr o Sm6 / mi or umult nr th pil sur. (,) Ault rllum stions immunostin or 6. In Sm6 +/,ll6-immunortiv grnul lls r oun in th n non in th molulr lyr (), whrs in Sm6 /, mny topi 6-positiv grnul lls r oun in th n nr th pil sur (rrowhs in ). (g,h) Ault rllum stion oul stin or CBP n 6 with Hohst ountrstining. In Sm6 +/, th molulr n Purkinj ll lyrs o not ontin 6-immunortiv lls. Th nuli o stllt n skt lls n osrv with Hohst in th molulr lyr (rrowhs in g). In Sm6 / mi, numrous 6-positiv grnul lls r oun in th molulr lyr (rrows in h). Intrnurons r lso osrv (rrowhs in h). Sl rs, 450 mm (,), 45 mm (,), 135 mm (,), 35 mm (g,h). Fig. 1). Ovrll, ths rsults suggst tht grnul ll migrtion within th EGL or rom th EGL is prtur in Sm6 / mi. Grnul ll prolirtion, irntition n survivl Although Sm6A is lry xprss uring th irst phs o tngntil migrtion o grnul ll prursors, in situ hyriiztion with th grnul ll prursor mrkrs Mth1 (lso known s Atoh1 n HATH1) n Brhl1 (r. 21) show tht th EGL ws inistinguishl in nworn Sm6 +/ n Sm6 / mi (Supplmntry Fig. 2 onlin). This suggsts tht th rly migrtion o grnul ll prursors is norml. W quntii grnul ll prolirtion in P0, n P21 rll using BrU (s Mthos) n ntiois to phospho-histon-h3. In Sm6A-iint mi, th numr o prolirting grnul lls (± s..m.; rrors r stt s s..m. throughout) in th uppr EGL GFAP TAG-1 P0 Mth1 P8 Csps-3 t. Sm6 +/ g k Sm6 +/ h Sm6 +/ PCL L1 Dx+ P16 i l Sm6 +/ j Crsyl violt CBP++Hohst g PCL (0.025 ± lls pr mm o EGL lngth t P0 n ± lls pr mm t ) ws similr to tht in htrozygous mi (0.024 ± lls pr mm o EGL lngth t P0 n ± lls pr mm t ; t not shown). Morovr, y P21, no prolirting lls wr tt in th molulr lyr in Sm6A-iint mi, monstrting tht th timing n lvl o grnul ll prolirtion wr lso norml. In th mous rllum, th pk o poptosis ours roun P8 (r. 22). To msur th lvl o poptosis in th vloping rllum, w us ntioy to tivt sps-3 (r. 23). No signiint (P 40.05) irn ws osrv in P8 Sm6A-iint mi (Fig. 3,). During thir irntition, grnul lls r known to squntilly xprss irnt gns, suh s th trnsription tors Mth1 n Px6 (r. 21). Th xprssion pttrns o ths two gns wr norml in Sm6A-iint mi (Fig. 3, n t not shown). Likwis, th xprssion pttrns o TAG-1 n L1, two moluls involv in grnul ll ril migrtion 24,25, wr norml (Fig. 3 h). h Figur 3 Norml poptosis, irntition n ril gli orgniztion in Sm6A knokouts. ( l) Sgittl rllum stions rom Sm6 +/ (,,,g,i,k) orsm6 / (,,,h,j,l) mi wr immunostin with ntiois ginst tivt sps-3 (,), L1 (,), oulortin (Dx; i,j), TAG-1 (g,h) or GFAP(k,l) or hyriiz with Mth1 riopros (,) n ountrstin with Hohst ( j). At P8, th numr o sps-3 ll lls in th EGL (rrowhs in n ) is similr in Sm6 +/ (3.9 ± 0.1 lls pr mm o EGL lngth) n Sm6 / (3.8 ± 0.5 lls/ pr mm o EGL lngth). In n, Mth1 signl hs n rtiiilly olor in r using Photoshop n suprimpos on th Hohst lling (in lu). Thr is no irn in mth1 xprssion twn Sm6 / n Sm6 +/.At, L1 (,) n TAG-1 (g,h) xprssion pttrns r intil in Sm6 +/ n Sm6 /.Ini n j, stions rom P16 rllum r oul stin or Dx n 6. In oth Sm6 +/ n Sm6 /, Dx is xprss in th p EGL (rrow in i n j) n in prpniulr prosss o rilly migrting grnul lls in th molulr lyr (rrowhs in i n j). In Sm6 /,mny6 positiv lls r oun in th molulr lyr. At, th ril orgniztion o GFAP-positiv Brgmnn gli lls (rrows in k n l) is similr in Sm6 +/ n Sm6 /. Sl rs, 115 mm (,), 55 mm (,), 30 mm ( l). PCL 1518 VOLUME 8 [ NUMBER 11 [ NOVEMBER 2005 NATURE NEUROSCIENCE

4 BDA BDA+CBP VGLUT2 VGLUT2+ Doulortin xprssion in rilly migrting grnul lls ws lso intil 26 (Fig. 3i,j). Finlly, topi grnul lls still xprss mrkrs o mtur grnul lls suh s 6 n CMKIV (Fig. 3i,j; n t not shown). Grnul ll migrtion ts in Sm6 / mi oul u to norml Brgmnn gli. This sm unlikly, howvr, s Sm6A is xprss in tngntilly migrting grnul lls in th EGL, ut not in rilly migrting grnul lls or in Brgmnn gli irs. Immunostining o th rllum y glil irillry ii protin (GFAP) onirm tht th numr, isposition n morphology o Brgmnn gli irs in Sm6 / mi wr similr to thos in Sm6 +/ mi (Fig. 3k,l). Thus th ts in migrtion sm to intrinsi to grnul lls. In wil-typ mi, grnul lls in th stlish synpti ontt with mossy irs 27. Th lrg mossy ir trminls r ll rostts. To ll mossy irs, w injt BDA into th ult rllum o wil-typ n Sm6A-iint mi (Fig. 4 ). In th wil typ, BDA-ll mossy irs wr onin to th (Fig. 4,). In ontrst, in Sm6A-iint mi, BDA-ll mossy irs wr lso osrv in th molulr lyr whr thy orm typil rostts (Fig. 4,). In ition, rostts n visuliz using immunoytohmistry or th vsiulr glutmt trnsportr VGLUT2 (r. 28; Fig. 4). VGLUT2 stining lso lls liming irs (Fig. 4), ut th siz n morphology o th two typs o trminls r lrly istinguishl. VGLUT2-positiv rostts wr oun in th molulr lyr (Fig. 4) los to topi 6-immunopositiv grnul lls (Fig. 4 g). Ovrll, ths rsults strongly suggst tht grnul ll irntition n mturtion r norml in Sm6A-iint mi. Norml prlll ir xtnsion n grnul ll polrity To urthr unrstn th rol o Sm6A in grnul ll migrtion, w ll iniviul grnul lls n thir xons (th prlll irs) with BDA. All prlll irs xtn long th mioltrl xis o th g PCL Figur 4 Etopi grnul lls r ontt y mossy irs in Sm6 /. ( ) Wil-typ (,) nsm6 / (,) ult mi wr injt into th rllum with BDA. Som stions (,) wr lso immunostin with ntioy to CABP to ll Purkinj lls. Th sh lin in n lints th pil sur. In wil-typ (,), ll lrg mossy ir trminls (or rostts) r lot in th intrnl grnulr lyr (; rrows in n rrowhs in ). No rostt is osrv in th molulr lyr (). By ontrst, in Sm6 / (s,), mny rostts r tt in th (rrows in n rrowhs in ), in ition to th. ( g) 1-mm onol imgs o VGLUT2 n 6 oul immunostining on Sm6 / rllum. VGLUT2 lls lrg mossy ir rostt (rrowhs in n g) n puntt liming ir trminls (rrow in ). Mossy ir rostts ontt topi 6-positiv grnul lls (rrowhs in n g). Sl rs, 100 mm (,), 35 mm (,), 45 mm ( g). rllr ortx 29. In ult wil-typ mi, BDA injtion in th molulr lyr ll prlll irs running ross th ntir lngth o th injt oli n grnul ll ois in th (Fig. 5). In Sm6A-iint mi, BDA lso ll prlll irs throughout th injt loul, ut BDA-ll grnul lls wr osrv in th molulr lyr in ition to th (Fig. 5). At highr mgniition, th morphology o topi grnul ll ws lrly visil (Fig. 5 h). Two min tgoris o lls wr osrv. Th irst typ ws oun throughout th molulr lyr (Fig. 5,,h). Ths lls xtn pross rsmling mtur grnul ll nrits towr th, n itionl nriti prosss mrg rom th ll oy. Ths prosss wr immunortiv or 6, whih is xprss only on grnul ll ois n nrits 18 (Fig. 5 h). Ths topi grnul lls h rgulr Tshp 3, xtning thinnr pross towr th pil sur (not immunortiv or 6) rom whih th prlll ir mrg. Th lngth o this upwr pross ws omprl or ll topi grnul lls. Th son typ o ll ws oun nr th pil sur within th 6-immunortiv lustrs (s Fig. 2). Ths lls h rthr ipolr shp (Fig. 5,) with prlll irs mrging irtly rom th ll oy, ut thy lso h nriti prosss, on o whih ws orint towr th. Anorml in vitro migrtion o Sm6 / grnul lls To trmin whthr Sm6A ontrols grnul ll nurit outgrowth, w s issoit grnul lls rom th rllum o P5 wil-typ mi onto Sm6A-xprssing COS7 lls n onto mok-trnst lls. Culturs wr ll tr 24 h, using phlloiin n -tuulin immunostining. In Sm6A-xprssing lls, th totl nuriti lngth (lngth o th longst nurit) n th numr o rnh points wr not signiintly irnt (P 4 0.5) rom thos in th ontrols (Supplmntry Fig. 3 onlin). Thus, xognous Sm6A os not inlun th longtion o grnul ll nurits. W nxt ompr th growth o rllr grnul ll nurits in wil-typ n Sm6 / mi. W oun tht nurit outgrowth rom Sm6 / grnul lls ws norml. Th totl nuriti lngth n th lngth o th longst nurit (137.3 ± 6.9 mm n ± 4.8 mm, rsptivly; n ¼ 127) wr not signiintly irnt (P 4 0.5) rom thos in th wil typ (126.3 ± 6.1 mm n 97.4 ± 4.9 mm, rsptivly; n ¼ 179). Th numr o rnh points ws lso similr (1.75 ± 0.04 mm orsm6 / nurons ompr to 1.70 ± 0.03 mm or wil typ, P 4 0.5; t not shown). To ssss ll migrtion mor irtly, w ultur EGL xplnts on polylysin n lminin or twn 18 h n 4.5 (r. 30). In ths ulturs, grnul lls migrt wy rom th xplnt in th sn o ny ril gli n ollow th xt squn o in vivo irntition. Th numr o migrting grnul lls, quntii using Hohst stining n phlloiin (Fig. 6), in ulturs riv rom EGL xplnts NATURE NEUROSCIENCE VOLUME 8 [ NUMBER 11 [ NOVEMBER

5 BDA+Hohst BDA+Hohst BDA g h Figur 5 In vivo lling o topi grnul lls n prlll irs in Sm6 /.(,g,h) Crllum stions o wil-typ n Sm6 / ult mi injt with BDA; in n, with Hohst ountrstining. (,) In wil-typ (wt; s ) nsm6 / (s ), BDA lls prlll irs (rrows in n ) in th molulr lyr () xtning ross th mioltrl xis o th injt oli. Howvr, whrs rtrogrly ll grnul ll ois in wt r xlusivly oun in th, in Sm6 /, mny r osrv in th (rrowhs). Pnls,g n h r onol imgs o topi grnul lls in th uppr prt o th molulr lyr o Sm6 / mi. Mny topi grnul lls hv typil morphology with T-shp xon (rrow in n h) n hook-n nrits (rrowhs in n h). Othr topi grnul lls los to th pil sur hv mor ipolr morphology (rrows in n ). Ths nrits r lso 6- immunortiv (rrows in g n h), whrs th xon is not (rrowh in h). () InultSm6 / rllum, most topi grnul lls xtn in th 6-positiv nrits (short rrows) orint towr th. Sl rs, 75 mm (,), 10 mm (,), 20 mm (,g,h), 35 mm (). BDA Phlloiin Hohst β-tuulin 108 h 36 h g Sm6 +/ Distn rom th xplnt (µm) * % 20% 30% Migrtion rt Figur 6 Anorml in vitro migrtion o Sm6A-iint grnul lls. ( ) Migrtion n nurit outgrowth rom P4 EGL miroxplnts tr 36 h (,,,) or 108 h (,). Hohst stining rvl tht tr 36 h, thr is signiintly lowr numr o migrting grnul lls in Sm6 / () ompr to Sm6 +/ xplnts (). Atr 108 h in vitro, phlloiin stining shows tht migrting grnul lls orm lrg ggrgts with Sm6 +/ xplnts (rrows in ), whrs th ggrgts r muh smllr with Sm6 / xplnts (rrows in ). Th numr o ggrgts is lso lowr. Atr 36 h in vitro, Tuj-1 immunostining rvls tht th numr o nurits utting lin t 150 mm rom th xplnt) ws similr in Sm6 +/ (s ) (367.4 ± 25.3 nurits/mm; n ¼ 24) n Sm6 / (s ) xplnts (379.6 ± 26.3 nurits/mm; n ¼ 10). (g) Quntiition o th istriution o migrting grnul lls roun EGL xplnts in Sm6 / n Sm6 +/ (s Mthos). Th strisk inits signiint irn twn th two (on-til t-tst; *P o 0.05). Sl rs, 225 mm ( ), 55 mm (,). * rom Sm6A-iint mi ws onsirly ru tr 36 h in vitro (Fig. 6,,g). Atr 36 h, th mximum migrtion istn (th xtrnl limit o th rgion tht ompris 90% o th grnul lls) ws lso signiintly lowr (P ¼ 0.02) or th Sm6 / grnul lls (127.9 ± 12.4 mm; n ¼ 5) thn or th Sm6 +/ grnul lls (189 ± 17.1 mm; n ¼ 6). Atr 4.5 in ultur, most grnul lls rom wil-typ mi ggrgt, orming lrg lustrs (Fig. 6). With xplnts rom Sm6 / rllum, ggrgts orm ut thir numr n siz wr smllr (Fig. 6). Th timing o TAG-1 n MAP2 xprssion ws similr in wil-typ n Sm6 / EGL ulturs, showing tht th sn o Sm6A os not t grnul ll mturtion (t not shown). Notly, th numr o prosss growing rom EGL xplnts (Fig. 6,) wssimilrinsm6 +/ n Sm6 /. Finlly, wil-typ EGL xplnts wr ultur on Sm6A sustrt (n ¼ 23) or in mium ontining Sm6A-F (n ¼ 27; s Mthos) or twn 36 n 96 h. In oth onitions, th numr o migrting grnul lls n grnul ll lustrs wr unhng (Supplmntry Fig. 3). Ths rsults suggst tht, in th sn o Sm6A, grnul lls oul xtn norml prosss ut tht th movmnts o th nulus, som, or oth wr prtur. To onirm this, w prorm timlps viomirosopy on EGL xplnts tr h in ulturs (Fig. 7,). In ontrol xplnts (n ¼ 3 xplnts; two inpnnt xprimnts), migrting lls rpily xtn long ling pross ring typil growth ons wy rom th xplnt. A lrg nlrgmnt t th rr ontin th nulus (Supplmntry Vio 1 onlin n Fig. 7). Tim-lps viomirosopy show tht nulr movmnt ws slttory n ws pr y th pprn o n longt swlling h o th nulus, suh s hs rntly n sri or ortil intrnurons 31 (Supplmntry Vio 2 onlin n Fig. 7). Although th nulus ws otn sttionry, th ovrll movmnt o th ll oy ws lwys in th opposit irtion rom th xplnt or. In 1520 VOLUME 8 [ NUMBER 11 [ NOVEMBER 2005 NATURE NEUROSCIENCE

6 β-tu+hohst α-tuulin 0:00 0:50 1:40 2:30 3:20 4:10 5:00 g h i j k l xplnts rom Sm6A-iint mi (n ¼ 8 xplnts; thr inpnnt xprimnts), long ling prosss with vry ynmi growth ons wr lso osrv, suggsting tht nurit xtnsion ws norml (Fig. 7 n Supplmntry Vio 3 onlin). Howvr, th orwr movmnt o th nulus ws prtur. Most ll ois ppr to osillt without moving sustntilly. Furthrmor, lls migrting towr th xplnt wr lwys osrv (Supplmntry Vios 3 5 n Fig. 7). Finlly, in th viinity o th xplnts, ll ois sm mor tightly ggrgt thn in th wil typ. To urthr hrtriz this norml migrtion, EGL ulturs t 36 h wr ix n ll with mrkrs o th ytosklton n ntrosom tht r importnt omponnts o th migrtion mhinry 31,32. Th ovrll isorgniztion o th ohort o migrting grnul lls ws lrly osrv using -tuulin stining (Fig. 7,). Howvr, th prinulr g o mirotuuls 32 ppr similr in wil-typ n Sm6A-iint lls (Fig. 7 h). Th ntrosom oul lso osrv using ntiois to g-tuulin 32 ; it h omprl position in th wil-typ n Sm6A-iint lls γ-tuulin Phlloiin Figur 7 Anorml grnul ll movmnt in sn o Sm6A. (,) Tim-lps imging o grnul ll migrtion rom P5 EGL miroxplnts ultur or 24 h or imging. Explnts r lot on th top o th rm (intrvl twn piturs is 50 min). In wil-typ (s ), grnul lls migrt opposit to th xplnt. Thy trnsintly show n longt swlling just h o th nulus (rrow). In ontrst, most Sm6 / grnul lls (s ) rmins sttionry (strisk) or mov k towr th xplnt (rrow). ( h) -tuulin immunostining o wil-typ (,) or Sm6 / (, h) EGL miroxplnts ultur or 36 h (with Hohst ountrstining). Explnts r on th lt si o th rm or n n in th ottom or h. Mny migrting Sm6A / grnul lls (rrows in ) ppr misorint ompr to wil typ (rrows in ). Howvr, th prinulr g o mirotuul ll with ntioy to -tuulin pprs similr in grnul lls rom wil-typ (s ) n Sm6A-iint ( h) mi. (i,j) g-tuulin immunostining o th ntrosom in grnul lls migrting rom EGL xplnts. In grnul lls riv rom oth wil-typ (i) n Sm6 / mi (j), th ntrosom is position h o th nulus. (k,l) Phlloiin-FITC stining o th tin ytosklton in grnul lls migrting rom EGL xplnts. No irn is osrv twn wil-typ (i) nsm6 / (j). Sl rs, 25 mm (,,k,l), 30 mm (,), 6 mm ( h), 15 mm (i,j). (Fig. 7i,j). Finlly, th tin ytosklton ll with phlloiin ppr similr in wil-typ n Sm6A-iint grnul lls (Fig. 7k,l). Non-ll-utonomous untion o Sm6A in grnul lls Sm6D n inst lss I smphorins t s ligns n rptors 10,12. To trmin whthr Sm6A untions s rptor or s lign in migrting grnul lls, w gnrt mous himrs. Sm6A mutnt mi wr irst ross with mi xprssing grn luorsnt protin (GFP) tth to th hikn At (-tin) promotr (At-GFP mi) 33 to llow th lls o h gnotyp to istinguish (s Mthos). GFP;Sm6 / morul wr thn ggrgt to wil-typ ( wt ) morul to otin GFP;Sm6 / :wt himri mi (n ¼ 2). As ontrol, w lso otin GFP;Sm6 +/ :wt himrs (n ¼ 5). Th proportion o Sm6A-iint lls, s trmin on th sis o GFP xprssion, ws twn 30% n 40% (n ¼ 2). GFP ws tt in ll typs o rllr lls suh s Brgmnn gli n Purkinj lls (Fig. 8 n t not shown). In GFP;Sm6 +/ :wt himrs, no CMKIV+GFP GFP; Sm6 +/ : GFP; : Figur 8 Non-ll-utonomous untion o Sm6A in migrting grnul lls. ( h) Crllum stions rom GFP; Sm6 +/ :wt (in ) n GFP; Sm6 / :wt (in h) mous himrs ll with ntioy to GFP (,g,h) ncmkiv(,,,h), or 6 (,). In GFP; Sm6 +/ :wt himr (s ), ll CMKIV- n GFP-positiv grnul lls (rrowhs) r in th. Som Purkinj lls lso xprss GFP (rrow). In ontrst, in GFP; Sm6 / :wt himr (s ), mny CMKIV-positiv topi grnul lls r osrv in th molulr lyr (rrowhs). ( h) 4-mm onol imgs o rllum stions rom GFP; Sm6 / :wt himr ll with 6 (in n ), CMKIV (,h) n GFP. Mny 6- or CMKIV-positiv grnul lls r lot in. Only sust o ths topi grnul lls r GFP positiv (n thus Sm6 / ; rrowhs), n th mjority r GFP ngtiv (n thus o wil-typ origin; rrows). Sl rs, 75 mm (,), 35 mm ( h). GFP; : +GFP +Hohst GFP CMKIV GFP g h CAMKIV +GFP NATURE NEUROSCIENCE VOLUME 8 [ NUMBER 11 [ NOVEMBER

7 topi 6- or CMKIV-positiv lls wr tt in th molulr lyr (Fig. 8). In ontrst, mny topi CMKIV- n 6-positiv grnul lls wr tt in th molulr lyr o GFP;Sm6 / :wt (Fig. 8,). Among GFP-xprssing grnul lls, out 30% wr topi. Conol mirosopy nlysis rvl tht only minority out 30 40% o th CMKIV- n 6-positiv topi lls wr lso GFP positiv n thus iint in Sm6A (Fig. 8 h). Ths lls wr isprs throughout th molulr lyr. Th rmining 6-positiv topi lls wr GFP ngtiv, n thus o wil-typ origin; lik th GFP-positiv lls, ths wr homognously istriut in th molulr lyr. DISCUSSION It hs n propos tht h phs o grnul ll vlopmnt is ontroll y oth xtrinsi tors n n intrinsi vlopmntl progrm 34. Som tors ting on grnul lls within th EGL hv gun to intii. Thus, th HLH trnsription tor Mth1 is nssry or ling trmintion o grnul lls 35. In th outr EGL, th prolirtion o grnul ll prognitors rquirs Purkinj ll riv Soni hghog 36. Th pssg rom th outr EGL to th pr EGL involvs th hmokin stroml-riv tor-1 n EphB2-phrinB2 rvrs signling 37. Th initition o grnul ll xon longtion in th pr EGL is rgult y intrtion twn th srin/thronin kins Un51.1, SynGAP n syntnin 38. Finlly, mny xtrllulr us suh s glutmt 39, nurgulin 40 n BDNF 41 moult th ril migrtion o grnul lls n my ooprt with Sm6A to triggr migrtion. Howvr, Sm6A untion irs rom ths tors in tht prolirtion, migrtion to th pr EGL n prlll ir xtnsion r norml in Sm6 / mi. Our t, togthr with prvious ntomil 2,4 n tim-lps nlyss 3, show tht Sm6A is xprss y tngntilly migrting grnul lls in th p EGL n is turn o whn thy initit ril migrtion. Sm6A untion sms to spiilly rquir or th initition o ril migrtion. W vor mol (Supplmntry Fig. 4 onlin) in whih Sm6A rgults intrtions twn grnul lls, rthr thn with Brgmnn gli. First, Sm6A is no longr xprss y grnul lls whn this intrtion ours n is nvr xprss y Brgmnn gli, whos morphology pprs norml in Sm6 / mutnts. Son, grnul ll migrtion rom EGL xplnts in vitro, whih ours without Brgmnn gli, is norml in sn o Sm6A. Notly, Sm6A os not t grnul ll nurit outgrowth in vitro n in vivo, whih might hv n its xpt rol. Our osrvtions suggst, rthr, tht Sm6A is spiilly involv in th rly stps o nulr n som trnslotion within th rilly orint grnul ll prosss (Supplmntry Fig. 4). Thus, Sm6A untion irs rom tht o othr known moluls xprss in th pr EGL (suh s Un51.1 n Px6) tht priniplly ontrol prlll ir xtnsion. Drosophil Sm1, whih is losly rlt to lss 6 smphorins, sms pl o ting s rptor 12, n Sm6D n t s rptor or plxin-a1 (r. 10). Howvr, th nlysis o Sm6A himrs rvls tht Sm6A untion in th EGL is primrily nonll-utonomous. This os not rul out possil rptor untion or Sm6A t rtin tim-points or in othr nurons. Th st nit rptors or Sm6A on grnul lls r typ A plxins 9,10, on o whih plxin-a4 mits Sm6A rpulsiv tivity on sympthti xons. Howvr, plxin-a4 mrna ws not tt in th vloping rllr ortx (unpulish t). Sm6A os not sm to lv n thus my work s ontt rpllnt, proviing sptil inormtion or unrlying lls in th pr EGL in prtiulr, or rilly migrting lls; thus Sm6A, togthr with grints o srt tors 37, my srv to orint grnul ll migrtion wy rom th EGL (Supplmntry Fig. 4). This is onsistnt with th ilur o mny grnul lls to migrt wy rom th EGL in Sm6A mutnts n with th osrvtions, rom tim-lps viomirosopy, tht mny grnul lls rom Sm6Amutnt xplnts migrt in th wrong irtion (towrs th xplnt). Aoringly, th prturtion o this sptil inormtion my lso xplin th prsn o mny topi wil-typ grnul lls in Sm6 / :wt himrs. Howvr, th xtintion o Sm6A xprssion with th initition o ril migrtion lso suggsts tht th sptiotmporl ontrol o Sm6A xprssion provis rully rgult prrin signl tht my initit ril migrtion or rrst tngntil migrtion. An ltrntiv hypothsis is tht Sm6A primrily ontrols th lvl o hsion twn tngntilly migrting lls n tht th ts in th initition o ril migrtion r sonry to inrs hsion twn grnul lls in th sn o Sm6A untion. Sm1A hs n shown to t siultion o motor xons t isrt hoi points 42 y ountring th ttrtiv tivity o homophili protins. Notly, in Sm6 / mi, mny topi grnul lls r lustr nr th pil sur, n th motility o grnul ll ois is lso mrkly ru in EGL ulturs. Thus, Sm6A oul untion s -hsiv molul in tngntilly migrting grnul lls, ilitting, through ontt rpulsion, grnul ll movmnt in th EGL n th rspons to nothr signl to initit ril migrtion. Howvr, in EGL xplnt ulturs, th Sm6A toomin os not sm to inlun grnul ll migrtion n lustring. Our tim-lps imging xprimnts show tht th primry onsqun o Sm6A iiny is t in th trnslotion o th grnul ll som or nulus. Orint nulr trnslotion, or nulokinsis, hs n sri in most migrting nurons n in othr ll typs 31,32,43. Migrting nurons r highly polriz, with ytoplsmi orgnlls suh s th ntrosom n th Golgi pprtus t thir ling g, n th nulus t thir rr. W oul not tt ny ovious ts in th ntrosom or ytosklton in Sm6 / grnul lls, suggsting tht thir norml migrtion my not rsult rom mjor isorgniztion o th migrtion mhinry. It will importnt to trmin whthr th xprssion or untion o th ntrosoml omponnts is ltr, n in prtiulr thos o Pr6, whih rgults ytoskltl ynmis n nulokinsis in rilly migrting grnul lls 32. Mny othr qustions rmin unnswr. Sm6A mrna is xprss in grnul lls in th, lthough ths lls r not immunortiv with Sm6A. This shows tht th xprssion o Sm6A protin is vlopmntlly rgult n sujt to pris trnsltionl moiitions, post-trnsltionl moiitions or oth. Th ts in initition n orinttion o grnul ll migrtion suggst tht th rgultion o Sm6A protin xprssion provis pris sptiotmporl signl. In ition, th osrvtion tht out 60% o grnul lls r still l to rh th suggsts ithr tht grnul lls r htrognous or tht othr moluls t togthr with Sm6A to ontrol th trnsition rom tngntil to ril migrtion. Sm6A-iint mi lso provi goo mol or stuying othr spts o rllr vlopmnt. During vlopmnt, mossy irs nvr inv th molulr lyr n rmin low th Purkinj lls 44. In vitro xprimnts hv lso suggst tht mossy ir growth is rgult y trgt-riv stop signls prou y grnul lls, n y inhiitory signls prou y Purkinj lls or prsnt in th molulr lyr 16.InSm6 / mi, topi grnul lls in th molulr lyr irntit n riv mossy ir innrvtion. Thus, 1522 VOLUME 8 [ NUMBER 11 [ NOVEMBER 2005 NATURE NEUROSCIENCE

8 th unusul llulr nvironmnt os not prlu n intrinsi irntition progrm. In ition to, n togthr with, othr prvious xmpls o mossy ir ontts osrv on topi grnul lls in wil-typ mi or s rsult o 6-hyroxyopmin trtmnts 45,46, this rsult shows tht thr r no inhiitors tht prvnt mossy irs rom ntring th molulr lyr. Rthr, th surprising pity o mossy irs to in n ontt grnul lls r rom th suggsts tht grnul lls srt long-rng hmottrtnts or mossy irs. METHODS Animls. Swiss n C57/Bl6 mi (Jnvir) wr us or xprssion stuis. Th Sm6A-iint lin hs n prviously sri 13 :rily,sstt noing CD4 trnsmmrn omin -gltosis nomyin phosphotrnsrs (TM--go) n humn plntl lklin phosphts (PLAP), sprt y n intrnl riosom ntry sit (IRES), ws insrt in th 17th intron o Sm6A 14. Th rmining N-trminl portion o th Sm6A protin up to mino i 623 (n thus lking th trnsmmrn n ytoplsmi omins) ws us to -gltosis n trpp in th noplsmi rtiulum (s Rsults). GFP;Sm6 mi wr otin y rossing Sm6A-iint mi with trnsgni mi xprssing GFP unr th hikn -tin promotr 33. P0 P5 mi wr nsthtiz on i n, tr P5, y inhltion o isoluorn Forn (Aott). Th y o irth orrspons to P0. All niml prours wr rri out in orn with institutionl guilins. Gnotyping o Sm6A-iint mi. DNA wr prpr rom tils using th R Extrt-N-Amp Tissu PCR kit (Sigm). Th Sm6A gnotyp ws trmin y PCR using th ollowing primrs: 5 -GAG ATG CAC AGC TAA CTT CTG GTG-3 (wil-typ lll orwr primr), 5 -TTG AAG CCT GCT CTT AGT GGC TCC-3 (rvrs primr) n 5 -GCT ACC GGC TAA AAC TTG AGA CCT-3 (mutnt lll rvrs primr), whih mplii 1.43-k prout or th wil-typ lll n 990-k prout or th mutnt lll. Immunoytohmistry. Brins wr ollt s hs n prviously sri 47. Brin stions wr inut with ntiois ginst hsm6a (1:200 ; R&D Systms), msm6a (1:200; R&D Systms), gltosis (1:1,000; Cppl), GABA A rptor 6 suunit (1:1,000; Chmion), CMKIV (1:500), TAG-1 (TG1; 1:3000), L1 (1:100; Chmion), Px6 (1:1,000; Chmion), CBP (1:1,000; SWANT), prvlumin (1:1,000; Sigm), zrin II Q113 (1:500), GFAP (1:400; Chmion), VGLUT2 (1:3,000; Chmion), phosphohiston-h3 (1:1,000; Cll Signling), tivt sps-3 (1:250; Cll Signling), Dx (1:1,000; Chmion), irontin (1:500; Sigm), GFP (1:300; Molulr Pros), GFP (1:200; US Biologil) or NCL-Ki67 (1:1,000; Novostr) ollow y spis-spii sonry ntiois (Jkson ImmunoRsrh). Stions wr ountrstin with Hohst (10 mg/ml, Sigm), mount in Mowiol (Cliohm) n xmin with luorsn mirosop (DMR, Li) or luorsn onol mirosop (DM IRBE, Li). Donvolution ws prorm y th 3D onvolution moul rom Mtmorph 6.2r2 sotwr (Univrsl Imging Corp.). BrU injtions n stining. P7 n mi wr injt intrpritonlly with BrU (Sigm; 15 mg/ml, 50 mg pr kg o oy wight) ilut in slin solution. Animls wr prus 3 h or 108 h tr injtion. Brin stions wr inut with rt ntioy to BrU (1:100; Hrln). In situ hyriiztion. Antisns riopros wr ll s sri prviously 47 y in vitro trnsription o DNAs noing mous Mth1 (git rom M. Wss 48 ), Brlh1 (git rom F. Qiu 21 )orsm6 15. In situ hyriiztion ws on s sri 47. BDA injtions. Ault mi wr nsthtiz with ktmin (Imlgn 500, 146 mg/kg; Mril) n xylzin (Rompun 2%, 7.4 mg/kg; Byr), tr whih 1 ml o 10% iotin-xtrn (BDA; 10,000 MW; Molulr Pros) ws injt into th rllum with glss miropipt. Mi wr kill 48 h ltr. BDA ws rvl with CY3-onjugt strptviin (1:400; Jkson Immunorsrh). Gnrtion o himri nimls. To otin himrs twn GFP;Sm6 / n wil-typ lls, morul rom GFP;Sm6 / GFP;Sm6 / rosss wr ggrgt in vitro with morul o wil-typ mi. As ontrols, w ggrgt morul rom wil-typ mi with thos rom GFP;Sm6 / GFP;Sm6 +/+ rosss. Morul ggrgtion ws prorm s hs n prviously sri 49, with w moiitions. Brily, tr rmovl o th zon pllui, two morul o pproprit gnotyps wr put in ontt in rop o 100 mg/ml phytohmgglutinin PHA-P (Sigm) in PBS, until thy wr physilly tth (out 1 min). Th mryo pirs wr thn trnsrr into prssion wlls ontining M16 mium (Sigm). Atr ovrnight ultur t 37 1C in7%co 2, lstoysts rsulting rom ggrgtions wr trnsrr into psuoprgnnt C57Bl/6/CBA mls. Dissoit grnul ll ulturs. Grnul lls wr purii s hs n prviously sri 50 n plt onto polylysin (0.2 mg/ml; Sigm) n lminin (20 mg/ml; Sigm), onto COS7 lls, or onto COS7 lls trnst with ull-lngth Sm6A, in ultur mium (BME (Invitrogn), 0.45% gluos, 10% hors srum, 5% tl ovin srum (Euroio)). Culturs wr ix tr 24 or 48 h with 4% prormlhy (PFA), 0.33 M suros n immunostin with mous ntioy to -tuulin (Tuj-1: 1:1,000; Eurognt). Miroxplnts ultur. Miroxplnts ulturs o P4 or P5 mi wr prpr s sri prviously 30. Culturs wr ix tr 18, 36, 72, 96 or 108 h with 4% PFA, 0.33 M suros. To tst Sm6A tivity, EGL xplnts wr ultur on polylysin (0.2 mg ml 1 ;Sigm),lminin(10mg/ml or 20 mg/ml) n rominnt Sm6-F (R&D Systms; 1,000 mg/ml) or in ultur mium ontining 1,000 ng/ml o Sm6A-F prlustr with ntioy to humn IgG1 (Jkson ImmunoRsrh). Culturs wr stin with phlloiin (Sigm) or immunostin with mous ntioy to -tuulin or mous ntioy to -tuulin (1:1,000; Sigm) n ountrstin with Hohst (10 mg/ml, Sigm). For ntrosom stinings, ulturs wr ix 10 min in 20 1C mthnol n immunostin with rit ntioy to g-tuulin (1:500; Sigm). Migrtion n outgrowth nlysis. Anlysis ws prorm with Mtmorph. To msur migrtion rts, w limit onntri rs o 50-mm with t inrsing istns rom th xplnt orr. Th numr o Hohst-ll pixls within h r ws ount n thn xprss s prntg o th totl numr o pixls. To vlut th ovrll rt o nuronl migrtion, th totl numr o Hohst-ll pixls surrouning h hl o th xplnt ws ount. Th numr o lustrs (ontining minimum o iv lls) surrouning th xplnt ws lso ount. Mximl nurit lngth wr stimt y msuring th thr longst Tuj-1 positiv nurits o h xplnt. Nuriti lngth ws stimt y lying out irl ontining pproximtly 90% o th Tuj-1 positiv nurits. W lso ount th totl numr o Tuj-1 positiv nurits within th 150-mm primtr roun th xplnt orr. Tim-lps viomirosopy. EGL miroxplnts wr ultur h s sri low, in Ptri ishs quipp with glss ovrslips. Bor imging, 20 mm HEPES ws to th ultur mium. Piturs wr quir with n invrt mirosop (DM IRBE, Li) quipp with Miromx CDD Printon mr rivn with Mtmorph Sotwr. Piturs wr ptur vry 5 min using 20 ry ojtiv quipp with phs optis, in 37 1C hmr. Hohst ws to ultur mium (1:10 6 ; Sigm) to visuliz th nulus in liv lls. Crsyl violt stining. Stions wr olor 7 min in 1% rsyl violt n 1% thionin solution n thn irntit in 80% thnol n ti i. Wstrn lotting. COS7 lls wr trnst with Lipotmin2000 (Invitrogn). Cll xtrts wr ollt 48 h ltr, sprt y SDS-PAGE n wstrn lott y stnr mthos. Protins wr immunott with ntiois to msm6a (1:500; R&D Systms), hsm6a (1:250; R&D Systms), hsm6a (1:500; R&D Systms) or my E910 (1:200; Snt Cruz), ollow y horsrish proxis link sonry ntiois n ECL kit (Amrshm). NATURE NEUROSCIENCE VOLUME 8 [ NUMBER 11 [ NOVEMBER

9 Sttistil nlysis. For ll sttistil nlysis, th signiin ws lult y ANOVA (Sttviw, Aus Conpts). Not: Supplmntry inormtion is vill on th Ntur Nurosin wsit. ACKNOWLEDGMENTS W thnk C. Sotlo or his onstnt support n ommnts on th mnusript; H. Skgmi n H. Kono (Tohoku Univrsity) or nti- isoorm o CMKIV ntioy; R. Hwks (Univrsity o Clgry, Cn) or Zrin II Q113 ntioy; D. Krgogos (Univrsity o Crt, Gr) or TAG-1 ntioy; F. Qiu (UMDNJ, Pistwy, USA) or Brhl1 DNA; M. Wss (ENS, Pris, Frn) or Mth1 DNA; K. Skurk n D. Rottkmp or hlp in lolizing th insrtion sit in th Sm6 gn trp lll; R. Shwrtzmnn n V. Gorgt (Srvi Imgri IFR83, Univrsité Pris 6, Frn) or thir hlp with onol n viomirosopy stuis; n M. Wss, C. Lrn, C. Métin n J.P. Buoin or isussions. A.C. is support y th Fontion pour l Rhrh sur l Crvu (FRC), th Shlumrgr Fountion n th Assoition pour l Rhrh sur l Cnr (ARC). K.J.M is Sin Fountion Irln (SFI) Invstigtor. This work ws support y SFI grnt 01/F.1/B006 to K.J.M; n grnts rom th 21 st Cntury COE Progrm, n rom th Cor Rsrh or Evolutionl Sin n Thnology (CREST) o th Jpn Sin n Thnology Agny (JST) to H.F. COMPETING INTERESTS STATEMENT Th uthors lr tht thy hv no ompting innil intrsts. Pulish onlin t Rprints n prmissions inormtion is vill onlin t rprintsnprmissions/ 1. Wingt, R.J. Th rhomi lip n rly rllr vlopmnt. Curr. Opin. Nuroiol. 11, (2001). 2. Ryr, E.F. & Cpko, C.L. Migrtion pttrns o lonlly rlt grnul lls n thir prognitors in th vloping hik rllum. Nuron 12, (1994). 3. Komuro, H., Yuov, E. & Rki, P. Mo n tmpo o tngntil ll migrtion in th rllr xtrnl grnulr lyr. J. Nurosi. 21, (2001). 4. Rmon y Cjl, S. Histologi u systèm nrvux l homm t s vrtérés (Mloin, Pris, 1911). 5. Yuov, E. & Komuro, H. Cllulr n molulr mhnisms o rllr grnul ll migrtion. Cll Biohm. Biophys. 37, (2003). 6. Fior, R. & Pushl, A.W. Th untion o smphorins uring nrvous systm vlopmnt. Front. Biosi. 8, s484 s499 (2003). 7. Qu, X. t l. 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Httn, M.E. Nuronl rgultion o stroglil morphology n prolirtion in vitro. J. Cll Biol. 100, (1985) VOLUME 8 [ NUMBER 11 [ NOVEMBER 2005 NATURE NEUROSCIENCE