Directional guidance of neuronal migration in the olfactory system by the protein Slit

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1 rtiles Diretionl guine of neuronl migrtion in the olftory system y the protein Wei Wu*, Kit Wong, Jin-hui Chen*, Zhi-hong Jing, Sophie Dupuis, Jne Y. Wu & Yi Ro * Lortory of Moleulr Neuroiology, Shnghi Reserh Center for the Life Sienes n The Ntionl Institute of Neurosiene, The Chinese Aemy of Sienes, 320 Yue-yng Ro, Shnghi, Chin Deprtment of Antomy n Neuroiology, Deprtments of Peitris, n Moleulr Biology n Phrmology, Wshington University Shool of Meiine, Box 8108, 660 S. Euli Avenue, St Louis, Missouri 63110, USA... Although ell migrtion is ruil for neurl evelopment, moleulr mehnisms guiing neuronl migrtion hve remine unler. Here we report tht the serete protein repels neuronl preursors migrting from the nterior suventriulr zone in the telenephlon to the olftory ul. Our results provie iret emonstrtion of moleulr ue whose onentrtion grient guies the iretion of migrting neurons. They lso support ommon guine mehnism for xon projetion n neuronl migrtion n suggest tht my provie moleulr tool with potentil therpeuti pplitions in ontrolling n ireting ell migrtion. Most neurons in the eveloping nervous system hve to migrte from their irthples to reh their finl positions. Stuies of neuronl migrtion re importnt for reveling the mehnisms unerlying the formtion of norml nervous system, for unerstning the etiology of humn iseses use y norml migrtion n for esigning therpeuti pprohes to neurologil iseses. Neuronl migrtion in the eveloping entrl nervous system (CNS) ws initilly inferre from histologil oservtions y lssil neuroemryologists inluing Vignl (1888), Rmon y Cjl (1891, 1911), Kolliker (1896), His (1904) n Hresty (1904) 1 (reviewe in refs 2 5). The possiility tht ells truly migrte, rther thn tht ells forme erlier re simply isple y ells forme lter 6, ws supporte initilly y ells moving in iretion opposite to tht expete from ell isplement through histologil exmintions in the spinl or of hik emryos 7 n lter y utoriogrphi tring in the ererum of roent emryos 8. The ft tht only nulei were tre in utoriogrphi stuies rise the possiility tht nulei, ut not entire ells, move in the highly struture nervous system. Eletron mirosopy n reonstrution provie strong eviene for the migrtion of neuronl ell oies 3 5, n oservtions of primry neurons n gli ulture in vitro emonstrte iretly tht neurons o migrte Using histologil, utoriogrphi, retrovirl tring, ye lelling n moern imging tehniques, it hs now een estlishe tht the mjority of neurons migrte throughout the eveloping nervous system Proper migrtion of neurons is essentil for the formtion n norml funtioning of the nervous system. In humns, efets in neuronl migrtion n use severl iseses inluing epilepsy 13,14. Migrtion is lso importnt for metstsis n invsion of neurolstom n gliom. Although it hs een known for some time tht ell migrtion is essentil for postntl ehviour hnges in irs, we now know tht neurogenesis n neuronl migrtion lso ontinue in the rins of postntl mmmls, inluing humns. These finings inite tht, for suessful pplition of ell-se therpies for neuroegenertive iseses, it is essentil to iret the orret migrtion of neurons or ells expressing therpeuti prouts to the trget region. Our unerstning of the moleulr mehnisms guiing neuronl migrtion is still limite. Geneti stuies in humns n mie hve ientifie mny moleules, efiienies of whih use efets in neuronl migrtion 15. Beuse most of these re intrellulr moleules, it is unlikely tht they t s guine ues for migrting neurons. One moleule ientifie from the geneti stuies is the serete protein Reelin Loss-of-funtion muttions in the reeler gene use efets in CNS lmintion, n Reelin hs een thought to ontrol ell ell intertions ritil for ell positioning. It is not ler, however, whether Reelin ts s stop signl or n hesive moleule for migrting neurons 15 18,21. Beuse moleules n regulte neuronl migrtion iniretly 22, it is not estlishe whether Reelin ts iretly or iniretly 15,21. Similrly, the preise roles of the other genetilly ientifie moleules involve in neuronl migrtion remin to e etermine 15,21. The olftory system is useful moel for stuying neuronl migrtion. The olftory ul relys olftory informtion from the olftory epithelium to the primry olftory ortex 23. The mjor types of interneuron in the olftory ul, inluing the grnule ells n the periglomerulr ells, re proue postntlly from the nterior suventriulr zone (SVZ) of the telenephlon in roents Neuronl progenitors thus hve to migrte in the rostrl migrtory strem (RMS) from the SVZ to the olftory ul 19,20, Co-ulture of explnts in ollgen gel mtries inite tht the septum, t the miline of the telenephlon n ul to the SVZ, seretes repulsive ftor(s) guiing the migrtion of SVZ neurons 31. However, experiments involving ifferent ulture metho using mtrigel foun no repellent tivity in the septum for migrting SVZ ells 32. Beuse the mtrigel llowe neurons to migrte on top of eh other, ehviour terme hin migrtion n thought to our in vivo 27,28,32, the signifine of the repulsive ue in the septum for guiing SVZ neurons ws oute y Wihterle et l. 32. Furthermore, the moleulr ientity of the puttive repellent(s) in the septum is not known. We show here tht the septum is repulsive to SVZ neurons in mtrigel. We foun the expression of two slit genes in the postntl septum, enoing proteins tht re repulsive ues ple of guiing the migrtion of SVZ ells. This guine epens on onentrtion grient, rther thn the solute mount, of the proteins. Moreover, repels neurons migrting from the SVZ into its nturl pthwy, the RMS. These results inite tht n t s iffusile moleule guiing the iretion of ell migrtion in the nervous system Mmilln Mgzines Lt NATURE VOL JULY

2 rtiles Repulsive tivity in the septum The existene of repulsive tivity in the septum for SVZ neurons ws inite y o-ultures of the SVZ n septum in ollgen gel mtries 31. Another ssy for stuying migrtion of SVZ neurons involves mtrigel, three-imensionl extrellulr mtrix gel of ollgen IV, lminin, heprn sulfte proteoglyns n enttin niogen 33. The morphology n ehviour of SVZ ells ulture in the mtrigel re thought to resemle those in vivo 27,28,32. To test whether the septum is repulsive to SVZ neurons, we isolte postntl SVZ n septl explnts n o-ulture them in mtrigel n ollgen gel mtries. The istriution of neurons migrting out of the SVZ explnts n revel repulsive or ttrtive tivities. When SVZ explnts were ple in mtrigel, the istriution of migrting ells ws symmetri roun the irumferene of eh explnt (Fig. 1). In the presene of the septum, more ells were foun in the qurnt istl to the septum thn those in the qurnt proximl to the septum (Fig. 1) (36/36 explnts). Thus, s in ollgen gel mtries 31 (Fig. 1), the septum is repulsive to SVZ neurons. The ventrl miline struture in the spinl or, the floorplte, ws lso repulsive to the SVZ in mtrigel (Fig. 1) (12/12 explnts). These results inite tht, lthough ells migrte on top of eh other in mtrigel 27,28,32, hins of migrting ells o respon to guine ues in the ventrl miline of the neurl tue, inluing the septum n the floorplte. Repulsion of migrting neurons y Genes enoing the serete proteins, whih re hemorepellents for xons 34 38, reexpresse inthe emryoni septum 34,35.Wehvenow foun tht slit genes re expresse in the postntl murine septum. Of the three slit genes, slit-1 (-1) nslit-2 (-2) re expresse in the postntl septum n the neoortex (Fig. 1e g), rising the possiility tht my e guine ue for SVZ neurons. To investigte whether proteins ffete migrtion of neurons from the SVZ, we first o-ulture SVZ explnts n -expressing ells in mtrigel. When SVZ explnts from postntl y 5 (P5) mie were o-ulture with humn emryoni kiney (HEK) ells stly trnsfete with ontrol plsmi 34, hins of migrting neurons were symmetrilly istriute (Fig. 2) (32/32 explnts). When SVZ explnts were o-ulture with HEK ells stly expressing the Xenopus protein (x), n orthologue of m-2, hin migrtion ws highly symmetri (Fig. 2) (42/42); there were more hins in the qurnt istl to the ells thn in the qurnt proximl to the ells. The sme effet ourre when SVZ explnts were o-ulture with m-1-expressing ells (Fig. 2e) (16/16). These results inite tht is repulsive for SVZ ells migrting in hins. We hve lso ssye SVZ migrtion using ollgen gel mtries 31. Although the istriution of migrting SVZ ells ws symmetri when explnts were o-ulture with ontrol ells (Fig. 2) (104/122 explnts), with x ells there were more SVZ ells in the istl qurnt (Fig. 2) (235/254 explnts). Cells trnsiently expressing m-1 hve similr effets (Fig. 2f) (31/31). The effet of x on SVZ ells ws quntifie, showing tht ells in the istl qurnt were not only more numerous, ut lso frther wy from the explnts (Fig. 3). Beuse single ells, rther thn hins of ells, migrte in ollgen gel mtries, the repulsive tivity of in the ollgen gel ssy inites tht n t on single ells. To fin out whether the migrting ells were neurons, we rrie out immunoytohemistry with the TuJ1 ntioy, whih reognizes the neuron-speifi -tuulin (Fig. 3). The migrting ells stine positive for TuJ1, onfirming tht they re neurons septum floor plte septum e f g x x e f septum septum septum Figure 1 Repulsion of SVZ neurons y the septum n the floorplte n expression of slit genes in the postntl septum., Chins of ells migrte out of SVZ explnts symmetrilly when ulture lone in mtrigel. In the presene of the septum () or floorplte (), there re more migrting ells in the qurnt of the SVZ explnt istl to the septum thn in the proximl qurnt., As reporte 31, the septum is repulsive to SVZ ells in ollgen gel. e, A oronl setion showing expression of -1 in the septum n the neoortex of postntl y 3 (P3) rts. f, A oronl setion showing expression of -2 in the septum, the horoi plexus n the neoortex of P3 rts. g, A sgittl setion showing expression of -1 in the septum n the neoortex of P3 rts. Rostrl is to the left n orsl is up. m-1 m-1 Mtrigel Collgen gel Figure 2 Effet of on neurons migrting from SVZ explnts.,, e, Distriution of hins of ells from the SVZ explnts, o-ulture with n ggregte of ontrol HEK ells (), with ells expressing x () or m-1 (e) in mtrigel for one (, ) or two ys (e). f, Distriution of ells migrting out of the SVZ explnts fter eing o-ulture with ontrol HEK ells (), with ells expressing x () or m-1 (f) in ollgen gel Mmilln Mgzines Lt 332 NATURE VOL JULY

3 rtiles (Fig. 3 ). The uneven istriution of TuJ1 stining within the SVZ explnts inites tht neurons migrte within eh explnt wy from the ells (Fig. 3). These finings oul e expline in two wys: either repels neurons, or inhiits migrtion. To istinguish etween them, we ple two SVZ explnts t ifferent istnes from single ggregte of ells. The istne from the ells to the proximl sie of the more istnt explnt ws equl to, or slightly frther thn, the istne from the ells to the istl sie of the loser explnt (Fig. 4). In oth explnts there were more ells in the istl qurnts. These results re est expline y repulsive tivity of, rther thn inhiition of migrtion y. Next we ple n SVZ explnt on top, rther thn to the sie, of -expressing ells. Neurons still migrte out of the SVZ (Fig. 4) (16 explnts), rguing ginst inhiition of neuronl migrtion y. These results inite tht is repulsive to, rther thn inhiitory of, migrting neurons. To test whether SVZ ells respon to the solute onentrtion of or to onentrtion grient of the protein, we ple SVZ explnts etween two ggregtes of ells. When n SVZ explnt ws ple t unequl istnes from two ggregtes, neurons migrte wy from the loser ggregte (Fig. 4e). When SVZ explnts were ple t n equl istne from two ggregtes, the ells migrte out symmetrilly (Fig. 4f). The simplest explntion for these oservtions is tht SVZ ells respon to onentrtion grient of the protein. To etermine the effetive istne of, multiple SVZ explnts were ple t ifferent istnes from ontrol or ells. When the explnts were within 1 mm of the ells, neurons migrte symmetrilly (Fig. 4h). n neuronl migrtion in the RMS Although n repel neurons migrting out of SVZ explnts in ollgen gels n in mtrigel, the question of whether n guie neurons migrting in their nturl pthwy ws not resse. We evelope n ssy to investigte the effet of on neurons migrting from the SVZ into their nturl pthwy, the RMS, towrs the olftory ul. Sgittl setions of postntl rins ontining the SVZ, the RMS n the olftory ul were isolte n ulture. Crystls of the lipophili ye 1,1 -ioteyl- 3,3,3,3 -tetrmethylinoroynine perhlorte (DiI) were ple into the SVZ to lel neuronl preursors. After 24 h in ulture, migrting neurons were foun in the RMS (Fig. 5). To test the effet of, ontrol HEK ells or HEK ells stly expressing were pre-lelle with nother lipophili ye 3,3 -ioteyloxroynine (DiO), n ple on top of the RMS. With ontrol HEK ells, SVZ neurons migrte into the RMS (Fig. 5 ) (14/14 explnts). In ontrst, when ells were ple on the RMS, few SVZ neurons migrte into the RMS (Fig. 5 f) (16/16). These results provie strong eviene initing tht n regulte neuronl migrtion in nturl pthwys. Role of enogenous in the septum The septum ontins repulsive tivity for SVZ neurons 31.We hve lso foun tht slit genes re expresse in the septum n tht proteins re repulsive to SVZ neurons. Together, these results inite tht enogenous my e involve in repelling SVZ neurons. To test iretly whether enogenous ontriutes to the repulsive tivity in the septum, we onstrute plsmi tht e f % of ells migrte further thn X istl proximl qurnt SVZ qurnt istl proximl Exp. Cells migrtion istne (µm) Figure 3 Neuronl nture n istriution of SVZ neurons., Cells migrting out of the SVZ explnt., The sme ells s in, stine with the TuJ1 ntioy. Uner ifferent fol plnes, essentilly ll ells re TuJ1 positive., Superimposition of right light n fluoresent views of nother explnt o-ulture with -expressing ells. Note the symmetri istriution of neurons within the SVZ explnt., Quntifition of the effet of on migrting SVZ neurons fter TuJ1 stining. X xis, istne of neurons from the ege of SVZ explnts; Y xis, perentge of neurons tht hve migrte further thn the istne inite on the X xis. Similr results hve een otine from five experiments. g h Figure 4 Sptil reltionship etween SVZ explnts n -expressing ells. Results of o-ultures in ollgen gels re shown.,, SVZ explnts ple next to HEK ells.,, SVZ explnts ple on top of HEK ells n migrting neurons revele y stining with the TuJ1 ntioy. e, When single SVZ explnt ws ple etween two ggregtes of ells, the symmetri istriution of ells shows tht neurons migrte oring to guine from the loser ggregte. f, When two SVZ explnts were ple t n equl istne from two ggregtes of ells, the istriution of SVZ neurons is symmetri. g, h, Plement of multiple SVZ explnts llows etermintion of the effetive istne of, with the most istl explnt t 1 mm Mmilln Mgzines Lt NATURE VOL JULY

4 rtiles Sep + Sep + RooN e Sep+C Sep+R Figure 5 Migrtion of neurons in the RMS. DiI rystls were ple into SVZ in sgittl setions of postntl rt rins to lel ells (re in,, e, f) migrting into the RMS. or HEK ells were lelle with DiO (green in,,, f)., ifferent views of the sme slie in whih n ggregte of ontrol HEK ells were ple on top of the RMS. f, Different views of the sme slie in whih n ggregte of ells were ple on top of the RMS. In ll pnels, the upper right orner is towrs the SVZ, the origin of the RMS, wheres the lower left orner is towrs to the olftory ul, the en of the RMS. expresse RooN, the extrellulr omin of Rounout (Roo), reeptor for 34, After omplementry DNA trnsfetion into HEK 293T ells, RooN protein ws serete into the meium n oul in to the protein (Fig. 6). Beuse RooN lks the trnsmemrne n intrellulr omins, it oul e ompetitive inhiitor of Roo signlling. To investigte whether RooN oul inhiit the tivity in the septum, we ple explnts of the septum on top of either ontrol HEK ells or ells expressing RooN, n then o-ulture them with explnts of the SVZ, n exmine the istriution of migrting SVZ neurons. When ulture on top of the ontrol ells, the septum ws effetive in repelling SVZ neurons (Fig. 6). RooN ells signifintly reue the numer of explnts with symmetri istriution of migrting neurons (Fig. 6, ). Although these results i not istinguish whih enogenous ws involve, they inite tht enogenous ontriutes to the repulsion of SVZ neurons y the septum. Disussion We hve shown tht is hemorepellent for neurons migrting from the SVZ n tht ts s iffusile moleule, the onentrtion grient of whih guies the iretion of migrting neurons. is, to our knowlege, the first moleule iretly shown to e suffiient for the iretionl sensing of migrting neurons. With previous finings tht is n xon repellent 34 38, these results lso support the ie tht there re t lest some moleulr guine mehnisms in ommon etween xon projetion n neuronl migrtion. is serete protein Our t inite tht n guie f R R+S (I) R+S (m) RN+S RN % of explnts with symmetri migrtion Figure 6 Inhiition of the repulsive tivity in the septum y RooN.,, Distriution of SVZ neurons fter o-ulturing with septl explnts on top of either ontrol HEK ells () or HEK ells expressing RooN ()., Bining of RooN to. Numers on the left inite reltive moleulr mss. HA-tgge full-length Roo (lne 1) n RooN (lne 5) were etete y n nti-ha ntioy; Roo ws o-inute either with My-tgge x lyste (lne 2) or with My-tgge x in the meium (lne 3) n Roo ws etete y the nti- HA ntioy fter immunopreipittion with the nti-my ntioy. Lne 4: RooN ws o-inute with My-tgge x in meium n etete y the nti-ha ntioy fter immunopreipittion with the nti-my ntioy., Perentge of SVZ explnts with symmetri migrtion. Sep þ C, results with the septl explnts ulture on top of ontrol ells (n ¼ 36), Sep þ R, results with the septl explnts ulture on top of RooN ells (n ¼ 73). The ifferene etween these two groups ws sttistilly signifint (P 0:05 in Stuent s t-test). neuronl migrtion without ontt etween the ells responing to n those prouing. It is unlikely tht ts iniretly y regulting the ifferentition of HEK ells or ells in the SVZ explnts. The repulsion of iniviul neurons y in ollgen gels further supports the ie tht ts iretly on migrting neurons. Our stuies hve shown tht is repulsive ue, rther thn n inhiitory ftor, for migrting neurons. The ehviour of SVZ explnts t ifferent istnes from two soures inites tht the onentrtion grient of is likely to e responsile for iretionl sensing. The ephrins n prevent neurl rest ells from migrting on ul somites Although the ft tht ephrins re memrne-nhore proteins mkes it unlikely tht they t s iffusile guine ues, it remins to e seen whether n ephrins shre ny moleulr mehnisms. In the olftory system, interneuron preursors migrte severl millimetres in the RMS from the SVZ to the olftory ul. The tivities of the septum n inite tht shoul e le to guie t lest the initil migrtion of ells from the SVZ. It is not ler whether grient of is responsile for guiing neuronl migrtion in the entire RMS. In our experiments with SVZ explnts, the ells oul not t t istnes greter thn 1 mm. However, it nnot e rule out tht n enogenous grient oul e li own y the septum over longer time over the entire RMS. It is lso possile tht other ues, ting in or roun the RMS, my ffet the migrtion of SVZ ells. Beuse signifint influene over the iretion of SVZ ell migrtion ws only exerte y the septum, ut not y the trget tissue (the olftory ul or y tissues surrouning the RMS 31, it seems resonle to suppose tht, if other moleules help to guie SVZ migrtion, they my t not just y themselves, ut in oorintion with. Moleules suh s the neurl ell hesion moleule (NCAM), whih is expresse in the RMS n is neessry for SVZ migrtion 30,42,43, my t lolly in the migrtion pthwy to 1999 Mmilln Mgzines Lt 334 NATURE VOL JULY

5 rtiles llow hesion of SVZ neurons. It will e interesting to test whether there is ny funtionl reltionship etween n NCAM. The emryoni septum n repel emryoni olftory ul xons n neontl SVZ neurons, wheres the neontl septum n repel only migrting neontl SVZ neurons ut not emryoni olftory ul xons 31. The expression n tivity of slit in oth emryoni n neontl septum inite tht either ifferent forms of proteins my e responsile for xon guine n neuronl migrtion, or there my e other moleules moulting the tivity of the sme proteins. Although only the ul, not the rostrl, septum hs een shown to repel SVZ neurons 31, our in situ hyriiztion hs not revele ovious ifferenes in the expression of slit genes in the rostrl n ul septum, gin rising the possiility of other moulting tivities. Funtionl stuies of mke it interesting to onsier the reltionship etween neuronl migrtion n xon guine. Both proesses involve ell motility, ut the entire ell oy moves in one se, wheres only xons move in the other se. Defets in oth xon pthwys n neuronl position n e use y muttions in the sme genes. Loss-of-funtion muttions in netrin, its Cenorhitis elegns homologue un-6 or their reeptors result in norml xon projetion n ell position in the mouse n C. elegns 47. Geneti n phenotypi nlyses in C. elegns inite tht un-6 is require for the migrtion of mesoerml ells 47. Experiments with ommissurl n other explnts hve emonstrte tht Netrin-1 n iretly guie xon projetions 44, n reent eviene from explnt stuies inites tht efets in pontine neuronl migrtion in mouse mutnts similrly reflet iret ttrtive tion of Netrin-1 on these neurons (K. Lee, H. Simon, M. Tessier-Lvigne n D. O Lery, personl ommunition). It will e interesting to see if the sme is true of other ell migrtion efets reporte in mie mutnt for Netrin or Netrin reeptors, or whether some of them re ue to iniret effets on the projetion of xons or glil fires long whih neurons my migrte (see ref. 48 for review of neurophili n gliophili migrtion). Geneti n explnt experiments inite tht ts iretly s hemorepellent for xons 34,35,37,38. Muttions in roo n slit in C. elegns n Drosophil result in efetive ell positioning, suggesting tht Roo signlling is require for ell migrtion 37,49. Geneti n phenotypi nlysis in Drosophil suggests tht slit is require for the migrtion of musle preursor ells 37 ; ertin musle preursor ells, whih normlly migrte wy from the emryoni miline, o not migrte in slit mutnts (T. Ki n C. S. Goomn, personl ommunition). Our present results hve shown iretly tht is suffiient to t s repellent for migrting neurons. hs therefore een iretly emonstrte to guie oth neuronl migrtion n xon projetion It seems tht whether guies the movement of ell oies or xons epens on the type of responing ell. It is unler, however, whether intrellulr mhinery or extrellulr mehnisms etermine the type of response. Is the sme form of protein involve? Are there more intrellulr omponents to enle neuron to move its ell oy? Or oes the sene of ritil omponents use neuron to move its ell oy rther thn its xon? The vilility of guine ue ting in vitro on oth migrting neurons n projeting xons offers the prospet of ressing these questions experimentlly. Cell migrtion is ruil in other evelopmentl n non-evelopmentl proesses, inluing migrtion of ortil neurons rilly long glil fires, migrtion of neurl-rest preursor ells, gstrultion, hert formtion, musle evelopment, ngiogenesis, leukoyte hemotxis n tumour metstsis. We hve otine eviene for funtion in other popultions of neurons 50, initing tht guine ues my e ruil in mny proesses involving neuronl migrtion. It will therefore e interesting to test whether the fmily or other guine ues n guie migrting ells in multiple systems in norml n norml situtions. For migrting ells tht o not respon to, it will e interesting to investigte whether the introution of Roo or other potentil reeptor omponents n onfer responsiveness to. This my roen potentil therpeuti pplitions of proteins in ontrolling unwnte ell migrtion n in elivering ells to the intene trget region.... Methos In situ hyriiztion. Brins were remove from P3 rts, n 16- m oronl setions were ut with ryostt, ollete on superfrost plus slies n ir rie. 35 S-lelle slit proes were me n ounts per minute (.p.m.) of rioproes ws pplie to eh slie with rin slies. The slies were overli with overslips n inute in humiifie hmers t 55 C overnight efore eing wshe with wshing uffer (50% formmie, 2 SSC n 0.1% -merptoethnol) t 65 C for 30 min, rinse for 10 min in RNse uffer (0.5 M NCl, 10 mm Tris-HCl, ph 7.5, 5 mm EDTA) n trete for 30 min t 37 C with 20 gml 1 RNse A. The slies were then ple t 65 C in the wshing uffer for 5 min t 37 C in2 SSC for 15 min n t 37 Cin0:1 SCC for 15 min efore eing ehyrte. The slies were ote with Kok NTB2 emulsion n store t 4 C for 3 weeks efore eing evelope in Kok D19 n ounterstine with hemtoxylin. Co-ulture of explnts. Brins from neworn Sprgue-Dwley rts (postntl ys 3 7) were emee in 7% low melting-point grose prepre in phosphte-uffere sline. Coronl n sgittl setions of 400 mm were ut with virtome. Tissue within the orers of the SVZ in oronl setions ws issete out to mke SVZ explnts of mm in imeter. We isolte septl explnts from sgittl setions n ple them into the ollgen gel or mtrigel s esrie 31,32,34. They were ulture for h uner 5% CO 2 in F-12 meium supplemente with 10% fetl ovine serum, n peniillin n streptomyin. Co-ulture of SVZ explnts with ell ggregtes. We hve two stle HEK ell lines 34. One ws trnsfete with plsmi expressing x, n the other with the vetor plsmi. Both lines went through similr seletion n suloning. The line ws onfirme y western lotting to express the full-length x protein tgge with the My epitope. We lso trnsiently trnsfete HEK 293 ells with either vetor or plsmi expressing fulllength My-tgge m-1. The expression of m-1 ws lso onfirme y western nlysis. Beuse x n m-1 ehve similrly in ll tests, we usully use the x stle line. We me ggregtes of the ontrol or -expressing ells y the hngingrop metho. They were ple into ollgen gels or mtrigel with SVZ explnts, n ulture s esrie ove. For immunohistohemistry, smples were fixe with 4% prformlehye n pre-inute with 10% got serum in 0.1 M phosphte uffer (ph 7.4) for 1 h. Inution with the primry ntioy (TuJ1, 1:200 ilution) ws rrie out overnight t 4 C. They were wshe n then inute with got ntimouse seonry ntioy onjugte to Cy3 for 1 h t room temperture, n wshe efore eing mounte with glyerol. For quntifition, we otine immunofluoresene imges of smples stine with TuJ1, using Zeiss mirosope. We quntifie ell istriution y nlysing the istne etween the ell oy n the nerest ege of the explnt, using the IPL progrm. To test whether the extrellulr omin of Roo (RooN) oul inhiit the septl tivity, ggregtes were me from HEK ells trnsiently trnsfete with plsmi expressing RooN. These ells were emee in thin lyer of ollgen gel. Explnts of the ul septum were ple on top of the RooN ells n o-ulture with SVZ explnts in ollgen gel mtries. Slie ssy with the RMS. Sgittl setions of postntl rts were ut with virtome n those ontining the SVZ, the RMS n the olftory ul were use. Crystls of DiI were inserte into the SVZ. The slies were ulture in similr wy to the SVZ explnts. or -expressing HEK ells were lelle with DiO. Aggregtes of these ells were ple on top of the RMS. We visulize DiI n DiO signls with ifferent filters uner the mirosope. Imges were tken with Spot mer n store on the omputer. Bining of RooN to. Full-length Roo hs 1,660 resiues; RooN ws me y tgging hemgglutinin (HA) epitope to mino-i resiue 718. Lystes of ells expressing HA-tgge full-length Roo or RooN were 1999 Mmilln Mgzines Lt NATURE VOL JULY

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Neuron 4, (1990). 48. Rki, P. Priniples of neurl migrtion. Experienti 46, (1990). 49. Zllen, J. A., Yi, B. A. & Brgmnn, C. I. The onserveimmunogloulin superfmily memersax-3/ Roo irets multiple spets of xon guine in C. elegns. Cell 92, (1998). 50. Zhu, Y., Li, H. S., Zhou, L., Wu, J. Y. & Ro, Y. Cellulrn moleulrguineof GABAergineuronl migrtion from n extrortil origin to the neoortex. Neuron (in the press). Aknowlegements. We thnk W. Yun n D. Ornitz for mouse slit DNAs; J. Xu, Q. Wng n L. Zhou for help with in situ hyriiztion; W. Gn n J. Lihtmn for help with onfol imging n nlysis; J. Brunstrom n A. Perlmn for isussions; C. S. Goomn n M. Tessier-Lvigne for omments; NIH, NSFC n SCST for support; n the John MerkFun, NSFC n the LeukemiSoiety of Ameri for sholr wrs (to Y.R. n J.Y.W.). Corresponenen requestsfor mterils shoul e resse to J.Y.W. or Y.R. (e-mil: jwu@phrmsun. wustl.eu; royi@thlmus.wustl.eu) Mmilln Mgzines Lt 336 NATURE VOL JULY