Development 127, 1359-1372 (2000) 1359 Printed in Great Britain © The Company of Biologists Limited 2000 DEV9687

Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system

S. Alcántara1,2,*, M. Ruiz1, F. De Castro2, E. Soriano1 and C. Sotelo2 1Department of Animal and Plant Cell Biology, Faculty of Biology, University of Barcelona, Barcelona E 08028, Spain 2Institut National de la Santé et de la Recherche Médicale U-106, Hôpital de la Salpétrière, 75651 Paris Cédex 13, France *Author for correspondence (e-mail: [email protected])

Accepted 14 January; published on WWW 7 March 2000

SUMMARY

Netrin 1 is a long-range diffusible factor that exerts postnatal cerebellum, netrin 1 repels both the parallel chemoattractive or chemorepulsive effects on developing fibres and migrating granule cells growing out from growing to or away from the neural midline. Here explants taken from the external germinal layer. The we used tissue explants to study the action of netrin 1 in the developmental patterns of expression in vivo of netrin 1 and migration of several cerebellar and precerebellar cell its receptors are consistent with the notion that netrin 1 progenitors. We show that netrin 1 exerts a strong secreted in the midline acts as chemoattractive cue for chemoattractive effect on migrating neurons from the precerebellar neurons migrating circumferentially along embryonic lower rhombic lip at E12-E14, which give rise the extramural stream. Similarly, the pattern of expression to precerebellar nuclei. Netrin 1 promotes the exit of in the postnatal cerebellum suggests that netrin 1 could postmitotic migrating neurons from the embryonic lower regulate the tangential migration of postmitotic rhombic lip and upregulates the expression of TAG-1 in premigratory granule cells. Thus, molecular mechanisms these neurons. In addition, in the presence of netrin 1, the considered as primarily involved in axonal guidance migrating neurons are not isolated but are associated with appear also to steer neuronal cell migration. thick fascicles of , typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell Key words: Netrin 1 receptors, Cerebellum, Pontine nucleus, progenitors, did not respond to netrin 1. Finally, in the Neuronal migration, Chemoattraction, , Migration

INTRODUCTION chemoattractive response to netrin 1 (Keino-Masu et al., 1996; Fazeli et al., 1997). Although the mechanisms that guide neurons during migration Loss-of-function mutations of either netrin 1 (Ntn1) or are not well understood, the existence of restricted migratory deleted in colorectal cancer (Dcc) in mice confirm their pathways suggests that neuronal migration is under the control involvement in the formation of commissural connections of specific guiding cues (Anderson et al., 1997; Soriano et al., (Serafini et al., 1996; Fazeli et al., 1997). A second DCC- 1997; Wichterle et al., 1997; Pearlman et al., 1998). In related , neogenin, also binds netrin 1 but its function addition, the heterogeneity of migratory routes followed by remains unknown (Keino-Masu et al., 1996). In addition, netrin migratory neurons that share their origin in a given germinal 1 also acts as a chemorepulsive cue for the axons of the matrix indicates that distinct migratory neurons may respond trochlear, trigeminal, facial and glossopharyngeal motor nuclei differentially to similar extracellular signals. (Colamarino and Tessier-Lavigne, 1995; Varela-Echavarria et are a family of -related secreted that al., 1997). Such chemorepulsive action is probably not may act either as chemoattractants or as chemorepellents for mediated by DCC, but by a second family of netrin 1 receptors, distinct developing axons (Kennedy et al., 1994; Serafini et al., the unc5-related proteins (Leung-Hagesteijn et al., 1992; 1994; Colamarino and Tessier-Lavigne, 1995). Netrin 1 is Hamelin et al., 1993; Leonardo et al., 1997). responsible for the attraction of commissural axons towards the During the development of the nervous system, considerable midline in the (Kennedy et al., 1994; Serafini et al., distances separate the final destination of migratory neurons 1996), although these axons also require the interaction of at from their proliferative matrices. Recent evidence, in both least two adhesion molecules, Axonin-1/TAG-1 and Ng- vertebrates and , suggests that some cues CAM/L1, to reach their targets (Stoeckli and Landmesser, governing axonal growth may also be involved in neuronal 1995). DCC (deleted in colorectal cancer), a neural cell migration (Culotti and Kolodkin, 1996; Goodman, 1996; Hu adhesion molecule of the Ig superfamily (Ig-CAMs), is a and Rutishauser, 1996; Tessier-Lavigne and Goodman 1996). component of the receptor complex that mediates the For instance, the lack of pontine nuclei and the severe atrophy 1360 S. Alcántara and others of the inferior olivary complex in both netrin 1 and Dcc repels parallel fibres and granule cells from postnatal EGL knockout mice suggest that netrin 1 provides cues not only for explants. We also show that the patterns of expression of netrin axonal guidance but also for neuronal migration (Serafini et al., 1 and its receptors (Dcc, neogenin, unc5h2 and unc5h3) are 1996; Keino-Masu et al., 1996; Bloch-Gallego et al., 1999). consistent with a role for this chemotactic molecule in the Moreover, mutations in the unc5h3 are responsible for the migration of neurons originated from the rhombic lip in vivo. murine “rostral cerebellar malformation”, leading to aberrant migration of granule and Purkinje cells in the rostral direction and loss of the rostral cerebellar boundary (Ackerman et al., MATERIALS AND METHODS 1997; Leonardo et al., 1997; Przyborski et al., 1998). The rhombic lip, the germinative neuroepithelium located Animals around the alar recess of the fourth ventricle (His, 1891), gives OF1 and postnatal mice (Iffa Credo, France) were used. The rise to neurons that follow distinct migratory pathways and mating day was considered embryonic day 0 (E0) and the day of birth substrates (Rakic, 1990). In the mouse, the upper rhombic lip postnatal day 0 (P0). After ether anaesthesia of the dams, embryos (uRL; the rostral aspect of the rhombic lip or germinal trigone; were dissected out and collected in 0.1 M phosphate-buffered saline Altman and Bayer, 1997) produces progenitor cells from E13 (PBS) and 0.6% D-glucose, and used for cultures or fixed with 4% onwards that spread in a rostromedial direction to cover the paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.3. Embryos older than E14 and postnatal animals were transcardially perfused whole cerebellar surface by E17 (neurophilic tangential with the above fixative. After postfixation, the brains were migration). These progenitors form the external germinal layer cryoprotected in 30% sucrose and frozen on dry ice. Coronal sections (EGL), which is the origin of the cerebellar granule cells (Gao 50 µm thick were collected in a cryoprotectant solution (30% glycerol, and Hatten, 1994; Mathis et al., 1997). During the first two 30% ethylene glycol, 40% 0.1 M PBS), and stored at −30¡C until use. postnatal weeks in mice, postmitotic neurons from the EGL migrate inwards along Bergmann (gliophilic radial Explant cultures migration) and differentiate into granule cells under the The uRL and lRL from E12-E15 embryos were dissected out as a Purkinje cell layer (Miale and Sidman, 1961; Rakic, 1971). single piece and cut into 150-300 µm tissue pieces with fine tungsten µ The lower region of the rhombic lip (lRL) gives rise to needles (Fig. 1A). 250 m parasagittal sections of postnatal cerebella precerebellar neurons in the inferior olive (IO), lateral reticular were cut with a tissue chopper. After removing the meninges, 150- 300 µm tissue pieces from the EGL were obtained (Fig. 1B). nucleus (LRN), external cuneate nucleus (ECN), nucleus Explants were cocultured at a distance (200-600 µm) with reticularis tegmenti pontis (NRTP) and basilar pons (BP). aggregates of EBNA-293 cells stably transfected with a construct Although there is some spatial and temporal overlap, neurons encoding netrin 1-c-myc, or with the vector alone (Keino-Masu et al., for each nucleus have a distinct temporal pattern of birthdates 1996). Explants and cell aggregates were embedded in rat-tail and, remarkably, distinct migratory routes. Thus, neurons collagen as described (Lumsden and Davies, 1986) and cultured in destined to IO are generated at E10-E11 and neurons fated to DMEM (Seromed) supplemented with L-glutamine, NaHCO3, D- the LRN and ECN become postmitotic at E11-E12. In contrast, glucose, and supplements B27 and N2 (all from Gibco Life pontine neurons (NRTP and BP) are generated during a Technologies), for 24-72 hours in a 5% CO2, 95% humidity incubator protracted period, extending from E12 to E16 with a late peak at 37¡C. To monitor the expression of netrin 1 and the distance at E14-E15 (Taber-Pierce, 1966). Although all precerebellar covered by the netrin 1 gradient, some cocultures were fixed and immunostained with the monoclonal 9E10 anti-c-myc antibody (Santa neurons follow a neurophilic type of migration along Cruz). Secreted netrin 1-c-myc was detected in a gradient of intensity circumferential routes at the periphery of the brainstem, IO over a distance of about 300-400 µm away from the EBNA-293 neurons migrate along the submarginal stream (Bourrat and aggregates after 2 days in vitro (div), indicating that a long-range Sotelo, 1988; Altman and Bayer, 1997), while LRN and gradient of netrin 1 is formed in our experimental conditions. In some ECN neurons migrate through the marginal stream (Bourrat experiments, explants were incubated with medium conditioned for and Sotelo, 1991; Altman and Bayer, 1997). NRTP and BP 36 hours with EBNA-293-netrin 1 or control cells diluted 1:1 with neurons follow a distinct rostral migratory route towards fresh medium. their respective pontine nuclei, named the pontobulbar or 28 experiments were done and up to 2000 explants analyzed. pontomedullary stream (Harkmark, 1954; Rakic, 1985; Ono Immunohistochemistry and Kawamura, 1990). Finally, while neurons destined to the Explants were fixed in 4% PFA for 1 hour and processed for the LRN and ECN cross the midline through the most ventral visualisation of neuronal bodies and neurites. After several rinses in region of the floor plate (Bourrat and Sotelo, 1991; Altman and PBS, cultures were incubated with monoclonal antibodies against Bayer, 1997), olivary and pontine neurons stop ipsilaterally on neuron-specific class III β-tubulin (1:3000; clone Tuj-1 Babco; the side where they were generated (Taber-Pierce, 1966; Moody et al., 1989), intracellular DCC (1:500; clone GA7 499, Ellerberger et al., 1969; Bourrat and Sotelo, 1988, 1990). Pharmigen), C-myc (1:200, Santa Cruz) or rabbit polyclonal The complexity of migratory routes and locations taken by antibodies against intracellular human DCC (1:1000, Fabre et al., rhombic lip-originated neurons suggests that multiple and 1999), L1 (1:2000, Persohn and Schachner, 1987), TAG-1 (1:2000, specific guiding and stopping factors are involved in their Karagogeos et al., 1991), calbindin D28k (1:10000, Swant) and GFAP migration and allocation. To understand the molecular bases of (1:1000, DAKO). The incubation with secondary antibodies (1:200; neuronal cell migration in the rhombic lip, here we study the Vector Labs.) was followed by the streptoavidin-peroxidase complex (1:400, Amersham). Enzymatic reaction was developed with effects of netrin 1 in vitro in explants taken from E12-E14 diaminobenzidine and H2O2. Alternatively, FITC-conjugated rhombic lip and from its postnatal derivative, the EGL. We secondary antibodies, or Texas Red-coupled streptoavidin were used, show that secreted netrin 1 exerts an attraction on both axons together with fluorescent nuclear markers (bisbenzimide or ethidium and neurons arising from embryonic lRL explants, whereas it bromide) to visualized both cell bodies and neurites. has no effect on uRL explants. In contrast, netrin 1 strongly Sections from different developmental stages, some of them Netrin 1 guides neuronal migration 1361 previously hybridised for netrin 1 expression, were immunolabeled background levels. For immunocytochemical controls, omission of with anti-DCC antibodies. the primary antibodies prevented immunostaining. In situ hybridisation Analysis and quantification In situ hybridisation was performed on free-floating sections Hybridised and immunostained sections were examined on a Zeiss essentially as described (Alcántara et al., 1998). Sections were Axiophot microscope. The delimitation of regional and laminar permeabilized in 0.2% Triton X-100 (15 minutes), treated with 2% boundaries was performed according to Jacobowitz and Abbott H2O2 (15 minutes), deproteinized with 0.2 N HCl (10 minutes), fixed (1998). Rhombic lip or EGL explants cultured alone displayed a in 4% PFA (10 minutes) and blocked in 0.2% glycine (5 minutes). radial pattern of outgrowth. Neurite quantification was Thereafter, sections were prehybridised at 60¡C for 3 hours in a obtained from explants immunostained with Tuj-1, whereas the solution containing 50% formamide, 10% dextran sulphate, 5× quantification of the neuronal migration was done on bisbenzimide- Denhardt’s solution, 0.62 M NaCl, 10 mM EDTA, 20 mM Pipes (pH stained explants. In both instances, explants were photographed 6.8), 50 mM DTT, 250 mg/ml yeast t-RNA and 250 mg/ml denatured (75× final magnification) and the field was divided into four salmon sperm DNA. netrin 1, Dcc, unc5h2, unc5h3 and neogenin quadrants as illustrated in Fig. 1C. For each explant, the areas riboprobes were labelled with digoxigenin-d-UTP (Boehringer- covered by either Tuj-1-positive neurites or by bisbenzimide-stained Mannheim) by in vitro transcription. A cDNA fragment of 2.7 kb cells (from the border of explants to the outer perimeter of the encoding the 3′ unstranslated region of mouse netrin 1 (Serafini et al., expanding processes or neurons) were measured in the distal and 1996) using T3 polymerase (Ambion), a cDNA fragment proximal quadrants using the IMAT image analysis program corresponding to the rat Dcc cytoplasmic region of 0.85 kb, and a (Scientific-Technical Services, University of Barcelona). Data were fragment of 0.72 kb encoding the rat neogenin cytoplasmic region presented as mean ± s.d. For the statistical analysis of the areas, (Keino-Masu et al., 1996) using SP6 polymerase (Promega). cDNA Anova and LSD tests were performed with the Statgraphic statistical fragments of the rat unc5h2 (1.5 kb) and rat unc5h3 (1.6 kb) package. To determine the preferential direction of the outgrowth, (Leonardo et al., 1997) were transcribed using T7 polymerase the ratio of the surface in the proximal and distal quadrants was (Ambion). Labelled antisense cRNA was added to the obtained for each explant, which gives a ratio of 1 for radial prehybridisation solution (250-500 ng/ml) and hybridisation was outgrowth. Ratios differing from the mean ratio obtained for carried out at 60 ¼C overnight. Sections were then washed in 2× SSC explants cultured with control cells by more than one s.d. were (30 minutes, room temperature), digested with 20 mg/ml RNAse A assigned therefore to the group attracted or repulsed by netrin 1. (37¡C, 1 hour), washed in 0.5× SSC/50% formamide (4 hours, 55¡C) and in 0.1× SSC/0.1% sarkosyl (1 hour, 60¡C). After rinsing in Tris- buffered saline (TBS)/0.1% Tween 20 (15 minutes), sections were RESULTS blocked in 10% normal goat serum (2 hours) and incubated overnight with an alkaline phosphatase-conjugated antibody to digoxigenin Netrin 1 effects on neurons and neurites derived (Boehringer-Mannheim, 1:2000). After washing, sections were from the lower rhombic lip developed with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro- The temporal sequence of proliferation of neuronal precursors 3-indolyl phosphate (BCIP) (Life Technologies), mounted on in the lRL allows us to pinpoint the classes of precerebellar gelatinised slides and coverslipped with MowiolTM. neurons affected by netrin 1 in their migration. In our Controls experiments, we selected rhombic lip explants taken from E12- Control hybridisations, including hybridisation with sense E14 embryos. At E12, all IO neurons have left the lRL and digoxigenin-labelled riboprobes or RNAse A digestion prior to most of the cells in these explants corresponded to LRN and the hybridisation, prevented alkaline phosphatase staining above ECN neurons. In contrast, the neurons generated at E14 are

Table 1. Quantitative analysis of the neuritic outgrowth (A) and cellular migration (B) in explants from the lower rhombic lip at different ages 2 Mean area ± s.d. (mm ) Statistical Number of Orientation Culture condition Proximal quadrant Distal quadrant significance explants + = − % + (A) Neurite outgrowth (Tuj-1 immunostaining) lRL E12 net 1 2 div 0.302±0.103 0.110±0.072 ** 23 21 2 0 91 lRL E12 cont 2 div 0.129±0.058 0.211±0.128 7 1 4 2 29 lRL E14 net 1 1 div 0.072±0.052 0.024±0.018 ** 17 10 7 0 59 lRL E14 cont 1 div 0.031±0.001 0.025±0.004 4 1 3 0 25 lRL E14 net1 2 div 0.296±0.178 0.097±0.039 ** 23 23 0 0 100 lRL E14 cont 2 div 0.115±0.073 0.131±0.083 7 0 7 0 0

(B) Neuronal cell migration (bisbenzimide nuclear staining) lRL E14 net1 2 div 0.380±0.177 0.056±0.025 ** 6 6 0 0 100 lRL E14 cont 2 div 0.056±0.018 0.045±0.014 6 0 6 0 0

div, days in vitro. The area occupied by the bulk of the neuritic processes and/or by the cell bodies in the proximal and distal quadrants in explants cocultured with aggregates of EBNA-293 control cells or with cells transfected with the netrin 1-c-myc construct was measured and expressed as means ± s.d. Significant differences between proximal and distal quadrants were calculated from crude data using the LSD test. **P=0.01. The preferential orientation of the outgrowth was determined for each explant dividing the area occupied by the neuritic outgrowth or cellular migration in the proximal quadrant into that of the distal quadrant, which gives a ratio of 1 for radial outgrowth. The limits between categories were calculated from the s.d. obtained for the ratios in explants cocultured with control cells. div, days in vitro. =, radial: ratio = 1±s.d.; +, attraction: ratio >1+s.d.; −, repulsion: ratio <1−s.d.; %+, percentage of the explants attracted by netrin 1. 1362 S. Alcántara and others A B E12-E14 Postnatal

EGL ML PL 4V IGL WM

uRL Rostral Caudal lRL

Fig. 1. Schematic drawings showing the anatomical location of the different types of explants used and the analysis applied. (A) Dorsal C view of an E12-E14 showing the location Distal Proximal of the rhombic lip around the IVth ventricle. The upper region or germinal trigone (uRL) is in purple and the lower region (lRL) is in yellow. (B) Sagittal section of a postnatal cerebellum showing the layers used for EGL explants. EBNA cells (C) Scheme illustrating the method used for the quantification of neuritic outgrowth and neuronal migration. For each explant, the area occupied respectively by neuritic processes and cell bodies was measured in the quadrants (marked in black) proximal and distal to the cell aggregates. exclusively destined to pontine nuclei (Taber-Pierce 1966, see were cultured with netrin 1-secreting cells, 91% of the E12 lRL also Discussion). explants (Table 1) showed a marked asymmetrical pattern of We first tested whether netrin 1 acts as an attractive or neurite outgrowth with far fewer processes in the distal than in repulsive cue for LRN and ECN neurons, by coculturing E12 the proximal quadrant (data not shown). This was so even when lRL explants with aggregates of netrin 1-secreting cells. tissue explants were located at long distances (around 600 µm) Explants cultured with control EBNA-293 cell aggregates for from the cell aggregates. Moreover, the neurites in the proximal 2 div showed no preferential orientation (radial) of outgrowing quadrant formed tight, prominent fascicles, which were neurites immunostained with the Tuj-1 mAb (Table 1; Fig. 2). associated with strands of migrating neurons. Most importantly, the outgrowing neurites were almost devoid To test the effect of netrin 1 on the migration of pontine of migrating cell bodies. In contrast, when E12 lRL explants precerebellar neurons, similar in vitro experiments were carried out using E14 lRL. In cocultures with control cell aggregates, lRL neurites grew symmetrically in 75% of the 0.35 explants after 1 div and in 100% of the explants after 2 div (Table 1; Fig. 3A). After both culture times, radial emerging ) 2 0.30 neurites were poorly fasciculated (Fig. 3A), and the few Tuj- proximal 0.25 distal 1-positive neurons leaving the explants showed long neuritic processes, probably corresponding to the leading process of 0.20 migrating pontine cells (Ono and Kawamura, 1990) and

0.15 roughly bipolar morphology (Fig. 3C,E). These neurons did not migrate for long distances and formed a narrow ring of less 0.10 than 100 µm thick around the explants (Table 1; Figs 3A, 4A). In contrast, 59% (1 div) and 100% (2 div) of the explants Area of neurite outgrowth (mm 0.05 cultured with netrin 1-secreting aggregates (Table 1A; Fig. 3B) 0.00 showed prominent neurite outgrowth directed towards the cell E12 net-1 E12 cont E14 net-1 E14 cont aggregates. Most of these attracted processes had fasciculated Fig. 2. Histograms illustrating the area of neuritic processes and cell and attained the netrin 1 producing cell masses (Fig. 3B,F). bodies in the proximal and distal quadrants of E12 and E14 lower Isolated migrating neurons were practically absent in rhombic lip explants cocultured with EBNA-293 control cells or with cocultures in which cell nuclei were visualized with DNA- cells secreting netrin 1 for 48 hours. Data are means ± s.e.m.; staining dyes. On the contrary, the migrating neurons were **P=0.01, LSD test. tightly apposed, following each other to form thick neuronal Netrin 1 guides neuronal migration 1363

Table 2. Quantitative analysis of the outgrowth in explants from the uRL and EGL 2 Mean area ± s.d. (mm ) Statistical Number of Orientation Culture condition Proximal quadrant Distal quadrant significance explants + = − % − uRL E14 net 1 1 div 0.058±0.017 0.059±0.023 22 5 8 9 41 uRL E14 cont 1 div 0.073±0.036 0.062±0.027 5 1 3 1 20 uRL E14 net 1 2 div 0.083±0.052 0.115±0.043 11 1 4 6 55 uRL E14 cont 2 div 0.133±0.053 0.149±0.062 5 1 3 1 20 EGL P0 net 1 3 div 0.069±0.025 0.099±0.030 * 18 2 4 12 67 EGL P0 cont 3 div 0.112±0.040 0.122±0.045 8 1 6 1 13 EGL P2 net 1 2 div 0.065±0.023 0.103±0.024 ** 22 0 3 19 86 EGL P2 cont 2 div 0.087±0.033 0.077±0.030 9 1 7 1 11 EGL P4 net 1 2 div 0.050±0.018 0.095±0.042 ** 32 0 3 29 91 EGL P4 cont 2 div 0.173±0.047 0.172±0.042 7 0 6 1 14 EGL P5 net 1 2 div 0.062±0.026 0.131±0.051 ** 31 1 3 27 87 EGL P5 cont 2 div 0.119±0.026 0.130±0.037 11 2 7 2 18

The area occupied for the bulk of the neuritic processes and cell bodies in the proximal and distal quadrants of explants cocultured with aggregates of EBNA- 293 control cells or with cells transfected with the netrin 1-c-myc construct were measured and expressed as means ± s.d. Significant differences between proximal and distal quadrants were calculated from crude data using the LSD test. *P=0.05; **P=0.01. The preferential orientation of the outgrowth was determined for each explant by dividing the area of the outgrowth in the proximal quadrant into that of the distal quadrant, which gives a ratio of 1 for radial outgrowth. The limits between categories were calculated from the s.d. obtained for the ratios in explants cocultured with control cells. div, days in vitro. =, radial: ratio = 1±s.d.; +, attraction: ratio >1+s.d.; Ð, repulsion: ratio <1−s.d.; %−, percentage of the cultures repelled by netrin 1. chains, which emerged from the explants, mainly in their proximal quadrants (Table 1B; Fig. 3B,D), but also occasionally in their lateral quadrants (Fig. 4B). In double-stained cocultures, most of the chains of migrating cells were intermingled with the fasciculated processes (Fig. 3B,D,F) as occurs in the pontine stream in vivo (Ono and Kawamura, 1990), and spanned the distance between the lRL explants and the EBNA-293 transfected cell masses (Fig. 3B). Thus, precerebellar neurons use a neurophilic type of migration (Rakic, 1990). Moreover, a few neurites also grew in the distal quadrants, but they were not associated with massive neuronal migration (Figs 3B, 4B), which supports the suggestion that, in the cocultures, neuritic outgrowth and neuronal migration are not a unique process. These results show that netrin 1 functions as a chemotactic cue for migrating precerebellar neurons, because it imposes directionality to their migration.

Fig. 3. Netrin 1 is chemoattractive for neurites and cells originating from E14 lRL explants. E14 lRL explants cocultured 48 hours with aggregates of EBNA-293 control cells (A,C,E) or cells transfected with the netrin 1-c-myc expression vector (B,D,F). Confocal images at a medium plane of the culture showing the neuronal processes immunostained with anti-β-tubulin III antibodies, in green, and the cellular nuclei stained with ethidium bromide, in red. (C,E) and (D,F) are respectively higher magnifications of the boxes shown in A and B, which illustrate the short distance and the random orientation of migrating neurons confronted with control cells (E) and the long chains of migrating cells oriented to the source of netrin 1 (F). c, aggregate of control cells; n, aggregate of netrin 1-secreting cells. Scale bars, 200 µm (A,B) and 40 µm (C-F). 1364 S. Alcántara and others

Fig. 5. Netrin 1 effects on embryonic granule cell progenitors and neonatal granule cells. Explants from E14 uRL (A,B), P0 EGL (C,D) were cocultured for 36-72 hours with EBNA-293 control cells (A,C) or with cells transfected with the netrin 1-c-myc construct (B,D), and immunostained with anti-β-tubulin III antibodies. Note that netrin 1 exerts inhibitory effects on the neuritic outgrowth from P0 EGL explants (D). Explants from P5 EGL cultured for 24 hours with conditioned medium from parental EBNA-293 cells (E) or from cells transfected with netrin 1-c-myc construct (F) labelled with anti-β- Fig. 4. The cells and neurites attracted by netrin 1 at E14 are TAG-1 tubulin III antibodies. Note that immunoreactive fibers are reduced in immunoreactive. E14 lRL explants cocultured for 48 hours with the presence of netrin 1. Perimeters of aggregates of control cells (c) and netrin 1-secreting cells (n), outlined with black circles, white plots aggregates of EBNA-293 control cells (A,C), or with cells secreting µ netrin 1 (B,D). Explants stained with bisbenzimide (A,B) illustrate outline perimeters of uRL and EGL explants. Scale bars, 200 m. the distribution of migrating cells, and the remarkable effect of netrin 1 on neuronal migration (B). Similar explants immunostained with TAG-1 antibodies (C,D) show that, in the absence of netrin 1, few secreting netrin 1 (Fig. 4E-H). Some of the lRL explants, symmetrically arranged neurites and cell bodies exit the explants; cultured either with aggregates of netrin 1-secreting cells or in whereas, they are much more numerous and oriented towards the conditioned medium, were immunostained with anti-GFAP source of netrin 1 when cocultured with cells secreting netrin 1. antibodies. GFAP-positive cells were missing, suggesting that Comparison of C and D indicates that netrin 1 upregulates TAG-1 the migration was not gliophilic. immunoreactivity (E-H). E14 lRL explants cultured for 72 hours These experiments demonstrate that netrin 1 is not only a either in conditioned medium from EBNA-293 control cells (E,G) or chemoattractant for neurites emerging from LRN, ECN, NRTP from cells transfected with netrin 1-c-myc construct, immunostained and BP neurons generated in the lRL, but also that it with anti-TAG-1 antibodies (F,H). In the presence of netrin 1, TAG-1 chemoattracts and promotes the massive cell migration of these immunoreactivity increases and cells migrate out of the explant forming long cellular strands. (G,H) Higher magnifications of the neurons. boxes shown in E,F, illustrating the chains of migrating cells. c, control; n, netrin 1. Scale bars, 200 µm (A-F) and 75 µm (G-H). Netrin 1 effect on neurons and neurites from the upper rhombic lip and external germinal layer To test the role of netrin 1 in the two modes of migration of To substantiate the notion that migrating neurons in E14 lRL the cerebellar granule cell precursors and neurons, in vitro explants are pontine neurons, cultures were immunostained experiments were carried out with either E14 uRL explants or with anti-TAG-1 antibodies (Wolfer et al., 1994). As shown in P0-P6 EGL explants. E14 uRL explants cocultured with Fig. 4C,D, TAG-1 antibodies stained thick strands of migrating control cells emitted neurites without preferential orientation neurons with long leading edges, which were directed towards after either 1 or 2 div (Table 2). When cultured with netrin 1- the netrin 1 aggregates. Finally, the strong migration of producing cells, the neuritic outgrowth from uRL explants was postmitotic pontine precerebellar neurons produced by netrin less homogeneous (Fig. 5A,B), although no significant changes 1 was reproduced when single E14 lRL explants were cultured were noticed in the area occupied by the emitted processes with medium conditioned for 36 hours with EBNA-293 cells (Fig. 6; Table 2). Netrin 1 guides neuronal migration 1365

0.20 ) 2 0.15

Fig. 6. Histograms illustrating the proximal area of neuritic outgrowth in the 0.10 distal proximal and distal quadrants of E14 upper rhombic lip and postnatal EGL explants cocultured for 48-72 hours with 0.05 EBNA-293 control cells or with Area of neurite outgrowth (mm cells transfected with the netrin 1- c-myc construct. Data are ± 0.00 presented as means s.e.m.; E14 E14 P0 P0 P2 P2 P4 P4 P5 P5 *P=0.05; **P=0.01, LSD test. net-1 cont net-1 cont net-1 cont net-1 cont net-1 cont

To dissect EGL explants from the parasagittal slices of 1 in our assay correspond to the parallel fibres. Finally, postnatal cerebella, we used the easiest cleavage plane, which together with the parallel fibers, TAG-1- or L1-positive cell passes just below the multicellular (P0-P2) or monocellular bodies were observed leaving the tissue explants (Fig. 7E). In (P4-P6) Purkinje cell layer. Thus, these EGL explants initially the presence of control EBNA-293 cells, few labelled cell contain the subpial basal and glial laminae, the entire EGL, the bodies were present near to the explants, in the radial- nascent molecular layer and most of the Purkinje cells emerging bundles of parallel fibres. In contrast, in the (Fig.1B). To favour the radial outgrowth of granule cell processes and neurons, explants were placed either on their upper subpial or their deeper Purkinje cell surface. The outgrowth from such EGL explants cultured in a tridimensional collagen gel matrix was symmetrical and radial, which allowed us to monitor the effects of netrin 1. To identify the classes of cerebellar neurons and processes that grew from EGL explants, cultures were immunostained with Tuj-1 mAb or with antibodies to different markers: TAG- 1 or L1 to visualise granule cell bodies and processes, calbindin for Purkinje cells and GFAP for Bergmann glia (Wassef et al., 1985; Buttiglione et al., 1996; Soriano et al., 1997). When P0 EGL explants were cocultured for 1 to 3 days with netrin 1-expressing cells, 67% of the explants showed Tuj- 1-positive emerging neurites that grew preferentially towards the distal quadrant (Table 2; Fig. 5C,D). Older EGL explants (P2, P4 or P5) showed a similar, although more dramatic, repulsive effect (Figs 6, 7A-D). For instance, P4 and P5 EGL explants showed a preferential outgrowth in the distal quadrant in about 90% of cases (Table 2). The repulsive effect of netrin 1 on neurite extension was manifested in preferential neuritic outgrowth in the distal quadrant rather than in veering away from the source of netrin 1, and was easily mimicked when EGL explants were cultured with conditioned medium from netrin 1-secreting cells, which reduced neurite outgrowth (Fig. 5E,F). The use of distinct cellular markers showed that calbindin- positive Purkinje cells did not survive in these conditions and degenerated during the second day in culture (not shown). Fig. 7. Netrin 1 repels neuritic outgrowth from postnatal mice EGL GFAP-immunostaining revealed positive cell bodies within explants. Explants from P2 (A,B), P4 (C,D) and P6 (E) cerebella the explants, most probably belonging to Bergmann glial were cocultured for 48 hours with EBNA-293 control cells (A,C) or with cells transfected with netrin 1-c-myc expression vector (B,D,E) cells, but no GFAP-positive processes grew out of the β explants (data not shown). In contrast, TAG-1 (Fig. 7E) and and immunostained with antibodies against -tubulin III (A-D) or TAG-1 (E). Note the strong chemorepulsion exerted by netrin 1 in L1 (not shown) antibodies, which in the developing B,D. (E) Photomicrograph showing TAG-1-positive granule cells cerebellum mark the most immature and mature parallel (arrows) migrating along fibres in the quadrant distal to the source of fibres, respectively (Buttiglione et al., 1996), labelled the netrin 1. Perimeters of aggregates of control cells (c) and netrin 1- majority of processes arising from EGL explants. This secreting cells (n), outlined with white circles. Scale bars, 250 µm indicates that most, if not all, the neurites repelled by netrin (A-D), 25 µm (E). 1366 S. Alcántara and others presence of netrin 1-expressing cells, the immunopositive cell stream (Fig. 8H). Unc5h2 and unc5h3 were faintly expressed bodies were more abundant and exclusively found in the in the pons at E14, and increased with age (Fig. 8I). quadrant distal to the netrin 1 cell aggregate (Fig. 7E). TAG- At E14, the expression of netrin 1 and its receptors was weak 1- or L1-immunoreactive cell bodies were very small (6-8 in the developing cerebellar plate. netrin 1 was virtually absent, µm) and therefore correspond to migrating granule cells. with the exception of a faint staining in deep nuclear regions Taken together, the above results show that netrin 1 has no effect on E14 uRL explants, indicating that this factor does not play a role in the initiation of the tangential migration of granule cell progenitors originating from the germinative trigone. In contrast, netrin 1 exerts a strong repulsive effect on the outgrowth of parallel fibres (the axons of granule cells) and on the intraneuritic translocation of granule cell nuclei in these axons. The netrin 1-generated repulsion is already evident at birth (P0) but is more dramatic at later postnatal stages. Developmental expression of netrin 1, Dcc, neogenin, unc5h2 and unc5h3 To examine whether in vivo netrin 1 mediates the attractive and repulsive effects observed in vitro, we studied by in situ hybridisation and immunohistochemistry the patterns of expression of netrin 1 and its receptors in the lower brainstem and cerebellum of embryonic and postnatal mice. At E12, netrin 1 was highly expressed in the epithelial cells at the origin of the floor plate. The expression spread mediolaterally (with a decreasing gradient) through the ventricular basal plate, up to the sulcus limitans. Dcc mRNA and protein were not expressed in the ventricular zone, but in the subventricular zone of both the basal and alar plates. DCC protein labelled fibres in the marginal and submarginal migratory streams. At this stage of development, the hybridisation signals for unc5h2 and unc5h3 were almost absent (not shown; Bloch-Gallego et al., 1999). At E14, the expression of netrin 1 was similar to E12 (Fig. 8A). Dcc expression remained high in the subventricular zone and along the pontomedullary migratory stream, up to the pons. Less intense expression was detected in Fig. 8. Embryonic expression of netrin 1 and its receptors in the lower brainstem and the nucleus of the solitary tract and the vestibular pons. (A-F) Coronal sections through the brainstem showing the lRL and the origin of the pontine migratory stream at E14. (A) netrin 1 mRNA (NET-1) is strongly region (Fig. 8C,D). neogenin mRNA was highly expressed in the floor plate and in the ventricular zone of the basal plate. expressed in cells of the ventricular zone in both (B) Neogenin mRNA (NEOG) is expressed in the ventricular zone of the basal plate the basal and alar plates and in two dorsalmost and less intensely in the ventricular zone of the alar plate including the lRL. (C) Dcc lines, underlying the lower rhombic lips (Fig. mRNA and (D) protein (DCC-P) are expressed in early postmitotic pontine neurons 8B). Thus, Dcc and neogenin genes show non- in the subventricular zone of the lRL and more intensely in cells migrating along the overlapping patterns of expression. Unc5h2 pontine migratory stream. (E) Unc5h2 mRNA is expressed in the ventricular zone of signals were high and occupied most of the the alar plate including the lRL. Note that the strongest expression is adjacent to the ventricular alar plates, including the lRL, area of netrin 1 expression and the small patches of unc5h2 delimitating the pontine whereas unc5h3 signals were practically absent migratory stream. (F) unc5h3 mRNA is not expressed at this age. (G-I) Coronal (Fig. 8E,F). The expression of unc5h2 was sections at the level of the pons at E14 (G,H) and E17 (I). (G) netrin 1 is strongly expressed in midline cells. (H) DCC protein is present in the pontine migratory complementary to that of netrin 1 and partially stream at the level of the pons. (I) at E17 unc5h2 is expressed in pontine neurons. overlapping with neogenin mRNA distribution AP, alar plate; BP, basal plate; FP, floor plate, lRL, lower rhombic lip; MFN, motor (Fig. 8A,B,E). At pontine levels, netrin 1 was facial nucleus; PN, pontine nuclei; PMS, pontine migratory stream; SL, sulcus expressed in the midline (Fig. 8G) and Dcc limitans; SVZ, subventricular zone; VL, nucleus of the ventral lemniscus; VZ, signals were intense in the pontine migratory ventricular zone; IV, IVth ventricle. Scale bars 250 µm. Netrin 1 guides neuronal migration 1367

Fig. 9. Expression of netrin 1 and its receptors in the cerebellar anlage at E14. (A) netrin 1 mRNA (NET-1) is almost absent in the cerebellar anlage. (B) Neogenin mRNA (NEOG) is strongly expressed in the ventricular zone with a mediolateral gradient of expression and also in the EGL and deep nuclei of the cerebellum. (C) Dcc mRNA and (D) protein (DCC- P), as neogenin, are localized in the ventricular zone and EGL. Note that several fibres tracts, probably cerebellar afferents and the trochlear nerve (arrow), are strongly immunoreactive for DCC. DN, deep nuclei; EGL, external granule layer; lRL, lower rhombic lip; VZ, ventricular zone; URL, upper rhombic lip; IV, IVth ventricle. Scale bars, 250 µm.

(Fig. 9A). Dcc mRNA and protein were found in the cerebellar primary neuroepithelium, the germinal trigone and the EGL. DCC protein present in fibre bundles might correspond to the vestibular fibres, which are the first extracerebellar projections to arrive (Fig. 9C,D). neogenin colocalized its expression with Dcc, mainly at the middle region of the ventricular zone. In addition, neogenin was also expressed in deep nuclear neurons (Fig. 9B). As development proceeded, the cerebellar expression of netrin 1 and its receptors changed. Thus, at P5 netrin 1 expression was prominent in the EGL and in both the stellate and basket cells (the inhibitory interneurons of the molecular layer), but almost absent in the IGL (Fig. 10A). neogenin mRNA was expressed in granule cells in both the EGL and IGL (Fig. 10B). Dcc mRNA was found throughout the EGL, whereas DCC protein was only found in the parallel fibres at the interface between the deep EGL and the nascent molecular layer. DCC-positive fibres were also observed in the granule cell layer (IGL) and in the white matter (Fig. 10C,D). Unc5h2 and unc5h3 mRNAs were confined to granule cell precursors (EGL) and postmigratory granule cells (IGL) (Fig. 10E,F).

DISCUSSION Fig. 10. Expression of netrin 1 and its receptors in the P5 cerebellum. (A) netrin 1 mRNA Netrin 1 is a guiding cue for the (NET-1) is expressed in the EGL and in the molecular layer interneurons. (B) neogenin migration of neurons fated to the mRNA (NEOG) is exclusively expressed in granule cells in the EGL and IGL. (C) Dcc pontine nuclei mRNA and (D) protein (DCC-P) are expressed in premigratory granule cells in the EGL and in the white matter. Arrows in D indicate parallel fibres. (E) unc5h2 and (F) unc5h3 are In the present study, we have addressed expressed in granule cells in both the EGL and IGL. EGL, external germinal layer; IGL, the role of netrin 1 in the migration internal granule layer; ML, molecular layer; PC, Purkinje cell layer; WM, white matter. Scale of precerebellar neurons by culturing bars, 250 µm (A-C, E-F), 50 µm (D). 1368 S. Alcántara and others explants of the inferior lateral recess of the IVth ventricle at Thus, although our findings show that netrin 1 acts as a E12-E14 (lRL explants). Although we cannot rule out the chemoattractive cue for the migration of pontine nuclei along possibility that some olivary neurons may be present in our the anterior extramural stream, as occurs for the navigation of explants, several arguments indicate that we have monitored ventrally growing axons (Kennedy et al., 1994; Shirasaki et al., the migration of neurons fated to the pontine precerebellar 1995, 1996), we cannot rule out additional mechanisms of nuclei. First, although a few olivary neurons are late-generated action of netrin 1 that may contribute to the generation of the at E12 in the lRL, this stage contributes mainly to the phenotype in netrin 1 mutant mice. generation of precerebellar neurons fated to the external The present observations also show that netrin 1 induces the cuneatus (EC), the lateral reticular (LR) and the raphe nuclei formation of thick strands and fascicles of migrating neurons (Taber-Pierce, 1966; Bourrat and Sotelo, 1988; Altman and and neurites, in which these cells attach closely to each other, Bayer, 1997). This is especially so at E14, when the lRL only which is reminiscent of the neurophilic migration that takes generates neurons destined to the pontine nuclei (PN) (Taber- place in the extramural migratory streams in vivo (Rakic, 1985; Pierce, 1966; Altman and Bayer, 1997). Second, during the Ono and Kawamura, 1989, 1990) and in other migratory formation of the precerebellar system, the surface glycoprotein pathways such as the rostral migratory stream to the olfactory TAG-1/Axonin 1 is strongly expressed along the extramural bulb (Jankovski and Sotelo, 1996; Lois et al., 1996; Wichterle migratory streams (which contains neurons destined to the EC, et al., 1997). Previous studies have reported the presence of LR and PN nuclei), but not along the intramural migratory TAG-1 in developing axons that respond to netrin 1 (Placzek stream, the pathway followed by migrating olivary neurons et al., 1990; Serafini et al., 1994; Wolfer et al., 1994; (Wolfer et al., 1994), which agrees with the present data Colamarino and Tessier-Lavigne, 1995; Guthrie and Pini, showing that the neurons that respond to netrin 1 are strongly 1995; Tamada et al., 1995; Shirasaki et al., 1996) and the immunoreactive for TAG-1. We thus conclude that the lRL participation of this molecule in the transduction of floor-plate- explants selected in our experiments correspond to the derived signals has been hypothesised (Shirasaki et al., 1996). precerebellar neuroepithelium that gives rise to the LR and EC The upregulation of TAG-1 protein in migrating cells by netrin nuclei at E12, and to the basilar pons at E14 (Taber-Pierce, 1 reported here represents the first direct link between TAG-1 1966). expression and a guidance protein secreted by the floor plate. Analysis of the precerebellar nuclei in mice deficient in TAG-1 forms homophilic trans-interactions and heterophilic expression of netrin 1 has revealed that basilar pontine neurons interactions with other IgCAMS, such as F3 or L1, which are missing (Serafini et al., 1996) and that less than 14% of results in changes in adhesivity and fasciculation (Lemmon et inferior olivary neurons reach their ventromedial position al., 1989; Felsenfeld et al., 1994; Buttiglione et al., 1998). (Bloch-Gallego et al., 1999), although it is not known how such TAG-1 may contribute to the formation of neuritic fascicles a phenotype arises. The present data show that netrin 1 acts as and, particularly, of the strands of migrating pontine neurons, a long-range chemoattractant factor for pontine nuclei similar to the participation of PSA-NCAM to the formation of migrating neurons, which represents the first direct evidence neuronal chains in the rostral migratory stream of the olfactory for netrin 1 influencing the direction of migrating neurons. Our bulb (Hu et al., 1996). data support the notion that the absence of pontine nuclei in netrin 1− hypomorphic mice directly reflects abnormal Correlation of netrin 1 effects with the pattern of neuronal migration from the lRL caused by the lack of netrin expression in vivo 1. Since Dcc knockout mice share similar defects in pontine The pattern of expression of netrin 1 and their receptors in the structures (Fazeli et al., 1997), it is likely that this receptor is early development of the pontine nuclei is summarised in Fig. involved in such chemoattraction. In favour of this conclusion 11. Some precerebellar neurons originating in the lRL migrate are the observations on inferior olivary neurons in netrin 1- along the marginal stream to cross the midline and allocate in deficient mice. The visualisation of these neurons by their Brn- the LRN and ECN contralateral to their site of origin. In 3b expression (Bloch-Gallego et al., 1999) suggested that the contrast, neurons migrating within the submarginal (inferior atrophy of the inferior olive is mainly the result of impaired olivary neurons) and pontomedullary streams (basilar pontine migration, because Brn-3b-expressing cells were distributed neurons) do not cross the midline, and populate nuclei along the submarginal migratory stream. Thus, the present ipsilateral to their side of origin (Taber-Pierce, 1966; Bourrat results, together with those in inferior olivary neurons (Bloch- and Sotelo, 1990). The present study shows that pontine nuclei Gallego et al., 1999), allow us to conclude that netrin 1 is an cell progenitors express unc5h2 and neogenin receptor genes essential cue for the circumferential migration of precerebellar but not Dcc, whereas, postmitotic pontine neurons along the neurons. anterior extramural migratory pathway express the opposite However, other mechanisms involving netrin 1 may combination of receptors, i.e., high Dcc and no neogenin, contribute to the mutant phenotype. For instance, DCC may unc5h2 or unc5h3. Finally, as these neurons end their induce in the absence of ligand-binding, but blocks circumferential migration near the midline, they re-express low cell death when engaged to netrin 1 (Mehlen et al., 1998). As levels of unc5h2 and unc5h3 genes. Conversely, netrin 1 is the early postmitotic cells in the lRL express DCC, the netrin expressed in the midline area near the target region (pontine 1/DCC interaction may regulate the survival of early pontine nuclei), which suggests that a gradient of netrin 1 produced in nuclei cells (Cho and Fearon, 1995). In addition to exerting a this area may be responsible for the unidirectional migration strong chemoattractive effect, our explants incubated with of neurons towards the precerebellar nuclei. Also, the pattern netrin 1-conditioned media show that this factor dramatically of receptor expression in vivo is consistent with current views promotes the exit of migrating neurons from the that DCC is a component of the receptor complex mediating neuroepithelium, even in the absence of well-defined gradients. chemoattractive responses by netrin 1 along the migratory Netrin 1 guides neuronal migration 1369 A C C B

SVZ VZ Fig. 11. Schematic drawings illustrating the pontine PMS migratory stream in relation to the space-temporal IC distribution of netrin 1 and its receptors. (A) Dorsolateral R view of an E14-E17 brain showing the localization of the B pontine migratory stream in green and the plane of the AP sections showed in B and C. The arrow in A indicate the lRL caudorostral direction of the pontine migration. (B) Scheme SVZ BP VZ of a coronal section at E14 illustrating the distribution of PMS RP ICP netrin 1 and its receptors at the origin of the pontine SL FP VL migratory stream (PMS). Note that the PMS only express STT Dcc and that areas of low unc5h2 and neogenin expression PMS PN surround the migratory pathway. (C) Scheme of a coronal section at the level of the midbrain and pons at E17 showing the destination of the neurons migrating along the E14 E17 PMS in the pontine nuclei. Pontine neurons start to express unc5h2 and unc5h3 receptors and downregulate Dcc expression at the end of their migration. AP, alar plate; BP, basal plate; FP, floor plate; IC, inferior colliculus; ICP, High Netrin-1 Low Netrin-1 inferior cerebellar peduncle; lRL, lower rhombic lip; PMS, High Neogenin Low Neogenin pontine migratory stream; PN, pontine nuclei; SL, sulcus High DCC Low DCC limitans; STT, spinal trigeminal tract; SVZ, subventricular High unc5H2 Low unc5H2 zone; R, red nucleus; RP, raphe nucleus; VL, ventral lemniscus; VZ, ventricular zone. High unc5H3 Low unc5H3 pathway. Finally, the re-expression of the unc5h2 and unc5h3 embryonic stages, the dorsal surface of the cerebellar anlage is receptor genes when migrating pontine neurons approach the crossed by DCC-containing axons that overlap with and run in target region suggests that these receptors may prevent these parallel with migrating granule cell progenitors (Fig. 10D). As neurons from crossing the midline, thereby allowing netrin 1 occurs with other IgCAMS such as L1 or NCAM (reviewed in to act as a stop signal, as proposed for olivary neurons (Bloch- Walsh and Doherty, 1997), homophilic trans DCC interactions Gallego et al., 1999). between axons and neurons may contribute to the tangential However, netrin 1 is also expressed in the floor plate region migration of embryonic granule cell precursors. In the unc5h3 close to the precerebellar neuroepithelium, so additional null mutant embryos, granule cell progenitors extend more mechanisms may prevent young postmitotic neurons from rostrally than in wild-type mice, indicating that unc5h3 migrating towards this source of netrin 1. Possible mechanisms receptors transduce a chemorepulsive signal for tangentially include the sequestration of netrin 1 by association with the migrating granule cell progenitors (Przyborski et al., 1998). or by neogenin receptors (Kennedy et al., The results in the unc5h3 mutant in vivo and our negative 1994; Serafini et al., 1994; Keino-Masu et al., 1996), which are finding with netrin 1 in vitro, suggest that unc5h3 receptors highly expressed near the site of netrin 1 expression, or the may bind additional ligands, as recently postulated in presence of a non-permissive substrate for neuronal migration (Colavita et al., 1998). in this brainstem area. Recent publications (Wu et al., 1999; Hu, 1999) have shown that tangentially migrating neuronal progenitors in the rostral Age-dependent chemorepulsion of granule cell migratory stream and some neurons in the cerebral cortex progenitors and axons mediated by netrin 1 might be guided by long-range repulsive factors, the During , granule cell progenitors are proteins. These results, together with those reported here for generated in the uRL and migrate tangentially over the precerebellar neurons, strongly suggest that chemotactic cerebellar anlage to form the EGL, where they re-enter the mechanisms exert a guidance effect on tangentially migrating mitotic cycle. The present data with E14-uRL explants show neurons. Although we have not observed chemotropic effects that embryonic granule cell progenitors do not respond to of netrin 1 on embryonic granule cell progenitors, it will be netrin 1, although they express the Dcc and neogenin receptor interesting to determine whether other diffusible factors, such genes. This is consistent with observations in the cerebellum as Slit proteins, are guiding these progenitors. of newborn netrin 1 defective mice showing an almost intact Starting at birth, postmitotic granule cells descend to the EGL layer (Bloch-Gallego et al., 1999). Since netrin 1 is not deep EGL and extend their axons, the parallel fibres (Ramón expressed in the cerebellar anlage at early embryonic stages, y Cajal, 1911; Miale and Sidman, 1961). Granule cell somata Dcc expression in granule cell progenitors derived from the then become oriented perpendicular to the axons and migrate uRL may reflect a netrin 1-independent action for this receptor. radially through the molecular layer, attached to the Bergmann It has been suggested that granule cell progenitors migrate glia, to form the IGL (Rakic, 1971, 1981, 1985; Hatten and along preexisting axonal tracts (Hynes et al., 1986). At Liem, 1981). The present data show that netrin 1 exerts a strong 1370 S. Alcántara and others chemorepulsive effect on axons growing from postnatal EGL data, together with our previous study in the olivary nucleus explants, which were characterised as parallel fibres on the (Bloch-Gallego et al., 1999) and the analysis of un5h3 mutants basis of their immunoreactivity for L1 and TAG-1 proteins (Przyborski et al., 1998), show a novel role for netrin 1 in (Persohn and Schachner, 1987; Yamamoto et al., 1990). Such neuronal migration, which, as for axonal growth, may be either a chemorepulsive effect is age-dependent, and thus more chemoattractant or chemorepellent. Thus, together with other dramatic at P5 than at P0, which again correlates with the molecules with a role in neuronal migration and axonal growth, period of parallel fibre formation in vivo (Altman and Bayer, such as reelin or semaphorin III (D’Arcangelo et al., 1995; Del 1997). In the postnatal cerebellum, netrin 1 is expressed in the Rio et al., 1997; Eickholt et al., 1999), netrin 1 is likely to have EGL and interneurons of the molecular layer. We suggest that a dual role in the construction of neural networks and in the netrin 1 contributes to the initiation of the correct parallel fibre guidance of migrating neurons towards their target nuclei and extension, which could be a prerequisite for the ordered exit of layers. postmitotic premigratory granule cells from the EGL (Engelkamp et al., 1999). The coexpression of Dcc, unc5h2 This work was supported by Grants SAF98-106 (Comision and unc5h3 receptor genes in granule cell precursors as well Interministerial de Ciencia y Tecnología, Spain), by the Human as in postmitotic granule cells is consistent with the view that Science Frontier Program and by Marató TV3 Foundation (Spain) to co-participation of DCC and unc5 receptors is required for E. S., and by the Institut National de la Santé et de la Recherche Médicale financial support to C. S.; S. A. is a recipient of MEC netrin 1 to exert a chemorepulsive response (Hong et al., 1999). postdoctoral fellowship (Spain). F. de C. has EC Fellowship ERB- In addition to axons, our data show migrating neurons 4001-GT-970077. We are indebted to Dr D. Karagogeos for the leaving the explants in the quadrants distal to the source of generous gift of anti-TAG-1 antibodies, Dr M. Fabre and Dr F. X. Real netrin 1, which are identified as granule cells because of L1 for anti-DCC antibodies, Dr M. Schachner for anti-L1 antibodies, D. and TAG-1 immunostaining. We believe that this observation Le Cren for photographic work and R. Rycroft for editorial assistance. reflects an action of netrin 1 on migrating neurons for the We are specially gratefull to Dr M. Tessier-Lavigne for providing us following reasons. (1) Granule cell perikarya outside the cDNA probes and cell lines. explants are exclusively observed in the distal quadrants, but not in the proximal quadrants where granule cell axons also grow, which rules out the possibility that migrating cells move REFERENCES passively through the parallel fibres. (2) The rostral cerebellar Ackerman, S., Kozak, L. P., Przyborski, S. A., Rund, L. A., Boyer, B. B. malformation, which results from the disruption of the unc5h3 and Knowles, B. B. (1997). The mouse rostral cerebellar malformation gene, leads to abnormal migration and positioning of granule gene encodes an UNC-5-like protein. Nature 386, 838-842. cells, which implies a possible role for netrin 1 signalling Alcántara, S., Ruiz, M., D’Arcangelo, G., Ezan, F., de Lecea, L., Curran, through this receptor in cerebellar migration. (3) unc5h2 and T., Sotelo, C. and Soriano, E. (1998). Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse. unc5h3 receptors are highly expressed in premigratory granule J. Neurosci. 18, 7779-7799. cell bodies. However, Bergmann fibres did not leave our EGL Altman, J. and Bayer, S. A. (1997). Development of Cerebellar System in explants and the granule cells migrate along fascicles of TAG- Relation to its Evolution, Structure and Functions. Boca Raton, FL: CRC 1-positive fibres belonging to parallel fibres. We thus interpret Press. our results as showing that the non-gliophilic migration Anderson, S. A., Eisenstat, D. D., Shi, L. and Rubenstein, J. L. R. (1997). Interneuron migration from basal forebrain to neocortex: dependence on observed in vitro mimics the tangential migration of granule DLX genes. Science 278, 474-476. cells within the deeper EGL, just before their inwards radial Bloch-Gallego, E., Ezan, F., Tessier-Lavigne, M. and Sotelo, C. (1999). migration (Ryder and Cepko, 1994), and that this tangential Floor plate and netrin-1 are involved in the migration and survival of inferior migration is under the control of netrin 1 and its receptors. olivary neurons. J. Neurosci. 19, 4407-4420. Bourrat, F. and Sotelo, C. (1988). Migratory pathways and neuritic differentiation of inferior olivary neurons in the rat embryo: Axonal tracing Netrin 1, a factor involved in axonal growth and study using the in vitro slab technique. Dev. Brain Res. 39, 19-37. neuronal migration Bourrat, F. and Sotelo, C. (1990). Early development of the precerebellar Netrin 1 secreted by the floor-plate acts as a chemoattractant system: Migratory routes, Selective aggregation and neuritic differentiation factor for ventrally directed axonal navigation (Kennedy et al., of the inferior olive and lateral reticular nucleus neurons. An overview. Arch. Ital. Biol. 128, 151-170. 1994; Keino-Masu et al., 1996; Shirasaki et al., 1995, 1996) Bourrat, F. and Sotelo, C. (1991). Relationships between neuronal birthdates and as a chemorepellent cue for dorsally oriented axons and cytoarchitecture in the rat inferior olivary complex. J. Comp. Neurol. (Colamarino and Tessier-Lavigne, 1995). The present data 313, 509-521. show that netrin 1 is involved in the growth and targeting of Buttiglione, M., Revest, J. M., Rougon, G. and Faivre-Sarrailh, C. (1996). F3 neuronal adhesion molecule controls outgrowth and fasciculation of intrinsic cerebellar circuits, which are independent of the cerebellar granule cell neurites: a cell type specific mediated by the Ig-like midline, by providing chemorepulsive signals to developing domains. Mol. Cell Neurosci. 8, 53-69. parallel fibres. Thus, together with reports on corticofugal Buttiglione, M., Revest, J. M., Pavlov, O., Karagogeos, D., Furley, A., fibres (Métin et al., 1997; Richards et al., 1997) and ongoing Rougon, G. and Faivre-Sarrailh, C. (1998). A functional interaction studies on the hippocampus (J. Barallobre, personal between the neuronal adhesion molecules TAG-1 and F3 modulates neurite outgrowth and fasciculation of cerebellar granule cells. J. Neurosci. 17, communication), the present data indicate that netrin 1 is also 6853-6870. involved in the guidance of some ipsilateral projections and in Cho, K. R. and Fearon, E. R. (1995). DCC: linking tumor suppressor genes target-layer selection. and altered cell surface interactions in cancer? Curr. Opin. Genet. Dev. 5, More importantly, the present results show that netrin 1 72-78. Colavita, A., Krishna, S., Zheng, H., Padget, R. W. and Culotti, J. G. exerts a direct chemoattractive influence on precerebellar (1998). Pioneer guidance by UNC-129. a C. elegans TGF-β. Science migrating neurons along the circumferential extramural stream, 281, 706-709. and a chemorepulsive effect on granule cell migration. These Colamarino, S. A. and Tessier-Lavigne, M. (1995). The axonal Netrin 1 guides neuronal migration 1371

chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. (1991). Developmental expression of the axonal glycoprotein TAG-1: Cell 81, 621-629. Differential regulation by central and peripheral neurons in vitro. Culotti, J. G. and Kolodkin, A. L. (1996). Functions of netrins and Development 112, 51-67. semaphorins in . Curr. Opin. Neurobiol. 6, 81-88. Keino-Masu K., Masu, M., Hinck, L., Leonardo, E. D., Chan, S. S. Y., D’Arcangelo, G., Miao, G. G., Chen, S.-C., Soares, H. D., Morgan, J. I. Culotti, J. G. and Tessier-Lavigne, M. (1996). Deleted in colorectal cancer and Curran, T. (1995). A protein related to extracellular matrix proteins (DCC) encodes a Netrin receptor. Cell 87, 175-185. deleted in the mouse mutant reeler. Nature 374, 719-723. Kennedy, T. E., Serafini, T., de la Torre, J. and Tessier-Lavigne, M. (1994). Del Río, J. A., Heimrich, B., Borrell, V., Förster, E., Drakew, A., Alcántara, Netrins are diffussible chemotropic factors for commissural axons in the S., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., Derer, P., embryonic spinal cord. Cell 78, 425-435. Frotscher, M. and Soriano, E. (1997). An essential role for Cajal-Retzius Lemmon, V., Farr, K. L. and Lagenaur, C. (1989). L1-mediated axon cells and reelin in the layer-specific targeting of hippocampal afferents. outgrowth occurs via a homophilic binding mechanism. Neuron 2, 1597- Nature 385, 70-74. 1603. Eickholt, B. J., Mackenzie, S. L., Graham A., Walsh, F. S. and Doherty, P. Leonardo, E. D., Hinck, L., Masu, M., Keino-Masu, K., Ackerman, S. L. (1999). Evidence for collapsin-1 functioning in the control of neural crest and Tessier-Lavigne, M. (1997). Vertebrate homologues of C. elegans migration in both trunk and hindbrain regions. Development 126, 2181- UNC-5 are candidate Netrin receptors. Nature 386, 833-837. 2189. Leung-Hagesteijn, C., Spence, A. M., Stern, B. D., Zhou, Y., Su, M.-W., Ellerberger, C., Hanaway, J. and Netsky, M. G. (1969). Embryogenesis of Edgecock, E. M. and Culotti, J. G. (1992). UNC-5, a transmembrane the inferior olivary nucleus in the rat: A radioautographic study and protein with immunoglobulin and thrombospondin type I domains, guides reevaluation of the rhombic lip. J. Comp. Neurol. 137, 71-88. cell and migrations in C. elegans. Cell 71, 289-299 Engelkamp, D., Rashbas, P., Seawright, A. and van Heyningen, V. (1999). Lois, C., García-Verdugo, J. M. and Alvarez-Buylla, A. (1996). Chain Role of Pax6 in development of the cerebellar system. Development 126, migration of neuronal precursors. Science 271, 978-981. 3585-3596. Lumsden, A. G. S. and Davies, A. M. (1986). Chemotropic effect of specific Fabre M., Martin, M., Ulloa, F. and Real, F. X. (1999). In vitro analysis of target in the development of the mammalian central nervous the role of DCC in mucus-secreting intestinal differentiation. Int. J. Cancer system. Nature 323, 538-539. 81, 799-807. Mathis, L., Bonnerot, C., Puelles, L. and Nicolas, J.-F. (1997). Retrospective Fazeli, A., Dickinson, S. L., Hermiston. M. L., Tighe, R. V., Steen, R. G., clonal analysis of the cerebellum using genetic lac Z/lacZ mouse mosaics. Small, R. G., Stoeckli, E. T., Keino-Masu, K., Masu M., Rayburn, H., Development 124, 4089-4104. Simons, J., Bronson, R. T., Gordon, J. I., Tessier-Lavigne, M. and Mehlen, P., Rabizadeh, S., Snipas, S. J., Ass-Munt, N., Salvesen, G. S. and Weinberg, R. A. (1997). Phenotype of mice lacking functional deleted in Bredesen, D. E. (1998). The DCC gene product induces apoptosis by a colorectal cancer (DCC) gene. Nature 386, 796-804. mechanism requiring receptor proteolysis. Nature 395, 801-804. Felsenfeld, D. P., Hynes M. A., Skoler, K. M., Furley, A. J. and Jessell, T. M. Métin, C., Deléglise, D., Serafini, T., Kennedy, T. E. and Tessier-Lavigne, (1994). TAG-1 can mediate homophilic binding but neurite outgrowth on M. (1997). A role for netrin-1 in the guidance of cortical efferents. TAG-1 requires a L1-like molecule and β1 integrins. Neuron 12, 257-267. Development 124, 5063-5074. Gao, W.-Q. and Hatten, M. E. (1994). Immortalizing subvert the Miale, I. and Sidman,R. L. (1961). An autoradiographic analysis of establishment of granule cell identity in developing cerebellum. histogenesis in the mouse cerebellum. Exp. Neurol. 4, 277-296. Development 120, 1059-1070. Moody, S. A., Quigg, M. S. and Frankfurter, A. (1989). Development of the Goodman, C. S. (1996). Mechanisms and molecules that control peripheral trigeminal system in the chick revealed by an isotype-specific guidance. Annu. Rev. Neurosci 19, 341-377. anti-beta-tubulin monoclonal antibody. J. Comp. Neurol. 279, 567-580. Guthrie, S. and Pini, A. (1995). Chemorepulsion of developing motor axons Ono, K. and Kawamura, K. (1989). Migration of immature neurons along by the floor plate. Neuron 14, 1117-1130. tangentially oriented fibers in the subpial part of the fetal mouse medulla Hamelin, M., Zhou, Y., Su, M. W. and Culotti, J. G. (1993). Expression of oblongata. Exp. Brain Res. 78, 290-300. the UNC-5 guidance receptor in the touch neurons of C. elegans steers their Ono, K. and Kawamura, K. (1990). Mode of neuronal migration of the axons dorsally. Nature 364, 327-330. pontine stream in fetal mice. Anat. Embryol. 182, 11-19. Harkmark, W. (1954). Cell migrations from the rhombic lip to the inferior Pearlman, A. L., Faust, P. L., Hatten, M. E. and Brunstrom, J. E. (1998). olive, the nucleus raphe and the pons. A morphological and experimental New directions for neuronal migration. Curr. Opin. Neurobiol. 8, 45-54. investigation on chick embryos. J. Comp. Neurol. 100, 115-205. Persohn, E., and Schachner, M. (1987). Immunoelectron microscopic Hatten, M. E. and Liem, R. K. H. (1981). Astroglia provide a template for localization of the neural adhesion molecules L1 and N-CAM during the positioning of developing cerebellar neurons in vitro. J. Cell Biol. 90, postnatal development of the rat cerebellum. J. Cell. Biol. 105, 569-576. 622-630. Placzek, M., Tessier-Lavigne, M., Jessell, T. M. and Dodd, J. (1990). His, W. (1891). Die Entwicklung des menschlichen Rautenhirns vom Ende des Orientation of commisural axons in vitro in response to a floor plate-derived ersten bis zum Beginn des dritten Monats. I. Verlängertes Mark. Abhandlung chemoattractant. Development 110, 19-30. der königlicher sächsischen Gesellschaft der Wissenschaften, Przyborski, S. A., Knowles, B. B. and Ackerman, S. L. (1998). Embryonic Mathematischephysikalische Klasse 29, 1-74. phenotype of Unc5h3 mutant mice suggest chemorepulsion during the Hong, K., Hinck, L., Nishiyama, M., Poo, M., Tessier-Lavigne, M. and formation of the rostral cerebellar boundary. Development 125, 41-50. Stein, E. (1999). A ligand-gated association between cytoplasmic domains Rakic, P. (1971). Neuron-glia relationship during cell migration in developing of UNC5 and DCC family receptors converts Netrin-induced growth cone cerebellar cortex: A Golgi and electron microscopic study in Macacus attraction to repulsion. Cell 97, 927-942. rhesus. J. Comp. Neurol. 141, 283-312 Hu, H. (1999). Chemorepulsion of neuronal migration by Slit2 in the Rakic, P. (1981). Neuron-glial interaction during brain development. Trends developing mammalian forebrain. Neuron 23, 703-711. Neurosci. 4, 184-187. Hu, H. and Rutishauser, U. (1996). A septum-derived chemorepulsive factor Rakic, P. (1985). Contact regulation of neuronal migration. In The Cell in for migrating olfactory interneuron precursors. Neuron 16, 933-940. Contact. (eds. G. E. Edelman and J. P.Thiery), pp 67-90. New York: J. Wiley. Hu, H., Tomasiewics, H., Magnuson, T. and Rutishauser, U. (1996). The Rakic, P. (1990). Principles of neural migration. Experientia 46, 882-891. role of polysialic acid in migration of interneuron precursors Ramón y Cajal, S. (1911). Histologie du Système Nerveux de l’Homme et des in the subventricular zone. Neuron 16, 735-743. Vértébres. Maloine, Paris. Hynes, R. O., Patel, R. and Miller, R. H. (1986). Migration of neuroblasts Richards, L. J., Koester, S. E., Tuttle, R. and O’Leary, D. D. M. (1997). along preexisting axonal tracts during prenatal cerebellar development. J. Directed growth of early cortical axons is influenced by a chemoattractant Neurosci. 6, 867-876. released from an intermediate target. J. Neurosci. 17, 2445-2458. Jacobowitz, D. M. and Abbott, L. C. (1998). Chemoarchitectonic Atlas of Ryder, E. F. and Cepko, C. L. (1994). Migration patterns of clonally related the Developing Mouse Brain. Boca Raton, FL: CRC Press. granule cells and their progenitors in the developing chick cerebellum. Jankovski, A. and Sotelo, C. (1996). Subventricular zone-olfactory bulb Neuron 12, 1011-1029. migratory pathway in the adult mouse: cellular composition and specificity Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. and as determined by heterochronic and heterotopic transplantation. J. Comp. Tessier-Lavigne, M. (1994). The Netrins define a family of axon outgrowth- Neurol. 371, 376-396. promoting proteins homologous to C. elegans UNC-6. Cell 78, 409-424. Karagogeos, D., Morton, S. B., Casano, F., Dodd, J. and Jessell, T. M. Serafini, T., Colamarino, S. A., Leonardo, E. D., Wang, E., Beddington, 1372 S. Alcántara and others

R., Skarnes, W. C. and Tessier-Lavigne, M. (1996). netrin-1 is required Tessier-Lavigne, M. and Goodman, C. S. (1996). The molecular biology of for commissural axon guidance in the developing vertebrate nervous system. axon guidance. Science 274, 1123-1132. Cell 87, 1001-1014. Varela-Echevarría, A., Tucker, A., Püschel, A. W. and Guthrie, S. (1997). Shirasaki, R., Tamada, A., Katsumata, R. and Murakami, F. (1995). Motor axon subpopulations respond differentially to the chemorepellents Guidance of cerebellofugal axons in the rat embryo: Directed growth toward netrin-1 and semaphorin D. Neuron 18, 193-207. the floor plate and subsequent elongation along the longitudinal axis. Neuron Walsh, F. S. and Doherty, P. (1997). Neural cell adhesion molecules of the 14, 961-972. immunoglobulin superfamily: Role in axon growth and guidance. Ann. Rev. Shirasaki, R., Mirzayan, C., Tessier-Lavigne, M. and Murakami, F. (1996). Cell Dev. Biol. 13, 425-456. Guidance of circumferentially growing axons by Netrin-dependent and - Wassef, M., Zanetta, J. P., Brehier, A. and Sotelo, C. (1985). Transient independent floor plate chemotropism in the vertebrate brain. Neuron 17, biochemical compartmentalization of Purkinje cells during early cerebellar 1079-1088. development. Dev. Biol. 111, 129-137. Soriano, E., Alvarado-Mallart, R. M., Dumesnil, N., Del Río, J. A. and Wichterle, H., García-Verdugo, J. and Alvarez-Buylla, A. (1997). Direct Sotelo, C. (1997). Cajal-Retzius cells regulate the radial glia phenotype in evidence for homotypic, glia-independent neuronal migration. Neuron 18, the adult and developing cerebellum and alter granule cell migration. Neuron 779-791. 18, 563-577. Wolfer, D. P., Henehan-Beatty, A., Stoeckli, E. T., Sondererger, P. and Stoeckli, E. T. and Landmesser, L. T (1995). Axonin-1, Nr.CAM and Ng- Lipp, H. P. (1994). Distribution of TAG-1/Axonin-1 in fibre tracts and CAM play different roles in the in vivo guidance of chick commissural migratory streams of the developing mouse nervous system. J. Comp. neurons. Neuron 14, 1165-1179. Neurol. 345, 1-32. Taber-Pierce, E. (1966). Histogenesis of the nuclei griseum pontis, corporis Wu, W., Wong, K., Chen, J-h., Jiang Z-h, Dupuis, S., Wu, J. and Rao, Y. pontobulbaris and reticularis tegmenti pontis (Bechterew) in the mouse. J. (1999). Directional guidance of neuronal migration in the olfactory system Comp. Neurol. 129, 219-240. by the protein Slit. Nature 400, 331-336. Tamada, A., Shirasaki, R. and Murakami, F. (1995). Floor plate Yamamoto, M., Hassinger, L. and Crandall, J. E. (1990). Ultrastructural chemoattracts crossed axons and chemorepels uncrossed axons in the localization of stage-specific neurite associated proteins in the developing vertebrate brain. Neuron 14, 1083-1093. rat cerebral and cerebellar cortices. J. Neurocytol. 19, 619-627.