CCN3 promotes maturation of cerebellar granule neuron precursors

Molecular and Cellular Neuroscience 43 (2010) 60–71

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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m c n e

NOV/CCN3 promotes maturation of cerebellar granule neuron precursors G. Le Dréau a,b, A. Nicot b, M. Bénard c, H. Thibout a, D. Vaudry c,d, C. Martinerie a,b, M. Laurent a,b,⁎ a

INSERM, UMR-S893, Hôpital Saint-Antoine, F-75012, Paris, France UPMC Univ Paris 06, UMR-S893, F-75005, Paris, France c Plate-Forme Régionale de Recherche en Imagerie Cellulaire de Haute-Normandie, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Université de Rouen, F-76821, Mont-Saint-Aignan, France d INSERM, UMR-S413, Laboratoire de Neuroendocrinologie Cellulaire et Moléculaire, F-76821, Mont-Saint-Aignan, France b

a r t i c l e

i n f o

Article history: Received 9 May 2008 Revised 9 February 2009 Accepted 26 February 2009 Available online 13 March 2009

a b s t r a c t A body of evidence points to the matricial CCN proteins as key regulators of organogenesis. NOV/CCN3, a founder CCN member, is expressed in the developing central nervous system but its functions during neural development have not been studied yet. Here we describe the pattern of NOV expression during rat cerebellar postnatal development and show that NOV expression increases during the second postnatal week, a critical period for the maturation of granule neuron precursors (GNP). NOV transcripts are specifically produced by Purkinje neurons and NOV protein localises extracellularly in the molecular layer and the inner part of the external granule layer, at a key position to control GNP proliferation and migration. In vitro, NOV reduces Sonic Hedgehog-induced GNP proliferation through β3 integrins and stimulation of GSK3-β activity whereas NOV stimulates GNP migration through distinct RGD-dependent integrins. These findings identify a new paracrine role of NOV in the development of cerebellar granule neurons. © 2009 Published by Elsevier Inc.

Introduction The cerebellum, which is involved in motor coordination and regulation of cognitive processes, is a useful model for studying the complexity of central nervous system development. Indeed, the cerebellum has a well-defined cytoarchitecture and its development continues throughout the whole developmental window, from early neural embryogenesis until postnatal completion of brain maturity (Goldowitz and Hamre, 1998; Sotelo, 2004). In mammals, growth of the cerebellum occurs mainly during postnatal development and involves a dramatic increase in volume (Goldowitz and Hamre, 1998). This event is predominantly due to the expansion of granule neurons, the most abundant neuronal population in the brain (Wang and Zoghbi, 2001). Studies over the last decade have demonstrated that proliferation of cerebellar granule neuron precursors (GNP) lying in the external granule layer (EGL) is driven by homotypic cell–cell contacts through the Notch pathway (Solecki et al., 2001) and by underlying Purkinje neurons through secretion of diffusible factors (Dahmane and Ruiz i Altaba, 1999; Smeyne et al., 1995; Ye et al., 1996). Among those, Sonic Hedgehog (SHH) represents so far the main mitogenic factor for GNP both in vitro and in vivo (Dahmane and Ruiz i Altaba, 1999; Lewis et al., 2004; Wechsler-Reya and Scott, 1999).

⁎ Corresponding author. INSERM, UMR-S893, équipe 1, Hôpital Saint-Antoine, bâtiment Kourilsky, 4è étage, F-75012, Paris, France. Fax: +33 1 43 43 10 65. E-mail address: [email protected] (M. Laurent). 1044-7431/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.mcn.2009.02.011

Medulloblastoma, the most common malignant childhood brain tumour, is thought to derive from dysregulated GNP proliferation (Marino, 2005). This has stimulated research to identify the factors directing GNP cell cycle exit. These factors include the pituitary adenylate cyclase-activating polypeptide (PACAP) (Nicot et al., 2002), several bone morphogenetic proteins (BMPs) (Alvarez-Rodriguez et al., 2007; Rios et al., 2004) and fibroblast growth factors (FGFs). Some evidence also indicate, that migration away from the mitogenic niche may also be required for GNP to exit the cell cycle successfully. Therefore, there is now a great interest in the identification of factors that could coordinate downregulation of GNP proliferation with stimulation of their migration from this mitogenic niche. Extracellular matrix proteins present in the molecular layer, just beneath the EGL, are attractive candidates for this role. NOV/CCN3 is a founder member of the CCN family (Cyr61/ccn1, Ctgf/ccn2, Nov/ccn3) of multitasking matricial proteins which are now emerging as key players during organogenesis (Chen and Lau, 2009). Recent reports evidence the importance of NOV during skeletal and cardiac development (Heath et al., 2008; Rydziel et al., 2007). However, although the central nervous system is a major site of NOV expression during development (Joliot et al., 1992; Kocialkowski et al., 2001), no study has yet tried to identify the putative functions of NOV during development of this tissue. Interestingly, the abundance of Nov mRNA increases throughout the postnatal period of rat brain development (Su et al., 2001). NOV is particularly abundant in the cerebellum of young adult rats and is specifically produced by Purkinje neurons at this stage (Su et al., 2001).

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NOV and the other cysteine-rich multimodular CCN proteins can regulate cellular processes as diverse as adhesion, migration, proliferation, differentiation and survival (Leask and Abraham, 2006). In particular, NOV regulates proliferation in various cellular contexts (Calhabeu et al., 2006; Ellis et al., 2000; Liu et al., 1999) and its overexpression leads to the growth arrest of several tumorigenic cell types (Benini et al., 2005; Gupta et al., 2001; McCallum et al., 2006). NOV can also stimulate the migration of several cell types including fibroblasts, endothelial and glioblastoma cells (Lin et al., 2005; Lin et al., 2003; Laurent et al., 2003). Interestingly, Nov overexpression inhibits proliferation and stimulates migration of a Ewing's Sarcoma cell line (Benini et al., 2005). Consistent with their multimodular structure and the diversity of their biochemical properties, NOV and other CCN members can modulate the activity of numerous signalling pathways including TGF-beta, TNF-alpha, VEGF, BMP, Wnt and Notch (Chen and Lau, 2009). In many cases, these modulations seem to result from direct interactions between CCN proteins and ligands, receptors or coreceptors of these pathways (Chen and Lau, 2009). However, it is currently assumed that NOV and other CCN members mainly act through integrin receptors (Chen and Lau, 2009). The specific integrin dimers described as possible NOV receptors include αvβ3 integrins (Ellis et al., 2003; Lin et al., 2005; Lin et al., 2003). Interestingly, two ligands of αvβ3 integrins, vitronectin and fibronectin, partially inhibit the mitogenic activity of SHH on GNP (Pons et al., 2001). NOV can also interact with several integrin dimers containing the β1 subunit which has been involved in the regulation of GNP proliferation in vivo and shown to be crucial for the synergistic effects of laminin on SHH-induced proliferation (Blaess et al., 2004; Graus-Porta et al., 2001). To study the roles of NOV during central nervous system development, we focused on the potential function of NOV during cerebellar development. We report that in rat cerebellum, NOV is specifically expressed by Purkinje neurons and its expression increases during the postnatal period. Moreover, we observed that NOV preferentially localises in the inner part of the external granule layer and in the molecular layer, at a key position to regulate both proliferation and migration of cerebellar granule neuron precursors. We therefore investigated whether NOV modulated these processes using GNP primary cultures. We found that NOV decreases SHHinduced proliferation by interacting with β3 integrins and triggering a pathway leading to the stimulation of GSK3-β activity and subsequent destabilization of N-Myc. In parallel, we demonstrate that NOV stimulates the migration of GNP through a distinct RGD-dependent integrin receptor.

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Fig. 1. NOV expression increases during postnatal cerebellar development. (A) Nov transcripts were quantified by real-time RT-PCR in rat cerebella collected on postnatal days (P) P01, P07, P14, P21 and P90. Mean values ± s.e.m. from five animals are expressed in arbitrary units and normalized to Gapdh mRNA. (B) Western blot analysis of NOV and Gapdh in rat cerebellar extracts from P01, P07, P14, P21 and P90. Results of two animals per stage representative of five animals are shown.

postnatal development. At P03, Nov transcripts were specifically detected in a cell population underlying the external granule layer (EGL) where Purkinje neurons reside (Fig. 2A). As revealed by ulterior detection of Calbindin-D-28K immunoreactivity (Fig. 2B), this cell population effectively corresponded to Purkinje neurons. We intriguingly observed that Nov transcripts were not detected in all Purkinje cell bodies at this early stage (Figs. 2C–D). These variations did not appear to correlate with developmental differences associated with foliation; thus, Nov expression may be induced in Purkinje neurons at an as yet unidentified maturation stage. At later developmental stages, such as P21 and at P90, Nov mRNA was specifically detected in all the Purkinje cell bodies which are organized in monolayers and are immunoreactive for Calbindin-D-28K (Figs. 2E, F, G). Even at high magnification, NOV mRNA was not detectable in the molecular layer or in the internal granule layer (Fig. 2H). This indicates that in the cerebellum Nov is specifically expressed by Purkinje neurons at birth and subsequently.

Results NOV expression increases during postnatal cerebellar development and specifically derives from Purkinje neurons Real-time RT-PCR experiments indicated that Nov transcript abundance was unchanged between postnatal days (P) 1 and 7 but then increased substantially between P07 and P14 (Fig. 1A). There were no obvious changes between P14 and P21 when cerebellar development is ending. Overall, the amount of Nov transcripts increased 3-fold between P01 and P21 (Fig. 1A) and it then doubled between P21 and P90. As assayed by western blotting on whole cerebellar extracts, NOV protein was already detected at P01, its expression level increased substantially between P07 and P14 and further increased by P21 and P90 (Fig. 1B). These findings demonstrate that NOV expression increases during the postnatal period of cerebellar development, with an induction mostly occurring between P07 and P14. We next performed in situ hybridization experiments to identify the cell types expressing Nov mRNA during cerebellar

The NOV protein localises preferentially in the inner external granule layer and the molecular layer NOV immunoreactivity was detected from P01 (Figs. 3A,B), when Purkinje neurons are committed and have exited the proliferation step but are still neither organized in a monolayer nor shaped (Sotelo, 2004). At this stage, NOV immunoreactivity overlapped with Calbindin-D-28K staining (Fig. 3C) but showed a broader distribution, spreading to the deeper region of the cerebellar cortex but not to the outer part of the external granule layer (EGL) containing the proliferating granule neuron precursors (Figs. 3A–C). We did not detect obvious signals using the flowthrough resulting from K19M purification (Figs. 3D–D′, see Experimental methods section). At P03, NOV immunoreactivity displayed a more intense signal at its most cortical location where it overlapped in the inner part of the EGL with postmitotic pre-migratory GNP revealed by TAG-1 immunoreactivity (Figs. 3E–H). From this stage, NOV immunoreactivity tends to be restricted to the molecular layer (ML), the region where DAPI staining is faint. This pattern was more obvious

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Fig. 2. Nov mRNA is specifically expressed by Purkinje neurons from birth. In situ hybridization using antisense (A,C,E,G,H) and sense (I) digoxigenin-labelled riboprobes specific for rat NOV mRNA at P03 (A,C), P21 (E) and P90 (G,H). Subsequent detection of Calbindin-D-28K immunoreactivity at P03 (B,D) and P21 (F). Bracket (A,B), star (C,D) and arrows (C,D,E, G) indicate Purkinje cell bodies. (C,D) High magnification of Nov in situ hybridization showing that Calbindin-immunoreactive Purkinje cell bodies do not all express NOV transcripts at P03 (positive/star vs negative/arrow cell bodies). (H) High magnification of Nov in situ hybridization at P90 showing that Nov transcripts are detected in Purkinje cell bodies but absent from soma of the other cell populations from the cerebellar cortex. Results representative of three animals per stage are shown.

on P07 (Figs. 3I, J), showing that NOV preferentially localises at the interface between proliferating granule neurons precursors (GNP) lying in the outer EGL and the monolayer of Purkinje cell bodies. NOV immunoreactivity coincided with the thickened ML at P14, when the EGL has become thinner due to the migration of numerous freshly postmitotic GNP towards the internal granule layer (Figs. 3K–L). In addition, NOV immunoreactivity overlaid the somas and the neo-forming dendritic arborescence of Purkinje neurons revealed by Calbindin-D-28K immunoreactivity (Fig. 3M). We used confocal microscopy to study the localisation of NOV more accurately: NOV immunoreactivity was in part colocalised with Calbindin-D-28K in both the somas and dendritic trees of Purkinje neurons (arrows, Fig. 3N), but some NOV immunoreactivity did not colocalise with Calbindin-D-28K (asterisk, Fig. 3N), indicating that NOV is also found in the extra-cellular matrix and thus secreted by Purkinje neurons. At P21 when the cerebellar cytoarchitecture is finalizing, NOV immunoreactivity still covered the molecular layer and Purkinje dendritic arborescence and extended to the limits of the cerebellar cortex while the EGL was disappearing (Figs. 3O–Q). We also observed some overlap between NOV and Calbindin-D-28K immunoreactivities in emerging Purkinje axons. The presence of NOV in Purkinje axons (arrows, Figs. 3P–R) was easy to detect at P21 but was also present at P14 (data not shown). This pattern of NOV localisation at various postnatal stages was confirmed using two other polyclonal anti-NOV antibodies: both gave similar patterns of labelling (data not shown). Moreover, the distribution of NOV did not change between P21 and P90 (data not shown). In summary, these data indicate that NOV localisation evolves with the postnatal shaping of the cerebellar cortex. NOV is secreted by Purkinje neurons and is present in the molecular layer and the inner part of the EGL, at the interface between Purkinje cell bodies and proliferating granule neuron precursors.

NOV reduces cerebellar GNP proliferation induced by Sonic Hedgehog in vitro The most cortical localisation of NOV overlaps with GNP exiting the cell cycle and NOV expression markedly increases when the rate of GNP proliferation decreases, suggesting that NOV may regulate GNP proliferation paracrinely. We therefore tested whether recombinant NOV protein modulated the proliferation of primary cultures of rat cerebellar GNP, having verified by western blot that cultured GNP do not produce detectable levels of NOV protein (data not shown). Immunohistochemical analysis of NOV expression and its affinity for heparin indicate that this CCN protein can be associated with the extra-cellular matrix (Leask and Abraham, 2006). Also, NOV is found in biological fluids, including blood serum and cerebrospinal liquid (Thibout et al., 2003), thereby suggesting that NOV can act on cells either as a substrate or soluble factor. Consequently, to investigate the function of NOV on GNP, we tested the effects of recombinant NOV both as a soluble and coated protein. We first performed Bromodeoxyuridine (BrdU) incorporation experiments and compared the effects of NOV to those mediated by vitronectin and laminin, two matricial components which oppositely modulate the proliferation of GNP induced by Sonic Hedgehog in vitro (Blaess et al., 2004; Pons et al., 2001). Cells were seeded on poly-lysine supports further coated with one of these various ECM proteins or PBS (control) and cultured for 48 h, the duration which has been shown to induce the maximal effect of SHH-N on GNP proliferation (WechslerReya and Scott, 1999). In the absence of mitogenic stimuli and despite the presence of B27 supplement, GNP proliferation was low as previously reported (Nicot et al., 2002; Pons et al., 2001). Neither NOV, nor vitronectin (VN) nor laminin (LN) affected this basal level of BrdU incorporation (Fig. 4A). By contrast, when GNP proliferation was stimulated by addition of SHH-N, the presence of NOV resulted in a 41% decrease in SHH-induced BrdU incorporation (14.0 ± 1.2 for

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SHH + NOV vs 21.9% ± 2.3 for SHH alone, P b 0.01, Fig. 4A). In comparison, the inhibition of SHH-N effects by vitronectin was only 26% (P b 0.05, Fig. 4A). By contrast, laminin strengthened the stimulatory effects of SHH-N on BrdU incorporation (46% increase of SHH effects, P b 0.01, Fig. 4A). Similar results were also observed when soluble NOV protein was added simultaneously with SHH-N (data not shown). In parallel, we analyzed GNP viability in response to NOV and SHH-N treatments by assessing DNA fragmentation with the TUNEL assay. As shown in Fig. 4B, treatments with NOV, SHH-N or both did not significantly modify the proportion of TUNELpositive cells. We neither observed any obvious effect of NOV or SHH-N treatments on the proportion of cells immunoreactive for cleaved caspase-3 (Fig. 4B), a key executioner of apoptosis in its active form (Fernandes-Alnemri et al., 1994). Therefore, by decreasing mitogenesis without modulating cell survival, NOV specifically reduces SHH-induced GNP proliferation. This was confirmed by

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analyzing the effects of NOV on the amounts of factors known to mediate SHH mitogenic activity in GNP (Kenney and Rowitch, 2000; Oliver et al., 2003). After 48 h of treatment, NOV significantly diminished the SHH-N-induced amounts of both N-Myc protein by 33% and Cyclin D1 protein by 35% (Figs. 4C,D). In addition, although NOV did not affect the basal levels of Cyclin D2 and E2F1 transcripts, the increase induced by SHH-N was significantly attenuated by the presence of NOV: these transcripts were 32 and 30% less abundant, respectively (P b 0.01, Fig. 4E). By maintaining GNP in a proliferative state, SHH delays their differentiation, as can be measured by counting the proportion of postmitotic neurons immunoreactive for NeuN (Pons et al., 2001; Wechsler-Reya and Scott, 1999). NOV did not affect per se the percentage of NeuN-immunoreactive cells but counteracted the inhibitory effect of SHH on GNP differentiation (45% inhibition of SHH effects, P b 0.05, Fig. 4F). These diverse experiments all

Fig. 3. Pattern of NOV localisation during postnatal cerebellar development. Immunohistochemical localisation of NOV on rat cerebellar cryosections obtained at P01 (A–D′), P03 (E– H), P07 (I–J), P14 (K–N) and P21 (O–R). DAPI staining (A, D′, E, I, K, O) and immunodetection of NOV (B, F, J, L, P), control antibody (D), TAG-1 (G) and Calbindin-D-28K (M, Q). (H) The most cortical part of NOV immunoreactivity overlays TAG-1 immunoreactivity. (N,R) Confocal microscopy shows the localisation of NOV in the extra-cellular matrix (asterisk) and a colocalisation with Calbindin-D-28K in soma, dendritic processes (arrows) and axons of Purkinje neurons (arrowheads). Results representative of three animals per stage are shown. External (EGL) and internal (IGL) granule layers. Molecular layer (ML).

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demonstrate that NOV is able to reduce SHH-induced GNP proliferation in vitro and consequently promote their differentiation. The anti-proliferative action of NOV requires GSK3-β activity Recent studies have established that the mitogenic action of SHH on GNP results from the transcriptional increase of N-myc gene triggered by activated GLI factors (Kenney and Rowitch, 2000; Knoepfler et al., 2002; Oliver et al., 2003). N-Myc activity is indeed necessary and sufficient to stimulate GNP proliferation both in vitro and in vivo (Kenney and Rowitch, 2000; Knoepfler et al., 2002; Oliver et al., 2003). As assessed by real-time RT-PCR experiments, NOV modulated N-myc transcripts level neither per se, nor by

antagonizing the slight but significant increase induced by a 3 h treatment with SHH-N (Fig. 5A). Similar results were obtained when measuring the abundance of Gli-1 mRNA (Fig. 5A), the induction of which is the best marker of SHH pathway activity (Ruiz i Altaba et al., 2002). These data thereby indicate that the anti-proliferative activity of NOV does not result from an inhibition of the early steps of SHH-GLI signalling and suggest that NOV acts downstream from Gli transcriptional activity. Interestingly, Kenney et al. demonstrated that the amount of NMyc protein in GNP is also influenced in a SHH-independent manner by the regulation of N-Myc protein stability (Kenney et al., 2004). Indeed, GSK3-β directly stimulates the hyperphosphorylation of the N-Myc protein leading to its subsequent degradation (Kenney et al.,

Fig. 4. NOV reduces SHH-induced proliferation of primary cultured GNP. (A) Cells were seeded on poly-lysine-coated supports (Ctrl) or supports additionally coated with 10 μg/ml of recombinant NOV (NOV), vitronectin (VN) or laminin (LN), and cultured without (− SHH) or with 3 μg/ml of recombinant mouse SHH-N (+SHH) for 48 h. Data represent the mean percentage of BrdU-labelled cells ± s.e.m. (B) TUNEL assays and immunocytochemical analysis of cleaved caspase 3-expressing (cCasp3+) cells were performed on GNP untreated (white bars) or treated with 10 μg/ml of recombinant NOV (black bars) in the absence (− SHH) or presence (+SHH) of 3 μg/ml SHH-N for 48 h. Data represent the mean percentage of TUNEL-positive or cleaved caspase 3-positive cells ± s.e.m. (C,D) Western blot analysis of N-Myc (C) and Cyclin D1 (D) in GNP untreated (white bars) or treated with NOV (black bars) in the absence (− SHH) or presence of SHH-N (+ SHH) for 48 h. Gapdh was used as control. Results representative of three independent experiments are shown. Quantifications by densitometry and statistical significances are reported. (E) Real-time RT-PCR assays of Cyclin D2 and E2F1 transcripts from GNP untreated (white bars) or treated with NOV (black bars) in the absence (− SHH) or presence of SHH-N (+SHH) for 48 h. Data represent the mean values ± s.e.m. of transcript levels for each gene normalized to endogenous β-actin mRNA and are expressed in arbitrary units. (F) Immunocytochemical analysis of NeuN-immunoreactive cells after GNP had been cultured without (white bars) or with NOV (black bars) in the absence (− SHH) or presence of SHH-N (+SHH) for 48 h. Results are expressed as mean percentages ± s.e.m. Statistical significance was tested by ANOVA followed by a Student–Newman–Keuls' test. (⁎P b 0.05; ⁎⁎P b 0.01; n.s. = non significant).

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2004). As shown in Fig. 5B, treatment of GNP with SHH-N for 45 h markedly increased the amount of N-Myc protein. Subsequent addition of NOV for 3 h resulted in a notable reduction of N-Myc abundance (45% reduction, Fig. 5B). This effect was associated with a decreased level of Ser9-phosphorylated GSK3-β (inactive GSK3-β) while the amount of total GSK3-β was unchanged (30% reduction of the Ser9-phosphorylated GSK3-β/total GSK3-β ratio, Fig. 5B). Interestingly, the effects triggered by NOV on GSK3-β phosphorylation and N-Myc levels were both antagonized by IGF-1 (Fig. 5B) which was reported to stabilize N-Myc protein by triggering the inhibition of

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GSK3-β activity through activation of the PI3K/Akt signalling (Kenney et al., 2004). Moreover, the decreased levels of phosphorylated GSK3β and N-Myc induced by NOV were abolished when cells were pretreated 15 min with lithium chloride (Fig. 5C), which is routinely used to inhibit GSK3-β activity (Cohen and Frame, 2001; Kenney et al., 2004). Finally, the anti-proliferative action of NOV was abolished when GSK3-β activity was blocked by lithium or SB216763, a selective ATP-competitive inhibitor of GSK3 (Fig. 5D) (Cross et al., 2001). This demonstrates that the anti-proliferative activity of NOV on SHHinduced proliferation requires the stimulation of GSK3-β activity.

Fig. 5. NOV decreases SHH-induced proliferation by stimulating GSK3-β-mediated N-Myc degradation. (A) Real-time RT-PCR of N-myc and Gli-1 transcripts from GNP cultured for 3 h without (white bars) or with 10 μg/ml of recombinant NOV (black bars) in the absence (− SHH) or presence of 3 μg/ml of SHH-N (+ SHH). Transcript levels for each gene were normalized to endogenous β-actin mRNA and are expressed in arbitrary units as the mean ± s.e.m. mRNA levels. (B) Western blot analysis of N-Myc, Ser9-phosphorylated GSK3-β (pSer9-GSK3-β), total GSK3-β and Gapdh. GNP were cultured without or with 3 μg/ml of SHH-N for 45 h. Cells were then further treated with 10 μg/ml of NOV and/or 100 ng/ml of IGF-1 for three additional hours. Results representative of two independent experiments are shown. Quantifications by densitometry are reported (C) GNP were treated as described in B except that 10 mM lithium chloride (LiCl) was added 15 min prior to NOV addition. Results representative of two independent experiments are shown. Quantifications by densitometry are reported. (D) GNP were cultured for 48 h without or with 3 μg/ml of SHH-N (SHH), 2.5 mM of lithium chloride (LiCl), 3 μM of the selective GSK-3 inhibitor SB216763 (SB21), in the absence or presence of 10 μg/ml of NOV. Data represent mean values of BrdU-labelled cells ± s.e.m. (compared to standardised values obtained for SHH-treated cells). Statistical significance was tested by ANOVA and a Student–Newman–Keuls' post hoc test. (⁎P b 0.05; ⁎⁎P b 0.01, n.s. = non significant).

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The anti-proliferative action of NOV is mediated by β3 integrins Studies which have reported a modulation of GSK3-β activity by CCN proteins all suggested that this results from their binding to integrins (Crean et al., 2004; Su et al., 2002; Xie et al., 2004). Interestingly, integrins are considered as essential mediators of the regulatory action of CCN proteins on cellular proliferation (Leask and Abraham, 2006). We thus wondered if some integrin receptors were responsible for the anti-proliferative activity of NOV on GNP. The specific integrin dimers described as possible NOV receptors include α6β1 and αvβ3 integrins (Ellis et al., 2003; Lin et al., 2005; Lin et al., 2003) which have already been linked to regulation of GNP proliferation (Blaess et al., 2004; Pons et al., 2001). We thus performed BrdU incorporation experiments in the presence of antibodies blocking the putative interaction of NOV with either β1 or β3 integrins. As shown in Fig. 6, the inhibitory effect of NOV on SHH-induced BrdU incorporation was abolished in the presence of anti-β3 antibodies whereas it was not affected by addition of anti-β1 antibodies. These data therefore indicate that the anti-proliferative action of NOV requires its interaction with integrin dimers involving the β3 subunit. NOV stimulates migration of cerebellar GNP in vitro through a distinct RGD-dependent integrin receptor The pattern of NOV expression and localisation during postnatal cerebellar development is also consistent with NOV being involved in granule cell migration. We therefore performed individual cell recordings by time-lapse videomicroscopy and tested whether the recombinant NOV protein modulated GNP motility in vitro. At seeding, cells displayed a small round-shaped morphology typical of the precursor stage and developed neuritis within 12 h as previously described (Falluel-Morel et al., 2005). Addition of various concentrations of NOV recombinant protein resulted in a dose-dependent increase of GNP mean velocity that was maintained throughout the recording period (13.8 ± 0.7 μm/h for 10 μg/ml NOV vs 8.9 ± 0.4 μm/h for control). Thus, highest doses of recombinant NOV protein resulted in a progressive and linear increase of GNP motility (P b 0.001 after 12 h, Fig. 7B, see movies added as Supplementary data). We further analyzed cell movements and calculated trajectories (example of the reconstruction of the trajectory of a single cell is shown in Fig. 7A); we observed a significant stimulation of the distance from the origin covered by cells in response to NOV (13.0 μm ± 1.7 for 10 μg/ml NOV vs 6.8 μm ± 0.8 for control after 12 h, P b 0.001, Fig. 7C). Similar results were obtained with cells seeded on NOV-coated dishes (data not

Fig. 6. The anti-proliferative activity of NOV requires its interaction with β3 integrins. GNP were cultured for 48 h without or with 3 μg/ml of SHH-N (SHH) and/or 10 μg/ml of NOV in the absence or presence of 50 μg/ml of blocking antibodies specific for β1 or β3 integrins. Data represent mean values of BrdU-labelled cells ± s.e.m. (compared to standardised values obtained for SHH-treated cells). Statistical significance was tested by ANOVA and a Student–Newman–Keuls' post hoc test. (⁎P b 0.05; ⁎⁎P b 0.01, n.s. = non significant).

Fig. 7. NOV stimulates GNP motility in vitro. Time-lapse videomicroscopy recordings. (A) Microphotographs at times 0 h and 12 h of culture showing individual cell recordings of GNP treated without (Ctrl) or with 10 μg/ml of recombinant NOV protein (NOV). Orthogonal frames are represented to show starting positions of cells. (B–C) Cumulated distance (B) and distance from the origin (C) covered by individual cells were measured for 12 h in the absence (black) or presence of 0.1 (green), 1.0 (yellow) or 10 (red) μg/ml of NOV. Data represent the mean ± s.e.m. of individual cell recordings. Statistical analysis was performed using ANOVA followed by a Tukey's test (⁎⁎⁎P b 0.001).

shown). These data indicate that NOV increases GNP motility in vitro, and further suggest that NOV stimulates oriented cell migration (chemotaxis). NOV belongs to the family of matricellular proteins. Those proteins are known to promote directed migration of cells on chemoattractant molecule bound on a surface (haptotaxis). Using Boyden chamber assay (Carnegie 1994; Borghesani et al. 2002) the putative haptotaxis activity of NOV was tested on NOV-coated membrane. As illustrated in Fig. 8A, we observed a marked increase in the mean number of GNP that migrated when seeded on NOVcoated membranes (73% ± 13 increase in PLL + NOV, P b 0.01, Fig. 8B). Subsequently, we tested the chemoattractant potential of NOV on GNP. Addition of NOV to the lower chamber wells significantly increased the number of migrating GNP by 68% ± 13 relative to controls (P b 0.01, Fig. 8C). When NOV was added to both upper and lower chambers, the chemoattractrant effect of NOV was abolished. These experiments further demonstrate that NOV is chemoattractant for GNP in vitro.

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data strongly suggest that the pro-migratory effect of NOV requires its interaction with an RGD-dependent integrin receptor which does involve neither the β1 nor the β3 subunits. Discussion To investigate the function of NOV during cerebellar development, we first established that NOV is specifically expressed and secreted by Purkinje neurons; we also demonstrated that NOV is mostly localised in the molecular layer and the inner part of the EGL, at the interface between Purkinje cell bodies and proliferating granule neuron precursors (GNP). Moreover, induction of NOV expression during postnatal cerebellar development coincides with the period of granule cell transition from the proliferative to the migratory stage. In addition, we report the first description of the effects of NOV on neural progenitor cells by demonstrating in vitro that NOV decreases SHH-induced proliferation and promotes migration of GNP, two effects mediated by distinct RGD-dependent integrin receptors. These findings strongly suggest that NOV/CCN3 may promote the maturation of granule neurons during postnatal development of the cerebellum. Note that NOV expression still increases after completion of proliferation and migration of granule cells and is maintained even after total disappearance of the EGL at P90. This is consistent with NOV having some role at later stages, in the dendritogenesis of Purkinje cells or in the synaptogenesis involving Purkinje dendrites and the axonal endings of the granular parallel fibres or climbing fibres. There is currently no data concerning the consequences of the invalidation of Nov gene on the development of the central nervous system. Recently, the mouse phenotype resulting from a Nov/ccn3 gene disruption was described (Heath et al., 2008). These mice present abnormal skeletal and cardiac development, cardiomyopathy, muscle atrophy and cataract but the phenotype of the central nervous system of these mutant mice has not been reported. In addition, the presence of a mutant NOV protein expressed at low levels in several tissues of this mouse model may have resulted in a phenotype distinct from that associated with complete invalidation of the Nov gene (Perbal, 2007). Therefore, our data are the first evidence for a function of NOV during development of the central nervous system and, particularly, the cerebellum. NOV could play a key role during cerebellar development by promoting cell cycle exit and migration of GNP

Fig. 8. NOV stimulates GNP migration in vitro through another RGD-dependent integrin receptor. Boyden chamber assays. Pictures (A) of granule cells that have migrated (arrows) after an overnight period on poly-lysine-coated (PLL) membranes or membranes additionally coated with 10 μg/ml of NOV (PLL + NOV) and (B) mean standardised values ± s.e.m. (C) Cells were seeded on PLL membranes and cultured in the absence (−) or presence of 10 μg/ml of NOV (NOV) added in lower and/or upper chambers. Results represent the mean standardised values ± s.e.m. of migrated cells. (D) Cells were pre-treated with 50 μM of the synthetic GRDGS peptide (RGD) or 50 μg/ ml of anti-β1 or -β3 integrins antibodies, then seeded on PLL membranes and cultured in the absence (white bars) or presence of 10 μg/ml of NOV (black bars) in the lower chambers. Results represent the mean standardised values ± s.e.m. of migrated cells, obtained from 8 (for B and C) or 3 (D) independent experiments. Statistical significance was assessed using ANOVA followed by a Student–Newman–Keuls' test (⁎⁎P b 0.01).

Based on our results indicating that the anti-proliferative action of NOV requires its interaction with integrins, we assessed the chemoattractant activity of NOV in the presence of either β1 or β3 integrins blocking antibodies. As shown in Fig. 8D, the pro-migratory effect of NOV was not affected by addition of either anti-β1 or anti-β3 antibodies but was abolished when cells were cultured in the presence of a synthetic GRGDS peptide (Fig. 8D) or echistatin, a RGD-containing disintegrin (data not shown). Therefore, these latter

After completion of GNP proliferation, the proper establishment of cerebellar granule neurons requires several steps, including exit from the cell cycle, induction of migration and differentiation. These events occur within an environment enriched in Purkinje-derived Sonic Hedgehog (SHH) (Choi et al., 2005) which is the main mitogenic signal for cerebellar GNP (Dahmane and Ruiz i Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999). Thus, the current view proposes that cell cycle exit of GNP and subsequent developmental steps require effectors to inhibit the mitogenic activity of SHH-GLI signalling. Some factors, including the pituitary adenylate cyclaseactivating polypeptide (PACAP) (Nicot et al., 2002), several bone morphogenetic proteins (BMPs) (Alvarez-Rodriguez et al., 2007; Rios et al., 2004) and fibroblast growth factors (FGFs) (Fogarty et al., 2007) reduce the proliferation rate of GNP in cerebellar explants or the mitogenic activity of SHH on cultured GNP. In this study, we showed that the expression of NOV markedly increases between P07 and P14, the period during which the EGL begins to thin down as the rate of granule neuron precursors proliferation decreases. From early stages of cerebellar postnatal development, the most cortical localisation of NOV overlaps with granule neuron precursors exiting the cell cycle. Our data demonstrate that in vitro NOV reduces SHH-induced GNP proliferation. These data strongly suggest that NOV favours cell cycle

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exit by GNP during development by reducing the mitogenic action of the SHH-Gli pathway. In mice deficient for the gene encoding Semaphorin 6A, many granule cells remain ectopically in the molecular layer but are still able to differentiate and connect to mossy fibres (Kerjan et al., 2005). However, in cases of impaired GNP migration due to genetic invalidation of Astrotactin, BDNF or rev-ErbA, GNP remain stuck in the EGL and continue to cycle beyond the normal temporal window of the proliferation step (Adams et al., 2002; Borghesani et al., 2002; Chomez et al., 2000). Those data indicate that the outer EGL represents a mitogenic niche for GNP (Choi et al., 2005) and that migration away from this niche may be necessary for GNP to exit the cell cycle successfully. We show here that NOV promotes GNP migration in vitro. Given its localisation in the inner part of the EGL and in the molecular layer, NOV may also favour continuing maturation of granule neurons by stimulating GNP migration. Interestingly, the ability of NOV to modulate both GNP proliferation and migration is similar to that reported for SDF-1α/CXC12 (Klein et al., 2001). This chemokine, which is secreted from the pia mater, whereas its receptor CXCR4 is expressed by proliferating GNP, both induces chemoattraction and enhances the mitogenic effects of SHH on cultured GNP (Klein et al., 2001). SDF-1α, by maintaining GNP proliferation in the outer EGL, appears to prevent their premature maturation (Klein et al., 2001). Thus, NOV located in the inner part of the EGL may act conversely, both repressing proliferation of GNP and stimulating their migration from the EGL so as to favour the continuing maturation of GNP during the appropriate temporal window. NOV decreases SHH-induced GNP proliferation through a mechanism involving β3 integrins and GSK3-β Having demonstrated that NOV reduces the mitogenic effects of SHH on GNP in vitro, we tested a possible regulation of N-Myc activity by NOV. N-Myc indeed represents a key mediator of SHH-GLI pathway in GNP and its activity is necessary and sufficient to drive GNP proliferation both in vitro and in vivo (Kenney et al., 2003; Knoepfler et al., 2002; Oliver et al., 2003). We showed that NOV has no effect on the early induction of N-myc (nor Gli-1) transcription by SHH, indicating that NOV does not counteract the initial steps of SHH-GLI signalling but rather acts downstream of Gli transcriptional activity. We next reported that NOV action results in a rapid decrease in N-Myc protein amount. This effect is correlated with a decreased proportion of inactive ser9-phosphorylated GSK3-β. GSK3-β is a multitasking serine/threonine kinase which downregulates GNP proliferation by stimulating hyperphosphorylation of N-Myc, thereby favouring its degradation by the proteasome (Kenney et al., 2004). The decrease of N-Myc amount caused by NOV was abolished in the presence of lithium chloride which is routinely used to inhibit GSK3-β activity (Cohen and Frame, 2001; Kenney et al., 2004), or by simultaneous addition of IGF-1 which was reported to stabilize N-Myc protein by triggering the inhibition of GSK3-β activity through activation of the IGF-1R/PI3K/Akt pathway (Kenney et al., 2004). Moreover, blocking GSK3-β activity in GNP by addition of either lithium or SB216763 abolished the reduction of SHH-induced proliferation caused by NOV. Taken together, these results suggest that NOV inhibits SHH-induced proliferation through a GSK3-β-mediated destabilization of N-Myc. We also determined which receptors could mediate the antiproliferative activity of NOV. Among others, CCN proteins can interact with integrins and as such regulate diverse cellular processes, including proliferation (Leask and Abraham, 2006). By using antibodies blocking the putative interaction of NOV with either β1 or β3 integrins, we determined that the anti-proliferative action of NOV requires its interaction with integrin dimers involving the β3 subunit. Previous studies have found that β1 is important for Shh-induced proliferation and for the synergistic effect of laminin (Blaess et al.,

2004). Our results indicate that β1 blocking antibodies do not affect GNP proliferation induced by SHH-N nor in the presence of NOV. Thus NOV and laminin activate different integrin dimers and modulate the proliferation of GNP through different mechanisms. Interestingly, vitronectin and fibronectin, two ligands of αvβ3 integrins, were also described to partially reduce the mitogenic activity of SHH-N on GNP (Pons et al., 2001). In addition, αvβ3 integrins immunoreactivity has been detected where we localised NOV: in the inner EGL and the ML (Pons et al., 2001). Therefore, αvβ3 integrins appear as an appealing candidate for mediating the anti-proliferative activity of NOV on GNP. Studies which have previously described the modulation of GSK3β activity by CCN proteins proposed a similar scheme of molecular events linking CCN, integrins and GSK3-β. Binding of CCN proteins to specific integrin receptors stimulates the activity of Integrin-Linked Kinase (ILK) which in turn inhibits GSK3-β activity either directly or indirectly through stimulation of Akt (Crean et al., 2004; Su et al., 2002; Xie et al., 2004). Interestingly, CNS restricted knock-out of the ilk gene results in the disruption of postnatal GNP proliferation and pharmacological inhibition of ILK kinase activity decreases SHHinduced BrdU labelling in cultured GNP (Mills et al., 2006). Therefore, inhibition of ILK activity in response to NOV is consistent with an activation of GSK3-β and its anti-proliferative activity on GNP. As previously reported, this indicates that NOV can act in the opposite way to other CCN proteins which after binding to integrin receptors activate ILK. A human fibronectin peptide has been shown to reorganize αvβ3 and αvβ5 integrins, to dissociate the β1ILK complex leading to inactivation of the ILK pathway. However the activation of GSK3β by NOV may also depend on other mechanisms. Several reports reveal that the regulation of GSK3β activity by dephosphorylation involves PP1, PP2A and PP2B phosphatases which interact with the α and β chains of integrin receptors. The binding of NOV to integrin receptors may therefore activate these phophatases or regulate their expression leading to activation of GSK3 and to N-Myc degradation. NOV stimulates GNP migration through another integrin receptor Beyond its effect on proliferation, we also demonstrated that NOV promotes GNP migration in vitro. Such pro-migratory effects of NOV have already been reported in several cellular contexts. NOV overexpression results in an increased migration of glioblastoma and Ewing's Sarcoma derived cell lines (Benini et al., 2005). A recombinant NOV protein has also been shown to stimulate the migration of fibroblasts and endothelial cells (Laurent et al., 2003; Lin et al., 2003, 2005). In these latter studies, the pro-migratory effects of NOV were mediated by integrin receptors (Laurent et al., 2003; Lin et al., 2003, 2005). As we demonstrated that the anti-proliferative activity of NOV on GNP depends on its interaction with β3 integrins, we thus investigated whether the stimulation of GNP migration by NOV was also mediated by β3 integrins. Unexpectedly, the chemoattractant activity of NOV was not inhibited by addition of anti-β3 integrins antibodies. NOV effects were not inhibited by addition of anti-β1 antibodies either. Some preliminary data obtained by time-lapse videorecording indicate that NOV promotes an increase of motility for GNP even when seeded in laminin-coated dishes (data not shown), thereby suggesting additive or synergistic effects of NOV and laminin on GNP migration. Given that laminin mediates most, if not all, of its effects through β1 integrins (Humphries et al., 2006), this observation reinforces the notion that NOV and laminin act through different molecular partners to stimulate GNP migration. Conversely, NOV promigratory effects were abolished in the presence of a synthetic GRGDS peptide or echistatin, a RGD-containing disintegrin (data not shown). These data thus strongly suggest that the pro-migratory effect of NOV requires its interaction with an RGD-dependent integrin receptor which is distinct from the one mediating its anti-proliferative activity.

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The possibility that NOV may mediate different cellular effects through distinct integrins receptors has already been reported in several systems. As such, NOV supports primary skin fibroblasts adhesion through integrins α5β1 and α6β1 whereas it induces fibroblast chemotaxis through integrins αvβ5 (Laurent et al., 2003; Lin et al., 2003, 2005). Considering all the integrins dimers already described as putative NOV receptors, the only one that does not involve either β1 or β3 subunits corresponds to αvβ5. Integrins αvβ5 thus appears as an appealing candidate for mediating the promigratory activity of NOV on GNP. Because of the lack of availability of blocking antibodies against rat integrins, we were not able to discriminate which RGD-dependent integrins are mediating this effect of NOV. Further experiments will thus be needed to determine precisely the molecular pathway mediating the pro-migratory action of NOV on GNP. Responses to integrin engagement depend not only on which ligand is bound to specific integrin but also the form in which the ligand is presented to the integrin. Several reports also indicate that the engagement of different integrins activates multiple intracellular signalling pathways which likely interact and coordinate each other to contribute to the different cellular activities of integrins. Acting as a matricellular protein, NOV functions through direct binding to disting integrin receptors. In endothelial cells and fibroblast NOV promotes cell adhesion and cell migration by the activation of distinct integrin dimers (Lin et al., 2005). Likely, in GNP the binding of NOV to different integrins may thus activate different signallings to promote chemotactism and to reduce the proliferative activity of Shh on GNP. In conclusion we report the first evidence of a role for NOV in CNS development involving its ability to regulate proliferation and migration by increasing chemotactic and chemokinetic activities on GNP in vitro. In vivo studies or work with cerebellum organic cultures will be required to elucidate further the mechanism of NOV-induced migration and proliferation of GNP Experimental methods Animals Wistar rats from Janvier were kept under standard housing, feeding and lighting conditions. All animal procedures were performed in accordance with the European Union Guidelines for the Care of Laboratory Animals. Antibodies and chemicals NOV immunoreactivity was detected using a commercial goat (R&D Systems) or the home-made affinity-purified rabbit (referred as K19M, (Chevalier et al., 1998) anti-human NOV antibodies. The flowthrough resulting from K19M purification was used as a negative control. For western blotting experiments, we used a rabbit antimouse NOV polyclonal antibody giving better signals. Other primary antibodies used were monoclonal mouse anti-Calbindin-D-28K (Sigma-Aldrich), anti-Cyclin D1 (Santa Cruz), anti-GAPDH (Chemicon), anti-NeuN (Chemicon), anti-N-Myc (Calbiochem), anti-O4 (AbCam), anti-TAG-1 (Developmental Studies Hybridoma Bank), and rabbit polyclonal anti-cleaved caspase-3, anti-total GSK3-β, antiSerine9 phosphorylated GSK3-β all purchased from Cell Signaling Technology or anti-GFAP (DakoCytomation). Recombinant human NOV protein was produced and purified as previously described (Doghman et al., 2007; Lafont et al., 2005). The effects of Sonic Hedgehog (SHH) were assessed by addition of the recombinant amino-terminal fragment of mouse SHH (referred as SHH-N, R&D Systems). IGF-1 was purchased from Preprotec. Laminin was a kind gift from Dr H. Kleinman (NIH, Bethesda, USA). Vitronectin from rat plasma and poly-L-lysine (70–150 kDa) were purchased from Sigma.

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Granule cell cultures Granule neuron precursors (GNP) were isolated from P06-P08 rat cerebella as previously described (Nicot et al., 2002). Cells were resuspended in Neurobasal medium (Invitrogen) containing 2% B27 supplement (Invitrogen), 0.5 mM Glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. We added 20 mM NaCl rather than 20–25 mM KCl because KCl can affect basal GNP proliferation (Borodinsky and Fiszman, 1998; Cui and Bulleit, 1998) and is not required for short cell survival in serum-free B27 supplemented media (Nicot et al., 2002). A pre-plating step on 10 μg/ml poly-L-lysine-coated dishes was performed to purify GNP further. Purified GNP were seeded on 10 μg/ml poly-L-lysine-coated supports or supports additionally coated with 10 μg/ml laminin, vitronectin or recombinant NOV protein for four hours at 37 °C in 5% CO2. After seeding, cells were allowed to adhere for 45 min and were then treated with recombinant SHH-N [3 μg/ml], NOV [10 μg/ml], IGF-1 [100 ng/ml] as appropriate. In some experiments, cells were treated with 3 μM of the selective GSK3 inhibitor SB216763 (Sigma), 50 μg/ml of purified hamster anti-CD29 (integrin β1chain, BD Biosciences) or mouse anti-CD61 (integrin β3 chain, BD Biosciences) monoclonal antibodies, or lithium chloride (10 mM for 3 h, 2.5 mM for 48 h treatments). Potential contaminating astrocytes, oligodendrocytes and Purkinje cells were assayed by GFAP, Rip, and Calbindin-D-28K immunoreactivities and made up 1.0% ± 0.2, 0.2% ± 0.1, 0.2% ± 0.1 respectively of the total cell population. In situ hybridization Rat brains were removed on postnatal days 1 to 90, postfixed in 4% paraformaldehyde (PFA) for 24 h and cryoprotected in 30% sucrose. Serial sagittal sections, 16 μm thick, were cut using a cryostat (Leica). Sense and antisense digoxigenin-labelled riboprobes specific for rat Nov mRNA were synthesized according to the manufacturer's protocol (Roche Diagnostic). Rat cerebellar cryosections were incubated for 18 h at 55 °C in hybridization buffer (50% formamide, 1× SSC, 1 mg/ml yeast tRNA, 1× Denhardt's) containing rat nov cRNA probes, then washed with 50% formamide, 1× SSC, 0.1% Tween 20 min at 65 °C. Hybridization was revealed by alkaline phosphatase-coupled anti-DIG Fab fragments according to the manufacturer's protocol (Roche Diagnostics) and with NBT/BCIP detection. Immunofluorescence analysis Immunostaining of cerebellar sections obtained as described for in situ hybridization or primary GNP fixed in 4% PFA for 15 min at room temperature was performed following standard procedures. Tissue sections were permeabilzed with 0.1% Tween or 0.1% triton prior to incubation with primary antibodies. Bound antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 or 594 (Invitrogen). Sections were finally mounted in the presence of DAPI to counterstain nuclei (Vector). To evaluate the GNP proliferation rate, cells were exposed to the S phase marker BrdU (10 μM, Sigma) for the final four hours of culture. Cells were fixed in 4% PFA for 20 min, rinsed in PBS prior exposure to HCl 2 N for 30 min, rinsed again and then incubated for one hour at 37 °C with a rat monoclonal anti-BrdU (Serotec). Cell death was evaluated by the TUNEL method according to the manufacturer's protocol (In Situ Cell Death Detection Kit, Fluorescein, Roche). Data are presented as means ± s.e.m. of immunoreactive cells counted from five random and non-overlapping fields for three replicates per experiment. At least three independent experiments were performed. ANOVA followed by the Student– Newman–Keuls' post hoc test were used for statistical analysis. Confocal analysis was carried out by the Service d'imagerie cellulaire de l'IFR65 (hôpital Tenon, Paris, France) using the TCS SP2 Leica (Lasertechnik GmbH) equipped with a 63× objective. For each optical section, triple fluorescence images were acquired in sequential

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mode and selected paired sections were then processed to produce single composite overlay images (color-merged). The focus step between the sections was generally 0.3 μm. Migration assays Real-time videomicroscopy For imaging of living cells, primary GNP were treated as indicated on the figures and cultured for 12 h at 37 °C in a controlled atmosphere (5% CO2) under an inverted microscope (Leica) equipped with an incubation chamber (Solent Scientific), standard phase-contrast optics, computer-controlled illumination shutters and filter wheels. Images were acquired every five minutes with a ×20 objective using a CoolSNAPfx camera (Princeton Instruments). Migration variables, including cell trajectories and distances covered, were analyzed using the METAMORPH software (Universal Imaging). Data are presented as means ± s.e.m. from at least 30 individual cell recordings per treatment obtained from four independent experiments. ANOVA followed by the Tukey's test were used for statistical analysis. Boyden chamber GNP migration was assayed in a modified Boyden chamber containing polyvinylcarbonate-free membranes (8–12 μm pores, Osmonic Inc.) coated with 10 μg/ml poly-L-lysine. To test for chemokinetic effects of NOV, membranes were further coated with 10 μg/ml of recombinant NOV protein. To study NOV chemotactic activity, 10 μg/ml of recombinant NOV protein was placed in the lower and/or upper chambers. Bovine serum albumin was used as a control. Cells were added to the upper chamber and cultured overnight at 37 °C in 5% CO2, then fixed with 4% PFA and stained with 0.2% Crystal Violet. Cells on the lower side of the filter were then counted. Data are expressed as mean standardised numbers of migrated cells per field ± s.e.m. from at least 30 replicate wells obtained from five to eight independent experiments. Statistical significance was assessed using ANOVA and the Student–Newman–Keuls' post hoc test. Real-time RT-PCR Total RNA was extracted from meninges-free cerebellar extracts or from primary GNP cultures using the Nucleospin RNA II kit (Macherey-Nagel). Reverse transcription and semi-quantitative realtime PCR amplification on the LightCycler apparatus were performed as previously described (Calhabeu et al., 2006). Specific primers (Proligo, Sigma-Aldrich) used for amplification of target genes were designed using the online Primer3 Input program and verified for specificity on BLAST. The comparative Ct method (Pfaffl, 2001) was used to calculate gene expression values. Gapdh and β-actin were used as housekeeping genes for cerebellar tissue extracts and primary cultures, respectively. PCR amplifications were assessed from two reverse transcriptions per experiment, for at least three independent experiments. Data are presented as mean standardised values ± s.e.m. ANOVA followed by the Student–Newman–Keuls' post hoc test was used for statistical analysis. Western blotting Total protein extracts were prepared from primary GNP cultures or rat cerebella collected on postnatal days 1 to 90 and were solubilised in lysis buffer in the presence of protease and phosphatase inhibitor cocktails (Sigma). The protein extracts were analyzed by Western blotting using 10% polyacrylamide gels and polyvinylidene difluoride (PVDF) membranes (Pharmacia Biotech). The membranes were incubated with appropriate primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Sigma). Enhanced chemiluminescence (Amersham, GE Healthcare) was used to develop the blots.

Acknowledgments This work was supported by l'Institut National de la Santé et de la Recherche Médicale (INSERM). G. Le Dréau was recipient of a fellowship from the Association pour la Recherche contre le Cancer (ARC). We are grateful to I. Renault and her team for devoted animal care. We also acknowledge Dr F. Calhabeu (Randall Division of Cell and Molecular Biophysics, King's college, Guy's Hospital, London, UK) for skilful assistance with real-time RT-PCR. We thank Dr C. Dubois (INSERM UMR-S 893, Hôpital Saint-Antoine, Paris, France) for fruitful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mcn.2009.02.011. References Adams, N.C., Tomoda, T., Cooper, M., Dietz, G., Hatten, M.E., 2002. Mice that lack astrotactin have slowed neuronal migration. Development 129, 965–972. Alvarez-Rodriguez, R., Barzi, M., Berenguer, J., Pons, S., 2007. 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Cross, D.A., Culbert, A.A., Chalmers, K.A., Facci, L., Skaper, S.D., Reith, A.D., 2001. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J. Neurochem. 77, 94–102. Cui, H., Bulleit, R.F., 1998. Potassium chloride inhibits proliferation of cerebellar granule neuron progenitors. Brain Res. Dev. Brain Res. 106, 129–135. Chen, C.C., Lau, L.F., 2009. Functions and mechanisms of action of CCN matricellular proteins. Int. J. Biochem. Cell Biol. 41 (4), 771–783. Chevalier, G., Yeger, H., Martinerie, C., Laurent, M., Alami, J., Schofield, P.N., Perbal, B., 1998. novH: differential expression in developing kidney and Wilm's tumors. Am. J. Pathol. 152, 1563–1575. Choi, Y., Borghesani, P.R., Chan, J.A., Segal, R.A., 2005. Migration from a mitogenic niche promotes cell-cycle exit. J. Neurosci. 25, 10437–10445. Chomez, P., Neveu, I., Mansen, A., Kiesler, E., Larsson, L., Vennstrom, B., Arenas, E., 2000. 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