Sonic hedgehog promotes the migration and proliferation of optic nerve oligodendrocyte precursors

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 36 (2007) 355 – 368

Sonic hedgehog promotes the migration and proliferation of optic nerve oligodendrocyte precursors Paloma Merchán, a,b,1 Ana Bribián, a,b,1 Cristina Sánchez-Camacho, c,1 Melissa Lezameta, d Paola Bovolenta, c and Fernando de Castro a,b,⁎ Grupo de Neurobiología del Desarrollo, Hospital Nacional de Parapléjicos, Finca “La Peraleda, s/n, E-45071-Toledo, Spain Instituto de Neurociencias de Castilla y León-INCyL, Universidad de Salamanca, Avda. de Alfonso X “el Sabio”, s/n, E-37007-Salamanca, Spain c Departamento de Neurobiología Celular, Molecular y del Desarrollo, Instituto Cajal, CSIC, Avda. Dr. Arce, 37, 28002 Madrid, Spain d Grupo de Neurobiología Comparada, Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universidad de Valencia, Polígono “La Coma”, s/n. E-46980-Paterna (Valencia), Spain a

b

Received 15 May 2007; revised 19 July 2007; accepted 24 July 2007 Available online 1 August 2007 Optic nerve (ON) oligodendrocyte precursors (OPCs) are generated under the influence of the Sonic hedgehog (Shh) in the preoptic area from where they migrate to colonise the entire nerve. The molecular events that control this migration are still poorly understood. Recent studies suggested that Shh is often used by the same cell population to control different processes, including cell proliferation and migration, raising the possibility that Shh could contribute to these aspects of OPC development. In support of this idea, we show here that Shh induces the proliferation of OPCs derived from embryonic mouse ON explants and acts as a chemoattractant for their migration. In ovo injections of hybridomas secreting Shh-specific blocking antibody decreases the number of OPCs present in chick ONs, particularly in the retinal portion of the nerve. Altogether these data indicate that Shh contributes to OPC proliferation and distribution along the ON, in addition to their specification. © 2007 Elsevier Inc. All rights reserved. Keywords: Migration; Oligodendrocyte; Chemotropism; Sonic hedgehog; Cell proliferation; Myelin

Introduction Oligodendrocytes, the myelin-producing cells of the central nervous system (CNS), derive from oligodendrocyte precursors (OPCs) generated from multiple restricted foci in the germinal

⁎ Corresponding author. Grupo de Neurobiología del Desarrollo-GNDe Hospital Nacional de Parapléjicos Finca “La Peraleda”, s/n E-45071Toledo, Spain. Fax: +34 925 247745. E-mail addresses: [email protected], [email protected] (F. de Castro). 1 These authors have equally contributed to this work. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.07.012

neuroepithelium (Yu et al., 1994; Spassky et al., 1998). Although a proportion of OPCs derive from the dorsal neural tube, most of them develop, under the influence of Sonic hedgehog (Shh) signalling, from a specialised oligodendrogenic domain in the ventral neuroepithelium, characterised by the expression of the Nkx2.2, Nkx6 and Olig2 transcription factors (Fu et al., 2002; Richardson et al., 2006). Once generated, OPCs proliferate in response to different mitogenic agents, such as PDGF-AA, FGF-2 and VEGF-C, and disperse throughout the prospective grey and white matter to populate the developing CNS prior to initiate their final differentiation into mature oligodendrocytes (Rowitch, 2004; de Castro and Bribián, 2005; Le Bras et al., 2006). The pattern of OPC migration is not random. Rather, OPCs follow precise routes of migration characteristic of each CNS region (Olivier et al., 2001), which suggests the existence of specific combinations of molecular cues that direct their movements. The vertebrate optic nerve (ON) has been one of the most useful models to study oligodendrocyte development. Indeed, ON oligodendrocytes derive from progenitors generated in the preoptic area (POA) under the influence of Shh (Gao and Miller, 2006), as shown for OPCs of other CNS regions (Orentas et al., 1999; Nery et al., 2001; Spassky et al., 2001; Murray et al., 2002; Gao and Miller, 2006). Thus, early eye removal or inhibition of Shh signalling reduces the presence of OPCs at the third ventricle, suggesting that Shh, either produced locally (Trousse et al., 2001) or secreted by retinal ganglion cell (RGC) axons (Gao and Miller, 2006), is required for the specification of these cells (Gao and Miller, 2006). Once specified, OPCs colonise the ON when the majority of RGC axons have already reached the forebrain (Small et al., 1987; Ono et al., 1997, 2001). Growth factors, such as PDGF-AA and FGF-2, have been shown to promote OPC motility (McKinnon et al., 1993; Rogister et al., 1999; Bribián et al., 2006) and gradients of Netrin-1, Sema3F, Sema3A and FGF-2 cooperate to provide directionality to the migration of OPCs along the ON

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(Sugimoto et al., 2001; Spassky et al., 2002; Bribián et al., 2006), while BMPs appear to prevent OPCs from invading the neural retina (Gao et al., 2006). In addition to these secreted factors, the transmembrane ligands ephrinB2/B3, the extracellular matrix molecule Tenascin-C and cell adhesion molecules, including integrins and PSA-NCAM, also modulate OPC movement (Payne and Lemmon, 1993; Milner and ffrench-Constant, 1994; Wang et al., 1994; Kiernan et al., 1996; Garcion et al., 2001; Prestoz et al., 2004; Zhang et al., 2004; Benson et al., 2005). Although these molecular cues appear to control the migration of a large proportion of OPCs, it is still ill-defined whether their activity is sufficient to support all the migratory processes or whether additional cues are needed.

Recent studies have suggested that a number of cell signalling molecules, including members of the Hedgehog (Hh), Wingless (Wnt), Transforming Growth Factor β (TGFβ) and Fibroblast Growth Factor (FGF) families, known for their key role in cell specification are often, in the same tissue, fundamental cues for other developmental events (Bovolenta and Martí, 2005). An example of this multiple use is provided by the Shh signalling activity in the neural tube, where it controls the specification of ventral neurons and oligodendrocytes (Martí and Bovolenta, 2002), acts as a mitogenic and survival agent for neuroepithelial cells (Cayuso et al., 2006) and controls the directionality of commissural axons at the floor plate (Charron et al., 2003; Bourikas et al., 2005). In a similar manner, Shh signalling is required at different steps of visual system development to control the specification of the eye field in its proximodistal axis, the proliferation and differentiation of RGCs (Esteve and Bovolenta, 2006) and the movement of their growth cones (Trousse et al., 2001). In the ON, Shh also contributes to the development of astrocytes (Dakubo et al., 2003) and, as mentioned before, to the specification of OPCs (Gao and Miller, 2006). In this study, we show that Shh has additional roles in the development of OPCs. Migrating OPCs express components of the Shh transduction pathway, and thus retain their competence to respond to Shh signalling even after their specification. A focalised source of Shh attracts OPC migration from embryonic ON explants and induces OPC proliferation, while interference with Shh activity hampers both processes in vitro and in vivo. These data demonstrate that Shh is a chemoattractant and a mitogen for migrating OPCs. Results Shh signalling components are expressed in OPCs as they colonise the ON In the mouse, OPCs derived from the preoptic area (POA), colonise the ON migrating in a gradient from the chiasm to the retina. Scattered precursors are first detected in the proximal end of the nerve at E14.5, reach the distal end at E16.5 and are homogenously distributed along the entire nerve by E17.5 (Spassky et al., 2002). During this period, Shh was expressed by RGCs (Figs. 1A–C; Wallace and Raff, 1999; Zhang and Yang, 2001; Dakubo et al., 2003). Shh transported along RGC axons is required

Fig. 1. Shh signalling components are expressed by ON OPCs. Coronal sections of mouse heads were hybridised with digoxigenin labelled probes specific for Shh (A–C), Ptc1 (D–E) and Gli1 (F–I) at embryonic day E14.5 (A, D, F), E16.5 (B, H, I) and E18.5 (C, E, G). Shh is expressed in the RGC layer at the three ages analysed (A–C). At E14.5 (D, F), but less so at E18.5 (E, G), the Shh signalling components Ptc1 (D, E) and Gli1 (F, G) are expressed in the preoptic area, where OPCs are generated. Doublelabelling of vibratome sections from E16.5 plp-sh ble-lacZ transgenic embryos show the distribution of β-galactosidase and Gli1 mRNA (H–I). Expression of Gli1 was detected by in situ hybridisation (blue) while βgalactosidase by immunohistochemistry (brown). Gli1 expression codistributes with β-gal+ cells in the ON (H) and preoptic area (I), indicating that OPCs are competent to respond to Shh signalling. (J–L) E16.5 mouse ON explants were grown over polylysine-laminin coated coverslips and immunostained with anti-Gli1 and A2B5. Images show an example of an OPC defined by the expression of A2B5 (in red; K, L) and expressing Gli1 (green, J, L). Abbreviations: och, optic chiasm; on, optic nerve; rgc, retinal ganglion cells; rpe, retina pigmented epithelium. Scale bar: 50 μm (H, I); 100 μm (A–G); 25 μm J–L.

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for the development of astroglial cells of the optic disc and nerve (Dakubo et al., 2003; Morcillo et al., 2006) and seems also to influence the generation of OPCs at the third ventricle in both chick and mouse embryos (Gao and Miller, 2006). Consistent with the idea that ON OPCs arise from Shh receiving cells, the ventral portion of the third ventricle of E14.5 mouse embryos expressed high levels of mRNA for the Shh receptor Ptc1 and its effector Gli1 (Figs. 1D, F). This expression was down-regulated at E18.5, when most OPC have presumably invaded the entire ON (Figs. 1E, G). Interestingly,

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expression of Gli1 was maintained also in committed ON OPCs, since positive in situ hybridisation signal was observed in β-gal+ cells in the surroundings of the third ventricle of E16.5 plp-sh blelacZ transgenic embryos (Fig. 1I). In these embryos, the expression of β-gal is under the control of the regulatory sequences of plp (proteolipid protein), one of the mayor CNS myelin components, that specifically identifies cells of the oligodendroglial lineage since early developmental stages (Spassky et al., 1998; Le Bras et al., 2005). More notably, scattered β-gal staining co-distributed with

Fig. 2. Shh attracts migrating ON OPCs in vitro. E16.5 ON explants were co-cultured with control (CT) PBS-soaked beads (A, B), floor plate (FP) explants or Shhsoaked beads (D–F). Explants were grown in culture medium alone (A–D) or in the presence of the 5E1 blocking antibody (E) or the Shh signalling inhibitor cyclopamine (Cyc; F). Cultures were immunostained with A2B5 antibody (red) to identify OPCs (A, C–F) or with anti-PAX2 (B) to identify astrocyte precursors. Explants were counterstained with Hoechst (blue). Dotted lines outline the beads or FP position. In CT, A2B5+ OPCs migrate out of explants in a radial fashion (A), while PAX2+ cells do not leave the explant (B). (G) The graph shows the number of A2B5+ OPCs in the proximal (PQ; black bars) and distal (DQ; grey bars) quadrants in the following conditions: CT (n = 63), Shh (n = 91), FP (n = 44), CT + 5E1 (n = 28), Shh + 5E1 (n = 51), CT + Cyc (n = 34) and Shh + Cyc (n = 18). Note that OPCs are significantly attracted towards Shh-beads and FP explants. This attraction is abolished by the addition of 5E1, but not with cyclopamine. FP attraction is preserved when the Netrin-1 receptor DCC was blocked (n = 51). (H) The graph illustrates the transmigrated ON OPCs in all conditions in comparison to control conditions (normalised to 100 %, CT: white dot; Shh: black dot; Shh + 5E1: blue dot; Shh + Cyc: dark blue dot; Shh + SU5402: red dot; Shh + SB402451: dark red dot). These data confirm the results obtained in collagen cultures. ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01 and ⁎P b 0.05 (paired t-test in G, and Student's t-test vs. control in H). Scale bar: 100 μm (A, C–F); 50 μm (B).

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Table 1 Shh increases the distance migrated by OPCs Maximal migrated distance (μm) Proximal quadrant Distal quadrant (PQ) (DQ) CT beads (n = 63) Shh-beads (n = 91) FP (n = 44) FP + anti-DCC (n = 51)

154 ± 7 227 ± 9 208 ± 14 277 ± 16

145 ± 7 160 ± 7 177 ± 11 160 ± 11

Statistics (PQ vs. DQ)

NS ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎

NS: non significant; ⁎P b 0.05; ⁎⁎⁎P b 0.001 (paired t-test between PQ and corresponding DQ).

Gli1 mRNA in the ON head of E16.5 embryos (Fig. 1H), supporting the idea that OPCs migrating along the ON are still competent to respond to Shh signalling, even after their induction in the POA (Gao and Miller, 2006). This idea was further supported by co-localisation studies using explants of E16.5 ON, where cells immunostained with the A2B5 antibody (Fig. 1K), an accepted marker for migrating OPCs (Bribián et al., 2006; Spassky et al., 2002), were also positive for Gli1 (Figs. 1J, L). These expression data are consistent with the hypothesis that RGC-derived Shh influences the migration of OPCs from the chiasm to populate the entire ON.

Shh attracts migrating OPCs To test this hypothesis, ON explants from E16.5 mouse embryos were cultured in collagen gel matrices in the presence of a source of Shh provided either by floor plate (FP) explants (Fig. 2C; Roelink et al., 1995) or by Shh-soaked beads (Fig. 2D). These cultures were grown in defined medium in the presence of FGF-2, which is necessary for ON OPCs to move (Bribián et al., 2006). The cells that migrated out of the ON explants in the presence of PBS-impregnated beads were immunostained with the A2B5 antibody (Fig. 2A). In contrast, only occasional cells were positive for PAX2, a marker for astrocyte precursors (Fig. 2B). In this condition, cells migrated radially out of the explants and no differences in their distribution in the proximal (PQ) and distal quadrants (DQ) were observed (Figs. 2A, G), nor in those lateral (not shown) to the bead position. This radial distribution was altered in the presence of Shh-soaked beads or FP explants, which significantly promoted the accumulation of OPCs in the PQ (Figs. 2C, D, G). Furthermore, in the presence of a Shh source, OPCs migrated significantly longer distances in the PQ than in the DQ, while no differences among quadrants were observed in the controls (Table 1). Consequently, comparison of the migrated distance in the PQ of control and Shh-exposed explants was significantly different (Student's t-test: P b 0.001 and P b 0.01, for Shh-beads and FP, respectively), while the extent of migration was comparable in the DQ in all conditions. To test the specificity of this effect, co-cultures of ON explants with Shh-beads or FP were grown in the presence of the 5E1 antiShh blocking antibody or cyclopamine, a Shh-signalling blocking compound (Chen et al., 2002). The 5E1 antibody abolished the attraction of OPCs towards the Shh source when compared with controls (Figs. 2E, G), while in the presence of cyclopamine, there was still a significant difference in the number of migrated OPCs between the PQ and DQ (Figs. 2F, G). Besides Shh, the FP secretes Netrin-1 (Kennedy et al., 1994), a molecular cue known to attract migrating OPCs via the DCC

receptor (Spassky et al., 2002). It was therefore possible that part of the FP-mediated effect on OPC migration implicated Netrin-1 activity. To exclude this possibility, co-cultures of ON and FP explants were grown in the presence of an anti-DCC blocking antibody. DCC blockage had no significant effect on the attraction exerted by the FP on the migration of OPCs (Fig. 2G). No statistical difference was also observed between the effect of the FP explants and that of the Shh-soaked beads for any of the parameters analysed to evaluate the migration of ON OPCs (Fig. 2G; Table 1). Together these data suggest that Shh has a strong activity sufficient to attract migrating OPCs, even in the absence of Netrin-1. FGF-2 is necessary for the migration of ON OPCs (Spassky et al., 2002; Bribián et al., 2006). We thus asked whether Shh chemotropic effect was dependent on FGF-2. For this purpose, we performed similar experiments without FGF-2 in the culture medium. In this condition, Shh-beads still promoted the migration out of the explants of a significant amount of OPCs that were attracted toward the beads. However, the total number of OPCs (49 ± 10) was lower than in the presence of FGF-2 (compare with Fig. 3A). This FGF-2independent attractive effect of Shh was confirmed with chemotaxis experiments, in which Shh beads consistently doubled the number of transmigrated OPCs as compared to controls (Fig. 2H). The 5E1 antibody and cyclopamine completely or partially interfered with this effect (Fig. 2H), confirming the differential effect observed with the two Shh signalling blocking agents in the collagen matrix cultures. A very recent report demonstrates that Shh activity maintains FGF-8 expression, which is otherwise lost in explanted chick optic nerves (Soukkarieh et al., 2007). Shh effect could thus be mediated by endogenously produced FGF. However, Shh-beads were effective in attracting OPCs transmigration even in the presence of SU5402 and SB402451, two compounds that block FGFR1 or all FGF receptors, respectively (Fig. 2H). Altogether these data support the conclusion that Shh can mobilise and attract the migration of a proportion of ON OPCs, independently of FGF activity. This partial independence from the motogenic activity of FGF-2 (or other FGFs) has not been observed with other cues that influence OPC migration, strengthening the relevance of Shh function. Shh increases cell proliferation in ON explants The total number of A2B5+ OPCs present in the four quadrants around the explants was always significantly higher in the presence of a Shh source than in control conditions (Figs. 2A, C, D, and 3A). In addition, a number of A2B5− cells were also observed in the surroundings of the explants. This number was particularly significant in the cultures containing Shh-beads (5-fold increase of control; Fig. 3C). These A2B5− cells preferentially showed a bipolar morphology characteristic of OPCs (Fig. 3B) and were attracted towards the Shh source (Fig. 3C). Notably, in the presence of Shh but not in controls, increasing numbers of A2B5− cells significantly correlated with the increasing number of A2B5+ OPCs (Fig. 3D). This correlation was even stronger in the PQ (P b 0.005, Pearson correlation test). The presence of A2B5− cells in the surroundings of the explant was also observed in the experiments performed without FGF-2 in the culture medium (not shown). The differences in cell number and distribution observed in the presence of Shh were not associated with differential cell death, as determined by propidium iodide and anti-caspase-3 staining (not shown), nor with a variability in the explant size, since the average length of the ON pieces used in the assays was comparable (control:

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352 ± 17 μm, n = 63; Shh-beads: 339 ± 15 μm, n = 91). Thus, we reasoned that Shh could promote cell proliferation in ON explants and dividing cells could be related to the migrating OPCs. To test this possibility, BrdU was added to the cultures and the number of proliferating cells was determined by immunocytochemistry. As previously reported (Bribián et al., 2006), in controls most of the BrdU+ cells were observed within the explant and only a limited number of proliferating cells was homogeneously distributed in all the quadrants (Fig. 4A). In contrast, a significantly higher number of BrdU+ cells was observed out of the explants

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when co-cultured with Shh-beads or FP explants (Figs. 4B, C, F). This increase was prevented by the addition of either 5E1 antibody or cyclopamine (Figs. 4D–F), both of which reverted the proportion of BrdU+ cells to control values (Figs. 4A, F). In all culture conditions, there was almost no sign of apoptosis in the cells migrating out of the explants, when chromatin was examined under electron microscopy (not shown), further supporting that cell proliferation is responsible of the larger number of cells observed in the presence of Shh and that Smo activity is normally required for cell cycling of ON cells.

Fig. 3. Shh induces the appearance of A2B5 cells in ON explants. (A) The graph represents the quantification of the total number of A2B5+ OPCs migrated out of the ON explants in the indicated experimental conditions. Note that both Shh and FP induce a significant increase in this number. This is reverted by the presence of 5E1 or cyclopamine. (B) Pairs of bright-field and A2B5 immunofluorescence images of cells migrated out of the explants in control (CT) and Shh-beads exposed ON cultures. A2B5− cells with a bipolar morphology (arrows), similar to that of OPCs (solid arrows), are present predominantly in Shh co-cultures. (C) The graph shows the number of A2B5− cells accumulated in the proximal (black) or distal (grey) quadrants of the explants. Note that cells are attracted by the Shh-beads. (D) Correlation between the total number of A2B5+ (OPCs) and A2B5− cells in control (white circles) and Shh treated (blue circles) cultures. The correlation is significant (Pearson correlation test) only for Shh. In the case of Shh, one value included in the correlation (322 OPCs, 1203 A2B5−cells) is out of scale. ⁎⁎⁎P b 0.001 (paired t-test in panels A and C). Scale bar is 20 μm.

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Fig. 4. Shh induces OPC proliferation. ON explants were grown in the presence of BrdU (see Experimental methods) in control (A, a′, a″), Shh (B, b′, b″, C), Shh + 5E1 (D) and Shh + cyclopamine (E) treated cultures. Cultures were immunostained with anti-BrdU antibody (green, A–E) alone or double-stained with A2B5 (red; A–B). a′, a″ and b′, b″ are enlargements of the boxed areas in panels A and B, respectively. Dotted lines in panels A and B outline the bead position. Note the increase in A2B5+/BrdU+ cells in the presence of Shh (B). Double labelled cells accumulated in the proximal quadrant (compare b′ with b″) in the presence of Shh but not in controls (a′, a″). Note that the presence of 5E1 (D, F) and cyclopamine (E, F) decreases the proliferative effect observed in the presence of Shh alone (C, F). (F) The histogram shows the percentage of BrdU+ cells over the total number of cells that had migrated out of the explants determined at 2DIV in the indicated experimental conditions (see Experimental methods for details). Co-cultures of ON explants with Shh-beads or FP explants significantly increase BrdU-incorporation respect to CT. ⁎⁎⁎P b 0.001 and ⁎P b 0.05 (Student's t-test in F). Scale bar: 100 μm (A–B), 50 μm (C–E).

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Newly generated cells are less-differentiated oligodendroglial precursors Because A2B5− cells accumulated near the Shh-impregnated beads, as A2B5+ OPCs did (Fig. 3C), and the number of both cell populations significantly correlated (Fig. 3D), we hypothesised that A2B5− cells could be related to the migrating A2B5+ OPCs. To verify this hypothesis, we characterised the population of A2B5− cells using different cell-specific markers in explant cultures exposed to Shh. The absence of βtubulin-III (not shown) ruled out the possibility that these cells were differentiating neurons. Staining with anti-PAX2 (Fig. 5D) and anti-GFAP (Fig. 5E), markers for undifferentiated and differentiated astrocytes respectively, was mostly localised within the explant and, as in controls (Fig. 2B), only few PAX2+ and even less GFAP+ cells were observed leaving the nerve

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stub (Fig. 5E and inset in D). Similarly, these cells were negative for more differentiated oligodendroglial markers as O4, which was observed in the cultures only after 5 or 6DIV (Fig. 5F), thus discarding the possibility of a premature Shh-induced oligodendroglial differentiation. It has been reported that OPCs suffer a discontinuity between the expression of early markers such as A2B5 and that of later ones, like O4. During this gap, OPCs appear to express GAP-43 (Fanarraga et al., 1995). However, lack of GAP-43 immunostaining in the migrated cells (data not shown) discarded the possibility that A2B5− cells represented OPCs in this transition phase. On the contrary, the vast majority of the cells that had migrated out of the explants (both A2B5+ and A2B5−) were stained with Nestin and plp (Figs. 5A–C). Both markers are expressed by cells of the oligodendroglial lineage before the appearance of A2B5 or O4 immunoreactivity (Spassky et al., 1998; Almazán et al., 2001; Liu et al., 2002). Altogether, these data

Fig. 5. A2B5 cells belong to the oligodendroglial lineage. ON explants from wild type (D–F) or plp-sh ble-lacZ embryos (A–C, G–H) were cultured in the presence of Shh beads (outlined by dotted lines) and immunostained with antibodies against Nestin (A, C), anti-β-gal (B–C) PAX2 (D), GFAP (E), and O4 (F). All cultures were counterstained with Hoechst (blue). PAX2+- and GFAP+-astrocytes remained mostly within the explants (D–E and inset in D). In contrast, the vast majority of the cells migrated out of the explants expressed Nestin (A, C) or β-gal in plp-sh ble-lacZ ON (B). Nestin+ cells were β-gal+ (C), thus cells of the oligodendroglial lineage. (G) Image of a toluidine blue-stained semi-thin section showing a chain of migrating OPCs. (H) Electron micrograph of the most frequent cell type observed. The cell is enriched in microtubules, a characteristic of OPCs. Scale bar represents 150 μm in A–B, D, F; 30 μm in C, E; 10 μm in G–H.

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indicated that, likely, A2B5− cells were less-differentiated OPCs resultant from Shh-induced cell division. The idea that most of the migrated cells belonged to the oligodendroglial lineage was further corroborated by electron

microscopic studies using ON explants derived from plp-sh blelacZ embryos. In both control and Shh-treated cultures migrating plp+ cells were often observed forming small chains (Fig. 5G). The cells were fusiform, rich in organuli, with an elongated nucleus

Fig. 6. OPCs require Shh activity to colonise properly the chick ON. Horizontal sections through the distal (retina, A, C, E) and proximal (chiasm B, D, F) end of E9 chick ONs from untreated (A–B), 3C2 control IgG (C–D) and 5E1 anti-Shh (E–F) treated embryos immunostained with anti-NKX2.2 antibody to identify OPCs. G– H: The graphs represent the relative distribution of OPCs along the nerve at E7 (G) and E9 (H), calculated as the number of Olig2+ and Nkx2.2+ cells, respectively, present in (proximal or distal) sections over the total number of Nkx2.2+ cells present along the entire ON length. (I) The graph represents the average number of Nkx2.2+ OPCs present in the proximal and distal end of the nerve at E9 in the indicated conditions. (J) Control and 5E1-treated E9 chick embryos were injected with BrdU 1 h before fixation. Horizontal sections through the ON were double labelled with anti-Olig2 and BrdU antibodies. The number of single and double positive cells was counted in the distal and proximal end of the nerve as above. The graph represents the relative distribution of BrdU+/Olig2+ cells along the nerve. Note that Shh blockade significantly reduces the number of OPCs (E–F, I) as compared to controls (A–D, I), particularly in the distal end of the nerve (E, H, J). ⁎⁎P b 0.05 (ANOVA test). Abbreviations: oc, optic chiasm; onh, optic nerve head. Scale bar: 100 μm.

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positioned along the major axis of the cell. The nuclei presented loose chromatin with several nucleoli. The cytoplasm was scattered with dictyosomes and was rich in short RER cisternae, polyribosomes and microtubules (Fig. 5H), typical of cells of the oligodendroglial lineage (Peters, 1996, 2002). Microvilli were also observed, whereas signs of exocytosis were rare. In agreement with immunocytochemical studies, in both control and Shh-treated cultures, only occasional cells presented an ultrastructure compatible with that of astroglial cells, with large cytoplasmic expansions and the presence of intermediate filaments (not shown), a characteristic of both differentiated and immature astrocytes (Bovolenta et al., 1987). Altogether, these data supported that most of the cells that had migrated out of the ON explants belonged to the oligodendroglial lineage, though at different step of differentiation: A2B5− or A2B5+ OPCs. OPCs loose chemoattractive but not proliferative response to Shh once they have colonised the ON By E18.5, OPCs have completely colonised the mouse ON (Spassky et al., 2002) and should no longer need migratory cues. Shh however is still expressed in RGCs (Fig. 1C). We thus asked whether cells of the oligodendroglial lineage were still responsive to Shh signalling at this stage. In chemotropic assays using E18.5 ON explants, A2B5+ as well as A2B5− OPCs migrated out of the explants but dispersed homogeneously in the presence of both control and Shhsoaked beads (not shown). Similarly, there was no statistically significant difference between controls and the Shh-treated explants in the chemotaxis experiments performed without FGF-2 (Supplementary Fig. 1C) nor in the total number of A2B5+ OPCs of Shh-treated or control ON cultures (Supplementary Fig. 1A). In contrast, Shh still induced an increase in the number of A2B5− OPCs (control: 19 ±4; Shh: 30 ± 5; Student's t-test: P b 0.05; Supplementary Fig. 1B), although this was lower than that observed at E16.5. By enlarge, these data suggests that OPCs no longer respond to Shh-induced chemoattraction once they have colonised the nerve. In ovo interference with Shh activity alters OPC migration in the chick ON Collectively, our data indicated that Shh, in addition to control the generation of ON oligodendrocyte progenitors (Gao and Miller, 2006), could also be crucial to achieve proper numbers and distribution of OPCs along the entire nerve. To test this hypothesis in vivo, we took advantage of the easy experimental manipulability of the chick embryos. As in the mouse, chick ON OPCs originate from the POA around E4.5, migrate along the ON when the majority of the Shh-expressing RGCs have extended their axons into the nerve (E6) and completely colonise it by E9 (Ono et al., 1997). Injecting hybridoma cells producing blocking antibodies into the amniotic sac is an effective method to interfere in ovo with the activity of secreted molecules (Bovolenta et al., 1996; Frade et al., 1997). Thus, endogenous Shh activity was blocked by inoculating 5E1 hybridoma cells (n = 10), and non-injected (n = 9) or 3C2 hybridoma (n = 4) injected embryos were used as controls. Injections were performed at E4.5, just before the beginning of OPC migration into the nerve but after the completion of the major eye patterning events. Embryos were analysed at E7 and E9. The efficiency of Shh blockade was clearly visible by morphological inspection of the treated embryos, which presented

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several defects (Supplementary Fig. 2), consistent with previously reported functions of Shh. These included the loss of pigmentation in a small ventral area of the eye (Zhang and Yang, 2001), the fusion of digits in the limb buds (Harfe et al., 2004) and defects in feather formation (Prin and Dhouailly, 2004). None of these defects were ever observed in controls or 3C2 treated embryos (Supplementary Fig. 2). Furthermore, immunostaining of spinal cord sections with antibodies against O4 and Nkx2.2, a homeobox protein selectively expressed in migratory chick OPCs (Qi et al., 2001), revealed a clear decrease in the number of positive cells in embryos treated with the anti-Shh antibody (Supplementary Figs. 3A–I, J), as expected for an effective interference with Shh activity, which is necessary for OPC generation in the neural tube (Pringle et al., 1996). Likewise, secondary antibodies directed against mouse IgGs highlighted the ventral midline (Supplementary Fig. 3K), indicating that the 5E1 IgGs were diffusing into the CNS of the embryos binding to Shh producing midline cells. Having proven that inoculation of the 5E1 hybridoma effectively blocked Shh, we quantified the presence of OPCs in the ON of E7 and E9 control and treated embryos using Nkx2.2 and Olig2 as markers. Both antibodies were used almost interchangeably, since in exploratory double labelling studies, we observed that all Nkx2.2+ cells were also Olig2+, while only very few cells expressed only Olig2+ (not shown), suggesting that Olig2 most likely slightly precedes Nkx2.2 expression during the differentiation of chick ON OPCs. Notably, treatment with the 5E1 antibody caused a clear decrease in the number of OPCs in the entire ON when compared with control non injected or 3C2 hybridoma-injected embryos (Fig. 6). These differences were particularly evident in the distal end of the nerve both in embryos analysed at E7 (not shown) and E9 (Figs. 6A, C, E, I). Consistently, analysis of the relative distribution of OPCs along the length of the nerve revealed that only roughly 2% and 17% of the total OPCs were localised to the distal region of the nerve in 5E1-treated embryos at E7 and E9, respectively, while the remaining 98% and 83% were in the proximal end (Figs. 6G, H). In contrast, in both non injected and 3C2-treated embryos, OPCs were more homogeneously distributed along the entire ON length at E9 (~ 35% in the distal and 65% in the proximal portion, Fig. 6H), while most of the OPCs have not yet entered the optic nerve at E7 (10% in the distal vs. 90% in the proximal portion, Fig. 6G). A similar α-Shhinduced impaired distribution of OPCs was observed also in the spinal cord (Supplementary Figs. 3C, F, I, J), where Nkx2.2+ or O4+ OPCs tend to accumulate in the ventricular zone and the white matter as compared with the distribution observed in control spinal cords (Supplementary Figs. 2A–B, D–E, G–H, J). Our in vitro studies indicate that Shh induces migration and proliferation of OPCs. We therefore asked whether the decreased number of OPCs observed in the 5E1-treated embryos was associated also to impairment in cell proliferation. To this end, we examined the distribution of BrdU, Olig2 (to avoid cross talk between secondary antibodies, see Experimental methods) or double positive cells in 5E1 treated and control embryos. Interference with Shh activity resulted in a clear reduction of the number of BrdU or Olig2-positive cells in both the proximal and distal portion of the ON (not shown). A small number of double positive cells was observed along the entire length of the nerve. However, blockade of Shh activity caused a change in the distribution of proliferating OPCs + + (BrdU /Olig2 ), which appeared to be more frequent in the proximal region of the nerve (Fig. 6J). Altogether, our results indicate that in vivo Shh activity controls the number and the migration of OPCs as they colonise the vertebrate ON.

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Discussion Shh is required for the specification of a large proportion of OPCs, including those that colonise the ON. In this study we have addressed whether Shh signalling has additional effects on ON oligodendrocyte development. Our data, based on explant cultures and in ovo interference with Shh activity, demonstrate that Shh promotes the migration and proliferation of ON OPCs. This chemotropic effect is independent from Netrin-1 and, at least partially, from FGF activity. Furthermore, Shh signalling increases the number of plp+/Nestin+ OPCs at two different stages of their development, defined by the absence or the presence of the A2B5 oligodendroglial marker. We thus propose that the activation of the Shh signalling pathway may be a potentially useful therapeutic strategy to favour OPC colonisation of CNS areas when affected by demyelinating diseases. Shh is a potent chemoattractant for ON OPCs Previous studies have demonstrated that OPCs, generated in specific domains of the CNS, colonise the grey and white matter following specific molecular cues, most of which are also used by growing axons, as for example Netrin-1 and semaphorins (Tsai and Miller, 2002; de Castro, 2003). Here, we have shown that Shh, which acts as an axon guidance molecule for different neuronal populations (Sánchez-Camacho et al., 2005), is an additional example. Our data indicate that Shh has a strong chemoattractive activity on migratory OPCs of the ON, which is observed, albeit at lower level, even in the absence of FGF-2, a motogenic agent for these cells (Spassky et al., 2002; Bribián et al., 2006) or after the blockade of FGF signalling. This idea is supported by chemotropic assays with ON explants, in which OPCs accumulate in the proximity of focalised sources of Shh, and corroborated by chemotaxis experiments, where OPCs transmigrate in response to Shh charged beads. Notably, Shh and Netrin-1, that cooperate as axon guidance cues (Charron et al., 2003), do not appear to provide synergistic information to migrating ON OPCs. Indeed, when the Netrin-1 receptor DCC was blocked, the FP explants still strongly promoted OPC migration. Furthermore, FP explants did not attract ON OPCs more than Shh-soaked beads alone, suggesting that Netrin1 and Shh act in parallel and, in absence of Netrin-1, Shh provides sufficient chemoattractive information to induce ON OPC migration. We cannot, however, exclude that the amount of Netrin-1 might be lower than that used in other studies (Spassky et al., 2002) and other molecules expressed by the FP may counteract or interfere with the combined Shh/Netrin-1 function. In this respect, however, none of the strong chemorepellent Slits, which are expressed at the FP (Brose et al., 1999), interfered with OPC migration when tested in our in vitro conditions (B. Le Bras and F. de Castro unpublished observations). PDGF-AA or FGF-2 are normally required for OPC movement (Armstrong et al., 1990; McKinnon et al., 1993; Milner et al., 1997; Osterhout et al., 1997; Bribián et al., 2006). We thus performed most of our in vitro experiments in the presence of exogenously added FGF-2. Interestingly, in absence of FGF-2 and in the presence of FGF signalling inhibitors, a proportion of ON OPCs still migrated in response to Shh. Although we cannot totally exclude that endogenously produced PDGF-AA may promote this migration, it is tempting to speculate that Shh may act also as a motogenic agent for a proportion of ON OPCs, thus suggesting their possible heterogeneity. Alternatively, Shh, like FGF-2, may activate mitogen-activated protein kinase signalling, as demonstrated for neocortical OPC

specification (Kessaris et al., 2004), and thus in part compensate for FGF-2 absence. The Shh-induced OPC migration is also strongly supported by our study with intact embryos. Co-distribution of a plp-reporter and Shh signalling components in cells dispersed along the ON indicates that OPCs, once generated, maintain their competence to respond to Shh. In ovo interference with Shh activity decreases the number of ON OPCs. This decrease in part reflects interference with the mitogenic effect of Shh on OPCs. In vivo, it is experimentally difficult to unequivocally separate Shh-induced migratory from proliferative effects. Nevertheless, we have shown that this reduction is comparatively stronger in the distal (retina) than in the proximal (chiasm) portion of the nerve, suggesting that, in vivo, Shh contribute to direct OPC migration along the nerve, besides promoting their specification (Gao and Miller, 2006). In our study, we have not directly addressed which is the physiological source of Shh that influences ON OPC migration. However, RGCs are the most likely possibility. Indeed, Shh is timely expressed by RGCs and the protein appears to be transported along their axons (Traiffort et al., 2001). Specific inactivation of Shh expression in the mouse RGCs interferes with proper ON astrocyte development (Dakubo et al., 2003) strongly indicating that Shh might be normally released by the growing axons to influence glial cells locally, as also suggested by the Western blot detection of the Shh protein in extract of postnatal rat optic nerves (Wallace and Raff, 1999). It is thus conceivable that RGC axon-derived Shh may attract OPCs generated in the POA. An open question is how newly generated OPCs are not retained in this region where Shh is also expressed (Trousse et al., 2001). The participation of additional motogenic factors (i.e. FGF-2) is a possibility. Alternatively, a timely and/or differential expression of receptors, as in the case of the different response of pre- and post-crossing commissural axons to Shh (Charron et al., 2003; Bourikas et al., 2005) or the timedependent response of ON OPCs to Netrin-1 (Spassky et al., 2002), are possible explanations. Additional explanation may involve the existence of alternative Shh signalling components mediating OPC migration. This idea may be supported by the observation that cyclopamine interference with Smo activity effectively inhibited Shh-induced OPC proliferation but could not totally abolish the chemotropic effect in our culture conditions. Whether control of cell movement induced by morphogens is mediated by the same signalling pathways used for patterning activity is an unresolved problem (Bovolenta, 2005; Bovolenta et al., 2006). Nevertheless, local changes in cAMP may underlie the effect of Shh on RGC growth cone movement (Trousse et al., 2001) and Boc, a Shhbinding cell-surface molecule of the Ig/fibronectin superfamily (Tenzen et al., 2006) may mediate Shh activity on spinal cord commissural axons (Okada et al., 2006). Similar alternative components might be involved in Shh-induced ON OPCs migration. Although OPCs in the spinal cord were not the main focus of our study, we noticed that in ovo interference with Shh activity causes alterations in their distribution as well. Indeed, in the presence of Shh + + blocking antibody, NKx2.2 - and O4 cells tended to accumulate preferentially around the ventral portion of the ventricle, where Shh is normally expressed. This raises the possibility that Shh signalling may provide opposite information to ON and neural tube OPCs: chemoattractive and chemorepellent, respectively. While highly speculative at the moment, this hypothesis is supported by the different behaviour (repellent in the spinal cord, attractive in the ON) of these same OPC populations in response to Netrin-1 (Spassky et al., 2002; Tsai et al., 2003).

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Shh as a proliferative agent for ON OPCs A direct role of Shh in the proliferation of neural cells has been extensively reported in both normal and pathological conditions (i.e., reviewed in: Ho and Scott, 2002; Palma et al., 2005), including in the case of adult neural stem cells that originated oligodendroglial cells in the adult spinal cord and brain (Bambakidis et al., 2003; Loulier et al., 2006). Here, we show that ON OPCs are additional targets of Shhinduced proliferation. This effect is rather strong since co-culture of ON explants with a Shh source doubled the basal rate of OPC proliferation observed in controls. Although Shh also induces the proliferation of ON astrocyte precursors (Jensen and Wallace, 1997; Wallace and Raff, 1999), these cells did not interfere with our analysis since most of them did not migrate out of the nerve explants, as confirmed by immunocytochemical and electron microscopic analysis. In vivo interference with Shh activity also significantly reduced the number of OPCs along the entire length of E9 chick ONs. Thus, in vivo, Shh released by the RGC axons may control the proliferation of both astrocyte and oligodendrocyte precursors. Notably, the effect of Shh on both proliferation and migration appears restricted to the initial phases of OPC development as suggested by the results obtained with E18.5 explants, which is consistent with the observed down-regulation of the expression of Shh signalling components in the POA. In conclusion, our data, together with previous studies, suggest that Shh signalling is required at different steps of ON oligodendrocyte development: specification (Danesin et al., 2006; Gao and Miller, 2006), proliferation and migration (present work). These activities in combination with those of other molecules, including PDGF-AA, FGF-2, Netrin-1 and semaphorins (Jarjour and Kennedy, 2004; Rowitch, 2004; de Castro and Bribián, 2005), should determine the distribution of oligodendrocytes in the ON. Potential implications of Shh effects on demyelinating diseases An additional interesting aspect of our findings is the potential therapeutic value of Shh signalling activation in demyelinating diseases. Alteration in Shh expression levels have been reported in both experimental models and patients of multiple sclerosis (Mastronardi et al., 2003; Fancy et al., 2004; Seifert et al., 2005). Furthermore, Shh signalling components appear among the targets of effective ameliorating treatments in animal models for demyelination (Moscarello et al., 2002; Mastronardi et al., 2003). It would be then interesting to test whether local administration of Shh or activation of its signalling components could facilitate the proliferation and invasion of OPCs at the lesion sites. Encouraging this line of thoughts, an increased number of OPCs has been reported after treatment of injured adult rat spinal cords with Shh (Bambakidis et al., 2003). Moreover, the combination of OPC transplantation and Shh administration results in improved functional performance of adult rats with moderate spinal cord contusions (Bambakidis and Miller, 2004). Experimental methods Animals Transgenic plp-sh ble-lacZ (Spassky et al., 1998) and CD1 mice embryos were used at E14.5, E16.5 and E18.5. The day of plug detection was E0.5. White-Leghorn chicken eggs were incubated at 38 °C in a 70% humidity atmosphere. Embryos were staged according to Hamburger and Hamilton (1951). Animals were used according to the Spanish (RD 223/88)

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and European (86/609/ECC) laws. The experimental procedures had been approved by the Animal Review Board (registered as SAPA001) of the Universidad de Salamanca and by the CSIC (Spain). Tissue culture procedures Mouse E16.5 embryos were collected in L15 medium (GibCo), and ONs were carefully dissected and divided into approximately 300 μm explants. Heparin acrylic beads (Sigma) were soaked for 2 h at 37 °C either in recombinant purified human N-Shh (1 mg/ml in phosphate-buffered saline, PBS) or in PBS alone (Trousse et al., 2001). FP explants were dissected from the rhombencephalon of E12.5 CD1 mice. Retina explants were obtained from E16.5 CD1 mice. ON explants were co-cultured in a three-dimensional rat tail collagen gel (BD Biosciences) in the presence of beads or FP explants positioned at a distance ranging from 150 to 500 μm. In some cases, explants were grown on 12 mm coverslips treated with polyL-lysine (10 mg/ml, Sigma-Aldrich) in borate buffer (0.1 M, pH 8.5), rinsed with distilled water and treated for 2 h at 37 °C with Laminin (10 mg/ml, Sigma-Aldrich). Cultures were grown at 37 °C, in 5% CO2 with a modified Bottenstein-Sato medium supplemented with FGF-2 (20 ng/ml; R&D Systems) for 3 days (3DIV), as previously reported (Spassky et al., 2002; Bribián et al., 2006). For blocking experiments, cyclopamine (3 μM; Sigma), 5E1 Shh blocking antibody (1:500 from ascitic fluid; Developmental Studies, Hybridoma Bank, DSHB, University of Iowa) or anti-DCC blocking antibody (10 μg/ml; R&D Systems) were added directly to the culture medium. After the 3DIV, cultures were incubated for 15 min with Hoechst 33342 (10 μg/ml, Sigma) to visualise the nuclei, rinsed with PBS and then fixed in 4% paraformaldehyde in phosphate buffer (PFA). When plp-sh ble-lacZ embryos were used, cultures were fixed in 2% PFA for immunostaining or in 3.5% glutaraldehyde in PBS (for 1 h, at 37 °C) for electron microscopy. For chemotaxis experiments, ONs from E16.5 or E18.5 CD1 embryos were dissociated in DMEM (GibCo) containing 1.14 U/ml papainWorthington (Serlabo), 12% collagenase (Sigma) and 0.48 mg/ml cysteine (Sigma). Cells were seeded at 5 × 104 in the upper chamber of transwells (Costar) in Bottenstein-Sato medium. Control or Shh impregnated beads were placed in the lower chamber. For blocking assays, cyclopamine (3 μM), SU5402 (10 μM; Calbiochem) or SB402451 (10 ng/ml; a gift of Dr. J. Hurlé and Dr. B. Zalc) were added to the upper chamber, while 5E1 antibody was added to the lower chamber (1:500). After 20 h, cultures were fixed with 4% PFA for 15 min and the presence of OPCs transmigrated in the lower chamber was evaluated by immunostaining with specific antibodies (Bribián et al., 2006; Le Bras et al., 2006). In situ hybridisation E14.5–18.5 mouse embryos were fixed by immersion in 4% PFA overnight at 4 °C. The embryos were embedded in a block of gelatin/ albumin and then sectioned (30 μm) in the coronal plane with a vibratome (Leica). Sections were hybridised as described (Esteve et al., 2000) with digoxigenin-labelled mRNA probes specific for the mouse Shh, Ptc1 and Gli1. Sections from E16.5 plp-sh ble-lacZ transgenic mice were hybridised and then processed for β-galactosidase detection. Immunohistochemical procedures E14.5–18.5 mouse embryos and E9 chick heads were fixed, washed in PBS and embedded for vibratome sectioning as above. Alternatively, embryos were cryoprotected in 30% sucrose in PBS, embedded in OCT compound and sectioned (20 μm) with a cryostat. Immunostaining of tissue sections and explant cultures were performed with standard protocols using the following primary antibodies: A2B5 mouse monoclonal (mAb; supernatant; diluted 1:5 in PBS plus 1% normal serum, PBSN), antiNkx2.2 (1:100 in PBTN), anti-O4 (1:1), G3G4 anti-BrdU (all obtained from DSHB, University of Iowa; 1:1000 in PBTN plus 0.2% gelatin), anti-GFAP rabbit polyclonal antibody (Dako; 1:200 in PBSN 0.1% Triton X100, PBTN), anti-nestin rabbit polyclonal antibody (a gift from Dr. C. Vicario;

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1:200 in PBTN), anti-Olig2 rabbit polyclonal antiserum (a gift from Prof. T. Jessell and diluted 1:8000 in PBTN), anti-PAX2 rabbit polyclonal antiserum (Zymed Laboratories; 1:2000 in PBTN), anti-β-galactosidase rabbit polyclonal antiserum (Cappel; 1:3000 in PBTN) and anti-Gli1 rabbit antiserum (Chemicon; 1:100). 488 or 594-Alexa™ (Molecular Probes), Rhodamnine-Red and FITC-conjugated fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) were used to visualise the different antigens. BrdU incorporation assays In explant cultures, cell proliferation was determined by the addition of BrdU (50 μM, Sigma) to the medium either between the 42–48 and the 66– 72 h of the culture period. Cultures were then washed and fixed with 4% PFA. The cultures were immunostained with the A2B5 mAb as above, fixed in 4% PFA for 15 min, washed in PBS and then treated with 2N HCl for 1 h, and neutralised in 0.1 M borate buffer (pH 8.5), before incubation with the anti-BrdU mAb. In E9 chick embryos, BrdU was added directly onto the amniotic sac 1 h before the end of the experiment. Chick embryos were then fixed and processed for immunocytochemistry. Electron microscopy For electron microscopy, plp-sh ble-lacZ explants were rinsed in 0.1 M sodium phosphate buffer and transferred to X-gal buffer containing 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6 and X-gal 0.5 mg/ml (Fermentas Life Sciences), for 8–12 h at 37 °C in the dark. βgal-stained tissue was postfixed in 1% osmium and 7% glucose in PB for 2 h, rinsed dehydrated and embedded in araldite (Durcupan, Fluka). Semi-thin sections (1.5 μm) were cut with a diamond knife and lightly stained with 1% toluidine blue. Semithin sections were then detached from the glass slide by repeated freezing (liquid nitrogen) and thawing and re-embedded on an araldite block. Ultrathin (70 nm) sections were cut with a diamond knife, stained with lead citrate and examined under an electron microscope (FEI Tecnai Spririt). In ovo interference with Shh activity Hybridoma cells producing anti-Shh blocking IgG (5E1) or the control hybridoma cell line (3C2) producing the anti-viral GAG IgG (DSHB, University of Iowa) were cultured in RPMI medium (GibCo) supplemented with 10% foetal calf serum. For in ovo injections, hybridoma cells were harvested by low-speed centrifugation, followed by two washes in RPMI and re-suspended at the density of 2 × 106 cells/μl in PBS. Approximately 20–30 μl of the cell suspensions were injected directly in the amniotic sac of E4.5 chick embryos using pulled glass pipettes, as described (Bovolenta et al., 1996). After 3 or 5 days of incubation (E7 and E9), embryos were dissected, fixed and processed for immunostaining with OPC-specific markers (Olig2 and Nkx2.2).

was counted in cryosections comprising the entire length of the ON. The nerve area was divided in two equal portions along the proximodistal axis and the number of positive cells was determined in the proximal and distal part. Sections comprising the entire thickness of the nerve were analysed in control, 5E1 or 3C2 hybridoma injected embryos. The frequency of OPCs in each section (proximal or distal) was calculated as the number of Nkx2.2+ or Olig2+ cells present in a section over the total number of positive cells present along the entire ON length. Data were analysed statistically using the Student's t-test, paired t-test, ANOVA test or their equivalent tests for non-parametric populations (SigmaStat, Jandel Scientific). Minimal statistical significance was fixed at P b 0.05. Data are expressed as mean ± SEM.

Acknowledgments We are indebted to Drs. Diego Clemente, Pedro Esteban, Virginia Vila del Sol, José Ángel Rodríguez-Alfaro and to Héctor Méndez, Maria Coelho and Jazmin Fermin for help with experimental work. This study was supported by grants from the following Spanish institutions: ‘Fondo de Investigaciones Sanitarias’-FIS (PI020768 and PI042591), Consejería de Sanidad de Castilla-La Mancha (ICS 06024/00), Ministerio de Educación y Ciencia-MEC (SAF200628387E), ‘Fundación Mutua Madrileña Automovilista-FMMA’, the ‘Federación de Cajas de Ahorro de Castilla y León’ to FdeC and by a grant from MEC (BFU-2004-01585) to PB. FdeC and CS-C respectively hold contracts from the ‘Ramón y Cajal’ and ‘Juan de la Cierva’ Programs of the MEC. PM and AB are supported by fellowships from the FMMA and the ‘Fundación del Hospital de Parapléjicos para la Investigación y la Integración-FUHNPAIIN’ (Spain), respectively. The G3G4, 74.5A5 antibodies against BrdU and Nkx2.2 developed by Stephen J. Kauffman and T.M. Jessell were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Science, Iowa City (IA-52242; USA). We are grateful to S. Morton and T. Jessell for providing the anti-Olig2 antiserum, and to J. Hurlé and B. Zalc for the SB402451.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mcn.2007.07.012.

References

Quantitation and statistical analysis To determine the number of migrating or proliferating OPCs, ON explants were examined with a Leica DMRD microscope and photographed with an Olympus DP50 camera. Double exposures were superimposed using Adobe Photoshop. The culture area was divided in four quadrants (proximal, distal and lateral quadrants facing up the bead, see Fig. 2A) as previously described (Spassky et al., 2002; Bribián et al., 2006). The number of A2B5+ OPCs and A2B5− cells was counted separately in each quadrant using Neurolucida (Microbright Field). The explant size along the proximodistal axis, the distance between the explant edge and the bead and the migration distance were measured. In chemotaxis assays, the lower chamber was observed with the microscope using a 20× objective and 10 random fields per well were photographed. The mean number of A2B5+ or Olig2+ cells was determined with Neurolucida in each condition. To determine the distribution of OPCs in control and in ovo treated chick embryos, the number of Nkx2.2, Olig2, BrdU or double positive cells

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