N-Cadherin Influences Migration of Oligodendrocytes on Astrocyte Monolayers

MCN

Molecular and Cellular Neuroscience 15, 288–302 (2000) doi:10.1006/mcne.1999.0819, available online at http://www.idealibrary.com on

N-Cadherin Influences Migration of Oligodendrocytes on Astrocyte Monolayers Oliver Schna¨delbach,*,†,1 Orest W. Blaschuk,‡ Matthew Symonds,‡ Barbara J. Gour,‡ Patrick Doherty,§ and James W. Fawcett*,† *Physiological Laboratory, Downing Street, Cambridge CB2 3EG, United Kingdom; †The Cambridge Centre for Brain Repair, Forvie Site, Cambridge, United Kingdom; ‡Division of Urology Research, Department of Surgery, Royal Victoria Hospital, Montreal, Canada; and §Molecular Neurobiology Grove, GKT School of Medicine Guy’s Hospital, London SE1 9RT, United Kingdom

Oligodendrocyte cell migration is required for the development of the nervous system and the repopulation of demyelinated lesions in the adult central nervous system. We have investigated the role of the calcium-dependent adhesion molecules, the cadherins, in oligodendrocyteastrocyte interaction and oligodendrocyte progenitor migration. Immunostaining demonstrated the expression of N-cadherin on the surfaces of both oligodendrocytes and astrocytes, and oligodendrocyte-like cells adhered to and spread on N-cadherin substrates. The blocking of cadherin function by antisera or specific peptides reduced adhesion of oligodendroglia to astrocyte monolayers, diminished contact time between oligodendrocyte processes and individual astrocytes, and significantly increased the migration of oligodendrocyte-like cells on astrocyte monolayers. Furthermore, a soluble cadherin molecule without adhesive properties increased oligodendroglial proliferation on various extracellular matrix substrates. These data suggest that cadherins are at least partially responsible for the poor migration-promoting properties of astrocytes and that decreasing cell–cell adhesion might effect repopulation of demyelinated multiple sclerosis lesions by oligodendrocyte progenitors.

INTRODUCTION Cells of the oligodendrocyte lineage must migrate considerable distances from their area of origin (for example, from the subventricular zone of the cerebel1 To whom correspondence and reprint requests should be addressed. Fax: 44-1223-333 840. E-mail: [email protected]. 1044-7431/00$35.00

288

lum and developing forebrain to their final destinations in future white matter areas) in order to fulfill their role as the myelinating cell type in the central nervous system (CNS). Similarly, in other areas of the brain (such as optic nerve or spinal cord), sites of oligodendroglial generation are frequently distant from their sites of myelination (Levison et al., 1993; Pringle and Richardson, 1993; Cameron-Curry and Le Douarin, 1995; Ben-Hur et al., 1998). Oligodendrocyte precursors (OP’s) encounter astrocytes as they migrate into the optic nerve and spinal cord white matter. Embryonic astrocytes have been shown to promote OP migration, while postnatal astrocytes are moderately inhibitory (Fok-Seang et al., 1995). In the demyelinating disease of the human CNS, multiple sclerosis (MS), oligodendroglia are destroyed by an autoinflammatory process (for review, see: Adams et al., 1998). Large areas in the brain are persistently demyelinated in patients with MS, although there is a degree of remyelination in most lesions, mostly around the margins. In order for spontaneous remyelination to occur, OPs must multiply, migrate to the affected axons, and myelinate them. Recently, it has been demonstrated that some OPs are present in demyelinated lesions of human and rat CNS (Woolswijk, 1998). Their number is probably insufficient to allow substantial repair of MS lesions. It is the outer region of MS plaques that generally remyelinates, suggesting that OPs migrating from surrounding CNS tissue are involved (Keirstead et al., 1998). In rodent demyelination models, OPs recruited into lesions can come from a distance of 2 mm (Nait-Oumesmar et al., 1995; Franklin and Blakemore, 1997). A variety of molecular determinants have been impli1044-7431/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.

289

Cadherin and Oligodendrocyte Migration

cated in migration of OPs. Among them are cell surface molecules (such as integrins and the recently discovered AN2 glycoprotein), extracellular matrix (ECM) glycoproteins (such as laminin, fibronectin and tenascin), proteoglycans, and sialylated carbohydrate structures found on the embryonic form of the neural cell adhesion molecule N-CAM (Wang et al., 1994; Fok-Seang et al., 1995; Frost et al., 1996; Milner et al., 1996; Schna¨delbach and Faissner, 1998; Niehaus et al., 1999). In addition, some regulatory cytokines and signalling molecules have been found to either promote or inhibit migration (Fok-Seang et al., 1998). Several proteases, which most likely alter matrix properties have also been found to affect migration (Amberger et al., 1997; Muir et al., 1998; Uhm et al., 1998). The family of calcium-dependent adhesion molecules, known as the cadherins, have been demonstrated to be involved in various aspects of cellular migration (Letourneau et al., 1990; Chen et al., 1997). Cadherins are expressed by most cell types of the CNS and in particular, oligodendrocytes have been shown to adhere to and spread on N-cadherin substrates (Payne and Lemmon, 1993; Payne et al., 1996). We have investigated the role of N-cadherin in the context of oligodendroglial migration. We report that N-cadherin is involved in the interaction between oligodendrocytes and astrocytes and contributes to the regulation of oligodendroglial migratory processes.

RESULTS Expression of N-Cadherin on Glial Cells The expression of N-cadherin on rat optic nerve oligodendrocytes has been demonstrated by Payne et al. (1996). Since our glial cell preparations used whole brain as a source, the distribution of N-cadherin expression was investigated in more detail. Shaken-off primary OP’s were labeled by double immunofluorescence with antibodies against N-cadherin (L7 antiserum) and oligodendrocyte lineage markers of various maturation stages such as A2B5, O4, or Galactocerebroside (GalC). Interestingly, the proportion of OPs labeled by the L7 antibody increased with maturation of the oligodendroglial lineage from 47% of the A2B5 expressing cells to 90% of the GalC expressing oligodendrocytes (Figs. 1A–1D). More than 90% of the rat OP cell line CG4 (Louis et al., 1992) was labeled by the L7 antibody (Figs. 1E and 1F) and N-cadherin, but not E- or P-cadherin mRNA could be detected in RNA preparations from CG4 cell cultures by reverse transcription-polymerase chain reaction

(RT-PCR) assays (data not shown). Maturation of the oligodendrocyte lineage also changed the expression pattern of N-cadherin within individual cells. Expression of N-cadherin in CG4 and primary OP process tips was measured using morphometric software on scanned photomicrographs of cultures stained with the L7 antibody. In both cell systems, the mean pixel intensity on process tips from cells with four or more processes was about 20% higher than that on process tips of cells with up to three processes (intensity values 111.3 6 6.0 vs 91.7 6 4.2 for CG4 cells, see Fig. 2, and 105.1 6 8.7 vs 83.9 6 4.5 for primary OPs). This increase was statistically significant (P values of 0.014 for CG4 cells and 0.035 for OPs in a Mann–Whitney rank sum test), possibly reflecting the above mentioned maturationdependent expression of N-cadherin on the oligodendroglial lineage. On the astrocyte lineage, a strong L7 immunostaining could be observed on cells costaining for GFAP (Figs. 1G and 1H).

N-Cadherin Does Not Support OP Migration Previous studies had demonstrated the ability of N-cadherin to support adhesion and spreading of OPs (Payne and Lemmon, 1993; Payne et al., 1996). In order to test whether N-cadherin also supports migration of OPs, an NCAD-Fc fusion protein was immobilized to an anti-Fc antibody bound to a tissue culture dish. On this substrate, CG4 cells cultured on a coverslip that was inverted onto the test surface completely failed to migrate, whereas migration on laminin in a control assay was rapid (data not shown and Fok-Seang et al., 1995). On a mixed substrate consisting of laminin (1 µg/ml) and increasing concentrations of NCAD-Fc, a concentration-dependent decrease of CG4 cell migration could be demonstrated (Fig. 3F), with 5 µg/ml of NCAD-Fc reducing the total migration rate by about 50%, as compared to a control substituting NCAD-Fc with human IgG (P 5 0.029 in a Mann–Whitney rank sum test; Figs. 3B, 3C, 3E, 3F). Thus, substrate-bound NCAD-Fc was found capable of counteracting the migration-promoting capabilities of laminin. The migrationinhibiting effect of NCAD-Fc could be neutralised by simultaneously adding cyclic peptides containing the classical cadherin cell adhesion recognition sequence HAV (which have previously been shown to be capable of blocking N-cadherin function; Makrigiannakis et al., 1999; Wilby et al., 1999; Figs. 3D and 3E). The peptides used to neutralise the effect of immobilized NCAD-Fc contained the amino acid sequences HAV and HAVD,

290

Schna¨delbach et al.

FIG. 1. N-cadherin is expressed in oligodendroglial and astroglial cell cultures and the CG4 cell line. Whole-brain preparations of newborn rats were cultivated for 1 week and loosely attached microglial cells and OPs were sequentially shaken off. Purified OP cultures and the remaining astrocytic monolayer were then subcultivated onto glass coverslips and processed for immunocytochemistry. A–D, purified primary oligodendrocyte cultures; E, F, CG4 cell culture; G, H, purifed astrocyte cultures. O4, A; GalC, C; Phase contrast, E; GFAP, G; L7 (anti N-cadherin), B, D, F, H. A, C, and G were developed with FITC-coupled secondary antibodies and B, D, F, and H with rhodamine-conjugated secondary antibodies. Magnification in A–D, G, H, 8003; E, F, 4003.

Cadherin and Oligodendrocyte Migration

291

FIG. 2. Relative N-cadherin expression on processes of CG4 cells with differential morphology. Photomicrographs of CG4 cell cultures stained with L7 primary and FITC-derivatised secondary antibody were analysed by image software and relative brightness of processes was determined. (A) N-cadherin expressed in CG4 cell processes (arrowheads denote stronger staining in oligopolar cell process tips, arrows point to weaker staining in tripolar cell process tips); (B) phase contrast. Magnification in A, B, 10503. (C) Individual intensity level on process tips from CG4 cells. Data were combined from multiple experiments.

respectively, and did not differ in their capabilities in this assay. Therefore their results were combined.

N-Cadherin and OP Migration on Astrocytes Both astrocytes and Schwann cells (SC) express N-cadherin (Tomaselli et al., 1988; Letourneau et al., 1990) and the migration of SCs on astrocytes is known to be mediated by this cell adhesion molecule (Fok-Seang et al., 1995). As OPs also express N-cadherin, we hypothesized that OP migration on astrocytic surfaces is slowed by an overly strong adhesive interaction between these two cell types. Therefore, the effects of agents that block N-cadherin function on OP migration on astrocyte monolayers were studied in greater detail. In a typical control culture, the ability of about 20,000 cells cultured on a coverslip fragment to migrate onto an astrocytic monolayer over a timeframe of 72 h was assessed. The majority of motile cells migrated between 100 and 400 µm and a small number of cells reached up to 1 mm of migration distance (Figs. 4A, control, 4B). In control cultures, the sum of the migration distances of all cells within the 1 3 1-mm span of the ocular graticule was about 90 mm after 24 h. When cultures were kept under the influence of the L7 antibody (1:20 dilution) or 100 µg/ml HAV-peptide, total migration was increased by 135 and 230%, respectively (P values of 0.008 and 0.036 compared to untreated controls in a Mann–Whitney

rank sum test, see Figs. 4A, 4B, 4D), whereas incubation with the HGV control peptide or an anti-GalC control antibody had no statistically significant effect (Figs. 4A, 4B, 4D). This effect of the blockade of N-cadherin function persisted, since in experiments lasting 72 h the incubation of migrating CG4 cells with the HAV-peptide still increased migration on astrocytes by about 60% compared to controls (Figs. 4C and 4D; P , 0.05 in a t test). The effect of N-cadherin blocking agents to increase CG4 cell migration extended into the whole population of cells, since in all equidistant fields of the counting graticule, cell numbers were increased under the influence of the HAV peptides. Similarly, the migration of primary OP cultures was increased by 140% after 3 days under incubation with HAV-containing peptides (Fig. 4D). Homophilic N-cadherin binding induces signalling events that can have profound effects on cell behavior (Williams et al., 1994; Walsh et al., 1997). We tested whether the effects of substrate-bound N-cadherin on migration could be duplicated by non-substrate-bound NCAD-Fc fusion protein, which will activate the same signalling pathways, but will not promote cell–substrate adhesion. Therefore, we tested these compounds in a CG4 cell migration assay on laminin. Nonspecific binding was blocked by coating the assay dish with BSA. In these experiments, neither soluble NCAD-Fc nor the HAV

292

Schna¨delbach et al.

FIG. 3. N-cadherin coated as a substrate inhibits laminin-promoted migration of CG4 cells. CG4 cells migrated on either 1 µg/ml laminin alone (A), laminin 1 5 µg/ml human IgG antibody (B), laminin 1 5 µg/ml NCAD-Fc (C), or laminin 1 5 µg/ml NCAD-Fc and 100 µg/ml soluble HAV peptide (D). Note that the migration-inhibiting effect of immobilised N-cadherin could be neutralized by the HAV peptide. (E) Distribution of CG4 cells migrating on laminin/NCAD-Fc after 24 h compared to laminin/human IgG controls. Numbers represent mean 6 SEM of triplicate experiments. (F) Dose-dependent reduction of total migration of CG4 cells on NCAD-Fc/laminin substrates after 72 h as compared to controls containing similar concentrations of human IgG. Bars represent mean 6 error of duplicate experiments. Scale bar in A, 100 µm.

FIG. 4. N-cadherin function-blocking agents increase migration of CG4 cells on astrocyte monolayers. (A) Photomicrographs of CG4 cell cultures after 3 days under control conditions, 100 µg/ml HAV peptide, or 100 µg/ml HGV peptide. Bar in A, 50 µm. (B) CG4 cell number distribution in relation to controls after 24 h under varying culture conditions. Values denote mean 6 SEM. CG4 cell number distribution in relation to controls after 72 h under varying culture conditions. Values are given as mean 6 SEM of multiple experiments. (D) Total migration performance compared to controls in percentage. Values denote mean 6 SEM of multiple experiments.

293

294

Schna¨delbach et al.

(Fig. 5, and data not shown). Since only the HAV peptide was capable of increasing migration of CG4 cells on astrocytes, we conclude that this function is not brought about by signalling-induced side effects of N-cadherin.

N-Cadherin Is Involved in the Adhesion of Oligodendrocytes to Astrocyte Monolayers

FIG. 5. Soluble NCAD-Fc is mitogenic for CG4 cells. CG4 cell cultures were subjected to a MTS proliferation assay for 72 h under control conditions or under coincubation with 5 µg/ml soluble NCAD-Fc or 100 µg/ml HAV peptide on laminin, a substrate inert to cadherin-mediated adhesion. Values are given as mean 6 SEM of multiple experiments.

peptide altered mean CG4 cell migration (data not shown). Another mechanism, by which N-cadherin might influence cell migration could be to increase cell numbers, thereby either pushing more cells out underneath the coverslip or inducing cell proliferation of migrated cells, giving the appearance of a higher number of migrated cells. To test this hypothesis, we included the soluble NCAD-Fc fusion protein and the HAV peptide in a proliferation assay over a period of 72 h, the longest time-span used for the migration assay. To exclude any adhesion-related effects, the neutral substrate laminin was used for the culture and binding of NCAD-Fc was blocked by coating the dishes with BSA. After 72 h, the soluble NCAD-Fc fusion protein significantly increased cell numbers by 240% over untreated controls (P 5 0.005 in a t test), whereas neither the HAV peptide nor a HGV control peptide or the use of human IgG antibody as a control for the NCAD-Fc fusion protein had any effect

The above mentioned results suggested that adhesive processes might be the reason for the antimigratory properties of N-cadherin. Thus, a coculture technique was developed to investigate the adhesive properties of glial cells. Primary astrocytes were grown to confluence in 24-well plates and OPs or CG4 cells that had previously been labeled with Dil were incubated in these plates for 1 h. When either the blocking antibody L7 (1:20 dilution) or N-cadherin blocking peptides (100 µg/ml) were added to the coculture, the numbers of adhering CG4 cells were reduced by 27 and 32%, respectively (P , 0.01 and P , 0.05 in a Mann–Whitney rank sum test). On the other hand, control antibodies such as GalC or a control peptide had no statistically significant effect on cell adhesion (Fig. 6A). Similarly, the adhesion of primary cultures of OPs to astrocyte monolayers was significantly reduced only when the N-cadherin blocking peptide was used, but not under the influence of the control peptide (Fig. 6B). Adhesion of OPs to astrocytes is therefore mediated at least in part by N-cadherin.

The Effect of N-Cadherin on Individual Contacts to Astrocytes Depends on the Morphology of OPs The experiments mentioned above deal with the behavior of a relatively large number of OPs in contact with a continuous monolayer of astrocytes. In order to investigate whether the influence of cadherin functiondisrupting agents could also be seen on an individual cell level, video microscopy of individual OP-astrocyte contacts in low-density cultures was performed (Figs. 7A–7C). In preliminary experiments, it was evident that the behavior of OPs or CG4 cells varied with morphology when they encountered astrocytes. The average duration of contacts between bipolar or tripolar CG4 cells and astrocytes was at 4632 6 1222 s. This was significantly shorter than the duration of contact between CG4 cells with four or more processes (multipolar) and astrocytes which lasted on average about 7038 6 966 seconds (Fig. 7D, black bars; P 5 0.007 in a Mann–Whitney rank sum test). Interestingly, the cadherin function-blocking HAV

Cadherin and Oligodendrocyte Migration

295

FIG. 6. N-cadherin is involved in astrocyte–oligodendrocyte adhesion. Cell numbers of primary oligodendrocytes (A) or CG4 cells (B) adhering to astrocytes after 1 h under varying culture conditions were counted and put in relation to untreated controls.

peptide reduced the mean duration of interactions between the multipolar CG4 cells and astrocytes by 55% (P 5 0.002, Mann–Whitney rank sum test), but had no significant effect on bipolar/tripolar CG4 cells contacting individual astrocytes (Fig. 7D, light gray bars). This apparent morphology-related effect might be correlated with higher expression levels of N-cadherin on process tips of quadripolar CG4 cells and OPs as compared to tripolar cells. Incubation of the cultures with the HGV control peptide had no influence on CG4 contact behavior irrespective of their morphology.

DISCUSSION Cell migration requires that the migrating cell be motile. Motility is a product of the intrinsic properties of the cells and of the various signalling pathways within them, some of which may be affected by adhesion molecules (McCarthy and Turley, 1993). Migration is also influenced by cell adhesion: cells must adhere to the surface in order to migrate, but must also release these adhesive contacts at the trailing edge of the cell in order to move ahead (Huttenlocher et al., 1995). In this study, the influence of cadherins on the

migratory and adhesive behaviour of OPs was analyzed. According to our data, OPs migrate rapidly on laminin, and their migration is inhibited by substrateattached NCAD-Fc, but not by NCAD-Fc in solution. Furthermore, N-cadherin seems partly responsible for the ability of astrocytes to inhibit OP migration. Inhibition of migration by both NCAD-Fc substrate and astrocytes could be perturbed by adding cadherin function-blocking agents, either antibodies or HAV-containing peptides to the cultures. The inhibition of OP migration by N-cadherin requires adhesive interaction with a surface-bound molecule. Signalling via N-cadherin alone does not inhibit migration, since a soluble N-cadherin–Fc fusion protein had a mitogenic effect on the immortalized oligodendrocyte cell line CG4 but did not affect migration. In several adhesion molecules, such as laminin and fibronectin, short amino acid sequences (IKVAV and RGDS, respectively) have been shown to be responsible for molecular interaction. Therefore, synthetic peptides have been successfully used for function blocking studies mainly by competing for ligands with the receptor (Ruoslathi and Pierschbacher, 1987; Reichardt and Tomaselli, 1991; Mu¨ller et al., 1995; Milner et al., 1996). In our experiments, the blocking of N-cadherin-mediated inter-

296

Schna¨delbach et al.

FIG. 7. N-cadherin is involved in CG4 process–astrocyte interaction. Cocultures of CG4 cells and primary astrocytes under varying culture conditions were recorded on time-lapse video and duration of interactions from initial contact (between A and B) until release of contact (C) was measured in relation to CG4 cell morphology. Enlargements have been drawn in camera lucida style for improved clarity. Arrowheads: exemplified contact. (D) Contact duration of multipolar CG4 cells to astrocytes is reduced upon N-cadherin block, whereas duration of bipolar/tripolar CG4 cells to astrocytes is not affected by blocking N-cadherin function. Data represent mean 6 SEM of at least triplicate experiments. Magnification in A–C, 5003.

actions was achieved by using synthetic cyclic peptides containing the classical cadherin cell adhesion recognition sequence, HAV. The binding specificity of the peptide can be enhanced by adding appropriate amino acids to the tripeptide sequence (Blaschuk et al., 1990; Willems et al., 1995; Noe et al., 1999; Williams et al., 2000).

Cadherins and Cell Migration Cadherin-mediated adhesion has been shown to be involved in the control of a variety of migratory processes. For example, neural crest cells lose their N-cadherin content prior to acquiring migratory status. N-cadherin is reexpressed by these cells upon reaggrega-

Cadherin and Oligodendrocyte Migration

tion into sensory ganglia (Hatta and Takeichi, 1986). Also, the loss of cadherin expression has been demonstrated to be necessary for the acquisition of motility in adult songbird forebrain neuronal precursor cells (Barami et al., 1994) and blocking cadherin function can increase the motility of tumor cells (Behrens et al., 1989). Apparently the existence of cadherins in these systems confers a high degree of adhesion between the cells so that motility is impaired. However, during development of the nervous system, cadherins seem to play an important role in the development of the retina and the guidance of optic nerve fibers (Matsunaga et al., 1988), as well as promoting the migration of sensory growth cones on Schwann cells (Letourneau et al., 1990). Thus, differential levels of adhesion and differential responses from the migrating cells or processes seem to be necessary to allow maximum migration or process outgrowth in diverse cellular systems. Experiments performed by Palecek and colleagues (1997) demonstrated the requirement of intermediate levels of adhesion to effect maximum migration of CHO cells, with either too little or too much adhesion being inhibitory. Along these lines, both adhesive and anti-adhesive molecules can increase migration (Huttenlocher et al., 1995). We have demonstrated that both OPs and astrocytes possess N-cadherin on their surface, and that adhesion between these two cell types is partially blocked by the N-cadherin function disrupting agents such as cyclic HAV-containing peptides and L7 antiserum. Although OPs adhered strongly to an N-cadherin substrate and extended processes, there was no migration on this surface. Moreover, N-cadherin inhibited OP migration: its presence reduced OP migration on laminin in a dose-dependent manner. An astrocyte cell surface, which also inhibits OP migration relative to migration on laminin, presents to OPs a mixture of migrationpromoting molecules such as laminin and the migrationinhibitor N-cadherin. We therefore investigated whether blocking N-cadherin would promote OP migration on astrocytes and found a significant increase in migration, although less than was observed in equivalent experiments on Schwann cell migration (Wilby et al., 1999). We conclude from these data that N-cadherin is one of a variety of molecules that are involved in the control of OP migration, such as the polysialylated form of N-CAM, the DSD-1-Proteoglycan, and ECM molecules (Wang et al., 1994; Frost et al., 1996; Schna¨delbach and Faissner, 1998). How then does N-cadherin inhibit OP cell migration and astrocytic contact? Our experiments show that only substrate-bound NCAD-Fc has an antimigratory effect, while NCAD-Fc in solution does not affect cell migra-

297 tion and instead acts as a mitogen. The adhesive functions of classical cadherins reside in the first extracellular domain (Takeichi, 1991; Shapiro et al., 1995; Overduin 1995). Laminin and N-cadherin mediate cell adhesion via different receptors and affect different signalling pathways. Laminin-related promigratory effects have been linked to the interaction of the molecule with integrins. For example, in the migration of OPs the binding to a6b1 integrin is important (Milner and ffrench-Constant, 1994). Integrins can exert their functions by linkage to the cytoskeleton and by stimulating aggregation of pp125 focal adhesion kinase and tyrosine phosphorylation (Miyamoto et al., 1995). N-cadherin, on the other hand, is known to affect cell behavior in several different ways, one of which is by binding to the intracellular signalling proteins, known as the catenins (Takeichi, 1991; Vleminckx and Kemler, 1999). The catenins link N-cadherin to the cytoskeleton. One of the catenins, known as b-catenin is also capable of translocating to the nucleus and affecting gene expression (Gumbiner, 1995). Cadherin–Catenin association and function seems to involve tyrosine phosphorylation of the catenins (Daniel and Reynolds, 1997; Levenberg et al., 1998) and the association of a protein tyrosine phosphatase with the cytoplasmic domain of N-cadherin has been correlated to the regulation of the linkage of cadherin to the cytoskeleton (Balsamo et al., 1996, 1998) and, thus, cadherin function (Soler et al., 1998). Interestingly, in another model system of cellular migration, the neural crest, a signalling interaction between N-cadherin and integrins has been found. Blocking of b1 and b3 integrin adhesion by RGD peptides caused signalling events involving Ca21 influx in neural crest cells and catenin phosphorylation that controlled distribution and activity of N-cadherin, resulting in N-cadherin-mediated cell aggregation (MonierGavelle and Duband, 1997). Furthermore, N-cadherin can cis-interact with the FGF-receptor and affect its signalling properties (Williams et al., 1994; Walsh et al., 1997). This interaction might be responsible for the apparent mitotic activity of N-cadherin on CG4 cells in our proliferation assay, since bFGF is known to increase proliferation of OPs (Bo¨gler et al., 1990). However, the HAV peptide did not act as a mitogenic signal in a proliferation assay and therefore presumably does not imitate FGF receptor signalling. It is interesting to note that in the development of the mouse brain, N-cadherin mRNA is downregulated from E16 onwards, only shortly after PDGFa-receptor immunopositive OPs appear. Limited amounts of N-cadherin mRNA are retained up to P6, but in the adult mouse

298

Schna¨delbach et al.

brain, N-cadherin mRNA is at low levels and cadherin protein is mainly found at synapses (Pringle and Richardson, 1993; Redies and Takeichi, 1993; Uchida et al., 1996; Benson and Tanaka, 1998). This time-course coincides with the period of OP migration and initiation of myelination in the CNS. However, OP migration in the damaged CNS appears to be limited to distances of about 2 mm, and N-cadherin might be responsible for this phenomenon. In the regenerating PNS, N-cadherin is upregulated at the contact sites between Schwann cells and peripheral nerve axons (Shibuya et al., 1995). However, the expression of N-cadherin on astrocytes and axons in CNS injuries has not been studied. Since N-cadherin can affect both migration and proliferation of OPs, it might be possible to promote the repopulation of demyelinated MS lesions with OPs by manipulating the function of this cell adhesion molecule. This approach could prove useful to promote remyelination.

overnight. The remaining monolayer consisted of about 95% pure astrocytes. B104 rat neuroblastoma cells were cultivated in large culture flasks in DMEM/FCS to near-confluency, where upon the medium was changed to nonsupplemented DMEM/ITS. Conditioning went on for 2 days and again, after a change to fresh DMEM/ITS, for another 2 days. The conditioned media were centrifuged at 1000 rpm, filtered through a sterile 0.22-µm syringe-filter and stored at 220°C until further use. After thawing, the medium was diluted 1:3 in DMEM/ITS and kept at 4°C until further use (referred to as B104CM). The CG4 cell line (kind donation of Dr. J. Louis, Department of Biology, University of California at San Diego) was established from newborn rat cortical cultures (Louis et al., 1992) and cultivated in B104CM. They resemble oligodendrocyte precursors and have almost indistinguishable migratory properties (Fok-Seang et al., 1995; Fok-Seang et al., 1998).

MATERIAL AND METHODS

Cadherin Function-Blocking Agents

Cell Culture Primary astrocyte and oligodendrocyte cultures were prepared from early postnatal rat brain as described (McCarthy and de Vellis, 1980; Fok-Seang et al., 1995). In brief, dissociated cortical cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-Life Technologies, Paisley, UK), supplemented with 10% (vol/vol) fetal calf serum (FCS, Harlan Serolab, Loughborough, UK), Penicillin-Streptomycine-Fungizone (Gibco), and ITS (Collaborative Biomedical Products, Becton-Dickinson, Bedford, UK; further referred to as DMEM/FCS). After 7–10 days, the cultures became confluent and loosely attached macrophages were removed by shaking the flasks on a Luckham R300 incubator shaker at 60 rpm for 1 h at 37°C. The supernatant was discarded and the cultures were then shaken in fresh culture medium for 2–4 h at 120 rpm to remove oligodendrocyte precursors from the cell monolayer. This supernatant was exposed to plastic surfaces for 30 min at 37°C to further purify the nonadhering oligodendrocyte precursors from macrophage contaminants. The oligodendrocyte precursors were then transferred to a culture flask conditioned with 0.1% poly-Dlysine (PDL, Sigma) in water and left to adhere overnight. From the next day on, cultures were maintained in B104CM (see below). To obtain highly purified astrocyte cultures, the flasks containing the residual cells after the shaking procedure were subjected to a vigorous shaking at 120 rpm

The L7 rabbit antiserum was produced against N-cadherin as described (Newton et al., 1993). It has previously been shown to specifically react with a 125-kDa band on Western blots of rat astrocytes and schwann cells (Wilby et al., 1999). Oligopeptides containing the HAV adhesion sequence or a HGV control sequence were a generous gift of Adherex Co. (Ottawa, Canada). The peptides were synthesized using solid phase peptide synthesis (Merrifield, 1963; Stewart and Young, 1969). The procedure has been described previously (Wilby et al., 1999; Makrigiannakis et al., 1999). In our experiments, mainly a peptide with the sequences HAV was used to block N-cadherin. Since the HAV sequence appears also in other adhesion molecules, a peptide with the sequence HAVD with potentially higher specificity for N-cadherin (Williams, et al., 2000) was also used in selected experiments. In all experiments, both peptides had the same effect and data were pooled.

Production of the NCAD-Fc Fusion Protein A 2.2-kb insert with the extracellular portion of chicken N-cadherin fused to the Fc part of human IgG was spliced into the plG plasmid (Walsh et al., 1997). Plasmid DNA was prepared from bacterial cultures according to established protocols (Sambrook et al., 1989). Tissue culture flasks containing semiconfluent cultures of Cos7 cells were then transfected with purified plasmid DNA using the DEAE-Dextran method

299

Cadherin and Oligodendrocyte Migration

(Sambrook et al., 1989). The culture medium was conditioned for 3 to 7 days and the fusion protein was purified from the culture supernatant by using protein A column chromatography (Pharmacia Ltd, Uppsala, Sweden). Integrity of the fusion protein was ascertained by SDS–PAGE and subsequent blotting onto nylon membranes (Hybond C pure, Amersham Life Science, UK), which were then developed with the L7 anti-N-cadherin primary and anti-rabbit HRP-coupled secondary antibody and visualised by the HRPL chemoluminescence method (National Diagnostics, Hull, UK), and purity of the preparation was checked by silver stain of an appropriate SDS–PAGE. The preparations usually yielded a major band at around 120 kDa and a minor degradation band at around 46 kDa (data not shown). Protein content was measured with the Coomassie1 protein stain (Pierce, Rockford, IL). In functional assays, human IgG was used as a control agent to exclude possible side effects from the Fc-tail of the fusion protein.

Immunocytochemistry Cell cultures used for immunocytochemistry were fixed in 4% paraformaldehyde for 10 min at room temperature. In case of intracellular antigens, permeabilization was achieved by submersing the coverslips in 96% ethanol at 220°C directly prior to the use of the respective antibody. Blocking of nonspecific binding sites was performed by incubating the cultures in a solution of 1% (w/vol) BSA. Primary antibodies against O4, Glial fibrillary acidic protein (GFAP, both Boehringer–Mannheim, Germany), and Galactocerebroside (GalC, Chemicon, Harrow, UK) have been described elsewhere. Antibodies were diluted in blocking buffer and applied for 30 min at room temperature. Secondary fluoresceine- or rhodamine-derivatized antibodies were obtained from Boehringer-Mannheim, Serotec (Oxford, UK), or Dako (Glostrup, Denmark). In some cases, biotin-coupled secondary antibodies were used and visualized with streptavidine-rhodamine (Serotec). The intensity of fluorescence on cell process tips was quantified using photomicrographs of immunofluorescence stainings, which were scanned with a Nikon LS-1000 Slide scanner. These pictures were then used to define areas of cell processes in which the brightness intensity could be averaged with the help of the Sigmascan program (Jandel Scientific).

Proliferation Assay CG4 cells were plated into 96-well plates coated with either 100 µg/ml poly-D-lysine or 10 µg/ml laminin at a

density of 1–2 3 104 cells per well and cultivated for 24 to 72 h. Then the staining solution from the Cell Titer AQueous kit (Promega, Madison, WI) was added and the cells were incubated for another hour, after which the extinction rate at 595 nm was compared to a standard curve with predefined cell densities ranging from 0.78 3 103 to 105 cells per well.

Cell Adhesion Assay To analyze determinants of oligodendrocyte adhesion to astrocyte monolayers, primary astrocyte cultures were subcultivated onto 24-well plates and grown to confluence. OPs and CG4 cells were dissociated and stained with 25 µg/ml Dil (of a 2.5 mg/ml stock solution in ethanol or DMSO, Molecular Probes, Leiden, The Netherlands) for 5 min at room temperature. 50,000 cells per well were then seeded onto the astrocyte monolayer and the cultures incubated for 1 h at 37°C on a rotary shaker at 60 rpm. Several treatments of these cultures were performed: unsupplemented control cultures, cultures supplied with 100 µg/ml of N-cadherinblocking or control peptides, or cultures to which the N-cadherin-blocking serum L7 (at a dilution of 1:20) or anti galactocerebroside control antibodies (at a dilution of 1:10) were added. After a wash, cultures were fixed in 4% paraformaldehyde for 10 min at room temperature and the number of adhering cells was determined by counting the number of stained oligodendrocytes in a counting grid under a 203 objective and repeating the counts in adjacent grids to cover one complete diameter of each well. Experiments were performed at least in triplicates and groups of data were statistically analysed by the Sigmastat program.

Astrocyte Monolayer Migration Assay The investigation of migratory properties of oligodendrocytes on astrocyte monolayers was carried out as described previously (Fok-Seang et al., 1995). In brief, astrocytes were cultivated in 4-well tissue culture plates to confluence. OPs or CG4 cells were dissociated and labelled with Dil as described above. Thereafter, the cells were cultivated onto small fragments of glass coverslips precoated with poly-D-lysine at a density of 2 3 104 per mm2 for 24 h. The coverslip fragments were carefully washed several times in HBSS and inverted onto the astrocyte monolayer. Cultures were checked for the absence of washed-out cells and cultivated for 24 to 72 h in DMEM/FCS with or without additives (100 µg/ml N-cadherin function-blocking or control peptides or cadherin-blocking or control antibodies at dilu-

300 tions described above. Each supplement was added every 24 h in longer assays). Migratory performance was assessed by counting the number of stained OPs or CG4 cells under a fluorescence microscope in individual strips of a 10 3 10 field-graticule grid with field size of 100 3 100 µm, which was aligned with an edge of the coverslip. The migration performance was assayed in two ways: first, the cell numbers in equidistant strips were counted in each well. From the collated data of four wells per assay and at least triplicate assays a mean value 6 SEM was calculated and used to plot a density distribution. Furthermore, in order to incorporate differences in numbers of migrating cells because of recruitment of an otherwise nonmigrating cell population we calculated the total migration rate, which was defined as the sum of the migration distances of all cells that had left one coverslip into the graticule grid. Statistical analyses as by t test or Mann–Whitney rank sum test were carried out with the help of the Sigmastat program.

Substrate Migration Assay To analyze the influence of cadherin functionblocking agents on migration of CG4 cells on noncadherin substrates, the migration assay was modified as follows: purified laminin (Sigma-Aldrich, Poole, UK) was coated onto 24-well petri dishes at 10 µg/ml and nonspecific binding sites were blocked with 1 mg/ml heat-inactivated BSA. For pure N-cadherin substrates, an anti-human Fc-specific antibody was separately immobilised onto the plates at 100 µg/ml and the NCAD-Fc fusion protein bound at 5 µg/ml and subsequently blocked as described above. Mixed substrates were generated by cocoating laminin and anti-human-Fcspecific antibody at the concentrations indicated above, with a subsequent overcoat of NCAD-Fc or human IgG in controls, followed by blocking as described above. Glass coverslips with confluent cultures of CG4 cells were then inverted onto the centre of the dish. Migration of the cells onto the substrate was followed every 24 h over several days by counting the cells in a grid composed of 10 fields of 100 3 100 µm in the ocular of the microscope. Data from at least triplicate experiments were pooled to determine the total migration rate and data were statistically analysed by the Sigmastat program (SPSS, Chicago, IL).

Video Microscopy To a low-density culture of primary astrocytes in a 3-cm petri dish 5 3 104 CG4 cells or primary OPs were

Schna¨delbach et al.

added under inclusion or exclusion of N-cadherin function-blocking or control peptides at 100 µg/ml in B104CM. This culture was transferred to a Nikon Diaphot inverted microscope inside a heated plexiglass chamber, to which a CO2-flask with regulator was attached. The microscopic picture from a 203 objective was fed into a Hitachi HV 720K video camera linked to a Panasonic NV 8051 single picture time-lapse video recorder. Eight pictures were taken every 30 s and filming continued for 12–24 h. Films were analyzed for duration of contacts between oligodendrocyte processes and astrocytes. Data were analyzed and processed with the help of Sigmastat and Sigmaplot programs (SPSS).

N-Cadherin mRNA Detection Total RNA was prepared from cell cultures using the acid guanidinium-phenol extraction method (Chomczynski and Sacchi, 1987). Quality was monitored by formaldehyde gel electrophoresis and subsequent ethidium bromide staining and the concentration was determined by measuring extinction at 260- and 280-nm wavelength. An RT-PCR approach was employed to assay the presence of N-cadherin mRNA transcripts in CG-4 cells. The specific primers used for rat N-cadherin were: forward 58-CAAGACAAAGAAACCCAGG-38 and reverse 58-CTGGTGCAGAAACTCAGG-38. The cDNA was synthesized from 1 mg of total RNA using 100 pmol of the reverse primer, in 20 ml 10 mM Tris (pH 8.3), with 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, and 400 units of M-MLV-Reverse Transcriptase (Gibco/BRL, Burlington, ON). PCR was performed using the contents of the first-strand reaction and 100 pmol of the forward primer in 100 ml 10 mM Tris (pH 8.3), with 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, and 2.5 U Taq polymerase (Boehringer-Mannheim, Laval, Quebec, Canada). The cycling program was as follows: denaturation at 95°C for 30 s; annealing at 57°C for 45 s; polymerization at 72°C for 1 min; repeat for 30 cycles. All PCR reactions were performed in parallel with reactions containing no cDNA as a control for contamination of PCR reagents. Products were identified by ethidium bromide-stained agarose gel electrophoresis. The expected product size is 492 bp.

ACKNOWLEDGMENTS The authors thank Ms. Dorothy Gibson, Ms. Clare Ellis, and Mr. David Brown for excellent technical assistance, Dr. Michelle Utton for support with the NCAD-Fc fusion protein and Drs. Elizabeth Muir and John Rogers for helpful comments.

Cadherin and Oligodendrocyte Migration

REFERENCES Adams, R. D., Victor, M., and Ropper, A. H. (1998). Multiple Sclerosis and allied demyelinative diseases. In Principles of Neurology (M. J. Wonsiewicz and M. Navrozov, Eds.), pp. 902–927. McGraw-Hill, New York. Amberger, V., Avellana-Adalid, V., Hensel, T., Baron-Van Evercooren, A., and Schwab, M. E. (1997). Oligodendrocyte-type 2 astrocyte progenitors use a metalloprotease to spread and migrate on CNS myelin. Eur. J. Neurosci. 9: 151–162. Balsamo, J., Leung, T., Ernst, H., Zanin, M. K., Hoffman, S., and Lilien, J. (1996). Regulated binding of PTP1b-like phosphatase to N-cadherin: Control of cadherin-mediated adhesion by dephosphorylation of beta-catenin. J. Cell Biol. 134: 801–813. Balsamo, J., Arregui, C., Leung, T., and Lilien, J. (1998). The nonreceptor protein tyrosine phosphatase PTP1B binds to the cytoplasmic domain of N-cadherin and regulates the cadherin-actin linkage. J. Cell Biol. 143: 523–532. Barami, K., Kirschenbaum, B., Lemmon, V., and Goldman, S. A. (1994). N-cadherin and Ng-CAM/8D9 are involved serially in the migration of newly generated neurons into the adult songbird brain. Neuron 13: 567–582. Behrens, J., Mareel, M. M., Van Roy, F. M., and Birchmeier, W. (1989). Dissecting tumor cell invasion: Epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell–cell adhesion. J. Cell Biol. 108: 2435–2447. Ben-Hur, T., Rogister, B., Murray, K., Rougon, G., and Dubois-Dalcq, M. (1998). Growth and fate of PSA-NCAM1 precursors of the postnatal brain. J. Neurosci. 18: 5777–5788. Benson, D. L., and Tanaka, H. (1998). N-cadherin redistribution during synaptogenesis in hippocampal neurons. J. Neurosci. 18(17): 6892– 6904. Blaschuk, O. W., Sullivan, R., David, S., and Pouliot, Y. (1990). Identification of a cadherin cell adhesion recognition sequence. Dev. Biol. 139: 227–229. Bo¨gler, O., Wren, D., Barnett, S. C., Land, H., and Noble, M. (1990). Cooperation between two growth factors promotes extended selfrenewal and inhibits differentiation of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells. Proc. Natl. Acad. Sci. USA 87: 6368–6372. Cameron-Curry, P., and Le Douarin, N. (1995). Oligodendrocyte precursors originate from both the dorsal and the ventral parts of the spinal cord. Neuron 15: 1299–1310. Chen, H., Paradies, N. E., Fedor-Chaiken, M., and Brackenbury, R. (1997). E-cadherin mediates adhesion and suppresses cell motility via distinct mechanisms. J. Cell Sci. 110: 345–356. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159. Daniel, J. M., and Reynolds, A. B. (1997). Tyrosine phosphorylation and cadherin/catenin function. Bioessays 19(10): 883–891. Fok-Seang, J., Mathews, G. A., ffrench-Constant, C., Trotter, J., and Fawcett, J. W. (1995). Migration of oligodendrocyte precursors on astrocytes and meningeal cells. Dev. Biol. 171: 1–15. Fok-Seang, J., DiProspero, N. A., Meiners, S., Muir, E., and Fawcett, J. W. (1998). Cytokine-induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth. Eur. J. Neurosci. 7: 2400–2416. Franklin, R. J., and Blakemore, W. F. (1997). To what extent is oligodendrocyte progenitor migration a limiting factor in the remyelination of multiple sclerosis lesions? Mult. Scler. 3(2): 84–87. Frost, E., Kiernan, B. W., Faissner, A., and ffrench-Constant, C. (1996).

301 Regulation of oligodendrocyte precursor migration by extracellular matrix: Evidence for substrate-specific inhibition of migration by tenascin-C. Dev. Neurosci. 18: 266–273. Gumbiner, B. M. (1995). Signal transduction of beta-catenin. Curr. Opin. Cell Biol. 7(5): 634–640. Hatta, K., and Takeichi, M. (1986). Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320: 447–449. Huttenlocher, A., Sandborg, R. R., and Horwitz, A. F. (1995). Adhesion in cell migration. Curr. Opin. Cell Biol. 7: 697–706. Keirstead, H. S., Levine, J. M., and Blakemore, W. (1998). Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 22: 161–170. Letourneau, P. C., Shattuck, T. A., Roche, F. K., Takeichi, M., and Lemmon, V. (1990). Nerve growth cone migration onto Schwann cells involves the calcium-dependent adhesion molecule, N-cadherin. Dev. Biol. 138: 430–442. Levenberg, S., Sadot, E., Goichberg, P., and Geiger, B. (1998). Cadherinmediated transmembrane interactions. Cell Adhes. Commun. 6: 161– 170. Levison, S. W., Chuang, C., Abramson, B. J., and Goldman, J. E. (1993). The migrational patterns and developmental fates of glial precursors in the rat subventricular zone are temporally regulated. Development 119: 611–622. Louis, J. C., Magal, E., Muir, D., Manthorpe, M., and Varon, S. (1992). CG4, a new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type-2 astrocytes. J. Neurosci. Res. 31: 193–204. Makrigiannakis, A., Coukos, G., Christofidou-Solomidou, M., Gour, B. J., Radice, G. L., Blaschuk, O., and Coutifaris, C. (1999). N-cadherin mediated human granulosa cell adhesion prevents apoptosis: A role in follicular atresia and luteolysis? Am. J. Pathol. 154(5): 1391–1406. Matsunaga, M., Hatta, K., Nagafuchi, A., and Takeichi, M. (1988). Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature 334: 62–64. McCarthy, J., and Turley, E. A. (1993). Effects of extracellular matrix components on cell locomotion. Crit. Rev. Oral Biol. Med. 4(5): 619–637. McCarthy, K. D., and de Vellis, J. (1980). Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85: 890–902. Merrifield, R. B. (1963). J. Am. Chem. Soc. 85: 2149. Milner, R., and ffrench-Constant, C. (1994). A developmental analysis of oligodendroglial integrins in primary cells: Changes in avassociated b subunits during differentiation. Development 120: 3497– 3506. Milner, R., Edwards, G., Streuli, C., and ffrench-Constant, C. (1996). A role in migration for the avb1 integrin expressed on oligodendrocyte precursors. J. Neurosci. 16(22): 7240–7252. Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995). Integrin function: Molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131: 791–805. Monier-Gavelle, F., and Duband, J. L. (1997). Cross talk between adhesion molecules: control of N-cadherin activity by intracellular signals elicited by beta1 and beta3 integrins in migrating neural crest cells. J. Cell Biol. 137(7): 1663–1681. Muir, E., Du, J., Fok-Seang, J., Smith-Thomas, L. C., Rogers, J., and Fawcett, J. W. (1998). Increased axon growth through astrocyte cell lines transfected with urokinase. Glia 23: 24–34. Mu¨ller, U., Bossy, B., Venstrom, K., and Reichardt, L. F. (1995). Integrin

302 a8b1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin. Mol. Biol. Cell 6: 433–448. Nait-Oumesmar, B., Vignais, L., Duhamel-Clerin, E., Avellana-Adalid, V., Rougon, G., and Baron-Van Evercooren, A. (1995). Expression of the highly polysialylated neural cell adhesion molecule during postnatal myelination and following chemically induced demyelination of the adult mouse spinal cord. Eur. J. Neurosci. 7: 480–491. Newton, S. C., Blaschuk, O. W., and Millette, C. F. (1993). N-cadherin mediates sertoli cell-spermatogenic cell adhesion. Dev. Dynam. 197: 1–13. Niehaus, A., Stegmu¨ller, J., Diers-Fenger, M., and Trotter, J. (1999). Cell-surface glycoprotein of oligodendrocyte progenitors involved in migration. J. Neurosci. 19: 4948–4961. Noe, V., Willems, J., Vandekerckhove, J., Roy, F. V., Bruyneel, E., and Mareel, M. (1999). Inhibition of adhesion and induction of epithelial cell invasion by HAV-containing E-cadherin-specific peptides. J. Cell Sci. 112: 127–135. Overduin, M., Harvey, T. S., Bagby, S., Tong, K. I., Yau, P., Takeichi, M., and Ikura, M. (1995). Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science 267: 386–389. Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A., and Horwitz, A. F. (1997). Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385: 537–540. Payne, H. R., and Lemmon, V. (1993). Glial cells of the O-2A lineage bind preferentially to N-cadherin and develop distinct morphologies. Dev. Biol. 159: 595–607. Payne, H. R., Hemperly, J. J., and Lemmon, V. (1996). N-cadherin expression and function in cultured oligodendrocytes. Dev. Brain Res. 97: 9–15. Pringle, N. P., and Richardson, W. D. (1993). A singularity of the PDGF alpha-receptor expression in the dorsoventral axis of the neural tube may define the origin of the oligodendrocyte lineage. Development 117: 525–533. Redies, C., and Takeichi, M. (1993). Expression of N-cadherin mRNA during development of the mouse brain. Dev. Dynam. 197: 26–39. Reichardt, L. F., and Tomaselli, K. J. (1991). Extracellular matrix molecules and their receptors: Functions in neural development. Annu. Rev. Neurosci. 14: 531–570. Ruoslathi, E., and Pierschbacher, M. D. (1987). New perspectives in cell adhesion: RGD and integrins. Science 238: 491–497. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning—A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schna¨delbach, O., and Faissner, A. (1996). TGF-b1 and motile behaviour of the oligodendroglial cell line Oli-neu: Evidence for involvement of DSD-1-Proteoglycan. J. Neurosci. Abstr. 22: 531. Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M. S., Gruebel, G., Legrand, J.-F., Als-Nielsen, J., Colman, D. R., and

Schna¨delbach et al.

Hendrickson, W. A. (1995). Structural basis of cell–cell adhesion by cadherins. Nature 374: 327–337. Shibuya, Y., Mizoguchi, A., Takeichi, M., Shimada, K., and Ide, C. (1995). Localization of N-cadherin in the normal and regenerating nerve fibers of the chicken peripheral nervous system. Neuroscience 67: 253–261. Soler, C., Rousselle, P., and Vincent, J. P. (1998). Cadherin-mediated cell–cell adhesion is regulated by tyrosine phosphatases in human keratinocytes. Cell Adhes. Commun. 5: 13–25. Stewart, J. M., and Young, J. D. (1969). Solid Phase Peptide Synthesis. W. H. Freeman, San Francisco. Takeichi, M. (1991). Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251: 1451–1455. Tomaselli, K. J., Neugebauer, K. M., Bixby, J., Lilien, J., and Reichardt, L. F. (1988). N-cadherin and integrins: Two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces. Neuron 1: 33–43. Uchida, N., Honjo, Y., Johnson, K. R., Wheelock, M. J., and Takeichi, M. (1996). The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones. J. Cell Biol. 126: 1527–1536. Uhm, J. H., Dooley, N. P., Oh, L. Y. S., and Yong, V. W. (1998). Oligodendrocytes utilize a matrix metalloproteinase, MMP-9, to extend processes along an astrocyte extracellular matrix. Glia 22: 53–63. Vleminckx, K., and Kemler, R. (1999). Cadherins and tissue formation: Integrating adhesion and signaling. Bioessays 21(3): 211–220. Walsh, F. S., Meiri, K., and Doherty, P. (1997). Cell signalling and CAM-mediated neurite outgrowth. Soc. Gen. Physiol. Ser. 52: 221–226. Wang, C., Rougon, G., and Kiss, J. Z. (1994). Requirement of polysialic acid for the migration of the O2A glial progenitor cell from neurohypophyseal explants. J. Neurosci. 14: 4446–4457. Wilby, M. J., Muir, E. M., Fok-Seang, J., Gour, B. J., Blaschuk, O. W., and Fawcett, J. W. (1999). N-cadherin inhibits schwann cell migration on astrocytes. Mol. Cell. Neurosci. 14: 66–84. Willems, J., Bruyneel, J., Noe, V., Slegers, H., Zwijsen, A., Mege, R. M., and Mareel, M. (1995). Cadherin-dependent cell aggregation is affected by decapeptide derived from rat extracellular superoxide dismutase. FEBS Lett. 363(3): 289–292. Williams, E. J., Furness, J., Walsh, F. S., and Doherty, P. (1994). Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13: 583–594. Williams, E., Williams, G., Gour, B. J., Blaschuk, O. W., and Doherty, P. (2000). A novel family of cyclic peptide antagonists suggest that N-cadherin specificity is determined by amino acids that flank the HAV motif. J. Biol. Chem. (in press). Woolswijk, G. (1998). Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells. J. Neurosci. 18: 601–609. Received September 29, 1999 Revised November 5, 1999 Accepted November 5, 1999