RESEARCH ARTICLE 415

Development 136, 415-426 (2009) doi:10.1242/dev.018234 and Dcc regulate oligodendrocyte process branching and membrane extension via Fyn and RhoA Sathyanath Rajasekharan, K. Adam Baker, Katherine E. Horn, Andrew A. Jarjour, Jack P. Antel and Timothy E. Kennedy*

The molecular mechanisms underlying the elaboration of branched processes during the later stages of oligodendrocyte maturation are not well understood. Here we describe a novel role for the chemotropic guidance cue netrin 1 and its receptor deleted in colorectal carcinoma (Dcc) in the remodeling of oligodendrocyte processes. Postmigratory, premyelinating oligodendrocytes express Dcc but not netrin 1, whereas mature myelinating oligodendrocytes express both. We demonstrate that netrin 1 promotes process extension by premyelinating oligodendrocytes in vitro and in vivo. Addition of netrin 1 to mature oligodendrocytes in vitro evoked a Dcc-dependent increase in process branching. Furthermore, expression of netrin 1 and Dcc by mature oligodendrocytes was required for the elaboration of myelin-like membrane sheets. Maturation of oligodendrocyte processes requires intracellular signaling mechanisms involving Fyn, focal adhesion kinase (FAK), neuronal Wiscott-Aldrich syndrome protein (N-WASP) and RhoA; however, the extracellular cues upstream of these proteins in oligodendrocytes are poorly defined. We identify a requirement for Src family kinase activity downstream of netrin-1-dependent process extension and branching. Using oligodendrocytes derived from Fyn knockout mice, we demonstrate that Fyn is essential for netrin-1-induced increases in process branching. Netrin 1 binding to Dcc on mature oligodendrocytes recruits Fyn to a complex with the Dcc intracellular domain that includes FAK and N-WASP, resulting in the inhibition of RhoA and inducing process remodeling. These findings support a novel role for netrin 1 in promoting oligodendrocyte process branching and myelin-like membrane sheet formation. These essential steps in oligodendroglial maturation facilitate the detection of target , a key step towards myelination.

KEY WORDS: Oligodendroglia, Myelination, Myelin, Netrin, Integrin, , Autocrine, Mouse, Rat, FAK (Ptk2), N-WASP (Wasl)

INTRODUCTION investigated the possibility that netrin 1, expressed in the developing To form a myelin sheath, an oligodendrocyte initially extends CNS and later by the oligodendrocytes themselves, might influence multiple branching processes that survey the local environment for late stages of oligodendrocyte differentiation. suitable axons (Hardy and Friedrich, 1996; Kirby et al., 2006). Upon The lamella elaborated by the tip of an extending oligodendrocyte contact with a target , oligodendrocyte processes coalesce to process has been compared to a neuronal (Fox et al., form a spreading sheet of membrane that begins to wrap the axon. 2006; Jarjour and Kennedy, 2004; Sloane and Vartanian, 2007). The extracellular cues that govern this process and the intracellular Interestingly, a number of the same intracellular signaling proteins mechanisms involved are poorly understood. have been implicated in netrin-1-mediated and the Laminin 2, which promotes oligodendrocyte maturation by development of oligodendrocyte processes required for myelination signaling through α6β1 integrins (Baron et al., 2005), has been (Fox et al., 2006; Jarjour and Kennedy, 2004). In both cases, the suggested to regulate process elaboration. Laminin-2-deficient mice reorganization of the actin cytoskeleton requires activation of the Src have dysmyelinated and hypomyelinated axons (Chun et al., 2003). family kinase (SFK) Fyn (Meriane et al., 2004; Osterhout et al., 1999; However, laminin 2 is not ubiquitous in myelinated CNS axon tracts Umemori et al., 1994) and involves Wiscott-Aldrich syndrome protein (Colognato et al., 2002) and transgenic mice lacking β1 integrin (N-WASP; Wasl – Mouse Genome Informatics) and Rho GTPases expression in oligodendrocytes exhibit no defects in CNS (Bacon et al., 2007; Liang et al., 2004; Shekarabi et al., 2005). myelination (Benninger et al., 2006). Thus, other ligand-receptor Here we provide evidence that netrin 1 and Dcc promote the interactions must also direct the changes in oligodendrocyte extension of oligodendrocyte processes in vivo. We used in vitro morphology required for myelination. assays to demonstrate that netrin 1 increases oligodendrocyte The axon-guidance cue netrin 1 (Ntn1) is a chemorepellent for process branching. Furthermore, expression of Dcc and netrin 1 by migrating oligodendrocyte precursors (OPCs) in the embryonic oligodendrocytes promotes the formation of myelin-like membrane (Jarjour et al., 2003; Tsai et al., 2003). These cells sheets. Addressing the signaling mechanisms involved, we show express the netrin 1 receptors Dcc, Unc5a and Unc5b, but not netrin that the SFK Fyn is required for netrin-1-induced process branching, 1 itself. In the adult CNS, netrin 1 is expressed by myelinating that netrin 1 recruits Fyn to Dcc in oligodendrocytes, increases SFK oligodendrocytes and is associated with non-compacted activity, and promotes process extension and branching associated oligodendroglial membranes (Manitt et al., 2001). We therefore with a decrease in RhoA activity. Our findings reveal a novel role for netrin 1 and Dcc in activating a signaling cascade in oligodendrocytes that directs process remodeling.

Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill MATERIALS AND METHODS University, Montreal, Quebec H3A 2B4, Canada. Animals and oligodendrocyte cultures *Author for correspondence (e-mail: [email protected]) Sprague Dawley rat pups and pregnant BALB/c mice were obtained from Charles River Canada (Montreal, Quebec, Canada). Ntn1–/+ and Dcc–/+ mice

Accepted 21 November 2008 were obtained from Marc Tessier-Lavigne (Genentech, South San Francisco, DEVELOPMENT 416 RESEARCH ARTICLE Development 136 (3)

CA, USA) and Robert Weinberg (Harvard University, Cambridge, MA, Analysis of phospho-Src puncta USA), respectively. Fyn KO (Fyntm1Sor) and control F2 hybrid (B6129SF2/J) The number of phospho-Src-positive puncta was measured using ImageJ. breeding pairs were obtained from Jackson Laboratories (Bar Harbor, ME, The brightness and contrast of the images of individual oligodendrocytes USA). All procedures were performed in accordance with the Canadian were modified to enhance puncta associated with extending processes; Council on Animal Care guidelines for the use of animals in research. modifications made were consistent across all images. Images were converted to a binary format and then the number of particles counted Cell culture automatically. Staining in the cell body and major processes was not OPCs were derived from mixed glial cultures from the cerebral cortices of punctate and thus counted as one particle, making variations under different postnatal day 0 (P0) rat pups and grown in oligodendrocyte defined medium conditions a direct indicator of changes in the number of distally located (OLDEM) as described previously (Armstrong, 1998; Jarjour et al., 2003), puncta. The number of puncta was divided by oligodendrocyte area to with 0.1% fetal bovine serum (FBS) to initiate differentiation. Cells were control for variance in oligodendrocyte size. seeded at 1.5ϫ104 cells/chamber in 8-well chamber slides coated with 10 μg/ml poly-L-lysine (Nalge Nunc, Rochester, NY, USA). For Quantification and statistical analyses immunoprecipitation, western blots and GST-rhotekin pulldowns, cells were Statistical significance was calculated by ANOVA followed by a post-hoc plated at 1.5ϫ106 cells/well in 6-well tissue culture dishes. Tukey test using Systat Software (San Jose, CA, USA). Analyses of oligodendrocyte morphology in vitro used three independent experiments Mouse oligodendrocyte cultures with a minimum of 30 cells per condition. In vivo analysis of process Culturing of mouse OPCs was similar to that of rat OPCs, but 10% horse extension and purification of oligodendrocytes from transgenic mice were serum was used instead of 10% FBS in mixed glial culture media. Each T75 performed using at least four pups of each genotype, derived from at least flask of mixed glial culture required two to three pups. Newborn Ntn1–/– and two different litters. Dcc–/– mice were identified by distinct behaviors. The genotype of individual pups was confirmed by PCR. Immunoprecipitation Cells in 6-well dishes were treated with netrin 1 for 5 minutes, then lysed in Antibodies RIPA lysis buffer (10 mM sodium phosphate pH 7.2, 150 mM NaCl, 1% Primary antibodies used in this study were: rabbit polyclonal anti-netrin 1 NP40, 0.1% SDS, 0.5% deoxycholate) and centrifuged at 13,000 rpm PN3 (Manitt et al., 2001), mouse monoclonal anti-Dcc (G97-449; BD (13,800 g) for 7 minutes. Supernatant was pre-cleared with 30 μl protein Biosciences Pharmingen, San Jose, CA, USA), goat polyclonal anti-Dcc A/G beads (Santa Cruz) for 30 minutes, incubated with 1 μg/ml anti-Dcc (Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit polyclonal anti-myelin (monoclonal) or anti-Fyn (rabbit polyclonal) for 1 hour, followed by addition μ basic protein (Mbp, Chemicon, Temecula, CA, USA), mouse monoclonal of 30 l protein A/G beads for 45 minutes. anti-Mbp (Chemicon), mouse monoclonal RIP antibody (Chemicon), mouse Ј Ј Ј GST pulldown assays monoclonal anti-2 ,3 -cyclic nucleotide 3 phosphodiesterase (Cnp, Fusion proteins comprising the RhoA-binding domain of rhotekin (a Sternberger Monoclonals, Lutherville, MD, USA), rabbit polyclonal anti- downstream substrate of RhoA) or Pak-CRIB and glutathione-S-transferase Mag (Chemicon), rabbit polyclonal anti-Nfm (Nefm – Mouse Genome (GST) were purified as described (Reid et al., 1996). Oligodendrocytes were Informatics) (Chemicon), rabbit polyclonal anti-Fyn (Upstate Cell treated with netrin 1 for 24 hours. Cells were then lysed and protein purified Signaling, Charlottesville, VA, USA) used for western blots, rabbit as described (Ren and Schwartz, 2000; Shekarabi et al., 2005). polyclonal anti-Fyn [gift of Dr Andre Veillete and described previously (Davidson et al., 1992)] used for immunoprecipitation and western blots, mouse monoclonal anti-FAK (BD Biosciences), rabbit polyclonal anti-N- RESULTS WASP (Santa Cruz), and rabbit polyclonal anti-phospho-Src (Cell Signaling), which recognizes the pY416 epitope in all SFK members. Oligodendrocytes express netrin 1 during For analyses of oligodendrocyte morphology in vivo, we crossed Ntn1 or myelination in the developing spinal cord Dcc heterozygous mice, obtained embryonic day 18 (E18) (plug Netrin 1, expressed by floor plate and neuroepithelial cells in the date taken as E1), fixed them in 4% paraformaldehyde and cut 18 μm early embryonic spinal cord, directs migrating OPCs away from the sections of the spinal brachial enlargement with a cryostat. Sections were ventral midline towards axons in the nascent white matter (Jarjour then stained with anti-Cnp (1:250), anti-Nfm (1:250), anti-netrin PN3 et al., 2003; Kennedy et al., 1994; Tsai et al., 2006; Tsai et al., 2003). (1:100), visualized using Alexa 546- or Alexa 488-conjugated secondary Although not expressed by OPCs, netrin 1 is widely expressed by antibodies (Jackson ImmunoResearch, West Grove, PA, USA), and nuclei spinal interneurons, motoneurons and by most, if not all, mature stained with Hoechst. Images were obtained with a Magnafire CCD camera myelinating oligodendrocytes in dorsal and ventral white matter in (Optronics, Goleta, CA, USA) and a Zeiss Axiovert 100 microscope the adult rat and mouse CNS (Manitt et al., 2001). (Toronto, Ontario, Canada). To determine when netrin 1 is expressed by differentiating Analysis of oligodendrocyte morphology oligodendrocytes, we performed a time-course analysis. Analyses of immature oligodendrocytes in vitro and in vivo were performed Premyelinating, postmigratory oligodendrocytes within the using the NeuronJ plugin for ImageJ (NIH, Bethesda, MD, USA). The corticospinal tract at brachial, thoracic and lumbar levels were length of the longest process was measured from the base of the process to examined. Netrin 1 expression was not detected in oligodendrocytes its tip (Fig. 3D; Fig. 4B). For Sholl analysis, the grid function in Northern at embryonic stages of development in mice (data not shown). For Eclipse (Empix) was used to draw concentric circles 15 μm apart around the postnatal stages, netrin-1-expressing cells were identified cell body of mature RIP-positive oligodendrocytes. The number of immunohistochemically. Oligodendrocytes in the developing intersections made by processes with each successive circle was counted. dorsolateral white matter tracts were identified by expression of β For studies using the 1 integrin subunit function-blocking antibody and 2Ј,3Ј-cyclic nucleotide 3Ј phosphodiesterase (Cnp), a marker that mouse oligodendrocyte cultures, a Sholl analysis plugin was used with labels oligodendrocyte cell bodies and processes in vivo, and by ImageJ (starting radius, 1.02 cm; step size, 1.02 cm; end, 5.08 cm; thickness, expression of the mature oligodendrocyte marker myelin basic 0.02 cm). The Mbp-positive myelin-like membrane sheets were outlined in ImageJ and the surface area reported in arbitrary units. protein (Mbp). At P8, before myelination begins in the corticospinal The pharmacological inhibitors PP2 and PP3 (Calbiochem) were used at tract, netrin 1 was not expressed by premyelinating oligodendrocytes 2 μM to inhibit SFK activity. Purified hamster anti-rat CD29 (β1 integrin) (Fig. 1A-C). Netrin-1-expressing neuronal cell bodies and neural- monoclonal and purified hamster anti-IgM monoclonal antibodies were used epithelial cells (Fig. 1D,E), but not astrocytes (Fig. 1I), were

at 2 μM to investigate β1 integrin function. detected immediately adjacent to differentiating oligodendrocytes. DEVELOPMENT Regulation of oligodendrocyte maturation RESEARCH ARTICLE 417

Fig. 1. Expression of netrin 1 by oligodendrocytes in vivo. (A,AЈ) Longitudinal section through a P8 rat spinal cord showing Mbp-immunopositive oligodendrocytes (red) that do not express netrin 1, intermingled with netrin-1-expressing cells (green). (B,BЈ) At P8, netrin 1 immunoreactivity (green) was detected in the immediate environment surrounding the cell bodies of Cnp-positive oligodendrocytes (red), but netrin 1 was not expressed by the oligodendrocytes themselves. (C,CЈ) An Mbp-positive netrin-1-negative process, surrounded by netrin-1- expressing neuroepithelial cells (arrows) in P8 spinal cord. (D,DЈ) Longitudinal section through a P8 rat spinal cord showing axons positive for neurofilament medium polypeptide (Nfm), surrounded by netrin-1- expressing cells. (E,EЈ) Cell bodies of netrin-1- immunopositive neurons. (F) Cross-section of a P12 rat dorsal spinal cord showing Cnp-positive, myelinating oligodendrocytes (red) expressing netrin 1 (green). (G-GЉ) Oligodendrocytes adjacent to the central canal (arrow) express netrin 1 in P12 spinal cord. (H,HЈ) Netrin 1 immunoreactivity (green) associated with a Cnp-immunopositive (red) myelinating oligodendrocyte in the P12 rat spinal cord. (I-IЉ) By P12, netrin 1 expression (green) colocalizes with Nfm- immunoreactive axons (blue), but is not detected in Gfap-positive astrocytes (red). (J,JЈ) Transverse section of a P22 thoracic spinal cord showing widespread expression of netrin 1 (green) by Cnp-positive, myelinating oligodendrocytes (red). (K-KЉ) Magnification of the boxed region from J showing Cnp-positive netrin-1-expressing cell bodies. A,F, 20ϫ0.5 n.a. objective; G,J, 40ϫ0.75 n.a. objective; D,E,I,K, confocal microscopy, 40ϫ0.75 n.a. objective; B,C,H, confocal microscopy, 100ϫ1.4 n.a. objective. Scale bars: 10 μm in BЈ,CЈ,H; 20 μm in AЈ,D,E,G,K; 40 μm in F,IЈ,J.

At P12, following the initiation of myelination in the rat 6-8 DIV and identified by expression of Mbp and Cnp (Watanabe corticospinal tract (Schwab and Schnell, 1989), netrin 1 was et al., 2006), extend highly branched processes that coalesce to detected in the cell bodies of a subset of oligodendrocytes double form cytoplasmic sheets (Fox et al., 2006). These sheets, which labeled with Cnp (Fig. 1F,G). At this stage of development, netrin 1 resemble unwrapped non-compacted myelin membrane (Knapp immunoreactivity was associated with axons (Fig. 1I). By P22, large et al., 1987), were immunopositive for netrin 1 (Fig. 2A). This numbers of mature myelinating oligodendrocytes expressing netrin staining was present on cells that were not permeabilized, 1 were readily detectable throughout the nascent white matter of the consistent with netrin 1 protein associated with the extracellular spinal cord (Fig. 1J,K). These findings indicate that netrin 1 begins face of the plasma membrane. To verify that the cells were not to be expressed by oligodendrocytes during early stages of permeabilized, the absence of Mbp staining was used as a myelination. negative control (not shown). We conclude that in the absence of neuronal contact in vitro, oligodendrocytes express netrin 1, and Oligodendroglial expression of netrin 1 and Dcc that netrin 1 protein is associated with the surface of myelin-like does not require neuronal contact membrane sheets. The timing of netrin 1 expression by oligodendrocytes suggested The netrin 1 receptor Dcc was detected along the processes of that expression might be regulated by axonal signals. To test this, immature and mature oligodendrocytes. In mature oligodendrocytes, purified rat oligodendrocyte progenitors were cultured in the Dcc was associated with major branches and present in small puncta absence of neurons in conditions that promote differentiation. at the leading edge of myelin-like membrane sheets (Fig. 2B). Immature oligodendrocytes in these cultures [4 days in vitro Addition of recombinant Myc-tagged netrin 1 to mature (DIV)] were defined as multipolar cells expressing Cnp and are oligodendrocytes revealed a preferential localization of exogenous the equivalent of the premyelinating cells in vivo. These cells did ligand at the branches and edges of sheets formed by the cells, Ј Ј not express netrin 1. More mature oligodendrocytes, cultured for similar to the distribution of Dcc (Fig. 2A ,B ). DEVELOPMENT 418 RESEARCH ARTICLE Development 136 (3)

the apparent influence of netrin 1 on oligodendrocyte differentiation in vivo, we determined whether the putative aberrant process extension detected in Ntn1–/– and Dcc–/– mice could be replicated in vitro in the absence of axonal influences or migration deficits.

Netrin 1 does not affect the differentiation of immature or mature oligodendrocytes in vitro Distinct changes in oligodendrocyte morphology accompany the differentiation of oligodendrocytes in vitro. Immature oligodendrocytes are characteristically multipolar cells that express Cnp but not Mbp (Fig. 4A,B). These cells differentiate into mature oligodendrocytes that elaborate myelin-like membrane sheets and express both Mbp and Cnp (Fig. 4A,D), and eventually myelin- associated glycoprotein (Mag). Immature cells (4-5 DIV) grown in the presence of netrin 1 (100 ng/ml) for 24 hours showed a modest (4%) decrease in the ratio of Cnp- to Mbp-positive cells. In more- mature Mbp/Mag-positive cultures (6-8 DIV), the ratio of Mbp- positive cells to Mag-positive cells was not affected by the addition of netrin 1 (Fig. 4A). We conclude that the addition of netrin 1 in vitro does not alter the acquisition of a mature phenotype, as Fig. 2. Expression of netrin 1 and Dcc by oligodendrocytes in assessed by the expression of standard markers. vitro. (A,AЈ) Netrin 1 protein (green) on the surface of myelin-like membrane sheets of non-permeabilized mature rat oligodendrocytes in culture. Recombinant netrin 1, labeled using a Myc epitope tag, Netrin 1 induces Dcc-dependent process extension preferentially localizes to branches formed by the cells (arrow). by immature oligodendrocytes in vitro (B,BЈ) Dcc (green) distributed along oligodendrocyte processes and at The effect of netrin 1 on process extension, as assayed by measuring puncta along the edge of Mbp-immunopositive (red) myelin-like the length of the longest process, was investigated in Cnp- membrane sheets (arrow). A,B, 40ϫ0.5 n.a. objective. Scale bars: immunopositive immature oligodendrocytes in culture (Fig. 4B). 40 μm in AЈ; 20 μm in BЈ. Application of 100 ng/ml netrin 1 for 24 hours to immature oligodendrocytes increased process length compared with control cells (Fig. 4C). To determine whether Dcc is required for netrin-1- induced extension of oligodendrocyte processes in vitro, a Dcc Netrin 1 and Dcc promote process extension by function-blocking antibody (Dccfb) was added for 24 hours to premyelinating oligodendrocytes in vivo immature oligodendrocytes treated with netrin 1. Consistent with the In the developing spinal cord, as premyelinating oligodendrocytes findings obtained in vivo and described above (Fig. 3F), disruption mature into myelinating oligodendrocytes, netrin 1 is widely of Dcc function blocked the netrin-1-induced increase in process expressed by neurons and neuroepithelial cells, but not by length, but did not significantly alter process extension when added oligodendrocytes (Fig. 1D,I; Fig. 3C). Mice lacking either netrin 1 alone (Fig. 4C). We conclude that the application of netrin 1 or Dcc die within hours of birth (Fazeli et al., 1997; Serafini et al., promotes Dcc-dependent oligodendrocyte process extension, and 1996). We therefore used E18 littermates to compare the that these changes occur independently of defects in migration and morphology of postmigratory, premyelinating oligodendrocytes in axon growth. tissue sections from the spinal cords of wild-type and Ntn1 and Dcc heterozygotes and knockout mouse embryos (Fig. 3A-C). At E18, Netrin 1 promotes Dcc-dependent increases in Cnp-immunoreactive premyelinating oligodendrocytes in the oligodendrocyte branching and myelin-like sheet dorsolateral spinal cord typically extend one or more processes (Fig. formation 3D, dashed lines). We found the length of these processes in both To investigate roles for netrin 1 during later stages of Ntn1–/– and Dcc –/– mice to be significantly shorter than in wild-type oligodendrocyte development, we characterized changes in littermates (Fig. 3E,F). These findings provide evidence that at the oligodendrocyte process branching and in the capacity of these cells end of precursor migration and at the initiation of oligodendrocyte to elaborate myelin-like membrane sheets in vitro. Mature differentiation, netrin 1 in the local environment of a postmigratory, oligodendrocytes cultured for 6-8 DIV were double labeled with RIP premyelinating oligodendrocyte promotes Dcc-dependent process antibody (against Cnp) to identify major processes, and Mbp extension in vivo, a phenomenon closely associated with the antibody to visualize extended myelin-like membrane sheets (Fig. capacity of an oligodendrocyte to contact target axons (Hardy and 4D, left). Oligodendrocytes grown in the presence of 100 ng/ml Friedrich, 1996; Kirby et al., 2006). netrin 1 for 24 hours exhibited a significant increase in the area of Interpretation of these findings could be confounded by two Mbp-positive sheets (Fig. 4E). The morphological complexity of factors. First, a subset of neurons in the spinal cords of Ntn1- and oligodendrocyte processes was quantified using Sholl analysis Dcc-null mice exhibits defects in axon guidance. However, this is (Ricard et al., 2001) (Fig. 4D). Cells treated with 100 ng/ml netrin 1 unlikely to exert a profound influence on the differentiation of for 24 hours exhibited a significant increase in branching compared individual oligodendrocytes as the majority of axons extend with the control (Fig. 4F,G). A dose-response analysis determined normally in the absence of netrin 1 function and the nascent white 100 ng/ml netrin 1 to be optimal (Fig. 4F). matter is well populated with axons. Second, a developmental delay As described above, immunocytochemical analyses detected Dcc, in OPC dispersal in the Ntn1 and Dcc mutants might delay process but little, if any, netrin 1 associated with the branches of

extension. In order to directly address the mechanisms underlying oligodendrocyte processes. Both Nfb (netrin function-blocking DEVELOPMENT Regulation of oligodendrocyte maturation RESEARCH ARTICLE 419

Fig. 3. Netrin 1 and Dcc regulate process outgrowth by immature oligodendrocytes in vivo. (A-C) Transverse sections of brachial spinal cord from E18 Ntn1–/– mice stained with antibodies to Cnp (green) and Nfm (red) to label premyelinating oligodendrocytes and neuronal cell bodies and axons, respectively. (A) Arrows indicate Cnp-immunopositive premyelinating oligodendrocytes. (B) As premyelinating oligodendrocytes search for and initially contact axons, they extend one or two major processes (arrows). (C) Netrin 1 expression, determined by β-galactosidase (β-Gal) reporter expression in a transgenic Ntn1+/– mouse, shown at the E18 spinal cord floor plate (arrow). Premyelinating oligodendrocytes are not immunopositive for β-galactosidase, but are adjacent to cells expressing netrin 1. (D) Dashed lines illustrate the measurement of extending processes of premyelinating oligodendrocytes in Ntn1+/+ and Ntn1–/– embryos. (E,F) Oligodendrocytes in the spinal cords of Ntn1–/– or Dcc–/– mice exhibit significantly shorter processes than wild-type or heterozygous littermates. *P<0.05, ***P<0.0001 versus control. The number of cells analyzed in each condition is indicated in parentheses. Error bars indicate s.e.m. A, 20ϫ0.5 n.a. objective; B-D, 40ϫ0.75 n.a. objective. Scale bars: 40 μm in A; 20 μm in B-D. antibody) and Dccfb antibodies blocked the increase in myelin-like lacking either Dcc or netrin 1 (Fig. 5B,C). We conclude that netrin membrane sheet formation and process branching induced by the 1 does not exert an autocrine effect on branching, and hypothesize addition of exogenous netrin 1 (Fig. 4E,G), indicating that the netrin- that this is a consequence of netrin 1 being sequestered to the 1-induced changes in oligodendrocyte morphology are Dcc- myelin-like membrane sheets (Fig. 2A), thereby exposing the dependent. Substantial netrin 1, but not Dcc, immunoreactivity was leading edges of processes to little, if any, endogenous netrin 1. detected in association with the myelin-like membrane sheets By contrast, oligodendrocytes lacking either netrin 1 or Dcc (Fig. 2A); however, function-blocking antibodies applied in the exhibited a significantly reduced surface area of the myelin-like absence of netrin 1 did not alter sheet formation or process membrane sheets compared with cells derived from wild-type or branching (Fig. 4E,G). We therefore tested the hypotheses that heterozygote littermates (Fig. 5D,E), thereby identifying an netrin 1 made by oligodendrocytes does not exert an autocrine autocrine role for netrin 1 in sheet formation. This is consistent with influence on the formation of myelin-like membrane sheets, and, our demonstration that the addition of exogenous netrin 1 increases alternatively, that the relatively short-term (24 hours) loss-of- myelin-like membrane sheet formation through a Dcc-dependent function assays described above are too brief to reveal a role for mechanism (Fig. 4E). These findings reveal differences between the endogenous netrin 1. response of oligodendrocytes to netrin 1 made by the cells themselves, which regulates myelin-like membrane sheet formation, Autocrine netrin 1 regulates the formation of and netrin 1 encountered in the local environment, which primarily myelin-like membrane sheets, but not process influences process branching. branching, in a Dcc-dependent manner To investigate an autocrine role for netrin 1, oligodendrocytes were Process elaboration induced by netrin 1 does not isolated from mixed glial cultures derived from P0 Ntn1–/– or Dcc–/– require β1 subunit-containing integrins mice (Fig. 5A). Application of netrin 1 for 24 hours to mature Oligodendrocytes express αvβ1, αvβ3, αvβ5 and α6β1 integrins. oligodendrocytes lacking Dcc did not increase process branching Engagement of integrins, specifically α6β1, by (Fig. 5B), indicating that oligodendrocyte processes require Dcc to (ECM) components activates SFKs to regulate changes in respond to exogenous netrin 1. Furthermore, in agreement with the oligodendrocyte morphology (Baron et al., 2005). Netrins are function-blocking antibody data described above (Fig. 4G), baseline members of the laminin family of ECM proteins, and netrin 1 has α β α β levels of process branching were not altered in oligodendrocytes been proposed to function as a ligand for 3 1 and 6 4 integrins DEVELOPMENT 420 RESEARCH ARTICLE Development 136 (3) in pancreatic cells (Yebra et al., 2003). We therefore investigated Application of a β1 integrin function-blocking antibody, whether netrin-1-mediated changes in the elaboration of which has been demonstrated to block -induced oligodendrocyte processes involve an interaction between netrin 1 changes in oligodendrocyte morphology (Liang et al., 2004), and integrins. did not significantly alter the netrin-1-induced increase in

Fig. 4. Netrin 1 induces Dcc-dependent process extension by immature oligodendrocytes, and Dcc-dependent process branching and myelin-like membrane sheet extension by mature oligodendrocytes, in vitro. (A) Addition of netrin 1 (100 ng/ml, 24 hours) to cultured rat oligodendrocytes results in a 4% decrease in the ratio of immature cells expressing Cnp only (arrowheads) as compared with more mature cells expressing Cnp and Mbp. Netrin 1 did not change the ratio of cells expressing Mbp only versus Mbp and Mag. (B) Cnp-positive immature oligodendrocytes are multipolar cells with one major process. Dashed red lines indicate examples of the processes measured. (C) The length of the major process increased following the addition of netrin 1 (100 ng/ml) for 24 hours. Application of Dcc function-blocking antibody (Dccfb) to immature oligodendrocytes blocks the netrin-1-induced increase in process extension, whereas Dccfb alone does not have a significant effect on process extension. (D) A mature Mbp- and RIP-positive oligodendrocyte extending both myelin-like membrane sheets and branched processes. Sheet area was quantified by tracing the Mbp-positive myelin-like membrane sheets (left, red outlines). Branching was quantified by measuring the number of intersections that processes made with concentric circles (right, red), which are numbered 1-5 to reflect increasing distance from the cell body (and as labeled on the x-axis of bar charts displaying branching complexity). (E) Netrin 1 (100 ng/ml, 24 hours) increased the area of Mbp- positive sheets compared with the control. This effect was blocked by Dccfb. (F) Processes of mature oligodendrocytes exposed to netrin 1 for 24 hours exhibited an increase in branching. Dose-response analysis indicated maximal branching at 100 ng/ml netrin 1. (G) Addition of Dcc function- blocking antibody together with netrin 1 prevented the netrin-1-dependent increase in branching, and decreased branching compared with controls. A, 20ϫ0.5 n.a. objective; B,D,F, 40ϫ0.75 n.a. objective. Scale bars: 40 μm in A; 20 μm in B,D,F. A.U., arbitrary units. *P<0.05, **P<0.005,

versus control. Error bars indicate s.e.m. DEVELOPMENT Regulation of oligodendrocyte maturation RESEARCH ARTICLE 421

Fig. 5. Oligodendrocytes from mice lacking netrin 1 or Dcc exhibit defects in process branching and in myelin-like membrane sheet formation. (A) Representative examples of the morphology of oligodendrocytes derived from wild- type and netrin-1-deficient or Dcc-deficient mice. 40ϫ0.75 n.a. objective. Scale bar: 20 μm. (B) Application of netrin 1 increased branching by oligodendrocytes from Dcc+/+ and Dcc+/–, but not from Dcc–/–, mice. (C) Oligodendrocytes from Dcc–/– or Ntn1–/– mice exhibit no difference in branching complexity compared with cells from wild-type and heterozygous littermates. (D) Ntn1–/– oligodendrocytes exhibit a significant decrease in myelin-like membrane sheet area compared with cells derived from wild-type and heterozygote littermates. (E) Dcc–/– oligodendrocytes elaborate smaller myelin-like membrane sheets than wild-type and heterozygous littermates. A.U., arbitrary units. ***P<0.0001 versus control. Error bars indicate s.e.m.

oligodendrocyte process branching (Fig. 6A). By contrast, this Netrin 1 binding to Dcc recruits Fyn to a complex antibody blocked HEK293T cell spreading on a fibronectin containing FAK substrate (Fig. 6B), verifying its efficacy. Yebra and colleagues We next assayed signaling proteins implicated as regulators of identified a 25 amino acid region within the C-terminus of netrin cytoskeletal organization downstream of netrin 1 and Dcc in axonal 1 that binds α6β4 and α3β1 integrins, and hypothesized that growth cones to determine whether similar signaling complexes potential interactions between netrin 1 and other integrins might might be engaged in oligodendrocytes. Oligodendrocytes express also occur through this region (Yebra et al., 2003). To determine the SFKs Src, Fyn and Lyn (Colognato et al., 2004). Of these, only whether such an interaction might contribute to netrin-1-induced mice lacking Fyn show defects in myelination (Sperber et al., 2001). changes in oligodendroglial morphology, we incubated cells with Furthermore, Fyn activation has been implicated in the regulation of a peptide comprising the putative integrin-binding sequence that process branching during the morphological maturation of functions as a competitive inhibitor of integrins binding netrin 1 oligodendrocytes (Osterhout et al., 1999; Umemori et al., 1994). In (Yebra et al., 2003). The netrin 1 peptide (20 μg/ml, a gift from axonal growth cones, application of netrin 1 recruits Fyn to the Dcc Dr V. Cirulli, UCSD, CA, USA) did not affect process complexity intracellular domain, where it is activated by focal adhesion kinase (Fig. 6A), nor did it disrupt the netrin-1-dependent increase in (FAK; Ptk2 – Mouse Genome Informatics) (Liu et al., 2004). To branching (Fig. 6A). We conclude that the novel role for netrin 1 determine whether Fyn might function downstream of Dcc in in regulating oligodendrocyte morphology occurs through Dcc oligodendrocytes, cells derived from newborn (P0) rats were independently of β1-containing integrins and the integrin-binding allowed to mature in culture for 3-4 days or for 5-6 days until they

region at the C-terminus of netrin 1. expressed Mbp. Co-immunoprecipitation studies carried out using DEVELOPMENT 422 RESEARCH ARTICLE Development 136 (3)

Fig. 6. Netrin 1 regulates oligodendrocyte morphology independently of integrins. (A) Application of β1 integrin function-blocking antibody (anti-β1, 2 μM) or a peptide encoding the netrin 1 integrin-binding domain (peptide, 20 μg/ml) failed to block the increase in oligodendrocyte process branching induced by netrin 1 (100 ng/ml). (B)The β1 integrin function-blocking antibody blocks the fibronectin-induced increase in the area (cell spreading) of HEK293T cells, whereas an anti-IgM control antibody does not. A.U., arbitrary units. *P<0.05, ***P<0.0001, versus control. Error bars indicate s.e.m. two different antibodies against Fyn revealed an interaction between branches (Fig. 8C,D). Colocalization with Dcc was observed, Fyn and Dcc in both immature (4 DIV) and mature (6 DIV) consistent with our immunoprecipitation results (Fig. 7B). To oligodendrocytes (Fig. 7A). Five minutes following application of determine whether changes in SFK phosphorylation occurred upon netrin 1, an increased amount of Fyn co-immunoprecipitated with netrin 1 stimulation, the relative number of SFK phospho-Y416- Dcc (Fig. 7A,D). A relatively minor Fyn-immunoreactive band of immunoreactive puncta was measured per unit area of the cell (Fig. slightly higher molecular weight was consistently detected 8E). Treatment of mature oligodendrocytes with netrin 1 (100 following immunoprecipitation and might reflect Fyn ng/ml) for 24 hours significantly increased the number of SFK phosphorylation as a result of activation by netrin 1. Our findings phospho-Y416-positive puncta per unit area, consistent with an also revealed a constitutive interaction between Dcc and FAK (Fig. association between netrin 1 stimulation and increased SFK activity 7A), suggesting that application of netrin 1 to oligodendrocytes (Fig. 8F). Treatment with the SFK inhibitor PP2 (2 μM) (Hanke et recruits Fyn into a complex with FAK that is bound to the al., 1996) blocked the netrin-1-induced increase in SFK phospho- intracellular domain of Dcc, as has been reported for neurons (Li et Y416-positive puncta (Fig. 8F), whereas the inactive SFK inhibitor al., 2004; Ren et al., 2004). Application of netrin 1 led to increased analog PP3 (2 μM) had no significant effect on the number of puncta phosphorylation of SFK tyrosine 416 (Y416), an event associated per unit area (Fig. 8F). Application of PP2 alone led to a decrease in with kinase activation (Smart et al., 1981), in the SFK associated the relative number of puncta per unit area, consistent with with Dcc (Fig. 7B,D). Analysis of SFK phosphorylation in whole- constitutive SFK activity contributing to the basal level of puncta cell lysates did not detect a global change in phospho-Y416, observed (Fig. 8F). To exclude the possibility that the increase in indicating that the netrin-1-induced change is specific to SFK SFK phospho-Y416-positive puncta was secondary to increased recruited into a complex with Dcc (Fig. 7C). Using an antibody branch formation, the same quantification was performed with cells specific for the SFK Src, an interaction with Dcc was not detected immunostained for Fyn. No difference was found in the number of (not shown). This provides evidence for specific recruitment of Fyn Fyn-immunopositive puncta per unit area in control and netrin-1- to Dcc; however, we do not rule out that other SFKs might be treated cells (Fig. 8G), consistent with the increase in SFK phospho- involved in netrin 1 signaling in oligodendrocytes. Y416-positive puncta resulting from an increase in SFK activity and not a netrin-1-induced increase in the number of branches. The netrin-1-induced increase in oligodendrocyte Our findings indicate that netrin 1 recruits Fyn to a complex with branching requires Fyn Dcc, increasing SFK activity in oligodendrocytes. We then tested the To determine whether Fyn is required downstream of netrin 1 in hypothesis that SFK activation is required for the morphological oligodendrocytes, we isolated cells from mice lacking Fyn changes induced by netrin 1. SFK activity was assessed in both [Fyntm1Sor, Fyn knockout (KO)] and from wild types of a matched immature (4 DIV, Cnp-positive) and mature (6 DIV, Mbp-positive) genetic background (B6129SF2/J, F2 hybrid) (Fig. 8A). Treatment oligodendrocytes. Treatment of oligodendrocytes with PP2 blocked of Fyn KO cells with netrin 1 did not result in increased branching, netrin-1-induced process extension in Cnp-immunoreactive in contrast to its effect upon cells isolated from control mice (Fig. immature cells and the netrin-1-dependent increase in branching in 8A,B). We conclude that Fyn is essential for these changes to occur. Mbp-expressing mature oligodendrocytes, whereas the inactive We therefore determined whether activation of SFKs is required to analog PP3 had no effect (Fig. 8H,I). Immature oligodendrocytes promote the morphological changes induced by netrin 1. treated with PP2 alone showed a small but significant decrease in process length (Fig. 8H), which was not seen with PP3 treatment. SFK activity is required for the netrin-1-induced Mature oligodendrocytes appeared less sensitive to the inhibition of increase in oligodendrocyte process length and basal SFK activity than immature cells, as PP2 alone did not affect branching branching (Fig. 8I). We conclude that netrin 1 binding to Dcc results Immunocytochemistry revealed SFK phospho-Y416 in the recruitment of the SFK Fyn, and that subsequent activation of immunoreactivity distributed within the oligodendrocyte cell body Fyn is required for the netrin-1-induced changes in oligodendrocyte

and proximal branches, and punctate staining within the distal morphology. DEVELOPMENT Regulation of oligodendrocyte maturation RESEARCH ARTICLE 423

Fig. 7. Netrin 1 binding to Dcc recruits Fyn. (A) Increased interaction between Dcc and Fyn is detected upon addition of exogenous netrin 1 (200 ng/ml, 5 minutes) to cultures of rat oligodendrocytes. This interaction was detected in both immature (4 DIV) and mature (6 DIV) oligodendrocytes and was observed when co-immunoprecipitations (IP) were performed with antibodies against Dcc or Fyn. A constitutive interaction between FAK and Dcc was also detected. IgG indicates immunoprecipitation with species-matched non-immune control IgG. (B) Increased active Src family kinase [phospho (p) SFK (pY416)] associated with Dcc in oligodendrocytes following application of netrin 1 (200 ng/ml, 5 minutes). (C) No detectable change in SFK activity in whole-cell lysates of cultured oligodendrocytes treated with netrin 1 (200 ng/ml) for either 5 minutes or 24 hours. (D) Quantification of the interaction between Fyn and Dcc, and between active SFK and Dcc. *P<0.05 versus control. Error bars indicate s.e.m.

Netrin 1 inhibits RhoA but does not affect Cdc42 netrin 1, a significant decrease in RhoA-GTP was detected or Rac1 in oligodendrocytes compared with controls (Fig. 9A). We conclude that netrin-1- We next asked what signals might act downstream of SFKs to trigger induced inhibition of RhoA, in the absence of altered Rac1 or Cdc42 the morphological changes induced by netrin 1 in oligodendrocytes. activity, promotes process elaboration by oligodendrocytes. Members of the Rho family of small GTPases, including RhoA, Rac1 and Cdc42, regulate the elaboration and branching of DISCUSSION oligodendrocyte processes (Liang et al., 2004). The effect of netrin Developing oligodendrocytes extend and retract their processes over 1 on Cdc42 and Rac1 activity was investigated using a GST-Pak- substantial distances, sampling the local environment to locate CRIB pulldown assay (Sander et al., 1998). Cell lysates of cultured unmyelinated axons (Hardy and Friedrich, 1996; Kirby et al., 2006). mature oligodendrocytes were incubated with GST-Pak-CRIB As the cell matures, these processes branch and eventually form fusion protein to quantify the levels of GTP-bound Cdc42 and Rac1. lamellae that ensheath target axons. Our findings indicate that netrin No significant change in the levels of GTP-bound Rac1 or Cdc42 1 and Dcc regulate oligodendrocyte process extension, branching was detected in oligodendrocytes following application of netrin 1 and myelin-like membrane sheet formation, which are all essential (Fig. 9B,C). We have reported that the Cdc42 effector protein N- events for initiating myelination. WASP is recruited into a protein complex with the intracellular domain of Dcc following the addition of netrin 1 to embryonic spinal Exogenous and autocrine netrin 1 contribute to commissural neurons (Shekarabi et al., 2005). Unlike in oligodendrocyte maturation oligodendrocytes, netrin 1 activates Cdc42 and Rac1 in commissural Our results support the conclusion that netrin 1, as expressed by neurons. In mature oligodendrocytes, N-WASP also constitutively neurons and neuroepithelial cells, when encountered by a co-immunoprecipitated with Dcc; however, addition of netrin 1 did premyelinating oligodendrocyte evokes Dcc-dependent extension not significantly alter the amount of N-WASP associated with Dcc of the motile processes of the cell. In more mature cells, exogenous (Fig. 9D), which is consistent with netrin 1 not increasing the netrin 1 promotes Dcc-dependent process branching and myelin-like activation of Cdc42 in oligodendrocytes. membrane sheet formation. Based on our findings in vitro and in Inhibiting RhoA, or its downstream effector Rho kinase vivo, we hypothesize that by promoting the morphological (Rock1/2), in oligodendrocytes increases process extension (Liang maturation of oligodendrocytes, netrin 1 facilitates the search for et al., 2004; Miron et al., 2007; Wolf et al., 2001). Interestingly, appropriate axonal targets. Interestingly, although exogenous netrin activation of Fyn in oligodendrocytes leads to inactivation of RhoA 1 promotes changes in oligodendrocyte morphology, at later stages (Wolf et al., 2001), and we therefore investigated the possibility that of maturation oligodendrocytes themselves begin to express netrin netrin 1 might regulate RhoA activity in oligodendrocytes. Levels 1. We identify a selective, Dcc-dependent autocrine role for netrin 1 of GTP-bound RhoA in mature Mbp-positive oligodendrocytes were in promoting the formation of myelin-like membrane sheets. assessed using a GST-rhotekin pulldown assay (Reid et al., 1996). Oligodendrocytes only begin to express netrin 1 in the developing

Following a 5 minute treatment of mature oligodendrocytes with spinal cord after myelination has begun. Our findings indicate that DEVELOPMENT 424 RESEARCH ARTICLE Development 136 (3) contact with axons is not essential for the initiation of netrin 1 Common signaling mechanisms in the regulation expression by these cells, but that it occurs coincident with myelin- of oligodendrocyte processes and axonal growth like membrane sheet formation. We hypothesize that autocrine cones by netrin 1 expression of netrin 1 specifically promotes later stages of The extending tips of oligodendrocyte processes in some ways maturation, facilitating the formation of large myelin-like membrane resemble motile axonal growth cones (Fox et al., 2006; Jarjour sheets by these cells. Netrin 1 expression by oligodendrocytes might and Kennedy, 2004), and increasing evidence suggests that also facilitate axon remodeling or prevent aberrant axonal sprouting common regulators of actin nucleation function in neuronal at later stages of development. growth cones and at the leading edge of oligodendroglial

Fig. 8. SFK activity is required for netrin-1-induced changes in oligodendrocyte morphology. (A) Oligodendrocytes were obtained from mice lacking Fyn (Fyn KO) and treated with netrin 1 (100 ng/ml, 24 hours). Cells obtained from wild-type mice of a matched genetic background were used as controls. (B) Netrin 1 increased branching in control oligodendrocytes (F2 hybrid), but not in Fyn KO oligodendrocytes. (C,CЈ) Phospho- SFK [P-SFK (pY416); red] immunoreactivity colocalizes (yellow) with Dcc (green) within the oligodendrocyte cell body and proximal branches. The confocal section shown is adjacent to the substrate and the appearance of staining throughout the cell body is not indicative of staining in the nucleus. (D,DЈ) Portions of the images in C and CЈ have been magnified to illustrate punctate staining present within distal branches. Arrows indicate colocalized enrichment of phospho-SFK (red) and DCC (green) at puncta. (E) Binary images of phospho-SFK immunostaining were used to quantify the number of phospho-SFK-positive puncta. (F) The relative number of phospho-SFK-positive puncta increased in the presence of netrin 1 (100 ng/ml, 24 hour treatment). PP2 (2 μM) blocked the netrin-dependent increase in puncta, and PP2 alone showed a significant decrease in puncta compared with the control. PP3 (2 μM) did not block the effects of netrin 1 and had no independent effect on the cells. (G) The number of Fyn-positive puncta per unit area was not significantly (n.s.) changed upon addition of netrin 1. (H) The netrin-1-dependent increase in process length of Cnp-positive immature oligodendrocytes was blocked by the addition of PP2 (2 μM). Addition of PP3 did not disrupt the effect of netrin 1 on the cells. Oligodendrocytes treated with PP2 alone (2 μM), but not PP3 alone, exhibit a small but significant decrease in process extension compared with the control. (I) Disruption of SFK activity in mature Mbp-positive oligodendrocytes prevented the increased branching observed in the presence of netrin 1. PP2 and PP3 did not independently affect oligodendrocyte morphology. *P<0.05, **P<0.005, ***P<0.001, versus control. ϫ Ј ϫ μ μ Ј Ј Error bars indicate s.e.m. A,E, 40 0.75 n.a. objective; C-D , confocal microscopy, 40 0.75 n.a. objective. Scale bars: 20 m in A,E; 40 m in C ,D . DEVELOPMENT Regulation of oligodendrocyte maturation RESEARCH ARTICLE 425

Netrin 1 activates a canonical signaling mechanism required for oligodendrocyte maturation To date, the candidate extracellular signals that might regulate the elaboration of oligodendroglial processes immediately preceding myelination have been limited to ECM proteins, of which laminin 2 and its receptor integrin α6β1 have been well characterized. Studies in vitro have shown that integrin-dependent activation of Fyn activates Cdc42 and Rac1 and deactivates RhoA, leading to process outgrowth (Liang et al., 2004; Osterhout et al., 1999). However, oligodendrocytes lacking the β1 integrin subunit mature and myelinate normally, demonstrating that this pathway is not essential in vivo (Benninger et al., 2006). Furthermore, laminin 2 is not ubiquitously present in myelinating axon tracts in the CNS, indicating that other ligand-receptor complexes must trigger these signaling mechanisms independently of β1 integrin function. Our results show that netrin 1, acting through Dcc, activates an intracellular signaling pathway that is required for the morphological maturation of oligodendrocytes. Crucially, the effects of netrin 1 do not require β1 integrin function and do not appear to act through netrin-binding integrins. Our findings identify a novel mechanism regulating oligodendrocyte morphology during later stages of differentiation. Interestingly, many oligodendroglial cells detected in multiple sclerosis lesions appear to have differentiated, but remain unable to Fig. 9. Treatment of mature oligodendrocytes with netrin 1 elaborate myelin (Chang et al., 2002). A better understanding of inhibits RhoA activity (A) Levels of GTP-bound RhoA in rat the mechanisms that promote myelination will advance the oligodendrocytes were assessed using GST-rhotekin pulldown. development of therapeutics that aim to promote the recovery of Treatment of mature oligodendrocytes with netrin 1 (100 ng/ml, 5 nervous system function. minutes) caused a significant decrease in RhoA-GTP compared with controls. (B,C) Similar treatment of cells resulted in no significant We thank Simon Moore, Sarah-Jane Bull and Adriana Di Polo for comments on change in the levels of GTP-bound Cdc42 or Rac1, as assessed by GST- the manuscript. S.R. and A.A.J. were supported by Multiple Sclerosis (MS) Pak-CRIB pulldown. (D) Addition of netrin 1 to mature oligodendrocytes Society of Canada studentships and a Canadian Institutes of Health Research (CIHR) Neuroinflammation Strategic Training Program award, K.A.B. by a (100 ng/ml, 5 minutes) did not alter the amount of N-WASP recruited Fonds de la Recherche en Santé du Québec (FRSQ) postdoctoral fellowship, to Dcc. *P<0.05 versus control. Error bars indicate s.e.m. and T.E.K. holds a FRSQ Chercheur Nationaux Award and is a Killam Foundation Scholar. The project was supported by grants (to T.E.K.) from the MS Society of Canada and the CIHR. processes and sheets (Sloane and Vartanian, 2007). Here, we References report that the similarities between growing axons and Armstrong, R. C. (1998). Isolation and characterization of immature oligodendrocyte lineage cells. Methods 16, 282-292. oligodendrocyte processes include the recruitment of similar Bacon, C., Lakics, V., Machesky, L. and Rumsby, M. (2007). N-WASP regulates intracellular signaling proteins downstream of netrin 1: Fyn, FAK, extension of filopodia and processes by oligodendrocyte progenitors, N-WASP and Rho GTPases (Li et al., 2004; Liu et al., 2004; Ren oligodendrocytes, and Schwann cells-implications for axon ensheathment at et al., 2004; Shekarabi and Kennedy, 2002). We demonstrate that myelination. 55, 844-858. Baron, W., Colognato, H. and ffrench-Constant, C. (2005). Integrin-growth Fyn is required for the netrin-1-dependent increase in factor interactions as regulators of oligodendroglial development and function. oligodendrocyte process branching. Netrin 1 recruits Fyn to a Glia 49, 467-479. complex that includes Dcc and FAK, resulting in SFK Benninger, Y., Colognato, H., Thurnherr, T., Franklin, R. J., Leone, D. P., Atanasoski, S., Nave, K. A., Ffrench-Constant, C., Suter, U. and Relvas, J. phosphorylation and activation. Importantly, previous studies B. (2006). Beta1-integrin signaling mediates premyelinating oligodendrocyte have implicated Fyn, Rac1, Cdc42, RhoA and N-WASP in the survival but is not required for CNS myelination and remyelination. J. Neurosci. mechanism governing the cytoskeletal changes in 26, 7665-7673. oligodendrocytes that lead to myelination (Simons and Trotter, Chang, A., Tourtellotte, W. W., Rudick, R. and Trapp, B. D. (2002). Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. N. Engl. 2007). J. Med. 346, 165-173. Activating Fyn results in RhoA inactivation in Chun, S. J., Rasband, M. N., Sidman, R. L., Habib, A. A. and Vartanian, T. oligodendrocytes (Wolf et al., 2001). We demonstrate reduced (2003). Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination. J. Cell Biol. 163, 397- levels of GTP-bound RhoA upon netrin 1 stimulation, but found 408. no evidence for the activation of Cdc42 or Rac1. Interestingly, Colognato, H., Baron, W., Avellana-Adalid, V., Relvas, J. B., Baron-Van transgenic mice specifically lacking Cdc42 and Rac1 in Evercooren, A., Georges-Labouesse, E. and ffrench-Constant, C. (2002). CNS integrins switch signalling to promote target-dependent oligodendrocytes exhibit normal oligodendroglial differentiation, survival. Nat. Cell Biol. 4, 833-841. but show eventual defects in myelin compaction (Thurnherr et al., Colognato, H., Ramachandrappa, S., Olsen, I. M. and ffrench-Constant, C. 2006). Our findings support the hypothesis that in a background (2004). Integrins direct Src family kinases to regulate distinct phases of of unchanging Rac1 and Cdc42 activity, modulation of RhoA oligodendrocyte development. J. Cell Biol. 167, 365-375. Davidson, D., Chow, L. M., Fournel, M. and Veillette, A. (1992). Differential function plays a key role in regulating the morphological regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine

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