RESEARCH ARTICLE
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Development 136, 415-426 (2009) doi:10.1242/dev.018234
Netrin 1 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 axons, a key step towards myelination.
INTRODUCTION
To form a myelin sheath, an oligodendrocyte initially extends
multiple branching processes that survey the local environment for
suitable axons (Hardy and Friedrich, 1996; Kirby et al., 2006). Upon
contact with a target axon, oligodendrocyte processes coalesce to
form a spreading sheet of membrane that begins to wrap the axon.
The extracellular cues that govern this process and the intracellular
mechanisms involved are poorly understood.
Laminin 2, which promotes oligodendrocyte maturation by
signaling through α6β1 integrins (Baron et al., 2005), has been
suggested to regulate process elaboration. Laminin-2-deficient mice
have dysmyelinated and hypomyelinated axons (Chun et al., 2003).
However, laminin 2 is not ubiquitous in myelinated CNS axon tracts
(Colognato et al., 2002) and transgenic mice lacking β1 integrin
expression in oligodendrocytes exhibit no defects in CNS
myelination (Benninger et al., 2006). Thus, other ligand-receptor
interactions must also direct the changes in oligodendrocyte
morphology required for myelination.
The axon-guidance cue netrin 1 (Ntn1) is a chemorepellent for
migrating oligodendrocyte precursors (OPCs) in the embryonic
spinal cord (Jarjour et al., 2003; Tsai et al., 2003). These cells
express the netrin 1 receptors Dcc, Unc5a and Unc5b, but not netrin
1 itself. In the adult CNS, netrin 1 is expressed by myelinating
oligodendrocytes and is associated with non-compacted
oligodendroglial membranes (Manitt et al., 2001). We therefore
investigated the possibility that netrin 1, expressed in the developing
CNS and later by the oligodendrocytes themselves, might influence
late stages of oligodendrocyte differentiation.
The lamella elaborated by the tip of an extending oligodendrocyte
process has been compared to a neuronal growth cone (Fox et al.,
2006; Jarjour and Kennedy, 2004; Sloane and Vartanian, 2007).
Interestingly, a number of the same intracellular signaling proteins
have been implicated in netrin-1-mediated axon guidance and the
development of oligodendrocyte processes required for myelination
(Fox et al., 2006; Jarjour and Kennedy, 2004). In both cases, the
reorganization of the actin cytoskeleton requires activation of the Src
family kinase (SFK) Fyn (Meriane et al., 2004; Osterhout et al., 1999;
Umemori et al., 1994) and involves Wiscott-Aldrich syndrome protein
(N-WASP; Wasl – Mouse Genome Informatics) and Rho GTPases
(Bacon et al., 2007; Liang et al., 2004; Shekarabi et al., 2005).
Here we provide evidence that netrin 1 and Dcc promote the
extension of oligodendrocyte processes in vivo. We used in vitro
assays to demonstrate that netrin 1 increases oligodendrocyte
process branching. Furthermore, expression of Dcc and netrin 1 by
oligodendrocytes promotes the formation of myelin-like membrane
sheets. Addressing the signaling mechanisms involved, we show
that the SFK Fyn is required for netrin-1-induced process branching,
that netrin 1 recruits Fyn to Dcc in oligodendrocytes, increases SFK
activity, and promotes process extension and branching associated
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
University, Montreal, Quebec H3A 2B4, Canada.
MATERIALS AND METHODS
*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
were obtained from Marc Tessier-Lavigne (Genentech, South San Francisco,
Accepted 21 November 2008
Animals and oligodendrocyte cultures
DEVELOPMENT
KEY WORDS: Oligodendroglia, Myelination, Myelin, Netrin, Integrin, Laminin, Autocrine, Mouse, Rat, FAK (Ptk2), N-WASP (Wasl)
RESEARCH ARTICLE
CA, USA) and Robert Weinberg (Harvard University, Cambridge, MA,
USA), respectively. Fyn KO (Fyntm1Sor) and control F2 hybrid (B6129SF2/J)
breeding pairs were obtained from Jackson Laboratories (Bar Harbor, ME,
USA). All procedures were performed in accordance with the Canadian
Council on Animal Care guidelines for the use of animals in research.
Cell culture
OPCs were derived from mixed glial cultures from the cerebral cortices of
postnatal day 0 (P0) rat pups and grown in oligodendrocyte defined medium
(OLDEM) as described previously (Armstrong, 1998; Jarjour et al., 2003),
with 0.1% fetal bovine serum (FBS) to initiate differentiation. Cells were
seeded at 1.5⫻104 cells/chamber in 8-well chamber slides coated with
10 μg/ml poly-L-lysine (Nalge Nunc, Rochester, NY, USA). For
immunoprecipitation, western blots and GST-rhotekin pulldowns, cells were
plated at 1.5⫻106 cells/well in 6-well tissue culture dishes.
Mouse oligodendrocyte cultures
Culturing of mouse OPCs was similar to that of rat OPCs, but 10% horse
serum was used instead of 10% FBS in mixed glial culture media. Each T75
flask of mixed glial culture required two to three pups. Newborn Ntn1–/– and
Dcc–/– mice were identified by distinct behaviors. The genotype of
individual pups was confirmed by PCR.
Antibodies
Primary antibodies used in this study were: rabbit polyclonal anti-netrin 1
PN3 (Manitt et al., 2001), mouse monoclonal anti-Dcc (G97-449; BD
Biosciences Pharmingen, San Jose, CA, USA), goat polyclonal anti-Dcc
(Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit polyclonal anti-myelin
basic protein (Mbp, Chemicon, Temecula, CA, USA), mouse monoclonal
anti-Mbp (Chemicon), mouse monoclonal RIP antibody (Chemicon), mouse
monoclonal anti-2⬘,3⬘-cyclic nucleotide 3⬘ phosphodiesterase (Cnp,
Sternberger Monoclonals, Lutherville, MD, USA), rabbit polyclonal antiMag (Chemicon), rabbit polyclonal anti-Nfm (Nefm – Mouse Genome
Informatics) (Chemicon), rabbit polyclonal anti-Fyn (Upstate Cell
Signaling, Charlottesville, VA, USA) used for western blots, rabbit
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-NWASP (Santa Cruz), and rabbit polyclonal anti-phospho-Src (Cell
Signaling), which recognizes the pY416 epitope in all SFK members.
For analyses of oligodendrocyte morphology in vivo, we crossed Ntn1 or
Dcc heterozygous mice, obtained embryonic day 18 (E18) embryos (plug
date taken as E1), fixed them in 4% paraformaldehyde and cut 18 μm
sections of the spinal brachial enlargement with a cryostat. Sections were
then stained with anti-Cnp (1:250), anti-Nfm (1:250), anti-netrin PN3
(1:100), visualized using Alexa 546- or Alexa 488-conjugated secondary
antibodies (Jackson ImmunoResearch, West Grove, PA, USA), and nuclei
stained with Hoechst. Images were obtained with a Magnafire CCD camera
(Optronics, Goleta, CA, USA) and a Zeiss Axiovert 100 microscope
(Toronto, Ontario, Canada).
Analysis of oligodendrocyte morphology
Analyses of immature oligodendrocytes in vitro and in vivo were performed
using the NeuronJ plugin for ImageJ (NIH, Bethesda, MD, USA). The
length of the longest process was measured from the base of the process to
its tip (Fig. 3D; Fig. 4B). For Sholl analysis, the grid function in Northern
Eclipse (Empix) was used to draw concentric circles 15 μm apart around the
cell body of mature RIP-positive oligodendrocytes. The number of
intersections made by processes with each successive circle was counted.
For studies using the β1 integrin subunit function-blocking antibody and
mouse oligodendrocyte cultures, a Sholl analysis plugin was used with
ImageJ (starting radius, 1.02 cm; step size, 1.02 cm; end, 5.08 cm; thickness,
0.02 cm). The Mbp-positive myelin-like membrane sheets were outlined in
ImageJ and the surface area reported in arbitrary units.
The pharmacological inhibitors PP2 and PP3 (Calbiochem) were used at
2 μM to inhibit SFK activity. Purified hamster anti-rat CD29 (β1 integrin)
monoclonal and purified hamster anti-IgM monoclonal antibodies were used
at 2 μM to investigate β1 integrin function.
Development 136 (3)
Analysis of phospho-Src puncta
The number of phospho-Src-positive puncta was measured using ImageJ.
The brightness and contrast of the images of individual oligodendrocytes
were modified to enhance puncta associated with extending processes;
modifications made were consistent across all images. Images were
converted to a binary format and then the number of particles counted
automatically. Staining in the cell body and major processes was not
punctate and thus counted as one particle, making variations under different
conditions a direct indicator of changes in the number of distally located
puncta. The number of puncta was divided by oligodendrocyte area to
control for variance in oligodendrocyte size.
Quantification and statistical analyses
Statistical significance was calculated by ANOVA followed by a post-hoc
Tukey test using Systat Software (San Jose, CA, USA). Analyses of
oligodendrocyte morphology in vitro used three independent experiments
with a minimum of 30 cells per condition. In vivo analysis of process
extension and purification of oligodendrocytes from transgenic mice were
performed using at least four pups of each genotype, derived from at least
two different litters.
Immunoprecipitation
Cells in 6-well dishes were treated with netrin 1 for 5 minutes, then lysed in
RIPA lysis buffer (10 mM sodium phosphate pH 7.2, 150 mM NaCl, 1%
NP40, 0.1% SDS, 0.5% deoxycholate) and centrifuged at 13,000 rpm
(13,800 g) for 7 minutes. Supernatant was pre-cleared with 30 μl protein
A/G beads (Santa Cruz) for 30 minutes, incubated with 1 μg/ml anti-Dcc
(monoclonal) or anti-Fyn (rabbit polyclonal) for 1 hour, followed by addition
of 30 μl protein A/G beads for 45 minutes.
GST pulldown assays
Fusion proteins comprising the RhoA-binding domain of rhotekin (a
downstream substrate of RhoA) or Pak-CRIB and glutathione-S-transferase
(GST) were purified as described (Reid et al., 1996). Oligodendrocytes were
treated with netrin 1 for 24 hours. Cells were then lysed and protein purified
as described (Ren and Schwartz, 2000; Shekarabi et al., 2005).
RESULTS
Oligodendrocytes express netrin 1 during
myelination in the developing spinal cord
Netrin 1, expressed by floor plate and neuroepithelial cells in the
early embryonic spinal cord, directs migrating OPCs away from the
ventral midline towards axons in the nascent white matter (Jarjour
et al., 2003; Kennedy et al., 1994; Tsai et al., 2006; Tsai et al., 2003).
Although not expressed by OPCs, netrin 1 is widely expressed by
spinal interneurons, motoneurons and by most, if not all, mature
myelinating oligodendrocytes in dorsal and ventral white matter in
the adult rat and mouse CNS (Manitt et al., 2001).
To determine when netrin 1 is expressed by differentiating
oligodendrocytes, we performed a time-course analysis.
Premyelinating, postmigratory oligodendrocytes within the
corticospinal tract at brachial, thoracic and lumbar levels were
examined. Netrin 1 expression was not detected in oligodendrocytes
at embryonic stages of development in mice (data not shown). For
postnatal stages, netrin-1-expressing cells were identified
immunohistochemically. Oligodendrocytes in the developing
dorsolateral white matter tracts were identified by expression of
2⬘,3⬘-cyclic nucleotide 3⬘ phosphodiesterase (Cnp), a marker that
labels oligodendrocyte cell bodies and processes in vivo, and by
expression of the mature oligodendrocyte marker myelin basic
protein (Mbp). At P8, before myelination begins in the corticospinal
tract, netrin 1 was not expressed by premyelinating oligodendrocytes
(Fig. 1A-C). Netrin-1-expressing neuronal cell bodies and neuralepithelial cells (Fig. 1D,E), but not astrocytes (Fig. 1I), were
detected immediately adjacent to differentiating oligodendrocytes.
DEVELOPMENT
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Regulation of oligodendrocyte maturation
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At P12, following the initiation of myelination in the rat
corticospinal tract (Schwab and Schnell, 1989), netrin 1 was
detected in the cell bodies of a subset of oligodendrocytes double
labeled with Cnp (Fig. 1F,G). At this stage of development, netrin 1
immunoreactivity was associated with axons (Fig. 1I). By P22, large
numbers of mature myelinating oligodendrocytes expressing netrin
1 were readily detectable throughout the nascent white matter of the
spinal cord (Fig. 1J,K). These findings indicate that netrin 1 begins
to be expressed by oligodendrocytes during early stages of
myelination.
Oligodendroglial expression of netrin 1 and Dcc
does not require neuronal contact
The timing of netrin 1 expression by oligodendrocytes suggested
that expression might be regulated by axonal signals. To test this,
purified rat oligodendrocyte progenitors were cultured in the
absence of neurons in conditions that promote differentiation.
Immature oligodendrocytes in these cultures [4 days in vitro
(DIV)] were defined as multipolar cells expressing Cnp and are
the equivalent of the premyelinating cells in vivo. These cells did
not express netrin 1. More mature oligodendrocytes, cultured for
6-8 DIV and identified by expression of Mbp and Cnp (Watanabe
et al., 2006), extend highly branched processes that coalesce to
form cytoplasmic sheets (Fox et al., 2006). These sheets, which
resemble unwrapped non-compacted myelin membrane (Knapp
et al., 1987), were immunopositive for netrin 1 (Fig. 2A). This
staining was present on cells that were not permeabilized,
consistent with netrin 1 protein associated with the extracellular
face of the plasma membrane. To verify that the cells were not
permeabilized, the absence of Mbp staining was used as a
negative control (not shown). We conclude that in the absence of
neuronal contact in vitro, oligodendrocytes express netrin 1, and
that netrin 1 protein is associated with the surface of myelin-like
membrane sheets.
The netrin 1 receptor Dcc was detected along the processes of
immature and mature oligodendrocytes. In mature oligodendrocytes,
Dcc was associated with major branches and present in small puncta
at the leading edge of myelin-like membrane sheets (Fig. 2B).
Addition of recombinant Myc-tagged netrin 1 to mature
oligodendrocytes revealed a preferential localization of exogenous
ligand at the branches and edges of sheets formed by the cells,
similar to the distribution of Dcc (Fig. 2A⬘,B⬘).
DEVELOPMENT
Fig. 1. Expression of netrin 1 by oligodendrocytes
in vivo. (A,A⬘) 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,B⬘) 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,C⬘) An Mbp-positive
netrin-1-negative process, surrounded by netrin-1expressing neuroepithelial cells (arrows) in P8 spinal
cord. (D,D⬘) Longitudinal section through a P8 rat
spinal cord showing axons positive for neurofilament
medium polypeptide (Nfm), surrounded by netrin-1expressing cells. (E,E⬘) Cell bodies of netrin-1immunopositive neurons. (F) Cross-section of a P12 rat
dorsal spinal cord showing Cnp-positive, myelinating
oligodendrocytes (red) expressing netrin 1 (green).
(G-G⬙) Oligodendrocytes adjacent to the central canal
(arrow) express netrin 1 in P12 spinal cord.
(H,H⬘) Netrin 1 immunoreactivity (green) associated
with a Cnp-immunopositive (red) myelinating
oligodendrocyte in the P12 rat spinal cord. (I-I⬙) By P12,
netrin 1 expression (green) colocalizes with Nfmimmunoreactive axons (blue), but is not detected in
Gfap-positive astrocytes (red). (J,J⬘) Transverse section
of a P22 thoracic spinal cord showing widespread
expression of netrin 1 (green) by Cnp-positive,
myelinating oligodendrocytes (red).
(K-K⬙) Magnification of the boxed region from J
showing Cnp-positive netrin-1-expressing cell bodies.
A,F, 20⫻0.5 n.a. objective; G,J, 40⫻0.75 n.a.
objective; D,E,I,K, confocal microscopy, 40⫻0.75 n.a.
objective; B,C,H, confocal microscopy, 100⫻1.4 n.a.
objective. Scale bars: 10 μm in B⬘,C⬘,H; 20 μm in
A⬘,D,E,G,K; 40 μm in F,I⬘,J.
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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 and Dcc promote process extension by
premyelinating oligodendrocytes in vivo
In the developing spinal cord, as premyelinating oligodendrocytes
mature into myelinating oligodendrocytes, netrin 1 is widely
expressed by neurons and neuroepithelial cells, but not by
oligodendrocytes (Fig. 1D,I; Fig. 3C). Mice lacking either netrin 1
or Dcc die within hours of birth (Fazeli et al., 1997; Serafini et al.,
1996). We therefore used E18 littermates to compare the
morphology of postmigratory, premyelinating oligodendrocytes in
tissue sections from the spinal cords of wild-type and Ntn1 and Dcc
heterozygotes and knockout mouse embryos (Fig. 3A-C). At E18,
Cnp-immunoreactive premyelinating oligodendrocytes in the
dorsolateral spinal cord typically extend one or more processes (Fig.
3D, dashed lines). We found the length of these processes in both
Ntn1–/– and Dcc –/– mice to be significantly shorter than in wild-type
littermates (Fig. 3E,F). These findings provide evidence that at the
end of precursor migration and at the initiation of oligodendrocyte
differentiation, netrin 1 in the local environment of a postmigratory,
premyelinating oligodendrocyte promotes Dcc-dependent process
extension in vivo, a phenomenon closely associated with the
capacity of an oligodendrocyte to contact target axons (Hardy and
Friedrich, 1996; Kirby et al., 2006).
Interpretation of these findings could be confounded by two
factors. First, a subset of neurons in the spinal cords of Ntn1- and
Dcc-null mice exhibits defects in axon guidance. However, this is
unlikely to exert a profound influence on the differentiation of
individual oligodendrocytes as the majority of axons extend
normally in the absence of netrin 1 function and the nascent white
matter is well populated with axons. Second, a developmental delay
in OPC dispersal in the Ntn1 and Dcc mutants might delay process
extension. In order to directly address the mechanisms underlying
Netrin 1 induces Dcc-dependent process extension
by immature oligodendrocytes in vitro
The effect of netrin 1 on process extension, as assayed by measuring
the length of the longest process, was investigated in Cnpimmunopositive immature oligodendrocytes in culture (Fig. 4B).
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-1induced extension of oligodendrocyte processes in vitro, a Dcc
function-blocking antibody (Dccfb) was added for 24 hours to
immature oligodendrocytes treated with netrin 1. Consistent with the
findings obtained in vivo and described above (Fig. 3F), disruption
of Dcc function blocked the netrin-1-induced increase in process
length, but did not significantly alter process extension when added
alone (Fig. 4C). We conclude that the application of netrin 1
promotes Dcc-dependent oligodendrocyte process extension, and
that these changes occur independently of defects in migration and
axon growth.
Netrin 1 promotes Dcc-dependent increases in
oligodendrocyte branching and myelin-like sheet
formation
To investigate roles for netrin 1 during later stages of
oligodendrocyte development, we characterized changes in
oligodendrocyte process branching and in the capacity of these cells
to elaborate myelin-like membrane sheets in vitro. Mature
oligodendrocytes cultured for 6-8 DIV were double labeled with RIP
antibody (against Cnp) to identify major processes, and Mbp
antibody to visualize extended myelin-like membrane sheets (Fig.
4D, left). Oligodendrocytes grown in the presence of 100 ng/ml
netrin 1 for 24 hours exhibited a significant increase in the area of
Mbp-positive sheets (Fig. 4E). The morphological complexity of
oligodendrocyte processes was quantified using Sholl analysis
(Ricard et al., 2001) (Fig. 4D). Cells treated with 100 ng/ml netrin 1
for 24 hours exhibited a significant increase in branching compared
with the control (Fig. 4F,G). A dose-response analysis determined
100 ng/ml netrin 1 to be optimal (Fig. 4F).
As described above, immunocytochemical analyses detected Dcc,
but little, if any, netrin 1 associated with the branches of
oligodendrocyte processes. Both Nfb (netrin function-blocking
DEVELOPMENT
Fig. 2. Expression of netrin 1 and Dcc by oligodendrocytes in
vitro. (A,A⬘) 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,
preferentially localizes to branches formed by the cells (arrow).
(B,B⬘) Dcc (green) distributed along oligodendrocyte processes and at
puncta along the edge of Mbp-immunopositive (red) myelin-like
membrane sheets (arrow). A,B, 40⫻0.5 n.a. objective. Scale bars:
40 μm in A⬘; 20 μm in B⬘.
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 myelinassociated 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 moremature Mbp/Mag-positive cultures (6-8 DIV), the ratio of Mbppositive 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
assessed by the expression of standard markers.
Regulation of oligodendrocyte maturation
RESEARCH ARTICLE
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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, 20⫻0.5 n.a. objective; B-D, 40⫻0.75 n.a. objective. Scale bars:
40 μm in A; 20 μm in B-D.
Autocrine netrin 1 regulates the formation of
myelin-like membrane sheets, but not process
branching, in a Dcc-dependent manner
To investigate an autocrine role for netrin 1, oligodendrocytes were
isolated from mixed glial cultures derived from P0 Ntn1–/– or Dcc–/–
mice (Fig. 5A). Application of netrin 1 for 24 hours to mature
oligodendrocytes lacking Dcc did not increase process branching
(Fig. 5B), indicating that oligodendrocyte processes require Dcc to
respond to exogenous netrin 1. Furthermore, in agreement with the
function-blocking antibody data described above (Fig. 4G), baseline
levels of process branching were not altered in oligodendrocytes
lacking either Dcc or netrin 1 (Fig. 5B,C). We conclude that netrin
1 does not exert an autocrine effect on branching, and hypothesize
that this is a consequence of netrin 1 being sequestered to the
myelin-like membrane sheets (Fig. 2A), thereby exposing the
leading edges of processes to little, if any, endogenous netrin 1.
By contrast, oligodendrocytes lacking either netrin 1 or Dcc
exhibited a significantly reduced surface area of the myelin-like
membrane sheets compared with cells derived from wild-type or
heterozygote littermates (Fig. 5D,E), thereby identifying an
autocrine role for netrin 1 in sheet formation. This is consistent with
our demonstration that the addition of exogenous netrin 1 increases
myelin-like membrane sheet formation through a Dcc-dependent
mechanism (Fig. 4E). These findings reveal differences between the
response of oligodendrocytes to netrin 1 made by the cells
themselves, which regulates myelin-like membrane sheet formation,
and netrin 1 encountered in the local environment, which primarily
influences process branching.
Process elaboration induced by netrin 1 does not
require β1 subunit-containing integrins
Oligodendrocytes express αvβ1, αvβ3, αvβ5 and α6β1 integrins.
Engagement of integrins, specifically α6β1, by extracellular matrix
(ECM) components activates SFKs to regulate changes in
oligodendrocyte morphology (Baron et al., 2005). Netrins are
members of the laminin family of ECM proteins, and netrin 1 has
been proposed to function as a ligand for α3β1 and α6β4 integrins
DEVELOPMENT
antibody) and Dccfb antibodies blocked the increase in myelin-like
membrane sheet formation and process branching induced by the
addition of exogenous netrin 1 (Fig. 4E,G), indicating that the netrin1-induced changes in oligodendrocyte morphology are Dccdependent. Substantial netrin 1, but not Dcc, immunoreactivity was
detected in association with the myelin-like membrane sheets
(Fig. 2A); however, function-blocking antibodies applied in the
absence of netrin 1 did not alter sheet formation or process
branching (Fig. 4E,G). We therefore tested the hypotheses that
netrin 1 made by oligodendrocytes does not exert an autocrine
influence on the formation of myelin-like membrane sheets, and,
alternatively, that the relatively short-term (24 hours) loss-offunction assays described above are too brief to reveal a role for
endogenous netrin 1.
RESEARCH ARTICLE
in pancreatic cells (Yebra et al., 2003). We therefore investigated
whether netrin-1-mediated changes in the elaboration of
oligodendrocyte processes involve an interaction between netrin 1
and integrins.
Development 136 (3)
Application of a β1 integrin function-blocking antibody,
which has been demonstrated to block fibronectin-induced
changes in oligodendrocyte morphology (Liang et al., 2004),
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 Mbppositive 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 functionblocking antibody together with netrin 1 prevented the netrin-1-dependent increase in branching, and decreased branching compared with
controls. A, 20⫻0.5 n.a. objective; B,D,F, 40⫻0.75 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
420
Regulation of oligodendrocyte maturation
RESEARCH ARTICLE
421
oligodendrocyte process branching (Fig. 6A). By contrast, this
antibody blocked HEK293T cell spreading on a fibronectin
substrate (Fig. 6B), verifying its efficacy. Yebra and colleagues
identified a 25 amino acid region within the C-terminus of netrin
1 that binds α6β4 and α3β1 integrins, and hypothesized that
potential interactions between netrin 1 and other integrins might
also occur through this region (Yebra et al., 2003). To determine
whether such an interaction might contribute to netrin-1-induced
changes in oligodendroglial morphology, we incubated cells with
a peptide comprising the putative integrin-binding sequence that
functions as a competitive inhibitor of integrins binding netrin 1
(Yebra et al., 2003). The netrin 1 peptide (20 μg/ml, a gift from
Dr V. Cirulli, UCSD, CA, USA) did not affect process complexity
(Fig. 6A), nor did it disrupt the netrin-1-dependent increase in
branching (Fig. 6A). We conclude that the novel role for netrin 1
in regulating oligodendrocyte morphology occurs through Dcc
independently of β1-containing integrins and the integrin-binding
region at the C-terminus of netrin 1.
Netrin 1 binding to Dcc recruits Fyn to a complex
containing FAK
We next assayed signaling proteins implicated as regulators of
cytoskeletal organization downstream of netrin 1 and Dcc in axonal
growth cones to determine whether similar signaling complexes
might be engaged in oligodendrocytes. Oligodendrocytes express
the SFKs Src, Fyn and Lyn (Colognato et al., 2004). Of these, only
mice lacking Fyn show defects in myelination (Sperber et al., 2001).
Furthermore, Fyn activation has been implicated in the regulation of
process branching during the morphological maturation of
oligodendrocytes (Osterhout et al., 1999; Umemori et al., 1994). In
axonal growth cones, application of netrin 1 recruits Fyn to the Dcc
intracellular domain, where it is activated by focal adhesion kinase
(FAK; Ptk2 – Mouse Genome Informatics) (Liu et al., 2004). To
determine whether Fyn might function downstream of Dcc in
oligodendrocytes, cells derived from newborn (P0) rats were
allowed to mature in culture for 3-4 days or for 5-6 days until they
expressed Mbp. Co-immunoprecipitation studies carried out using
DEVELOPMENT
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 wildtype and netrin-1-deficient or Dcc-deficient mice.
40⫻0.75 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.
422
RESEARCH ARTICLE
Development 136 (3)
two different antibodies against Fyn revealed an interaction between
Fyn and Dcc in both immature (4 DIV) and mature (6 DIV)
oligodendrocytes (Fig. 7A). Five minutes following application of
netrin 1, an increased amount of Fyn co-immunoprecipitated with
Dcc (Fig. 7A,D). A relatively minor Fyn-immunoreactive band of
slightly higher molecular weight was consistently detected
following immunoprecipitation and might reflect Fyn
phosphorylation as a result of activation by netrin 1. Our findings
also revealed a constitutive interaction between Dcc and FAK (Fig.
7A), suggesting that application of netrin 1 to oligodendrocytes
recruits Fyn into a complex with FAK that is bound to the
intracellular domain of Dcc, as has been reported for neurons (Li et
al., 2004; Ren et al., 2004). Application of netrin 1 led to increased
phosphorylation of SFK tyrosine 416 (Y416), an event associated
with kinase activation (Smart et al., 1981), in the SFK associated
with Dcc (Fig. 7B,D). Analysis of SFK phosphorylation in wholecell lysates did not detect a global change in phospho-Y416,
indicating that the netrin-1-induced change is specific to SFK
recruited into a complex with Dcc (Fig. 7C). Using an antibody
specific for the SFK Src, an interaction with Dcc was not detected
(not shown). This provides evidence for specific recruitment of Fyn
to Dcc; however, we do not rule out that other SFKs might be
involved in netrin 1 signaling in oligodendrocytes.
The netrin-1-induced increase in oligodendrocyte
branching requires Fyn
To determine whether Fyn is required downstream of netrin 1 in
oligodendrocytes, we isolated cells from mice lacking Fyn
[Fyntm1Sor, Fyn knockout (KO)] and from wild types of a matched
genetic background (B6129SF2/J, F2 hybrid) (Fig. 8A). Treatment
of Fyn KO cells with netrin 1 did not result in increased branching,
in contrast to its effect upon cells isolated from control mice (Fig.
8A,B). We conclude that Fyn is essential for these changes to occur.
We therefore determined whether activation of SFKs is required to
promote the morphological changes induced by netrin 1.
SFK activity is required for the netrin-1-induced
increase in oligodendrocyte process length and
branching
Immunocytochemistry
revealed
SFK
phospho-Y416
immunoreactivity distributed within the oligodendrocyte cell body
and proximal branches, and punctate staining within the distal
branches (Fig. 8C,D). Colocalization with Dcc was observed,
consistent with our immunoprecipitation results (Fig. 7B). To
determine whether changes in SFK phosphorylation occurred upon
netrin 1 stimulation, the relative number of SFK phospho-Y416immunoreactive puncta was measured per unit area of the cell (Fig.
8E). Treatment of mature oligodendrocytes with netrin 1 (100
ng/ml) for 24 hours significantly increased the number of SFK
phospho-Y416-positive puncta per unit area, consistent with an
association between netrin 1 stimulation and increased SFK activity
(Fig. 8F). Treatment with the SFK inhibitor PP2 (2 μM) (Hanke et
al., 1996) blocked the netrin-1-induced increase in SFK phosphoY416-positive puncta (Fig. 8F), whereas the inactive SFK inhibitor
analog PP3 (2 μM) had no significant effect on the number of puncta
per unit area (Fig. 8F). Application of PP2 alone led to a decrease in
the relative number of puncta per unit area, consistent with
constitutive SFK activity contributing to the basal level of puncta
observed (Fig. 8F). To exclude the possibility that the increase in
SFK phospho-Y416-positive puncta was secondary to increased
branch formation, the same quantification was performed with cells
immunostained for Fyn. No difference was found in the number of
Fyn-immunopositive puncta per unit area in control and netrin-1treated cells (Fig. 8G), consistent with the increase in SFK phosphoY416-positive puncta resulting from an increase in SFK activity and
not a netrin-1-induced increase in the number of branches.
Our findings indicate that netrin 1 recruits Fyn to a complex with
Dcc, increasing SFK activity in oligodendrocytes. We then tested the
hypothesis that SFK activation is required for the morphological
changes induced by netrin 1. SFK activity was assessed in both
immature (4 DIV, Cnp-positive) and mature (6 DIV, Mbp-positive)
oligodendrocytes. Treatment of oligodendrocytes with PP2 blocked
netrin-1-induced process extension in Cnp-immunoreactive
immature cells and the netrin-1-dependent increase in branching in
Mbp-expressing mature oligodendrocytes, whereas the inactive
analog PP3 had no effect (Fig. 8H,I). Immature oligodendrocytes
treated with PP2 alone showed a small but significant decrease in
process length (Fig. 8H), which was not seen with PP3 treatment.
Mature oligodendrocytes appeared less sensitive to the inhibition of
basal SFK activity than immature cells, as PP2 alone did not affect
branching (Fig. 8I). We conclude that netrin 1 binding to Dcc results
in the recruitment of the SFK Fyn, and that subsequent activation of
Fyn is required for the netrin-1-induced changes in oligodendrocyte
morphology.
DEVELOPMENT
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.
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, a significant decrease in RhoA-GTP was detected
compared with controls (Fig. 9A). We conclude that netrin-1induced inhibition of RhoA, in the absence of altered Rac1 or Cdc42
activity, promotes process elaboration by oligodendrocytes.
DISCUSSION
Developing oligodendrocytes extend and retract their processes over
substantial distances, sampling the local environment to locate
unmyelinated axons (Hardy and Friedrich, 1996; Kirby et al., 2006).
As the cell matures, these processes branch and eventually form
lamellae that ensheath target axons. Our findings indicate that netrin
1 and Dcc regulate oligodendrocyte process extension, branching
and myelin-like membrane sheet formation, which are all essential
events for initiating myelination.
Exogenous and autocrine netrin 1 contribute to
oligodendrocyte maturation
Our results support the conclusion that netrin 1, as expressed by
neurons and neuroepithelial cells, when encountered by a
premyelinating oligodendrocyte evokes Dcc-dependent extension
of the motile processes of the cell. In more mature cells, exogenous
netrin 1 promotes Dcc-dependent process branching and myelin-like
membrane sheet formation. Based on our findings in vitro and in
vivo, we hypothesize that by promoting the morphological
maturation of oligodendrocytes, netrin 1 facilitates the search for
appropriate axonal targets. Interestingly, although exogenous netrin
1 promotes changes in oligodendrocyte morphology, at later stages
of maturation oligodendrocytes themselves begin to express netrin
1. We identify a selective, Dcc-dependent autocrine role for netrin 1
in promoting the formation of myelin-like membrane sheets.
Oligodendrocytes only begin to express netrin 1 in the developing
spinal cord after myelination has begun. Our findings indicate that
DEVELOPMENT
Netrin 1 inhibits RhoA but does not affect Cdc42
or Rac1 in oligodendrocytes
We next asked what signals might act downstream of SFKs to trigger
the morphological changes induced by netrin 1 in oligodendrocytes.
Members of the Rho family of small GTPases, including RhoA,
Rac1 and Cdc42, regulate the elaboration and branching of
oligodendrocyte processes (Liang et al., 2004). The effect of netrin
1 on Cdc42 and Rac1 activity was investigated using a GST-PakCRIB pulldown assay (Sander et al., 1998). Cell lysates of cultured
mature oligodendrocytes were incubated with GST-Pak-CRIB
fusion protein to quantify the levels of GTP-bound Cdc42 and Rac1.
No significant change in the levels of GTP-bound Rac1 or Cdc42
was detected in oligodendrocytes following application of netrin 1
(Fig. 9B,C). We have reported that the Cdc42 effector protein NWASP is recruited into a protein complex with the intracellular
domain of Dcc following the addition of netrin 1 to embryonic spinal
commissural neurons (Shekarabi et al., 2005). Unlike in
oligodendrocytes, netrin 1 activates Cdc42 and Rac1 in commissural
neurons. In mature oligodendrocytes, N-WASP also constitutively
co-immunoprecipitated with Dcc; however, addition of netrin 1 did
not significantly alter the amount of N-WASP associated with Dcc
(Fig. 9D), which is consistent with netrin 1 not increasing the
activation of Cdc42 in oligodendrocytes.
Inhibiting RhoA, or its downstream effector Rho kinase
(Rock1/2), in oligodendrocytes increases process extension (Liang
et al., 2004; Miron et al., 2007; Wolf et al., 2001). Interestingly,
activation of Fyn in oligodendrocytes leads to inactivation of RhoA
(Wolf et al., 2001), and we therefore investigated the possibility that
netrin 1 might regulate RhoA activity in oligodendrocytes. Levels
of GTP-bound RhoA in mature Mbp-positive oligodendrocytes were
assessed using a GST-rhotekin pulldown assay (Reid et al., 1996).
Following a 5 minute treatment of mature oligodendrocytes with
RESEARCH ARTICLE
contact with axons is not essential for the initiation of netrin 1
expression by these cells, but that it occurs coincident with myelinlike membrane sheet formation. We hypothesize that autocrine
expression of netrin 1 specifically promotes later stages of
maturation, facilitating the formation of large myelin-like membrane
sheets by these cells. Netrin 1 expression by oligodendrocytes might
also facilitate axon remodeling or prevent aberrant axonal sprouting
at later stages of development.
Development 136 (3)
Common signaling mechanisms in the regulation
of oligodendrocyte processes and axonal growth
cones by netrin 1
The extending tips of oligodendrocyte processes in some ways
resemble motile axonal growth cones (Fox et al., 2006; Jarjour
and Kennedy, 2004), and increasing evidence suggests that
common regulators of actin nucleation function in neuronal
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,C⬘) PhosphoSFK [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,D⬘) Portions of the images in C and C⬘ 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.
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 μm in A,E; 40 μm in C⬘,D⬘.
DEVELOPMENT
424
Fig. 9. Treatment of mature oligodendrocytes with netrin 1
inhibits RhoA activity (A) Levels of GTP-bound RhoA in rat
oligodendrocytes were assessed using GST-rhotekin pulldown.
Treatment of mature oligodendrocytes with netrin 1 (100 ng/ml, 5
minutes) caused a significant decrease in RhoA-GTP compared with
controls. (B,C) Similar treatment of cells resulted in no significant
change in the levels of GTP-bound Cdc42 or Rac1, as assessed by GSTPak-CRIB pulldown. (D) Addition of netrin 1 to mature oligodendrocytes
(100 ng/ml, 5 minutes) did not alter the amount of N-WASP recruited
to Dcc. *P<0.05 versus control. Error bars indicate s.e.m.
processes and sheets (Sloane and Vartanian, 2007). Here, we
report that the similarities between growing axons and
oligodendrocyte processes include the recruitment of similar
intracellular signaling proteins downstream of netrin 1: Fyn, FAK,
N-WASP and Rho GTPases (Li et al., 2004; Liu et al., 2004; Ren
et al., 2004; Shekarabi and Kennedy, 2002). We demonstrate that
Fyn is required for the netrin-1-dependent increase in
oligodendrocyte process branching. Netrin 1 recruits Fyn to a
complex that includes Dcc and FAK, resulting in SFK
phosphorylation and activation. Importantly, previous studies
have implicated Fyn, Rac1, Cdc42, RhoA and N-WASP in the
mechanism governing the cytoskeletal changes in
oligodendrocytes that lead to myelination (Simons and Trotter,
2007).
Activating Fyn results in RhoA inactivation in
oligodendrocytes (Wolf et al., 2001). We demonstrate reduced
levels of GTP-bound RhoA upon netrin 1 stimulation, but found
no evidence for the activation of Cdc42 or Rac1. Interestingly,
transgenic mice specifically lacking Cdc42 and Rac1 in
oligodendrocytes exhibit normal oligodendroglial differentiation,
but show eventual defects in myelin compaction (Thurnherr et al.,
2006). Our findings support the hypothesis that in a background
of unchanging Rac1 and Cdc42 activity, modulation of RhoA
function plays a key role in regulating the morphological
differentiation of oligodendrocyte processes.
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
elaborate myelin (Chang et al., 2002). A better understanding of
the mechanisms that promote myelination will advance the
development of therapeutics that aim to promote the recovery of
nervous system function.
We thank Simon Moore, Sarah-Jane Bull and Adriana Di Polo for comments on
the manuscript. S.R. and A.A.J. were supported by Multiple Sclerosis (MS)
Society of Canada studentships and a Canadian Institutes of Health Research
(CIHR) Neuroinflammation Strategic Training Program award, K.A.B. by a
Fonds de la Recherche en Santé du Québec (FRSQ) postdoctoral fellowship,
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.
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