RESEARCH ARTICLE 1329
Development 138, 1329-1337 (2011) doi:10.1242/dev.058677
© 2011. Published by The Company of Biologists Ltd
N-WASp is required for Schwann cell cytoskeletal dynamics,
normal myelin gene expression and peripheral nerve
myelination
Fuzi Jin1,2,*, Baoxia Dong1,2,*, John Georgiou3, Qiuhong Jiang2, Jinyi Zhang2, Arjun Bharioke2,†, Frank Qiu2,
Silvia Lommel4, M. Laura Feltri5, Lawrence Wrabetz5, John C. Roder3, Joel Eyer6, Xiequn Chen1,
Alan C. Peterson7,‡,§ and Katherine A. Siminovitch2,‡,§
SUMMARY
Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although
actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined.
Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in
myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the
onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin.
Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia
formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the
mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an
essential role in Schwann cell maturation and myelin formation.
INTRODUCTION
Axons ensheathed with myelin propagate action potentials in a
saltatory manner resulting in significantly accelerated conduction
velocity and reduced energy requirements. In the peripheral
nervous system (PNS), myelin is elaborated by Schwann cells.
Their precursors emerge from the neural crest early in development
and distribute amongst elongating axons, where they proliferate
and migrate with advancing axons (Jessen et al., 1994; Wanner et
al., 2006). As development proceeds they form discrete Schwann
cell families that encircle bundles of axons, where they continue to
proliferate as well as extend and retract radially oriented processes
between axons, ultimately ensheathing all large caliber axons in a
process referred to as radial sorting (Webster et al., 1973; Grove et
al., 2007; Woodhoo et al., 2009) (for a review, see Woodhoo and
Sommer, 2008).
1
Department of Haematology, Xijing Hospital, Fourth Military Medical University,
Xi'an, China. 2Departments of Medicine, Immunology and Molecular Genetics,
University of Toronto, Samuel Lunenfeld and Toronto General Research Institutes,
600 University Avenue, Toronto, ON M5G 1X5, Canada. 3Samuel Lunenfeld
Research Institute, Mount Sinai Hospital, and Department of Molecular Genetics,
University of Toronto, Toronto, ON M5G 1X5, Canada. 4Institute for Cell Biology,
University of Bonn, Ulrich-Haberland-Str. 61A, 53121 Bonn, Germany. 5Division of
Genetics and Cell Biology, San Raffaele Scientific Institute, 20132 Milan, Italy.
6
Laboratory Neurobiologie et Transgenese, UPRES-EA3143, INSERM, Centre
Hospitalier Universitaire, 49033 Angers, France. 7Laboratory of Developmental
Biology, Royal Victoria Hospital, MUHC, McGill University, Montreal, QC H3A 1A1,
Canada.
*These authors contributed equally to this work
†
Present address: Howard Hughes Medical Institute, Janelia Farm Research Campus,
19700 Helix Drive, Ashburn, VA 20147, USA
‡
These authors co-directed this work
§
Authors for correspondence ([email protected]; [email protected])
Accepted 4 January 2011
To initiate axon ensheathment, a Schwann cell develops a
longitudinal groove that deepens as it extends processes
circumferentially to fully enwrap the axon (Webster, 1971; Webster
et al., 1973; Webster, 1984). Through mechanisms that remain
poorly understood (Bunge et al., 1989), the Schwann cell
membrane spirally wraps around the axon, following which,
interlamellar Schwann cell cytoplasm is extruded and the apposing
membrane layers are stabilized by myelin-associated proteins
(Wood et al., 1990). In addition to their seemingly lamellipodialike mesaxons, Schwann cells generate microvilli at the
electrophysiologically specialized nodes of Ranvier that are located
between successive myelin sheaths (Gatto et al., 2003).
The migratory, membrane-extending and microvilli-generating
capacities of Schwann cells suggest an integral role for cytoskeletal
remodeling in Schwann cell maturation. This possibility is
supported by the growth cone-like properties displayed by the
terminal processes of migrating Schwann cell precursors in vivo
(Wanner et al., 2006) and by both the structure and composition of
the mesaxons that are elaborated by myelinating Schwann cells in
vitro (Bacon et al., 2007). Also, in Schwann cell-neuron cocultures, myelin formation is attenuated when actin polymerization
is disrupted by cytochalasin D (Fernandez-Valle et al., 1997).
Similarly, in mice, myelin formation is attenuated when the Cdc42
or Rac1 GTPase cytoskeletal regulators are deleted (Nodari et al.,
2007; Benninger et al., 2007). Additionally, Rho GTPase and its
kinase, ROCK, have been implicated in Schwann cell- and/or
oligodendrocyte-mediated myelination (Melendez-Vasquez et al.,
2004) (for a review, see Feltri et al., 2008).
Although a link between cytoskeletal dynamics and myelination
is widely appreciated, the specific effectors that modulate
cytoskeletal reorganization in developing and myelinating Schwann
cells are not well defined. However, one likely effector is the
neuronal Wiskott-Aldrich syndrome protein [N-WASp; also known
DEVELOPMENT
KEY WORDS: Myelination, Schwann cells, N-WASp (Wasl), Mouse
1330 RESEARCH ARTICLE
MATERIALS AND METHODS
Conditional knockout and transgenic mice
N-WASp floxed (N-WASpflx/flx), mPoTOTA-Cre (P0-Cre) and Z/EG reporter
mice have been reported by Lommel et al. (Lommel et al., 2001), Feltri et
al. (Feltri et al., 1999a; Feltri et al., 1999b) and Novak et al. (Novak et al.,
2000), respectively. C57BL/6 mice heterozygous for the N-WASp floxed
allele were crossed with C57BL/6/P0-Cre mice to derive N-WASpflx/+/
P0-Cre. These were crossed with N-WASpflx/flx mice to generate NWASpflx/flx/P0-Cre (N-WASp–/– mutant) mice. Z/EG homozygous mice were
mated to P0-Cre mice to derive Z-EG/P0-Cre mice for assessing P0-Cre
activity. Genotypes were determined by PCR analysis of tail and/or sciatic
nerve genomic DNA using primers that yield amplicons of 200 bp (wildtype), 306 bp (floxed) or 408 bp (recombined) from the N-WASp allele: 5⬘TAACTCACATCCATAGTATC-3⬘, 5⬘-ACTTGGTCTGCTATGTATCGG3⬘ and 5⬘-TTGCACAGAGAGAATGAATG-3⬘.
To test gait abnormalities, paint was applied to paws of mutants and
control littermates and the footprint patterns generated by walking across
a 12⫻2 inch Plexiglas track evaluated. Tremor activity was assessed using
an SDI Tremor Monitor (SD Instruments), which detects movement
amplitude via a force transducer. For rotarod testing, mice were placed on
a rod rotating at 5-7 rotations per minute (Columbus Instruments) and
tested three times per day over 3 days for the length of time that they
remained on the rod.
Reagents
Reagents included rabbit monoclonal (Cell Signaling Technology) and
polyclonal (Lommel et al., 2004) anti-N-WASp antibodies; rat monoclonal
Mbp antibody (Millipore); mouse monoclonal P0 and neurofilament
antibodies (Developmental Studies Hybridoma Bank); mouse monoclonal
S100 and -actin antibodies (Sigma); rat monoclonal BrdU and mouse
monoclonal Mag antibodies (Abcam); goat polyclonal antibodies to
Krox20 (Santa Cruz Biotechnology), Oct6 (Abcam) and Sox10 (R&D
Systems); FITC- or Cy3/5-conjugated secondary antibodies (Jackson
ImmunoResearch); FITC-phalloidin (Molecular Probes); laminin (Sigma);
and DAPI (Roche).
Immunofluorescence analyses and primary cultures
Sciatic nerve sections were fixed in 4% paraformaldehyde (PFA), blocked
for 1 hour with 10% goat serum/0.1% Triton X-100 in PBS and incubated
overnight at 4°C with primary antibodies. After washing in PBS, tissue
sections were incubated for 1 hour with secondary antibody. Alternatively,
Schwann cells were isolated from P4 sciatic nerves following nerve
digestion with 1 mg/ml collagenase/dispase and 2.5% trypsin and Thy1.2
antibody/complement-mediated fibroblast lysis (Honkanen et al., 2007) and
maintained in culture in DMEM supplemented with 10% fetal calf serum
(FCS)/antibiotics. Dorsal root ganglia (DRG) were dissected as previously
described from E13.5 wild-type mice (Päiväläinen et al., 2008) and
cultured for 2 days in neural basal medium containing 10% FCS followed
by supplementation with 10–5 M uridine/5⬘-fluoro-2⬘-deoxyuridine
(Sigma). Schwann cells and DRG were then co-cultured in myelinationpromoting media containing 50 g/ml ascorbic acid (Eldrige et al., 1987).
For staining, Schwann cells or Schwann cell/DRG co-cultures were fixed
in 4% PFA for 10 minutes at room temperature, the cells then
permeabilized with 0.2% Triton X-100 for 5 minutes at room temperature
and incubated with primary antibody in blocking buffer (1% BSA in PBS)
overnight at 4°C, followed by incubation with secondary antibodies in
blocking buffer for 1 hour at room temperature. Staining was visualized
using an Olympus FluoView 1000 inverted confocal microscope equipped
with fluorescence optics and Olympus FluoView FV100 software.
Immunoblotting analysis
Sciatic nerves were homogenized with a chilled mortar and pestle in RIPA
buffer containing protease inhibitors (Roche). The lysate was suspended in
loading buffer, electrophoresed through a 10% polyacrylamide gel and
transferred to nitrocellulose. After overnight blocking, as previously
described (Badour et al., 2004), membranes were incubated for 1 hour with
primary antibody followed by incubation for 1 hour with a 1/3000 dilution
of horseradish peroxidase-conjugated secondary antibody (Bio-Rad), then
ECL substrate (Amersham Biosciences) for 1 minute, followed by a 1- to
10-minute exposure to X-ray film (Eastman Kodak).
Morphological analysis
Sciatic nerves or spinal roots were immersion-fixed in either 4% PFA or
by trans-cardiac perfusion with 2.5% glutaraldehyde/0.5% PFA in 0.1 M
phosphate buffered saline (pH 7.4) and subjected to alcohol dehydration
and Epon embedding. Alternatively, specimens were fixed for 2 hours in
2% glutaraldehyde in 0.1 M sodium cacodylate buffer, dehydrated and
embedded in Spurrs resin (Electron Microscopy Sciences). Semi-thin
sections were stained with Toluidine Blue or Methylene Blue and examined
by light microscopy. Ultrathin sections were stained with uranyl acetate and
lead citrate and examined by electron microscopy (JEOL JEM2010
electron microscope).
Proliferation and TUNEL assays
Mice were injected intraperitoneally with BrdU (300 mg/kg body weight)
and sacrificed 24 hours later. The labeled sciatic nerves were then fixed
with 4% PFA, embedded in paraffin and 5 m sections stained with BrdU
antibody. Apoptotic cell death was measured in transverse sciatic nerve
sections by TUNEL labeling using calf thymus TdT, biotin-labeled UTP
and horseradish peroxidase-streptavidin (Boehringer). Counterstaining was
performed with Haematoxylin. Sections were dehydrated and coverslipped
and the percentage of TUNEL-positive cells in a sample of 500 was
determined.
Gene expression assays
Total RNA (200 ng) prepared from sciatic nerve samples using the Qiagen
RNeasy Kit was reverse transcribed with Superscript II (Invitrogen)
according to the manufacturer’s protocol. SYBR Green-based qRT-PCR
was performed using the ABI 7900HT system (Applied Biosystems).
Primers were: Mag forward 5⬘-GTGGAGCTGAGTGTCATGTATG-3⬘ and
DEVELOPMENT
as Wiskott-Aldrich syndrome-like (Wasl) – Mouse Genome
Informatics], a member of the WASp family of cytoskeletal
regulators. Like other family members, N-WASp links extracellular
stimuli and actin polymerization (Wegner et al., 2008) via the
binding of its verprolin homology/connecting/acid region domain
to the Arp2/3 complex. In cultured Schwann cells, N-WASp
localizes to the leading edge of extending processes where its
activity depends upon interaction with Cdc42, a protein with
essential roles in Schwann cell development (Benninger et al.,
2007; Bacon et al., 2007). Further, the myelinating capacity of
cultured rat Schwann cells is impaired by an inhibitor of N-WASp
activity (Bacon et al., 2007), as is oligodendrocyte-mediated
myelination in the central nervous system (CNS) when another
WASp family member, Wave1, is deleted in vivo (Kim et al., 2006).
Additionally, process remodeling in mature oligodendrocytes
involves a complex including FAK, Fyn, the intracellular domain
of Dcc and N-WASp (Rajasekharan et al., 2009).
Here, to investigate the in vivo role of N-WASp during
myelination, we used conditional gene targeting to generate mice
in which N-WASp (Wasl) is deleted in Schwann cells as they initiate
myelin elaboration. N-WASp-deficient Schwann cells achieve a
normal 1:1 relationship with large caliber axons, albeit on a delayed
schedule. However, their capacity to elaborate myelin sheaths
appears to be arrested completely and their expression of myelinrelated genes is severely reduced. Additionally, the production of
lamellipodia-like processes in vitro is markedly impaired. Although
these mutant mice manifest motor deficits characterized by tremor
and mild ataxia, they are viable into advanced age. These
observations demonstrate that N-WASp is required for normal
Schwann cell maturation and that it plays an essential role in
myelin formation, potentially by regulating the cytoskeletal
remodeling that is required for the spiral extension of Schwann cell
processes around axons.
Development 138 (7)
N-WASp is required for PNS myelination
RESEARCH ARTICLE 1331
reverse 5⬘-CATAGATGACCGTGGCTAGGA-3⬘; Mbp forward 5⬘-GTGCCACATGTACAAGGACTC-3⬘ and reverse 5⬘-TTGAAGAAATGGACTACTGGGTT-3⬘; and P0 forward 5⬘-GATTCGGTACTGCTGGCTG-3⬘ and reverse 5⬘-CATACAGCACTGGCGTCTG-3⬘. Levels of
mRNA were calculated by the comparative cycle threshold method (Pfaffl,
2001) and expressed as fold increase relative to Gapdh levels.
RESULTS
N-WASp is expressed in myelinating Schwann cells
N-WASp is expressed in many regions of the developing and mature
brain and also in cultured neurons, oligodendrocytes and Schwann
cells (Tsuchiya et al., 2006; Bacon et al., 2007). To determine when
N-WASp might participate in Schwann cell development in vivo, we
first evaluated the developmental programming of N-WASp
accumulation in the PNS of postnatal mice. N-WASp was readily
detectable by immunocytochemistry in sciatic nerves at postnatal day
(P) 28, a time when myelin sheaths are approaching a mature state.
As demonstrated by its co-localization with S100, the N-WASp
signal was localized largely to a cytoplasmic domain adjacent to the
external Schwann cell membrane. Intensely labeled profiles
frequently contained Schwann cell nuclei with the N-WASp signal
extending from the perinuclear cytoplasm to define much, but not all,
of the fiber circumference. Similarly intense N-WASp labeling was
not observed in either the Schwann cell cytoplasm adjacent to the
axon nor in nuclei (Fig. 1A).
To determine when N-WASp expression initiates, we
investigated sciatic nerve samples obtained from mice at earlier
stages of postnatal development (Bray et al., 1977). As early as P1,
coinciding with the initiation of PNS myelin elaboration, N-WASp
was readily detectable by immunofluorescence imaging (Fig. 1B).
A broad ring of N-WASp staining partially overlapping that of
myelin basic protein (Mbp) and extending to the cell periphery was
the most characteristic labeling pattern observed at this age (see
Fig. S1 in the supplementary material). Staining intensity appeared
DEVELOPMENT
Fig. 1. N-WASp expression is
developmentally regulated in Schwann
cells and its conditional deletion is
associated with motor impairment.
(A)Confocal images of sciatic nerve transverse
sections from wild-type mouse reveal N-WASp
to be localized around individual axons in a
pattern distinct from those of S100 and
DAPI. (B)N-WASp immunofluorescence
increases between P1 and P5, diminishes by
P13 and remains low at P28. (C)qRT-PCR
analysis shows changes over time in N-WASp
(left) and Mbp (right) mRNA levels in wild-type
sciatic nerves (mean of three independent
experiments expressed relative to Gapdh +
s.d.). (D)Two-month-old (left) and 7-monthold (right) mutant mice have a wider and
outward-pointing hindpaw stance. Footprint
patterns recorded from forepaws (red) and
hindpaws (blue) reveal a widened ataxic gait
compared with the narrow, even stride of
controls (see Movies 1 and 2 in the
supplementary material). (E)Rotarod tests
performed on three consecutive days (three
trials per day) showing significantly reduced
mean (± s.e.m.) hold times in 2-month-old
mutant mice. (F)Tremor activity in 2-monthold mutant mice shown as a representative
recording of movement amplitude and a
graph indicating numbers of tremor events at
each frequency (mean ± s.e.m.) (data
representative of three independent
experiments).
to increase to peak levels near P5 and moderated by P13, at which
time a distribution pattern characterized by thinner and incomplete
peripheral rings, typical of more mature fibers, was common (Fig.
1B). Quantitative real-time PCR (qRT-PCR) assays revealed that
N-WASp and Mbp mRNA accumulated on similar programs and
indicated that N-WASp expression was coincident with, or
preceded, the initiation of myelination (Fig. 1C).
Mice with N-WASp-deficient Schwann cells
To explore the role of N-WASp in Schwann cell-mediated
myelination, we derived mice in which N-WASp was specifically
inactivated in premyelinating Schwann cells. Mice homozygous for
an N-WASp floxed allele (N-WASpflx/flx) were bred with N-WASpflx/+
mice carrying a P0-Cre transgene (Feltri et al., 1999a; Lommel et
al., 2001). As the P0 (Mpz – Mouse Genome Informatics) sequence
drives expression in Schwann cells during the perinatal period
(Feltri et al., 1999a; Feltri et al., 1999b), we expected accumulated
Cre to delete floxed N-WASp at the beginning of PNS myelination.
In sciatic nerve samples, both a Cre-sensitive GFP reporter and Cre
expression followed the expected developmental program, both
being detectable at P1, increasing through P5 and stabilizing
thereafter (see Fig. S2 in the supplementary material). Evaluation
of Cre expression revealed ~65% of all DAPI-labeled nuclei to be
Cre-positive at P1, increasing to 99% by P28, thus suggesting that
most Schwann cells express effective levels of Cre.
Crosses involving floxed N-WASp and P0-Cre alleles resulted
in the predicted Mendelian ratios of wild-type, floxed,
heterozygous and homozygous mutant (N-WASpflx/flx/P0-Cre,
referred to as N-WASp–/–) offspring. The deleted N-WASp allele
was detected only in Schwann cells from mice bearing the P0Cre transgene and the floxed N-WASp allele (see Fig. S3A in the
supplementary material). Consistent with widespread
inactivation of the N-WASp gene in Schwann cells, N-WASp
protein levels were profoundly reduced in mutant sciatic nerves
(see Fig. S3B in the supplementary material). Moreover, in
immunolabeled sciatic nerve samples, the perineurium labeled
intensely, whereas N-WASp was undetectable in all but rare
Schwann cells (see Fig. S3C in the supplementary material).
Motor impairment in N-WASp–/– mice
N-WASp–/– mice appeared normal at birth but, by P14, showed
locomotor anomalies including a wide-based ataxic gait with
extraneous steps and impaired rotarod endurance (Fig. 1D,E and
see Movies 1 and 2 in the supplementary material). Mutants also
manifested an abnormally wide hindlimb stance while sitting and
low frequency (10-20 Hz) tremors (Fig. 1D,F). Interestingly, these
mice achieved normal body weights and their behavioral
phenotypes progressed minimally over an 8-month follow-up
period.
N-WASp-null Schwann cells fail to elaborate
myelin
As shown in Fig. 2, all large caliber (>2 m) axons coursing
through mutant sciatic nerves (Fig. 2A,B) and ventral roots (Fig.
2C) were ensheathed. However, most fibers were amyelinated
(>99% in sciatic nerve; see Table S1 in the supplementary material)
and, as seen in many demyelinating or amyelinating conditions,
axons without normal myelin sheaths failed to undergo normal
radial growth (see Table S1 in the supplementary material).
Exceptional myelinated fibers were encountered in the mutants
(<1%; see Table S1 in the supplementary material) and, consistent
with the enhanced radial growth of myelinated axons, such fibers
Development 138 (7)
appeared as prominent components in nerve transverse sections
(Fig. 2C). Such myelin sheaths appeared qualitatively normal and
revealed normal interlamellar spacing (13.17±0.48, versus
13.41±0.46 nm in controls) suggesting that these rare Schwann
cells might have escaped N-WASp inactivation. Nonetheless,
relative to the calibers of their associated axons, these sheaths were
of modestly reduced thickness, achieving g-ratios of 0.79±0.06,
versus 0.67±0.06 in controls (mean of 31 myelinated fibers).
Finally, regional heterogeneity may exist in the frequency at which
myelin sheaths arise in this model as they were encountered in
most spinal roots but were virtually absent in sciatic nerves (Fig.
2A,B). Interestingly, enlarged blood vessels were also prominent
in mutant spinal roots (Fig. 2C).
At birth, many axons in normal sciatic nerves remain closely
packed into bundles surrounded by Schwann cell families. Through
a complex program involving process extension/retraction and cell
division, Schwann cells segregate such bundles to arrive at a 1:1
relationship with large caliber axons. This radial sorting was
successfully achieved in sciatic nerves of mature N-WASp–/–
animals. However, in mutant nerves, axon bundles were still
prominent in the sciatic nerves of P5 mice, whereas in control mice
at this age radial sorting was largely complete (Fig. 2A,B; see Fig.
S4A and Table S1 in the supplementary material). Thus, the mutant
Schwann cells remained capable of completing the radial sorting
process, albeit over an extended period.
Mutant fibers that failed to elaborate myelin sheaths
demonstrated numerous ultrastructural features shared with
promyelinating fibers in normal perinatal mice (Webster, 1971).
Processes enwrapping axons often comprised inner- and outerfacing Schwann cell membrane with little cytoplasm between.
Where their processes confronted each other, no evidence for
continued circumferential movements was apparent. Instead, such
mesaxon contacts remained in apposition (Fig. 2E,F) or developed
multiple short interdigitations (Fig. 2G). Also common were one
or more Schwann cell processes extending away from the axon into
interfiber space (Fig. 2E,F). All outward-oriented Schwann cell
membrane was associated with a continuous and non-redundant
basal lamina and collagen fibrils were deposited densely between
the amyelinated fibers. In Remak fibers, where Schwann cells
ensheath multiple small diameter C-fibers, but elaborate no myelin,
qualitative anomalies were not obvious (see Fig. S4B and Table S1
in the supplementary material) and neither the number nor caliber
of the ensheathed axons was remarkable (see Table S1 in the
supplementary material).
With the exception of Schwann cells, N-WASp was expressed
widely in mutant mice bearing both the P0-Cre transgene and
floxed N-WASp alleles (data not shown). Therefore, the
amyelination observed in the PNS of N-WASp mutants was likely
to be the result of a Schwann-cell-autonomous defect. To test this
conclusively, we used mixed cultures in which mutant or wild-type
Schwann cells were assessed for their capacity to myelinate axons
extending from wild-type dorsal root ganglia neurons. Consistent
with a Schwann-cell-autonomous defect, myelination, as assessed
by Mbp immunoreactivity, was detected only in cultures containing
wild-type Schwann cells (Fig. 2H).
N-WASp deficiency alters Schwann cell numbers
and proliferation
We next compared Schwann cell numbers (Brown and Asbury,
1981), proliferation and apoptosis in mutant versus control-type
nerves. Cell numbers were equivalent at P1 (Fig. 3A,B), but by P7
and at all later ages examined the mutant nerves contained
DEVELOPMENT
1332 RESEARCH ARTICLE
N-WASp is required for PNS myelination
RESEARCH ARTICLE 1333
Fig. 2. N-WASp is required for Schwann cell
myelination. (A-C)Light microscopy of semithin transverse Toluidine Blue-stained sections
from control and mutant P5 (A) and P60 (B)
sciatic nerves and P60 L3 ventral roots (C). At
P5, axons in control nerves are sorted and many
have myelin sheaths, whereas in mutant
samples bundles of unsorted axons remain
within Schwann cell families. Asterisks indicate
unsorted axon bundles. At P60, all large caliber
axons in control nerves are myelinated, whereas
in the mutant only rare myelinated fibers are
observed amongst ensheathed amyelinated
axons (see Table S1 in the supplementary
material). (D-G)Electron micrographs of control
(D) and mutant (E-G) fibers in P60 samples.
Although axon membranes are covered
completely in both control and mutant samples,
mutant Schwann cells do not show spiral
membrane wrapping. Where mesaxons abut,
they show little evidence of further
circumferential extension (F), often elaborating
multiple short interdigitating processes (G). In
both mutant and control nerves, all fibers are
surrounded by basal lamina and the interfiber
space is richly invested with collagen fibrils.
(H)Immunofluorescence analysis of control and
mutant Schwann cells co-cultured with control
dorsal root ganglia neurons co-stained with
DAPI and antibodies to neurofilament and Mbp.
The numbers of DAPI-stained nuclei and
neurofilament-stained axons are equivalent in
both culture types, but mutant cells do not
produce Mbp-stained internodes (data
representative of five independent
experiments).
N-WASp modulates myelin-related gene
expression
Genes encoding a group of myelin proteins are coordinately
upregulated as Schwann cells initiate myelination. To determine
whether the lack of N-WASp modulates this expression program,
levels of three such myelin proteins were assessed in P5 sciatic
nerve samples. In the hypercellular mutant samples, both
immunoblotting and qRT-PCR analyses revealed reduced
accumulation of Mag and P0 (Fig. 4A,B), whereas only trace
amounts of transcript and negligible protein were apparent for the
later-accumulating Mbp (Fig. 4A-C). Notably, these findings
cannot be explained by a generalized transcription defect, as
expression of Oct6 (Pou3f1 – Mouse Genome Informatics),
Krox20 (Egr2 – Mouse Genome Informatics) and Sox10, which are
all promyelinating transcription factors with key roles in Schwann
cell differentiation and myelin gene activation (Svaren and Meijer,
2008; Ghislain and Charnay, 2006), was increased in the mutant
compared with control samples (Fig. 4D).
N-WASp modulates lamellipodia formation
Cultured N-WASp-deficient Schwann cells were assessed for their
capacity to form the actin-based processes that are thought
necessary for spiral membrane wrapping and myelin formation
(Bunge et al., 1989; Feltri et al., 2002). After 16 hours in culture,
similar numbers of processes extended from the main cell axis of
both mutant and wild-type cells, but those extending from the
mutant cells were only ~60% the normal length (Fig. 4E).
Although phalloidin staining revealed that an equivalent number of
lamellipodia formed at cell termini, only wild-type cells elaborated
well-defined, F-actin-rich leading edges (Fig. 4F,G). Additionally,
the number of processes extending from the periphery of the cell
body was reduced in N-WASp–/– cells.
DEVELOPMENT
significantly more cells and these demonstrated greater
proliferation as revealed by Ki67 labeling and bromodeoxyuridine
(BrdU) incorporation (Fig. 3B,C). By contrast, TUNEL assays
revealed no differences in the percentages of TUNEL-positive cells
in mutant compared with control (6.58±0.54% versus 6.64±0.83%,
P0.81) nerves (data not shown). As the extent of caspase 3
staining was also similar in mutant and control samples (data not
shown), the enhanced Schwann cell proliferation observed in
mutant nerves does not appear to be in response to increased
Schwann cell death. Rather, their increased proliferation is
consistent with the limited capacity of non-myelin-forming, as
compared with myelin-forming, Schwann cells to extend for long
distances along their associated axons. Thus, relatively more nonmyelin-forming Schwann cells are required to ensheath a
comparable length of axon.
1334 RESEARCH ARTICLE
Development 138 (7)
DISCUSSION
Our study of Schwann cell maturation in the context of N-WASp
deficiency identifies crucial roles for N-WASp in the elaboration
of PNS myelin. The amyelination phenotype manifested by the
mutant Schwann cells studied here is likely to be attributable, at
least in part, to loss of N-WASp actin polymerization activity. The
cytoskeleton supporting the growth cones elaborated by migrating
and elongating Schwann cells is normally enriched in actin, and
perturbation of the actin cytoskeleton is associated with defective
Schwann cell morphology and myelin production (Fernandez-Valle
et al., 1997; Melendez-Vasquez et al., 2004; Nodari et al., 2007;
Benninger et al., 2007) (for a review, see Feltri et al., 2008). Thus,
N-WASp deficiency might result in an impaired cytoskeletal
capacity to evoke the inner Schwann cell mesaxon movement that
drives the elaboration of spirally wrapped membrane needed for
myelin sheath production. This possibility is supported by the
absence of mesaxon-mediated plasma membrane circumnavigation
following axon ensheathment in the N-WASp-deficient cells in
vivo and also by the multiple cytoskeletal anomalies manifested by
the N-WASp-deficient Schwann cells cultured from mutant nerves.
Thus, in contrast to the actin-rich growth cones observed in both
axial and radial lamellipodia of cultured wild-type Schwann cells,
the axial processes elaborated by cultured N-WASp-deficient cells
were reduced in length and lacked defined F-actin-rich leading
edges. Numbers of radial lamellipodia also were decreased
dramatically in the mutant cells. These findings are consistent with
the aberrant process extension observed in cultured Schwann cells
and oligodendrocytes treated with an N-WASp inhibitor (Bacon et
al., 2007) and suggest that the crucial role for N-WASp in Schwann
cell-mediated myelination reflects, at least partially, the regulation
of actin rearrangements that are integral to the formation and
function of Schwann cell lamellipodia.
In addition to actin regulatory roles for N-WASp at the Schwann
cell membrane, the localization of N-WASp to multiple cellular
compartments, including the perinuclear cytoplasm external to the
myelin sheath, suggests that the influence of N-WASp on
myelination might be realized through multiple effects on Schwann
cell biology. When co-cultured with neurons, for example,
Schwann cells normally undergo long extensions along the axon of
contact prior to initiating the circumferential mesaxonal movement
that drives deposition of spirally wrapped plasma membrane
(Bunge et al., 1989). By contrast, as is consistent with the extensive
Schwann cell hypercellularity observed in mutant nerves,
longitudinal extension of N-WASp-deficient Schwann cells is
markedly foreshortened. The consequent failure of pre-myelinating
Schwann cells to achieve significantly extensive apposition with
DEVELOPMENT
Fig. 3. Increased number and proliferation of N-WASpdeficient Schwann cells. (A)Haematoxylin- and Eosinstained sciatic nerve transverse sections show increased
numbers of Schwann cell nuclei in mutant nerves at all
timepoints studied (bar chart). (B,C)The number of
proliferating cells is increased in mutant compared with
control nerve sections as assessed by DAPI and Ki67 staining
at P5 and P28 (B) and by BrdU staining of cells 24 hours
after BrdU intraperitoneal injection at P5 and P24 (C). Data
are expressed as mean + s.e.m. of at least four independent
experiments.
N-WASp is required for PNS myelination
RESEARCH ARTICLE 1335
the axon membrane may limit the signal exchanges necessary to
support transition to the myelination program (Lemke and Chao,
1988). Such signals might involve, for example, induction of other
cytoskeletal regulatory pathways or effectors key to the
myelination process, such as Rho kinase (ROCK). Notably, ROCK
deficiency leads to aberrant Schwann cell branching and
elaboration of myelin with multiple shortened internodes, which is
suggestive of a failure to form functionally cohesive mesaxons
(Melendez-Vasquez, 2004). Similarly, the association of integrin
1, Rac1 or Cdc42 deficiency with defects not only in myelination,
but also in Schwann cell proliferation and axonal sorting
(Benninger et al., 2007; Nodari et al., 2007), implies that complex
signaling interactions that enable Schwann cells to regulate their
developmental relationship with axons are integrally involved in
the myelination program. Rho family GTPases are also known to
play key roles in F-actin-mediated Schwann cell membrane
exocytosis processes (Ory and Gasman, 2010) and N-WASp effects
on myelination may thus also reflect associations with the effectors
that modulate the membrane synthesis and assembly needed for
mesaxons to drive productive circumferential travel. Improved
understanding of the basis for N-WASp effects on myelination will
therefore require definition of the extent to which N-WASp
interactions with these and other effector pathways influences
Schwann cell maturation.
Of note, the in vivo effects of Cre-mediated N-WASp deletion
were not restricted to myelin elaboration, but also included a
transient delay in the earlier developmental events associated with
axon radial sorting. Thus, the P0-regulated Cre transgene used in
DEVELOPMENT
Fig. 4. Myelin gene expression and
lamellipodia formation are
impaired in N-WASp-deficient
Schwann cells. (A)Western blot
analysis reveals diminished myelin
protein accumulation in mutant P10
sciatic nerves. (B)qRT-PCR analysis also
shows reduced expression of myelinrelated genes in the N-WASp–/– sciatic
nerves (values are expressed relative to
Gapdh transcript levels and represent
mean + s.d. of three independent
experiments). (C)Confocal images
showing absence of Mbp staining in
nerves from P5 and P28 mutants.
(D)Western blot showing modest
increases in expression of Oct6,
Krox20 and Sox10 in mutant relative
to control P10 sciatic nerves.
(E)Phase-contrast micrograph
showing reduced lengths of the
filamentous processes extended by
mutant compared with control
Schwann cells cultured over laminin 2.
(F)Immunofluorescence analysis
showing lack of well-formed F-actinenriched process tips in mutant
compared with control FITCphalloidin-stained Schwann cells.
(G)Immunofluorescence analysis
showing that the numbers of axial
lamellipodia (white arrows) present at
the terminus of the Schwann cell long
axis are equivalent in control and
mutant Schwann cells, whereas the
number of radial lamellipodia (red
arrows) formed outside this region is
dramatically reduced in the mutant
samples. (E-G)Measurements of
process length, number of process tips
with intact leading edges and number
of processes per cell are mean + s.d.
of 100 representative Schwann cells
per sample.
deriving the mutant Schwann cells investigated here must be
expressed at levels sufficient to cause deletion of floxed N-WASp
alleles prior to the initiation of myelination, when genes such as P0
are upregulated. Although the sorting anomaly implies a role for
N-WASp in early Schwann cell maturation events, the eventual
realization of the mature relationship between each Schwann cell
and a single axon demonstrates that Schwann cells deficient in NWASp do successfully complete radial sorting. Schwann cell
mitosis is also not adversely affected by N-WASp deficiency, as
intense Schwann cell proliferation accompanies the normal sorting
process. In further support of this conclusion, the size of the
Schwann cell population in the amyelinated nerves of mature
mutant mice was markedly increased.
N-WASp deficiency in Schwann cells was also associated with
diminished expression of multiple myelin-associated genes,
including Mag, P0 and Mbp. This finding was, however, not
indicative of a global transcription deficit as expression of the
promyelinating transcription factors Oct6, Knox20 and Sox10
appeared normal or increased. Thus, failure of the N-WASpdeficient Schwann cells to realize a normal myelin gene expression
program could reflect their apparent developmental immaturity
(Fernandez-Valle et al., 1997). However, evidence is growing for
direct roles of nuclear actin and its modulating partners in
transcriptional regulation (for a review, see Gieni and Hendzel,
2009); both N-WASp and related WASp molecules are now
directly implicated in transcriptional activities relevant to their
cellular functions. WASp, for example, has recently been shown to
directly regulate epigenetic changes crucial to the transcriptional
induction of the TBX21 gene required for the differentiation of T
helper cells (Taylor et al., 2010). Similarly, N-WASp has been
identified within a transcription complex that is recruited in
response to retinoic acid to the HoxB2 gene enhancer, and the actin
polymerization evoked by N-WASp is required for the induction of
these genes (Ferrai et al., 2009). The extent to which transcriptional
regulatory roles for N-WASp might contribute to the impaired
expression of myelin genes in N-WASp-deficient Schwann cells
requires further investigation.
Interestingly, the multiple Schwann cell deficits associated
with N-WASp deficiency appear to be tolerated relatively well
by the mutant mice, in which neither the amyelinated PNS
phenotype nor the motor abnormalities significantly evolved
with age. Such stability is remarkable as it contrasts with the
severe axon pathology and progressive neuropathies associated
with many myelin gene mutants (for a review, see Nave, 2010).
The stability of this phenotype provides a unique opportunity to
investigate the regulation of peripheral nerve fiber maturation in
the amyelination context and also renders these mutant mice of
particular value in delineating the molecular pathways that
connect N-WASp and/or N-WASp-modulated cytoskeletal
remodeling to myelination and the control of myelin gene
expression.
Acknowledgements
This work was supported by grants from the Canadian Institute for Health
Research (MOP12136) to K.A.S. and the Multiple Sclerosis Society of Canada
to K.A.S. and A.C.P. K.A.S. holds a Canada Research Chair and the Sherman
Family Chair in Genomic Medicine.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058677/-/DC1
Development 138 (7)
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N-WASp is required for PNS myelination