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The Journal of Neuroscience, March 15, 2001, 21(6):2039-2047
A Unique Role for Fyn in CNS Myelination
Brian R.
Sperber1, 2,
Éilis A.
Boyle-Walsh1,
Mark J.
Engleka1,
Paul
Gadue1,
Alan C.
Peterson4,
Paul L.
Stein1,
Steven S.
Scherer3, and
F. Arthur
McMorris1, 2
1 The Wistar Institute, Philadelphia, Pennsylvania
19104, 2 Graduate Group in Neuroscience and
3 Department of Neurology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104, and 4 Department of
Neurology and Neurosurgery, Royal Victoria Hospital, McGill University,
Montreal, Canada H3A 1A1
 |
ABSTRACT |
We analyzed the role of Fyn tyrosine kinase in CNS myelination by
using fyn
/ null mutant mice,
which express no Fyn protein. We found a severe myelin deficit in
forebrain at all ages from 14 d to 1 year. The deficit was maximal
at 1 month of age and was similar regardless of mouse strain background
or whether it was determined by bulk isolation of myelin or by
quantitation of myelin basic protein. To determine the cellular basis
of the myelin deficit, we counted oligodendrocytes in tissue sections
of mice expressing oligodendrocyte-targeted 
-galactosidase, and we
used light and electron microscopy to examine the number and morphology
of myelinated fibers and size of myelinated CNS structures. All of
these parameters were reduced in
fyn/ mice. Unexpectedly, there
were regional differences in the myelin deficit; in contrast to
forebrain, fyn/ cervical spinal
cord exhibited no reduction in myelin content, number of
oligodendrocytes, or number of myelinated fibers, nor was myelination
delayed developmentally. We found that oligodendrocytes express Src,
but there was no significant reduction of myelin content in null
mutants lacking the Fyn-related kinases Src, Yes, or Lyn. Finally, we
investigated the molecular features of Fyn that are required for
myelination and found that a single amino acid substitution, which
abolishes the tyrosine kinase activity of Fyn, resulted in a myelin
deficit as great as that observed in the complete absence of Fyn
protein. These results demonstrate that Fyn plays a unique role in
myelination, one that requires its kinase activity.
Key words:
myelin; oligodendrocyte; corpus callosum; spinal cord; tyrosine kinase; Fyn; Src; Lyn; Yes; development; knock-out
| |
INTRODUCTION |
The regulation of myelination
by oligodendrocytes in the CNS and Schwann cells in the PNS is poorly
understood. We studied the possible role of Fyn, Src, Yes, and Lyn,
four nonreceptor protein tyrosine kinases of the Src family, in
myelination in the CNS.
Src family kinases have long been implicated in the regulation of cell
growth and differentiation, and the family members Src, Fyn, Yes, Lyn,
and Lck are expressed in the nervous system (Thomas and Brugge, 1997
).
Recent studies indicate that Fyn plays an important role in brain
development and physiology; Fyn mutant mice exhibit disrupted
hippocampal architecture, abnormal long-term potentiation, impaired
spatial learning, and increased fearfulness and sensitivity to ethanol
(Grant et al., 1992; Miyakawa et al., 1994, 1997). Fyn also plays a
role in CNS myelination; mice homozygous for a Fyn--galactosidase
(-gal) fusion protein are hypomyelinated at 1 month of age
(Umemori et al., 1994). Fyn is found in many brain areas, including
glial cells in white matter tracts and in cultured oligodendrocytes,
and its activity in brain is highest during the developmental period
approximately corresponding to the peak of myelination (Bare et al.,
1993; Yagi et al., 1993; Umemori et al., 1994; Osterhout et al., 1999).
Evidence suggests that the cell surface proteins myelin-associated
glycoprotein (MAG), F3, and 120 kDa neural cell adhesion molecule
(NCAM120) may act as receptors coupled to Fyn (Umemori et al., 1994;
Kramer et al., 1999).
Although Fyn has been shown to be important for CNS myelination,
many important questions have not been addressed. Previous studies on
Fyn mutants have been restricted to the first 2 months of age, raising
the possibility that the hypomyelination only represents a temporary
developmental delay. Also, previous studies have used Fyn mutants,
which still express a fragment of the Fyn protein, raising the
possibility that the fragment may bind to other molecules and disrupt
their function rather than act as a true null. Furthermore, it is not
known whether different CNS regions are equally affected in
fyn mutant animals, nor is it known whether the myelin
deficit results from a decrease in oligodendrocyte cell number or a
reduction in the amount of myelin produced per oligodendrocyte.
Finally, the possible role of other Src family kinase members in
myelination has not been studied.
In the present study, we analyzed myelination in mice containing null
mutations in each of four Src family kinases. In the fyn/ mice, we investigated
myelination at multiple time points, both by isolation and quantitation
of myelin and by quantitation of the major CNS myelin protein, myelin
basic protein (MBP). We used light and electron microscopy to analyze
the number and morphology of myelin sheaths in several CNS regions, and
we counted oligodendrocytes in several CNS regions to determine whether
the hypomyelination in fyn/ mice
is the result of decreased oligodendrocyte numbers or a reduction in
the amount of myelin produced by each oligodendrocyte. Finally, we
investigated whether the tyrosine kinase activity of Fyn is essential
for normal myelination.
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MATERIALS AND METHODS |
Mouse mutants and genotyping. Mice with mutations of
fyn (Stein et al., 1992), src (Soriano et al.,
1991), or yes (Stein et al., 1994) were bred, and homozygous
offspring were used for experiments. lyn/ mice (Chan et al., 1997)
were kindly provided by Dr. Ellen Puré (The Wistar Institute,
Philadelphia, PA). Homozygous mice in which the wild-type
fyn gene was replaced with a kinase-inactive mutant fyn gene (FynK296R) by homologous recombination have been
described elsewhere (P. L. Stein, unpublished data).
Homozygous transgenic mice that express Escherichia coli
-galactosidase under the control of the mouse myelin basic protein
promoter (referred to as Mbp-lacZ mice; A. C. Peterson,
unpublished data) were intercrossed with homozygous
fyn/ mice to produce
fyn/ mice that also express the
Mbp-lacZ transgene. Control mice for experiments
involving these mutant mice were littermates, which were wild type at
the fyn locus
(fyn+/+) and hemizygous or
homozygous for lacZ (expressing -gal). The specificity of
-galactosidase expression exclusively within oligodendrocytes was
verified by demonstrating the coexpression of -galactosidase with
multiple oligodendrocyte-specific markers in CNS tissue sections (A. C. Peterson, unpublished data). Wild-type 129/Sv,
C57BL/6J, or wild-type littermates of mutant mice were used as
controls, as described in the individual experiments. Genotypes of
src/ mice were determined
according to methods described previously (Thomas et al., 1995).
fyn/ mutant mice were genotyped
using the following primers: Fyn reverse, 5'-GCAAAACAACCCACACAGAG-3';
Fyn forward, 5'-AGCGAAACTGACAGAGGAGA-3'; and Neo2,
TGGCTACCCGTGATATTGCT. The wild-type allele produces a 600 bp product,
whereas a 450 bp product is indicative of the mutant allele. Mice
bearing the Mbp-lacZ transgene were identified by PCRbased
genotyping using the following primers coding for a
PCRamplification product of ~600 bp: Rob-1,
5'-GAAAACCCTGGCGTTACCCAACTT-3'; and Rob-4,
5'-CTGAACTTCAGCCTCCAGTACAGC-3'. Mice were housed and all
experimental procedures were conducted according to institutional, National Institutes of Health, and Institutional Animal Care and Use
Committee guidelines and regulations.
Statistical analysis. Statistical analysis of data were
performed using Student's t test.
Determination of brain myelin content. Myelin from mouse
forebrain was purified and quantitated by sucrose density gradient centrifugation, essentially as described by Norton and Poduslo (1973). In brief, mice were killed by
CO2 inhalation, and the forebrain was quickly
removed, weighed, and frozen in liquid nitrogen. The forebrain was
defined by a coronal cut at the anterior border of the cerebellum. All
brain structures rostral to that cut were included for determination of
myelin content. For myelin isolation, the frozen brains were
homogenized in 0.32 M sucrose and centrifuged on
a 0.32:0.85 M discontinuous sucrose gradient at
82,500 × g for 30 min. Myelin was collected from the
interface, osmotically shocked in deionized water to release
contaminants from within myelin vesicles, and recentrifuged over a
second discontinuous sucrose gradient as above. The myelin was again
collected from the interface and repeatedly washed with deionized water
and pelleted to remove residual sucrose. The myelin was frozen at
80°C, lyophilized in tared tubes, and weighed.
Western blot analysis. Mice were killed by
CO2 inhalation, and the forebrain was removed and
homogenized in lysis buffer (1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 57 µg/ml aprotinin, 1 mM sodium orthovanadate, and 100 µM leupeptin, in PBS). Homogenate was
then incubated on ice for 30 min and centrifuged at 10,000 rpm for 10 min at 4°C. Equal amounts of lysate (40 µg of protein) were
separated by electrophoresis on 12% SDS-PAGE and transferred to
polyvinylidene difluoride membranes. After blocking overnight in
blocking solution (75 mM NaCl, 3% BSA, and 30 mM Tris, pH 7.6), the membranes were probed with
antibodies against MBP (McMorris et al., 1981), neurofilament light
chain (NF-L) (Dr. Virginia Lee, University of Pennsylvania), and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Chemicon, Temecula,
CA), followed by treatment with
125I-labeled protein A (NEN Life Science
Products, Boston, MA). The amount of MBP, NF-L, and GAPDH was
quantified using a Molecular Dynamics (Eugene, OR) PhosphorImager.
For Western blot analysis of Src, immunopurified oligodendroglial cells
(see below) were washed with cold PBS, and protein extracts were
prepared in the lysis buffer described above. Equal amounts of lysate
(70 µg of protein) were separated on 8% SDS-PAGE and transferred as
above. After blocking overnight, the filters were probed with a
monoclonal antibody against Src (1:500) (Oncogene Sciences, Uniondale,
NY) and then incubated with 15 µg/ml rabbit anti-mouse IgG (Rockland
Immunochemicals, Gilbertsville, PA). Then membranes were incubated with
125I-labeled protein A, and bands were
visualized using a Molecular Dynamics PhosphorImager.
-Galactosidase staining. Mice were deeply
anesthetized and transcardially perfused with 0.9% NaCl, followed by
fixative (0.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5). Brains and
cervical spinal cords were then removed, further dissected to isolate
only the desired tissue, and incubated in fixative for 1 hr at 4°C.
Tissue blocks were then immersed in 30% sucrose in 0.1 M sodium phosphate buffer, pH 7.5, for 48 hr at
4°C, embedded in OCT freezing medium (Tissue Tek, Miles Inc.,
Elkhart, IN), and frozen in an acetone-dry ice slurry. Cryosections (6-µm-thick) were cut and allowed to adhere to glass slides
(SuperFrost Plus; Fisher Scientific, Pittsburgh, PA). Sections then
were dried for 15 min at 37°C and incubated in
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal) staining solution [0.4 mg/ml X-gal (Boehringer
Mannheim, Indianapolis, IN), 3 mM potassium
ferricyanide, 3 mM potassium ferrocyanide, and 2 mM MgCl2, in 0.1 M sodium phosphate buffer, pH 7.5] for 30 min at
37°C, after which they were washed with PBS, mounted under coverslips
in aqueous mounting medium, and observed by conventional light
microscopy. The cross-sectional areas of spinal cord and corpus
callosum were determined by capturing a light microscope image of the
structure with a digital camera and determining its area in pixels,
using Adobe Photoshop (Adobe Systems, San Jose, CA). A microruler was
observed and measured by the same procedure to convert area in pixels
to square millimeters.
Electron microscopy. Postnatal day 0 (P0), P14, and P28 mice
were deeply anesthetized and transcardially perfused with 0.9% NaCl,
followed by 3% glutaraldehyde in 0.1 M sodium
phosphate buffer, pH 7.5. Brain, optic nerve, and cervical spinal cord
were then removed, trimmed to minimize excess tissue, and incubated overnight in 3% glutaraldehyde in 0.1 M sodium
phosphate buffer, pH 7.5. Tissue blocks were then washed in 0.1 M sodium phosphate buffer and incubated 1.5 hr in
2% osmium tetroxide. Blocks were washed in 0.1 M
sodium phosphate buffer, pH 7.5, dehydrated in ethanol, and
equilibrated in Epon (44.2% Embed 812, 35.4% dodecenylsuccinic anhydride, 17.7% nadic methyl anhydride, and 2.7%
benzyldimethylamine). Sections (0.5-µm-thick) for light microscopy
were cut using an ultramicrotome and stained with toluidine blue. Thin
sections for electron microscopy were cut using an ultramicrotome,
stained with 4 mg/ml uranyl acetate in 95% ethanol and 0.4 mg/ml lead citrate, and analyzed using a Zeiss (Oberkochen, Germany) electron microscope. For quantitative analysis of the number of myelinated fibers in the corpus callosum, we counted myelinated fibers and the
total number of axons in 10 closely spaced, nonoverlapping fields of
1680 and 340 µm2, respectively, from
sagittal sections in the genu of the corpus callosum.
In vitro preparation of purified mouse oligodendroglial
cells. Postnatal day 1 C57BL/6J or
fyn/ mouse pups were
decapitated, and their forebrains were removed and minced in wash
medium (25 mM HEPES, pH 7.3, 100U/ml penicillin, and 100 µg/ml streptomycin, in HBSS) containing 14.3 µg/ml
DNase I. Minced suspension was then digested with 1% trypsin in
calcium-free PBS for 70 min in a 37°C incubator with shaking at 250 rpm. The suspension was then centrifuged at 500 rpm (75 × g) for 10 min, resuspended in wash medium containing 14.3 µg/ml DNase I, and centrifuged again for 10 min. Cells were then
resuspended in OM-5 culture medium (Raible and McMorris, 1990), which
contains 10% serum, sequentially passed through two 70 µm pore-sized
cell strainers (Falcon Labware, Lincoln Park, NJ), plated in 75 cm2 tissue culture flasks at 30 × 106 cells per flask, and incubated in 10%
CO2-90% air at 37°C.
Oligodendrocytes and their precursors were purified by immunopanning
using monoclonal antibodies against cell surface antigens, essentially
as described previously (Barres et al., 1992; Vemuri and McMorris,
1996). Ran-2, an antibody that recognizes astrocytes (Eisenbarth et
al., 1979; Sommer and Schachner, 1981), was prepared as a tissue
culture supernatant from hybridoma cells. Briefly, an enriched
population of oligodendrocytes and precursor cells was isolated by
differential shake off of 7-d-old mouse glial cell cultures essentially
as described by McCarthy and de Vellis (1980). The cell
suspension was then incubated for 30 min intervals over a series of
three bacteriological Petri dishes (Fisher Scientific) coated with
Ran-2 monoclonal antibody. During this time, astrocytes (which express
the Ran-2 surface antigen) and microglia [which are very adhesive and
bind to immunoglobulins via their F(c) receptors] attached to the
Ran-2 antibody on the surface, whereas oligodendroglia remained in
suspension. A sample of nonadherent cells was incubated for 30 min with
1 µM prostaglandin E1
(PGE1) and 1 µM
PGE2 and immunostained with antibody against cAMP
to determine whether astrocytes or microglia were still present.
Previous work from our laboratory has shown that oligodendroglia do not
elevate cAMP in response to this treatment, whereas
non-oligodendroglial cells in the cultures (astrocytes and microglia)
respond robustly and are intensely immunostained (A. P. Wiemelt and
F. A. McMorris, unpublished observations). No cells (<1
of 2000) were positive for cAMP immunofluorescence. The
remaining nonadherent cells were centrifuged at 500 rpm (75 × g) for 10 min and prepared for Western blot analysis as
described above.
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RESULTS |
Mice that lack Fyn are irreversibly myelin-deficient
As described in detail previously (Stein et al., 1992),
fyn/ mutant mice were derived by
disrupting the normal fyn gene using a targeting construct
that deleted exon 2, the first coding exon, which contains the
initiator methionine. The resulting
fyn/ mice express no detectable
Fyn protein (Stein et al., 1992). We analyzed myelin content of
forebrain in fyn/ and
age-matched C57BL/6J (B6) control mice at time points ranging from P14
to P385 and found a myelin deficit at all time points analyzed (Fig.
1A). The deficit was
most pronounced at postnatal day 26 (52%) and persisted even at ages
beyond 1 year. From P55 to P385, the myelin deficit remained relatively
constant and was significantly different from controls at all time
points, with the exception of P283 (Fig. 1A).
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Figure 1.
Myelin content is reduced in forebrain
of fyn/ mice but not in
src/,
yes/, or
lyn/ mice. A,
Myelin content is reduced in fyn/
mouse forebrain. Myelin from C57BL/6J,129Sv
fyn/ or C57BL/6J
fyn+/+ control mouse forebrain was
isolated by sucrose density centrifugation and quantified at P14, P26,
P55, P95, P283, or P385. Each point represents at least
six mice. Data are expressed as milligrams of myelin (dry weight) per
gram of brain (wet weight). fyn/
values are significantly different from control at days P26, P55, P95,
and P385 (p < 0.05). B,
Western blot analysis of MBP content in
fyn/ forebrain and spinal cord.
Samples of lysate containing 30 µg of protein from P28
fyn/ and control mouse forebrain
or spinal cord were separated on a 12% polyacrylamide gel and
transferred to a PVDF membrane, and the blot was probed with antibodies
against NF-L, GAPDH, and MBP, followed by 125I-protein A. Bands were visualized by autoradiography. C, The amount
of radioactivity in individual bands of the blot shown in
B was quantified by PhosphorImager analysis, and
the amount of MBP or NF-L was normalized to the amount of GAPDH in the
same lane. Each bar represents the
mean ± SEM normalized MBP or NF-L from four different
animals, expressed as percent of that in
fyn+/+ controls. The amount of MBP
and NF-L is significantly different from controls in forebrain of
fyn/ mice
(p < 0.01 and p < 0.05, respectively) but not in spinal cord
(p > 0.5). D, Src is
expressed in oligodendrocytes. Samples of lysate containing 80 µg of
protein from B6 mouse forebrain (WT brain),
src/ mouse forebrain, highly
purified B6 oligodendrocytes (WT oligos), and highly
purified fyn/ oligodendrocytes
were separated on a 12% polyacrylamide gel and transferred to a PVDF
membrane, and the blot was probed with monoclonal antibody against Src.
Bands were visualized by autoradiography. Src immunoreactivity is
evident in all samples except lysate from
src/ mouse forebrain.
E, Myelin is reduced in forebrain of
fyn/ mice but not in forebrain of
src/,
yes/, or
lyn/ mice. Myelin was isolated
from forebrain of fyn/,
src/,
yes/,
lyn/, and control mice by sucrose
density gradient centrifugation and quantified. Data, calculated as
milligrams of myelin (dry weight) per gram of brain (wet weight), are
shown as percent of control. Each bar represents the
mean of at least five mice; error bars represent SEM. The myelin
content of B6,129 fyn/ mouse
forebrain at P26 is shown relative to age-matched B6 control mice. In
addition, myelin content of strain 129Sv
fyn/ mouse forebrain at P26 is
shown compared with age-matched 129Sv control mice. In both genetic
backgrounds, myelin content was significantly less in
fyn/ mice than in controls
(p < 0.05). Myelin content of
src/,
yes/, and
lyn/ mice at day P28 is shown
compared with that of age-matched control mice. There is no significant
difference in myelin content when comparing mice lacking Src, Yes, or
Lyn and control mice (p > 0.05).
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The fyn/ mutant mice we used
were derived from 129Sv embryonic stem cell chimeras that were crossed
to B6 mice to produce hybrid animals, which were then maintained by
interbreeding. Thus, the genotype of the mutant mice contained
contributions from the B6 and 129/Sv (129) strains but was not inbred
or even uniform between littermates, making it problematic to select an
appropriate strain for the control animals. For most experiments, we
used age-matched inbred B6 mice as controls. Because the myelin content
data demonstrated a clear difference between the inbred B6 controls and
the non-inbred fyn/ B6,129 mice,
and because the SEs at a given age were of a similar magnitude
within the control and fyn/
groups, we thought it unlikely that the observed myelin deficit was
primarily attributable to strain differences. To address this issue further and determine whether the myelin deficit is indeed the
result of differences at the fyn locus, we also quantitated myelin content of fyn/ and
fyn+/+ mice that were maintained on
the inbred 129Sv background (Stein et al., 1992). These
fyn/ 129 mice were analyzed for
myelin content at P26 and compared with inbred 129 control mice of the
same age. As shown in Figure 1E, the
fyn/ 129 mice had a 37% deficit
in forebrain myelin content at P26 (p < 0.005),
in agreement with the 52% deficit observed in the fyn/ B6,129 mice at the same age
(Fig. 1E). Thus, the difference in myelin content
observed in fyn/ mice compared
with controls is attributable to differences at the fyn
locus and not to differences in genetic background.
Myelin content was also assessed in whole homogenates by quantitation
of the amount of MBP, a myelin-specific protein that accounts for
30-35% of the total protein of CNS myelin (Lees and Brown, 1984). MBP
was assayed on Western blots using P28 forebrain or spinal cord
homogenate from fyn/ and B6
control mice. Blots were probed with a combination of antibodies
against MBP, the neuron-specific protein NF-L and the ubiquitously
expressed GAPDH. Brain GAPDH content per milligram of protein was
unchanged in fyn/ mouse brain
and spinal cord (both, p > 0.1) when compared with B6
controls (data not shown). When the amount of MBP was normalized to
GAPDH, MBP was found to be significantly reduced in
fyn/ brain compared with B6
controls (p < 0.01) (Fig.
1B,C), and the magnitude of the
deficit (40%) was comparable with that determined by myelin isolation
(52%). A similar deficit was observed when the amount of MBP was
normalized to protein loading (data not shown). Therefore, the myelin
deficit determined by quantitation of purified myelin cannot be
attributed to errors encountered during myelin isolation and
purification. Unexpectedly, the amount of NF-L per milligram of protein
was reduced by 18.3% in brains of
fyn/ mice compared with B6
controls (p < 0.01) (Fig.
1B,C).
Both methods of quantitation of myelin content revealed a deficit of
myelin content in forebrain. In contrast, MBP content in P28
fyn/ mice was not significantly
reduced in spinal cord, regardless of whether the data were normalized
to GAPDH (p > 0.5) (Fig.
1B,C) or to total protein
(p > 0.1) (data not shown).
Fyn is unique among Src-family kinases in being essential
for myelination
Fyn is one of nine known members of the Src family of protein
tyrosine kinases, which are highly similar in amino acid sequence, except in the N-terminal "unique" domain (Brown and Cooper,
1996). In addition to Fyn, four other Src family members, Src, Yes,
Lyn, and Lck, have been shown to be expressed in brain (Thomas and Brugge, 1997); Fyn and Lyn are expressed in oligodendrocytes (Kramer et
al., 1999; Osterhout et al., 1999). Therefore, we sought to determine
whether Src, Yes, or Lyn are essential for normal myelination. Here we
demonstrate, by Western blot analysis of lysates of highly purified
cultures of B6 mouse oligodendrocytes, that Src is also expressed in
oligodendrocytes (Fig. 1D). In these cultures, no astrocytes or microglia could be detected (less than 1 of 2000 cells
scored) by the highly sensitive method of cAMP immunofluorescence of
PGE1/PGE2-stimulated cells
(see Materials and Methods). The specificity of the Src antibody was
demonstrated by the lack of a signal in
src/ brain samples (Fig.
1D).
Analysis of the myelin content of
src/,
yes/, and
lyn/ mice at P28 revealed that
there is no significant myelin deficit in these mutants (Fig.
1E). Therefore, among Src family kinases, Fyn plays a
unique role in myelination.
Morphological analysis of myelin in
fyn/ CNS
To determine whether myelin structure, as well as myelin content,
is compromised in fyn/ animals,
we analyzed P28 fyn/ mouse
myelin profiles in three separate CNS white matter areas: corpus
callosum, optic nerve, and spinal cord. To ensure that we were
analyzing similar regions in each animal, we focussed our analysis on
discrete locations within each structure; in the brain, we analyzed
sagittal sections of the genu of the corpus callosum, and in the spinal
cord, cross-sections at the level of the cervical enlargement. The
optic nerve is sufficiently small and homogeneous so that we analyzed
entire cross-sections of its intracranial segment.
In sagittal sections of corpus callosum from P28
fyn/ mice, we observed a
widespread reduction in the number of myelin sheaths (Fig.
2). In 10 closely spaced, nonoverlapping
fields from the genu, we observed a 77% reduction in the number of
myelin profiles (p < 0.0001) (Fig.
2E). This reduction was not the result of an overall decrease in the number of axons, because the percent of axons
that were myelinated also was markedly reduced, by 89%. Although the
number of myelin sheaths was reduced, they had a normal ultrastructure
(Fig. 2C). Moreover, the size distribution of axons appeared
to be normal, and myelin sheath thickness appeared appropriate for
axonal caliber (Fig. 2).
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Figure 2.
The corpus callosum is hypomyelinated in
fyn/ mice. Electron micrographs
of the genu of the corpus callosum, in midline sagittal sections of
control (B, D) and
fyn/ (A,
C) mice at 28 d of age. The reduction in number of
myelin sheaths in the fyn/ mice
is evident (compare A with B, and
C with D; arrows indicate
representative myelin sheaths). Numerous unmyelinated axons
(arrowheads) are evident in both
fyn/ and
fyn+/+ mouse corpus callosum
(C, D), but they account for a greater
proportion of total axons in the
fyn/ mice. Scale bars:
A, B, 10 µm; C,
D, 2 µm. E, Myelinated fibers and the
total number of axons were counted in 10 randomly selected,
nonoverlapping fields of 1680 µm2 (myelinated
fibers) and 340 µm2 (axons) from midline sagittal
sections in the genu of the corpus callosum. The number of myelinated
axons per square millimeter and the percent of total axons that are
myelinated were calculated and are shown in the figure as percent of
control. Values represent the mean ± SEM of data from three
different animals of each genotype. Both the number of myelin profiles
and the percent of axons that are myelinated are significantly reduced
in the fyn/ mouse corpus callosum
when compared with controls (p < 0.0001).
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Analysis of optic nerve from
fyn/ mice revealed a 15%
reduction in cross-sectional area relative to controls
(p < 0.005), consistent with a reduction in
myelin content, although no striking reduction in abundance of myelin
profiles comparable with that in corpus callosum was apparent (Fig.
3). However, in morphology,
fyn/ mouse myelin profiles were
indistinguishable from control (Fig. 3).
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Figure 3.
Myelination in the optic nerve of
fyn/ and control mice. Light
micrographs of the intracranial portion of the optic nerve, in
0.5-µm-thick toluidine blue-stained cross-sections of
fyn/ (A,
C) and control (B, D) mice
at 28 d of age. The significant reduction in cross-sectional area
in fyn/ mice
(p < 0.005) is evident at low-power
magnification (A, B). The morphology of
myelin from fyn/ mice appears
indistinguishable from control at higher magnification
(C, D). Each micrograph is representative
of sections from three different animals. Scale bars: A,
B, 50 µm; C, D, 10 µm.
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To determine whether there were structural abnormalities in
myelin from the spinal cord of
fyn/ mice, we analyzed white
matter tracts at the level of the cervical enlargement. At P28, we
observed no decrease in the abundance of myelin sheaths, and no
morphological deficits (Fig.
4E,F). The dorsal, ventral, and lateral funiculi all appeared normal. Similar
results were observed at P14 (Fig. 4C,D).
Although much less myelin was present in both controls and knock-outs
at this earlier time point, no morphological abnormalities could be
detected. To determine whether there is a delay in the onset of
myelination in the absence of Fyn,
fyn/ and control, mouse spinal
cords were analyzed at P0, the age at which myelin profiles can
first be detected in control mouse spinal cord. Myelin profiles
were present in fyn/ spinal
cord at P0 (Fig. 4A,B) and appeared
indistinguishable from control in both their abundance and
morphology. Therefore, the development, onset, and maturation of myelin
sheaths in the cervical spinal cord was unaffected in
fyn/ mice.
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Figure 4.
Myelination in spinal cord of
fyn/ and control mice. Light
micrographs of the ventral funiculus in the cervical enlargement of the
spinal cord, in 0.5-µm-thick toluidine blue-stained cross-sections
from fyn/ (A,
C, E) and control (B,
D, F) mice. Myelin profiles are
evident in both fyn/ and control
mouse ventral funiculus at P0 (arrows in
A and B). Similarly, sections of ventral
funiculus from P14 (C, D) and P28
(E, F) mice demonstrate that there
is no appreciable difference in myelin sheath abundance or morphology
in fyn/ mice (C,
E) compared with controls (D,
F). Each micrograph is representative of sections
from three different animals. Scale bar, 20 µm.
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Oligodendrocyte numbers are reduced in
fyn/ corpus callosum but not in
spinal cord
The deficit in myelin content of
fyn/ mice might be the result of
a reduction in the number of oligodendrocytes, a reduction in the
amount of myelin elaborated by an individual oligodendrocyte, or both.
To investigate this issue, we analyzed oligodendrocyte number in
selected CNS areas of fyn/ and
control mice. To facilitate identification and accurate counting of
oligodendrocytes, we used transgenic mice in which expression of
bacterial lacZ gene (which encodes -gal) was
targeted specifically to oligodendrocytes with a mouse MBP promoter
transgene. A nuclear localization signal was placed in-frame with
-gal, and as shown in Figure 5,
-gal activity was clearly evident in the nuclei of cells expressing
the transgene and was also detected in the soma and to a lesser extent
in cellular processes and myelin. Mbp-lacZ transgenic mice
were intercrossed with fyn/ mice
to produce mice that express -gal in their oligodendrocytes and are
null (/) at the fyn locus. Cryosections of forebrain and
spinal cord were stained with X-gal, and the total number of
oligodendrocytes was counted in each section.
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Figure 5.
Oligodendrocyte number and
myelination are reduced in the corpus callosum of
fyn/ mice. A-D,
Light micrographs of 10-µm-thick coronal cryosections from
fyn/ (A,
C) and control (B, D)
mice. Control and fyn/ mice carry
an Mbp-lacZ transgene that targets -galactosidase
expression to oligodendrocytes, resulting in dark staining of
oligodendrocyte nuclei and cell bodies and lighter staining of myelin
in the X-gal-stained sections. Darkly stained oligodendrocytes are
concentrated in the corpus callosum and surrounding white matter
regions (arrows in A and
B). Fewer oligodendrocytes are apparent in
fyn/ corpus callosum
(cc) at the level of the ventral hippocampal commissure
(compare A with B). Similarly, reduced
numbers of oligodendrocytes are evident at the level of the habenula,
in which a reduction in corpus callosum thickness is also apparent
(compare C with D). Micrographs are
representative of sections from three different animals. Scale bar, 100 µm. E, The number of oligodendrocytes is reduced in
fyn/ corpus callosum but not in
spinal cord. Oligodendrocytes were counted in cervical spinal cord and
corpus callosum of control and
fyn/ mice, which expressed
-galactosidase from the Mbp-lacZ transgene. In spinal
cord, every oligodendrocyte was counted in 10 nonadjacent 6-µm-thick
cross-sections of the cervical enlargement of the spinal cord. The
number of oligodendrocytes in corpus callosum was determined by
counting oligodendrocytes in five nonadjacent 6-µm-thick coronal
cross-sections. Counts were made in
fyn/ and control sections at
corresponding levels along the rostrocaudal axis of the corpus
callosum. The cross-sectional areas of spinal cord and corpus callosum
were determined by capturing a light microscope image of the structure
with a digital camera and determining its area in pixels, using Adobe
Photoshop. A microruler was observed and measured by the same procedure
to convert area in pixels to square millimeters. There was a
significant reduction in the number of oligodendrocytes per square
millimeter in fyn/ mouse corpus
callosum (p < 0.005) but not in spinal cord
(p > 0.5) (left). Similarly,
there was no reduction in the number of
fyn/ spinal cord
oligodendrocytes when represented as the total number of
oligodendrocytes per cross-section (p > 0.1) (right). Each bar represents the
mean of data from 5 or 10 sections from each of three different animals
of each genotype. Error bars represent SEM.
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In coronal sections of forebrain, we observed a significant 29%
reduction in the average number of oligodendrocytes per section within
a fixed width (0.8 mm at the midline) of the corpus callosum (p < 0.005) (Fig. 5E). In addition,
the thickness of the corpus callosum was reduced in all
fyn/ mice analyzed (Fig.
5A,C), but the number of mice
analyzed was insufficient for statistical testing of this parameter. In
contrast, in cervical spinal cord, the number of oligodendrocytes was
not detectably different between
fyn/ and littermate control mice
(Fig. 5E). When expressed either as the number of
oligodendrocytes per cross-section of spinal cord or the number of
oligodendrocytes per square millimeter, there was no significant
difference between fyn/ mice and
littermate controls (p > 0.2 and
p > 0.4, respectively) (Fig. 5E).
The myelin deficit in fyn/ mice
results from the absence of the kinase activity of Fyn
Because of the important role of protein phosphorylation in
signaling and cellular regulation, studies on the function of Fyn and
other protein tyrosine kinases usually focus on their kinase activity.
However, there is increasing evidence that many kinases, including Src
family kinases, also may have other important functions in cells
[e.g., as adapter molecules, via their Src homology 2 (SH2),
SH3, or "unique" domains, or by competing with other molecules for
binding to putative partners]. To determine whether the tyrosine
kinase activity of Fyn is essential for normal myelination, we analyzed
myelin content in mice expressing full-length Fyn containing a point
mutation at amino acid 296 (lysine to arginine), which prevents ATP
binding, thus abolishing kinase activity. Brains from these Fyn K296R
mice were analyzed for myelin content. Relative to B6 control, myelin
content in Fyn K296R mouse brain was reduced by 66%
(p < 0.0005) (Fig.
6), which is similar to the deficit observed in the complete absence of Fyn protein (Fig. 1).
Therefore, the kinase activity of Fyn is necessary for normal
myelination.
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Figure 6.
Fyn kinase activity is required for normal
myelination. Myelin was isolated and quantified from kinase-deficient
FynK296R mutant and age-matched B6 control mouse forebrain at 28 d
of age. Myelin content, calculated as milligrams of myelin (dry weight)
per gram of brain (wet weight), is shown as percent of control. Each
bar represents the mean ± SEM value from six
animals.
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DISCUSSION |
In the present study, we analyzed myelination in the complete
absence of Fyn, using fyn/ mice
that make no detectable Fyn protein (Stein et al., 1992). We
demonstrated that Fyn is unique among Src kinases in that it is
necessary for normal forebrain myelination, whereas the closely related
molecules Src, Yes, and Lyn are not. We have further demonstrated that
the corpus callosum of fyn/ mice
has fewer oligodendrocytes and drastically reduced numbers of myelin
sheaths, whereas spinal cord has a normal number of oligodendrocytes
and is normally myelinated. Finally, we show that Fyn kinase activity
is required for normal myelination.
The myelin deficit in fyn/
mice varies by CNS region
Using several different approaches, analysis of myelin content,
analysis of MBP content, and light and electron microscopy, we
demonstrated that the fyn/
forebrain is hypomyelinated. Surprisingly, the myelin in cervical spinal cord appears unaffected. At all ages analyzed, there were no
evident abnormalities in myelination of the spinal cord in fyn/ mice, demonstrating that
the time of onset and rate of myelination are not affected. Our
observation of normal myelination in spinal cord is in disagreement
with a recent report that myelin sheath thickness is dramatically
decreased in the spinal cord of mice expressing a different Fyn allele
(Umemori et al., 1999). Because different tracts within the spinal cord
are myelinated at different times during development (Schwab and
Schnell, 1989) and because the timing of myelination is highly
dependent on position along the length of the spinal cord (Schwab and
Schnell, 1989), we made all of our measurements in the ventral
funiculus within the cervical enlargement. Because the anatomical
location of the measurements was not specified in the previous study
(Umemori et al., 1999), it is unclear whether the differences in
myelination were attributable to the effect of the fyn
mutation or simply to normal developmental differences. Alternatively,
it is possible that the abnormal myelin morphology in the spinal cord
from the fyn mutant mouse described by Umemori et al. (1999)
represents a more severe phenotype resulting from the expression, in
those mice, of a truncated Fyn protein that may have dominant-negative
or toxic activity, which would not be present in our
fyn/ mice that lack any
expression of Fyn protein (Stein et al., 1992).
Despite normal myelin content within the spinal cord of
fyn/ mice, we found a large
deficit in forebrain. Analysis of
fyn/ corpus callosum revealed a
29% reduction in the number of oligodendrocytes and a more extensive,
77%, reduction in the number of myelinated fibers. This discrepancy
suggests that the fyn/
oligodendrocytes in the corpus callosum are deficient in their ability
to myelinate the appropriate number of axons. This possibility is
consistent with the observation that Fyn is important for the development of the extensive network of processes characteristic of
mature oligodendrocytes (Osterhout et al., 1999; Sperber and McMorris,
2001). Thus, a deficit in process outgrowth could result in a decrease
in the number of axons that each oligodendrocyte could successfully
myelinate. Three oligodendroglial cell-surface molecules have been
identified that may contribute to this oligodendrocyte-autonomous phenotype: MAG (Umemori et al., 1994), NCAM120, and F3 (Kramer et al.,
1999). In addition, Fyn is required for normal insulin-like growth
factor I signaling in oligodendrocytes (Sperber and McMorris, 2001). It is possible that the absence of Fyn results in deficient signaling through some or all of these adhesion and signaling molecules, thus disrupting normal oligodendrocyte function.
Recently, Biffiger et al. (2000) reported that the optic nerve of
fyn/ mice is hypomyelinated,
consistent with our observation of a significant decrease in optic
nerve cross-sectional area and of forebrain hypomyelination, and also
reported that myelin sheaths of
fyn/ mice are indistinguishable
from control by light and electron microscopy, in agreement with our
findings. No apparent reduction in oligodendrocyte number was observed
in optic nerve, similar to our observation in spinal cord but very
different from our observation in corpus callosum. Additionally, in
double-mutant mice
(fyn//mag/),
Biffiger et al. (2000) observed regional differences in phenotype; myelination was unaffected in fasciculus gracilis and cuneatus or
ventral funiculus of spinal cord but was reduced in corticospinal tract
and optic nerve.
It is not clear, however, why the absence of Fyn from oligodendrocytes
would give rise to a dramatic myelin deficiency within some regions of
CNS (corpus callosum) and not others (spinal cord). Although the
initiation of myelination occurs in the spinal cord at a much earlier
time than in the corpus callosum, we found no myelin abnormalities,
even at early stages of myelination in spinal cord; therefore, the
regional variation cannot be attributed to intrinsic differences in the
timing of myelination. Interestingly, mice deficient in
platelet-derived growth factor A (PDGF-A) exhibit a different pattern
of regional differences: PDGF-A null mice are more severely
hypomyelinated in optic nerve and spinal cord than in corpus callosum
and cerebral cortex, the reverse of what we observed in our
fyn null mice (Fruttiger et al., 1999).
One possible explanation for the regional differences we observed is
that oligodendrocytes in forebrain require Fyn for normal myelination,
whereas those in spinal cord do not. In classic morphological studies,
Del Rio-Hortega (1928) proposed that there are distinct subpopulations
of oligodendroglia, and there are additional findings consistent with
this view. Forebrain and spinal cord oligodendrocytes have distinct
morphologies in neuron-free cultures, those in forebrain having a
greater number of processes (Bjartmar, 1998). Evidence has been
presented for two independent populations of oligodendrocyte precursors, one characterized by expression of PDGF 
-receptors and
the other by expression of mRNA for the myelin protein DM20 (Spassky et
al., 1998, 2000) (but see Fruttiger et al., 1999). If there are
separate lineages of oligodendrocytes, they may differ in the growth
factors and signaling pathways that regulate their development and function.
However, most evidence indicates that local neuronal factors dictate
which and how many axons are myelinated, as well as the amount of
myelin they receive. When optic nerve oligodendrocytes, which
ordinarily myelinate numerous small axons, are transplanted into spinal
cord, they myelinate only a few large axons (Fanarraga et al., 1998).
Additionally, the formation of compact myelin does not occur in the
absence of axons (Lubetzki et al., 1993). Thus, we must consider what
role neurons might be playing in the
fyn/ mouse myelin phenotype.
Because neurons also express relatively high levels of Fyn and because
there is an intimate reciprocal regulation between oligodendrocytes and
neurons, we cannot rule out the possibility that the lack of Fyn in
neurons contributes to the hypomyelinating phenotype, and the regional
differences in myelination arise from neuronal influences. Nonetheless,
because Fyn is active in oligodendrocytes (Osterhout et al., 1999) and is important for the normal morphological development of
oligodendrocytes in the absence of neurons (Osterhout et al., 1999;
Sperber and McMorris, 2001), we believe that the myelin deficit of
fyn/ mice results, at least in
part, from the absence of Fyn in oligodendrocytes.
The role of Fyn in myelination is unique among the Src family
of kinases
Of the nine known Src family kinase members, five family members,
Src, Fyn, Yes, Lyn, and Lck, are expressed in brain (Thomas and Brugge,
1997); of these, Fyn (Umemori et al., 1994; Osterhout et al., 1999) and
Lyn (Osterhout et al., 1999) have been reported to be expressed within
oligodendrocytes. A recent study performed in rat was unable to detect
Src or Yes in oligodendrocytes (Osterhout et al., 1999), but we
detected expression of Src in purified mouse oligodendroglial cells.
The apparent discrepancy in expression of Src is likely attributable to
differences in the methods rather than to species differences. Taking
these data together, at least three members of the Src family, Fyn,
Lyn, and Src, are expressed in oligodendrocytes. However, we found
that, in sharp contrast to the striking myelin deficit when Fyn is
absent, there is no detectable deficit in CNS myelination in the
absence of Lyn, Src, or Yes.
Fyn kinase activity is required for normal myelination
Fyn and the other Src family members possess protein tyrosine
kinase activity that plays a prominent role in their function as
signaling molecules. In comparison, many important signal transduction molecules are devoid of kinase or other known enzymatic activity but
instead act as adapter proteins; by virtue of SH2, SH3, or other
binding motifs, they assemble other proteins into specific functional
signaling complexes. Src family kinases, and indeed many other
signaling molecules with catalytic activity, also have binding motifs,
such as SH2 and SH3 domains, and there is accumulating evidence that
they can perform signaling functions independent of their enzymatic
activity (Xu and Littman, 1993; Kaplan et al., 1995; Schwartzberg et
al., 1997; Katsuta et al., 1998). In addition to its kinase domain, Fyn
has an SH2, an SH3, and a unique domain, all of which are believed to
be important for normal Fyn activity. In the case of CNS myelination,
however, we have shown that full-length, catalytically inactive Fyn is
not sufficient to rescue the
fyn/ mouse myelin deficit,
indicating that the function of Fyn in myelination requires its kinase activity.
| |
FOOTNOTES |
Received Aug. 7, 2000; revised Dec. 20, 2000; accepted Jan. 5, 2001.
This work was supported by grants from The National Multiple Sclerosis
Society and by National Institutes of Health Grant CA09171. S.S.S. was
supported by National Institutes of Health Grant NS34528. P.L.S. is
supported by National Institutes of Health Grants CA72806 and CA73796
and is a recipient of an Arthritis Foundation Investigator Award. We
thank Dr. Ted Xu, Suzanne McGettigan, Neil Morton, and Trina Fernandes
for their invaluable assistance.
Dr. Boyle-Walsh's present address: 1 Athlumney Castle, Navan, County
Meath, Ireland.
Dr. Engleka's present address: Graduate Group in Cellular and
Molecular Biology, University of Pennsylvania, Philadelphia, PA 19104.
Drs. Gadve's and Stein's present address: Department of Dermatology,
University of Pennsylvania, Philadelphia, PA 19104.
Correspondence should be addressed to Dr. F. Arthur McMorris, The
Wistar Institute, Philadelphia, PA 19104. E-mail:
mcmorris{at}wistar.upenn.edu.
| |
REFERENCES |
-
Bare DJ,
Lauder JM,
Wilkie MB,
Maness PF
(1993)
p59fyn in rat brain is localized in developing axonal tracts and subpopulations of adult neurons and glia.
Oncogene
8:1429-1436[ISI][Medline].
-
Barres BA,
Hart IK,
Coles HS,
Burne JF,
Voyvodic JT,
Richardson WD,
Raff MC
(1992)
Cell death and control of cell survival in the oligodendrocyte lineage.
Cell
70:31-46[ISI][Medline].
-
Biffiger K,
Bartsch S,
Montag D,
Aguzzi A,
Schachner M,
Bartsch U
(2000)
Severe hypomyelination of the murine CNS in the absence of myelin-associated glycoprotein and Fyn tyrosine kinase.
J Neurosci
20:7430-7437[Abstract/Free Full Text].
-
Bjartmar C
(1998)
Morphological heterogeneity of cultured spinal and cerebral rat oligodendrocytes.
Neurosci Lett
247:91-94[ISI][Medline].
-
Brown MT,
Cooper JA
(1996)
Regulation, substrates and functions of src.
Biochim Biophys Acta
1287:121-149[Medline].
-
Chan VW,
Meng F,
Soriano P,
DeFranco AL,
Lowell CA
(1997)
Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation.
Immunity
7:69-81[ISI][Medline].
-
Del Rio-Hortega P
(1928)
Tercera aportacion al conocimiento morfologica e interpretacion functional de la oligodendroglia.
Mem Real Soc Exapn Hist Nat
14:5-122.
-
Eisenbarth GS,
Walsh FS,
Nirenberg M
(1979)
Monoclonal antibody to a plasma membrane antigen of neurons.
Proc Natl Acad Sci USA
76:4913-4917[Abstract/Free Full Text].
-
Fanarraga ML,
Griffiths IR,
Zhao M,
Duncan ID
(1998)
Oligodendrocytes are not inherently programmed to myelinate a specific size of axon.
J Comp Neurol
399:94-100[ISI][Medline].
-
Fruttiger M,
Karlsson L,
Hall AC,
Abramsson A,
Calver AR,
Bostrom H,
Willetts K,
Bertold CH,
Heath JK,
Betsholtz C,
Richardson WD
(1999)
Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice.
Development
126:457-467[Abstract].
-
Grant SG,
O'Dell TJ,
Karl KA,
Stein PL,
Soriano P,
Kandel ER
(1992)
Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice.
Science
258:1903-1910[Abstract/Free Full Text].
-
Kaplan KB,
Swedlow JR,
Morgan DO,
Varmus HE
(1995)
c-Src enhances the spreading of src/ fibroblasts on fibronectin by a kinase-independent mechanism.
Genes Dev
9:1505-1517[Abstract/Free Full Text].
-
Katsuta H,
Tsuji S,
Niho Y,
Kurosaki T,
Kitamura D
(1998)
Lyn-mediated down-regulation of B cell antigen receptor signaling: inhibition of protein kinase C activation by Lyn in a kinase-independent fashion.
J Immunol
160:1547-1551[Abstract/Free Full Text].
-
Kramer EM,
Klein C,
Koch T,
Boytinck M,
Trotter J
(1999)
Compartmentation of Fyn kinase with glycosylphosphatidylinositol-anchored molecules in oligodendrocytes facilitates kinase activation during myelination.
J Biol Chem
274:29042-29049[Abstract/Free Full Text].
-
Lees MB,
Brown D
(1984)
Proteins in myelin.
In: Myelin (Morell P,
ed), pp 197-224. New York: Plenum.
-
Lubetzki C,
Demerens C,
Anglade P,
Villarroya H,
Frankfurter A,
Lee VM,
Zalc B
(1993)
Even in culture, oligodendrocytes myelinate solely axons.
Proc Natl Acad Sci USA
90:6820-6824[Abstract/Free Full Text].
-
McCarthy KD,
de Vellis J
(1980)
Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.
J Cell Biol
85:890-902[Abstract/Free Full Text].
-
McMorris FA,
Miller SL,
Pleasure D,
Abramsky O
(1981)
Expression of biochemical properties of oligodendrocytes in oligodendrocyte × glioma cell hybrids proliferating in vitro.
Exp Cell Res
133:395-404[ISI][Medline].
-
Miyakawa T,
Yagi T,
Watanabe S,
Niki H
(1994)
Increased fearfulness of Fyn tyrosine kinase deficient mice.
Brain Res Mol Brain Res
27:179-182[Medline].
-
Miyakawa T,
Yagi T,
Kitazawa H,
Yasuda M,
Kawai N,
Tsuboi K,
Niki H
(1997)
Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function.
Science
278:698-701[Abstract/Free Full Text].
-
Norton WT,
Poduslo SE
(1973)
Myelination in rat brain: method of myelin isolation.
J Neurochem
21:749-757[ISI][Medline].
-
Osterhout DJ,
Wolven A,
Wolf RM,
Resh MD,
Chao MV
(1999)
Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase.
J Cell Biol
145:1209-1218[Abstract/Free Full Text].
-
Raible DW,
McMorris FA
(1990)
Induction of oligodendrocyte differentiation by activators of adenylate cyclase.
J Neurosci Res
27:43-46[ISI][Medline].
-
Schwab ME,
Schnell L
(1989)
Region-specific appearance of myelin constituents in the developing rat spinal cord.
J Neurocytol
18:161-169[ISI][Medline].
-
Schwartzberg PL,
Xing L,
Hoffmann O,
Lowell CA,
Garrett L,
Boyce BF,
Varmus HE
(1997)
Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src/ mutant mice.
Genes Dev
11:2835-2844[Abstract/Free Full Text].
-
Somm
TOP
ABSTRACT
INTRODUCTION
