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The Journal of Neuroscience, February 1, 2003, 23(3):883
Fibroblast Growth Factor Receptor 3 Signaling Regulates the Onset
of Oligodendrocyte Terminal Differentiation
Luke Y. S.
Oh1,
Adam
Denninger1,
Jennifer S.
Colvin3,
Aditee
Vyas2,
Shubha
Tole2,
David M.
Ornitz3, and
Rashmi
Bansal1
1 Department of Neuroscience, University of Connecticut
Medical School, Farmington, Connecticut 06030, 2 Department
of Biological Sciences, Tata Institute of Fundamental Research, Mumbai,
400 005 India, and 3 Department of Molecular Biology
and Pharmacology, Washington University School of Medicine, St. Louis,
Missouri 63110
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ABSTRACT |
Fibroblast growth factor receptor (FGFR) signaling is essential for
nervous system development. We have shown that, in the normal postnatal
brain, the spatial and temporal expression pattern of FGFR3 parallels
the appearance of differentiated oligodendrocytes and that in culture
FGFR3 is expressed maximally at the critical stage in the
lineage at which oligodendrocyte late progenitors (Pro-OLs) enter
terminal differentiation. Therefore, FGFR3 expression is positioned
ideally to have an impact on oligodendrocyte differentiation. In
support of this we show that, during the onset and active phase of
myelination in FGFR3-deficient mice, there are reduced numbers of
differentiated oligodendrocytes in the forebrain, cerebellum, hindbrain, and spinal cord. Furthermore, myelination is delayed in
parallel. Delay of oligodendrocyte differentiation also is observed in
primary cell culture from this mutant. On the other hand, no
differences are observed in the survival or proliferation of
oligodendrocyte progenitors. This suggests that the decrease in the
number of differentiated oligodendrocytes is attributable to a delay in
the timing of their differentiation process. Astrocytes also express
FGFR3, and in mice lacking FGFR3 there is an enhancement of the
astrocytic marker glial fibrillary acidic protein expression in a
region-specific manner. Thus our findings suggest that there are cell
type- and region-specific functions for FGFR3 signaling and in
particular emphasize a prominent role for FGFR3 as part of a system
regulating the onset of oligodendrocyte terminal differentiation.
Key words:
oligodendrocyte; myelin; FGF; astrocyte; cerebellar
neuron; FGF receptor
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INTRODUCTION |
Fibroblast growth factors (FGFs), a
family of 23 known members, play central roles in nervous system
development (Vaccarino et al., 1999 ; Ford-Perriss et al., 2001; Ornitz
and Itoh, 2001). FGFs signal through four high-affinity tyrosine kinase
receptors (FGFR1-FGFR4) (Johnson and Williams, 1993). FGFRs are
expressed differentially during development and in the adult brain
(Orr-Urtreger et al., 1991; Peters et al., 1992, 1993; Asai et al.,
1993; Yazaki et al., 1994; Miyake et al., 1996; Ford-Perriss et al.,
2001). FGFs modulate a variety of biological activities including
proliferation, migration, and survival of neurons and glial cells
(Vaccarino et al., 1999; Ford-Perriss et al., 2001). Furthermore, FGFs
influence specification of neuronal and glial cell fate, regional
patterning of neocortex and midbrain-hindbrain boundaries, cerebellar
development, and cerebral cortex size (Crossley et al., 1996; Qian et
al., 1997; Ye et al., 1998; Fukuchi-Shimogori and Grove, 2001). For example, mice lacking FGF-2 exhibit decreased numbers of neurons and
glia, whereas the injection of FGF-2 into the embryonic subventricular zone produces the opposite effect (Vaccarino et al., 1999). In mice
lacking both FGF-17 and one allele of FGF-18 the anterior lobe of the
cerebellar vermis does not develop (Xu et al., 2000), whereas in mice
lacking FGF-14 recent studies have identified a function for this
molecule in neuronal signaling in the adult brain (Wang et al., 2002).
However, the role of FGFs in early postnatal development is less well
understood, especially with respect to oligodendrocyte and astrocyte differentiation.
Oligodendrocyte (OL) progenitors originate at specific locations in the
ventral ventricular zones. As they migrate to their final destinations,
they mature through a series of stages that include proliferative and
migratory early progenitors (EPs) and proliferative and nonmigratory
late progenitor or pro-oligodendrocytes (Pro-OLs). Ultimately, the
Pro-OLs become postmitotic and enter terminal differentiation, leading
to myelination (Warrington and Pfeiffer, 1992; Pfeiffer et al., 1993;
Miller, 1996; Woodruff et al., 2001). OL development is regulated by a
number of growth factors, including FGF-2 (McMorris and McKinnon,
1996). FGF-2 affects multiple responses including proliferation and
migration of OL progenitors and the conversion of mature OLs to a novel phenotype (for review, see Oh and Yong, 1996; Bansal and Pfeiffer, 1997; Bansal, 2002). During OL maturation FGF receptor transcripts are
expressed in a developmentally regulated manner that could account at
least in part for the variety of responses of OL lineage cells to
FGF-2. Specifically, although FGFR1 is expressed at all stages, FGFR2
appears only in differentiated OLs, and FGFR3 expression peaks in
Pro-OLs and then is downregulated as cells enter terminal differentiation (Bansal et al., 1996).
On the basis of the temporal expression pattern of FGFR3, we
hypothesize that FGFR3 transduces signals important for the regulation of the critical interface between proliferation and differentiation. We
report here that, in mice lacking FGFR3, the appearance of terminally
differentiated OLs is retarded initially, whereas the expression of the
astrocytic marker glial fibrillary acidic protein is enhanced. Because
neither survival nor proliferation of OL progenitors is altered, we
conclude that FGFR3 signaling is involved in the regulation of the
onset of terminal differentiation of Pro-OLs and in the negative
regulation of astrocytic differentiation and/or function.
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Materials and Methods |
FGFR3 null mice
FGFR3 null mice exhibit a characteristic phenotype including
crooked tail, curvature of vertebrae, and abnormal long bone growth
(Colvin et al., 1996; Deng et al., 1996). FGFR3 null and wild-type mice
were obtained from heterozygous crosses of breeders received from Dr.
David Ornitz (Washington University, St. Louis, MO). Genotypes were
determined by PCR of tail DNA by using three sets of primers: (1)
5'-GGGCTCCTTATTGGACTCGC-3', (2) 5'-AGGTATAGTTGCCACCATCGGAGGG-3', and (3) 5'-TGCTAAAGCGCATGCTCCAGACTGC-3'. Products of 322 bp (wild-type gene) and 221 bp (homozygote gene) were amplified by using primers 1 and 2 and 1 and 3, respectively (35 cycle PCR reactions: 2 min at
94°C, 30 sec at 94°C, 30 sec at 55°C, and 10 sec at 72°C). Initially, three groups of mice were studied: wild types (+/+), heterozygotes (+/ ), and FGFR3 null mice (/). However, no
differences were observed between +/+ and +/, in agreement with
previous studies (Colvin et al., 1996).
In situ hybridization
Mice older than postnatal day 7 (P7) were perfused with 4%
paraformaldehyde (PFA), and the brains were removed, postfixed in 4%
PFA at 4°C overnight, and cryoprotected by consecutive incubations in
10% sucrose and 30% sucrose overnight at 4°C (mice at P2 and P7
were not perfused but followed the same postfixation procedure). Brains
were immersed in cryo-embedding media and were quick frozen at
-80°C. Using a cryostat, we cut whole brain sections (with a small
portion of the cervical spinal cord attached) parasagittally (15 µm
thick) and collected them on RNase-free Superfrost glass slides
(Fisher Scientific, Pittsburgh, PA), stored them at
-20°C, and used them for in situ hybridization and immunohistochemistry.
A riboprobe specific for proteolipid protein (PLP) mRNA was designed to
cover the entire coding region (a gift from B. Fuss and W. B. Macklin, Cleveland, OH). A platelet-derived growth factor receptor  (PDGF-R) mRNA probe was transcribed from a 1637 bp EcoRI
cDNA fragment encoding most of the extracellular domain of mouse
PDGF-R, and a FGFR3 probe was transcribed from a 900 bp
EcoRI cDNA fragment encoding the extracellular domain and a part of intracellular tyrosine kinase domain (a gift from Bill Richardson and Nigel Pringle, London, UK). Hybridization for PLP mRNA
was performed as described previously (Bansal et al., 1999). Briefly,
sections were dried for 2 hr at room temperature and fixed with 3% PFA
for 30 min. The sections were treated with 0.1 M
HCl (5 min), followed by acetylation with 0.1 M
triethanolamine, pH 8, in acetic anhydride (10 min). After the sections
were washed in 2× SSC and air-dried, hybridization was performed
overnight at 50°C by using digoxigenin-labeled sense and antisense
cRNA probes in hybridization solution [containing 50% formamide and (in µM) 350 NaCl, 10 dithiothreitol, 20 Tris-Cl, pH 7.5, 1 EDTA plus 1× Denhardt's, 500 µg/ml tRNA, and 100 µg/ml poly(A) RNA]. Then the sections were washed in 2× SSC three
times and digested in RNase solution (20 µg/ml RNase and 1 U RNase T1
at 37°C for 30 min), followed by washing in 0.2× SSC at 50°C (5 min) and at room temperature (5 min). After equilibration in 100 mM Tris-HCl plus 150 mM
NaCl (10 min) and blocking for nonspecific binding in 1% blocking
buffer (Boehringer Mannheim, Indianapolis, IN) and 0.5%
bovine serum albumin (BSA; 1 hr), the bound cRNA was detected via an
alkaline phosphatase-coupled anti-digoxigenin antibody (1:1000 for 2 hr; Boehringer Mannheim). After a washing in equilibrium
buffer containing (in mM) 100 Tris-HCl, pH 9.5, 100 NaCl, and 50 MgCl2, color development
in the presence of 4-nitroblue tetrazolium chloride,
5-bromo-4-chloro-3-indolylphosphate, and levamisole was performed in
the dark at room temperature. The sections were washed in PBS
and incubated in Hoechst blue dye 33342 (1 µg/ml; Sigma,
St. Louis, MO) to counterstain the nuclei, were air-dried, and were
mounted with 90% glycerol.
In situ hybridization for PDGFR- or FGFR3 mRNAs was
performed with a slight modification. Briefly, air-dried and PFA-fixed sections were hybridized directly with these probes overnight at
65°C. Then the slides were rinsed and incubated in preheated wash
buffer (1× SSC, 50% formamide, 0.1% Tween 20) at 65°C (15 min),
washed twice with wash buffer at room temperature (30 min) and twice
with MABT solution (30 min) [100 mM maleic acid,
pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20],
blocked in MABT containing 2% blocking reagent (Boehringer
Mannheim) and 1% BSA (1 hr), incubated in alkaline
phosphatase-coupled anti-digoxigenin antibody, and developed as
described above. To increase sensitivity, we included 50% (w/v)
polyvinyl alcohol in the final color reaction and developed it
overnight at 37°C.
Immunohistochemistry
Tissue preparation and sectioning were performed as described
above. For myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) immunolabeling, whole brain parasagittal cryosections were permeabilized in 100% ethanol (10 min), washed in PBS, and blocked for 1 hr in a buffer consisting of 10% normal goat serum (NGS), 5% BSA, 0.05% NaN3, and 0.1% gelatin in
PBS. For NG2 immunolabeling the sections were blocked in 3% NGS/0.1%
Triton X-100 in PBS. Sections were incubated overnight at 4°C in
polyclonal rabbit anti-MBP (1:3000; Dr. S. E. Pfeiffer,
Farmington, CT), monoclonal anti-rat GFAP (1:50; Dr. Virginia Lees,
University of Pennsylvania, Philadelphia, PA), or polyclonal rabbit
anti-NG2 (1:100; Chemicon, Temecula, CA). After being
washed in PBS, the sections were incubated for 1 hr with either goat
anti-rabbit IgG conjugated to Oregon green (1:100; Molecular
Probes, Eugene, OR) or goat anti-rat IgG conjugated to
fluorescein (1:100; Chemicon), washed in PBS, mounted in
DABCO [1,4-diazobicyclo-(2,2,2)-octane in glycerol], and analyzed with an epifluorescent microscope (Axiovert microscope, Carl
Zeiss, Thornwood, NY).
Detection of cell proliferation
So that we could identify cells that were in the S phase of the
cell cycle, mice received an intraperitoneal injection of bromodeoxyuridine (BrdU; 100 µg/gm body weight) for incorporation into newly synthesized DNA and were killed 3 hr later. After perfusion, postfixation, and sectioning as described above, the sections were
rinsed in PBS for 10 min, fixed with acid alcohol (95% ethanol/5% acetic acid, 2 min, -20°C), washed in PBS, denatured with 2N HCl (10 min), neutralized with 0.1 M sodium borate buffer, pH 8.5 (10 min), blocked with 3% NGS/PBS (1 hr), incubated in mouse
monoclonal anti-BrdU antibody (overnight at 4°C; 1:50; Becton
Dickinson, Lincoln Park, NJ), and then washed in PBS, followed
by incubation in goat anti-mouse IgG conjugated to Cy3 (1:500;
Jackson ImmunoResearch, West Grove, PA) and Hoechst Blue
33342 (1:1000; 1 hr). Slides were washed and mounted in DABCO. In
double-labeling experiments the sections were immunolabeled first with
anti-NG2 as described above, followed by anti-BrdU labeling.
Detection of apoptotic cells
Apoptotic cells were detected by using terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay
(Apoptag kit; Intergen, Purchase, NY) according to the
manufacturer's protocol. In brief, brain cryosections were incubated
in 4% PFA for 30 min, treated with 0.3%
H2O2 to quench endogenous
peroxidase, washed in equilibrium buffer, and incubated in reaction
buffer containing digoxygenin-dNTP and terminal deoxynucleotidyl
transferase (30 min, 37°C). The sections were washed and incubated
with anti-digoxygenin-peroxidase conjugate for 30 min. The
TUNEL+ cells were identified by reaction
with 3,3-diaminobenzidine (DAB; Research Genetics,
Huntsville, AL) and analyzed by epifluorescence microscopy.
Immunoblotting
Forebrains, cerebella, hindbrains, and spinal cords were
harvested and stored at 80°C before analysis. Mixed primary
cultures were harvested in RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% deoxycholate, and 1% NP-40, pH
7.4) with protease inhibitors (2 mM PMSF, 2 µg/ml
leupeptin, 2 µg/ml aprotinin) on ice and were cup sonicated (30 sec;
4°C). Tissue samples were homogenized similarly. Then the homogenates
were incubated (30 min, on ice) and centrifuged (15,000 × g for 10 min at 4°C). The protein concentration was assayed with the DC Protein Assay kit (Bio-Rad, Hercules,
CA). Aliquots of total protein were electrophoresed on 12% SDS
polyacrylamide gels and transferred onto polyvinylidene difluoride
membranes. The membranes were blocked for 1 hr (Tris-buffered saline,
5% powder milk, 0.2% Tween 20) and incubated for 1 hr in monoclonal anti-myelin oligodendrocyte glycoprotein (MOG; 1:5000; Dr. C. Linington, Max Planck Institute, Munich, Germany), polyclonal anti-MBP (1:10,000), polyclonal anti-2',3'-cyclic nucleotide
3'-phosphodiesterase (CNP; 1:5000; Dr. S. Pfeiffer, Farmington, CT),
polyclonal anti-FGFR3 (1:1000; Dr. D. Ornitz, Washington University,
St. Louis, WA), or polyclonal anti-GFAP (1:5000; Dako,
Carpinteria, CA). Then the membranes were incubated for 30 min in
either anti-rabbit IgG (1:10,000; Santa Cruz Biotech, Santa Cruz, CA)
or anti-mouse IgG (1:10,000; Transduction Laboratories,
Lexington, KY), both conjugated to horseradish peroxidase. The
membranes were developed with the ECL Plus kit (Amersham,
Arlington Heights, IL). The NIH Image analysis program (Bethesda, MD)
was used for quantification of the bands.
Electron microscopy
Animals were anesthetized and perfused with 2% PFA/2.5%
glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.4. The brains were removed and postfixed overnight in the same fixative.
The medial parts of corpus callosum were dissected, treated with 1% osmium tetroxide, stained in block in 0.5% uranyl acetate, dehydrated in ethanol, cleared in propylene oxide, and embedded in Polybed (Polysciences, Warrington, PA). The sections were stained
in uranyl acetate and lead citrate and then mounted on a 200 mesh grid; fields were sampled randomly, and axons within these fields were chosen
randomly for analysis. Axon diameters were determined by tracing the
parameters, using the NIH Image program; myelin thickness was measured
similarly and quantified.
Cell culture
Mixed primary cultures. Mixed primary cultures of
neonatal (P2) mice telencephala were prepared as described previously
(Bansal et al., 1999). Briefly, dissociated cells were plated in 10%
fetal calf serum in DMEM (FCS/DMEM) at a density of 2.5 × 105 cells/cm2
into poly-L-lysine-coated (50 µg/ml; Sigma)
35 mm tissue culture plates for protein isolation. After 1 d the
cultures were changed to defined medium mN2 [DMEM with 4.5 gm/l
D-glucose, 50 µg/ml human transferrin, 5 µg/ml bovine
pancreatic insulin, and (in nM) 15 3,3,5-triiodo-L-thyronine, 30 sodium selenium, 10 D-biotin, 10 hydrocortisone, plus 0.11 mg/ml sodium
pyruvate, penicillin/streptomycin (10 IU/ml and 100 µg/ml,
respectively), and 0.1% BSA (all ingredients from Sigma)
plus 1% FCS and 1% horse serum].
Purified populations. Purified populations of
developmentally synchronized OL lineage cells were prepared at three
stages: EPs, Pro-OLs, and terminally differentiated OLs; we
characterized the purity and phenotype of each population extensively
by immunolabeling the cells with a panel of antibodies (Pfeiffer et
al., 1993; Bansal et al., 1996). Briefly, progenitors were obtained
from mixed primary cultures from neonatal rat telencephalon by
overnight shaking, followed by differential adhesion and complement
lysis with anti-galactocerebroside to remove astrocytes, macrophages,
and terminally differentiated OLs. Cells were plated in 5% FCS/DMEM in
tissue culture plates coated with 50 µg/ml
poly-D,L-ornithine (Sigma). After cell
attachment for 2-3 hr the medium was changed to serum-free defined
medium mN2 (see above). Cultures were grown for 7 d to produce
terminally differentiated OLs or were expanded and arrested at either
the EP stage by growth in PDGF-BB plus FGF-2 (10 ng/ml each;
Upstate Biotechnology, Lake Placid, NY) for 2 d or at
the Pro-OL stage by growth in FGF-2. For some experiments the
progenitors also were prepared without growth factor treatment (similar
results were obtained with cells grown in both conditions).
Astrocyte cultures. Astrocyte cultures prepared from
monolayer cultures remaining after releasing OL progenitors from mixed primary cultures (see above; Bansal et al., 1996) were 99% positive for the astrocytic marker GFAP.
Comparative analyses of wild-type and mutant mice. These
assays were done as described previously (Bansal et al., 1999).
Briefly, three to nine mice from at least three separate litters were
analyzed from both control and mutant groups at each time point. Whole brains (plus the most rostral portion of the cervical spinal cord) were
cut parasagittally (15-µm-thick sections) in the sampling plane,
which was located ~300 µm from the midline. Wild-type and FGFR3
null sections were matched by comparing landmarks observed by
counterstaining with Hoechst dye so that they were equidistant from the
midline and in the hippocampal region. Cell counts were obtained from
sections from the sampling plane through the entire brain region (such
as cortex, corpus callosum, cerebellar white matter, or cerebellar
cortex), and all of the cells in each area were counted in these
sections, except in cases for which specific matched subregions are
specified. Cells in the sections were counted systematically with a
grid and 20× objective. Cell counts and observations were made by
double-blind analysis by at least two independent investigators.
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Results |
Developmental expression of FGFR3 in normal postnatal brain
OL differentiation and myelination in the mouse CNS occur in a
caudal to rostral manner. For example, at birth small numbers of
differentiated OL are already present in the spinal cord but only
subsequently appear sequentially in the hindbrain, cerebellum, corpus
callosum, and finally in the cerebral cortex. This spatial and temporal
pattern of development can be visualized by immunohistochemical and
in situ hybridization analyses of parasagittal sections of the whole brain. Although expression of FGFR3 has been demonstrated in
multiple cell types in different regions of the CNS, including astrocytes in the adult brain (Yazaki et al., 1994; Miyake et al.,
1996), OL progenitors in the adult spinal cord (Messersmith et al.,
2000), spinal motor neurons (Philippe et al., 1998), embryonic neuroepithelium (Peters et al., 1992, 1993), forebrain-derived cultured
OL progenitors and astrocytes (Bansal et al., 1996), and, more
recently, spinal cord astrocytes and their neuroepithelial precursors
(W. Richardson, personal communications), its expression pattern
as a function of postnatal brain development had not been addressed.
Because OL development in the brain takes place postnatally and because
FGFR3 is expressed by OL progenitors in vitro (Bansal et
al., 1996), we considered the possibility that FGFR3 signaling may be
important for OL development. We first analyzed the spatiotemporal pattern of expression of FGFR3 mRNA in vivo and related it
to the appearance of OL progenitors and differentiated OLs at P2, P4,
P7, P9, P13, and P31. Typical results are shown for P2 and P9
schematically and as representative sections taken from the forebrain
and hindbrain (Fig. 1).
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Figure 1.
Spatiotemporal expression of FGFR3 mRNA in normal
brain. Parasagittal sections of wild-type mouse brain were analyzed by
in situ hybridization for PDGFR, FGFR3, and PLP mRNA
at P2 and P9. The regional distributions of positive cells are shown
diagrammatically as dots and as representative sections
taken from forebrain and hindbrain from regions marked by a
box. Higher magnifications are shown as
insets. The temporal wave of FGFR3 mRNA expression moves
from caudal to rostral brain in parallel to that for PLP but not
PDGFR. CC, Corpus callosum; CX,
cortex. Scale bars: (in l) a-l,
100 µm; insets, 50 µm.
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At P2, PDGFR mRNA (a marker for OL early progenitors; Pringle et
al., 1992) expression already was widespread over the entire brain, and
the pattern remained the same at P9 (Fig.
1P2a,P2b,P9g,P9h). In contrast, at P2, FGFR3 was
expressed only in the spinal cord, pons, and medulla (hindbrain), with
very little expression in the midbrain and none in the forebrain (Fig.
1P2c,P2d). By P4 the FGFR3 mRNA expression had
increased rostrally into the midbrain and cerebellum, and by P7 it
appeared in the forebrain where it continued to be expressed until
adulthood (Fig. 1P9i,P9j). The expression of PLP mRNA
(a marker for differentiated OLs; Hudson et al., 1989) follows a caudal
to rostral progression with a developmental age similar to FGFR3 (Fig.
1P2e,P2f,P9k,P9l), although initially it was
more restricted, appearing first in the corpus callosum by P7 before
spreading progressively into the cerebral cortex with time. These
results show that there is a wave of FGFR3 mRNA expression in the
normal postnatal brain that spreads from the hindbrain to the forebrain
in parallel to markers for terminally differentiated OLs rather than
for OL early progenitors.
FGFR3 protein is expressed maximally by Pro-OLs and
downregulated as the Pro-OLs differentiate into oligodendrocytes; FGFR3
protein also is expressed by astrocytes
Purified populations of OL early progenitors (EPs) from rat
forebrain growing in culture express low levels of FGFR3 mRNA. This
mRNA expression increases twofold as EPs mature to the Pro-OL stage and
then is downregulated dramatically as the cells enter terminal
differentiation (Bansal et al., 1996). Because protein expression does
not necessarily follow mRNA expression resulting from
post-transcriptional regulation, we examined FGFR3 protein expression
from purified populations of EPs, Pro-OLs, OLs, and astrocytes by
immunoblotting (Fig.
2A). Consistent with
the data for mRNA expression, FGFR3 protein was present in EPs but was increased in amount in Pro-OLs, and it was virtually absent in OLs.
Astrocytes (AST) also expressed FGFR3 protein. Analyses of homogenates
(2 µg) from transfected cell lines (3T3 and PC12) overexpressing
FGFR1, FGFR2, or FGFR3 demonstrated the specificity of the antibody
used for FGFR3. We conclude that FGFR3 protein is expressed maximally
at the Pro-OL stage and that its expression is downregulated as the
cells differentiate into OLs even more dramatically than at the mRNA
level.
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Figure 2.
The expression of FGFR3 protein is regulated
during the maturation of OL lineage cells. A, Purified
cells from rat forebrain, at three stages of OL maturation and
astrocytes, were analyzed by immunoblotting for FGFR3. Equal amounts of
total protein (50 µg) were loaded in each lane. FGFR3 protein is
expressed by early progenitors (EP), is increased
substantially as EPs mature to Pro-OLs, and is downregulated
dramatically to undetectable levels with the terminal differentiation
of progenitors into OLs (OL). Astrocytes
(AST) also express FGFR3 protein. Analyses of
homogenates (2 µg) from transfected cell lines (3T3 and PC12)
overexpressing FGFR1, FGFR2, or FGFR3 demonstrate the specificity of
the antibody used for FGFR3. B, Immunoblots of
homogenates (50 µg) from P9 wild types (+/+), heterozygotes (+/),
and FGFR3 null (/) hindbrains with anti-FGFR3 show the loss of
FGFR3 protein to undetectable levels in FGFR3 null animals.
Representative experiments of three are shown.
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Myelination is delayed in FGFR3 null mice in vivo
Because the FGFR3 expression pattern in vivo and
in vitro was consistent with a role in OL terminal
differentiation, we postulated that FGFR3 signaling is important for
myelination. We therefore analyzed mice lacking FGFR3 to evaluate the
effect of eliminating FGFR3 function on myelin formation. Consistent
with the null-genotype (Colvin et al., 1996), no FGFR3 protein was
detected in the mutant mice (Fig. 2B); in contrast,
the levels of FGFR1 and FGFR2 proteins were unchanged (data not shown).
We examined MBP expression as a myelin marker by immunohistochemistry
on parasagittal sections of whole brain from wild-type (+/+) and FGFR3
null (/) mice as a function of development (Fig.
3A,B). Myelin formation in the
corpus callosum of normal mice was initiated at approximately P7 when a
few MBP+ fibers appeared (data not shown).
At P9 (Fig. 3Aa) more apparent myelin developed, and
myelination progressed rapidly by P13 (Fig. 3Ac). In
contrast, in FGFR3 null mice there were no myelinated fibers at P7
(data not shown); at P9 there were fewer myelinated fibers, and they
were arranged more loosely (Fig. 3Ab,Ad). These differences
became progressively less marked with age, and by 1 month of age both
normal and mutant mice appeared to be myelinated similarly (Fig.
3Ae,Af). Reduced myelination also was observed in the
cerebellum at these ages (P7 is shown) (Fig. 3B). This difference in myelination between the wild-type and mutant mice was
confirmed by immunoblotting (Fig. 3C) for the myelin markers CNP and MBP in homogenates of forebrain at P13 (active myelination) and
5 months (myelination completed). Consistent with the
immunohistochemical results, the expression of both proteins was
reduced ~1.5-fold in the FGFR3 null mice compared with wild type
(P13) and became comparable in the adult (5 months). Although the
myelin from adult FGFR3 mice appeared normal by immunohistochemistry,
it did not ensure that the myelin formed was also normal
ultrastructurally. Therefore, we further analyzed adult FGFR3
null mice by electron microscopy (Fig.
4A-D). Cross sections
from the corpus callosum of 2-month-old wild-type and FGFR3 null
littermates showed no significant differences in the number of
myelinated and unmyelinated axons (Fig. 4E) or in the
thickness of myelin sheaths (Fig. 4F).
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Figure 3.
Myelin formation is delayed in FGFR3 null mice
brain. Parallel parasagittal sections from forebrain
(A) and cerebellum (B) of
wild-type (+/+) and FGFR3 null (/) mice were analyzed by
immunohistochemistry for MBP, a marker for myelinated fibers. In the
forebrain of FGFR3 null mice, when compared with wild-type mice, fewer
myelinated fibers developed initially, as seen at both P9 (Aa,
Ab; higher magnifications are shown in insets)
and P13 (Ac, Ad); this difference became comparable with
wild type by P31 (Ae, Af). B, In
cerebellar white matter there were also fewer myelinated fibers in the
FGFR3 null mice when compared with the wild type, as shown in a
representative section taken at P7 (Ba, Bb). The
boxed regions are shown at higher magnification
(Bc, Bd). A, B, Two to four sections each
from three to nine mice from each group and age were analyzed; similar
results were obtained. CC, Corpus callosum;
CX, cortex; HC, hippocampus. Scale bars:
Af, 100 µm; insets (for A, Bc,
Bd), 50 µm. C, Immunoblot analysis of
forebrain homogenates from wild-type (+/+) and FGFR3 null (/) mice.
Compared with wild-type mice, in FGFR3 null mice the levels of CNP and
MBP isoforms were reduced at P13; however, they reached wild-type
levels by adulthood.
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Figure 4.
Ultrastructural analysis of myelinated axons in
FGFR3 null mice. Cross sections of corpus callosum from 2-month-old
littermate wild-type (+/+) and FGFR3 null (/) mice were analyzed by
electron microscopy at low (A, C; 8230×) and high
(B, D; 193,000×) magnification. E,
Numbers of myelinated and unmyelinated axons were counted from the two
groups (~70 randomly chosen axons each). Wild-type number are set to
100%, and FGFR3 null levels are shown relative to that. Error bars
indicate SEM. F, Thickness of myelin sheath and size of
axon (arbitrary units) were measured from 30 randomly chosen myelinated
axons from each group (NIH Image software). No apparent differences
were observed in the ultrastructure of myelin between the two groups of
mice.
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These results show that myelination is delayed in FGFR3 null mice but
appears structurally normal. We conclude that FGFR3 signaling is
important for OLs to progress to the stage of myelination.
Reduced numbers of differentiated oligodendrocyte appear
in FGFR3 null mice
We hypothesized that the reduced myelination at early postnatal
ages is attributable to a reduction in the number of terminally differentiated OLs. To examine this possibility, we determined the time
course of OL differentiation in parallel parasagittal sections from +/+
and / littermates, using in situ hybridization for PLP
mRNA to identify and count the number of OLs (Fig.
5; the estimation of OL cell numbers by
immunohistochemistry with myelin protein markers is difficult because
of the background of highly immunolabeled myelinated fibers). During
the onset and active phase of myelination there were markedly fewer
differentiated OLs in all regions of the brains from FGFR3 null mice,
including spinal cord at P2 (Fig. 5A; the most dorsal
cervical region is shown), and in the corpus callosum and cortex (Fig.
5B) and cerebellum (Fig. 5C) at P9.
Quantification of the data for the corpus callosum and cortex (Fig.
5D) showed that there were twofold to threefold fewer OLs
expressing PLP mRNA in the FGFR3 null than in wild types at P9 and P13.
The difference between wild type and mutant continued to become
progressively smaller with increasing age, and the numbers of
PLP+ cells reached control levels with
subsequent development (P31) (Fig. 5D), consistent with the
normal level of myelination observed at this age (Fig.
3Ae,Af). Similar differences in the number of OLs
also were observed in spinal cords from wild-type and mutant mice (data
not shown). These results suggest that, during the period when
myelination is initiated and is progressing actively, fewer
differentiated OLs appear in FGFR3 null brain compared with wild-type
mice.
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Figure 5.
Oligodendrocyte differentiation is delayed in
FGFR3 null mice in vivo. Parasagittal sections taken
from parallel regions of wild-type (+/+) and FGFR3 null (/) mice
from spinal cord at P2 (A), forebrain at P9, P13,
and P31 (P9; B, D), and cerebellum at P9
(C) were analyzed by in situ
hybridization for the OL marker PLP mRNA. Fewer PLP
mRNA+ cells were present in FGFR3 null compared with
wild-type mice of the same age in all three regions of the brain
(A-C); representative sections are shown.
Sections hybridized with sense cRNA probe showed no labeling (data not
shown). Scale bar, 100 µm. D, Quantification of the
number of PLP mRNA+ cells that differentiated as a
function of time in the corpus callosum (CC) and the
cortex (CX) of wild-type and FGFR3 null mice. All
PLP mRNA+ cells in the entire cortical or corpus
callosum region were counted for each section. Two to four sections
each from three to nine mice from each group and age were counted.
Wild-type numbers (average of all +/+ animals) were set to 100%; FGFR3
null (/) numbers are shown relative to that. The numbers of PLP
mRNA+ OLs were reduced at P9 and P13 in FGFR3 null
corpus callosum and cerebral cortex, which became comparable with that
of wild type by P31. HC, Hippocampus; d,
dorsal region of the spinal cord; v, ventral region of
the spinal cord. Error bars indicate SEM (n = 3-9); *p < 0.005, unpaired Student's
t test.
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The decrease in oligodendrocyte number in FGFR3 null mice is not
attributable to either reduced proliferation or survival of
oligodendrocyte progenitors
The reduced number of OLs expressing PLP mRNA observed in FGFR3
null mice during the active phase of myelination could be caused by a
reduction in the number of OL progenitors available for differentiation
to mature OLs, attributable in turn to reduced proliferation and/or
increased cell death or, alternatively, to a decreased efficiency of
terminal differentiation by OL progenitors. We investigated these
mechanisms by analyzing whole brain parasagittal sections at P2, P7,
P9, and P13 from wild-type (+/+) and FGFR3 null (/) mice.
The proliferative capacity of OL progenitors was studied by injecting
BrdU intraperitoneally into these mice 2 hr before the brains were
harvested for double-immunofluorescence microscopy with anti-BrdU and
anti-NG2, a marker for OL progenitors (Fig. 6A,B). No differences
in the total number of BrdU+ cells were
observed between wild-type and FGFR3 null mice in either the cortex or
corpus callosum (Fig. 6Ab-Ad). Further, ~40-50% of the BrdU+ cells in the cerebral cortex
were also NG2+ at P9 (Fig.
6Bd), consistent with previous observations (Dawson et al., 2000; Mallon et al., 2002), in both wild-type and FGFR3 null
mice. These data suggest that the proliferation of OL progenitors is
not affected in the forebrain of FGFR3 null mice and cannot account for
the reduced number of differentiated OLs.
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Figure 6.
Proliferation and survival of OL progenitors is
not altered in FGFR3 null mice. P9 sagittal sections taken from
parallel regions of wild-type (+/+) and FGFR3 null (/) mice were
analyzed. A, B, BrdU incorporation analyzed by
immunohistochemistry was used as a measure of proliferation
(Ab-Ad). The sections were counterstained with Hoechst
dye (Aa) to show the region of the brain that was
analyzed. Scale bar, 100 µm. The total number of
BrdU+ cells (Ad) was obtained by
counting all BrdU+ cells in either the entire
cerebral cortex (CX) or splenium of the corpus
callosum region (CC) for each section. B,
Cells double-immunolabeled with NG2 (Ba) and BrdU
(Bb) are shown as a merged image (Bc) at
higher magnification than in A. The percentage of
BrdU+ cells colabeled with anti-NG2 estimates the
number of proliferating OL progenitors (Bd). Neither the
total numbers of proliferating cells nor the number of
NG2+/BrdU+ cells showed
significant differences between wild-type and FGFR3 null forebrain.
C, TUNEL assay was used as a measure of cell death.
TUNEL+ cells in the subependymal germinal zone of P9
forebrain are shown. Inset, High magnification
(Ca, Cb, arrows). Two to four sections
from three animals from each group were counted. The total number of
TUNEL+ cells in either the entire cortex plus corpus
callosum regions (CC) or in the whole cerebellar white
matter (WM/CB; Cc) was counted for each
section. Wild-type numbers were set to 100%; FGFR3 null levels are
shown relative to that. No statistically significant differences were
observed between the two groups. Scale bars: 100 µm;
insets, 30 µm. D, PDGFR mRNA
in situ hybridization was used to identify OL
progenitors. No obvious differences were observed between the numbers
of PDGFR mRNA+ cells at P2 in the forebrain of
the wild-type and FGFR3 null mice. Scale bar, 100 µm. Error bars
indicate SEM (n = 3).
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OL cell death was studied in the white matter of FGFR3 null and
wild-type mice via the TUNEL assay (a measure of apoptotic cell death).
In the normal mouse brain cell death occurs in the subependymal
germinal zone in the forebrain and cerebellum at an early postnatal
period; then it is reduced progressively to barely detectable levels in
the adult brain (Levison et al., 2000). Although a few
TUNEL+ cells were observed at P9 in the
forebrain (Fig. 6Ca,Cb) and in the corpus callosum and white
matter of the cerebellum (WM/CB) (Fig. 6Cc), the numbers did
not differ between wild-type and FGFR3 null mice.
TUNEL+ cells were not observed in the
spinal cord or hindbrain (data not shown). We conclude that reduced
survival of OL progenitors or newly formed OLs is not the cause of
reduced numbers of differentiated OLs seen in FGFR3 null mice during
early postnatal differentiation.
Consistent with the absence of any changes in the proliferative or
survival capacities, the numbers of OL early progenitors, determined by
in situ hybridization for the progenitor markers PDGFR
(Fig. 6D) or Olig-1 (data not shown), were similar in
wild-type and FGFR3 null mice at P2. We conclude that changes in OL
progenitor population size do not account for the reduced number of
terminally differentiated OLs in FGFR3 null mice.
In the absence of changes in OL progenitor survival,
proliferation, or cell number, we conclude that the decrease in the
number of terminally differentiated OLs in the FGFR3 null mice is
attributable to delays in the timing of their differentiation process.
Oligodendrocyte differentiation also is inhibited in mixed primary
cultures from FGFR3 null forebrain
OL development in vivo is orchestrated by complex
interactions among at least three major cell types, OLs, astrocytes,
and neurons. We studied the roles of these interactions on the
regulation of OL number by examining the timing and extent of OL
differentiation in mixed primary cultures grown in defined medium
initiated from P1 forebrains of either wild-type (+/+) or FGFR3 null
(/) littermates (Fig. 7). These
cultures are devoid of neurons, as indicated by the virtual absence of
the neuron-specific marker tubulin III (data not shown). FGF2, a
ligand for FGFR3, is present in these cultures (immunoblot analyses;
data not shown), consistent with the reported expression of FGF2 by
astrocytes (Araujo and Cotman, 1992). Immunoblot analyses of marker
proteins for OL terminal differentiation, which are expressed
sequentially as CNP, MBP, and MOG, were performed as a function of time
in culture. In FGFR3 null cultures the expression of all three markers
lagged behind that seen in normal control cultures at P13, P17, and P24
d in vitro (DIV). Although the expression of CNP and MBP
reached control levels by 28 DIV (Fig. 7A,B), the late
marker MOG (Fig. 7C) still lagged behind control levels at
P28 (the latest point that was studied). As an example of the reduced
expression of OL markers, quantification of MOG protein levels is
shown, demonstrating an approximate threefold decrease at 17 DIV in
FGFR3 null compared with control cells (Fig. 7C). This is
consistent with a reduced number of MOG+
cells observed by immunofluorescence microscopy (data not shown). It is
noteworthy that the expression of even the earliest marker of terminal
differentiation, CNP, was reduced. This indicates that the inhibition
of differentiation occurs at or near the onset of terminal
differentiation (a transient increase in the number of cells at a stage
just before the CNP+ stage of the lineage
would be predicted); however, this fleeting stage is difficult to
observe under the conditions of these analyses [i.e., "pre-GalC
stage" (Bansal and Pfeiffer, 1992)]. The levels of total protein and
the astrocytic marker GFAP were comparable in the two groups (data not
shown), suggesting that the decrease in OL markers was not attributable
to overall changes in the culture system. Note that these cultures were
initiated from forebrain, where no change in GFAP expression was
observed in FGFR3 null in vivo (see below). We conclude that
the lower levels of OL marker proteins are attributable to a reduction
in the rate of OL differentiation and that a similar delay of OL
differentiation in the absence of FGFR3 signaling observed in
vivo also occurs in culture in the absence of neurons, effectively
ruling out the possibility that the effect on OL differentiation occurs
indirectly via neurons.
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Figure 7.
Analysis of OL differentiation in mixed primary
cell cultures from FGFR3 null forebrain. Mixed primary cell cultures
initiated from P2 forebrain were analyzed by immunoblotting as a
function of time in culture for wild-type (+/+) and FGFR3 null (/)
littermate mice for the OL differentiation markers. A,
CNP; B, MBP; C, MOG. D,
Representative examples for the time courses of CNP, MBP, and MOG and
quantification for MOG (NIH Image analysis) at one time point (17 DIV)
are shown (n = 3 for wild type;
n = 5 for mutants); p < 0.05. Then 10 µg of total protein was loaded in each lane. Note that,
similar to in vivo, the expression of OL differentiation
markers was delayed in vitro in the FGFR3 null mice
compared with wild type.
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There is increased expression of the astrocytic marker GFAP in the
hindbrain and cerebellum of FGFR3 null mice, but not in the
forebrain
Astrocytes express FGFR3 (Fig. 2) (Bansal et al., 1996; Miyake et
al., 1996) and proliferate in response to FGFs. During development the
differentiation of astrocytes is accompanied by the expression of GFAP,
a marker for astrocytic maturation (Goldman, 2001). In adults the
astrocytes then downregulate GFAP expression in a region-specific manner (DiProspero et al., 1997; Reilly et al., 1998). GFAP null mice
display abnormalities in myelination, suggesting a link
between astrocytic function and myelination (Liedtke et al., 1996).
Hypothesizing that the elimination of FGFR3 would have an impact on
astrocytic function, we analyzed GFAP expression in astrocytes by
immunohistochemistry on whole brain parasagittal sections (including
the most rostral region of cervical spinal cord) at P7, P9, P13, and
P31 (Fig. 8A-H; P9 and
P31 are shown). In the FGFR3 null brain at P7 and P9 there was an
increase in GFAP expression in the spinal cord, hindbrain, and
cerebellum when compared with wild-type mice. This increase in the
spinal cord and hindbrain persisted at all of the ages that were
examined and continued into adulthood, unlike the wild type, in which
GFAP normally is downregulated. In contrast, no increase was seen in
the forebrain. GFAP immunoblot analysis of these regions confirmed
these observations (Fig. 8I,J). It is
noteworthy that FGF-2 mediated the downregulation of astrocytic gap
junction communication, and upregulation of dopamine receptor also
occurs in a region-specific manner (Reuss et al., 1998, 2000). The
GFAP+ astrocytes appeared to be
morphologically normal and did not exhibit the stocky, ramified
processes characteristic of reactive glia. These results suggest that
the downregulation of GFAP by FGF-2 in culture (Reilly et al., 1998)
involves FGFR3 signaling and that during development the upregulation
of GFAP that normally occurs transiently in the hindbrain and spinal
cord is accelerated and persists in the absence of FGFR3 signaling.
Therefore, FGFR3 signaling appears to regulate negatively the
astrocytic differentiation and/or function.
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Figure 8.
Astrocytic protein, GFAP, is increased in FGFR3
null mice. P9 and P31 parasagittal sections taken from parallel regions
of wild-type (+/+) and FGFR3 null (/) spinal cord
(A-D) at the most dorsal cervical region (just
below the medulla), P9 cerebellum (E, F), and P9
forebrain (G, H) were immunolabeled with
anti-GFAP. Two to four sections each from three to five mice from each
group were analyzed. Representative sections are shown. Scale bars: (in
F), A-F, 50 µm; (in
H),G, H, 100 µm.
BG, Bergmann glia; CX, cortex;
CC, corpus callosum; EGL, external
granular layer; HC, hippocampus; WM,
white matter. A-D, Spinal cord orientation: dorsal
(right), ventral (left), rostral
(top), caudal (bottom). I,
J, GFAP immunoblot analysis of homogenates from hindbrain
(HB), spinal cord (SC), cerebellum
(CB), and forebrain (FB) from wild-type
(+/+) and FGFR3 null (/) mice is shown at P9, P17, and P31.
Compared with wild type, in mutant mice there was an increased
expression of GFAP in the spinal cord, cerebellum, and
hindbrain. In contrast, no increase was seen in the forebrain. The
increase in spinal cord and hindbrain GFAP immunolabeling continued
into adulthood.
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Increased cell death in cerebellar cortex of FGFR3 null mice
Because FGFs act as survival factors for many neuronal populations
in vitro, we asked whether FGF signaling via FGFR3 played a
role in their survival, using TUNEL labeling of parallel whole brain
parasagittal sections at P7, P9, P13, and P31 (Fig.
9; P9 is shown). Increased
TUNEL+ cells were, in fact, observed in
the Purkinje, molecular, and external granular cell layers of the
cerebellar cortex of FGFR3 null mice (Fig. 9A,B) (Fig.
9C,D, sections counterstained with Hoechst dye). The cell
death was particularly prominent in the first few folia, including many
cells with the morphology of Purkinje neurons (Fig. 9D,
inset). Quantification showed that the increase in the
numbers of TUNEL+ cells was approximately
twofold (Fig. 9E). FGFR3 mRNA expression was also present in
the Purkinje cell layer (Fig. 9F). In contrast to the
cerebellum, other areas, such as the hippocampus and cerebral cortex,
did not show an increase in cell death in the FGFR3 null mice (data not
shown). Moreover, general hippocampal development was normal
(data not shown). We conclude that there is a region-specific increase in cell death in the absence of FGFR3.
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Figure 9.
Cell death in the cerebellar cortex is increased
in FGFR3 null mice. A, B, Parallel parasagittal sections
of cerebella from P9 wild-type (+/+) and FGFR3 null (/) mice were
analyzed by TUNEL assay. C, D, These sections were
counterstained with Hoechst dye to show the regions analyzed in
A and B. Scale bars: (in
B), A-D, 100 µm; inset,
25 µm. E, TUNEL+ cells were counted
from two to four sections each in the area covering the entire
cerebellar cortex from three separate animals in each group. The
wild-type values are set to 100%, and mutant numbers are plotted as a
percentage of wild type. There was a significant increase in cell death
in the Purkinje (PKL) and granule cell layers. Error
bars indicate SEM (n = 3). *p < 0.1, unpaired Student's t test. F,
In situ hybridization for FGFR3 mRNA shows a signal in
Purkinje cell layer (arrows). Scale bars:
F, 50 µm; inset, 25 µm.
EGL, External germinal layer; WM,
white matter.
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Discussion |
Three FGF receptors, FGFR1, FGFR2, FGFR3, are regulated
differentially during the course of OL lineage progression. The main finding from this study is that FGFR3 signaling is needed for the
timely generation of differentiated OLs. This conclusion is based on
two principal observations. First, FGFR3 is positioned ideally both
spatially and temporally to impact OL differentiation: that is, it is
expressed by Pro-OLs at the brink of the onset of OL terminal
differentiation and then is downregulated; further, in postnatal brain
the spatial and temporal expression pattern of FGFR3 parallels the
appearance of differentiated OLs. Second, the absence of FGFR3
expression in FGFR3 null mutants results in reduced numbers of
differentiated OLs during the active phase of myelination; this deficit
is corrected in mature animals, and there is no apparent abnormality in
the ultrastructure of mature myelin. We therefore conclude that FGFR3
signaling is transient and part of a system regulating the onset of
terminal differentiation of Pro-OL but that it is not needed for
maintenance of the differentiated state of OLs or further events of
myelination per se. This is consistent with the downregulation of FGFR3
in differentiated OLs.
The mechanism by which FGFR3 signaling influences the number of
terminally differentiated OLs could involve the regulation of OL
progenitor survival, proliferation, or differentiation. OL progenitor
survival is influenced by a competition for survival factors. However,
in contrast to other trophic factors such as PDGF, insulin-like growth
factor-1 (IGF-1), ciliary neurotrophic factor (CNTF), neuregulin, and
neurotrophin-3 (Barres et al., 1993; Trapp et al., 1997; Fernandez et
al., 2000; Flores et al., 2000), there is conflicting evidence
regarding the role of FGF in OL progenitor survival (McKinnon et al.,
1991; Barres et al., 1993; Yasuda et al., 1995; Ebner et al., 2000). We
did not observe a significant change in the survival capabilities of OL
progenitors or OLs in FGFR3 null mice compared with wild-type mice. On
the other hand, FGFs are known to be potent mitogens for OL progenitors (Bogler et al., 1990; McKinnon et al., 1990; Gard and Pfeiffer, 1993;
Bansal and Pfeiffer, 1994; Fok-Seang and Miller, 1994; Ben-Hur et al.,
1998; Baron et al., 2000). Therefore, the loss of one of the FGF
receptors could affect proliferation adversely. However, we did not
observe a change in the proliferative capacity of OL progenitors in
FGFR3 null mice. These data suggest that FGFR3-mediated signaling is
not required for either OL progenitor survival or proliferation in the
postnatal brain; therefore, the decreased numbers of OLs are probably
not attributable to alterations in either of these developmental parameters.
In the absence of effects on progenitor survival or proliferation, it
seems likely that FGFR3 signaling is regulating the onset of terminal
differentiation. Although proliferation and differentiation
traditionally have been cast as opposing activities whereby progenitors
need to cease proliferation to enter terminal differentiation, it is
becoming apparent that cessation of proliferation is necessary but not
sufficient to enable terminal differentiation. This predicts a
transition state in which progenitors stop dividing but do not enter
terminal differentiation automatically; that is, a second, temporally
related but independent, signal is required. A stage with these
characteristics has been observed (Bansal and Pfeiffer, 1992; Tang et
al., 1998; Tikoo et al., 1998; Ghiani et al., 1999; Huang et al.,
2002). Thus FGFR3 may provide one of the signals that promote OL
terminal differentiation.
Because proliferation and survival of OL progenitors proceeded normally
in FGFR3 null mice, they may be regulated instead by FGFR1 or FGFR2;
alternatively, FGFR1 and/or FGFR2 could compensate for the absence of
FGFR3 (FGFR4 is not expressed by OLs). These three receptors in fact do
have many overlapping ligand-binding specificities and significant
homology in their signaling domains. Nevertheless, receptor-specific
differences in their signaling patterns, leading to different
physiological responses, are also present. For example, in
gene-targeted disruption of specific FGF receptors, signals mediated by
one receptor are not rescued by those mediated by another (Deng et al.,
1994; Arman et al., 1998); proliferation and Ras/MAP kinase pathway
activation are promoted less effectively by activation of FGFR3 than of
FGFR1 in cell lines (Wang et al., 1994; Shaoul et al., 1995) and OL progenitors (our unpublished observations), presumably because of
differences in the levels of tyrosine kinase domain signaling, such as
the absence in FGFR3 of one of the two specific tyrosine residues
essential for the mitogenicity of FGFR1 (Wang and Goldfarb, 1994, 1997;
Lin et al., 1996; Naski et al., 1996). On the other hand, FGFR3 does
induce strong signals for promoting differentiation, e.g., via
Ras/MAPK-independent signaling pathways (Choi et al., 2001;
Rozenblatt-Rosen et al., 2002). In chondrocytes FGFR3 activates Stat-1
(Su et al., 1997; Sahni et al., 1999), which in turn upregulates the
expression of the cell cycle protein
p21CIP1 (Chin et al., 1996), leading to
differentiation; consistent with this, OL differentiation is disrupted
in p21 null mice (Zezula et al., 2001). Similarly, FGFR3 may activate
pathways that promote OL terminal differentiation and not proliferation.
It seems likely that the inhibition of differentiation of OLs in FGFR3
null mice reflects signaling intrinsic to OL progenitors. Nevertheless,
because astrocytes (Fig. 2) (Asai et al., 1993; Bansal et al., 1996;
Miyake et al., 1996) and certain neurons (Philippe et al., 1998) also
express FGFR3, the observed perturbation of OL development could result
indirectly via these cells. However, because the inhibition of OL
differentiation also is seen in neuron-free cultures, an indirect
effect from neurons probably can be ruled out. Astrocytes, on the other
hand, remain a possibility.
Could changes in glial cell fate account for the differences in the
number of OLs? In FGFR3 null mice there is an increase in GFAP
expression relative to wild-type littermates in the cerebellum, hindbrain, and spinal cord that continues into adulthood (Fig. 8). This
increase in GFAP could reflect either an increase in the number of
differentiated astrocytes expressing GFAP or to an increase in the
amount of GFAP per astrocyte, such as is seen in reactive astrocytes
after injury and cell death. There is, in fact, an elevated level of
cell death in the cerebellum, so the latter mechanism is possible.
However, in the hindbrain and spinal cord there is no apparent increase
in cell death, and the morphology of FGFR3 null astrocytes remains
normal (i.e., not reactive); therefore, the increase in the levels of
GFAP in these regions may reflect an increase in the number of
astrocytes. Thus in these regions there could be a relationship between
the opposing effects of the numbers of OLs (decreased) and astrocytes
(increased). For example, bone morphogenetic proteins
(BMPs) have been shown to favor the differentiation, at least in
culture, of certain progenitors into astrocytes rather than OLs (Mabie
et al., 1997; Grinspan et al., 2000; Mehler et al., 2000). However,
because lineage relationships between OLs and astrocytes in
vivo remain a point of debate (Rao and Mayer-Proschel, 1997;
Herrera et al., 2001; Lu et al., 2002; Rowitch et al., 2002; Zhou and
Anderson, 2002) and evidence for the elevation of BMPs in FGFR3 null
brain currently is lacking, the speculation that FGFR3 affects OL
numbers by a mechanism related to glial cell fate decisions remains open.
FGF signaling also is involved in the regulation of glial
migration. For example, disruption of FGF signaling in
Drosophila blocks glial migration (Klambt et al., 1992), and
OL progenitors expressing a dominant-negative FGFR1 transgene failed to
migrate in a transplantation model (Osterhout et al., 1997). However, because heterodimerization of different FGFRs has been reported in cell
lines (Bellot et al., 1991), these studies do not distinguish definitively between the relative importance of FGFR1 or FGFR3 (recall
that FGFR2 is not expressed by OL progenitors) for migration of OL
progenitors. Our current studies show that, in mice lacking FGFR3
signaling, OL progenitors still reach their final destinations, supporting the conclusion that OL progenitor migration is regulated by
FGFR1 rather than by FGFR3.
Why does the absence of FGFR3 signaling lead to only a partial and
transient inhibition of OL differentiation? A number of other mutant
mice null for specific growth factors/receptors also exhibit defects in
myelinogenesis, including mice null for CNTF (Barres et al., 1996),
neuregulin receptor (erbB2) (Park et al., 2001), IGF-1 (Beck et al.,
1995), PDGF-A (Fruttiger et al., 1999), and thyroid hormone (T3)
receptors (Baas et al., 2002). These defects are also mostly partial or
transient, involving reduced extents of differentiation and
myelination. Thus there appears to be an orchestrated set of growth
factors that regulate myelinogenesis. In a simple model two factors,
both of which are stimulatory for OL differentiation, act together.
Thus the loss of one merely reduces, rather than eliminates, terminal
differentiation. Several studies suggest that FGF-2 does, in fact, act
in concert with other trophic factors known to influence OL
progenitors. For example, the mitogenic effect of FGF-2 on OL
pre-progenitors and progenitors is enhanced by T3 (Ben-Hur et al.,
1998) and IGF-1 (Jiang et al., 2001) and on astrocytes by CNTF
(Albrecht et al., 2002); in addition, FGF-2 upregulates the expression
by OL progenitors of PDGFR (McKinnon et al., 1990), Notch-1
(Bongarzone et al., 2000), and the AMPA glutamate receptor GLUR4 (Gallo
et al., 1994). How these interactions may relate to the regulation of
OL differentiation in vivo is a challenging question.
In summary, we have demonstrated that, in the absence of FGFR3
signaling, the early events of the OL lineage progress normally, including proliferation, survival, and migration, but that the number
of terminally differentiated OLs is reduced significantly. Combined
with the "temporal location" of FGFR3 expression in the OL
developmental pathway, these data suggest that FGFR3 signaling regulates the probability of terminal differentiation by Pro-OLs and
thus specifically influences the timing of the onset of OL terminal
differentiation. In addition, these studies take advantage of the full
complement of signals present in vivo, allowing for the
investigation of the overall effect of the loss of FGFR3 signaling on
OL development within the context of the combinatorial interactions of
other signals.
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FOOTNOTES |
Received July 16, 2002; revised Oct. 31, 2002; accepted Nov. 1, 2002.
This work was supported by National Institutes of Health Grant NS
38878. We thank Drs. W. D. Richardson and N. P. Pringle (University College, London, UK) for communicating their unpublished data, for a critical reading of this manuscript, and for the PDGFR and FGFR3 cRNA probe; we also thank Dr. W. B. Macklin (Cleveland Clinic, Cleveland, OH) for the generous gift of the PLP cRNA probe. We
are pleased to acknowledge the contributions to cell culture by S. Winkler, manuscript processing by J. Seagren, and insightful manuscript
reviewing by Drs. S. E. Pfeiffer, M. Rasband, and K. Morest
(University of Connecticut Medical School). We especially appreciate
the valuable advice and encouragement of Dr. Morest during the course
of this work.
Correspondence should be addressed to Dr. Rashmi Bansal, Department of
Neuroscience, University of Connecticut Medical School, 263 Farmington
Avenue, Farmington, CT 06030-3401. E-mail: bansal{at}neuron.uchc.edu.
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ABSTRACT
INTRODUCTION
