1 Wolfson Institute for Biomedical Research and Department of Biology,
University College London, Gower Street, London WC1E 6BT, UK
2 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore
117609
3 Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, St. Louis, MO 63110, USA
* Author for correspondence (e-mail: w.richardson{at}ucl.ac.uk)
Accepted 3 October 2002
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SUMMARY |
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Key words: Fgfr3, Targeted deletion, Astrocyte, Reactive gliosis, CNS, Neuroepithelium
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INTRODUCTION |
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After neurones, the VZ switches to producing glial cells. Oligodendrocytes,
the myelinating glial cells of the CNS, develop from the ventral VZ. Small
numbers of oligodendrocyte progenitors (OLPs), which express the
platelet-derived growth factor receptor- (Pdgfra), first appear at the
ventricular surface on embryonic day 12.5 (E12.5) in the mouse, then
proliferate and migrate away into the grey and white matter before starting to
differentiate into myelin-forming oligodendrocytes
(Miller, 1996
;
Rogister et al., 1999
;
Richardson et al., 2000
;
Spassky et al., 2000
). In
rodents, OLPs are generated from the same part of the neuroepithelium as
somatic motoneurones (MNs) but not until after MN production has ceased
(Sun et al., 1998
;
Lu et al., 2000
) (for a
review, see Rowitch et al.,
2002
). This prompted us to suggest that there is a pool of shared
neuroglial precursors that first generates MNs, then switches to OLPs
(Richardson et al., 1997
;
Richardson et al., 2000
). This
idea has been supported recently by the finding that the bHLH proteins Olig1
and Olig2 are expressed and required in pMN for production of both
motoneurones and OLPs (Lu et al.,
2002
; Zhou and Anderson,
2002
; Takebayashi et al.,
2002
) (reviewed by Rowitch et
al., 2002
).
Where do astrocytes, the other major class of CNS glia, originate in the
neuroepithelium? It is believed that at least some astrocytes are generated by
transdifferentiation of radial glia
(Bignami and Dahl, 1974;
Choi et al., 1983
;
Benjelloun-Touimi et al., 1985
;
Voigt, 1989
;
Culican et al., 1990
). Others
are formed from multipotent precursors in the subventricular zones (SVZ) of
the postnatal brain. However, the origins of astrocytes in the developing
spinal cord are unclear, so we looked for an astrocyte lineage marker that
might be helpful in following the development of astrocytes from their
earliest precursors in the VZ. Previous expression studies of the fibroblast
growth factor receptor 3 (Fgfr3) suggested that this receptor might
be expressed in glial cells, possibly astrocytes
(Peters et al., 1993
;
Miyake et al., 1996
). Our own
studies, reported here, support this conclusion and suggest that
Fgfr3-positive astrocytes develop from Fgfr3-positive
precursor cells in the neuroepithelium. Fgfr3 is not expressed
equally in all parts of the neuroepithelium but is reduced or absent from pMN,
suggesting that astrocytes and OLPs have separate neuroepithelial origins. We
also found that astrocytes are formed in vitro in the absence of hedgehog
signalling unlike oligodendrocytes, which require sonic hedgehog from
the ventral midline. This reinforces the notion that at least some astrocytes
develop independently of OLPs.
To investigate the function of Fgfr3 in astrocytes, we examined
mice with a targeted deletion in the Fgfr3 locus
(Colvin et al., 1996). The
number of Fgfr3-expressing cells was normal in the knockout,
suggesting that Fgfr3 does not mediate a mitogenic or
survival-promoting effect for these cells. However, Gfap was markedly
upregulated in grey matter astrocytes, which normally have little or no Gfap
unlike their counterparts in white matter. Our results imply that
signalling through Fgfr3 normally represses Gfap expression in grey matter
astrocytes and suggest that white matter astrocytes might preferentially
express Gfap because ligands for Fgfr3 are not normally available in axon
tracts.
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MATERIALS AND METHODS |
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For dissociated cell cultures, E17 rat cervical spinal cords were digested in 0.25% (w/v) trypsin in Earle's buffered saline (Ca2+ and Mg2+ free; Gibco) for 15 minutes at 37°C, then FBS was added to a final concentration of 10% (v/v) and the tissue physically dissociated by trituration. Cells were washed by centrifugation and resuspended in BS medium before plating in a 50 µl droplet on poly-D-lysine-coated glass coverslips (5x104 cells/coverslip). Both explants and dissociated cell cultures were cultured at 37°C in 5% CO2 in a humidified atmosphere.
Neutralising Shh activity in vitro
Monoclonal Shh neutralising antibody 5E1
(Ericson et al., 1996) was
concentrated by ammonium sulphate precipitation from hybridoma supernatants
(Harlow and Lane, 1988
).
Monoclonal anti-NG2 proteoglycan #4.11
(Stallcup and Beasley, 1987
)
was used as a negative control. Precipitated antibodies were dissolved in a
small volume of PBS and dialysed first against PBS and then Dulbecco's
modified Eagle's medium (DMEM, Gibco). The final volumes were approximately
tenfold less than the starting volumes and were assumed to be ten time as
concentrated. Explants were incubated in the presence of either anti-Shh or
control antibodies at twice the final concentration. Antibodies were added at
the start of the experiment and fresh medium and antibody were added each day
thereafter. In some experiments cyclopamine (1 µM; from William Gaffield)
instead of anti-Shh was added to cultures daily.
BrdU labelling in vivo
E18 pregnant mice were injected intraperitoneally with BrdU at 50 µg
BrdU per gram bodyweight. Two injections were given, 6 hours apart. Mice were
sacrificed 3 hours after the second injection and the embryos were processed
for Fgfr3 in situ hybridisation and BrdU immunolabelling.
Preparation of tissue sections
C57B1/6 mice were obtained from Olac and bred in-house. Noon on the day of
discovery of the vaginal plug was designated embryonic day 0.5 (E0.5). We also
used Fgfr3-null mice (Colvin et
al., 1996) bred at UCL. Mid-gestation embryos were staged
according to the morphological criteria of Theiler
(Theiler, 1972
). Rats
(Sprague-Dawley) were obtained from the UCL breeding colony and staged
according to Long and Burlingame (Long and
Burlingame, 1938
). Fertilised White Leghorn chicken eggs were
obtained from Needle Farm (Cambridge, UK). They were incubated at 38°C and
the chicken embryos staged according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951
).
Embryos were decapitated and immersion-fixed in cold 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours before cryoprotecting in cold 20% (w/v) sucrose in PBS for at least 24 hours. In sections processed for immunohistochemistry after in situ hybridisation, the fixation time was reduced to 1 hour to preserve epitope integrity. Tissues were immersed in OCT embedding compound (BDH), frozen on solid CO2 and stored at -70°C before sectioning. Frozen sections (15 µm) were cut on a cryostat and collected on 3-aminopropyl-triethoxysilane (APES)-coated glass microscope slides. Sections were air-dried for 2 hours before storing dry at -70°C.
Immunohistochemistry
Anti-Gfap monoclonal ascites, clone G-A-5 (Sigma), was used at a dilution
of 1:400. Anti-BrdU (monoclonal BU209)
(Magaud et al., 1989) was used
at 1:5 dilution. Monoclonal O4 (Sommer and
Schachner, 1981
) was used as cell culture supernatant diluted 1:5.
Secondary antibodies were rhodamine- or fluorescein-conjugated goat
anti-rabbit or goat anti-mouse immunoglobulin (all from Pierce) diluted 1:200.
All antibodies were diluted in PBS containing 0.1% (v/v) Triton X-100 and 10%
(v/v) normal goat serum, except O4, which was diluted in PBS alone. Sometimes
diaminobenzidine (DAB) labelling (ABC kit, Vector Laboratories) was used
instead of fluorescence detection.
In situ hybridisation
Our in situ hybridisation procedures have been described
(Pringle et al., 1996;
Fruttiger et al., 1999
);
detailed protocols are available at
http://www.ucl.ac.uk/~ucbzwdr/richardson.htm.
Digoxigenin (DIG)- or fluorescein (FITC)-labelled RNA probes were transcribed
in vitro from cloned cDNAs. The rat Fgfr3 probe was transcribed from
a
900 bp partial cDNA encoding most of the tyrosine kinase (TK) domain
(W.-P. Yu, PhD thesis, University of London, 1995) and the chicken
Fgfr3 probe from a
440 bp partial cDNA encoding part of the TK
domain (from Ivor Mason, King's College London). The mouse Pdgfra
probe was made from a
1600 bp cDNA encoding most of the extracellular
domain_(from Chiayeng Wang, University of Chicago). The chicken
Pdgfra probe was made from a
3200 bp cDNA covering most of the
3' untranslated region of the mRNA (from Marc Mercola, Harvard Medical
School, Boston).
For double in situ hybridisation, two probes one FITC labelled and the other DIG labelled were applied to sections simultaneously. The FITC signal was visualised with alkaline phosphatase (AP)-conjugated anti-FITC Fab2 fragments before developing in p-iodonitrotetrazolium violet (INT) and 5-bromo-4-chloro-3-indolyl phosphate (toluidine salt) (BCIP), which produces a magenta/brown reaction product. The sections were photographed, then the AP was inactivated by heating at 65°C for 30 minutes followed by incubating in 0.2 M glycine (pH 2) for 30 minutes at room temperature. The INT-BCIP reaction product was removed by dehydration through graded alcohols, concluding with 100% ethanol for 10 minutes at room temperature. The DIG signal was then visualised with AP-conjugated anti-DIG Fab2 fragments and a mixture of nitroblue tetrazolium (NBT) and BCIP (all reagents from Roche Molecular Biochemicals) and the sections re-photographed. No labelling with NBT/BCIP was observed when we omitted either the DIG labelled probe or the anti-DIG antibody (data not shown).
For the Fgfr3-Pdgfra double in situ hybridisation of Fig. 4 we visualised the FITC (Pdgfra) signal with hoseradish peroxidase (HRP)-conjugated anti-FITC Fab2 fragments (Roche) before developing in fluorescein-tyramide reagent (NENTM Life Science Products, Boston) according to the manufacturer's instructions. The HRP-conjugate was inactivated by incubating in 2% (v/v) hydrogen peroxide for 30 minutes at room temperature. The DIG (Fgfr3) signal was then visualised with HRP-conjugated anti-DIG Fab2 fragments followed by rhodamine-tyramide, and the sections photographed under fluorescence optics. As specificity controls we omitted either the FITC-labelled Pdgfra probe or the HRP-conjugated anti-FITC antibody, which gave no staining other than for Fgfr3 (not shown).
|
Combined immunolabelling and in situ hybridisation
For the experiment of Fig.
7, cultured cells were first subjected to in situ hybridisation
with a [35S]-labelled RNA probe against Fgfr3 then
immunolabeled with anti-Gfap and biotinylated goat-anti-mouse Ig. The Gfap
signal was developed with DAB and the slides dehydrated through ascending
alcohols, dipped in nuclear emulsion (Ilford K5), exposed in the dark for
several days and developed in Kodak D19.
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RESULTS |
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Individual Fgfr3+ cells were also present outside the VZ after stage 34 (E8), both lateral and dorsal to the Fgfr3+ neuroepithelial domains. Often the individual cells appeared to be streaming away from the VZ into the parenchyma. This is evident in Fig. 1C, for example. By stage 37 (E11) Fgfr3 expression was no longer detectable in the VZ but scattered Fgfr3+ cells were present throughout the grey and white matter of the cord (Fig. 1D). Fgfr3 expression followed a similar progression in mouse and rat (Fig. 1F,G and not shown). In rodents, however, the ventral gap in Fgfr3 expression was not as pronounced as in chicks (Fig. 1G, arrow).
A scattered population of Fgfr3-expressing cells is found throughout most regions of the late embryonic and postnatal mouse brain, both in white and in grey matter. As in the embryonic spinal cord, there appear to be specific regions of the embryonic brain VZ that give rise to Fgfr3+ cells that stream away from the VZ into the parenchyma (not shown).
Fgfr3-expressing cells originate mainly outside the pMN domain of the
neuroepithelium
In the developing spinal cord, neuroepithelial precursors at different
positions along the dorsoventral axis generate distinct neuronal subtypes. The
ventral half of the spinal cord VZ is divided into five neuroepithelial
domains known as (from ventral to dorsal) p3, pMN, p2, p1 and p0
(Briscoe et al., 2000). Of
these, pMN is known to generate motoneurones followed by oligodendrocyte
progenitors (OLPs). It seemed to us that the ventral gap in Fgfr3
expression (Fig. 2A) might
correspond to pMN. To test this, we performed double in situ hybridisation for
Fgfr3 and Olig2 (which defines pMN)
(Lu et al., 2000
;
Zhou et al., 2000
). At stage
35, the Olig2 in situ hybridisation signal was within the gap in the
Fgfr3 signal (Fig. 2B,
arrow). Therefore, Fgfr3 is preferentially downregulated in pMN where
oligodendrocyte lineage cells originate, but is expressed both ventral and
dorsal to pMN.
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Fgfr3- expressing cells are glia
The fact that most of the scattered Fgfr3+ cells are
generated after stage 34 (E8) in the chick, E13.5 in mouse, is itself a strong
argument that they are glial cells, not neurones, because most spinal neurones
are born before this (Altman and Bayer,
1984). That some of the Fgfr3+ cells are found
in axon tracts also suggests that they are glia, for there are very few
neuronal cell bodies in fibre tracts.
Another indication that they are glial cells is that they continue to divide after they leave the VZ. We showed this by injecting BrdU into a pregnant mouse at 18 days gestation. The embryos were removed 3 hours later and processed by in situ hybridisation for Fgfr3 followed by immunolabelling for BrdU. We found many (Fgfr3+, BrdU+) cells scattered throughout the white and grey matter of the cord (Fig. 3, arrows). This confirms that Fgfr3-expressing cells divide in vivo and are therefore unlikely to be neurones or neuronal progenitors, which leave the VZ as postmitotic cells. This strengthens the idea that the Fgfr3-expressing cells are glia. There was also a population of (BrdU+, Fgfr3-) cells in both grey and white matter (Fig. 3C, arrowheads), so there is a distinct population(s) of dividing cells that do not express Fgfr3.
|
Fgfr3-expressing cells are distinct from Pdgfra+
oligodendrocyte progenitors
To determine whether the Fgfr3+ cells that we detect
are oligodendrocyte progenitors (OLPs), we double labelled mouse E18 and P2
spinal cord sections for Fgfr3 and Pdgfra, an established
marker of early OLPs. Both in situ hybridisation probes labelled similar
numbers of cells that were scattered throughout the spinal cord grey and white
matter, but the two cell populations were completely non-overlapping
(Fig. 4). This also held true
throughout the postnatal brain (N. P. P., unpublished). We also looked in
newborn Pdgfa knockout mice which contain far fewer
Pdgfra+ OLPs than normal
(Fruttiger et al., 1999).
Despite the lack of OLPs, there were normal numbers of
Fgfr3+ cells at this age
(Fig. 5). Clearly, the
Fgfr3+ cells detected by our in situ hybridisation
procedures are not early OLPs but a different cell population. This is
consistent with the fact that in mice lacking Fgfr3, early events of
oligodendrocyte lineage progression occur normally and the numbers of
Pdgfra+ cells remains unchanged (R. Bansal, personal
communication) (N. P. P., unpublished).
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Fgfr3-expressing cells are astrocytes and astrocyte precursors
To test whether the Fgfr3+ cells might be astrocytes,
we double labelled E18 mouse spinal cord sections for Fgfr3 and
Gfap mRNAs. At E18, white matter astrocytes begin to express
Gfap mRNA, which initially remains in the astrocyte cell bodies and
allows identification of individual astrocytes. (As astrocytes mature further,
both Gfap mRNA and protein are relocated to the extending cell
processes, making individual cells difficult to distinguish.)
All the Gfap+ astrocytes in developing white matter at E18 also expressed Fgfr3 (Fig. 6, arrows). This result clearly identifies many of the Fgfr3-expressing cells as astrocytes. Nevertheless, the majority of Fgfr3-expressing cells in the grey matter (Fig. 6B, arrowheads) are Gfap-negative. We presume that these represent Gfap-negative, possibly immature, astrocytes.
|
In an attempt to label all astrocytes, including Gfap-negative astrocytes, we used an in situ hybridisation probe against glutamine synthetase mRNA (Glns) (EC 6.3.1.2). Glns is widely regarded as an astrocyte marker, although there have been reports that it is also present in mature oligodendrocytes and even OLPs. We found that Glns transcripts were present in the VZ of the E15 mouse spinal cord and in cells outside the VZ in a pattern that was very similar that of Fgfr3 (Fig. 7). This is consistent with the view that Fgfr3 and Glns mark astrocytes and their precursors. This conclusion was further strengthened by studies of cultured astrocytes (see below).
Cultured astrocytes co-express Gfap and Fgfr3
When CNS cells are dissociated and placed in culture, astrocytes in the
culture upregulate Gfap and are easily recognisable. We dissociated and
cultured cells from E17 rat cervical spinal cord and labelled them by in situ
hybridisation for Fgfr3 and by immunocytochemistry for Gfap. Almost
all of the Gfap+ astrocytes also expressed Fgfr3
(Table 1;
Fig. 8, arrows). There was also
a small population of flat, fibroblast-like Fgfr3+ cells
that did not express Gfap (Fig.
8, arrowheads). The number of these cells decreased with time in
culture; at 3 days they were 6% of all cells, by 9 days less than 1%
(Table 1). These
(Fgfr3+, Gfap-) cells might be astrocyte
precursors or immature astrocytes that have not yet upregulated Gfap. In any
case, this experiment provides clear evidence that most or all
Gfap+ astrocytes in culture co-express Fgfr3.
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Gfap is upregulated in grey matter astrocytes in Fgfr3 null mice
If Fgfr3 is expressed by astrocytes, we might expect to see
specific effects on astrocytes in transgenic mice homozygous for a targeted
disruption of Fgfr3. These mice have previously been shown to have
skeletal and inner ear defects but no CNS defects have yet been reported
(Colvin et al., 1996).
We visualised astrocytes in spinal cord sections of 3-month-old Fgfr3 null mice, together with their heterozygous Fgfr3+/- and wild-type littermates, by immunolabelling with anti-Gfap. Heterozygous and null mutant mice all displayed the normal pattern of Gfap expression up to 6 weeks of age. Gfap expression was observed in the white matter around the circumference of the spinal cord, many Gfap-labelled processes being oriented in a radial direction (Fig. 9A). By comparison, there was little or no Gfap expression in the grey matter, except in astrocytes associated with blood vessels. Between 6 weeks and 2 months of age, a striking up-regulation of Gfap expression occurred in the grey matter of Fgfr3 null mice, though not in their heterozygous or wild-type littermates (Fig. 9B). Astrocytes lining blood vessels also had increased Gfap immunoreactivity.
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The number of cells that contain Fgfr3 mRNA was not noticeably different in Fgfr3-null spinal cords compared with wild type (data not shown). This suggests that Fgfr3 does not normally mediate a signal for proliferation or survival of astrocytes, although further experiments (e.g. BrdU labelling in vivo) would be required to substantiate this.
Astrocyte development in vitro does not depend on Hedgehog
signalling
In the spinal cord, production of ventral cell types motoneurones,
ventral interneurones and OLPs is dependent on Shh signalling
(Ericson et al., 1996;
Orentas et al., 1999
) (for a
review, see Jessell, 2001
). We
wanted to know whether production of astrocytes from the ventral neural tube
is also dependent on Shh. We microdissected stage 12/13 (E2) chick spinal cord
into thirds along the dorsoventral axis and cultured the ventral-most
fragments in collagen gels with either a control antibody or an anti-Shh
neutralising antibody (see Materials and Methods). After 48 hours in culture
we labelled explants with monoclonal antibody 4D5, which recognises
homeodomain proteins Isl1 and Isl2 in motoneurones. Control explants contained
numerous Isl+ cells, whereas none were observed in explants
incubated with anti-Shh (data not shown). After a further 10 days in culture
(12 days total) we visualised OLPs with monoclonal antibody O4
(Sommer and Schachner, 1981
)
(Fig. 10C,D) and astrocytes
with anti-Gfap (Fig. 10A,B).
All of the explants incubated with control antibody (19/19) contained large
numbers (>300) of O4+ late-stage OLPs
(Fig. 10C). As expected, OLP
production was markedly decreased by anti-Shh
(Fig. 10D); 14/22 explants
contained no O4+ cells and, of the remaining eight explants, seven
contained fewer than ten positive cells and the other one contained 38
positive cells. By contrast, all explants contained numerous (>300)
Gfap+ astrocytes whether they had been incubated with control
antibody (19/19) or anti-Shh (22/22) (Fig.
10A,B).
|
Similar results were obtained with explants from stage 25 (E5) embryos from which we were able to dissect the ventral one-quarter of the neural tube and discard the floor plate. Once again, large numbers of Gfap+ astrocytes developed in explants cultured with control antibody (22/22) and with anti-Shh (25/25), even though OLP production in these explants was inhibited by anti-Shh (not shown).
To test the possibility that other hedgehog (Hh) proteins (Desert Hh,
Indian Hh) control astrocyte production in ventral explants, we inhibited the
activity of all isoforms with the alkaloid cyclopamine
(Cooper et al., 1998;
Incardona et al., 1998
). This
gave similar results as Shh neutralising antibodies (data not shown). Thus, we
conclude that astrocyte induction in ventral spinal cord does not require
Hedgehog signalling.
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DISCUSSION |
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Fgfr3 is also expressed transiently by a subpopulation of
motoneurones (Philippe et al.,
1998) and by late oligodendrocyte progenitors (late OLPs) just
prior to terminal differentiation in vitro
(Bansal et al., 1996
). We are
convinced that the Fgfr3+ cells that we detect are not
OLPs, however. First and foremost, double labelling for Fgfr3 and
Pdgfra (a marker of early OLPs) demonstrates that these mark separate
populations of cells. The Fgfr3+ and
Pdgfra+ cell populations appear at different times and
initially their distributions are different. Moreover, the number and
distribution of Fgfr3+ cells was unaltered in neonatal
Pdgfa-null spinal cords, which have very few
Pdgfra+ OLPs and oligodendrocytes
(Fruttiger et al., 1999
). This
argues strongly that the large majority of Fgfr3+ cells
revealed by our in situ hybridisation protocol are not OLPs. Bansal et al.
(Bansal et al., 1996
) have
shown that OLPs do express Fgfr3 mRNA in culture but only at a low
level during the earlier stages of the lineage. Presumably this is below our
limit of detection in situ. OLPs upregulate Fgfr3 strongly just prior
to oligodendrocyte differentiation (Bansal
et al., 1996
) but these presumably represent a small subset of
OLPs in the embryonic spinal cord and do not feature in our analysis.
Fgfr3-positive cells co-expressed mRNA encoding glutamine
synthetase (Glns; EC 6.3.1.2). In the CNS, Glns is an accepted marker of
mature astrocytes (Norenberg and
Martinez-Hernandez, 1979;
Stanimirovic et al., 1999
) but
it is also expressed in oligodendrocytes
(Domercq et al., 1999
) and
OLPS (Baas et al., 1998
). Glns
has not previously been ascribed to neuroepithelial precursors or immature
astrocytes in the embryo, although Glns transcripts have been
detected in the rat brain by northern blot as early as E14. To our knowledge,
Glns has not been described in neurones except in pathological situations such
as Alzheimer's disease (Robinson,
2000
). Therefore, we are confident that the
(Fgfr3+, Glns+) double-positive cells
described here are glial cells. Taken together with the evidence against them
being OLPs (see above), it seems likely that they correspond to immature and
mature astrocytes. This is strongly supported by the observations that
Fgfr3+ cells co-express Gfap protein and/or mRNA in (1)
the formative white matter of the normal developing spinal cord and (2)
cultures of dissociated spinal cord cells.
Neuroepithelial origins of astrocytes
Fgfr3 was expressed in two domains of the spinal cord
neuroepithelium separated by an Fgfr3-negative region. This was true
of both rodent and avian embryos though it was more obvious in the latter. The
Fgfr3-negative region corresponds roughly to the pMN domain of the VZ
that generates somatic motoneurones followed by Pdgfra+
OLPs (Sun et al., 1998;
Rowitch et al., 2002
).
Therefore, our data indicate that OLPs and astrocytes originate from separate
precursors that reside in different parts of the VZ. How does this fit with
other ideas about the origin of astrocytes? One hypothesis is that astrocytes
arise by transdifferentiation of radial glia, after the latter have fulfilled
their role as cellular substrates for radial migration of neuronal progenitors
(Bignami and Dahl, 1974
;
Choi et al., 1983
;
Benjelloun-Touimi et al., 1985
;
Voigt, 1989
;
Culican et al., 1990
). This
could be compatible with our Fgfr3 expression data, as radial glia
have their cell bodies close to the ventricular surface. However, radial glia
are distributed all around the spinal cord lumen, unlike Fgfr3, so
one would have to postulate that only a subset of radial glia express
Fgfr3.
In double-knockout mice that lack the two basic helix-loop-helix (bHLH)
transcriptions factors Olig1 and Olig2, the pMN domain of the VZ undergoes a
homeotic transformation into p2, its immediate dorsal neighbour
(Rowitch et al., 2002). As a
result, pMN no longer generates motoneurones followed by OLPs, but instead
produces V2 interneurones followed by astrocytes
(Zhou and Anderson, 2002
;
Takebayashi et al., 2002
). By
implication, this is the usual fate of p2 precursors in wild-type mice. This
is consistent with our observation that Fgfr3+ astrocytes
apparently originate within an extended part of the ventral VZ, including p2
but excluding pMN. Our Fgfr3 expression data are also consistent with
previous fate mapping experiments in chick-quail chimeras, which indicated
that astrocytes are generated from dorsal as well as ventral parts of the VZ,
whereas OLPs are generated only from ventral territory
(Pringle et al., 1998
). It
remains to be seen whether astrocytes that are generated from distinct
neuroepithelial domains (p3 or p2, say) have identical properties or whether
they are functionally specialised for modulating synaptic activity or
interacting with blood vessels, for example.
Production of ventral cell types such as motoneurones, interneurones and
OLPs is dependent on Shh signalling. As many Fgfr3-expressing
astrocyte precursors appear to originate in p3, p2 and other ventral domains,
we might expect that production of astrocytes might also depend on Shh
signalling. However, we found that astrocytes developed in explant cultures of
ventral neural tube either in the presence or absence of Shh activity. Our
data imply that astrocytes are specified by different mechanisms than OLPs
at least, they demonstrate that astrocyte and OLP production are not
obligatorily linked. In fact, there is evidence that more than one signalling
pathway can lead to astrocyte development in vitro
(Rajan and McKay, 1998).
Because astrocytes can be formed from dorsal as well as ventral
neuroepithelium, it remains possible that `ventral' astrocytes might normally
be under Shh control, but that by blocking Shh signalling we uncover an
alternative `dorsal' pathway for astrocyte development.
It has been reported that there are glial-restricted precursor cells (GRPs)
in the embryonic rat spinal cord that are dedicated to the production of
astrocytes and oligodendrocytes (Rao and
Mayer-Proschel, 1997; Herrera
et al., 2001
). This seems to conflict with current evidence that
oligodendrocytes and astrocytes are generated from different precursors in the
embryonic spinal cord (Lu et al.,
2002
; Rowitch et al.,
2002
; Zhou and Anderson,
2002
) (this paper). A possible reconciliation might be that GRPs
with the potential to generate both astrocytes and oligodendrocytes are formed
in all parts of the spinal cord VZ but are constrained in vivo to generate
only astrocytes or only oligodendrocytes, depending on the signals in their
local environment (i.e. where they are located) (for a review, see
Rowitch et al., 2002
).
Fgfr3 regulates Gfap expression in grey matter astrocytes
Astrocytes with distinct, heritable morphologies have been described in
cultures of rat spinal cord cells
(Fok-Seang and Miller, 1992).
Astrocytes in different parts of the CNS differ in morphology or function in
vivo too, suggesting that they might fulfil different, region-specific
functions. In addition, astrocytes in white matter tracts generally have
smaller cell bodies with more and longer processes compared to their
counterparts in grey matter (Connor and
Berkowitz, 1985
). For this reason, white matter astrocytes are
sometimes referred to as `fibrous' and those in grey matter as `protoplasmic'
or `velous'. White matter astrocytes also express high levels of Gfap, whereas
grey matter astrocytes contain little or no immunoreactive Gfap.
Fibrous and protoplasmic astrocytes might develop from separate lineages
(Connor and Berkowitz, 1985).
However, our observation that Gfap is upregulated in grey matter astrocytes of
Fgfr3-null mice provides strong in vivo evidence that extracellular
signals might be required to maintain their normal Gfap-negative phenotype.
This is consistent with a report that adding Fgf2 to cultured astrocytes
downregulates Gfap mRNA and protein and causes their morphology to change
(Reilly et al., 1998
). Fgf2
and other known Fgfr3 ligands such as Fgf9 are made by, and presumably
released from, many CNS neurones
(Eckenstein et al., 1991
;
Cotman and Gomez-Pinilla,
1991
; Woodward et al.,
1992
; Gomez-Pinilla et al.,
1994
; Kuzis et al.,
1995
). One possible reason that white matter astrocytes express
high levels of Gfap in wild-type mice might be that they are denied exposure
to Fgfr3 ligands in axon tracts perhaps because Fgf, like
Pdgf, is secreted from neuronal cell bodies but not from axons
(Fruttiger et al., 2000
).
Upregulation of Gfap in the Fgfr3-null mouse is mindful of the
astrocyte response to CNS injury or disease so-called reactive gliosis
or astrocytosis (for reviews, see Ridet et
al., 1997; Norton,
1999
). It would be interesting to know whether interruption of
signalling through Fgfr3 is somehow involved in the astrocyte reaction to
injury. However, it is unlikely to be straightforward, because Gfap
upregulation in the Fgfr3-null animals does not occur until around 2
months of age, suggesting that it is an indirect effect. In addition, the data
from the Fgfr3-null mouse are difficult to square with the
observation that intra-ventricular injection of Fgf2 has been reported to
increase the number of Gfap+ reactive astrocytes (Unsicker,
1993).
Most grey matter (protoplasmic) astrocytes possess many short sheet-like
processes containing little, if any, Gfap
(Connor and Berkowitz, 1985).
It has been suggested that this morphology might help them to infiltrate the
neuropil and surround axonal terminals, synapses and neuronal cell bodies,
consistent with one of their proposed roles in neurotransmitter metabolism
(Martinez-Hernandez et al.,
1977
; Norenberg and
Martinez-Hernandez, 1979
). It will be interesting to see if the
reactive astrocytes in Fgfr3-null mice are defective in
neurotransmitter metabolism and whether this contributes to the premature
death of the animals.
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ACKNOWLEDGMENTS |
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