1 Cambridge Centre for Brain Repair, University of Cambridge, ED Adrian
Building, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK
2 The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
3 Neural Development Unit, Institute of Child Health, 30 Guilford Street, London
WC1N 1EH, UK
4 Waisman Center Stem Cell Research Program, University of Wisconsin-Madison,
1500 Highland Avenue, Madison, WI 53705, USA
* Author for correspondence (e mail: sc222{at}cam.ac.uk)
Accepted 16 September 2003
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SUMMARY |
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Key words: Stem cell, Oligodendrocyte, FGF2, Spinal cord, Sonic hedgehog, OLIG2, NKX2.2
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Introduction |
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The spinal cord has served as a valuable model for the study of
oligodendrocyte origins. During normal development, oligodendrocytes originate
from restricted foci within the ventral neural tube under the influence of
ventral midline-derived (notochord and floorplate) SHH signalling
(Pringle and Richardson, 1993;
Orentas et al., 1999
). Graded
SHH signalling patterns the ventral neural tube by influencing the expression
of various transcription factors, including the basic helix-loop-helix (bHLH)
genes Olig1 and Olig2
(Lu et al., 2000
;
Zhou et al., 2000
;
Takebayashi et al., 2000
).
Olig1 and Olig2 represent the earliest cell type specific
genes within the oligodendrocyte lineage, and their expression precedes
markers of the oligodendrocyte precursor cell (OPC), including PDGF receptor
(PDGFR
) and DM20/PLP (Lu et
al., 2000
). In addition to SHH, increasing evidence suggests that
other factors are important in determining cell fate along the dorsoventral
axis of the developing spinal cord
(McMahon et al., 1998
;
Barth et al., 1999
;
Pierani et al., 1999
;
Liem et al., 2000
;
Ishibashi and McMahon, 2002
).
More specifically, two recent studies have examined the idea that dorsally
derived signals play a role in controlling OPC specification
(Wada et al., 2000
;
Mekki-Dauriac et al., 2002
).
Mekki-Dauriac and coworkers provide evidence that bone morphogenetic proteins
(BMPs) negatively regulate spinal cord oligodendrocyte specification
(Mekki-Dauriac et al.,
2002
).
Several lines of evidence suggest that, within the spinal cord, OPCs arise
exclusively from the ventral ventricular zone, and that oligodendrocyte
development in the dorsal cord results only from a ventral to dorsal OPC
migration (Warf et al., 1991;
Pringle and Richardson, 1993
;
Yu et al., 1994
;
Timsit et al., 1995
;
Hall et al., 1996
;
Pringle et al., 1998
).
However, it remains possible that the lack of OPCs and oligodendrocytes in
isolated rat embryonic day 14 (E14) dorsal spinal cord-derived cultures
results from a failure to provide signals necessary for the generation of
oligodendrocytes from neural precursors that are intrinsic to the dorsal cord.
This interpretation is consistent with the demonstration that dorsal-derived
cell populations have the capacity to generate oligodendrocytes in vitro upon
exposure to notochord or SHH, and following transplantation
(Trousse et al., 1995
;
Hardy and Friedrich, 1996
;
Orentas et al., 1999
). In
addition, dissociated E14 dorsal cultures stimulated with FGF2 and EGF
generate oligodendrocytes following mitogen withdrawal
(Chandran et al., 1998
). The
latent oligodendroglial potential of E14 dorsal cord suggests that stimulation
and proliferation of a dorsal-derived cell may generate oligodendrocytes in
response to these and other mitogens, without the need for inward migration of
cells from the ventral cord.
Here, we characterise the relationship of the rat E14 dorsal-derived precursor to oligodendrocytes, and the role of SHH in mediating oligodendrogenesis following expansion of these precursors in response to FGF2. We show, using clonal analysis, that the embryonic dorsal spinal cord contains an FGF2-responsive stem cell, and provide evidence for the existence of a SHH-independent pathway for the generation of oligodendrocytes. Analysis of uncultured and FGF2-treated embryonic spinal cords from mice homozygous for a null mutation of SHH reveals a hedgehog-independent pathway for the induction of Olig2 and Nkx2.2 genes and oligodendrocytes. Finally, we show that FGF2-mediated induction of oligodendrocytes requires MAP kinase signalling and is inhibited by BMP4.
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Materials and methods |
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Precursor and clonal cultures
Following enzymatic dissociation of dorsal spinal cord (rat E14 or mouse
E12.5) or whole SHH-null cord (E12.5), tissue was seeded into uncoated T25
culture flasks (Nunclon) at a density of 2x105 live
cells per ml of proliferation medium [DMEM/F12 (3:1) (Gibco) containing B27
supplement with FGF2 and heparin (5 ug/ml)]. Characteristic multicellular
aggregates or neurospheres formed by 7 days, at which time cells were
harvested by centrifugation at 1000 g for 3 minutes, then
re-suspensed and dissociated using mechanical trituration to a single cell
suspension. Aliquots of cells were plated, as described above, in the absence
of mitogens to observe differentiation. Hedgehog requirement for
oligodendrogenesis was assessed using cyclopamine and the cyclopamine
derivative 3-keto, N-aminoethyl aminocaproyl dihydrocinnamoyl
cyclopamine (KAAD; Toronto Research Chemicals)
(Taipale et al., 2000
). The
pathway inhibitors LY294002 (PI3-kinase) and U0126 (MAP kinase) were obtained
from Sigma, UK and Calbiochem. For clonal studies, primary cultures were
enzymatically and mechanically dissociated to a single cell suspension at 24
hours after seeding in proliferation medium. The resulting cell suspension was
filtered and sorted (Becton Dickinson FACS Vantage SE, San Jose, CA) into
single live cells in uncoated 96-well plates containing 200 µl of clonal
proliferation medium composed of DMEM/L15 (1:1) FGF2, B27 and N2. Cultures
were maintained for up to 21 days and then plated in control medium
supplemented with B27. Cultures were immunostained for phenotypic potential at
7 days post-plating.
PCR analysis
The yolk sac was removed from each embryo, washed in phosphate-buffered
saline and stored frozen before genotyping. Genomic DNA was extracted and PCR
amplification performed using primers for the neomycin resistance gene and SHH
as previously described (Chiang et al.,
1996). Semi-quantitative cross comparison of cultures or tissues
was performed by RT-PCR using SMART technology (Clontech). Gene expression
comparisons were made using matched samples normalised for the level of GADPH
expression, quantified by SYBR Green (Molecular Probes) staining and band
intensity measurements using FLA-3000 Imager System (Fujifilm). PCR conditions
for all primer pairs used in this study were optimised for cDNA derived from
foetal tissue:
nestin, 5'-agtcagagcaagtgaatgg-3' and 5'-agaaacaagatctcagcagg-3';
Shh, 5'-tcacccccaattacaacc-3' and 5'-acgtaagtccttcaccagc-3';
Ihh, 5'-cagtgatgtgcttattttcc-3' and 5'-tagagtcccttcagcttcc-3';
Dhh, 5'-tgcctctgctatacaagc-3' and 5'-gtagacccagtcgaatcc-3';
Nkx6.1, 5'-gctctactttagccccagc-3' and 5'-tgtaatcgtcgtcatcctcc-3';
Nkx6.2, 5'-ctgcacaacatggctgagat-3' and 5'-gtcatgcccagagagtaggc-3';
Olig1, 5'-gacctcagccaatcttcc-3' and 5'-taacacccttgatgtttgtacc-3';
Olig2, 5'-tcagagcacaggagcaagc-3' and 5'-aacgacacagaaagaaaacagc-3';
Nkx2.2, 5'-cattcgctacaagatgaaacg-3' and 5'-agaggcaaagaagcaaagc-3';
Nkx2.9, 5'-gagacaagccctgcagactc-3' and 5'-ggtgctaagtgctggtaggc-3';
Pax7, 5'-cgatttctcatctctagacc-3' and 5'-cagacagattcacaaaagc-3';
Hb9, 5'-gacaacttcccgtacagc-3' and 5'-ataacacctcactccactacc-3';
Lhx3, 5'-cccagacaccaacttgagc-3' and 5'-gccagtaaagaaagagaaatgc-3'; and
Gapdh, 5'-accacagtccatgccatcac-3' and 5'-tccaccaccctgttgctgta-3'.
Immunohistochemistry
Live cells were stained for the surface markers O4 (supernatant diluted
1:5) and galactocerebroside (GC; supernatant diluted 1:10)
(Sommer and Schachner, 1981)
(IC-07 cell line, ECACC, UK) to assess glial lineages. Intracellular antigens
studied were: glial fibrillary acidic protein (GFAP) as an astrocyte marker
(rabbit polyclonal, 1:250; Dako, UK); ß-tubulin type III as a neuronal
marker (1:250; Sigma); and myelin basic protein (1:500, Serotec) and actin
(1:250, Sigma) to identify smooth muscle. All antibodies were made up in 5%
serum and DMEM. The surface antigens were stained with primary antibody for 60
minutes at 37°C, followed by the appropriate conjugated secondary antibody
for 60 minutes. After fixation with 4% PFA for 10 minutes and permeabilisation
with absolute methanol at -20°C for 15 minutes, the primary antibodies to
intracellular antigens were added for 60 minutes at 37°C, followed by the
appropriate secondary conjugated antibody. All secondary antibodies (Sigma and
Harlan Sera-Lab) were used at a dilution of 1:100, and Hoechst nuclear stain
(1:5000) was included in the final antibody application. Cryostat sections of
PFA fixed neurospheres were also stained for OLIG2 (1:3000; kindly provided by
Dr Takebayashi) and NKX2.2 (1:50; Developmental Hybridoma Study Bank). Removal
of primary antibodies resulted in no specific staining. Coverslips were
mounted on glass slides using Vectashield (Vector Labs, UK), and viewed under
a Leitz inverted microscope with appropriate filters for cell identification
and counting. Using a grid, five consecutive fields were counted for each
coverslip. The number of positive cells was expressed as a mean±s.e.m.
from 2-4 coverslips and from a minimum of three separate experiments, unless
stated otherwise. Statistical significance was assessed by ANOVA with post hoc
Neuman-Keuls and Student's t-test using a standard statistical
package (Graphpad, Prism).
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Results |
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Clonal analysis was next undertaken to characterise the developmental relationship of the FGF2-responsive cell and the GC-positive oligodendrocyte, in particular whether this cell represents a stem cell or a fate-committed cell of the oligodendrocyte lineage. Twenty-four hours after seeding of primary dorsal cultures in proliferation medium, individual cells were FACS sorted into a single well of a 96-well plate. Clonal neurospheres were generated in response to FGF2 under substrate and serum free conditions, and assessed for phenotypic potential. Expression of GFAP (astrocyte), ß-tubulin III (neurones), and O4 or GC (oligodendrocytes) was used to identify the three principal CNS phenotypes. Twenty-two clones were studied: the predominant clonal cell type was macroglial, with 16 clones generating oligodendrocytes and astrocytes. Six clones generated neurones, oligodendrocytes and astrocytes, and could be considered to be stem cells (Fig. 1A). These findings provide in vitro evidence for the existence of an FGF2-responsive CNS stem cell within the population derived from E14 dorsal spinal cord.
|
In order to establish whether endogenous hedgehog was responsible for
oligodendrocytes generated from dorsal neurosphere cultures following FGF2
stimulation, we performed experiments with cyclopamine. Cyclopamine is a
plant-derived teratogen that acts on the general hedgehog signal transducer
smoothened to inhibit hedgehog signalling, and also the generation of
oligodendrocytes from primary embryonic culture
(Incardona et al., 1998;
Taipale et al., 2000
;
Tekki-Kessaris et al., 2001
;
Alberta et al., 2001
). Primary
cultures derived from E14 dorsal spinal cord were grown in the presence of
both FGF2 and cyclopamine. Differentiation of these cultures demonstrated that
cyclopamine did not inhibit the appearance of O4- and GC-positive
oligodendrocytes (Fig. 1B,C).
Importantly, following cyclopamine treatment, longer-term cultures (14-21 days
in vitro) express the mature oligodendrocyte marker myelin basic protein
(Fig. 1C). In order to further
examine the contribution of hedgehog signalling to the generation of
oligodendrocytes from FGF2-stimulated cultures, we studied the effect of the
addition of KAAD to FGF2 expansion medium. KAAD is a cyclopamine derivative of
significantly greater potency in blocking the activation of the hedgehog
signalling pathway (Taipale et al.,
2000
). KAAD had no significant effect on oligodendrocyte
generation from the FGF2-treated cultures
(Fig. 1B). By contrast,
cyclopamine and KAAD at a concentration of 1 µM did result in a significant
reduction in oligodendrocytes derived from dissociated primary (without FGF2
treatment) E12 rat whole spinal cord cultures, an observation consistent with
the requirement of hedgehog for the induction of spinal cord oligodendrocytes
(Orentas et al., 1999
;
Soula et al., 2001
)
(Table 1). The generation of a
significant number of oligodendrocytes despite the hedgehog signalling
blockade following FGF2 treatment suggests that an additional
hedgehog-independent pathway for oligodendrocyte formation may exist.
|
|
|
Recent observations of unexpected Indian hedgehog (IHH) expression in
dissociated embryonic cultures led us to look for Ihh and
Dhh transcripts (Alberta et al.,
2001; Tekki-Kessaris et al.,
2001
). Ihh was present in some conditions, notably
wild-type untreated dorsal cultures, and was consistently found to be
differentially regulated by FGF2 in SHH mutant compared with wild-type tissue.
Significantly the pattern of Ihh expression did not correlate with
gliogenic gene expression. Nonetheless, in order to exclude the possibility
that IHH may account for Olig and Nkx2.2 expression in
dissociated cultures, both SHH null E12.5 whole cord and wild-type E12.5
dorsal mouse spinal cord were grown in the presence of FGF2 and cyclopamine.
Significantly, Olig1, Olig2 and Nkx2.2 expression was again
evident following FGF2 exposure, a finding consistent with the demonstration
of O4+ oligodendrocytes in cyclopamine-treated cultures
(Fig. 2C,
Fig. 3). Taken together, these
findings demonstrate a hedgehog-independent pathway for induction of the
oligodendrogenic genes Olig2 and Nkx2.2 in the presence of
FGF2.
FGF2 mediated oligodendrocyte induction is dependent on MAP kinase
signalling and is inhibited by BMP4
In order to explore whether FGF2 has an active role in the generation of
oligodendrocytes from dorsal spinal cord precursors, or whether
oligodendrogenesis results from a passive process - for example, by dilution
of inhibitory factors present in primary dorsal cultures - we next cultured
dorsal-derived cells in the presence of both FGF2 and the respective
PI3-kinase and MAP kinase pathway inhibitors LY294002 and UO126. MAP kinase
inhibition resulted in a significant reduction of oligodendrocytes, suggesting
an active role for FGF2 in the derivation of oligodendrocytes from the
cultures (Fig. 4). PI3-kinase
inhibition had no significant effect on oligodendrocyte numbers.
|
|
In view of the known effect of BMPs on astroglial differentiation of neural
precursors, we also examined expression of GFAP, a marker of astrocytes
(Gross et al., 1996;
Mabie et al., 1999
;
Zhu et al., 1999
). To our
surprise, BMP4-treated cultures contained reduced numbers of astrocytes, in
contrast to cultures treated with FGF2 alone
(Fig. 4). On account of the
dorsal inductive capacity of BMPs we also examined cultures for the expression
of smooth muscle actin, a marker of neural crest derivatives
(Liem et al., 1995
;
Shah et al., 1996
;
Mujtaba et al., 1998
;
Panchision et al., 2001
).
Significantly, BMP4-treated cultures generated smooth muscle actin expressing
cells, although a large number of cells remained negative for the studied
markers (Fig. 4).
The respective phenotypic profiles of FGF2 cultures treated with BMP4 (10 ng/ml) or U0126 were similar, and revealed negligible numbers of oligodendrocytes, reduced astrogenesis and induction of smooth muscle actin upon differentiation. These findings are suggestive of crosstalk between FGF2-activated MAP kinase and BMP signalling.
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Discussion |
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The E14 dorsal spinal cord contains an FGF2-responsive CNS stem
cell
FGF2 alone was able to stimulate the generation of neurospheres from rat
E14 dorsal spinal cord, and these neurospheres in turn were able to produce
significant numbers of oligodendrocytes following differentiation. This
observation is consistent with previous reports showing FGF2-dependent
oligodendrocyte formation from the embryonic whole spinal cord
(Kalyani et al., 1997).
Single-cell analysis of E14 dorsal cultures revealed cells that were capable
of generating clones. Significantly, monoclonal derivation of neurones,
astrocytes and oligodendrocytes was confirmed. The observation of multipotent
cells within the E14 dorsal spinal cord is compatible with reports of
FGF2-responsive stem cells isolated from both the embryonic and adult whole
spinal cord (Rao and Mayer-Proschel,
1997
; Shihabuddin et al.,
2000
). Rao and coworkers have characterised the potential of whole
spinal cord-derived stem cells, and have further identified neural- and
glial-restricted progenitors derived from the developing spinal cord
(Rao and Mayer-Proschel, 1997
;
Mayer-Proschel et al., 1997
).
A recent study has extended these observations, and has identified differences
between glial-restricted progenitors derived from the developing ventral and
dorsal spinal cord (Gregori et al.,
2002
). However, the E14 dorsal stem cell described in this study
and the dorsal glial-restricted progenitor reported by Gregori and co-workers
(Gregori et al., 2002
)
self-evidently differ in their capacity to generate neurones, although both
require FGF2. Further characterisation requires a more extensive range of
markers than is presently available.
Hedgehog-dependent and -independent pathways for oligodendrocyte
formation
The restricted ventral origin of spinal cord oligodendrocytes in vivo
reflects the importance of notochord and floor plate-derived signals. The
morphogen SHH represents the primary ventralising signal and is sufficient to
induce ectopic oligodendrocyte formation
(Pringle et al., 1996).
Blocking SHH inhibits the induction of oligodendrocytes
(Orentas et al., 1999
;
Soula et al., 2001
;
Alberta et al., 2001
). Recent
studies have extended our understanding of the genetic requirements underlying
spinal cord oligodendrocyte specification and its relationship to
neurogenesis. Oligodendrocytes emerge from the same area of the ventral spinal
cord that initially generate MNs and/or V3 interneurones
(Richardson et al., 2000
;
Tekki-Kessaris et al., 2001
;
Soula et al., 2001
).
SHH-regulated OLIG2 specifies MNs and, subsequently, oligodendrocytes
(Novitch et al., 2001
). This
is dependent on the differential temporal association of OLIG2 with
neurogenin2 and later with NKX2.2
(Mizuguchi et al., 2001
;
Zhou et al., 2001
;
Qi et al., 2001
). Analysis of
mice lacking Olig1 or Olig2, and double-homozygous
Olig1/Olig2 mutants, has further shown that OLIG expression
is obligatory for oligodendrocyte and motoneurone formation
(Lu et al., 2002
;
Zhou and Anderson, 2002
).
We provide separate lines of evidence that support an FGF-dependent
SHH-independent pathway for the generation of oligodendrocytes. Analysis of
the SHH null mouse spinal cord showed that at E12.5, an equivalent stage of
development to the E14 rat, no oligodendrocytes were identified from primary
cultures by comparison with wild type, an observation consistent with the
known requirement of SHH signalling for oligodendrocyte generation. However,
significant numbers of oligodendrocytes did develop in FGF2-stimulated E12.5
SHH-null spinal cord cultures. This is unlikely to be due to redundancy with
IHH or DHH for two reasons (Tekki-Kessaris
et al., 2001; Alberta et al.,
2001
). First, the absence of constitutive oligodendrocytes (and
Olig2) in isolated mouse E12.5 dorsal cultures that contain
Ihh transcripts suggests that IHH is insufficient to induce
oligodendrocytes. Second, the induction of Olig2 and Nkx2.2
genes, and the subsequent generation of oligodendrocytes in SHH null cultures
following FGF2 treatment in the presence of cyclopamine, provides evidence for
an in vitro hedgehog-independent pathway for the induction of
oligodendrocytes. These observations are also compatible with recent studies
showing hedgehog-independent generation of oligodendrocytes from mouse
embryonic stem cell cultures in the presence of FGF2 (H.K., S.C. and N.D.A.,
unpublished), and from SHH null forebrain progenitors
(Nery et al., 2001
).
Overall, our findings are consistent with recent studies that provide
evidence for factor(s) additional to SHH having a role in determining ventral
cell fates (Pierani et al.,
1999). The partial rescue of ventral neuronal subtypes in
double-mutant SHH/GLI3 and SHH/opb (RAB23 - Mouse Genome Informatics) mice
suggests that other signalling pathways contribute to ventral specification
(Litingtung and Chiang, 2000
;
Eggenschwiler et al., 2001
).
The absence of pMN and ventral interneurone progenitor domain derivatives in
noggin mutant mice, despite the presence of normal SHH expression and
upregulation of patched transcription, suggests that, in addition to SHH,
modulation of BMP signalling is necessary for ventral patterning
(McMahon et al., 1998
).
Specification of motoneurones and oligodendrocytes from the same progenitor
pool is temporally regulated (Lu et al.,
2002; Zhou and Anderson,
2002
). In the present case, the induction of Nkx2.2 and
Olig1/Olig2, but not Nkx2.9, Nkx6.1, Nkx6.2, Hb9 or Lim3, in
expanded dorsal cultures suggests the generation of cells with late pMN- but
not early pMN-domain characteristics. The precise regulation of OLIG genes is
unclear. A recent study provides evidence that, in the early pMN domain, SHH
regulates Olig2 expression through the class II protein
Nkx6.1 (Novitch et al.,
2001
). However, our finding of OLIG gene induction in the absence
of Nkx6.1 and Nkx6.2 expression, following FGF2 treatment,
provides indirect support for an additional SHH-independent pathway that
regulates OLIG gene expression. This idea is supported by the identification
of co-expressing OLIG2 and NKX2.2 cells in dorsal neurospheres, in numbers
consistent with GC-positive cells observed in differentiating cultures.
Furthermore, although oligodendrocyte and motoneurone progenitors share a
lineage relationship in vivo, this relationship is not obligate for the
generation of oligodendrocytes in other contexts
(Fig. 6).
|
In addition to promoting dorsal cell fate, BMPs are thought to influence
ventral patterning of the neural tube by modulating the hedgehog signalling
pathway (Basler et al., 1993;
McMahon et al., 1998
;
Liem et al., 2000
). BMP
antagonism of SHH also negatively regulates spinal cord OPC specification
(Mekki-Dauriac et al., 2002
).
However, in the present study addition of noggin, follistatin and chordin to
primary dorsal cultures was insufficient to generate oligodendrocytes. This
argues against the idea of a loss or dilution of extracellular inhibitory
factors in FGF2-derived expanded dorsal cultures.
The absence of oligodendrocytes in our study following co-treatment with
FGF2 and BMP4 is consistent with reports of BMPs suppressing oligodendrocyte
differentiation from neural precursors
(Zhu et al., 1999). The
comparable levels of expression of BMP4 in primary (uncultured) and
FGF-treated cultures, and the dose-response nature of BMP4 suppression of
oligodendrocytes, is compatible with a balance between inductive and negative
signalling requirements for oligodendrogenesis in our system.
BMPs can also promote astrocyte formation from embryonic neural precursors
(Gross et al., 1996;
Mabie et al., 1999
). However,
in our dorsal spinal cord cultures an elimination of oligodendrocytes was
accompanied by a reduction in astrocyte numbers. The reason for this reduction
in astrocyte numbers is unclear. Multiple mechanisms appear to underly the
generation of astrocytes from neural precursors
(Johe et al., 1996
;
Gross et al., 1996
;
Rajan and McKay, 1998
).
Evidence suggests that CNTF/LIF-mediated instruction of astrogenesis is
through activation of the JAK-STAT pathway, in contrast to BMP4 and EGF.
Recent studies showing the opposing actions of FGF and BMP in positively and
negatively regulating EGF receptors (EGFR) in neural precursors may help to
explain our findings (Lillien and Raphael,
2000
). Following FGF2 treatment there was a significant induction
of EGFR expression, consistent with previously reported observations
(Ciccolini and Svendsen, 1998
).
Interestingly a recent study demonstrated that BMP4 suppresses oligodendrocyte
formation from the developing spinal cord without an increase in astrocyte
formation (Mekki-Dauriac et al.,
2002
). In our study, treatment with BMP4 also resulted in the
generation of smooth muscle actin, a neural crest derivative, a finding
consistent with the demonstration of a peripheral neural potential of spinal
cord neural precursors following BMP treatment
(Mujtaba et al., 1998
;
Panchision et al., 2001
).
An interpretation of our findings is that FGF is opposing inhibitory
effects of endogenous BMP signalling, a recognised interaction in other
developmental processes, including limb, tooth, lung and submandibular gland
morphogenesis (Niswander and Martin,
1993; Neubuser et al.,
1997
; Weaver et al.,
2000
; Hoffman et al.,
2002
). The mechanism of such antagonism is unclear. In the
developing vertebrate limb there is evidence that FGF antagonism of BMP
signalling is mediated in part through SHH
(Zuniga et al., 1999
).
However, recent studies suggest alternative mechanisms that could underly the
hedgehog-independent FGF antagonism of BMP observed in our system
(Kretzschmar et al., 1997
;
Lo et al., 2001
).
In summary, we describe a novel in vitro developmental pathway by which FGF-mediated signalling can influence dorsoventral neural precursor cell fate, and specifically oligodendrogenesis, by mechanisms that are independent of SHH. In future, it will be important to clarify the extent to which hedgehog-independent oligodendrocyte generation contributes to normal developmental myelination, and also to re-myelination, as this is likely to have clinical implications for the design of strategies to enhance myelin repair following disease in the adult CNS.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Alberta, J. A., Park, S. K., Mora, J., Yuk, D., Pawlitzky, I., Iannarelli, P., Vartanian, T., Stiles, C. D. and Rowitch, D. H. (2001). Sonic hedgehog is required during an early phase of oligodendrocyte development in mammalian brain. Mol. Cell Neurosci. 18,434 -441.[CrossRef][Medline]
Barth, K. A., Kishimoto, Y., Rohr, K. B., Seydler, C.,
Schulte-Merker, S. and Wilson, S. W. (1999). Bmp activity
establishes a gradient of positional information throughout the entire neural
plate. Development 126,4977
-4987.
Basler, K., Edlund, T., Jessell, T. M. and Yamada, T. (1993). Control of cell pattern in the neural tube: regulation of cell differentiation by dorsalin-1, a novel TGF beta family member. Cell 73,687 -702.[Medline]
Boilly, B., Vercoutter-Edouart, A. S., Hondermarck, H., Nurcombe, V. and Le Bourhis, X. (2000). FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Factor Rev. 11,295 -302.[CrossRef][Medline]
Briscoe, J., Sussel, L., Serup, P., Hartigan-O'Connor, D., Jessell, T. M., Rubenstein, J. L. and Ericson, J. (1999). Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398,622 -627.[CrossRef][Medline]
Chandran, S., Svendsen, C., Compston, A. and Scolding, N. (1998). Regional potential for oligodendrocyte generation in the rodent embryonic spinal cord following exposure to EGF and FGF-2. Glia 24,382 -389.[CrossRef][Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Ciccolini, F. and Svendsen, C. N. (1998).
Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth
factor (EGF) responsiveness in mouse striatal precursor cells: identification
of neural precursors responding to both EGF and FGF-2. J.
Neurosci. 18,7869
-7880.
Dunnett, S. B. and Bjorklund, A. (1992). Staging and dissection of rat embryos. In Neural Transplantation (ed. S. B. Dunnett and A. Bjorklund), pp.1 -18. Oxford: IRL Press.
Eggenschwiler, J. T., Espinoza, E. and Anderson, K. V. (2001). Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway. Nature 412,194 -198.[CrossRef][Medline]
Gage, F. H. (2000). Mammalian neural stem
cells. Science 287,1433
-1438.
Gregori, N., Proschel, C., Noble, M. and Mayer-Proschel, M.
(2002). The tripotential glial-restricted precursor (GRP) cell
and glial development in the spinal cord: generation of bipotential
oligodendrocyte-type-2 astrocyte progenitor cells and dorsal-ventral
differences in GRP cell function. J. Neurosci.
22,248
-256.
Gross, R. E., Mehler, M. F., Mabie, P. C., Zang, Z., Santschi, L. and Kessler, J. A. (1996). Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells. Neuron 17,595 -606.[Medline]
Hall, A., Giese, N. A. and Richardson, W. D.
(1996). Spinal cord oligodendrocytes develop from ventrally
derived progenitor cells that express PDGF alpha-receptors.
Development 122,4085
-4094.
Hardy, R. J. and Friedrich, V. L. (1996).
Oligodendrocyte progenitors are generated throughout the embryonic mouse
brain, but differentiate in restricted foci.
Development 122,2059
-2069.
Hoffman, M. P., Kidder, B. L., Steinberg, Z. L., Lakhani, S., Ho, S., Kleinman, H. K. and Larsen, M. (2002). Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms. Development 129,5767 -5778.[CrossRef][Medline]
Incardona, J. P., Gaffield, W., Kapur, R. P. and Roelink, H.
(1998). The teratogenic Veratrum alkaloid cyclopamine inhibits
sonic hedgehog signal transduction. Development
125,3553
-3562.
Ishibashi, M. and McMahon, A. P. (2002). A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo. Development 129,4807 -4819.[Medline]
Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M. and McKay, R. D. (1996). Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev. 10,3129 -3140.[Abstract]
Kalyani, A., Hobson, K. and Rao, M. S. (1997). Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterisation, and clonal Analysis. Dev. Biol. 186,202 -223.[CrossRef][Medline]
Kessaris, N., Pringle, N. and Richardson, W. D. (2001). Ventral neurogenesis and the neuron-glial switch. Neuron 31,677 -680.[CrossRef][Medline]
Kretzschmar, M., Doody, J. and Massague, J. (1997). Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature 389,618 -622.[CrossRef][Medline]
Lee, K. and Jessell, T. M. (1999). The specification of dorsal cell fates in the vertebrate central nervous system. Annu. Rev. Neurosci. 22,261 -294.[CrossRef][Medline]
Liem, K. F. J., Tremml, G., Roelink, H. and Jessell, T. M. (1995). Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82,969 -979.[Medline]
Liem, K. F. J., Tremml, G. and Jessell, T. M. (1997). A role for the roof plate and its resident TGF beta-related proteins in neuronal patterning in the dorsal spinal cord. Cell 91,127 -138.[CrossRef][Medline]
Liem, K. F. J., Jessell, T. M. and Briscoe, J.
(2000). Regulation of the neural patterning activity of sonic
hedgehog by secreted BMP inhibitors expressed by notochord and somites.
Development 127,4855
-4866.
Lillien, L. and Raphael, H. (2000). BMP and FGF
regulate the development of EGF-responsive neural progenitor cells.
Development 127,4993
-5005.
Litingtung, Y. and Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between shh and gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Lo, R. S., Wotton, D. and Massague, J. (2001).
Epidermal growth factor signaling via Ras controls the Smad transcriptional
co-repressor TGIF. EMBO J.
20,128
-136.
Lu, Q. R., Yuk, D., Alberta, J. A., Zhu, Z., Pawlitzky, I., Chan, J., McMahon, A. P., Stiles, C. D. and Rowitch, D. H. (2000). Sonic hedgehog-regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system. Neuron 25,317 -329.[Medline]
Lu, Q. R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C. D. and Rowitch, D. H. (2002). Common developmental requirement for olig function indicates a motor neuron/oligodendrocyte connection. Cell 109,75 -86.[Medline]
Mabie, P. C., Mehler, M. F. and Kessler, J. A.
(1999). Multiple roles of bone morphogenetic protein signaling in
the regulation of cortical cell number and phenotype. J.
Neurosci. 19,7077
-7088.
Mayer-Proschel, M., Kalyani, A. J., Mujtaba, T. and Rao, M. S. (1997). Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 19,773 -785.[Medline]
McMahon, J. A., Takada, S., Zimmerman, L. B., Fan, C. M.,
Harland, R. M. and McMahon, A. P. (1998). Noggin-mediated
antagonism of BMP signaling is required for growth and patterning of the
neural tube and somite. Genes Dev.
12,1438
-1452.
Mekki-Dauriac, S., Agius, E., Kan, P. and Cochard, P. (2002). Bone morphogenetic proteins negatively control oligodendrocyte precursor specification in the chick spinal cord. Development 129,5117 -5130.[Medline]
Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako, H., Nagao, M., Yoshida, S., Nabeshima, Y., Shimamura, K. and Nakafuku, M. (2001). Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31,757 -771.[Medline]
Mujtaba, T., Mayer-Proschel, M. and Rao, M. S. (1998). A common neural progenitor for the CNS and PNS. Dev. Biol. 200,1 -15.[CrossRef][Medline]
Neubuser, A., Peters, H., Balling, R. and Martin, G. R. (1997). Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90,247 -255.[Medline]
Nery, S., Wichterle, H. and Fishell, G. (2001).
Sonic hedgehog contributes to oligodendrocyte specification in the mammalian
forebrain. Development.
128,527
-540.
Niswander, L. and Martin, G. R. (1993). FGF-4 and BMP-2 have opposite effects on limb growth. Nature 361, 68-71.[CrossRef][Medline]
Novitch, B. G., Chen, A. I. and Jessell, T. M. (2001). Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron 31,773 -789.[Medline]
Orentas, D. M., Hayes, J. E., Dyer, K. L. and Miller, R. H.
(1999). Sonic hedgehog signaling is required during the
appearance of spinal cord oligodendrocyte precursors.
Development 126,2419
-2429.
Panchision, D. M., Pickel, J. M., Studer, L., Lee, S. H.,
Turner, P. A., Hazel, T. G. and McKay, R. D. (2001).
Sequential actions of BMP receptors control neural precursor cell production
and fate. Genes Dev. 15,2094
-2110.
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. M. (1999). A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97,903 -915.[Medline]
Poncet, C., Soula, C., Trousse, F., Kan, P., Hirsinger, E., Pourquie, O., Duprat, A. M. and Cochard, P. (1996). Induction of oligodendrocyte progenitors in the trunk neural tube by ventralizing signals: effects of notochord and floor plate grafts, and of sonic hedgehog. Mech. Dev. 60,13 -32.[CrossRef][Medline]
Pringle, N. P. and Richardson, W. D. (1993). A
singularity of PDGF alpha-receptor expression in the dorsoventral axis of the
neural tube may define the origin of the oligodendrocyte lineage.
Development 117,525
-533.
Pringle, N. P., Yu, W. P., Guthrie, S., Roelink, H., Lumsden, A., Peterson, A. C. and Richardson, W. D. (1996). Determination of neuroepithelial cell fate: induction of the oligodendrocyte lineage by ventral midline cells and sonic hedgehog. Dev. Biol. 177,30 -42.[CrossRef][Medline]
Pringle, N. P., Guthrie, S., Lumsden, A. and Richardson, W. D. (1998). Dorsal spinal cord neuroepithelium generates astrocytes but not oligodendrocytes. Neuron 20,883 -893.[Medline]
Qi, Y., Cai, J., Wu, Y., Wu, R., Lee, J., Fu, H., Rao, M., Sussel, L., Rubenstein, J. and Qiu, M. (2001). Control of oligodendrocyte differentiation by the Nkx2.2 homeodomain transcription factor. Development 128,2723 -2733.[Medline]
Rajan, P. and McKay, R. D. G. (1998). Multiple
routes to astrocytic differentiation in the CNS. J.
Neurosci. 18,3620
-3629.
Rao, M. S. and Mayer-Proschel, M. (1997). Glial restricted precursors are derived from multipotent neuroepithelial stem cells. Dev. Biol. 188,48 -63.[CrossRef][Medline]
Richardson, W. D., Smith, H. K., Sun, T., Pringle, N. P., Hall, A. and Woodruff, R. (2000). Oligodendrocyte lineage and the motor neuron connection. Glia 29,136 -142.[CrossRef][Medline]
Shah, N. M., Groves, A. K. and Anderson, D. J. (1996). Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members. Cell 85,331 -343.[Medline]
Shihabuddin, L. S., Horner, P. J., Ray, J. and Gage, F. H.
(2000). Adult spinal cord stem cells generate neurons after
transplantation in the adult dentate gyrus. J.
Neurosci. 20,8727
-8735.
Sommer, I. and Schachner, M. (1981). Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev. Biol. 83,311 -327.[Medline]
Soula, C., Danesin, C., Kan, P., Grob, M., Poncet, C. and
Cochard, P. (2001). Distinct sites of origin of
oligodendrocytes and somatic motoneurons in the chick spinal cord:
oligodendrocytes arise from Nkx2.2-expressing progenitors by a Shh-dependent
mechanism. Development
128,1369
-1379.
Svendsen, C. N. and Smith, A. G. (1999). New prospects for human stem-cell therapy in the nervous system. Trends.Neurosci. 22,357 -364.[CrossRef][Medline]
Taipale, J., Chen, J. K., Cooper, M. K., Wang, B., Mann, R. K., Milenkovic, L., Scott, M. P. and Beachy, P. A. (2000). Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406,1005 -1009.[CrossRef][Medline]
Takebayashi, H., Yoshida, S., Sugimori, M., Kosako, H., Kominami, R., Nakafuku, M. and Nabeshima, Y. (2000). Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3. Mech. Dev. 99,143 -148.[CrossRef][Medline]
Tekki-Kessaris, N., Woodruff, R., Hall, A. C., Gaffield, W.,
Kimura, S., Stiles, C. D., Rowitch, D. H. and Richardson, W. D.
(2001). Hedgehog-dependent oligodendrocyte lineage specification
in the telencephalon. Development
128,2545
-2554.
Tessier-Lavigne, M., Placzek, M., Lumsden, A. G., Dodd, J. and Jessell, T. M. (1988). Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336,775 -778.[CrossRef][Medline]
Timsit, S., Martinez, S., Allinquant, B., Peyron, F., Puelles, L. and Zalc, B. (1995). Oligodendrocytes originate in a restricted zone of the embryonic ventral neural tube defined by DM-20 mRNA expression. J. Neurosci. 15,1012 -1024.[Abstract]
Trousse, F., Giess, M. C., Soula, C., Ghandour, S., Duprat, A. M. and Cochard, P. (1995). Notochord and floor plate stimulate oligodendrocyte differentiation in cultures of the chick dorsal neural tube. J. Neurosci. Res. 41,552 -560.[Medline]
Wada, T., Kagawa, T., Ivanova, A., Zalc, B., Shirasaki, R., Murakami, F., Iemura, S., Ueno, N. and Ikenaka, K. (2000). Dorsal spinal cord inhibits oligodendrocyte development. Dev. Biol. 227,42 -55.[CrossRef][Medline]
Warf, B. C., Fok-Seang, J. and Miller, R. H. (1991). Evidence for the ventral origin of oligodendrocyte precursors in the rat spinal cord. J. Neurosci. 11,2477 -2488.[Abstract]
Weaver, M., Dunn, N. R. and Hogan, B. L.
(2000). Bmp4 and Fgf10 play opposing roles during lung bud
morphogenesis. Development
127,2695
-2704.
Xu, X., Cai, J., Fu, H., Wu, R., Qi, Y., Modderman, G., Liu, R. and Qiu, M. (2000). Selective expression of Nkx-2.2 transcription factor in chicken oligodendrocyte progenitors and implications for the embryonic origin of oligodendrocytes. Mol. Cell. Neurosci. 16,740 -753.[CrossRef][Medline]
Yu, W. P., Collarini, E. J., Pringle, N. P. and Richardson, W. D. (1994). Embryonic expression of myelin genes: evidence for a focal source of oligodendrocyte precursors in the ventricular zone of the neural tube. Neuron 12,1353 -1362.[Medline]
Zhou, Q. and Anderson, D. J. (2002). The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. Cell 109,61 -73.[Medline]
Zhou, Q., Wang, S. and Anderson, D. J. (2000). Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25,331 -343.[Medline]
Zhou, Q., Choi, G. and Anderson, D. J. (2001). The bHLH transcription factor Olig2 promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31,791 -807.[Medline]
Zhu, G., Mehler, M. F., Zhao, J., Yu, Y. S. and Kessler, J. A. (1999). Sonic hedgehog and BMP2 exert opposing actions on proliferation and differentiation of embryonic neural progenitor cells. Dev. Biol. 215,118 -129.[CrossRef][Medline]
Zuniga, A., Haramis, A. P., McMahon, A. P. and Zeller, R. (1999). Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401,598 -602.[CrossRef][Medline]