1 Wolfson Institute for Biomedical Research and Department of Biology,
University College London, Gower Street, London WC1E 6BT, UK
2 Curis, 61 Moulton Street, Cambridge, MA 02138, USA
Author for correspondence (e-mail:
w.richardson{at}ucl.ac.uk)
Accepted 5 December 2003
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SUMMARY |
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Key words: FGF, SHH, MAPK, Embryonic neural stem cells, Cell fate specification, Neural development, Oligodendrocyte progenitors, Mouse
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Introduction |
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SHH is also expressed in the ventral forebrain where it induces formation
of neurons (Ericson et al.,
1995) and OLPs (Nery et al.,
2001
; Tekki-Kessaris et al.,
2001
). The OLPs then appear to migrate into all parts of the
developing forebrain including the cerebral cortex
(Tekki-Kessaris et al., 2001
).
There might also be local production of OLPs within the mammalian cortex
(Gorski et al., 2002
) (but not
avian cortex: see Discussion). If so, cortical OLP production must begin after
E17 in the mouse because significant numbers of OLPs are not found in the
cortex before then, whereas OLPs are generated in large numbers in the ventral
forebrain as early as E13. If cortical precursor cells are removed and
cultured at E13, they do not generate OLPs for at least four days in vitro
(DIV4) (Tekki-Kessaris et al.,
2001
). However, E13 neocortical precursors can be induced to
generate OLPs within a couple of days of SHH treatment in vitro
(Tekki-Kessaris et al., 2001
;
Alberta et al., 2001
;
Murray et al., 2002
).
Olig genes are also expressed and required for oligodendrogenesis in
the ventral forebrain, because Olig1/2 double-knockout mice lack OLPs
in the forebrain, and indeed anywhere else in the central nervous system (CNS)
(Zhou and Anderson, 2002
).
In addition to SHH, fibroblast growth factor 2 (FGF2) also stimulates the
generation of oligodendrocytes from cultured cortical precursors
(Qian et al., 1997;
Hall, 1999
). Since SHH and
FGF2 share this property, the question arises whether SHH and FGF2 use some of
the same intracellular signalling pathways or operate along entirely
independent lines. This is the main question addressed by the work described
here. FGF2 is routinely added to neural stem cell (neurosphere) cultures
derived from embryonic or adult forebrain, so understanding the interactions
between FGF and other factors will help us to understand the behaviour of stem
cells in these cultures.
FGF proteins often act as mitogenic growth factors but can also signal cell
survival and differentiation (reviewed by
Yamaguchi and Rossant, 1995;
Ornitz and Itoh, 2001
). SHH
was originally identified as a morphogen and cell fate determinant
(Roelink et al., 1995
) but
more recently has been shown to influence axonal outgrowth
(Charron et al., 2003
), cell
survival and proliferation (Teillet et
al., 1998
; Ahlgren and
Bronner-Fraser, 1999
; Marcelle
et al., 1999
; Rowitch et al.,
1999
; Yu et al.,
2002
; Thibert et al.,
2003
). Moreover, over-activity of the Hedgehog signalling pathway
is associated with tumour growth (Oro et
al., 1997
; Dahmane et al.,
2001
). SHH is now known to be a mitogen for neural precursors from
several regions of the CNS including cerebellum
(Dahmane and Ruiz i Altaba,
1999
; Wechsler-Reya and Scott,
1999
; Kenney and Rowitch,
2000
) and retina (Jensen and
Wallace, 1997
).
The biochemical basis for these activities of SHH is poorly understood. SHH
binds to the transmembrane receptor Patched (PTC), causing disinhibition of
its co-receptor Smoothened (SMO), a seven-pass transmembrane G-protein coupled
receptor (GPCR). In Drosophila, this eventually promotes nuclear
translocation of a proteolytic fragment of the transcription factor Cubitus
interruptus (Ci). The mammalian equivalents of Ci are the GLI proteins, which
are transcriptionally activated as a result of SHH signalling. The downstream
targets of Ci/GLI proteins include G1- and S-phase cyclins
(Kenney and Rowitch, 2000;
Duman-Scheel et al., 2002
) and
N-MYC (Kenney et al., 2003
;
Oliver et al., 2003
), linking
directly to the cell cycle machinery. FGF (of which there are >20 known
mammalian family members) triggers an entirely different set of early
signalling events. FGF binds to one of four closely related receptors
(FGFR1-4) at the extracellular surface, causing receptor clustering and
autophosphorylation of their intracellular tyrosine kinase (TK) domains. The
phosphorylated receptor then acts as a nucleation centre for signalling
molecules, which bind either directly or indirectly to individual
phosphotyrosine residues. The main pathways that are activated by FGFR include
the mitogen-activated protein kinase (MAPK) pathway, phosphoinositol 3-kinase
(PI 3-kinase) pathway, phospholipase C
and elevation of
intracellular calcium (reviewed by Klint
and Claesson-Welsh, 1999
). Despite their apparently divergent
signalling mechanisms there is evidence of intracellular cross-talk between
Hedgehog and FGF signalling pathways. For example, the GLI proteins seem able
to integrate SHH and FGF signalling in some circumstances
(Brewster et al., 2000
).
Moreover, it is established that receptor TKs can be trans-activated inside
cells by GPCRs (Schwartz and Baron,
1999
; Ferguson,
2003
; Wetzker and Bohmer,
2003
), raising the possibility that SMO might be able to
trans-activate FGFR.
Here, we investigate the signalling pathways used by SHH in cell fate specification and lineage progression of embryonic neocortical precursors. Our starting point was the apparent connection between SHH and FGF2 biology. We searched for- but found no evidence of - trans-activation of FGFR by SHH in cortical cells. However, we did find that the shared OLP-inducing activities of SHH and FGF2 involve overlapping intracellular signals. Notably, SHH has a requirement for a low basal level of MAPK phosphorylation that results from constitutive FGFR signalling - since both basal MAPK activity and SHH activity could be blocked by a selective inhibitor of FGFR-TK. Infection of cultures with a retrovirus encoding constitutively active RAS protein demonstrated that SHH signalling and MAPK activity were required in the same cells for OLP induction. Our results raise the possibility that cooperation with receptor or non-receptor TK pathways might be a more general requirement for cell fate specification by SHH.
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Materials and methods |
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Target specificity of PD173074
PD173074 has been described as a selective inhibitor of FGFR1
(Skaper et al., 2000) but it
probably blocks all four high-affinity FGF receptors (FGFR1-4). It might
conceivably inhibit other closely related RTKs or non-RTKs as well. PD173074
inhibits FGFR1 at
25 nM but does not inhibit PDGRß, EGFR or SRC
significantly at this concentration
(Dimitroff et al., 1999
).
Skaper et al. (Skaper et al.,
2000
) showed that PD173074 effectively blocked the neurotrophic
action of FGF2 on cerebellar granule neurons and dorsal root ganglion neurons
at nM concentrations, but had no effect on the activities of insulin-like
growth factor 1, nerve growth factor, brain-derived neurotrophic factor or
ciliary neurotrophic factor.
The concentration of PD173074 required for half-maximal inhibition of
SHH-induced NG2 expression was 25 nM, similar to that reported for
inhibition of FGFR1 itself. This strongly suggests that the target of PD173074
in our experiments was indeed FGFR, not another related kinase. As an
additional test of specificity we wanted to confirm that PD173074 does not
inhibit the platelet-derived growth factor alpha-receptor (PDGFR
),
since NG2-positive OLPs express PDGFR
and proliferate in response to
PDGFAA (Hall et al., 1996
;
Fruttiger et al., 1999
). We
cultured E13 ventral forebrain cells in defined medium plus recombinant PDGFAA
(10 ng/ml) and either PD173074 or tyrophostin A9 (a selective inhibitor of
PDGFR-TK) (Levitzki and Gilon,
1991
) for 48 hours before immunolabelling with anti-NG2 and
counting OLPs. In the presence of PDGFAA alone, there was a large increase in
the number of NG2-positive OLPs (not shown). Tyrphostin A9 effectively
neutralised PDGF-driven OLP proliferation (not shown) but PD173074 had no
significant effect. Thus, PD173074 selectively inhibits FGFR over the closely
related PDGFR
.
Chick neural tube cultures
Chick neural tubes from Hamburger-Hamilton stage 11 (E2) embryos
(Hamburger and Hamilton, 1951)
were isolated in MEM-Hepes following a gentle treatment with 1 mg/ml dispase
to remove surrounding tissues. Using a flame-sharpened tungsten needle the
neural tube was divided into dorsal and ventral halves. Approximately 100
µm3 explants were cultured in three-dimensional collagen gels as
previously described (Guthrie and Lumsden,
1994
) in defined medium
(Bottenstein and Sato, 1979
)
lacking transferrin but containing concanavalin A, 0.25% (v/v) foetal calf
serum (FCS) and antibiotics.
Retroviral vectors
To identify transfected cells we used the pBird retroviral vector, which
encodes enhanced green fluorescent protein (eGFP) driven by the
cytomegalovirus (CMV) promoter. The pBird-RasV12 vector
co-expresses eGFP and a constitutively active form of RAS
(Tang et al., 2001).
Recombinant retroviruses were produced and concentrated as described
previously (Kondo and Raff,
2000
). Neocortical cultures were infected for 3 hours with
concentrated retroviral supernatant, starting 1 day after plating the cells.
Infected cells were identified by eGFP fluorescence.
Immunocytochemistry
Cells on coverslips or explants were lightly fixed in 4% (w/v)
paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 5 minutes at
room temperature and washed in PBS. The following primary antibodies were
used: anti-NG2 rabbit serum (1:350 dilution, Chemicon) or monoclonal anti-NG2
(clone N11.4) (Levine and Stallcup,
1987; Stallcup and Beasley,
1987
), monoclonal antibody O4
(Sommer and Schachner, 1981
),
anti-OLIG2 rabbit IgG (DF308, 1:4000 dilution, a gift from David Rowitch) and
monoclonal anti-MAPK (diphosphorylated ERK1/ERK2; Sigma-Aldrich, 1:200
dilution). For OLIG2 staining, the cells were made permeable with 0.1% (v/v)
Triton X-100 in PBS. Primary antibody treatments were for 1 hour or overnight
in a humid chamber at 4°C. Fluorescent secondary antibodies (Perbio
Science, UK) were applied for 60 minutes at room temperature. Following
staining of the nuclei with Hoechst (Sigma) the cells were post-fixed for 5
minutes in 4% (w/v) PFA in PBS and mounted on slides in Citifluor (City
University, UK).
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Results |
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Rapid induction of OLIG2 by FGF2 or SHH
Activation of NG2 expression is a relatively late event in oligodendrocyte
lineage progression, an earlier lineage marker being OLIG2. We looked at
induction of OLIG2 expression in response to FGF2 or SHHAg. OLIG2-positive
cells first appeared within 20 hours of either FGF2 or SHHAg treatment,
peaking around 48 hours (Fig.
3). Control cultures without added FGF2 or SHH did not develop any
OLIG2-positive cells for at least 70 hours
(Fig. 3G).
|
To test these predictions we cultured E13.5 neocortical precursors at high
density in the presence of FGF2 or SHHAg, with or without cyclopamine or
PD173074, and looked for induction of OLIG2 at DIV2. We found that the
OLIG2-inducing activity of FGF2 (10 ng/ml) was strongly inhibited by PD173074,
as expected, but was unaffected by cyclopamine
(Fig. 4A,B). Moreover,
cyclopamine did not inhibit the OLP-inducing activity of FGF2 in dorsal spinal
cord cultures (Fig. 1F). In
contrast, the OLIG2-inducing activity of SHHAg in cortical cultures was
strongly inhibited by both cyclopamine
(Fig. 4B) and PD173074
(Fig. 4A,B). We also looked at
induction of NG2-positive OLPs at DIV4, with analogous results, i.e.
NG2-induction by FGF was blocked by PD173074 (not shown) but not by
cyclopamine (Fig. 4A), whereas
induction by SHHAg was sensitive to both reagents
(Fig. 4A and not shown). The
concentration of PD173074 required for half-maximal inhibition of SHHAg was
25 nM, similar to that reported for FGFR1 itself
(Dimitroff et al., 1999
)
(Fig. 4C). We also determined
that PD173074 does not inhibit the closely related PDGFR (see Materials and
methods). Our data suggest that SHH ultimately relies on activation of FGFR,
either directly or indirectly, for its OLIG2- and OLP-inducing abilities.
|
Induction of OLIG2 expression by SHHAg or FGF2 requires MAPK activity
If SHH and FGF both act through FGFR as implied above, one would expect
them to trigger the same intracellular signalling pathways. FGFR activation
leads to autophosphorylation of the TK domains, which in turn can initiate
MAPK and pathways and elevation of intracellular calcium. We investigated the
involvement of MAPK and PI 3-kinase signalling pathways, using synthetic drugs
that inhibit MEK1/2 (U0126) or PI 3-kinase (LY294002).
We found that induction of OLIG2 by either FGF2 or SHHAg was strongly inhibited by U0126, but not by LY294002, at either DIV1 (Fig. 5A) or DIV2 (not shown), indicating that the MAPK pathway but not the PI 3-kinase pathway is crucial for this first step of lineage specification. We visualised MAPK activation directly by immunofluorescence microscopy with an antibody that specifically recognises the phosphorylated form of the protein. Within 1 hour of FGF2 exposure there was a marked increase in MAPK immunolabelling over control (compare Fig. 5Ba with Bk). Surprisingly (given the data of Fig. 5A), we could detect no increase in MAPK immunolabelling after SHHAg treatment (compare Fig. 5Ba and 5Bg).
|
FGFR maintains a constitutive low level of active MAPK that is required for SHH activity
The inability of SHHAg to activate MAPK argues against trans-activation of
FGFR, since direct stimulation by FGF2 causes robust MAPK activation. What,
then, is the essential role of FGFR in the activity of SHH? In the absence of
added SHH or FGF2 there is a background of active MAPK in our cultures (Fig.
5Ba and
5C lanes 1, 4), but this
background is abolished by adding PD173074
(Fig. 5Bc). Even in the
presence of SHH, the basal level of active MAPK is obliterated by PD173074
(Fig. 5Bi, and lane 6 in C).
Therefore, it seems likely that the steady-state level of active MAPK in our
cultures is caused by low, constitutive FGFR activity and that this basal
activity is absolutely required for OLIG2 induction by SHH.
Cell-autonomous requirement for MAPK activation in SHH-responding cells
The experiments described above showed that MAPK phosphorylation is
required in neocortical cultures for the OLP-inducing activity of SHH but did
not distinguish between a direct or indirect effect of MAPK. For example, MAPK
might stimulate release of a diffusible factor that acts secondarily on
neighbouring cells to render them responsive to SHH. Alternatively, MAPK might
be required within the same cells that respond to SHH.
We addressed this question by infecting neocortical precursors with a retrovirus vector encoding a mutated form of RAS that constitutively activates the MAPK pathway. The retrovirus also encodes the enhanced green fluorescent protein (eGFP) so that infected cells can be positively identified using the fluorescence microscope. Unsurprisingly, we found that constitutively active RAS was not by itself sufficient to activate OLIG2 expression in the absence of SHH signalling (added cyclopamine; Fig. 6Ab-d). However, in the presence of SHHAg and PD173074 (to block MAPK activation via FGFR) the only cells that expressed OLIG2 were those that also expressed activated RAS (Fig. 6Af-g,B). Note that not all cells that expressed active RAS also expressed OLIG2 (Fig. 6Af-h). These observations allow us to conclude, (1) the MAPK pathway is necessary but not sufficient for OLIG2 induction as SHH signalling is also required, and (2) MAPK activation is required cell-autonomously, i.e. it acts directly in the SHH-targeted cells.
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Discussion |
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We previously reported that cortical precursor cell cultures have the
ability to generate OLPs in the absence of added SHH or FGF2. This inherent
potential takes a long time to manifest itself (DIV6) and can be blocked by
cyclopamine, implying that endogenous Hedgehog activity in the cultures is
largely responsible (Tekki-Kessaris et
al., 2001). Consistent with this, we found that mRNAs encoding SHH
and its relative Indian Hedgehog (IHH) were up-regulated in the cultures
(Tekki-Kessaris et al., 2001
).
Recently, Gabay et al. (Gabay et al.,
2003
) reported that neocortical cells in monolayer or neurosphere
culture up-regulate SHH in response to FGF2 (0.2 ng/ml) and that the
OLIG2-inducing activity of this low concentration of FGF2 can be blocked by
cyclopamine. This suggests that the up-regulation of Hedgehog transcripts that
we observed previously (Tekki-Kessaris et
al., 2001
) might be due to endogenous FGFR activation and that
part of the OLP-inducing activity of added FGF might be mediated indirectly
through Hedgehog proteins. However, that cannot account for all of the effect
of FGF when added at the higher concentrations (10 ng/ml) used in our present
study, because in our hands FGF-mediated OLP induction was not inhibited
significantly by cyclopamine. On the contrary, we found that the OLP-inducing
activity of SHHAg is dependent on FGFR and MAPK. However, MAPK alone is not
sufficient to induce OLPs - SHH signalling is also required. The additional
obligatory signal that is triggered by SHH is presumably also triggered by
FGFR, since FGF2 can induce OLPs independently of SHH.
Despite its critical role in cortical precursors, we found that FGFR is not required for SHH-mediated cell fate specification in ventral spinal cord or forebrain, even though ventral precursors are known to express FGFR1-3 in vivo. It is possible that receptor TKs other than FGFR collaborate with SHH in non-cortical cells.
Do FGF2 and SHH act on the same population of cortical precursors?
FGF2 activated the MAPK pathway rapidly in all, or nearly all, E13 cortical
precursors (Fig. 5Bk). This is
consistent with the fact that FGFR1-3 are expressed in most cortical cells at
this age. However, only a minority of the MAPK-active cells -around 10% - went
on to express OLIG2 at DIV2 (not shown). A similar proportion of precursors
expressed OLIG2 after SHHAg stimulation. What distinguishes the precursor
cells that are competent to express OLIG2 from their OLIG2-incompetent
neighbours is a mystery.
The FGF2 and SHH-responsive cells could belong to the same or different populations. The simplest interpretation of our data - the one we prefer - is that SHH and FGF2 act directly on the same sub-population of cortical precursors to activate OLIG2. This interpretation is strengthened by our finding that MAPK and SHH act together in the same precursors.
Is FGF involved in oligodendrocyte generation in vivo?
We and others have presented evidence that OLPs are generated in the
ventral spinal cord and forebrain during embryogenesis and migrate from there
into more dorsal territories including the cerebral cortex
(Warf et al., 1991;
Pringle and Richardson, 1993
;
Noll and Miller, 1993
;
Timsit et al., 1995
;
Spassky et al., 1998
;
Nery et al., 2001
;
Tekki-Kessaris et al., 2001
).
Chick-quail grafting experiments suggest that, in birds, all oligodendrocytes
in the cortex develop from ventrally derived, migratory OLPs
(Olivier et al., 2001
).
However, cell fate analysis in mice, using a Emx1-Cre transgene to
activate a conditional lacZ reporter, showed that the majority of
cortical oligodendrocytes were descended from Emx1-expressing
precursors - presumably indigenous cortical precursors
(Gorski et al., 2002
). It is
possible that as ventrally derived progenitors migrate into the cortex they
turn on Emx1, so that the Emx1-Cre fate mapping experiments
erroneously score them as cortex derived. Alternatively, there could be two
populations of OLPs: a primitive population that is derived from ventral
precursors and a later-developing population that is indigenous to the cortex.
This second wave might be present in rodents but not birds, because of the
need for greater numbers of OLPs in the much expanded mammalian cortex. It is
conceivable that FGF signalling might be involved in the putative second wave
of oligodendrogenesis in the mammalian cortex. It might be possible to address
this question in future by studying OLP production in neocortex-specific
Fgfr1 knockout mice.
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ACKNOWLEDGMENTS |
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Footnotes |
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