|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 16, 2007
doi: 10.1242/10.1242/dev.000216
Division of Cell and Developmental Biology, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
* Author for correspondence (e-mail: k.g.storey{at}dundee.ac.uk)
Accepted 28 March 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Neurogenesis, FGF, Retinoic acid, Wnt, Differentiation, Stem cells, Chick
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Schier, 2001;
Stern et al., 2006). In
particular, in the chick embryo, FGF and Wnt have been shown to act together
in a dose-dependent manner to specify midbrain and hindbrain regions of the
CNS (Nordstrom et al., 2002)
whereas specification of spinal cord identity additionally involves retinoid
signalling (Delfino-Machin et al.,
2005; Muhr et al.,
1999; Nordstrom et al.,
2006). FGF, Wnt and RA signalling have also been shown in various
contexts to regulate caudal (Cdx) genes, which are part of the mechanism that
defines rostro-caudal identity in the body axis via regulation of Hox gene
expression (Nordstrom et al.,
2006) (reviewed in Lohnes,
2003). However, how all three pathways interact to assign caudal
identity is still not clear. Several studies have addressed the regulatory
relationships between FGF, Wnts and RA pathways
(Blumberg et al., 1997;
Domingos et al., 2001;
Kudoh et al., 2002;
McGrew et al., 1997;
Moreno and Kintner, 2004;
Shiotsugu et al., 2004), but
these experiments involve misexpression of signalling factors or their
antagonists throughout the early frog or fish embryo, making it difficult to
determine a precise sequence of tissue interactions and signalling events (see
Diez del Corral and Storey,
2004; Stern et al.,
2006). Importantly, in higher vertebrates patterning of caudal
neural tissue does not simply involve subdivision of the neural plate but is
intrinsically linked to the progressive generation of new neural tissue as the
body axis extends. This at once makes the problem more complex, but has the
advantage that the temporal sequence of events underlying differentiation and
pattern progression becomes spatially separated in the extending axis and the
regulation of these steps can therefore be more easily investigated.
As caudal hindbrain and spinal cord regions are generated sequentially, the
finding that increasing levels of FGF and Wnt signalling lead to more caudal
character (Nordstrom et al.,
2002; Nordstrom et al.,
2006) can also be viewed in terms of the progressive assignment of
rostro-caudal character; cells that reside close to the caudally regressing
source of FGF and Wnt signals (the primitive streak) remain undifferentiated
and acquire progressively more caudal fates (see
Vasiliauskas and Stern, 2001).
Importantly, FGF, Wnt and RA stimulate distinct cell behaviours as well as
inducing expression of caudal marker genes. FGF and Wnt can both stimulate
proliferation (Chenn and Walsh,
2002; Dickinson et al.,
1994; Lee et al.,
1997; Megason and McMahon,
2002; Qian et al.,
1997; Zechner et al.,
2003), while in contrast, RA signalling drives differentiation and
can promote cell cycle exit (reviewed by
Diez del Corral and Storey,
2004), and these behaviours are important when we consider the
roles of these signals in the extending body axis. We have shown recently that
FGF-dependent Notch signalling maintains an undifferentiated cell state in
stem zone (caudal neural plate) cells that lie adjacent to the regressing
primitive streak at the tail end of the embryo
(Akai et al., 2005;
Diez del Corral et al., 2002;
Mathis et al., 2001). This
cell population progressively gives rise to new neural progenitors
(Brown and Storey, 2000) and
has been shown to harbour a resident stem cell-like population in the mouse
(Cambray and Wilson, 2002;
Mathis and Nicolas, 2000) and
most likely in the chick (Mathis et al.,
2001; Delfino-Machin et al.,
2005). As cells leave this stem zone, FGF-dependent Notch
signalling declines and cells enter the transition zone (preneural tube) where
they encounter RA, which is synthesised by Raldh2 in the adjacent
rostral presomitic mesoderm. We have found that retinoid signalling
downregulates Fgf8, in both the presomitic mesoderm and the
neuroepithelium and in this way drives and coordinates the differentiation of
these tissues. Conversely, FGF signalling represses Raldh2 expression
in caudal regions protecting stem zone and caudal presomitic mesoderm cells
from precocious differentiation (Diez del
Corral et al., 2003). FGF and RA pathways therefore act
antagonistically in this context and we have shown that they have opposing
effects on neuronal differentiation and ventral patterning onset in the newly
formed neural tube (Diez del Corral et
al., 2003; Novitch et al.,
2003). In addition, the opposition of these two pathways in the
presomitic mesoderm defines the position of the future somite boundary during
the process of segmentation [(Diez del
Corral et al., 2003); although this appears to be restricted to
early stages in the mouse embryo (see
Sirbu and Duester, 2006)]. The
transition from FGF to retinoid signalling thus serves as a differentiation
switch in the extending body axis. Importantly, a similar relationship between
these pathways has been observed in the extending limb and in some cancer cell
lines, indicating that this is a fundamental and conserved molecular mechanism
that regulates differentiation progression (reviewed by
Diez del Corral and Storey,
2004).
There is growing evidence that caudal Hox gene expression depends on FGF
and not retinoid signalling in the stem zone or caudal neural plate
(Bel-Vialar et al., 2002;
Delfino-Machin et al., 2005;
Liu et al., 2001). However,
later, more rostral domains of caudal Hox gene expression then switch to
dependence on RA (Muhr et al.,
1999; Oosterveen et al.,
2003). As onset of more caudal Hox genes commences in the stem
zone we have proposed a model in which caudal Hox genes are progressively
induced by FGF signalling and the expression of these genes then becomes
`fixed' as cells leave the stem zone and encounter retinoid signals
(Diez del Corral and Storey,
2004). This also implies that the switch from FGF to RA regulates
both differentiation status and progressive assignment of rostro-caudal
character.
Here we focus on the regulatory relationships between FGF, Wnt and RA pathways during this critical transition from FGF to retinoid signalling. We demonstrate that FGF promotes expression of a caudal Wnt and that Wnt signalling acts, following decline of FGF activity in the body axis, to permit RA activity in the neuroepithelium and to promote RA synthesis in the neighbouring presomitic mesoderm. Wnt signals thus function as an intermediary between FGF and RA signalling and facilitate the spatial and temporal separation of signalling events in the extending body axis.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
)
were set up in EC culture (Chapman et al.,
2001) for operations or bead grafts. Presomitic mesoderm was
removed unilaterally from HH7-9 embryos after brief exposure to 0.1%
trypsin.
In vitro explant culture
Explants (indicated in each figure) were isolated from HH7-8 chick embryos
and cultured in collagen as described previously
(Diez del Corral et al., 2002;
Placzek and Dale, 1999). In
all experiments control and experimental explants were derived from the same
embryo, processed individually and scored as pairs. Two quail somites (last
formed somites HH7-8) were combined with single chick caudal neural plate
explants.
Manipulating FGF signalling
Heparin beads soaked in human FGF4 (200 µg/ml) or mouse FGF8 (250
µg/ml) (R&D Systems) were grafted under the caudal neural plate and the
embryos allowed to develop for 6 to 18 hours. Explants were treated with human
FGF4 at 200 ng/ml or mouse FGF8 at 250 ng/ml in the presence of heparin (0.1
ng/ml) and BSA (0.0001%). Explants were exposed to SU5402 (10 µM)
(Calbiochem) or to DMSO only in controls.
Retinoic acid treatments
Explants were treated with 9-cis RA (10 µM, Sigma) or the RA receptor
(RAR) agonist TTNPB (1 µM, a kind gift from C. Tickle) or DMSO only for
controls. Vitamin A-deficient quails (VAD) have been described previously
(Dersch and Zile, 1993).
Manipulating Wnt signalling
COS7 cells (ECACC) were transiently transfected with either the empty
vector (control cells) or Wnt8c-IRES-GFP/PCINeo (Wnt8c cells) (kindly provided
by R. Lahder and C. Hume, NCBI #AB193181). Transfection efficiency was
measured as the percentage of GFP-positive cells (typically 55-75%),
immediately before making aggregates by the hanging drop culture technique;
each experiment was carried out in parallel with a positive control for Wnt8c
protein activity (either loss of NeuroM in preneural tube explants or
induction of Raldh2 in caudal presomitic mesoderm). Explants were
cultured with either LiCl (5 mM) (Klein
and Melton, 1996), DKK1 (1 µg/ml, R&D Systems)
(Glinka et al., 1998) or the
casein kinase I inhibitor CKI-7 (Chijiwa
et al., 1989) (200 µM, United States Biological). Neither LiCl
(5 mM) nor CKI-7 (200 µM) increased cell death in explants as assessed with
the LIVE/DEAD Viability/Cytotoxicity kit (Molecular Bioprobes) (data not
shown). Affiblue gel beads soaked in mouse sFRP2 (R&D Systems, 2
µg/µl) were grafted in contact with caudal presomitic mesoderm, and
embryos incubated for 8 hours. Embryos HH8-10 were cultured on filters for 4
hours as described previously
(Delfino-Machin et al., 2005)
in culture media with either LiCl (10 mM), SU5402 (60 µM in DMSO), or CKI-7
(400 µM in ethanol 100%) or the corresponding control media.
In ovo electroporation
The Wnt8c-IRES-GFP/PCINeo construct or the empty vector were introduced in
the caudal neural plate and preneural tube at 9-10 HH using standard in ovo
electroporation. After 10-18 hours embryos were processed for double in situ
hybridisation (ISH). NeuroM-positive cells and total nuclei (DAPI)
were counted in the electroporated half of the neural tube in at least ten
sections per embryo.
In situ hybridisation, immunocytochemistry
Standard methods for whole-mount ISH and double ISH were used. Automated in
ISH was carried out for explants using a robotic InsituPro machine (protocol
available on request). Quail cells were detected with QCPN antibody
(DSHB).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Hume and Dodd,
1993) as a likely regulator of differentiation onset in the
extending neural axis. In particular, Wnt8c expression overlaps
caudally with Fgf8 (Fig.
1A-B'), but extends more rostrally into the preneural tube
and is then sharply downregulated at the level of the somites where
Raldh2 is expressed (Fig.
1C,C'). We therefore next examined the regulation of
Wnt8c by FGF and retinoid signals.
FGF signalling is required to maintain Wnt8c expression
To test whether Wnt8c expression depends on signals from the
presomitic mesoderm (a source of FGF signals), this tissue was unilaterally
removed in HH7-9 embryos (Fig.
1D). After 4-6 hours in culture, Wnt8c levels were
reduced in half the cases (11/22) (Fig.
1E), suggesting that signals from the presomitic mesoderm may
promote Wnt8c. Many Fgfs are expressed by the caudal
presomitic mesoderm and primitive streak including Fgf4 and
Fgf8 (reviewed by Diez del Corral
and Storey, 2004), and FGF4- or FGF8-soaked beads grafted under
the caudal neural plate (Fig.
1F) maintain Wnt8c expression rostrally into the neural
tube (FGF4, 9/11; FGF8, 8/10) (Fig.
1G,H), whereas control PBS-soaked beads have no effect (six
control embryos) (Fig. 1I). In
contrast, the other caudal Wnt ligands, Wnt3a and Wnt5a,
were not maintained more rostrally by FGF4 beads in this same assay (Wnt3a,
0/5 and 3 control PBS beads; Wnt5a, 0/5 and 3 control PBS beads)
(Fig. 1J,K). These experiments
therefore indicate that Wnt8c, but not other caudally expressed Wnt
genes, is promoted by FGF signalling.
|
Finally, Wnt8c might reciprocally maintain expression of Fgf8; however, this seems unlikely because Wnt8c transcripts persist after Fgf8 has declined in the neural axis (compare Fig. 1A and 1B). Fgf8 is detected in caudal neural plate explants after 24 hours in culture but is not present in caudal presomitic mesoderm explants derived from the same embryo (compare Fig. 1P and 1Q and see details Fig. S2 in the supplementary material). Exposure to Wnt8c (provided by Wnt8c secreting COS7 cells) does not sustain or lead to more extensive Fgf8 in either caudal neural plate (4/4) (Fig. 1P') or caudal presomitic mesoderm (4/4) (Fig. 1Q') (while the same Wnt8c cells grafted on the same day promoted expression of Wnt regulated genes, see below Fig. 5A-B'). Together these findings define the regulatory relationship between FGF signalling and Wnt8c expression in the extending axis. Although Wnt8c does not promote Fgf8, caudal FGF signals, provided in part by the presomitic mesoderm, are required specifically within the neuroepithelium to maintain Wnt8c expression.
Somite-derived retinoic acid downregulates Wnt8c via the FGF pathway
Wnt8c has a sharp rostral boundary in the neural tube at the level
of somitogenesis onset (Hume and Dodd,
1993) (Fig. 1B) and
somitic RA signalling accelerates the loss of Fgf8 transcripts
(Diez del Corral et al., 2003)
and so we next assessed whether somites and RA signalling are also responsible
for Wnt8c downregulation. Wnt8c expression was examined in
pairs of chick-derived caudal neural plate explants cultured either alone or
in contact with quail somites (Fig.
2A). In most cases, Wnt8c is reduced by somite signals
(after 4 hours, 4/9; 12 hours, 3/6 and 24 hours, 6/8)
(Fig. 2B,B'). Consistent
with this and compared with the DMSO-treated controls, 9-cis RA (8/8)
or the RA agonist TTNPB (5/5) inhibited dramatically Wnt8c in caudal
neural plate explants cultured for 24 hours
(Fig. 2C,C'). These
findings suggest that somite-derived RA suppresses Wnt8c expression
in the neuroepithelium.
|
) and perdurance of stem zone Wnt8c. These findings
thus strongly suggest that RA is required for downregulation of Wnt8c
as the neural tube forms.
It is possible that retinoid signals inhibit Wnt8c indirectly by
attenuating FGF signalling. We therefore tested whether the persistent
Wnt8c domain in VAD neural tube is still dependent on FGF signalling
[even though Fgf8 transcripts are not detected in VAD neural tube
(see Diez del Corral et al.,
2003)], or if it resulted from the direct loss of retinoid
signalling. In the majority of cases, VAD neural tube explants still expressed
Wnt8c after 6 hours in culture (3/5)
(Fig. 2G,H) and in these, the
contralateral explant pair treated with SU5402 showed a dramatic reduction in
Wnt8c (3/3) (Fig.
2H'). This indicates that RA is unlikely to repress directly
Wnt8c expression, which is FGF-dependent even in the VAD neural tube.
This suggests that during normal development RA indirectly represses
Wnt8c via its ability to attenuate FGF signalling.
Wnt8c inhibits neuronal differentiation
As Wnt8c is induced by FGF signalling, this molecule likely
mediates some FGF activities. We therefore tested whether maintaining
Wnt8c expression could delay onset of neuronal differentiation, as
indicated by expression of NeuroM. Electroporation of a
Wnt8c-IRES-GFP/PCINeo expression vector into the caudal neural plate and
preneural tube HH8-10 lead to a significant reduction in the number of
NeuroM-positive cells in the neural tube (11/12, embryos) compared
with the control empty vector (nine controls)
(Fig. 3A-C). A similar
reduction was also observed for the proneural gene homologue
Neurogenin1 (3/4 embryos and 3 controls, data not shown). These in
vivo data are further supported by the effects of Wnt8c on explanted neural
tissue; comparison of explants of the preneural tube
(Fig. 3D) combined with COS7
cells transiently transfected either with the Wnt8c-IRES-GFP/PCINeo vector
(Wnt8c cells) or the empty control vector (control cells) shows that after 24
hours explants combined with control cells consistently express
NeuroM (9/9) (Fig. 3E)
(Diez del Corral et al.,
2002), but contralateral explants cultured with Wnt8c cells
contain less NeuroM-positive cells than the controls (6/9)
(Fig. 3E'). Wnt8c is
generally thought to signal through the ß-catenin pathway, so we next
cultured preneural tube explants in the presence of LiCl, an inhibitor of
GSK3ß that can mimic canonical Wnt signalling
(Klein and Melton, 1996).
After 24 hours, NeuroM expression was reduced in LiCl-treated
explants compared with untreated controls (11/15)
(Fig. 3F,F'). This result
is comparable to the effects of Wnt8c cells and is consistent with previous
work identifying canonical/ß-catenin signalling as an inhibitor of
neuronal differentiation (Megason and
McMahon, 2002). Wnt signalling thus appears to be a relay of FGF
activity in the newly formed neuroepithelium, where it acts to prevent
precocious neuronal differentiation.
FGF signalling can inhibit neurogenesis independently of Wnt signalling
It is, however, unclear whether Wnt and FGF signalling act via the same
mechanism to inhibit neuronal differentiation. Indeed, whereas FGF
dramatically inhibits NeuroM expression in neural tube explants
[17/17; after Diez del Corral et al. (Diez
del Corral et al., 2002)] (Fig.
3G,G'), stimulating Wnt signalling with LiCl inhibits
NeuroM in just half the cases (6/12) and leads to only a modest
reduction in NeuroM-positive cells
(Fig. 3H,H'). Similarly,
Neurogenin1 expression was dramatically reduced in neural tube
explants treated with FGF4 (12/13) (Fig.
3I,I') but only partially inhibited by LiCl in half the
cases (5/9) (Fig. 3J,J').
This suggests that Wnt signalling is a less efficient repressor of
neurogenesis. Furthermore, in neural tube explants (which no longer express
Fgf8 or Wnt8c at the time of excision) exposure to FGF4 does
not re-activate Wnt8c (8/8) (Fig.
4A,A'), indicating that Wnt8c is not necessary for FGF to
inhibit neurogenesis. It is possible that FGF suppresses neuronal
differentiation in this context via other canonical Wnt ligands Wnt1
and Wnt3a, which are normally present in the dorsal neural tube
(Hollyday et al., 1995).
Indeed, neural tube explants express both Wnt1 (4/4) and
Wnt3a (3/4) after 24 hours in culture
(Fig. 4B,C) but treatment with
FGF4 strongly inhibits Wnt1 (4/4)
(Fig. 4B') and can
downregulate Wnt3a expression in some cases (2/4)
(Fig. 4C'). To test
directly whether FGF relies on Wnt signalling to inhibit neuronal
differentiation we next simultaneously exposed neural tube explants to FGF and
blocked Wnt signalling, using the casein kinase I inhibitor CKI-7
(Chijiwa et al., 1989;
Price, 2006) (see below).
CKI-7 by itself has no consistent effect on NeuroM (8/8)
(Fig. 4D,D') and neural
tube explants in the presence of FGF4 and CKI-7 still lose NeuroM
expression (3/4) (Fig.
4E,E'). These findings indicate that inhibition of neuronal
differentiation by FGF is unlikely to rely on canonical Wnt signalling in the
neural tube and further, strongly suggest that FGF and Wnt act via different
mechanisms to interfere with neurogenesis. To address this possibility we next
assessed the regulatory relationship between these two signalling pathways and
the retinoid pathway, which is necessary for neuron production in this context
(Diez del Corral et al.,
2003).
|
). To dissect the mechanism(s) by which FGF and Wnt exert
their effects on neuronal differentiation we therefore tested whether these
signals act by attenuating RARß levels. RARß
transcripts are normally detected in the neural tube
(Fig. 4F,F') and as
expected are present in explants of this tissue cultured for 24 hours (8/8)
(Fig. 4G). Strikingly, FGF
treatment strongly inhibits RARß expression in neural tube
explants (8/8) (Fig. 4G')
indicating that FGF can interfere with RA signal transduction within the
neuroepithelium. As Wnt8c is expressed in the preneural tube, we used
this tissue to test whether Wnt signalling also acts by repressing
RARß. In contrast to FGF treatment, Wnt8c/control cells or LiCl
produce no consistent change in levels of RARß in preneural tube
explants, after 8 or 24 hours culture (Wnt8c cells, 8h, 6/6; LiCl, 24 hours,
4/4, data not shown; Wnt8c cells, 24 hours, 6/6
Fig. 4H,H'). These
observations suggest that FGF and canonical Wnt signalling repress neuronal
differentiation via different mechanisms: FGF, but not Wnt signalling,
interferes with the retinoid pathway, either at the level of
RARß transcription and/or RA signal transduction.
Wnt signalling promotes Raldh2 onset in the presomitic mesoderm
We reasoned that although Wnt signalling does not inhibit RA transduction
in the preneural tube it might interfere with this process or retinoid
synthesis in the mesoderm, as Lef1, a key component of the Wnt signal
transduction machinery, is expressed at high levels in the rostral presomitic
mesoderm (Schmidt et al.,
2004) (see Fig. S1E,E' in the supplementary material). We
therefore tested whether Wnt8c could inhibit RARß expression in
the presomitic mesoderm. However, unexpectedly half of the presomitic mesoderm
explants cultured in contact with Wnt8c cells showed enhanced
RARß expression compared with the control cells (4/8, 8 hours)
(Fig. 4I,I'). This
suggests that in this tissue Wnt signalling actually promotes RA signalling.
One way in which the Wnt pathway might promote RARß is by
increasing RA synthesis. We therefore next manipulated Wnt signalling in the
presomitic mesoderm and examined the onset of Raldh2 in this tissue.
Caudal presomitic mesoderm explants (Fig.
5A) weakly express Raldh2 after 8 hours in culture with
control cells (3/11 are slightly positive)
(Fig. 5B) whereas in the
presence of Wnt8c cells, Raldh2 is clearly upregulated in most
explant pairs (9/11) (Fig.
5B'), indicating that Wnt signalling can promote
Raldh2 onset.
|
) it may be that in vivo Raldh2 expression requires
Wnt activity in the context of low or no FGF signalling (a condition achieved
in explanted caudal paraxial mesoderm, which rapidly loses Fgf8
expression, see Fig. S2 in the supplementary material). Treatment of embryos
with the FGF receptor inhibitor SU5402 results in the loss of the FGF
responsive gene Sprouty2 (SU5402, 5/5 and 5 control embryos)
(Fig. 5G,H) whereas
Raldh2 expression is largely unaffected (SU5402, 5/6 and 6 control
embryos) (Fig. 5I,J). However,
exposure to both SU5402 and LiCl consistently enhances Raldh2
expression (SU5402 + LiCl, 11/12 and 9 control embryos)
(Fig. 5K,L) and a subset of
these embryos (4/11) exhibits a caudal expansion of the Raldh2 domain
(Fig. 5L). These data therefore
suggest that Wnt signals promote Raldh2, once FGF signalling has
declined.
We next carried out a series of experiments to test whether Raldh2
onset requires Wnt signalling. To inhibit this pathway in whole embryos we
first grafted beads soaked in SFRP2 protein between the neuroepithelium and
presomitic mesoderm and embryos were allowed to develop for 8 hours. In most
cases Raldh2 onset is shifted rostrally with respect to the
unoperated side of the embryo (Fig.
6A,B) (SFPR2 beads 7/9, control PBS beads n=7),
indicating that local inhibition of Wnt signalling delays Raldh2
expression. Consistent with this finding, Lef1 expression in the
presomitic mesoderm is also attenuated by sFRP2 beads (3/3, 3 control PBS
beads, data not shown). These data suggest that there is a specific
requirement for Wnt signalling for mesoderm maturation. SFRP2 can inhibit both
canonical and non-canonical Wnt signals (for a review, see
Kawano and Kypta, 2003) and we
therefore next tested the effects of CKI inhibition, as this molecule is a key
mediator of the canonical Wnt pathway
(Chijiwa et al., 1989;
Price, 2006). For treatment
with this drug embryos were prepared in filter culture, exposed to media
containing CKI-7 and processed in parallel with control embryos, as above.
This revealed that in most cases CKI-7 attenuates both Lef1
(Fig. 6C,D) (CKI-7, 4/5 and 5
control embryos) and Raldh2 expression
(Fig. 6E,F) (CKI-7, 5/9 and 11
control embryos), supporting a requirement for canonical Wnt signalling for
Raldh2 onset.
|
|
), which,
despite our assessment of effects in embryos on filters after only 4 hours,
might contribute to the observed reduction of Raldh2 in the whole
embryo when Wnt signalling is blocked. To assess directly the effects of Wnt
signalling on the maturation of the presomitic mesoderm we therefore next
manipulated this pathway in explants of this tissue. Incubation of caudal
presomitic mesoderm explants for a long period leads eventually to
Raldh2 expression (Diez del
Corral et al., 2003). We therefore cultured these explants for 18
hours either alone or in the presence of a Wnt inhibitor. As expected,
explants cultured in control medium eventually came to express Raldh2
by themselves (16/16) (Fig. 6G)
whereas significantly, treatment with either CKI-7 (7/8) or the secreted
canonical Wnt signalling inhibitor DKK1 (14/16)
(Glinka et al., 1998)
inhibited endogenous Raldh2 onset
(Fig. 6G'). Altogether
these findings indicate that in the embryo, once FGF signalling has declined
(which happens more rapidly in the mesoderm than in the neuroepithelium, see
Fig. S2 in the supplementary material) canonical Wnt signalling acts
specifically in the presomitic mesoderm to promote Raldh2
expression. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
;
Dickinson et al., 1994;
Ikeya et al., 1997;
Megason and McMahon, 2002;
Zechner et al., 2003). FGF
signalling also promotes proliferation in many contexts, including neural
progenitors in which it can additionally accelerate the cell cycle
(Lukaszewicz et al., 2002;
Wilcock et al., 2007). Our
data further support this distinction between Wnt and FGF signalling and
suggest that Wnt signalling is a milder inhibitor of neurogenesis than FGF as
indicated by the expression of NeuroM and Ngn1. These
findings are consistent with data showing that although activation of
ß-catenin in the neuroepithelium maintains proliferation, some cells can
still differentiate into neurons in this context
(Zechner et al., 2003).
Although FGF does not depend on Wnt signalling to inhibit neuronal
differentiation in our neural tube explant assay, it is still possible that
FGF regulates Wnt activity in the neuroepithelium. FGF can influence the
outcome of ß-catenin activity in cortical progenitor cells maintained in
vitro; when ß-catenin is overexpressed with FGF it promotes proliferation
whereas in the absence of FGF it can enhance neuronal differentiation
(Israsena et al., 2004). There
is also some evidence that Wnt signals promote neuron production by regulating
Ngn1 expression in cortical progenitors, however, this is stage
dependent (Hirabayashi et al.,
2004; Israsena et al.,
2004) and appears not to correspond to neurogenesis in our neural
tube assay, where Wnt signalling mildly reduces neuron production.
The difference in the impact of Wnt and FGF signalling on neuronal
differentiation may well be explained by our finding that FGF, but not
canonical Wnt signalling, can inhibit the retinoid pathway, which is required
for neuronal differentiation. Indeed, there is evidence in cell lines that
association of RARß and ß-catenin proteins can elicit activity at
RAREs in the promoters of RA-responsive genes and additionally, that there can
be competition for ß-catenin association with either RARß or
TCF/Lef1, which could reduce either RA or Wnt activity
(Easwaran et al., 1999). This
suggests that direct interactions between RA and Wnt pathways could help to
regulate neuronal differentiation within the neural tube. Together these
observations underscore a key conclusion of this work: although Wnt signalling
can restrain neuronal differentiation it permits RA activity, whereas FGF, as
indicated by its dramatic inhibition of RARß, abolishes RA
signalling. The regulatory relationships between FGF, Wnt and RA pathways
defined in this study may also help to explain why a combination of FGF and
Wnt signalling leads to the acquisition of more caudal spinal cord character,
as these signals are characteristic of the stem zone where progressively more
caudal genes are expressed, and why exposure to retinoid signalling, which
represses FGF/Wnt activity, gives more rostral spinal cord character (see
Nordstrom et al., 2006).
Wnt signalling controls the timing of presomitic mesoderm maturation
Canonical Wnt signals are critical for multiple steps in the mesodermal
lineage. These include: mesoderm induction
(Szeto and Kimelman, 2004;
Takada et al., 1994;
Yamaguchi et al., 1999;
Yoshikawa et al., 1997);
regulation of cyclic gene expression and maintenance of Fgf8 and
hence the maturation wavefront underpinning segmentation
(Aulehla et al., 2003;
Dubrulle and Pourquie, 2004a;
Ishikawa et al., 2004); as
well as the promotion of myogenesis (reviewed by
Tajbakhsh and Buckingham,
2000). Here we identify a new role for canonical Wnt signalling in
controlling the timing of retinoid production in the extending body axis. We
demonstrate in whole embryos that canonical Wnt signalling is required for
Raldh2 expression and that blocking this pathway with either a small
molecule inhibitor or an endogenous secreted LRP5/6 co-receptor antagonist
(DKK1) specifically in explants of the caudal paraxial mesoderm inhibits
Raldh2 onset. Furthermore, we show that Wnt signals are sufficient to
accelerate onset of this gene in explanted caudal paraxial mesoderm.
Importantly, such explants cultured for a long period without exposure to
additional Wnt ligand do eventually express Raldh2 and this is most
likely due to prior exposure to Wnts, as blocking canonical Wnt signalling in
this tissue inhibits Raldh2 onset. This indicates that Wnt signalling
acts normally in the presomitic mesoderm to control the timing of
Raldh2 expression.
However, in our short-term in vivo assay, acceleration of Raldh2
expression requires both stimulation of canonical Wnt signalling and loss of
FGF signalling. This reflects the ability of FGF to repress Raldh2
(Diez del Corral et al.,
2003), while the difference between in vivo and in vitro assays
may be explained by the very rapid loss of Fgf8 expression observed
in caudal paraxial mesoderm explants (see Fig. S2 in the supplementary
material). This requirement for Wnt signalling in addition to attenuation of
FGF for Raldh2 onset in vivo is also consistent with previous work
showing that inhibiting FGF signalling alone is insufficient for onset of
paraxis, a later marker of somitic tissue
(Delfini et al., 2005).
Finally, by placing Wnt inhibitor-presenting beads between the neuroepithelium
and the paraxial mesoderm we localize this requirement for Wnt signalling for
Raldh2 expression in vivo. This experiment also supports the
possibility that it is Wnt signals provided by the neuroepithelium that
regulate Raldh2 onset. Importantly, Wnt8c is a good
candidate to mediate this step as it is expressed by the neuroepithelium and
is the only known canonical Wnt expressed in the vicinity of the
Raldh2 domain. We also demonstrate that Wnt8c can induce
Raldh2 in caudal presomitic mesoderm explants. So, although there may
be a contribution from persisting Wnts transcribed more caudally in the
mesoderm (Wnt3a, Wnt5a and Wnt8c) (see
Nakaya et al., 2005), our
experiments strongly suggest that local stimulation of Wnt signalling, as
indicated by raised Lef1 expression in the rostral paraxial mesoderm,
is most likely provided by Wnt8c during normal development.
This conclusion further suggests that timely Raldh2 onset depends
on the differential loss of FGF signalling in presomitic mesoderm and caudal
neuroepithelium, which would allow Wnt8c expression maintained by
low-level FGF signalling to persist and act on the rostral presomitic
mesoderm. We demonstrate the differential loss of FGF signalling by comparing
Fgf8 transcript levels in the caudal presomitic mesoderm and caudal
neural plate explants taken from the same embryo (see Fig. S2 in the
supplementary material). Our finding that Fgf8 is lost more rapidly
from the mesodermal layer together with a previous study which shows that this
tissue contains only degrading Fgf8 transcripts
(Dubrulle and Pourquie,
2004b), suggests that transcription is only ongoing in the upper
layer, the caudal neural plate. The sensitivity of Wnt8c to FGF
signalling is demonstrated by its continued expression once Fgf8
transcripts have declined in the neuroepithelium; Wnt8c is only
downregulated at the level of the somites concomitant with Sprouty2
(Chambers and Mason, 2000), a
reporter of FGF signalling via MAPK
(Minowada et al., 1999).
Furthermore, we show that Wnt8c remains sensitive to loss of FGF
signalling in the VAD neural tube, where even Sprouty2 and activated
MAPK are beneath detection levels (Diez
del Corral et al., 2003), suggesting that Wnt8c is able
to respond to very low levels of FGF signalling.
It is likely that Wnt signals are transduced via Lef1 in the
presomitic mesoderm, as this appears to be the main TCF expressed in this
tissue (Schmidt et al., 2004).
Previous studies indicate that Lef1 expression in the rostral
presomitic mesoderm is elicited by a combination of Shh and Wnt/ß-catenin
signalling and that this leads to induction of MyoD and subsequent
myogenesis (Schmidt et al.,
2000). We have shown previously that Shh expression in
the neural plate is attenuated by FGF signalling and documented the onset of
Shh in the floor plate at the level of somites
(Diez del Corral et al.,
2003), both of which suggest that Shh activity rises as FGF
signalling declines. So, as FGF signalling diminishes, Shh levels increase and
act together with Wnt8c to promote Lef1 expression, which may then
lead to the discrete onset of Raldh2 (see
Fig. 7). Interestingly, as well
as Wnts, retinoid signalling promotes myogenesis in the embryo
(Hamade et al., 2005;
Maden et al., 2000) and can
drive cell cycle exit and differentiation of myoblasts in vitro
(Puri and Sartorelli, 2000).
So, an early step in Wnt-directed myogenesis may be the promotion of RA
synthesis in the presomitic mesoderm.
Crucially, once RA begins to be produced by the presomitic mesoderm, it
acts back to inhibit Wnt8c. Our results support the idea that caudal
Wnt8c is indirectly repressed by RA via its attenuation of FGF
signalling; although Wnt8c/8a are dramatically expanded into the
neural tube in the absence of retinoid signalling (this work)
(Dupe and Lumsden, 2001;
Niederreither et al., 2000),
blocking FGF signalling in retinoid-deficient neural tube explants still leads
to loss of Wnt8c. However, this does not rule out the possibility
that RA also directly inhibits Wnt8c. Interestingly, caudal Wnt3a is
also inhibited by exposure to retinoid signalling
(Iulianella et al., 1999;
Shum et al., 1999), suggesting
that this regulatory loop may commence even earlier in the primitive streak
where Wnt3a is required for maintenance of Fgf8 expression
(Aulehla et al., 2003).
Significantly, although Fgf8 then promotes Wnt8c, Fgf8
cannot induce Wnt3a (Kengaku et
al., 1998) and we show here that Wnt8c does not induce
Fgf8. These regulatory relationships are therefore directional and
indeed a similar directional relay of Wnt and FGF signalling has been
described during the initiation and outgrowth of the vertebrate limb bud
(Kawakami et al., 2001).
Wnt-FGF signalling relays therefore appear to be conserved mechanisms which
underpin the spatial and temporal separation of signalling events during axis
extension. In the case of the body axis described here, this directional
signalling determines the precise spatial regulation of retinoid production
and in this way controls the timing of the FGF/RA differentiation switch.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2125/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akai, J., Halley, P. A. and Storey, K. G.
(2005). FGF-dependent Notch signaling maintains the spinal cord
stem zone. Genes Dev.
19,2877
-2887.
Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B. and Herrmann, B. G. (2003). Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4,395 -406.[CrossRef][Medline]
Bel-Vialar, S., Itasaki, N. and Krumlauf, R. (2002). Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development 129,5103 -5115.[Medline]
Blumberg, B., Bolado, J., Jr, Moreno, T. A., Kintner, C., Evans, R. M. and Papalopulu, N. (1997). An essential role for retinoid signaling in anteroposterior neural patterning. Development 124,373 -379.[Abstract]
Brown, J. M. and Storey, K. G. (2000). A region of the vertebrate neural plate in which neighbouring cells can adopt neural or epidermal fates. Curr. Biol. 10,869 -872.[CrossRef][Medline]
Cambray, N. and Wilson, V. (2002). Axial progenitors with extensive potency are localised to the mouse chordoneural hinge. Development 129,4855 -4866.[Medline]
Chambers, D. and Mason, I. (2000). Expression of sprouty2 during early development of the chick embryo is coincident with known sites of FGF signalling. Mech. Dev. 91,361 -364.[CrossRef][Medline]
Chapman, S. C., Collignon, J., Schoenwolf, G. C. and Lumsden, A. (2001). Improved method for chick whole-embryo culture using a filter paper carrier. Dev. Dyn. 220,284 -289.[CrossRef][Medline]
Chenn, A. and Walsh, C. A. (2002). Regulation
of cerebral cortical size by control of cell cycle exit in neural precursors.
Science 297,365
-369.
Chijiwa, T., Hagiwara, M. and Hidaka, H.
(1989). A newly synthesized selective casein kinase I inhibitor,
N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification
of casein kinase I from bovine testis. J. Biol. Chem.
264,4924
-4927.
Cui, J., Michaille, J. J., Jiang, W. and Zile, M. H. (2003). Retinoid receptors and vitamin A deficiency: differential patterns of transcription during early avian development and the rapid induction of RARs by retinoic acid. Dev. Biol. 260,496 -511.[CrossRef][Medline]
de The, H., Tiollais, P. and Dejean, A. (1990). The retinoic acid receptors. Nouv. Rev. Fr. Hematol. 32, 30-32.[Medline]
Delfini, M. C., Dubrulle, J., Malapert, P., Chal, J. and
Pourquie, O. (2005). Control of the segmentation process by
graded MAPK/ERK activation in the chick embryo. Proc. Natl. Acad.
Sci. USA 102,11343
-11348.
Delfino-Machin, M., Lunn, J. S., Breitkreuz, D. N., Akai, J. and
Storey, K. G. (2005). Specification and maintenance of the
spinal cord stem zone. Development
132,4273
-4283.
Dersch, H. and Zile, M. H. (1993). Induction of normal cardiovascular development in the vitamin A-deprived quail embryo by natural retinoids. Dev. Biol. 160,424 -433.[CrossRef][Medline]
Dickinson, M. E., Krumlauf, R. and McMahon, A. P. (1994). Evidence for a mitogenic effect of Wnt-1 in the developing mammalian central nervous system. Development 120,1453 -1471.[Abstract]
Diez del Corral, R. and Storey, K. G. (2004). Opposing FGF and retinoid pathways: a signalling switch that controls differentiation and patterning onset in the extending vertebrate body axis. BioEssays 26,857 -869.[CrossRef][Medline]
Diez del Corral, R., Breitkreuz, D. N. and Storey, K. G.
(2002). Onset of neuronal differentiation is regulated by
paraxial mesoderm and requires attenuation of FGF signalling.
Development 129,1681
-1691.
Diez del Corral, R., Olivera-Martinez, I., Goriely, A., Gale, E., Maden, M. and Storey, K. (2003). Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron 40, 65-79.[CrossRef][Medline]
Domingos, P. M., Itasaki, N., Jones, C. M., Mercurio, S., Sargent, M. G., Smith, J. C. and Krumlauf, R. (2001). The Wnt/beta-catenin pathway posteriorizes neural tissue in Xenopus by an indirect mechanism requiring FGF signalling. Dev. Biol. 239,148 -160.[CrossRef][Medline]
Dubrulle, J. and Pourquie, O. (2004a). Coupling
segmentation to axis formation. Development
131,5783
-5793.
Dubrulle, J. and Pourquie, O. (2004b). fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature 427,419 -422.[CrossRef][Medline]
Dupe, V. and Lumsden, A. (2001). Hindbrain patterning involves graded responses to retinoic acid signalling. Development 128,2199 -2208.[Medline]
Easwaran, V., Pishvaian, M., Salimuddin and Byers, S. (1999). Cross-regulation of beta-catenin-LEF/TCF and retinoid signaling pathways. Curr. Biol. 9,1415 -1418.[CrossRef][Medline]
Gamse, J. and Sive, H. (2000). Vertebrate anteroposterior patterning: the Xenopus neurectoderm as a paradigm. BioEssays 22,976 -986.[CrossRef][Medline]
Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C. and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391,357 -362.[CrossRef][Medline]
Hamade, A., Deries, M., Begemann, G., Bally-Cuif, L., Genet, C., Sabatier, F., Bonnieu, A. and Cousin, X. (2005). Retinoic acid activates myogenesis in vivo through Fgf8 signalling. Dev. Biol. 289,127 -140.[CrossRef][Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the develoment of the chick embryo. J. Morphol. 88,49 -92.[CrossRef]
Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama,
T., Masuyama, N. and Gotoh, Y. (2004). The Wnt/beta-catenin
pathway directs neuronal differentiation of cortical neural precursor cells.
Development 131,2791
-2801.
Hollyday, M., McMahon, J. A. and McMahon, A. P. (1995). Wnt expression patterns in chick embryo nervous system. Mech. Dev. 52,9 -25.[CrossRef][Medline]
Hume, C. R. and Dodd, J. (1993). Cwnt-8C: a novel Wnt gene with a potential role in primitive streak formation and hindbrain organization. Development 119,1147 -1160.[Abstract]
Ikeya, M., Lee, S. M., Johnson, J. E., McMahon, A. P. and Takada, S. (1997). Wnt signalling required for expansion of neural crest and CNS progenitors. Nature 389,966 -970.[CrossRef][Medline]
Ishikawa, A., Kitajima, S., Takahashi, Y., Kokubo, H., Kanno, J., Inoue, T. and Saga, Y. (2004). Mouse Nkd1, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling. Mech. Dev. 121,1443 -1453.[CrossRef][Medline]
Israsena, N., Hu, M., Fu, W., Kan, L. and Kessler, J. A. (2004). The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev. Biol. 268,220 -231.[CrossRef][Medline]
Iulianella, A., Beckett, B., Petkovich, M. and Lohnes, D. (1999). A molecular basis for retinoic acid-induced axial truncation. Dev. Biol. 205, 33-48.[CrossRef][Medline]
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Rodriguez Esteban, C. and Izpisua Belmonte, J. C. (2001). WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104,891 -900.[CrossRef][Medline]
Kawano, Y. and Kypta, R. (2003). Secreted
antagonists of the Wnt signalling pathway. J. Cell
Sci. 116,2627
-2634.
Kengaku, M., Capdevila, J., Rodriguez-Esteban, C., De La Pena,
J., Johnson, R. L., Belmonte, J. C. and Tabin, C. J. (1998).
Distinct WNT pathways regulating AER formation and dorsoventral polarity in
the chick limb bud. Science
280,1274
-1277.
Klein, P. S. and Melton, D. A. (1996). A
molecular mechanism for the effect of lithium on development. Proc.
Natl. Acad. Sci. USA 93,8455
-8459.
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129,4335 -4346.[Medline]
Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997). Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. Development 124,959 -969.[Abstract]
Liu, J. P., Laufer, E. and Jessell, T. M. (2001). Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron 32,997 -1012.[CrossRef][Medline]
Lohnes, D. (2003). The Cdx1 homeodomain protein: an integrator of posterior signaling in the mouse. BioEssays 25,971 -980.[CrossRef][Medline]
Lukaszewicz, A., Savatier, P., Cortay, V., Kennedy, H. and
Dehay, C. (2002). Contrasting effects of basic fibroblast
growth factor and neurotrophin 3 on cell cycle kinetics of mouse cortical stem
cells. J. Neurosci. 22,6610
-6622.
Maden, M., Graham, A., Zile, M. and Gale, E. (2000). Abnormalities of somite development in the absence of retinoic acid. Int. J. Dev. Biol. 44,151 -159.[Medline]
Mathis, L. and Nicolas, J. F. (2000). Different clonal dispersion in the rostral and caudal mouse central nervous system. Development 127,1277 -1290.[Abstract]
Mathis, L., Kulesa, P. M. and Fraser, S. E. (2001). FGF receptor signalling is required to maintain neural progenitors during Hensen's node progression. Nat. Cell Biol. 3,559 -566.[CrossRef][Medline]
McGrew, L. L., Hoppler, S. and Moon, R. T. (1997). Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus. Mech. Dev. 69,105 -114.[CrossRef][Medline]
Megason, S. G. and McMahon, A. P. (2002). A
mitogen gradient of dorsal midline Wnts organizes growth in the CNS.
Development 129,2087
-2098.
Minowada, G., Jarvis, L. A., Chi, C. L., Neubuser, A., Sun, X., Hacohen, N., Krasnow, M. A. and Martin, G. R. (1999). Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 126,4465 -4475.[Abstract]
Moreno, T. A. and Kintner, C. (2004). Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis. Dev. Cell 6, 205-218.[CrossRef][Medline]
Muhr, J., Graziano, E., Wilson, S., Jessell, T. M. and Edlund, T. (1999). Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos. Neuron 23,689 -702.[CrossRef][Medline]
Nakaya, M. A., Biris, K., Tsukiyama, T., Jaime, S., Rawls, J. A.
and Yamaguchi, T. P. (2005). Wnt3alinks left-right
determination with segmentation and anteroposterior axis elongation.
Development 132,5425
-5436.
Niederreither, K., Vermot, J., Schuhbaur, B., Chambon, P. and Dolle, P. (2000). Retinoic acid synthesis and hindbrain patterning in the mouse embryo. Development 127, 75-85.[Abstract]
Nordstrom, U., Jessell, T. M. and Edlund, T. (2002). Progressive induction of caudal neural character by graded Wnt signaling. Nat. Neurosci. 5, 525-532.[CrossRef][Medline]
Nordstrom, U., Maier, E., Jessell, T. M. and Edlund, T. (2006). An early role for Wnt signaling in specifying neural patterns of Cdx and Hox gene expression and motor neuron subtype identity. PLoS Biol. 4,e252 .[CrossRef][Medline]
Novitch, B. G., Wichterle, H., Jessell, T. M. and Sockanathan, S. (2003). A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron 40, 81-95.[CrossRef][Medline]
Oosterveen, T., Niederreither, K., Dolle, P., Chambon, P., Meijlink, F. and Deschamps, J. (2003). Retinoids regulate the anterior expression boundaries of 5' Hoxb genes in posterior hindbrain. EMBO J. 22,262 -269.[CrossRef][Medline]
Placzek, M. and Dale, K. (1999). Tissue recombinations in collagen gels. Methods Mol. Biol. 97,293 -304.[Medline]
Price, M. A. (2006). CKI, there's more than
one: casein kinase I family members in Wnt and Hedgehog signaling.
Genes Dev. 20,399
-410.
Puri, P. L. and Sartorelli, V. (2000). Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and post-transcriptional modifications. J. Cell. Physiol. 185,155 -173.[CrossRef][Medline]
Qian, X., Davis, A. A., Goderie, S. K. and Temple, S. (1997). FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells. Neuron 18, 81-93.[CrossRef][Medline]
Roztocil, T., Matter-Sadzinski, L., Alliod, C., Ballivet, M. and Matter, J. M. (1997). NeuroM, a neural helix-loop-helix transcription factor, defines a new transition stage in neurogenesis. Development 124,3263 -3272.[Abstract]
Schier, A. F. (2001). Axis formation and patterning in zebrafish. Curr. Opin. Genet. Dev. 11,393 -404.[CrossRef][Medline]
Schmidt, M., Tanaka, M. and Munsterberg, A. (2000). Expression of (beta)-catenin in the developing chick myotome is regulated by myogenic signals. Development 127,4105 -4113.[Abstract]
Schmidt, M., Patterson, M., Farrell, E. and Munsterberg, A. (2004). Dynamic expression of Lef/Tcf family members and beta-catenin during chick gastrulation, neurulation, and early limb development. Dev. Dyn. 229,703 -707.[CrossRef][Medline]
Shiotsugu, J., Katsuyama, Y., Arima, K., Baxter, A., Koide, T.,
Song, J., Chandraratna, R. A. and Blumberg, B. (2004).
Multiple points of interaction between retinoic acid and FGF signaling during
embryonic axis formation. Development
131,2653
-2667.
Shum, A. S., Poon, L. L., Tang, W. W., Koide, T., Chan, B. W., Leung, Y. C., Shiroishi, T. and Copp, A. J. (1999). Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo. Mech. Dev. 84, 17-30.[CrossRef][Medline]
Sirbu, I. O. and Duester, G. (2006). Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm. Nat. Cell Biol. 8,271 -277.[CrossRef][Medline]
Stern, C. D., Charite, J., Deschamps, J., Duboule, D., Durston, A. J., Kmita, M., Nicolas, J. F., Palmeirim, I., Smith, J. C. and Wolpert, L. (2006). Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems? Int. J. Dev. Biol. 50,3 -15.[CrossRef][Medline]
Szeto, D. P. and Kimelman, D. (2004).
Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm
formation. Development
131,3751
-3760.
Tajbakhsh, S. and Buckingham, M. (2000). The birth of muscle progenitor cells in the mouse: spatiotemporal considerations. Curr. Top. Dev. Biol. 48,225 -268.[Medline]
Takada, S., Stark, K. L., Shea, M. J., Vassileva, G., McMahon,
J. A. and McMahon, A. P. (1994). Wnt-3a regulates somite and
tailbud formation in the mouse embryo. Genes Dev.
8, 174-189.
Vasiliauskas, D. and Stern, C. D. (2001). Patterning the embryonic axis: FGF signaling and how vertebrate embryos measure time. Cell 106,133 -136.[CrossRef][Medline]
Wilcock, A. C., Swedlow, J. R. and Storey, K. G.
(2007). Mitotic spindle orientation distinguishes stem cell and
terminal modes of neuron production in the early spinal cord.
Development 134,1943
-1954.
Yamaguchi, T. P. (2001). Heads or tails: Wnts and anterior-posterior patterning. Curr. Biol. 11,R713 -R724.[CrossRef][Medline]
Yamaguchi, T. P., Takada, S., Yoshikawa, Y., Wu, N. and McMahon,
A. P. (1999). T (Brachyury) is a direct target of Wnt3a
during paraxial mesoderm specification. Genes Dev.
13,3185
-3190.
Yoshikawa, Y., Fujimori, T., McMahon, A. P. and Takada, S. (1997). Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Dev. Biol. 183,234 -242.[CrossRef][Medline]
Zechner, D., Fujita, Y., Hulsken, J., Muller, T., Walther, I., Taketo, M. M., Crenshaw, E. B., 3rd, Birchmeier, W. and Birchmeier, C. (2003). beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev. Biol. 258,406 -418.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Wilson, I. Olivera-Martinez, and K. G. Storey Stem cells, signals and vertebrate body axis extension Development, May 15, 2009; 136(10): 1591 - 1604. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Ribes, I. Le Roux, M. Rhinn, B. Schuhbaur, and P. Dolle Early mouse caudal development relies on crosstalk between retinoic acid, Shh and Fgf signalling pathways Development, February 15, 2009; 136(4): 665 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Strate, T. H. Min, D. Iliev, and E. M. Pera Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system Development, February 1, 2009; 136(3): 461 - 472. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
