Department of Molecular and Cellular Biology, University of California at Berkeley, CA 94720, USA
* Author for correspondence (e-mail: monsoro{at}uclink.berkeley.edu)
Accepted 30 March 2003
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SUMMARY |
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Key words: FGF, WNT, FGF8, Paraxial mesoderm, Xenopus embryo, Neural crest, Neural patterning
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INTRODUCTION |
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In the amphibian embryo, the current analysis of the molecular basis of
ectoderm-neural tissue interactions results in a two-step model of neural
crest induction detailed below (reviewed by
Aybar and Mayor, 2002;
Knecht and Bronner-Fraser,
2002
). Slug was generally used in these studies as a
specific marker gene for neural crest development
(Nieto et al., 1994
;
Mayor et al., 1995
). In the
first step of the model, in parallel to what happens during amphibian neural
induction (Harland, 2000
), BMP
activity in the ectoderm must be attenuated by BMP antagonists. Neural crest
forms after moderate BMP inhibition while neural tissue induction requires
higher levels of inhibition (Marchant et
al., 1998
). However, the levels of Slug induction in
these assays, using BMP antagonists alone, are very low compared with
endogenous levels (LaBonne and
Bronner-Fraser, 1998
; Marchant
et al., 1998
). This suggests that in the embryo, additional
factors are required for normal levels of Slug expression and neural
crest induction/maintenance.
Co-injection of BMP antagonists with molecules such as Wnts (Wnt7b or
Wnt8), fibroblast growth factors (eFGF or bFGF) or retinoic acid (RA) results
in strong neural crest formation in ectodermal explants (animal caps)
(Mayor et al., 1995;
Chang and Hemmati-Brivanlou,
1998
; LaBonne and
Bronner-Fraser, 1998
;
Villanueva et al., 2002
).
Although these molecules do not induce neural crest by themselves in vitro,
the in vivo overexpression of positive regulators of the Wnt, FGF or RA
pathways expands neural crest-forming domains, whereas blocking these pathways
prevents normal neural crest induction in both embryo and explant assays
(Mayor et al., 1997
;
Chang and Hemmati-Brivanlou,
1998
; LaBonne and
Bronner-Fraser, 1998
;
Villanueva et al., 2002
).
Together, these data suggest a second phase of induction where partially
neuralized ectoderm is specified to become neural crest either by Wnts, FGF,
RA or a combination. However, this model does not specifically address the
mechanism by which paraxial mesoderm might induce the neural crest.
Furthermore, both FGF and Wnt proteins play important roles in mesoderm
induction and paraxial mesoderm development
(Cornell and Kimelman, 1994
;
LaBonne and Whitman, 1994
;
Fisher et al., 2002
;
Vonica and Gumbiner, 2002
) and
reagents that affect neural crest induction might do so indirectly by their
effects on the mesoderm (Mayor et al.,
1995
; Mayor et al.,
1997
; Chang and
Hemmati-Brivanlou, 1998
;
LaBonne and Bronner-Fraser,
1998
). Finally, all three classes of molecules implicated in
neural crest induction are also important neural posteriorizing agents
(Lamb and Harland, 1995
;
Bang et al., 1997
;
Bang et al., 1999
;
Kiecker and Niehrs, 2001
;
Kudoh et al., 2002
). BMP
antagonism results in the formation of anterior neural tissue that is not
expected to form neural crest (Lamb et
al., 1993
; Knecht and Harland,
1997
). This raises the possibility that posteriorization of this
area into a neural crest-producing tissue would account for the Slug
induction recorded after co-injecting Noggin/Chordin with Wnt/FGF/RA
molecules. This correlation of neural crest induction with posterior identity
has recently been demonstrated in embryos
(Villanueva et al., 2002
).
Thus, whether induction of neural crest can occur independently from neural
induction and patterning remains unclear.
In this study, we address two questions. First, what is the nature of the
mesodermal signal(s) inducing neural crest in the ectoderm? Second, how is
neural crest induction related to early anteroposterior (AP) patterning of the
neural plate? To study the molecular mechanisms of neural crest induction by
the paraxial mesoderm in the Xenopus laevis embryo, we focused on the
neural crest-inducing properties of the dorsolateral marginal zone (DLMZ) on
animal cap explants. Using various neural crest markers, we show that the DLMZ
and the dorsal marginal zone (DMZ) exhibit qualitative differences in their
inducing properties. In order to study the role of specific growth factor
signaling in neural crest induction, we then took advantage of previously
characterized molecular tools, consisting of broad range or more specific
inhibitors of the Wnt and FGF pathways. These reagents include NFz8, GSK3,
dnTCF3 and a truncated form of Dishevelled (Xdd1) for Wnt signaling, and
SU5402, XFD and dnFGFR4a for FGF signaling
(Amaya et al., 1993;
Sokol, 1996
;
Mohammadi et al., 1997
;
Deardorff et al., 1998
;
Hongo et al., 1999
;
Deardorff et al., 2001
), for
reviews see (Galzie et al.,
1997
; Brantjes et al.,
2002
; Moon et al.,
2002
). We have also used these reagents in vivo to address whether
neural crest formation can be uncoupled from repatterning of the mesoderm or
changes in AP patterning of the neural plate.
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MATERIALS AND METHODS |
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To block the response of the ectoderm to endogenous Wnt molecules, we
injected mRNAs encoding either xNFz8, Glycogen Synthase Kinase 3 (GSK3),
dnTCF3 or Xdd1. The pCS2-xNFz8 encodes a wide spectrum
dominant-negative Wnt receptor (Deardorff
et al., 1998), Xdd1 is a truncated form of Dishevelled, which acts
as a dominant-negative in both the canonical and the non canonical planar cell
polarity (PCP) pathways (Sokol,
1996
; Wallingford and Harland,
2002
). The pCS2-xGSK3, pT7Ts-dnTCF3,
p64T-XWnt8 and pCS2-dnXWnt8 plasmids have been described
previously (Christian et al.,
1991
; Molenaar et al.,
1996
; Pierce and Kimelman,
1996
; Hoppler and Moon,
1998
). We blocked FGF signaling in the responding ectoderm using
either a dominant-negative form of xFGFR1, constructed by S. Dougan
(pCS2-XFD-GFP) similar to the XFD construct published by Amaya et al.
(Amaya et al., 1991
), or a
truncated FGFR4a (p64T-dnXFGFR-4a)
(Hongo et al., 1999
),
subcloned into pCS108. XFGF8
(Christen and Slack, 1997
) was
subcloned into pCS107.
Tissue recombination, SU5402 treatment of the recombinants
Stage 10-10.5 DLMZ or DMZ were recombined with stage 8-9 animal caps
(Fig. 1A)
(Bonstein et al., 1998).
Dissections and culture were performed in 3/4 Normal Amphibian Medium (NAM)
containing gentamycin (100 µg/ml). The recombinants were harvested when
sibling embryos reached stage 18. For inhibition of FGF signaling by the
SU5402 (Calbiochem) (Mohammadi et al.,
1997
), the recombinants were cultivated in 50 µM SU5402 diluted
into 3/4 NAM (Shinya et al.,
2001
; Maroon et al.,
2002
). Controls were grown in DMSO diluted in 3/4 NAM.
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The probes for Slug, Twist, Snail, Krox20, Cpl-1 and Otx2
have been described elsewhere (Richter et
al., 1988; Hopwood et al.,
1989
; Bradley et al.,
1993
; Lamb et al.,
1993
; Mayor et al.,
1993
; Grammer et al.,
2000
). The Sox9 probe was a kind gift of R. Spokony and
J-P. Saint-Jeannet (Spokony et al.,
2002
). Zic5 and FoxD3 in situ probes were
derived from a X. tropicalis library made by A. Zorn
(Khokha et al., 2003
).
RNA isolation and Reverse Transcriptase-PCR assay
Preparation of total RNA and RT-PCR assay were carried out as described
previously (Condie et al.,
1990). For each lane of one given experiment, 15-20 animal caps or
six to eight recombinants were pooled and analyzed. One non-injected sibling
embryo serves as a positive control in the first lane of each PCR gel. The
absence of DNA contamination was verified by omitting the reverse
transcriptase in an equivalent total embryo sample (lane 2 of the PCR gels).
EF1
was used as a cDNA loading control. Primers for
EF1
, muscle actin, Krox20, Otx2, Xnot, MyoD and
Twist have been described elsewhere
(Rupp and Weintraub, 1991
;
von Dassow et al., 1993
;
Ribisi et al., 2000
)
(Xenopus MMR database
http://www.xenbase.org/XMMR/Welcome.html).
Specific primers used in this study are described in
Table 1. Each of them was
designed using MacVector 6.5.3 from the sequences published in GenBank so that
they do not crossreact with related genes.
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RESULTS |
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If slightly larger DMZ explants were cut, extending beyond the stage 10.25 dorsal lip, they variably expressed muscle actin and Slug upon recombination (not shown). Thus, for consistency in the experiments illustrated in this study, we dissected the DMZ as a narrow band of tissue taken at stage 10-10.5, and cut DLMZs that contained robust Slug inducing activity.
To characterize the neural crest induced by the DLMZ in this explant assay
in more detail, we analyzed several other genes in addition to Slug,
all expressed mainly by the neural crest around stage 18
(Fig. 1C,D). Snail
(Essex et al., 1993;
Mayor et al., 1993
),
Twist (Hopwood et al.,
1989
), Zic5 (Nakata
et al., 2000
), Sox9
(Spokony et al., 2002
) and
FoxD3 (Pohl and Knochel,
2001
; Sasai et al.,
2001
) were all upregulated when the DLMZ was recombined with
animal caps (Fig. 1C, lane 5).
FoxD3 responded in a very similar manner to Slug: in
particular, neither was induced in the AC-DMZ recombinants
(Fig. 1B, lane 7). Both showed
weak expression in the mesoderm, corresponding to what was observed in vivo
(Fig. 1C, lanes 4 and 6)
(Linker et al., 2000
;
Sasai et al., 2001
). By
contrast, Sox9, Zic5 and Snail expression were also
upregulated in the AC-DMZ, although at a low level in the case of
Sox9. Interestingly, Snail and Zic5 induction was
as strong with the DMZ as with the DLMZ, perhaps reflecting the normal
expression of these genes in the midline of the anterior neural fold
(Fig. 1D)
(Linker et al., 2000
;
Nakata et al., 2000
).
This analysis suggests that neural crest induction observed in this recombination assay reproduces the complexity of in vivo mechanisms. Because of their basal expression in the isolated animal caps and/or mesoderm explants, Snail and Twist were not analyzed further in this study. We focused on Slug, FoxD3, Sox9 and Zic5, which were specifically upregulated in the recombinants.
Blocking Wnt signaling does not prevent induction of neural crest by
the DLMZ
The canonical Wnt pathway has been shown to be important in neural crest
formation in other systems. In addition, the Slug promoter contains
LEF-TCF binding sites suggesting a direct regulation by this pathway
(Vallin et al., 2001). To test
the hypothesis that the DLMZ requires Wnt signals to induce neural crest, we
blocked the response of the ectoderm to Wnt signaling using the antagonists
NFz8, GSK3 and dnTCF3. The xFz8 receptor has been shown to mediate the
activity of Wnt1, Wnt2c, Wnt3a, Wnt5a, Wnt7b, Wnt8 and Wnt11 efficiently
(Deardorff et al., 2001
).
NFz8, a truncated and diffusible form of xFz8, acts on gastrulation movements
and neural plate patterning as expected for a Wnt antagonist, but does not
prevent dorsal mesoderm specification
(Deardorff et al., 1998
). In
contrast to NFz8, glycogen synthase kinase 3 (GSK3) and dnTCF3 prevent Wnt
signaling in a cell autonomous manner
(Brantjes et al., 2002
;
Moon et al., 2002
).
In this series of experiments, positive controls of Wnt inhibiting activity
showed that 400 pg of NFz8 mRNA efficiently blocked XWnt8-induced
secondary axis formation (100% reversal of double axis formation, after
co-injecting 400 pg of NFz8 and 50 pg p64T-XWnt8 mRNAs,
n=31, not shown). Moreover, the injected embryos displayed defects in
dorsal neural tube closure, as shown when Wnt signaling is blocked
(Wallingford and Harland,
2002). Thus, injections of 400 to 800 pg of NFz8 mRNA per embryo
were generally used in the next experiments, although doses above 1 ng were
also tested. Moreover, as Wnt antagonists, NFz8 and GSK3 overexpression is
expected to anteriorize the neural plate and, later, increase cement gland
formation (Deardorff et al.,
1998
; Kiecker and Niehrs,
2001
). After injecting GSK3 or NFz8 (400 to 1600 pg) in the animal
hemisphere of two- or four-cell stage embryos, the cement gland was enlarged
in more than 96% (n>53) of the embryos
(Fig. 2A). This phenotype was
used as a routine control, when sibling embryos were analyzed for neural crest
formation as described below.
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We first blocked signaling by putative endogenous Wnt molecules using NFz8. After injections of 800 pg of NFz8 mRNA, a similar proportion of the recombinants exhibited Slug staining, being virtually identical to controls (Fig. 2E). This observation was confirmed by RT-PCR analysis. After recombination, control explants strongly expressed Slug and other neural crest markers (n=151, on average, 8-10 recombinants were used for each lane; Fig. 2F, lane 7). Moderate to high doses of NFz8, which are fully active in the biological tests described above, did not prevent the induction of any of the neural crest markers tested (400 pg/n=81 and 800 pg/n=76, Fig. 2F, lanes 8 and 9). This was also true when the explants were analyzed at stage 12, shortly after initial neural crest induction (not shown). In some cases, however, the induction of Slug and Sox9 was reduced compared with controls (lane 9).
Massive doses of NFz8 resulted in inhibition of Slug, FoxD3 and Sox9, but not of Zic5 (1200-1600 pg/n=21, Fig. 2F, lanes 10). This was correlated with a striking lack of elongation of the recombinants, suggesting that these higher doses affect the development of the mesoderm itself rather than the response of the ectoderm (Fig. 2I). Although Xnot was expressed normally in the recombinants, muscle actin and MyoD, which were expressed at the same levels in the 0-800 pg NFz8 injected recombinants, were slightly diminished in the 1600 pg NFz8 injections (Fig. 2F-lane 10 and not shown). This suggests that other aspects of the specification of the DLMZ could also be perturbed by the highest doses of NFz8. Such perturbation could secondarily alter the DLMZ signaling activity and account for the reduction of neural crest induction seen in lane 10.
We thus focused on 400-800 pg NFz8 doses (lanes 8 and 9): the decrease in Slug and Sox9 neural crest markers expression, in lanes 9, could either reflect the requirement for a Wnt signal acting directly on the ectodermal cells or a change in the DLMZ-inducing properties. To avoid Wnt-dependent changes in the signaling properties of the DLMZ, we blocked the response to the canonical and non canonical Wnt pathways intracellularly in the ectoderm, by injecting either GSK3 (300-400 pg/n=38 and 800-1000 pg/n=40), dnTCF3 (1 ng/n=10) or Xdd1 (1 ng/n=10) (Fig. 2G-H and not shown). None of these blocked the induction of neural crest markers by the DLMZ (Fig. 2G, lanes 6-8 and Fig. 2H). However, the injection of NFz8 or GSK3 did modulate the expression of other genes, such as Krox20 or Otx2, but not Pax3 (not shown). We conclude that neither canonical nor PCP Wnt-dependent pathways are required directly for the ectoderm to respond to the DLMZ neural crest-inducing activity. Blocking Wnt signals by diffusible antagonists perturbs DLMZ development and most probably its signaling properties. However, if Wnt signaling is not perturbed in the mesoderm, the DLMZ can induce neural crest in the ectoderm, suggesting alternative or redundant pathways for neural crest induction.
FGF signaling is required for neural crest induction by the DLMZ
FGFs bind to one of four tyrosine-kinase receptors, FGFR1-FGFR4, which lead
to activation of MAP kinase or phosphatidyl inositol pathways, eventually
modulating target gene expression (for a review, see
Galzie et al., 1997). Blocking
signaling by FGFRs, in vivo or in vitro, has employed either a truncated
dominant-negative form of FGFR1, XFD (Amaya
et al., 1993
) or a synthetic inhibitor (SU5402) that binds to the
kinase domain of FGFRs (Mohammadi et al.,
1997
).
In the first approach, we blocked FGF signaling in the explants by growing them in presence of 50 µM SU5402. Two DLMZs were dissected out of each stage 10 embryo and used to make two recombinants, one was cultivated in the SU5402 solution, the other in the control DMSO medium. RT-PCR analysis (Fig. 3A) showed that the SU5402 treatment completely suppressed Slug induction (Fig. 3A-lane 4, n=19). However, it also prevented normal development of the paraxial mesoderm from the DLMZ as shown by the lack of muscle actin expression. Under these conditions, the lack of Slug induction could be a secondary effect caused by abnormal DLMZ development.
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FGFs and FGFRs are expressed in the recombinants
We analyzed the expression of FGF3, FGF4 (eFGF) and
FGF8 in explants during the period of neural crest induction, i.e.
stages 10.25-14, using semi-quantitative RT-PCR
(Aybar and Mayor, 2002).
FGF3, FGF4 and FGF8 were detected in the isolated DLMZ but
not in the isolated animal caps at all stages analyzed
(Fig. 4A,C). In the DLMZ, the
expression of FGF genes preceded that of myotome markers such as MyoD
and muscle actin, which appeared around stage 12
(Fig. 4A, lanes 5 and 6),
similar to Slug in the ectoderm
(Linker et al., 2000
). Thus,
FGF genes and FGF8 in particular are expressed in the DLMZ during
gastrulation and early neurulation, and this expression is maintained without
the need for external signals.
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FGF8 enhances neural crest formation in embryos and is sufficient to
induce neural crest markers in explants
As FGF8 has recently been shown to be involved in neurogenesis without
inducing mesoderm (Hardcastle et al.,
2000), we decided to focus on this member of the family and
analyze its potential activity in neural crest formation. We examined
FGF8 gene expression at gastrula and early neurula stages.
FGF8 appears initially as a ring around the blastopore and is
reinforced dorsally by stage 11-11.5, when neural crest induction is thought
to begin (Fig. 5E)
(Christen and Slack, 1997
).
FGF8 expression level is then enhanced in the dorsolateral mesoderm
at stage 13 and onwards, whereas it is downregulated in the dorsal midline
(Fig. 5E). FGF8 is thus a good
candidate to mediate the FGF-dependent DLMZ activity on neural crest
induction. To test this hypothesis in whole embryos, we analyzed Slug
expression after FGF8 mRNA injections. Compared with control sibling
embryos (Fig. 5A), 50 pg of
FGF8 mRNA injections were followed by a strong increase in
Slug expression (Fig.
5B, yellow arrows indicate the injected side). This upregulation
was not correlated to an expansion of the MyoD domain
(Fig. 5C,D, small red arrow).
Interestingly, when the injected cells (lacZ staining) were located
in the anterior part of the neural plate, this region expressed Slug,
suggesting that these injections transformed the anterior neural fold into a
more posterior structure (Fig.
5B, red arrow) (Christen and
Slack, 1997
). However, in the embryo, co-factors from the
surrounding tissues, such as the mesoderm or the ectoderm, could also be
recruited for FGF8 activity on the neural crest.
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In conclusion, these data suggest that FGF8 alone is sufficient to mediate both the DLMZ-specific induction of FoxD3 and the common DMZ/DLMZ induction of Zic5 and Sox9. Second, because, in vivo, FGF8 injections show a potent Slug upregulation, we conclude that this aspect of FGF8 activity requires interactions with other DLMZ-specific factors. Moreover, in the AC-DLMZ or AC-DMZ recombinants, the expression of neural crest markers is induced and maintained, indicating that other molecules must reinforce and sustain FGF8 inductive activity.
In vivo inhibition of Wnt or FGF signaling result in anteriorization
of the neural plate prior to neural crest induction and affects paraxial
mesoderm development
Previous studies have shown that both Wnt and FGF signals are required for
normal expression of Slug in the Xenopus embryo
(Mayor et al., 1997;
LaBonne and Bronner-Fraser,
1998
; Villanueva et al.,
2002
). However, these signaling molecules are also required for
multiple steps of early development, such as mesoderm formation or neural
plate AP patterning (Ribisi et al.,
2000
; Kiecker and Niehrs,
2001
). We repeated the analysis of Slug expression under
similar experimental conditions (Fig.
6A) and also tested expression of the rhombencephalon marker
Krox 20 in parallel (Fig.
6B) (Bradley et al.,
1993
). Moreover, we analyzed mesoderm development in these assays,
by staining the embryos simultaneously for Slug mRNA and with the
monoclonal antibody 12-101, which stains differentiated muscle
(Kintner and Brockes,
1984
).
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To avoid the diffusible effects of NFz8, we also inhibited Wnt signaling cell-autonomously using GSK3 injected either in one half of the embryo or into the prospective neural fold at the 16-cell stage. Control injections did not alter Slug expression (Fig. 7A,D) or paraxial mesoderm formation (Fig. 7A). However, in both types of GSK3 injections, the decrease or a lack in Slug expression was correlated with altered paraxial mesoderm and neural patterning (Fig. 7B-F). Thus, in these in vivo assays, we have not been able to dissociate the effects of Wnt signaling on neural crest formation from those on neural plate and mesoderm patterning.
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DISCUSSION |
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The paraxial (but not the axial) mesoderm, induces a whole range of
neural crest-specific genes in the ectoderm
Elegant experiments using albino Xenopus embryos have shown that
the ectoderm can form neural crest in response to DLMZ signals and that the
DMZ was a less efficient Slug inducer than the DLMZ
(Bonstein et al., 1998;
Marchant et al., 1998
). We
show here that the induction of Slug by mesoderm explants is closely
correlated to the presence of muscle actin in the inductive tissue,
i.e. to the presence of some paraxial tissue
(Fig. 1). When DMZs are cut
medially, they consistently fail to induce Slug. This suggests that
the quantitatively lower activity of the DMZ reported previously might reflect
some variability in the width of the explants. We also show that the DLMZ is
able to induce a whole range of neural crest markers: Slug, FoxD3,
Sox9 and Zic5 (Fig.
1C, lane 5). By contrast, the DMZ does not induce Slug or
FoxD3 but upregulates Zic5 and Sox9 expression at
various levels (Fig. 1C, lane
7). The distinct inducing activities of the DLMZ and of the DMZ might be due
either to a dorsal-to-lateral increasing gradient of neural crest inducing
activity or to a different combination of inducing molecules produced by each
kind of tissue. According to the first hypothesis, Zic5 and
Sox9 genes would be upregulated by low levels of this inducer,
whereas Slug and FoxD3 activation would require a higher
concentration. According to the second hypothesis, the DMZ would express a
molecule able to induce Zic5 and Sox9 whereas the DLMZ would
express an additional signal(s) required for inducing either the complete
range of neural crest markers or Slug and FoxD3
specifically. We therefore consider Slug and FoxD3 to be
most characteristic of neural crest induction because they are specifically
induced by the DLMZ and because their in vivo expression pattern is mostly
restricted to the neural crest (Fig.
1D).
Neural crest induction by the paraxial mesoderm requires functional
FGF-FGFR1 signaling
Previous studies have shown that neural crest formation can be induced by a
combination of BMP antagonists plus Wnt/FGF signals in Xenopus animal
cap assays (Chang and Hemmati-Brivanlou,
1998; LaBonne and
Bronner-Fraser, 1998
). Moreover, the same classes of molecules
regulate the expression of Slug and FoxD3. FoxD3 is induced
by a combination of either chordin+bFGF or chordin+Wnt3a
(Sasai et al., 2001
).
Similarly, Slug is upregulated in animal caps by combining chordin
with either eFGF or XWnt8 (LaBonne and
Bronner-Fraser, 1998
). The regulation of Zic5 and
Sox9 genes has not yet been studied, although these genes are
required for neural crest development in vivo
(Nakata et al., 2000
;
Spokony et al., 2002
). Both
Wnt and FGF signals are expressed in the paraxial mesoderm. They might play a
role in mesoderm development itself, as well as mediating mesodermal signaling
activities toward the ectoderm. These activities could be redundant and do not
exclude the possibility that alternative mechanisms may also be active.
To analyze the mechanisms of action of the DLMZ, we blocked the response of
the ectoderm to either endogenous Wnt or FGF signals, in the DLMZ-AC
recombination assay (Fig. 2).
Many previous studies have used secreted antagonists such as dnWnt8 to block
Wnt signals in embryos or in explants: this results in downregulation of
Slug in Xenopus (LaBonne
and Bronner-Fraser, 1998) and blocks Pax3 induction by
the chick paraxial mesoderm (Bang et al.,
1999
). After NFz8 injections in the ectoderm of the recombinants,
we only saw a moderate downregulation of Slug, Sox9 and Pax3
at high doses (Fig. 2; data not
shown). By blocking the intracellular downstream canonical and PCP Wnt
pathways, we show that none of the four neural crest markers analyzed depend
directly on Wnt signaling to be induced by the DLMZ. Therefore, the effects of
diffusible antagonists observed in these recombination assays might reflect a
Wnt-dependent modulation or maintenance of the paraxial mesoderm-inducing
activity, or indicate that the Wnt pathway may have an overlapping
activity.
By contrast, blocking FGF-FGFR1 signaling, by injecting XFD in the
ectoderm, strongly reduced the induction of Slug, FoxD3 and to a
lesser extent Sox9 (Fig.
3). The induction of Zic5 was unaffected by the XFD
injections. The most affected genes corresponded to those specifically induced
by the DLMZ but not by the DMZ. This suggests that the DLMZ-specific aspect of
neural crest inducing activity requires functional FGF signaling, probably
through FGFR1. FGF signaling is also required in vivo for normal neural crest
formation as XFD injections strongly downregulate Slug expression
(Mayor, 1997) (this work). Interestingly, we found that dnFGFR4a did not
affect neural crest induction. In contrast to this observation, FGFR4a plays a
prominent role in neurogenesis (Hardcastle
et al., 2000). This raises the attractive possibility that
different FGFRs might display different roles in neuronal versus neural crest
development.
FGF8 induces neural crest
We show that the DLMZ expresses FGF3, FGF4 and FGF8 at
gastrula and early neurula stages (Fig.
4). We further show that FGF8 can account for the neural crest
induction by the DLMZ, either alone or in cooperation with other DLMZ factors
(Fig. 5). First, in vivo
FGF8 expression is detected at early gastrula stages as a ring around
the blastopore, it is then reinforced in the DLMZ area. Second, FGF8
mRNA injections in vivo are followed by a large increase in
Slug expression without expansion of the paraxial mesoderm. Finally,
FGF8 injections in the animal cap assay induce the expression of
neural crest markers without inducing mesoderm formation. This contrasts with
previous studies using FGF4 (eFGF) or bFGF in similar assays, which showed (1)
mesoderm induction, (2) absence of neural crest induction by FGF4 or bFGF
alone and (3) requirement for co-expression with a BMP antagonist
(LaBonne and Bronner-Fraser,
1998; Mizuseki et al.,
1998
). The unique properties of FGF8 on neural crest can be
compared with its ability to induce neurogenesis without mesoderm induction,
when it is expressed from blastula stages
(Hardcastle et al., 2000
).
Other FGF molecules can also be direct neural inducers, but only if they are
expressed after the period of competence to form mesoderm, and in tissue that
has attenuated BMP signaling (Lamb and
Harland, 1995
). FGF8 is thus a good candidate for mediating FGF
neural-specific roles during the period of early neural crest development
defined by Aybar and Mayor (Aybar and
Mayor, 2002
).
Is FGF8 a neural crest inducer?
To be considered a physiologically significant activity, a neural crest
inducer must satisfy the following properties. First, it should be expressed
by tissue(s) with a neural crest inducing potential, in early neurula stage
embryos. FGF8, which is expressed in the paraxial mesoderm as early
as stage 10, satisfies this first condition
(Fig. 5E). By contrast,
Wnt1 expression is detected by stage 14, i.e. after Slug
induction (Deardorff et al.,
2001). Thus, Wnt1 and Wnt 3a, which act via Xfrizzled-3 and
Kermit, are more likely to play later roles in neural crest development, such
as maintenance of the induction or fate choice
(Dorsky et al., 1998
;
Basch et al., 2000
;
Dorsky et al., 2000
;
Deardorff et al., 2001
;
Jin et al., 2001
;
Tan et al., 2001
). Thus, in
amphibians, the activity of an ectoderm-restricted Wnt, equivalent to the
chick Wnt6 gene, remains to be found
(Garcia-Castro et al., 2002
).
The activity of ß-catenin on early neural crest formation
(LaBonne and Bronner-Fraser,
1998
) could rather reflect a role of Wnt7b and Wnt 8, which are
present in the early ectoderm/neurectoderm and paraxial mesoderm, respectively
(Bang et al., 1999
;
Wu et al., 2003
).
Second, the activity of the inducer should be necessary to obtain neural
crest formation, although this can be missed if redundant pathways are
activated in the same assay. We show here that FGF signaling is required to
mediate paraxial mesoderm induction of Slug and FoxD3
(Fig. 3). Active FGFR1
signaling is also necessary in vivo (Mayor
et al., 1997). Further analysis by a selective knockdown of FGF8
will determine if FGF8 is specifically required in the DLMZ for neural crest
induction or if other FGFs have overlapping activity.
In addition to these two properties, the neural crest-inducing activity
could be mediated either by a single factor or a combination of molecules.
Tested separately, these molecules might be able to evoke neural crest
formation even if the robust induction of neural crest markers and further
development of neural crest cells might require additional inputs. In
Xenopus animal cap assay, FGF8 induces FoxD3, Sox9 and
Zic5 (but Slug is only very slightly upregulated)
(Fig. 5F,G). Moreover, the
induction by FGF8 in this assay is transient, showing the requirement for
other factors to maintain and complete the induction of the full range of
neural crest markers. It has been shown by similar experiments that, although
they do not induce neural crest markers by themselves, Wnts, eFGF and bFGF
synergize with noggin or chordin to induce neural crest
(Chang and Hemmati-Brivanlou,
1998; LaBonne and
Bronner-Fraser, 1998
; Mizuseki
et al., 1998
). The cooperation of FGF8 with other molecules such
as BMP antagonists or Wnts in the maintenance of neural crest induction
remains to be explored.
FGF8 activity must be modulated to become a potent crest-inducing activity,
as FGF8 is expressed in both dorsal and dorsolateral marginal zones
(Fig. 5), and these have
qualitatively different neural crest-inducing activity
(Fig. 1). FGF8 might account
for DMZ-DLMZ common induction of Zic5 and Sox9. However, in
the recombinant assay, neither XFD nor dn FGFR4a injections prevented
Zic5 or Sox9 induction, supporting the idea that redundant
inducing mechanisms are provided by the DLMZ
(Fig. 3). In addition,
Zic5 and Sox9 are not restricted to the neural crest, but
also expressed in the anterior neural fold and the prospective otic placode,
respectively. They are thus expected to respond to neural crest specific
inducers as well as to other signals (Fig.
1). Our study also revealed distinct regulation for Slug
and FoxD3. Both genes were considered specifically induced by the
DLMZ (Fig. 1) and this
induction requires FGF signaling (Fig.
3). However, FGF8 is sufficient to induce expression of
FoxD3 but not of Slug. Cooperation of FGF8 with additional
signals could account for the expansion of the Slug domain observed
in the embryo (Fig. 5).
Alternatively, our in vitro conditions might not induce the right relative
levels of FoxD3/Sox9/Zic5: each of these factors is necessary for
normal neural crest development and/or Slug expression. In
particular, overexpression of FoxD3 can either increase or prevent
Slug activation, suggesting that a fine balance is controlled in the
embryo (Pohl and Knochel,
2001; Sasai et al.,
2001
). Finally, we cannot rule out the possibility that the neural
crest induction we observed in the isolated ectoderm occurred secondarily to
FGF8-induced neural tissue (Hardcastle et
al., 2000
), secondary to the formation of a border between the
ectoderm and induced neural tissue. Further experiments will test if FGF8 is a
direct neural crest inducer or if it switches on a developmental program
eventually resulting into neural crest induction. However, by its neural crest
inducing activity in the animal cap assay, FGF8 stands as an excellent
candidate inducer when compared with previously proposed ones such as WNT8 or
WNT7b, which do not act alone in this assay
(Chang and Hemmati-Brivanlou,
1998
; LaBonne and
Bronner-Fraser, 1998
).
In the chick embryo, the ectoderm can induce neural crest from early neural
tissue and WNT6 signaling seems necessary and sufficient to mediate this
activity (Garcia-Castro et al.,
2002). In Xenopus, blocking Wnt signaling strongly
downregulates neural crest formation in vivo, whereas in animal cap assay, Wnt
signals require additional downregulation of BMPs to act on Slug
induction (LaBonne and Bronner-Fraser,
1998
). Combined with our data, this suggests that, in vivo, both
the ectoderm and the mesoderm participate in inducing the neural crest and
that they have different requirements to achieve neural crest induction. The
coordinate activity of both Wnt and FGF pathways may account for the robust
neural crest formation observed in normal embryos.
Neural crest induction and neural plate posteriorization
Neural crest induction is achieved experimentally by combining the same
classes of molecules as those required for neural plate patterning: BMP
antagonists, Wnts and FGFs. All three kinds of molecules have been shown to
downregulate Bmp4 expression or BMP4 activity, either in
Xenopus or in chick embryos (Lamb
et al., 1993; Lamb and
Harland, 1995
; Baker et al.,
1999
; Wilson et al.,
2000
). In addition, FGF and Wnts also posteriorize the neural
plate (Lamb and Harland, 1995
;
Domingos et al., 2001
;
Kiecker and Niehrs, 2001
). We
show here: (1) that Slug expression in vivo strongly correlates to
proper neural and mesoderm development (Figs
6,
7), but (2) that blocking
FGFR4a signaling strongly affects the AP neural pattern without preventing
robust Slug expression (Fig.
8). We conclude that although the AP position of the
Slug-positive domain might vary under these conditions, Slug
induction can occur independently of AP neural patterning. Thus, we postulate
that the loss of Slug expression observed after blocking Wnt or FGFR1
signaling (Mayor et al., 1997
;
LaBonne and Bronner-Fraser,
1998
) (this work) reflects a role of these pathways in neural
crest formation, on top of their role on neural patterning
(Villanueva et al., 2002
).
In conclusion, our study shows that, in the Xenopus embryo, (1) normal early development of the neural crest can occur in a context of abnormal AP neural patterning in vivo, (2) the paraxial mesoderm induces neural crest by an FGF-dependent pathway and (3) FGF8 is likely to mediate this activity. Our data still agree with the two-signal model of neural crest induction, and even suggest a multiple-signal model: in this model, the neural crest would arise in a location where a 'cocktail' of positive regulators is expressed. We propose that simultaneous moderate downregulation of BMP4 signaling, upregulation of ectodermal-derived factors (Wnt) and mesoderm-produced FGFs provides this suitable environment.
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ACKNOWLEDGMENTS |
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