1 Neuroscience Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST) Central 6, 1-1-1 Higashi, Tsukuba 305-8566,
Japan
2 Department of Anatomy, Institute of Basic Medical Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
3 Faculty of Human Environmental Science, Fukuoka Women's University,
Kasumigaoka Higashi-ku, Fukuoka, 813-8529, Japan
Author for correspondence (e-mail:
h-okamoto{at}aist.go.jp)
Accepted 27 June 2003
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SUMMARY |
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Key words: Fgf, Xcad3, Ets, Tcf/Lef, Sox, Neural patterning, Xenopus
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Introduction |
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Among the candidate signaling molecules for neural induction and
patterning, Fgf is of particular interest. Fgf can change the developmental
fate of Xenopus ectoderm cells in culture from epidermal to neural
(Kengaku and Okamoto, 1993),
and induce these cells to express position-specific neural markers along AP
axis in a dose-dependent manner, with higher doses eliciting more posterior
neural marker genes (Kengaku and Okamoto,
1995
). Furthermore, loss-of-function experiments have shown that
Fgf signaling is required for both anterior
(Hongo et al., 1999
) and
posterior (Holowacz and Sokol,
1999
; Pownall et al.,
1996
; Ribisi et al.,
2000
) neural development, as judged by expression of positional
marker genes. However, a recent report indicates that dose-dependent Wnt
signaling is both necessary and sufficient for AP neural patterning
(Kiecker and Niehrs, 2001
),
although another report indicates that Wnt signaling posteriorizes neural
tissue through elevating the level of Fgf signaling
(Domingos et al., 2001
). Thus,
the precise role of each of these signaling pathways for the establishment of
AP neural pattern and how they are integrated with signaling in the preceding
neural induction step is still not clear.
To address these questions, we may need not only loss- or gain-of-function
experiments, but also an approach to directly identify cis-acting
sequence elements in positional marker genes, which respond to the neural
patterning signals. In this study, we investigated the nature of the Fgf
response element (FRE) in a posterior marker gene, Xcad3
(Northrop and Kimelman, 1994).
Xcad3, a Xenopus caudal homologue encoding a homeodomain
transcription factor, lies downstream of Fgf signaling and functions as an
upstream activator of several Hox genes that regulate posterior embryonic
development (Isaacs et al.,
1998
; Northrop and Kimelman,
1994
; Pownall et al.,
1996
), as has been implicated for some members of the
caudal gene family of other vertebrate species (Cdx genes in mammals
and chicken) (Deschamps et al.,
1999
).
We have isolated an Xcad3 genomic clone containing regulatory
elements that drive Xcad3 expression in the posterior neural tube in
response to Fgf signaling. We provide evidence that FREs of Xcad3 are
widely dispersed in the first intron and we demonstrate that these multiple
FREs comprise Ets-binding and Tcf/Lef-binding motifs (EBMs and TLBMs) that lie
in juxtaposition. Functional analysis shows that Ets family transcription
factors are indeed involved in the Fgf response of Xcad3 activation.
This indicates that Xcad3 is directly targeted by Fgf signaling, as
Ets proteins are nuclear effectors of Fgf/Ras/mitogen-activated kinase (Mapk)
pathway (Wasylyk et al.,
1998). By contrast, XTcf3, a nuclear effector of
Wnt/ß-catenin pathway (Molenaar et
al., 1996
), functions as a repressor of Xcad3
(Nusse, 1999
). Furthermore,
Sox2, a Sry-related transcription factor that shares the cognate DNA-binding
motif with Tcf/Lef family members (Kamachi
et al., 2000
) is shown to cooperate with Ets proteins, possibly by
competing with XTcf3 for TLBMs in the composite FREs. Direct interaction of
these proteins with some EBMs and TLBMs were demonstrated in gel mobility
shift assays. Sox2 is de-repressed in the neurogenic region by Bmp antagonists
during the neural induction step (Mizuseki
et al., 1998a
). Our results thus indicate that signaling pathways
of Bmp, Fgf and Wnt are integrated on the FREs to regulate the expression of
Xcad3 in the posterior neural tube during neural patterning.
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Materials and methods |
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Plasmid construction
The 5' flanking and first intron of Xcad3 were subcloned
into a luciferase reporter plasmid pGL3-Basic Vector (Promega), separately or
in combination. The 5' flanking sequence was inserted upstream of the
luciferase coding sequence, although the first intron sequence was inserted
downstream as follows. An EcoRI fragment of a genomic clone
(1741 to +389) was subcloned into the EcoRI site of pBluscript
II SK-(Stratagene); sequence 1741 to +174 (one nucleotide upstream of
the translation start site) was amplified by PCR and cloned into blunted
NcoI site of pGL3-Basic Vector. SacI (in the phage
arm)-EcoRI fragment (7000 to 1741) was then cloned into
the SacI-EcoRI site of pGL3-Basic that contained 5'
fragment 1741 to +175. The first intron sequence was amplified by PCR
and cloned into the BamHI site of pGL3-Basic. To generate chimeric
constructs with SV40 sequences, pGL3-Promoter Vector that contains SV40
promoter sequence upstream of luciferase sequence, or pGL3-Enhancer Vector
that contains SV40 enhancer sequence downstream of it was used instead of
pGL3-Basic Vector. To generate constructs containing 5' flanking and
intronic sequence deletion, respective PCR fragments were cloned into
pGL3-Basic as above. For a GFP reporter plasmid, the luciferase-coding region
was removed from pGL3 vector sequence by NcoI-XbaI digestion
and replaced by a NcoI-XbaI fragment from pEGFP-N3
(Clontech), containing the EGFP-coding sequence. Mutations of EBMs and TLBMs
were introduced by Ex-Site mutagenesis kit (Stratagene). The entire or deleted
coding sequences of XEts1 (Meyer et al.,
1997), XER81 (Chen et al.,
1999
; Munchberg and
Steinbeisser, 1999
), human ELK1
(Chen et al., 1999
), XTcf3
(Molenaar et al., 1996
), XLef1
(Molenaar et al., 1998
) and
ß-catenin (Molenaar et al.,
1996
) were amplified by PCR and subcloned into pSP64T. Vp16-XTcf3
was as published (Kim et al.,
2000
). Sox2-EnR was made by in-frame C-terminal fusion of the
Drosophila engrailed repressor region
(Conlon et al., 1996
) to Sox2.
Sox2, Sox2 BD() and SoxD BD() were kindly provided by Y. Sasai
(Kishi et al., 2000
;
Mizuseki et al., 1998a
;
Mizuseki et al., 1998b
).
Microinjection and transgenesis
Microinjection of reporter and internal standard plasmids with or without
synthetic mRNA of various transcription factors was performed as previously
described (Hongo et al.,
1999). Injected plasmids were adjusted to the same on a molar base
and they were injected at 3x1018 moles in 1.6
nl/blastomere of eight-cell stage Xenopus embryos. Transgenic embryos
were generated as described (Kroll and
Amaya, 1996
).
Microculture and quantitative RT-PCR
Injected or uninjected Xenopus gastrula embryos were used. Methods
for culturing ectoderm cells were essentially as previously described
(Kengaku and Okamoto, 1993).
RNA was extracted from 20 cultures for each experimental point and subjected
to quantitative RT-PCR as previously described
(Hongo et al., 1999
;
Kengaku and Okamoto,
1995
).
Luciferase assay
The luciferase assay was performed using the Dual-Luciferase Reporter Assay
System (Promega), in which firefly luciferase in pGL3 was used for the
reporter gene assay, whereas Renilla luciferase in the internal standard
plasmid pRL-CMV was used for normalization. After a group of injected embryos
or cultures were incubated up to the desired stage, they were homogenized in
Passive Lysis buffer (Promega). For the embryonic cell culture assay, half the
culture medium in each culture well was replaced by 2xPassive Lysis
buffer and 20 cultures were collected and homogenized for each experimental
point. The lysate was centrifuged at 18,000 g for 1 minute at
4°C. The clear supernatant was assayed with firefly luciferase substrate
and Renilla luciferase substrate separately to avoid possible interference.
Each luciferase activity was measured three times, and the mean value was
used. All of the injection experiments were carried out at least three times
and gave reproducible results. One representative experiment was shown for
each figure.
Gel mobility shift assay
V5-epitope-tagged XTcf3, Sox2 and XEts1 proteins were made by in vitro
translation with a rabbit reticulocyte lysate (Promega), and gel mobility
shift assays were performed as described
(Huang et al., 1995). DNA
fragments used as probes were 3' end-labeled with digoxygenin-11-ddUTP
according to the manufacturer's recommendations (Roche Diagnostics; DIG Gel
Shift Kit). Supershifts were generated by adding 1 µl of monoclonal
antibody directed against V5 epitope (Invitrogen). DNA-protein complexes were
separated by electrophoresis through 3.5% polyacrylamide gel containing
0.5xTBE and 2.5% glycerol. Gels were further processed according to the
manufacturer's recommendation (Roche Diagnostics; DIG Gel Shift Kit).
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Results |
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To locate regulatory elements in the 5' flanking sequence, deletion analysis was carried out using the luciferase assay system. We found that a truncation down to 185 (relative to the transcription initiation site) did not largely affect the spatial specificity and the extent of luciferase expression (details not shown, but see Fig. 1D for an example).
We then tested the ability of the 5' immediate upstream sequence and
intron1 to regulate spatiotemporal expression pattern of Xcad3. For
this we replaced luciferase with green fluorescent protein (GFP) as a
reporter, and generated transgenic embryos carrying the GFP construct
(185/GFP/intron1 as depicted in Fig.
1C). GFP expression was first detected at stage 11 in the marginal
zone, the prospective mesoderm region. During neurula and tail bud stages, GFP
was expressed primarily in the posterior of the developing neural tube
(Fig. 1C). This spatiotemporal
expression pattern of GFP was consistent with that of the endogenous
Xcad3 as determined by in situ hybridization, except for a stripe of
GFP expression above the eye (Northrop and
Kimelman, 1994). The ectopic expression may reflect a high level
of Fgf signals at the midbrain/hindbrain boundary
(Christen and Slack, 1997
;
Tannahill et al., 1992
). It is
likely that additional elements would be necessary to suppress GFP expression
in this region.
We further asked whether the reporter activity induced by the 5'
upstream elements and intron1 of Xcad3 was dependent on Fgf
signaling. For this, mRNA encoding a dominant-negative Xenopus Fgf
receptor type 4a (XFgfR4a) (Hongo
et al., 1999
) were co-injected into the PNT site at the eight-cell
stage. We found that overexpression of
XFgfR4a caused a strong
suppression of luciferase activity induced by 7000/LUC/intron1 or
185/LUC/intron1 construct (Fig.
1D). Although the extent of suppression varied somewhat in several
series of experiments, it averaged more than 80%.
Collectively, our results indicate that regulatory elements present in 5' upstream and intron1 sequences are sufficient to drive Xcad3 expression in the posterior neural tube and they include FREs, which are indispensable for Xcad3 expression.
High-dose-dependent FREs are present in intron1
Fgf can induce Xenopus gastrula ectoderm cells in culture to
express position-specific neural marker genes along the anteroposterior axis
in a dose-dependent manner; with lower doses eliciting more anterior marker
genes such as XeNK2 or En2 and higher doses more posterior
marker genes such as XlHbox1 (Hoxc6) or XlHbox6
(Hoxb9) (Kengaku and Okamoto,
1995). Indeed endogenous Xcad3 was activated in
considerably higher Fgf dose range than En2, when examined in the
embryonic cell culture assay (Fig.
2A,B).
|
Where, then, are the FREs of Xcad3, in the 5' upstream
sequence or in intron1? To answer this question, we prepared chimeric
constructs in which the 5' sequence and intron1 were replaced with SV40
promoter and enhancer sequences, respectively, as depicted in
Fig. 2E,F. These SV40 elements
were used to enhance reporter activities, as 5' sequence or intron1
alone could not induce sufficient reporter activities for quantitative
analysis, as shown in Fig. 1B. We found that a chimeric construct containing intron1 (SV40 promoter/Luc/
intron1) exhibited a dose-dependent response to Fgf
(Fig. 2E, ), but any
other constructs examined including 7000/LUC/SV40 enhancer, SV40
promoter/LUC/SV40 enhancer (Fig.
2F,
,
), or 185/LUC/SV40 enhancer (not shown)
did not show such dose-dependence on Fgf. However, the Fgf dose-response
profile of the active construct (SV40 promoter/LUC/intron1) did not coincide
well with that of the intact construct
(Fig. 2E;
,
7000/LUC/intron1) in this and other series of experiments. It is likely
that the FREs of Xcad3 are primarily located within the intron1 but
their interaction with 5' upstream elements through specific
transcription factors is required to mediate the proper dose-dependent
response of Xcad3 to Fgf.
FREs comprise Ets-binding and Tcf/Lef-binding motifs
To identify the FREs in the intron1, deletion analysis was carried out
using the embryonic cell culture assay. A control construct containing the
full-length intron1 exhibited at least 20-fold activation of the reporter
activity by the addition of bFgf as exemplified in
Fig. 3A(i). Several series of
experiments showed that the 5'-most 1200 bp and 3'-most 600 bp
sequences were dispensable (not shown). Further serial deletion of 1100 bp
from the 5' side resulted in progressive loss of Fgf responsiveness
[Fig. 3A(ii-iv)]. Similar
gradual change was reproducibly observed by deleting this region
consecutively, indicating that multiple FREs are distributed within it (domain
1 in Fig. 3A). Notably, the
sequence 3' to the domain 1 still retained Fgf responsiveness that
amounted to about 5-fold activation as exemplified in
Fig. 3A(iv). Indeed, several
series of deletion experiments from the 3' side
[Fig. 3A(v), (vi), for example]
showed that there was another domain that contained FREs (domain 2 in
Fig. 3A). Progressive loss of
Fgf responsiveness by serial deletions within the domain 2 was exemplified in
Fig. 3B (see below for more
detail). These results indicate that FREs are not localized in a narrow region
within intron 1 but rather widely dispersed throughout it, conferring the full
Fgf responsiveness to intron 1 in a coordinated manner.
|
|
Ets transcription factors mediate the response of Xcad3 to
Fgf
We next explored the transcription factors that interact with the FREs of
Xcad3 to mediate the Fgf response. Obvious candidates are Ets family
and Tcf/Lef family proteins. Notably, transcripts of some of the Ets genes
including Ets1, Ets2 (Meyer et
al., 1997) and ER81
(Chen et al., 1999
;
Munchberg and Steinbeisser,
1999
) as well as that of XTcf3
(Molenaar et al., 1996
) are
expressed in early Xenopus embryos. These transcription factors would
act cooperatively through binding to respective motifs in the composite
FREs.
We first assessed the functional role of Ets proteins, as they are
potential nuclear effectors of the Fgf/Ras/Mapk signal transducing pathway
(Wasylyk et al., 1998). A mRNA
encoding a dominant-negative form of Xenopus Ets1 (dnXEts1), ER81
(dnXER81) or human Elk1 (dnElk1) was co-injected with a reporter construct.
These dominant-negative constructs lack the N-terminal (dnXEts1, dnXER81) or
C-terminal (dnElk1) regions of the respective wild-type proteins, which
include the activation domain (Fig.
5, bottom right). These mutants thus mainly comprise the
DNA-binding ets domain, thereby potentially competing with endogenous Ets
proteins for the EBMs (Wasylyk et al.,
1994
). Overexpression of dnElk1 and dnEts1 to a lesser extent
caused a suppression of the response of the reporter construct to Fgf, while
that of dnXER81 did not suppress the response
(Fig. 5A). Increasing the
amount of co-injected mRNA suppressed more effectively the Fgf response for
dnEts1, but not for dnXER81 (not shown).
|
Collectively, our results indicate that some of the Ets transcription factors are involved as activators in the response of Xcad3 to Fgf. It is highly likely that these Ets proteins bind to EBMs in composite FREs in intron 1, and that Fgf signaling enhances their ability to activate transcription by phosphorylating them through Mapk.
XTcf3 represses Fgf response of Xcad3
We next assessed the functional role of XTcf3, a nuclear target of the
Wnt/ß-catenin pathway, using a dominant-negative XTcf3 (dnXTcf3)
construct. dnXTcf3 lacks the N-terminal region that is required for binding of
ß-catenin, a co-activator of XTcf3, but retains the ability to bind its
cognate DNA motif, thereby abrogating transcriptional activation by the
endogenous ß-catenin-XTcf3 complex
(Molenaar et al., 1996). In
embryonic cell culture assays, overexpression of dnXTcf3 resulted in a
suppression of the response to Fgf of a reporter construct in a dose-dependent
manner (Fig. 6A). Surprisingly,
however, overexpression of wild-type XTcf3 also caused a profound suppression
of the Fgf response instead of an activation
(Fig. 6B). This was unexpected,
because Wnt signaling was suggested to be involved in activation of posterior
neural genes (Kiecker and Niehrs,
2001
; McGrew et al.,
1995
) and we had anticipated that XTcf3 functioned as an activator
of Xcad3 by binding ß-catenin. It should be noted, however, that
XTcf3 has also been shown to function as a transcriptional repressor instead
of an activator by binding co-repressors such as Groucho
(Roose et al., 1998
) or XCtBP
(Brannon et al., 1999
) in place
of ß-catenin. Overexpression of XLef1, another Tcf/Lef family member that
lacks the repressing function of XTcf3, but retains the activating function by
binding ß-catenin (Brannon et al.,
1999
; Molenaar et al.,
1998
) had no effect on the Fgf response
(Fig. 6C). It is possible that
the endogenous pool of ß-catenin is considerably smaller compared with
those of XCtBP, Groucho or other co-repressors in Xenopus ectoderm
cells, and XTcf3 (VP16-dXTcf3) may act primarily as a repressor in these
cells. Indeed, overexpression of ß-catenin itself or a mutant construct
in which the VP16 activation domain was fused to truncated XTcf3 counteracted
the repressing action of endogenous XTcf3
(Kim et al., 2000
): in
embryonic cell culture assays, they induced robust luciferase activity
(Fig. 6C,D). By contrast,
overexpression of Wnt8 protein, which would facilitate endogenous
ß-catenin to complex with XTcf3
(Domingos et al., 2001
;
Molenaar et al., 1996
) did not
affect the Fgf response (not shown). Collectively, our results indicate that
XTcf3 functions primarily as a repressor of Xcad3. This raises the
possibility that Ets proteins overcome this repression by cooperating with
other transcription factors that bind to TLBMs in place of XTcf3.
|
Possible involvement of Sox2 in the Fgf response of Xcad3 was
examined in the embryonic cell culture assay using a dominant-negative version
of Sox2 in which the engrailed repressor domain was fused with Sox2 (Sox2-EnR)
(Conlon et al., 1996).
Overexpression of Sox2-EnR resulted in a suppression of the Fgf response of a
reporter construct containing the full-length of intron 1
(Fig. 7A). By contrast,
overexpression of wild-type Sox2 caused an activation of the response
(Fig. 7B). Similar suppression
by Sox2-EnR and activation by wild-type Sox2 were observed with a reporter
construct containing the well characterized domain 2* fragment
(Fig. 4A) and wild-type Sox2
counteracted the suppression caused by Sox2-EnR
(Fig. 7C).
|
Collectively, our functional analysis shows that endogenous Sox2 is
required for the Fgf response of Xcad3. The most plausible partner
factors of Sox2 are Ets proteins, which are also known to require interaction
with partner factors to direct signals to specific target genes
(Wasylyk et al., 1998). It is
highly likely that Sox2 competes with XTcf3 for TLBMs in the composite FREs
and cooperate with Ets proteins that bind to adjacent EBMs. To test this idea,
an intronic fragment containing one EBM and two TLBMs (overlined in
Fig. 4A; probe T2/E4/T3 in
Fig. 8A) was examined for its
ability to interact with the XTcf3, Sox2 and XEts1 proteins in the gel
mobility shift assay. V5-tagged XTcf3 alone shifted the end-labeled probe,
yielding three bands (bands 1, 2 and 3 in
Fig. 8A, lane 2). All the three
bands were supershifted by antibody against V5-epitope (lane 3) and competed
by a 125-fold molar excess of the unlabelled probe (lane 4). When the two
TLBMs in the probe were mutated (probe E4), no bands emerged (lane 5), but
mutation in either TLBM3 (probe T2/E4) or TLBM2 (probe E4/T3) alone gave rise
to two bands that co-migrated with bands 1 and 3 (lane 6 and 8), respectively.
All these bands were supershifted by the anti-V5 antibody (lane 7 and 9).
These results indicated that band 3 was derived from binding of XTcf3 to
either TLBM2 or TLBM3, while band 2 was derived from binding of XTcf3 to both
motifs. Band 1 was probably formed by binding of multimerized XTcf3 to these
motifs. Binding of XTcf3 to TLBMs appeared to be competed by the presence of
an increasing amount of Sox2 in the binding reaction (lane 10 to 12).
V5-tagged Sox2 alone gave rise to a single band (lane 13), that was abolished
by the anti-V5 antibody (lane 14), and did not emerge with the probe E4 (lane
15). We could not detect binding of Ets proteins used in this study with the
wild-type probe, but we found that XEts1 was capable of binding to the
TLBM3-mutated probe (band 5; lane 2 in Fig.
8B) to which Sox2 also bound (band 6; lane 1). XEts1 and Sox2
proteins appeared to form a ternary complex with this mutated probe (band 4;
lane 3), which was supershifted by the anti-V5 antibody (lane 4). These
observations strongly support the idea of the direct regulatory function of
XTcf3, Sox2 and XEts1 proteins in the Fgf response of Xcad3.
|
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Discussion |
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TLBMs could serve as the binding sites for Tcf/Lef family transcription
factors that are nuclear effectors of the Wnt/ß-catenin pathway
(Molenaar et al., 1996). We
had anticipated that XTcf3 functioned as a co-activator of Ets proteins, as
Wnt signaling was suggested to be involved in activation of posterior neural
genes (Kiecker and Niehrs,
2001
; McGrew et al.,
1995
). Surprisingly, however, functional analysis reveals that
XTcf3 acts as a repressor of Xcad3. Our data suggest that the
endogenous pool of ß-catenin in ectoderm cells is considerably smaller
compared with that of XTcf3 co-repressors such as XCtBP and Groucho. This in
turn implies that Wnt signaling could activate Xcad3 expression in
embryonic cells, when they were provided with larger pool of ß-catenin.
Marginal zone cells of the early gastrula embryo, where Xcad3 is
initially expressed, are among such candidate cells, as a relatively large
amount of ß-catenin is translocated into the nucleus in these cells
(Schohl and Fagotto, 2002
).
Recently, an mutant function of Tcf3 as a repressor is revealed in the
zebrafish headless mutant that carries a mutation in Tcf3
(Kim et al., 2000
). In this
mutant, expression of midbrain-hindbrain boundary genes such as En2 and Pax2
are de-repressed in more anterior neural region, leading to severe head
defects. It would be interesting to know whether similar anterior expansion is
seen in Cdx gene expression in this mutant.
Sox2 is de-repressed by Bmp antagonists in the neurogenic region of
ectoderm during neural induction (Mizuseki
et al., 1998a). We show that Sox2 which shares a cognate DNA
bindings motif with Tcf/Lef family members, is required as a co-activator for
the Fgf response of Xcad3. Sox2 is likely to compete with XTcf3 for
TLBMs in the composite FREs to cooperate with Ets proteins that bind to
adjacent EBMs. Physical analysis supports this idea. Both Sox and Ets family
transcription factors interact with specific partner factors to direct signals
to target genes (Kamachi et al.,
2000
; Wasylyk et al.,
1998
), but direct partnership between them has not been reported.
Collectively, our results indicate that signaling pathways of Fgf, Bmp and Wnt
are integrated on the FREs to regulate the expression of Xcad3 in the
posterior neural tube through positively acting Ets and Sox proteins and
negatively acting Tcf protein (Fig.
9B).
Fgf as a morphogen
Ets (Chen et al., 1999) and
Sox (Mizuseki et al., 1998a
;
Mizuseki et al., 1998b
)
proteins are ubiquitously expressed in the neurogenic region during gastrula
stages when neural patterning is initiated. Posteriorly biased Xcad3
expression could, therefore, be primarily due to similarly biased expression
of Fgf proteins. Indeed, several Fgf genes are activated in the posterior
ectoderm and mesoderm during late gastrula and early neural stages
(Christen and Slack, 1997
;
Isaacs et al., 1992
;
Tannahill et al., 1992
). In
this and previous studies (Kengaku and
Okamoto, 1995
), we have shown that Fgf can induce gastrula
ectoderm cells to express position-specific neural marker genes along the AP
axis in a dose-dependent manner, with higher doses eliciting more posterior
neural genes. Interestingly, functional analysis indicated that Sox-mediated
signaling (Kishi et al., 2000
;
Mizuseki et al., 1998b
) and
Fgf signaling (Hongo et al.,
1999
) were also required for the expression of anterior neural
genes. These studies raise the possibility that regulatory mechanisms
underlying the transcriptional activation of anterior neural genes possess
common features with that for Xcad3 activation except for higher
sensitivity to Fgf. Differential sensitivity of position-specific neural genes
to Fgf would then imply that Fgf acts as a morphogen during neural patterning
(Kengaku and Okamoto, 1995
).
To explore this issue, we need to identify cis-elements in anterior
neural genes, and the present embryonic cell culture assay system will be
useful for this purpose.
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ACKNOWLEDGMENTS |
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Footnotes |
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