From the Department of Cell and Developmental Biology, Graduate
School of Biostudies, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan
The fibroblast growth factor (FGF)/MAPK pathway
plays an important role in early Xenopus developmental
processes, including mesoderm patterning. The activation of the MAPK
pathway leads to induction of Xenopus Brachyury (Xbra),
which regulates the transcription of downstream mesoderm-specific genes
in mesoderm patterning. However, the link between the FGF/MAPK pathway
and the induction of Xbra has not been fully understood. Here we
present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early
embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the
formation of the trunk and tail structures. Overexpression of Ets-2
resulted in the induction of Xbra, and expression of the
dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in
vitro. These results taken together indicate that
Xenopus Ets-2 plays an essential role in mesoderm
patterning, lying between the FGF/MAPK pathway and the Xbra transcription.
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INTRODUCTION |
During Xenopus early embryonic development, mesoderm
arises from ectoderm by induction that requires signals from the
vegetal hemisphere of the embryo (1, 2). The
FGF1/MAP kinase (MAPK)
pathway has been shown to be involved in early mesodermal patterning
(3-8). One of the genes that are thought to be regulated directly by
FGF via the MAPK signal transduction pathway is Xenopus
Brachyury (Xbra). Brachyury is an important regulatory gene in early
vertebrate development (9-14). Loss of Brachyury function in mouse,
zebrafish, and Xenopus embryos causes defects in posterior
mesoderm and notochord differentiation (9, 15-17). However, the link
between the FGF/MAPK pathway and the induction of Xbra expression has
not been fully defined.
The ETS family of transcription factors, comprising more than 30 different members, has been found to play a crucial role in controlling
transcription of a variety of genes involved in important cellular
processes, such as proliferation and differentiation (19, 20). They
share a unique DNA binding domain, the ETS domain, which interacts
specifically with GGA(A/T)-based recognition sites (18). As targets of
the Ras-MAPK signaling pathway, Ets transcription factors are
phosphorylated by MAPK and function as critical nuclear integrators of
ubiquitous signaling cascades. The ETS family is divided into
subfamilies by sequence similarity based on the ETS domain or
additional sequence motifs. Ets-2 is a member of the ETS subfamily,
which consists of three members: Ets-1, Ets-2, and
Drosophila Pointed (21, 22). Studies with mammalian cultured
cells have shown that Ets-2 is activated by MAPK-dependent
phosphorylation of threonine 72 in an N-terminal regulatory domain (the
Pointed domain) (21, 23). Xenopus Ets-2 is maternally
expressed in both the animal pole and the intermediate zone (24, 25).
On the basis of these results, we hypothesized that Ets-2 relays the
FGF/MAPK signaling and induces mesoderm by inducing Xbra gene
transcription. In this study, we have presented several lines of
evidence indicating that Ets-2 plays an essential role in mesodermal
patterning, lying between the FGF/MAPK pathway and the Xbra transcription.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Xenopus Ets-2 cDNA was
obtained by screening a
ZAP II cDNA library made from
stage 10.5 embryos. To construct CS2-En-N, the 888-bp fragment coding
for amino acids 1-294 of Drosophila engrailed protein was
inserted into the StuI site of CS2+. To
generate Ets
N En-R (the repressor domain of Drosophila
Engrailed), the DNA binding domain of Ets-2 (amino acid residues
310-468) was subcloned into CS2-En-N. In vitro synthesis of
capped mRNA was performed using mMESSAGE mMACHINE kit (Ambion)
according to the manufacturer's instructions.
Embryo Manipulation and Animal Cap Assay--
Embryos were
in vitro fertilized, dejellied, and cultured in 0.1×
modified Barth's saline (1.5 mM HEPES, pH 7.4, 8.8 mM NaCl, 0.1 mM KCl, 0.24 mM
NaHCO3, 0.082 mM MgSO4, 0.03 mM Ca(NO3)2, and 0.041 mM CaCl2). Embryos were staged according to
Nieuwkoop and Faber (26). Embryos at the four-cell stage were injected with mRNA as described in the text and figure legends. Animal caps
were dissected from the injected embryos at stages 8-8.5 and cultured
in 1× Steinberg solution containing 0.1% bovine serum albumin to
various stages for further analysis as described in the figure legends.
RT-PCR experiments were performed according to standard protocol. The
primer pairs used here for RT-PCR have been described elsewhere (27,
28). For morpholino oligonucleotide injections, an Ets-2 antisense
oligonucleotide with the sequence 5'-AGCTGAGGGAGGGTATGTCCTTCC-3' was obtained from Gene Tools, LLC. Oligonucleotides were resuspended in sterile, filtered water and injected into four-cell stage embryos with the indicated amounts (29).
Luciferase Assay--
Embryos were injected with 200 pg of
Xbra-pOLUC (provided by Dr. K. W. Y. Cho) and 100 pg of
pCMV-
-galactosidase together with 1 ng of Ets-2 mRNA into animal
poles at the two-cell stage. Dissected animal caps were assayed for
luciferase and
-galactosidase activities at stage 11 (13, 14). The
ratio of luciferase to
-galactosidase activity provides a normalized
measure of luciferase expression. In all cases, fold activation was
calculated using the results from only Xbra pOLUC- and
CMV-
-galactosidase-injected embryos as background. Each experiment
was performed three times to ensure reproducibility of results.
Gel Mobility Shift Assay--
To obtain the recombinant
GST-Ets-2 protein, the entire coding region of Ets-2 was subcloned into
pGEX-6P. Production of GST-Ets-2 and GST protein was performed as
described (30). A DNA sequence for oligonucleotides of the Ets binding
site was 5'-CAGGTGTCAGTTCTTACTGGATGTAAGTTTATTGAAGGCA-3'. A gel mobility
shift assay was carried out as described (31). For the
competition assay, the Ets binding site was mutated using a
site-directed mutagenesis kit (QuikChangeTM; Stratagene.
The Ets binding site was mutated using forward primer 5'-CAGGTGTCAGTTCTTACTGGCCGTAAGTTTATTGAAGGC-3' and reverse primer 5'-TGCCTTCAATAAAC TTACGGCCAGTAAGAACTGACACCTG-3'.
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RESULTS |
Ets-2 Is Required for Mesoderm Patterning in FGF Signaling--
We
first examined the effect of overexpression of wild type
Xenopus Ets-2 mRNA on early embryonic development. Ets-2
mRNA was injected into the dorsal or ventral marginal zones of
four-cell stage Xenopus embryos. When compared with normal
uninjected control embryos at tadpole stage 35, embryos with dorsal
injections of Ets-2 showed anterior truncation and shortened body axis.
The embryos injected with relatively high doses of Ets-2 mRNA (1-2 ng) lacked eyes and cement glands (Fig.
1A). About 70% of embryos injected with 2 ng of Ets-2 mRNA showed the severe anterior defects (n = 34). A typical image is shown in Fig.
1A. About 40% of embryos injected with 1 ng of Ets-2
mRNA showed similar severe defects (n = 45). A
typical phenotype is shown (Fig. 1A, Ets-2 1 ng
DMZ (dorsal marginal zone)). Moreover, injections with high doses of mRNA sometimes resulted in embryos with tail-like protrusions (data not shown). The ventral injections had little or no effect on the
development of the embryos (Fig. 1A, Ets-2 1 ng
VMZ (ventral marginal zone)). To investigate these embryos in more
detail, we examined the wide range of molecular markers in Ets-2
mRNA-injected embryos. The results of the RT-PCR analysis carried
out on stages 26 and 37 whole embryos injected with Ets-2 mRNA of 1 ng are shown (Fig. 1B). Sibling embryos were used as
control. Overexpression of Ets-2 mRNA resulted in remarkable
reduction of expression of a forebrain marker Otx2 and a cement gland
marker XAG. Moreover, expression of a pan-neural marker neural
cell adhesion molecule was also reduced by injection of Ets-2 mRNA.
Thus, Ets-2 overexpression suppressed expression of anterior markers.
Moreover, Ets-2 overexpression increased expression of the posterior
markers such as Xbra and Xcad3 (Fig. 1B). A transverse
section through the anterior of the posteriorized embryo showed no
development of brain ventricles (data not shown). These data suggest
that overexpression of Ets-2 causes anterior truncation and induction
of posterior mesoderm. Next, to examine whether Ets-2 functions
downstream of FGF signaling, we tested whether expression of Ets-2
could rescue the defects caused by inhibition of FGF signal in
Xenopus embryos. Dominant-negative FGF receptor (XFD)
mRNA with or without Ets-2 mRNA was injected into dorsal
marginal zones of the four-cell stage. Overexpression of XFD caused a
severe posterior defect (18 out of 18, Fig.
2). This defect is thought to result from
inhibition of both gastrulation movement and posterior mesoderm
formation (32). Co-injection of Ets-2 rescued this morphological
defect, and the injected embryos developed almost normally (22 out of
47) (Fig. 2). This rescue of the XFD-induced phenotype by Ets-2
suggests that Ets-2 functions in mesoderm patterning downstream of
FGFs.

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Fig. 1.
Overexpression of Ets-2
induces posteriorized embryos. A, tadpole stage (stage
35) embryos and sibling embryos that have been injected with Ets-2
mRNA into dorsal marginal zones (DMZ) or ventral
marginal zones (VMZ) at four-cell stage at indicated
doses. B, expression of marker genes in whole embryos that
were injected with 1 ng of Ets-2 mRNA into the dorsal marginal
zones. Injected embryos were cultured until sibling embryos reached
stage 26 or 37. Expression of indicated marker genes was analyzed by
RT-PCR. EF1- served as a loading control. RNA from whole embryo
(indicated as embryo) provides a positive control. No signal was
observed in the absence of reverse transcription ( RT).
NCAM, neural cell adhesion molecule.
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Fig. 2.
Ets-2 rescues the defects by XFD.
Embryos were injected into the dorsal marginal zones at four-cell stage
and cultured until stage 35. 0.5 ng of XFD mRNA was injected with
or without 1.5 ng of Ets-2 mRNA.
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To confirm the effects of Ets-2, we analyzed various markers. Animal
caps were dissected from Ets-2 mRNA-injected embryos and cultured
to stage 10.5 or 22. In stage 10.5 animal cap explants, Ets-2 induced
the expression of the pan-mesodermal marker Xbra in a
dose-dependent manner (Fig.
3A). The dorsal mesoderm
marker Goosecoid and the ventral mesoderm marker Xwnt8 were not induced by Ets-2 overexpression (data not shown). In stage 22 animal caps, Ets-2 strongly induced the expression of the posterior markers Xcad3
and Xhox3 (Fig. 3B). The expression of Xlhbox6
(HoxB9), which is expressed in lateral mesoderm and spinal chord, was
little induced by the injection of Ets-2 mRNA.

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Fig. 3.
Ets-2 induces expressions of mesodermal
markers in animal cap explants. As shown in A, Ets-2
mRNA was injected into animal poles of two-cell stage embryos at
the indicated doses. Animal caps were dissected at blastula stage and
cultured until sibling embryos reached stage 11. Expression of
indicated marker genes was analyzed by RT-PCR. RT, absence
of reverse transcription. As shown in B, Ets-2 mRNA was
injected as in panel A. Animal caps were dissected at
blastula stage and cultured until sibling embryos reached stage 26. Indicated markers were analyzed by RT-PCR.
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Inhibition of Ets-2 Causes Posterior Mesoderm Defects in
Embryos--
To examine whether Ets-2 is necessary for mesoderm
patterning, we generated a dominant-negative form of Ets-2. The
schematic diagram of the Ets-2-based construct used here was shown
(Fig. 4A). Ets-2 possesses a
highly conserved N-terminal regulatory domain (the Pointed domain) and
a C-terminal motif that comprises the DNA binding domain (the ETS
domain). Ets
N En-R was made consisting of the C-terminal region of
Ets-2, which contains the DNA binding domain but lacks the activation
function, fused to the transcription En-R. We examined the
effect of overexpression of Ets
N En-R (Fig. 4B). Ets
N
En-R mRNA was injected into dorsal marginal zones of four-cell
stage embryos, and these embryos were cultured until stage 34. A
typical image is shown in Fig. 4B. Ets
N En-R-injected embryos showed the severe posterior defect (47 out of 52). Body axis
truncation and dorsal bending of embryos were also observed. These
features of the injected embryos were similar to those of the
XFD-injected embryos (Fig. 2). Embryos injected ventrally developed
almost normally (Fig. 4B).

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Fig. 4.
Inhibition of Ets-2 causes defects in
mesodermal patterning. A, a schematic diagram of Ets-2
construct fused with an En-R. B, tadpole stage (stage 35)
embryos that have been injected with 1 ng of mRNA encoding Ets N
En-R into dorsal or ventral marginal zones at the four-cell stage.
DMZ, dorsal marginal zone; VMZ, ventral marginal
zone. C, an inhibitory effect of Ets N En-R on FGF- or
MAPKK SESE-induced Xbra expression in isolated animal caps. Animal caps
were dissected at blastula stage from embryos that had been injected
with Ets N En-R mRNA (1 ng) together with MAPKK SESE mRNA
(0.1 ng) at the two-cell stage and were cultured until sibling embryos
reached stage 11. On FGF treatment, animal caps were cultured in the
medium including 50 ng/ml bFGF. Expression of Xbra was analyzed by
RT-PCR. D, wild type Ets-2 rescued the reduction of Xbra
expression caused by Ets N En-R. Ets N En-R mRNA (50 pg)
was injected together with wild type Ets-2 mRNA (1 ng) into
marginal zones of the two-cell embryo. Injected embryos were cultured
until sibling embryos reached stage 11. Expression of Xbra was
analyzed by RT-PCR with injected whole embryos.
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We then examined whether Ets
N En-R is able to block FGF- or
constitutively active MAPKK (MAPKK SESE)-induced expression of Xbra
(Fig. 4C). MAPKK (MEK) is a specific activator of classical MAPK (ERK MAPK) (33-36). Although uninjected animal caps with FGF treatment expressed a high level of Xbra, Ets
N En-R-injected animal
caps with FGF treatment did not induce Xbra expression. Although
injection of active MAPKK alone induced Xbra expression strongly,
co-injection of Ets
N En-R resulted in great reduction of Xbra
expression. These results suggest that the FGF/MAPK pathway induces
Xbra expression via Ets-2.
To test the specificity of the Ets
N En-R construct, Ets
N En-R
mRNA was injected with wild type Ets-2 mRNA, and expression of
Xbra was analyzed by RT-PCR with whole embryos (Fig. 4D).
Ets
N En-R-injected whole embryos showed greatly reduced expression of Xbra when compared with uninjected whole embryos. The wild type
Ets-2 co-injected embryos showed recovered expression of Xbra,
indicating that wild type Ets-2 rescued the effect brought by Ets
N
En-R.
Next, we tested the effect of expression of antisense morpholino
oligonucleotides (MO) against Ets-2. We injected the Ets-2 MO into
dorsal marginal zones in four-cell stage embryos. About 80% of embryos
injected with the Ets-2 MO in dorsal sides showed a severe posterior
defect (n = 42). A typical phenotype is shown in Fig.
5. Expression of control MO in dorsal
sides of embryos had no effect (Fig. 5). Embryos injected with the
Ets-2 MO in ventral sides were almost normal (data not shown). To
confirm that the Ets-2 MO-induced phenotype is specifically caused by blocking the Ets-2 function, we co-injected Ets-2 mRNA with
Ets-2 MO. Co-injection of Ets-2 mRNA rescued about 60% of embryos from the defects in posterior structures induced by Ets-2 MO (n = 28). A typical image is shown (Fig. 5, lower panel).
Taken together, these results suggest that Ets-2 is necessary for
patterning of posterior mesoderm.

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Fig. 5.
Ets-2 is necessary for patterning of
posterior mesoderm. Embryos were injected with Ets-2 MO (50 ng) or
control MO (50 ng) into dorsal marginal zones at the two-cell stage and
cultured until stage 33. For rescue of Ets-2 depletion, Ets-2 mRNA
(1.5 ng) was co-injected with Ets-2 MO.
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Xbra Transcription Is Regulated by Ets-2--
Our RT-PCR analysis
showed that expression of Ets-2 is able to induce expression of Xbra.
We supposed that Ets-2 regulates the Xbra transcription. To test the
role of Ets-2 in regulation of Xbra transcription, we performed a
reporter assay using the luciferase reporter plasmid containing the
1562-bp fragment of the Xbra promoter region (13, 14). The Xbra
reporter construct was co-injected with Ets-2 mRNA and the internal
control
-galactosidase into animal poles of two-cell stage embryos.
Animal caps were dissected at stage 8 and assayed for luciferase and
-galactosidase activities at stage 11. The ratio of luciferase to
-galactosidase activity provides a normalized measure of luciferase
expression. Ets-2 activated luciferase expression about 4-fold relative
to control (Fig. 6A). With FGF
treatment, the luciferase activity by Ets-2 was slightly increased. On
the contrary, Ets
N En-R repressed the luciferase activity markedly.
Similarly, Ets
N En-R strongly inhibited the FGF-induced luciferase
activity (Fig. 6A). These results are in good accordance
with our hypothesis that Ets-2 regulates the transcription of Xbra.
Then, we investigated whether Ets-2 protein binds to the promoter
region of Xbra. A recent study has shown that a restricted upstream
region of the Xbra promoter is necessary for its expression (37). We
searched for the Ets binding sites within this region of Xbra promoter.
We found two Ets binding motifs in the Xbra promoter and tested their
ability to bind to Ets-2 in the gel mobility shift assay. The gel
mobility shift assay was performed on
310 to
271 and
259 to
219
fragments of the Xbra promoter using the bacterially expressed
glutathione S-transferase (GST)-Ets-2 fusion protein or GST
alone. The
259/
219 fragment bound only very weakly to GST-Ets-2
(data not shown), whereas the
310/
271 region of the Xbra promoter
bound to Ets-2 especially (Fig. 6C). Oligonucleotide probes
corresponding to the wild type and the mutated
310/
271 region were
designed for the gel mobility shift assay (Fig. 6B). The
GST-Ets-2 fusion protein bound to the wild type oligonucleotide probe
strongly, whereas GST alone did not bind to the probe (Fig.
6C). The binding was inhibited by preincubation with an
excess of unlabeled oligonucleotide (Fig. 6C,
competitor). However, the binding persisted with an excess
of unlabeled mutant oligonucleotide (Fig. 6C, mutant
competitor). Thus, Ets-2 is functional in forming a specific
DNA-protein complex with a Xbra promoter region.

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Fig. 6.
Ets-2 is essential for Xbra
transcription. A, Ets-2-regulated expression of
reporter gene construct containing a 1.5-kb fragment of the Xbra
promoter region. The Xbra promoter construct was co-injected with Ets-2
mRNA or Ets N En-R mRNA into animal poles of two-cell stage
embryos. Animal caps were dissected at stage 8 and cultured with or
without FGF (50 ng/ml). Cultured animal caps were assayed for
luciferase activity at stage 11. B, the sequence of the wild
type and the mutant Ets binding sites. C, Ets-2 bound to the
310/ 271 region of the Xbra promoter. The gel mobility shift assay
was performed using radiolabeled double-strand oligonucleotide probe of
the Ets-binding site. After incubation of the radiolabeled probe with
protein extracts, DNA-protein complex was analyzed by autoradiography
following electrophoresis of binding reactions on 4% polyacrylamide
gels. The upper arrow indicates the position of DNA-protein
complex. The recombinant GST or GST-Ets-2 fusion protein was incubated
with radiolabeled oligonucleotide probe containing the Ets-binding
site. For competition assay, binding reactions were preincubated with a
200-fold molar excess of unlabeled oligonucleotide probe as competitor,
and then binding reactions with GST-Ets-2 and labeled probe were
incubated as described above. The same competition assay was performed
with unlabeled mutant oligonucleotide.
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DISCUSSION |
It is known that FGFs can induce mesoderm via the Ras/MAPK pathway
in early Xenopus development (3-8). Previous studies have shown that members of at least six subfamilies of ETS proteins (ETS, YAN, ELG, PEA3, ERF, TCF) are nuclear targets of the
Ras/MAPK pathway. Elk-1 is a member of the ternary complex factor
subfamily, and it is well known that phosphorylation of Elk-1 by MAPK
enhances activities of Elk-1. However, the FGF-induced Xbra expression was not reduced by overexpression of dominant-negative Elk-1 in animal
cap assay (38). The other member of ETS family, ER81, which belongs to
the PEA3 subfamily, was identified in Xenopus. Although
XER81 has been reported to be a target of FGF signaling, XER81 alone
did not induce Xbra expression in animal cap explants (39). Moreover,
overexpression of XER81 did not change the expression pattern of Xbra
transcript (40). Our results here have demonstrated that Ets-2 induces
Xbra expression and plays an essential role in mesoderm patterning
downstream of the FGF/MAPK pathway. Furthermore, overexpression of
Ets-2 caused posteriorized embryos. Similar phenotypes were reported to
be induced by expressing Xcad3 that is required for posterior
development downstream of FGF signaling (41). Both Ets-2 and Xcad3
appear to posteriorize anterior neural tissue. How Ets-2 cooperates
with Xcad3 and the Hox gene pathway in posterior development remains to
be studied (42).
In Xenopus mesoderm patterning, no transcription factor that
would relay the FGF signal at the Xbra promoter has been identified (43). Our results suggest that Ets-2 regulates the transcription of
Xbra downstream of the FGF/MAPK pathway. Mullick et al. (44) have recently shown that Ets-2 protein binds to a "weak" Ets-like site of the cytochrome P-450c27 promoter. They provide new insights on
the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators (44). Because the putative weak
Ets consensus sites are widely distributed on the Xbra promoter, it is
possible that interactions with several Ets-like sites synergistically regulate Xbra transcription. Furthermore, other transcription factors,
such as AP-1, potentially participate in transcriptional regulation of
Xbra (45). It is likely that other transcription factors in
coordination with Ets-2 regulate Xbra transcription. Elucidation of the
precise relationship of Ets-2 with other transcription factors will be
a necessary step toward understanding the regulation of Xbra.
Our results reported here strongly suggest that Ets-2 plays an
essential role in mesoderm patterning. Previously, HpEts has been
identified as a sea urchin homologue of Ets-2. Overexpression of HpEts
in sea urchin embryos caused primary mesenchyme cells to extinguish
cellular adhesion and to migrate (46, 47). In Xenopus,
it is likely that Ets-2 is also involved in cellular adhesion and
migratory cell guidance. We are currently investigating other roles of
Ets-2 in Xenopus developmental processes.
We thank Dr. Ken W. Y. Cho for the
Xbra-Luc (luciferase) plasmid. We also thank H. Hanafusa and M. Kusakabe in our laboratory for technical advice and support.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M211054200
The abbreviations used are:
FGF, fibroblast growth factor;
bFGF, basic fibroblast growth factor;
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
ERK, extracellular signal-regulated kinase;
Xbra, Xenopus brachyury;
En-R, repressor domain of
Drosophila Engrailed;
XFD, Xenopus
dominant-negative form of FGF receptor;
RT-PCR, reverse
transcription-coupled polymerase chain reaction;
GST, glutathione
S-transferase;
MO, morpholino oligonucleotides.
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