We have previously demonstrated the involvement
of AP-1/Jun in fibroblast growth factor (FGF) signaling by
loss-of-function assay (Dong, Z., Xu, R.-H., Kim, J., Zhan, S.-N., Ma,
W.-Y., Colburn, N. H., and Kung, H. (1996) J. Biol. Chem.
271, 9942-9946). Further investigations by gain-of-function are
reported in this study. AP-1 transactivation activity was increased by
the treatment of animal cap explants with FGF. Ectopic overexpression
of two components of AP-1 (c-jun and c-fos
together, but not alone) produced posteriorized embryos and induced
mesoderm formation in animal cap explants, indicating that both AP-1
heterodimers are required for mesoderm induction. Since Ras/AP-1
functions downstream of FGF signaling, we then tested the involvement
of Ras/AP-1 in mesoderm maintenance mediated by embryonic FGF/Xbra
using dominant-negative mutants. Mesoderm maintenance mediated by
embryonic FGF/Xbra was blocked by dominant-negative mutants of
Ras/AP-1, and AP-1 enhanced the expression of Xbra. Further studies
demonstrated the inhibition of Ras/AP-1-mediated mesoderm formation by
dominant-negative mutants of the FGF receptor and Xbra. These results
indicate that Ras/AP-1 and FGF/Xbra signals are involved in the
mesoderm maintenance machinery and mesoderm formation through the
synergistic action of the diversified signal pathways derived from the
FGF/Xbra autocatalytic loop.
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INTRODUCTION |
In early vertebrate development, one of the most important events
is the mesoderm-inducing process, and several mesoderm-inducing factors
have been identified using an amphibian embryo system (1-6). In
Xenopus, normal mesoderm formation in the embryo largely depends on FGF1 signaling
(7-13). Experiments using the dominant-negative FGF receptor
demonstrate that FGF signaling is required for posterior mesoderm
formation in whole embryos (7, 8) and is also required for mesoderm
induction in response to members of the transforming growth factor-
superfamilies in animal cap culture (9-11). While two members of the
FGF family, basic FGF (bFGF) (14, 15) and embryonic FGF (eFGF) (16),
are present in mesoderm formation stages during early embryonic
development, eFGF is more likely to be an endogenous inducer since it
is efficiently secreted. bFGF, however, lacks a signal sequence for
secretion. Xenopus eFGF is expressed maternally, and its
expression increases significantly during gastrulation, suggesting its
roles in mesoderm induction as well as mesoderm maintenance (17).
The components of the FGF signal transduction pathway required for
mesoderm induction include Ras (18, 19), the protein kinase cascade of
Raf (20), and the mitogen-activated protein kinase pathway (21-24).
However, the downstream FGF signal(s) in the nucleus is largely
unknown. One nuclear target of FGF signaling seems to be Xbra, a
Xenopus homologue of Brachuary (25). Xbra has been used as a
panmesodermal marker, and its expression is increased by treatment with
mesoderm inducers including members of the FGF and transforming growth
factor-
families. While ectopic overexpression of Xbra is enough to
induce the differentiated mesoderm, mesoderm formation in response to
Xbra requires FGF signaling (13). Xbra and eFGF have been shown to be
involved in the autoregulatory loop, in which Xbra induces the
expression of eFGF, which is required for maintenance of the expression
of Xbra (17).
In our laboratory, we have been interested in the roles of AP-1 during
Xenopus early embryonic development (26, 27). We recently
reported the involvement of AP-1/c-Jun in the FGF signaling pathway
using dominant-negative jun (DN-jun) (26).
Although the requirement for AP-1/c-Jun in FGF-induced mesoderm
induction has been demonstrated by loss-of-function activity using
DN-jun, these experiments cannot discriminate between the
involvement of homodimeric and heterodimeric AP-1 in mesoderm
induction. Also, we cannot conclude whether AP-1 is a necessary or a
sufficient component in the pathway leading to mesoderm induction.
Since the DNA-binding activity of the homodimeric Jun complex and the heterodimeric Jun-Fos complex to the AP-1 site is different in vitro (28) and the phenotypes of c-jun and
c-fos null mutants are different in mammalian embryonic
development (29-31), we examined the roles of c-Fos in the AP-1
complex in mesoderm formation during Xenopus early embryonic
development.
Here, we report, for the first time, the microinjection of a
heterodimeric AP-1 (c-jun/c-fos) transcription
factor causing posteriorized embryos and leading to the induction of
mesoderm in ectoderm explants from Xenopus embryos. In
addition, we demonstrate that AP-1 (c-Jun/c-Fos) functions downstream
of FGF signaling and is an essential component of the mesoderm
maintenance machinery mediated by eFGF/Xbra. Furthermore, we found that
FGF induces mesoderm formation and maintenance through the synergistic
action of the diversified signals derived from the FGF/Xbra
autocatalytic loop.
 |
EXPERIMENTAL PROCEDURES |
DNA and RNA Preparation--
The c-jun cDNA was
inserted into pGEM (28), and c-fos and antisense
c-fos were subcloned into the pSP65 vector (32). The dominant-negative FGF receptor (DN-FR) (7), Xbra, and Xbra-Engrailed were inserted into the pSP64T vector (33, 34). Xbra-Engrailed (dominant-negative mutant of Xbra (DN-Xbra)) is a dominant-interfering Xbra generated by replacing the activation domain of Xbra with the
repressor domain of the Drosophila Engrailed protein (34). DN-jun was subcloned into the pSP64TEN vector (26, 27). The constitutively active ras ([Val12]Ha-Ras) and
dominant-negative ras (DN-ras;
[Asn17]Ha-Ras) cDNAs were inserted into the pSP64
vector (18). Each of the cDNAs were linearized and used for
in vitro synthesis of capped mRNA using an Ambion
transcription kit in accordance with the manufacturer's instructions.
The synthetic RNA was quantitated by ethidium bromide staining in
comparison with a standard RNA.
Embryo Injection and Explant Culture--
Xenopus
laevis embryos were obtained by in vitro fertilization
(35). Developmental stages were designated according to Nieuwkoop and
Faber (36). Embryos at the two-cell stage were injected in the animal
pole with messenger RNA or cDNA as described in the figure legends.
Animal caps were dissected from the injected embryos at stages 8.5-9
and cultured to various stages for further analysis as described in the
figure legends.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Total RNA was extracted from cultured explants with
TRIzol reagent (Life Technologies, Inc.) following the manufacturer's instructions. RT-PCR was performed with a Superscript preamplification system (Life Technologies, Inc.). Primer sets and PCR conditions for
Xbra, muscle actin, and EF-1
were as described previously (26,
36).
Analysis of AP-1 and NF-
B Activities in Animal Cap
Culture--
AP-1- or NF-
B-luciferase plasmid DNA (26, 37, 38) was
injected alone or together with the designated RNA into two blastomers of the two-cell stage embryo as described in the figure legends. After
injection, the animal caps were excised from the embryos (stages
8.5-9) and cultured in the presence of different dosages of bFGF
(2-100 ng/ml) until stages 10.5 and 13. AP-1-dependent luciferase activity in the animal cap explants was measured after homogenization in lysis buffer as described previously (26).
 |
RESULTS AND DISCUSSION |
bFGF Stimulates AP-1 Activity in Animal Cap Explants during
Mesoderm Induction (Stage 10.5) and Mesoderm Maintenance (Stage 13)
Stages--
Previously, we reported high levels of
AP-1-dependent transactivation activity during
Xenopus early embryonic development and inhibition of
FGF-induced mesoderm formation in animal cap explants from embryos
injected with DN-jun RNA (26). To determine whether AP-1 is
a target transcription factor of the FGF signal, we performed the
following experiments. Embryos were injected with AP-1- or
NF-
B-luciferase reporter genes at the two-cell stage. The animal
pole tissue was then dissected at stages 8.5-9 and incubated with
different dosages of bFGF. The bFGF-treated animal caps were harvested
at stages 10.5 and 13, and the luciferase activity in the animal caps
was measured. The luciferase activity in the animal caps injected with
the NF-
B-luciferase reporter gene was not affected by bFGF (Fig.
1B). On the other hand, the luciferase activity in the animal caps injected with the
AP-1-luciferase reporter plasmid was increased 2-8-fold by bFGF
treatment in a dose-dependent manner (Fig.
1A).

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Fig. 1.
AP-1 activation by bFGF. a,
dose-dependent AP-1 activation by bFGF (2-100 ng). Ten pg
of AP-1-luciferase construct was injected at the two-cell stage (26,
27), and the animal caps obtained from the embryos (stages 8.5-9) were
cultured in the presence of different dosages of bFGF (2-100 ng/ml)
until stages 10.5 and 13. b, effects of bFGF (2-100 ng) on
NF- B activity. The same procedure was followed as described for
a, except that the NF- B-luciferase reporter plasmid was
used instead of the AP-1-luciferase construct. c, effect of
bFGF and AP-1 on AP-1 activation. AP-1-luciferase reporter plasmids (10 pg) were coinjected with the mRNAs encoding c-Jun (0.5 ng) and
c-Fos (0.5 ng) at the two-cell stage, and the luciferase activity was
compared with the animal cap explants treated with 10 ng/ml bFGF.
Treatment with bFGF was as described for a. The experiments
were repeated three times with similar results. The data are presented
as luciferase activity relative to the average luciferase activity in
at least 20 animal caps. The luciferase activity in individual animal
caps was measured after homogenization in 20 µl of lysis buffer
as described previously (26).
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In separate experiments, AP-1-luciferase reporter plasmids were
coinjected with the mRNAs encoding c-Jun and c-Fos, and the luciferase activity was compared with the animal cap explants treated
with bFGF. AP-1 RNA (c-jun/c-fos)-injected animal
cap explants showed similar luciferase activity as bFGF-treated animal cap explants at stages 10.5 and 13 (Fig. 1C). The AP-1
activity in FGF-treated animal cap explants was inhibited by
DN-ras RNA injection (data not shown), suggesting that FGF
signals through the Ras pathway for AP-1 activation. However,
AP-1-dependent luciferase activity was not affected in
animal cap explants injected with noggin or dominant-negative BMP-4
receptor mRNA (data not shown), which caused neuralization in the
ectoderm.
Heterodimeric AP-1 Induces Mesoderm Formation--
The
mesoderm-inducing activity of AP-1 (c-jun and
c-fos) was then examined by injection of RNA encoding c-Jun
and/or c-Fos at the two-cell stage. The animal pole tissue was
dissected at the blastula stage (stages 8.5-9) and incubated until the
animal cap explants were harvested. Injection of
-galactosidase,
c-jun, or c-fos RNA alone or c-jun
plus antisense c-fos RNA did not induce any morphological
changes in the animal cap explants cultured until the tadpole stage
(Fig. 2a, panel A).
On the other hand, animal caps from embryos injected with
c-jun and c-fos RNAs together showed
morphological changes (the animal caps were swollen and elongated)
similar to those induced by FGF treatment in a
dose-dependent manner (Fig. 2a, panels
B-D).

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Fig. 2.
Mesoderm induction in blastula animal caps by
AP-1 (c-jun/c-fos). a, morphological
changes in animal cap explants. X. laevis embryos were
obtained by in vitro fertilization (35). Embryos at the
two-cell stage were injected in the animal pole with mRNAs of
control -galactosidase (1 ng), c-jun (1 ng),
c-fos (1 ng), or c-jun (1 ng) plus antisense
c-fos (1 ng) (panel A); c-jun/c-fos (0.25 ng each) (B);
c-jun/c-fos (0.5 ng each) (C); or
c-jun/c-fos (1 each ng) (D).
c-jun, c-fos, and antisense c-fos were
derived from rat clones (28, 32). Animal caps were dissected from the injected embryos at stages 8.5-9 and cultured until stage 30 for the picture. The experiment was repeated three times in over 120 embryos with similar results. Developmental stages were designated
according to Nieuwkoop and Faber (36). b, molecular markers
of mesoderm induction induced by AP-1 (c-jun and
c-fos together). Embryos were injected, and animal caps were
dissected and cultured as described for a. RNA was isolated
from animal caps injected with the following: lane 1,
-galactosidase ( -gal; 1 ng); lane 2,
c-jun (1 ng); lane 3, c-fos (1 ng);
lane 4, c-jun (1 ng) plus antisense
c-fos (1 ng); lane 5, c-jun (1 ng)
plus sense c-fos (1 ng); lane 6, control
(No Injection). The RNA was analyzed by RT-PCR (26, 35) to
determine the expression of the molecular markers Xbra and EF-1 with
stage 11 animal caps and muscle actin and EF-1 with stage 30 animal
caps. Embryos at equivalent stages were used as a positive control
(lane 7), and the same embryo sample processed for RT-PCR in
the absence of reverse transcriptase (No RT) was used as a
negative control (lane 8). c, immunohistochemical
analysis of animal caps generated by injection of -galactosidase RNA
(1 ng) (panel A) or AP-1 RNAs (1 ng of c-jun and
1 ng of c-fos) (panel B) and by treatment with bFGF (100 ng/ml) (panel C). Animal caps were harvested and
sectioned at stage 30. The slides were then fixed and stained with
specific antibody to muscle actin. Panels B and C
show the muscle actin-stained section (dark brown color)
with unorganized muscle tissue. Panel A shows an unstained
section with a typical epidermis structure. The animal caps from
embryos injected with c-jun (1 ng), c-fos (1 ng),
or c-jun (1 ng) plus antisense c-fos (1 ng) were
examined with the same procedure as described for panels A,
B, and C of Fig. 2c. Typical epidermis
structures similar to -galactosidase-treated animal caps were found.
d, dose-dependent mesoderm formation as measured
by the expression of Xbra (stage 11) and actin (stage 24). Injection of
c-jun alone (0.5-4 ng in lanes 1-4 in the
upper panels), c-jun + c-fos (0.5-4
ng; 0.5 ng = 0.25 ng of each in lane 1, 1 ng = 0.5 ng of each in lane 2, 2 ng = 1 ng of each in lane
3, and 4 ng = 2 ng of each in lane 4 in the
lower panels), and -galactosidase (lane 5).
Lane 6 is the positive control of the embryo, and lane
7 is the negative control with no reverse transcriptase. Embryos
were injected, and animal caps were dissected and cultured as described
for a. RT-PCR was performed as described for b.
Expression of EF-1 was used as a control for equal loading of
reverse transcriptase samples.
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To confirm the mesoderm induction in animal caps, we examined early and
late molecular markers for mesoderm by RT-PCR. Xbra, an immediate-early
marker for mesoderm, and muscle-specific actin, a late marker for
mesodermal differentiation, were expressed in AP-1
(c-jun/c-fos)-injected caps, but not in
-galactosidase, c-fos, or c-jun RNA-injected
caps or in c-jun plus antisense c-fos RNA-injected caps (Fig. 2b). Immunohistochemical analysis
showed that the animal cap injected with c-jun and
c-fos together generated unorganized muscle tissues similar
to the tissues generated by treatment with bFGF. Actin was detected by
specific antibody to muscle actin in the sections of c-jun-
and c-fos-injected animal cap samples and bFGF (100 ng)-treated samples, but not in the other samples (Fig. 2c).
For mesoderm formation, injection of 0.5 ng of c-jun RNA and
0.5 ng of c-fos RNA together was sufficient to induce Xbra
at the early stage (stage 11) and muscle actin at the later stage
(stage 24) in ectoderm cells. However, animal cap explants injected
with up to 4 ng of c-jun alone did not show any detectable
morphological changes or increased expression of the mesodermal markers
Xbra and actin. A very small amount of Xbra was detected in
c-jun-injected animal cap explants as determined by RT-PCR
(Fig. 2d).
These results clearly demonstrate that heterodimeric AP-1
(c-jun and c-fos) is sufficient to induce
mesoderm in animal cap explants, similar to FGF and its signal
molecules, Ras/Raf/mitogen-activated protein kinase, but homodimeric
AP-1 (c-Jun) is not able to induce mesoderm. The previous results (26)
for the AP-1/Jun involvement in FGF-mediated mesoderm induction by
loss-of-function assay with DN-jun were possibly caused by
the blocking of heterodimeric AP-1 activity. Although we have
demonstrated the requirement of both c-Jun and c-Fos for mesoderm
formation, it remains to be determined whether the requirement of
c-Jun/c-Fos can be replaced by other members of the Jun/Fos families.
In the present study, we have focused only on the direct roles of
c-Jun/c-Fos.
AP-1 Causes Posteriorized Embryos--
Whole embryos injected with
-galactosidase, c-jun, or c-fos RNA alone at
the two-cell stage did not show any morphological changes (data not
shown). Another transcription factor (AP-2 RNA) was also injected, and
no morphological change was observed (data not shown). However,
c-jun and c-fos RNAs injected together caused morphological changes in a dose-dependent manner (Fig.
3a). Embryos injected with
AP-1 mRNAs (0.5 ng of c-jun and 0.5 ng of
c-fos) at the two-cell stage developed normally through the
mid-gastrula stage, but the blastopore closure was delayed (Fig.
3b, panel B). A similar phenotype of abnormal
gastrulation defects was observed with injection of other mesoderm
inducers, Xbra (0.5 ng) or ras RNA (1 ng) (Refs. 12 and 33
and data not shown). Injection of low doses of AP-1 RNAs (0.125 ng of
c-jun and 0.125 ng of c-fos) caused an enlarged
proctodeum at the posterior part of the embryo at the tadpole stage,
which is a typical phenotype of CSKA-eFGF plasmid-injected embryos
(17). The embryos injected with AP-1 exhibited a posteriorized
phenotype with diminished anterior structures including cement gland
and forebrain. The extent of morphological changes depends greatly on
AP-1 dosages up to 2 ng. Histological analysis showed that unorganized
muscle-like structure accumulated in the posterior part of
AP-1-injected embryos (data not shown). Interestingly, in animal cap
explants excised from embryos coinjected with AP-1 and chordin,
posterior neural markers were
induced.2 The caudalization
of neural tissues with FGF or Xbra has recently been reported (39-41),
indicating that AP-1 is involved in a broad range of FGF
activities.

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Fig. 3.
Morphological changes in whole embryos
induced by ectopic expression of AP-1 (c-jun and
c-fos). a, dose-dependent
morphological changes in whole embryos by injection of AP-1 (0.125-1
ng of c-jun/c-fos). Panel A, 0.125 ng
each; panel B, 0.25 ng each; panel C, 0.5 ng each; panel D, 1 ng each. The RNAs of c-jun and
c-fos were injected into the animal pole of the two-cell
stage embryos. The injected embryos were allowed to develop until the
tadpole stage. The embryos injected with AP-1 show posteriorized
phenotypes with diminished anterior structures including cement gland
and forebrain. The extent of morphological changes was
dose-dependent. In the same experiments, the morphological
changes were never observed in embryos injected with -galactosidase
(1 ng), c-jun (1-4 ng), c-fos (1-4 ng), or
c-jun (1 ng) plus antisense c-fos (1 ng).
b, gastrulation defect induced by injection of AP-1. The
blastopore of an embryo injected with 1 ng of -galactosidase was
closed (panel A). The embryos injected with AP-1 (0.5 ng of
c-jun and 0.5 ng of c-fos) developed normally
through the blastula and early gastrula stages (not shown). However,
the blastopore failed to close as shown by a round circle in the
picture (panel B). Pictures were taken at stage 15. c, effects on embryonic development of injection of AP-1
into the VMZ or DMZ. AP-1 mRNAs (0.5 ng of c-jun and 0.5 ng of c-fos) were injected at the four-cell stage into the
DMZ or VMZ. Injection of AP-1 into the VMZ caused a minor posterior
defect (panel A, lower picture) compared with a
normal tadpole (panel A, upper picture). In
contrast, AP-1 injection into the DMZ of embryos caused a gastrulation
delay and severe defects in body patterning including lack of head
structure (~10% embryos) (panel B, lower
picture).
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Injection of AP-1 RNAs (c-jun and c-fos) into the
dorsal marginal zone (DMZ) or the ventral marginal zone (VMZ) in
embyros at the four-cell stage resulted in different phenotypes (Fig. 3c). Injection of AP-1 into the VMZ caused a minor posterior
defect with normal head structure (Fig. 3c, panel
A). However, injection of AP-1 RNAs into the DMZ caused a
gastrulation delay and consequently more severe defects in the
anterior, including lack of a head structure, but rather normal
posterior structure (Fig. 3c, panel B).
While this work was in progress, an implantation experiment with eFGF
beads in gastrulas was published (42). When an eFGF bead was implanted
into the dorsal lip of a stage 11.5 embryo, it resulted in the loss of
eyes and forebrain as well as other anterior structures, with a normal
proctodeum in the tail bud-stage embryo. However, when an eFGF bead was
implanted into the ventral lip at stage 11.5, the embryos developed
normally, including the head structure. The results were similar to
those of the present study of AP-1 (see above). The similarity in
morphological changes further suggests that AP-1 (c-Jun/c-Fos) may be a
mediator in the FGF signaling pathway.
Ras/AP-1 Functions Downstream of FGF Signaling in Mesoderm
Formation--
AP-1 transactivation activity in animal cap explants
was increased by treatment with bFGF in culture media. Furthermore,
AP-1 induced mesoderm formation and the expression of Xbra and muscle actin similar to bFGF. These results suggest that AP-1 may be a target
molecule of FGF signaling in the nucleus. Although Xenopus bFGF produced from the injected mRNA has a potent mesodermalizing effect on animal hemisphere cells, bFGF has no signal sequence that may
be necessary for cell-cell signaling events such as mesoderm induction.
Furthermore, virtually no phenotypic change by bFGF treatment is
observed in intact embryos (43). In contrast to bFGF, eFGF plays an
important role in maintaining the properties of mesoderm in the
gastrulas of Xenopus embryos in addition to its
mesoderm-inducing activity during the blastula stages. The expression
of eFGF increases significantly during gastrulation. In addition, eFGF
caused similar morphological changes in whole embryos
(posteriorization) as observed with AP-1 mRNA injection. Although
Xbra and eFGF have been reported to require each other for mesoderm
maintenance (13, 17), the roles of Ras/AP-1 have not been clearly
demonstrated in mesoderm maintenance mediated by eFGF/Xbra. Therefore,
it is interesting to test whether signaling components of FGF
(Ras/AP-1) are involved in mesoderm maintenance mediated by the
FGF/Xbra autocatalytic loop.
The injection of Xbra RNA or the CSKA-eFGF plasmid into animal caps
caused morphological changes (swelling and elongation) as shown in Fig.
4a. The CSKA-eFGF plasmid
becomes transcriptionally active after mid-blastula transition (44),
and eFGF mRNA does not accumulate until stage 10 (17). The animal
cap explants start to lose competence for mesoderm induction in
response to bFGF after stage 10. However, the animal cap injected with
the CSKA-eFGF plasmid showed a consistently potent mesoderm-forming ability, indicating that the period of competence of ectoderm cells for
mesoderm induction might be longer than what is usually expected.

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Fig. 4.
Inhibition of eFGF- and Xbra-induced mesoderm
induction by DN-ras and DN-jun. a,
reversal of CSKA-eFGF- or Xbra-induced morphological changes in animal
caps by DN-jun at stage 13. Animal caps from embryos
injected with 25 pg of CSKA-eFGF plasmid and 1 ng of -galactosidase
RNA (panel A), 25 pg of CSKA-eFGF plasmid and 1 ng of
DN-jun RNA (panel B), 0.5 ng of Xbra RNA and 1 ng of -galactosidase RNA (panel C), and 0.5 ng of Xbra RNA
and 1 ng of DN-jun RNA (panel D) were dissected
at stages 8.5-9 and cultured until stage 13, when the pictures were
taken. b, inhibition of eFGF- and Xbra-induced actin
expression by DN-ras and DN-jun. Animal caps were
dissected and cultured until stage 24 as described for Fig.
2a. RT-PCR was performed with the RNAs isolated from animal
caps injected with the following: lane 1, Xbra (0.5 ng) and
-galactosidase ( -gal; 1 ng); lane 2, Xbra
(0.5 ng) and DN-ras (1 ng); lane 3, Xbra (0.5 ng)
and DN-jun (1 ng); lane 4, CSKA-eFGF (25 pg) and
-galactosidase (1 ng); lane 5, CSKA-eFGF (25 pg) and
DN-ras (1 ng); lane 6, CSKA-eFGF (25 pg) and
DN-jun (1 ng); lane 7, -galactosidase (1 ng).
Embryos at equivalent stages were used as a positive control
(lane 8), and the same embryo sample without reverse
transcriptase (No RT) was used as a negative control (lane 9) as described for Fig. 2b.
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Coinjection of RNAs encoding DN-Ras significantly reduced elongation
movements of animal cap explants in response to eFGF or Xbra during the
gastrulation stages (data not shown), suggesting that maintenance of
mesoderm depends on the downstream FGF signal, Ras. As expected,
coinjection of DN-jun with Xbra or eFGF inhibited elongation
movements similar to DN-ras (Fig. 4a). Since the
maintenance of mesoderm in the gastrulating embryo is crucial for the
formation of differentiated mesoderm-like muscle (45), we examined the expression of muscle actin. Animal caps derived from coinjection of
DN-ras or DN-jun with eFGF or Xbra were cultured
until the tadpole stage and assayed for muscle actin expression by
RT-PCR. Consistent with the morphological data, DN-ras and
DN-jun significantly inhibited the expression of muscle
actin in animal caps derived from coinjected samples (Fig.
4b). Since Ras/AP-1 activates Xbra expression, and Xbra is
able to induce the differentiated mesoderm, it was unclear whether
Ras/AP-1 was involved only in mesoderm induction or also in later
events after mesoderm induction. The results suggest that Ras and AP-1
are involved in mesoderm maintenance in addition to mesoderm
induction.
AP-1-induced Mesoderm Is Inhibited by DN-Xbra--
FGF/Ras/AP-1
had an activity to induce an early mesodermal marker, Xbra. While
ectopic overexpression of Xbra itself is enough to generate the
differentiated mesoderm, Xbra still requires endogenous FGF/Ras/AP-1
signals for mesoderm maintenance and differentiated mesoderm formation.
On the other hand, FGF-mediated mesoderm formation and maintenance are
dependent on Xbra. Since AP-1 was found to be involved in the mesoderm
maintenance machinery mediated by eFGF/Xbra, we examined whether Xbra
is required for AP-1-mediated mesoderm formation. AP-1-mediated
mesoderm formation was abolished by coinjection of DN-Xbra. DN-Xbra
inhibited the expression of muscle actin as well as Xbra itself in
animal caps derived from coinjected samples (Fig.
5). The RT-PCR product of Xbra shown in
Fig. 5 has been designed not to detect injected DN-Xbra. The results
indicate that intact Xbra is required for its own expression and
mesoderm induction. Furthermore, the effect of DN-Xbra was not reversed
by AP-1. Although it remains to be investigated how Xbra regulates its
own transcription, we postulate that Xbra is required to keep the
mesoderm character of the tissue and that DN-Xbra blocks the competence
of the animal cap for mesoderm induction. We extended the mesoderm
maintenance machinery of eFGF/Xbra to include Ras/AP-1, and the
blocking of one of the components is enough to abolish the machinery of
mesoderm formation, suggesting that positive feedback is necessary to
maintain mesoderm (see Fig. 7).

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Fig. 5.
Inhibition of AP-1-induced mesoderm induction
by coinjection of DN-Xbra. Embryos were injected as described
below, and animal caps were dissected and cultured as described for
Fig. 2a. RNA was isolated from animal caps injected with the
following: lane 1, c-jun (1 ng) plus
c-fos (1 ng); lane 2, c-jun (1 ng), c-fos (1 ng), and DN-Xbra (1 ng); lane 3, DN-Xbra
(1 ng); lane 4, -galactosidase ( -gal; 2 ng). The RNA was analyzed by RT-PCR as described for Fig. 2b
(Xbra and EF-1 with stage 11 animal caps and muscle actin and
EF-1 with stage 30 animal caps). Embryos at equivalent stages were
used as a positive control (lane 5), and the same embryo
sample processed for RT-PCR in the absence of reverse transcriptase
(No RT) was used as a negative control (lane 6)
as described for Fig. 2b.
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Mesoderm Formation by FGF Signaling Is a Synergistic Action of
Diversified Signal Pathways--
FGF and Xbra require each other for
mesoderm formation and maintenance, and the downstream signal molecules
of the FGF receptor are involved in these events. However, it is not
clear whether FGF signals for mesoderm induction and maintenance
through a linear array of signals or through the synergistic action of
the diversified signals. These two possibilities were examined by
coinjecting an activated form of Ras with DN-FR. Constitutively active
ras was coinjected with DN-FR, and the animal cap explants
were analyzed. The elongation movement of animal cap explants caused by
the overexpression of the constitutively active form of ras
was inhibited by coinjection of DN-FR (Fig.
6a). Consistent with
morphological data, the expression level of the early mesodermal marker
(Xbra) and the differentiated mesodermal marker (muscle actin) was
reduced or totally abolished by coinjection of DN-FR (Fig.
6b). This result suggests that Ras-mediated mesoderm
formation requires endogenous FGF signaling and that FGF triggers at
least two different signals including Ras for mesoderm formation.
Consistently, AP-1-induced mesoderm formation was also inhibited by
coinjection of DN-FR (Fig. 6c). Furthermore, AP-1-induced
mesoderm formation was inhibited by coinjection of DN-ras
(Fig. 6c). Our results suggest that mesoderm formation induced by FGF signaling is the synergistic action of the diversified signals. Based on these results, a model is proposed. As shown in Fig.
7, the signals derived from the FGF/Xbra
autocatalytic loop contribute synergistically to the formation of
mesoderm. The blocking of any signals by dominant-negative mutants
resulted in inhibited mesoderm formation. In this paper, we show, for
the first time, that heterodimeric AP-1 (c-Jun/c-Fos) induces mesoderm formation in animal cap explants of Xenopus embryos.
Additionally, we found that eFGF- or Xbra-mediated mesoderm maintenance
was dependent on the downstream signals of FGF, Ras, and AP-1. Also, AP-1-mediated mesoderm formation required FGF/ras/Xbra, and
Xbra expression was activated by AP-1. Furthermore, we found that FGF induced mesoderm formation and maintenance through the synergistic action of the diversified signals derived from the FGF/Xbra
autocatalytic loop.

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Fig. 6.
Requirement of endogenous signaling in Ras-
or AP-1-mediated mesoderm formation. a, morphological
reversion of activated Ras-mediated mesoderm formation by DN-FR at
stage 13. Animal caps from embryos injected with -galactosidase RNA
(1 ng) (panel A), constitutively active ras RNA
(1 ng) (panel B), and constitutively active ras
RNA (1 ng) and DN-FR RNA (1 ng) (panel C) were dissected at
stages 8.5-9 and cultured until stage 13, when the pictures were
taken. b, inhibition of Ras-mediated mesoderm formation by DN-FR. Embryos were injected as described below, and animal caps were
dissected and cultured as described for Fig. 2a. RNA was isolated from animal caps injected with the following: lane
1, ras (1 ng) and DN-FR (1 ng); lane 2,
ras (1 ng) and -galactosidase ( -gal; 1 ng);
lane 3, -galactosidase (2 ng). The RNA was analyzed by
RT-PCR as described for Fig. 2b. 6, inhibition of
AP-1-mediated mesoderm formation by DN-FR or DN-ras. Embryos
were injected, dissected, and cultured as described for Fig.
2a. RT-PCR was performed as described for Fig. 2b
with animal caps injected with the following: lane 2,
c-jun (1 ng) and c-fos (1 ng); lane 3,
c-jun (1 ng), c-fos (1 ng), and DN-FR (1 ng);
lane 4, c-jun (1 ng), c-fos (1 ng), and DN-ras (1 ng); lane 5, -galactosidase (2 ng); lane 6, DN-FR (1 ng); lane 7,
DN-ras (1 ng). Lane 1 is the non-injected
control. Xbra and EF-1 were measured with stage 11 animal caps, and
muscle actin and EF-1 were measured with stage 24 animal caps.
Embryos at equivalent stages were used as a positive control
(lanes 4 and 8 in b and c,
respectively), and the same embryo sample processed for RT-PCR in the
absence of reverse transcriptase (No RT) was used as a
negative control (lanes 5 and 9, respectively) as
described for Fig. 2b.
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Fig. 7.
Proposed model for mesoderm formation
mediated by the FGF/Xbra autoregulatory loop. Ras/AP-1 is involved
in the FGF/Xbra autoregulatory loop, and Xbra is activated by AP-1.
Diversified signals derived from each component (FGF/Ras/AP-1/Xbra) of
the autoregulatory loop are required for mesoderm induction and
maintenance. The blocking of any one component of the loop by the
dominant-negative mutants results in the inhibition of mesoderm
formation.
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We thank Dr. T. Curran for c-jun
and sense and antisense c-fos cDNAs; Drs. F. L. Conlon, V. Cunliffe, and J. C. Smith for the Xbra and
Xbra-Engrailed cDNAs; Drs. H. V. Isaacs and J. M. Slack
for the eFGF plasmid; Drs. E. Amaya and M. Kirschner for the
dominant-negative FGF receptor cDNA; and Drs. M. Whitman and D. A. Melton for the constitutively active ras
([Val12]Ha-Ras) and dominant-negative ras
([Asn17]Ha-Ras) cDNAs. We also thank Dr. D. L. Newton for synthesis of the oligonucleotide primers, Drs. Nancy H. Colburn and Ira Daar for helpful comments on the manuscript, and Annie
Rogers for editing the manuscript.