(Received for publication, December 11, 1995; and in revised form, February 13, 1996)
From the
In Xenopus, normal mesoderm formation depends on
signaling through the fibroblast growth factor (FGF) tyrosine kinase
receptor. An important signaling pathway from receptor tyrosine kinases
involves Ras/Raf/MAP kinase. However, the downstream pathway that
occurs in the nucleus to finally trigger gene expression for mesoderm
formation remains unknown. We report here that a high level of
activator protein-1 (AP-1)-dependent transcriptional activity is
detected during the early development of Xenopus embryos.
Injection of a dominant negative mutant jun (DNM-jun or TAM67) RNA into the two-cell stage embryos inhibited endogenous
AP-1 activity and blocked normal embryonic development with severe
posterior truncation in tadpoles. The inhibition of AP-1 activity and
the phenotypic change induced by TAM67 was rescued by co-injection of
wild-type c-jun RNA, but not by the control
-galactosidase RNA. The FGF-stimulated mesoderm induction was
markedly inhibited in animal cap explants from the embryos injected
with TAM67. Activin induction of mesoderm, on the other hand, was
normal in the embryos injected with TAM67 RNA. These findings suggest
that AP-1 mediates FGF, but not activin, receptor signaling during
mesoderm induction and the AP-1/Jun is a key signaling molecule in the
development of posterior structure.
Using stage 8 Xenopus embryo animal pole explants, it
has been shown that mesoderm induction occurs at an early stage of
vertebrate embryogenesis and is stimulated by growth factors, e.g., bFGF ()and members of the FGF
family(1) . Experiments with a dominant-negative mutant of FGF
receptor indicated that FGF signaling is required in the early embryo
for the formation of posterior and lateral mesoderm(2) .
Similar experimental results were obtained for activin receptor
signaling (3, 4, 5) . Disruption of activin
signaling blocks mesoderm formation(3, 4) . Although
the detailed molecular mechanism is not clear, it appears that both Ras
and Raf are involved in FGF signaling, while only Ras is involved in
the activin-stimulated signaling pathway(6, 7) . More
recently, it has been reported that MAP kinase is required for
FGF-induced mesoderm formation(8, 9) . AP-1 activity
has been reported to be modulated through the Ras/Raf and MAP kinase
signaling pathways in many cell lines(10) . Stimulators of AP-1
include the protein kinase C activator phorbol 12-myristate 13-acetate
(TPA), growth factors such as platelet-derived growth factor, epidermal
growth factor, FGF, and interleukins and oncogene
products(11, 12, 13, 14, 15, 16, 17) .
The AP-1 complex consists of dimers of jun and fos multigene families and is a sequence-specific DNA binding
transcription factor that is part of a pathway by which intracellular
signals are converted into changes in gene activity(10) .
Although AP-1 is downstream of the signal transduction pathway of Ras/Raf in many biological systems and Ras/Raf and MAP kinase are involved in FGF-induced mesoderm induction(7, 10) , these experiments do not reveal whether Ras/Raf and MAP kinase do act through AP-1 to induce mesoderm or whether pathways involving other transcription factors are implicated. In the present study, we investigated the role of AP-1 activity in the Xenopus development in response to FGF.
In order to study the role of AP-1 activity in the development of Xenopus, the two-cell stage Xenopus embryos were injected with (AP-1)4-Luc, a construct that contains four tandem AP-1 binding sites (TPA-responsive cis-enhancer elements, TRE) linked to a luciferase reporter gene(22, 23) . The embryos were collected at various developmental stages, and AP-1-dependent luciferase activity of the embryo lysates was measured. As shown in Fig. 1, AP-1 activity was detectable at stage 10 and increased with increasing developmental stage. The corresponding vector Al-Luc without AP-1 binding sites was used as a control, and the luciferase activity was found to remain at the background level (Table 1). The high levels of AP-1 activity in the early embryos suggest that AP-1 might play an important role in early development. It has been reported that fos was expressed at a low level at the mid-blastula, late neurula, and tadpole stage(24) . By using a specific antibody (Ab-1, Oncogene Sciences), we detected a c-Jun protein band that can compete with a c-Jun peptide at stages 10 and 33 (data not shown)(22, 23) .
Figure 1: Time course of endogenous AP-1 activity in embryonic development of X. laevis and inhibition of AP-1 activity in Xenopus embryo by DNM-ras, DNM-raf, or DNM-jun. Fifty pg of (AP-1)4-Luc plasmid DNA alone (A) or with 1 ng mRNA encoding DNM-ras or DNM-raf (A) or DNM-jun (B) was injected into the two blastomeres of two-cell stage embryos. After injection, five embryos per group were pooled at each developmental stage and homogenized to measure AP-1-dependent luciferase activity expressed as relative light units by using the Promega luciferase assay reagent and a luminometer (Monolight 2010, Analytical Luminescence Laboratory) for 10 s after mixing extract with assay reagent. Results are expressed as the mean of three experiments.
If AP-1 is a mediator of
FGF/Ras/Raf signaling for the induction of mesoderm, then DNM-Ras or
DNM-Raf should inhibit AP-1 activity in the Xenopus embryo
system, and blocking of AP-1 activity should inhibit the FGF-induced
mesoderm and the process of development. Indeed, when the embryos were
injected with either DNM-ras or -raf mRNA, AP-1
activity was inhibited (Fig. 1A). Next, we used a DNM
of human c-jun to attempt to block AP-1 activity. This
DNM-jun, which has an integral leucine zipper and DNA binding
domain but lack a transactivation domain, is understood to act by
sequestering endogenous Jun and Fos family proteins into AP-1 complexes
having reduced activity (29, 31) . The high degree of
conservation of jun and fos family genes allows the
human DNM to function across species. The DNA binding region at the
carboxyl-terminal of Xenopus c-Jun protein product shares
77-79% nucleotide homology and 93% amino acid homology with the
DNA binding region of mouse and human c-jun, and contains an
intact leucine zipper motif(25) . The Xenopus c-fos cDNA and protein also have extensive homology with
their mammalian counterparts. We and others have previously shown that
a dominant-negative mutant c-jun (TAM67) lacking the
transactivation domain of c-jun can (a) dimerize with jun or fos family members to inhibit AP-1
activity(22, 23, 26) ; (b)
specifically block tumor promoter-induced AP-1 activity and
transformation in JB6 cells(22) ; (c) block
transformation of rat embryo cells by ras and jun, fos or SV40 large T antigen(26, 27) ; and (d) revert transformed
phenotype(22, 23, 29, 30) . In order
to specifically inhibit AP-1 activity in the Xenopus embryo
system, we injected TAM67 mRNA into the two-cell stage embryos. The 29 K TAM67 protein was detected at stages 10 and 33,
then declined after the tadpole stage (data not shown). As shown in Fig. 1B, TAM67 inhibited endogenous AP-1 activity at
all developmental stages. This inhibition appears specific, because
TAM67 did not affect the Rous sarcoma virus promoter-dependent
luciferase activity. Injection of an equal amount of
-galactosidase (
-gal) RNA had no significant effect on AP-1
activity (Fig. 2), nor did
-gal reverse the TAM67
inhibition of AP-1 activity (Table 1). Injection of c-jun RNA caused enhancement of AP-1 activity (Fig. 2). Moreover,
co-injections of the synthetic mRNAs and control luciferase vector
without AP-1 binding sites, did not generate a significant difference
in the luciferase activity of the injected embryos (Table 1),
excluding the possibility of a nonspecific effect of injected mRNA on
luciferase expression.
Figure 2:
Enhancement of AP-1 activity in Xenopus embryo by c-jun. Fifty pg of (AP-1)4-Luc DNA
was injected into embryos alone or with 1 ng mRNA encoding c-jun or -gal (Ctrl-RNA), and AP-1 activity was measured
as described in the legend to Fig. 1.
When we injected dominant negative TAM67 mRNA at the two-cell stage and allowed embryonic development to proceed, a striking phenotypic change was observed in the free-feeding tadpole form (stage 45). The most obvious defect in the TAM67 embryos was severe posterior truncation, resulting in the loss of tail structure (Fig. 3). All the TAM67 embryos developed cement glands, heart, and eyes. Careful examination of these TAM67-induced truncations suggests that expression of TAM67 leads to the development of embryos with histologically abnormal posterior patterning. Although the blastopore formed normally, its closure was delayed, and thereafter there was little or none of the elongation of the embryo that normally occurs during the neurula stage. All displayed a truncated axis posteriorly, with the posterior remnant bent upwards. Head development was often remarkably normal. In conclusion, the anterior pattern formation was relatively normal in TAM67 embryos, although posterior development in the same embryos was consistently abnormal. Similar results of abnormal posterior patterning were reported for a dominant negative FGF receptor, a dominant negative Raf-1 or MAP kinase phosphatase(2, 7, 8) , suggesting that AP-1 acts in the signal transduction pathway mediated by FGF-Ras-Raf-MAP kinase in Xenopus development.
Figure 3: DNM-jun TAM67-induced phenotype of stage 45 Xenopus embryos. Both cells of the two-cell embryos were injected with water or TAM67 RNA, and development was allowed to proceed for 7 days, until the embryos had reached the free-feeding tadpole stage.
To investigate whether the
TAM67-induced defects occur through the specific inhibition of
AP-1/jun function, we performed the following experiments with
wild-type c-jun. Co-expression of the wild-type c-jun rescued the defects induced by TAM67 (Table 2). The majority
(65%) of the TAM67-injected embryos were abnormal. Most of these
abnormal embryos had truncated tails (39%) and bent anterior-posterior
axis (21%), similar to the lithium chloride-induced phenotype. A small
fraction (5%) of the water-injected embryos displayed bent axis as a
result of mechanical damage during injection. The incidence of
abnormality of TAM67-injected embryos was significantly higher than
that of the water-control group. Co-injection of wild-type c-jun with TAM67 at 2:1 ratio of RNA clearly rescued the TAM67 defects.
The bent axis embryos were also reversed to the normal background level
(7%). As a control, overexpression of a wild-type c-jun alone
resulted in an essentially normal phenotype (Table 2). Moreover,
co-injecting TAM67 with wild-type c-jun RNA also rescued
TAM67-inhibited AP-1 activity (Table 3). At stages 16, 18, 20,
and 22, AP-1 activity was significantly inhibited by TAM67 (p < 0.05, or p < 0.01). After co-injection of 2 ng of
c-jun RNA with TAM67, AP-1 activity in these stages was not
significantly different from the corresponding -gal controls (Table 3). These experiments show that the effects of TAM67 on
embryonic development were specifically due to the inhibition of AP-1
and/or other Jun-containing transcription factor activity. There are
several mesoderm-inducing factors including Vg1, FGF, activin, and bone
morphogenetic proteins(31, 32) . Their functions
differ in the induction of different parts of mesoderm. For example,
FGF signaling is required for formation of posterior and lateral
mesoderm, while activin induces anterior dorsal mesodermal
tissues(5, 27, 28, 32, 33, 34, 35) .
Bone morphogenetic protein-4, on the other hand, induces ventral
mesodermal tissues and antagonizes dorsal and neural inducing
signals(18, 19, 32, 36, 37, 38) .
As discussed above, DNM-jun (TAM67)-induced posterior
truncations were similar to those observed when a dominant negative FGF
receptor or a DNM-raf mRNA was introduced into Xenopus embryos(5, 11) . Therefore, AP-1/jun appears to be involved in the FGF-ras/raf signaling. This
model was further tested by an animal pole explant experiment. As shown
in Fig. 4, after injection of TAM67 mRNA into the two-cell stage
embryos, bFGF-induced elongation of animal caps was inhibited by TAM67
mRNA. Moreover, the bFGF-induced muscle formation was completely
blocked in TAM67 explants. By contrast, the explants derived from
TAM67-explants responded well to activin, a dorsal-type mesoderm
inducer. By RT-PCR analysis, a muscle-specific actin signal was
completely blocked in TAM67 explants after induction by bFGF, but only
slightly decreased after induction by activin (Fig. 4). After
normalization with internal control EF-1
, expression of the
activin-induced early dorsal marker goosecoid was not affected by TAM67 (Fig. 4).
Figure 4:
TAM67 inhibits FGF induced- but not
activin induced-mesoderm. A-F, morphological analysis
demonstrating that TAM67 inhibits elongation characteristic of mesoderm
induction at early neurula stage. A, animal caps derived from
embryos injected with -gal RNA; C and E, animal
caps derived from embryos injected with water; B, D,
and F, animal caps derived from embryos injected with TAM67
RNA; C and D, animal caps treated with activin; E and F, animal caps treated with bFGF; G, RT-PCR
analysis of actin and EF-1
; H, RT-PCR analysis of
goosecoid (gsc) and EF-2
. Embryos were injected at the
two-cell stage with RNA encoding TAM67 or
-gal. Animal caps were
explanted at stage 8.5 and harvested at stage 10.5 for RT-PCR analysis
using primers specific for EF-1
, muscle actin (G), or gsc (H).
In summary, this report provides evidence for a role of AP-1/jun in Xenopus development and suggests that AP-1 mediates mesoderm induction by FGF. By contrast, AP-1 appears not to mediate activin-stimulated mesoderm induction, implying that the AP-1 pathway is relatively specific for a particular group of growth factors, typified by FGF receptors. These findings demonstrate that AP-1/jun is a key signaling molecule, possibly downstream of FGF-Ras/Raf in the development of Xenopus posterior structure.