AP-1/jun is required for early Xenopus development and mediates mesoderm induction by fibroblast growth factor but not by activin.

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 beta-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 1 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 exper-imental 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.

EXPERIMENTAL PROCEDURES
DNA and RNA Preparation-The dominant negative c-jun (DNMjun or TAM67) and c-jun were subcloned into SP64TEN (18,19). DNMras, and DNM-raf were inserted into Bluescript II SKϩ. These vectors contained SP6, T7, or T3 promoter which is required for in vitro RNA transcription. Each of these constructs was linearized and used for in vitro transcription by using an in vitro transcription kit (Ambion, Austin, TX), and capped mRNAs were prepared as described by Moon and Christian (20).
mRNA Injection and Explant Culture of Embryonic Tissues-Xenopus laevis embryos were obtained by artificial insemination after induction of females with 500 units of human chorionic gonadotropin. Developmental stages were designated according to Nieuwkoop and Faber (21). The jelly layer was removed with 2.5% thioglycolic acid (pH 8.1). The two-cell stage embryos were injected with fixed amount of synthetic capped mRNAs. The injected embryos were allowed to develop for up to 45 stages.
Analysis of AP-1 Activity-We have used an AP-1 luciferase reporter (AP-1)4-Luc in this study. (AP-1)4-Luc containing four AP-1 binding sequences (TGAC/GTCA) was inserted into a luciferase construct with the minimal promoter sequences from the albumin gene. (AP-1)4-Luc plasmid DNA was injected at 50 pg/embryo alone or together with various mRNAs into two blastomeres of the two-cell stage embryos (22). After injection, five embryos per group were pooled at various developmental stages and homogenized to prepare the cell extract. AP-1-dependent luciferase activity in the extract was determined using the * This work was supported in part by The Hormel Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § The first two authors contributed equally to the manuscript. ¶ To whom correspondence should be addressed: The Hormel Institute, University of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.: 507-437-9640; Fax: 507-437-9606; E-mail: zgdong@wolf.co.net. 1 The abbreviations used are: bFGF, basic fibroblast growth factor; FGF, fibroblast growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA-responsive element; AP-1, activator protein 1; DNM, dominant negative mutant; TAM67, dominant negative mutant jun; ␤-gal, ␤-galactosidase; MAP, mitogen-activated protein; RT, reverse transcriptase; PCR, polymerase chain reaction; EF-1␣, elongation factor-1␣; Luc, luciferase. luciferase assay reagent from Promega and a luminometer from Analytical Luminescence Laboratory (Monolight 2010) for 10 s after mixing the extract and assay reagent (22).
Detection of Molecular Markers-Total RNA was isolated from animal caps and oligonucleotide primers for the reverse transcriptionpolymerase chain reaction (RT-PCR) were as described previously (18,19). Molecular markers for development including muscle actin, elongation factor-1␣ (EF-1␣), Xbra, and goosecoid were analyzed by RT-PCR as described previously (18,19).

RESULTS AND DISCUSSION
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 I). 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).
If AP-1 is a mediator of FGF/Ras/Raf signaling for the induc-

AP-1/Jun in Xenopus Development
tion 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 d 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 I). 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 I), excluding the possibility of a nonspecific effect of injected mRNA on luciferase expression. 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.
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. Coexpression of the wild-type c-jun rescued the defects induced by TAM67 (Table II). The majority (65%) of the TAM67-injected embryos were abnormal. Most of these abnormal embryos had

Rescue of TAM67-inhibited AP-1 activity by wild-type c-jun
Fifty pg of (AP-1)4-Luc plasmid DNA with 1-3 ng of mRNA were injected into two blastomers of two-cell stage embryos, and AP-1 activity was measured at different developmental stages. Results are mean Ϯ standard error for three independent experiments. p values are determined by Student's t test.

AP-1/Jun in Xenopus Development
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 II). Moreover, co-injecting TAM67 with wild-type c-jun RNA also rescued TAM67-inhibited AP-1 activity (Table III). At stages 16, 18, 20, and 22, AP-1 activity was significantly inhibited by TAM67 (p Ͻ 0.05, or p Ͻ 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 III). 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 -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).
In summary, this report provides evidence for a role of AP- FIG. 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). 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.