The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3. However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, we examined the growth factor-dependent serine phosphorylation of STAT1 and STAT3. We provide in vitro and in vivo evidence that the ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylate STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process.The JAK/STAT pathways are activated by various cytokines and growth factors such as interleukin-6 (IL-6), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) (reviewed in references 5 and 15). Once these growth factor or cytokine receptors are occupied by their ligands, tyrosine residues in the cytoplasmic domain of the receptors become phosphorylated by cytokine receptor-associated JAK family tyrosine kinases (19,32) or by the growth factor-activated receptor tyrosine kinases (16,26,34). The phosphorylated tyrosine residue, positioned within specific amino acid sequences, provides docking sites for the recruitment of specific STATs via their SH2 domains (13,33). STATs associated with the receptors are consequently phosphorylated at conserved tyrosine residues. This induces the STATs to dimerize via their own SH2 domains (29), translocate into the nucleus, and activate STAT target genes (30). To date, six STAT proteins have been characterized at the molecular level (reviewed in reference 15).EGF and PDGF activate primarily STAT1 and STAT3 (27,28,31,41). STAT1 and STAT3 can form three distinct dimers to activate target genes: STAT1 or STAT3 homodimers and STAT1-STAT3 heterodimers. Although STAT1 and STAT3 are simultaneously activated by various growth factors and cytokines, the results of knockout-mouse experiments suggest a specific role of STAT1 as a target for interferons (10, 21). However, STAT3 is expected to play a broader role. Recent results which show that STAT3 is activated by the expression of Src oncogenes (39) or infection by human T-cell leukemia virus type 1 (22) raise the possibility that STAT3 is involved in tumorigenic cell growth.Mitogen-activated protein (MAP) kinase pathways play important roles in the regulation of cell growth and differe...

Platelet-derived growth factor receptor (PDGF-R) phosphorylation at tyrosines 740/751 and insulin receptor phosphorylation of insulin receptor substrate-1 effects the recruitment and activation of phosphatidylinositol-3-OH kinase (PI(3)K). Changes in PI(3)K activity correlate with cell growth but its downstream signal transducers are unknown. Activation of the 70/85K S6 kinases (pp70S6k) by serine phosphorylation results in 40S ribosomal protein S6 phosphorylation and is important for G1 cell-cycle transition in a variety of cells. Although receptor tyrosine kinases activate the microtubule-associated protein kinase cascade through SH2-/SH3-adaptor proteins, Sos and c-Ras, it is unclear how tyrosine kinases are coupled to the pp70S6k phosphorylation cascade. Here we report that PI(3)K mediates PDGF or insulin receptor signalling to pp70S6k. PI(3)K-mediated activation of pp70S6k is independent of conventional protein kinase C isoforms. Additionally, rapamycin blocks pp70S6k activation by all mitogens, without inhibiting PI(3)K, and acts downstream in this signalling system.

The frog Xenopus laevis has provided significant insights into developmental and cellular processes. However, X. laevis has an allotetraploid genome precluding its use in forward genetic analysis. Genetic analysis may be applicable to Xenopus (Silurana) tropicalis, which has a diploid genome and a shorter generation time. Here, we show that many tools for the study of X. laevis development can be applied to X. tropicalis. By using the developmental staging system of Nieuwkoop and Faber, we find that X. tropicalis embryos develop at similar rates to X. laevis, although they tolerate a narrower range of temperatures. We also show that many of the analytical reagents available for X. laevis can be effectively transferred to X. tropicalis. The X. laevis protocol for whole-mount in situ hybridization to mRNA transcripts can be successfully applied to X. tropicalis without alteration. Additionally, X. laevis probes often work in X. tropicalis-alleviating the immediate need to clone the X. tropicalis orthologs before initiating developmental studies. Antibodies that react against X. laevis proteins can effectively detect the X. tropicalis protein by using established immunohistochemistry procedures. Antisense morpholino oligonucleotides (MOs) offer a new alternative to study loss of gene activity during development. We show that MOs function in X. tropicalis. Finally, X. tropicalis offers the possibility for forward genetics and genomic analysis.

Transplanted Spemann's organizer induces dorsal embryonic cell fates such as the nervous system and somites, but in normal development, elimination of individual organizer signals (such as the bone morphogenetic protein [BMP] antagonists) has surprisingly modest effects on these tissues. Thus, the role of BMP antagonists may be limited to fine tuning the size of the dorsal domain. However, at least five BMP antagonists are specifically expressed in the organizer, and all can mimic aspects of organizer function, suggesting overlapping functions. Here, we deplete the function of three BMP antagonists, chordin, noggin, and follistatin, in Xenopus tropicalis. We demonstrate that this results in catastrophic failure of dorsal development and expansion of ventral and posterior fates. We conclude that BMP antagonists are required for formation of the neural plate and dorsal mesoderm. In addition, our results show that neural specification requires the continuous activity of BMP antagonists from blastula through gastrula stages.

Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (␣II and ␣I isoforms, referred to collectively as pp70 S6k ) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70 S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C T cells are activated, representing G 0 -to-G 1 transition, by antigen presentation to the multimeric T-cell receptor. This results in the transcription, production, and secretion of the 15-kDa glycoprotein lymphokine, interleukin-2 (IL-2). Antigen stimulation also induces expression of the IL-2 receptor (IL-2R) ␣ subunit (p55) and increases the level of IL-2R ␤ subunit (p75). Together with the ␥ subunit, they form the high-affinity IL-2R (reviewed in references 75 and 103). IL-2 then stimulates activated T cells in an autocrine/paracrine fashion, driving G 1 -S transition and cell proliferation. The IL-2R has no intrinsic kinase activity, yet ligand binding increases tyrosine phosphorylation of many proteins, including the IL-2R ␤ chain. IL-2-dependent signalling also results in activation of c-Ras and phosphatidylinositol 3-kinase (PI3K) and increased serine/ threonine protein phosphorylation. Although much is known about Ras-regulated signal transduction (see references 12, 39, and 68 for reviews), the identities of the signalling proteins lying downstream of PI3K remain to be established.PI3K is a novel signal transducer composed of an 85-kDa SH2-and SH3-domain-containing regulatory subunit and a 110-kDa catalytic subunit with specificity toward the D3 hydroxyl in the inositol ring of phosphatidylinositol (37,54,81,98). Numerous studies provide evidence that PI3K, in association with various mitogenically active receptor and nonreceptor protein tyrosine kinases, mediates the transmission of growth-regulatory information within cells (reviewed in references 18, 21, and 82). These studies suggest that the activation of PI3K contributes a positive, but undefined, cell proliferation signal.The activity of the 70/85-kDa S6 protein kinases (␣I and ␣II isoforms, referred to collectively as pp70 S6k ) is also stimulated by IL-2 in IL-2-responsive cells (16,63,106) as well as in other cell types by many growth factors and oncogenes (reviewed in reference 36). However, the cytosolic mediators involved in its signal cascade have been previously unknown. pp70S6k was identified on the basis of its ability to phosphorylate the 40S ribosomal protein S6 in vitro. A number of other kinases, including the growth-regulated 90-kDa ribosomal S6 kinases (RSKs), can also phosphoryl...

The mitogen-activated protein kinases (MAPKs) ERK-1 and ERK-2 are activated by a wide variety of oncogenes and extracellular stimuli. The MAPKs participate in a signalling cascade downstream of growth factor/cytokine receptors, Ras, Raf, and MEK. However, MAPK activation is more complicated than a simple linear pathway, and the evidence presented here supports a model of multiple, temporally distinct pathways converging on MAPK which are differentially utilized by various stimuli and cell types. In addition to MEK-dependent MAPK activation, we provide evidence for MEK-independent regulation of the MAPKs. Our results suggest that phosphatidylinositol-3-kinases (PI(3)K) or conventional protein kinase C isoforms (cPKCs) partially contribute to MEK-dependent activation. Importantly, we also find that PI3K and cPKCs play a major role in the MEK-independent, prolonged MAPK activation by platelet-derived growth factor signalling. This finding is of interest as the maintained activation of MAPK has been correlated by others to the regulation of cell proliferation and differentiation.

Background: Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization ~40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.