Regulated expression of the retinoblastoma gene product by fibroblast growth factor but not by activin during mesoderm induction in Xenopus

 The retinoblastoma (RB) gene is a tumor suppressor gene that plays an important role in cell cycle arrest and in the terminal differentiation of skeletal myoblasts. Differentiation into muscle occurs in Xenopus embryo explants during mesoderm induction by fibroblast growth factor (FGF) or activin A. We examined expression of the RB gene product (pRB) during mesoderm induction in vivo and in vitro. We show that hypo- and hyper-phosphorylated forms of pRB are present during early development and that expression of both forms increases significantly during the blastula stage, concomitant with mesoderm induction. Further investigation revealed that pRB is enriched in the presumptive mesoderm of the blastula stage embryo. In animal cap explants induced by Xenopus bFGF (XbFGF), pRB expression levels increased approximately tenfold while no increase was observed in explants induced by activin. However, when explants were induced by XbFGF in the presence of sodium orthovanadate, a compound previously shown to synergize with FGF to produce more dorsal ”activin-like” inductions than FGF alone, only a slight increase in pRB expression was observed. Furthermore, upregulation of pRB during mesoderm induction in vitro displayed an inverse correlation with expression of XFKH1, a marker for notochord. These results suggest that pRB may be important for patterning along the dorsoventral axis. Received: 22 February 1996 / Accepted: 20 September 1996     

Induction of dorsal and ventral mesoderm by ectopically expressed Xenopus basic fibroblast growth factor.

Peptide growth factors from the fibroblast growth factor (FGF) and transforming growth factor-beta families are likely regulators of mesoderm formation in the early Xenopus embryo. Although basic FGF is found in the Xenopus embryo at the correct time and at sufficient concentrations to suggest that it is the FGF-type inducer, the lack of a secretory signal sequence in the basic FGF peptide has raised questions as to its role in the inductive process. We show here that Xenopus basic FGF can ectopically induce mesoderm when translated from injected synthetic RNA within the cells of a Xenopus embryo. Basic FGF produced in this manner is able to induce the formation of both dorsal and ventral mesoderm with the type of mesoderm formed dependent on the inherent dorsal-ventral polarity of the animal hemisphere. Surprisingly, although Xenopus basic FGF produced from the injected mRNA has a potent mesodermalizing effect on animal hemisphere cells, virtually no phenotypic effect is observed with intact embryos. These results suggest that the role of Xenopus basic FGF is to specify the size of the marginal zone, and synergistically with a dorsally localized prepatterning signal, to initially establish the dorsal-ventral axis of the mesoderm.     

Direct activation of phospholipase C-gamma by fibroblast growth factor receptor is not required for mesoderm induction in Xenopus animal caps.

Members of the fibroblast growth factor (FGF) family induce mesoderm formation in explants of Xenopus embryonic ectoderm (animal caps). Recent studies have been directed at determining signaling pathways downstream of the FGF receptor that are important in mesoderm induction. We have recently shown that a point mutation in the FGF receptor changing tyrosine 766 to phenylalanine (Y/F mutation) abolishes phospholipase C-gamma (PLC-gamma) activation in mammalian cells. To explore the role of PLC-gamma activation in FGF-stimulated mesoderm induction, we constructed two chimeric receptors, each consisting of the extracellular portion of the platelet-derived growth factor beta receptor, with one having the transmembrane and intracellular portions of the wild-type FGF receptor 1 (PR-FR wt) and the other having the corresponding region of the Y/F766 mutant FGF receptor 1 (PR-FR Y/F766). When expressed in Xenopus oocytes, only PR-FR wt was able to mediate PLC gamma phosphorylation, inositol-1,4,5-trisphosphate accumulation, and calcium efflux in response to platelet-derived growth factor stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps as measured by cap elongation, muscle-specific actin mRNA induction, and skeletal muscle formation. These results demonstrate that PLC gamma activation by the FGF receptor is not required for FGF-stimulated mesoderm induction.     

Nuclear translocation of cell-surface receptors: lessons from fibroblast growth factor

The nuclear localization of a number of growth factors, cytokine ligands and their receptors has been reported in various cell lines and tissues. These include members of the fibroblast growth factor (FGF), epidermal growth factor and growth hormone families. Accordingly, a number of nuclear functions have begun to emerge for these protein families. The demonstration of functional interactions of these proteins with the nuclear import machinery has further supported their functions as nuclear signal transducers. Here, we review the membrane- trafficking machinery and pathways demonstrated to regulate this cell surface to nucleus-trafficking event and highlight the many remaining unanswered questions. We focus on the FGF family, which is providing many of the clues as to the process of this unusual phenomenon.     

Fibroblast growth factor during mesoderm induction in the early chick embryo

A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.     

Activin as a morphogen in Xenopus mesoderm induction.

Activin, a member of the Transforming Growth Factor beta (TGF-beta) superfamily, can behave as a morphogen in cells of the early Xenopus embryo by inducing a range of mesodermal genes in a concentration-dependent manner. This review examines the behaviour of activin as it forms a morphogen gradient. It also discusses how a cell can perceive its position in a concentration gradient in order to activate appropriate mesodermal gene responses.     

SNT-1/FRS2alpha physically interacts with Laloo and mediates mesoderm induction by fibroblast growth factor.

Members of the fibroblast growth factor (FGF) ligand family play a critical role in mesoderm formation in the frog Xenopus laevis. While many components of the signaling cascade triggered by FGF receptor activation have been identified, links between these intracellular factors and the receptor itself have been difficult to establish. We report here the characterization of Xenopus SNT-1 (FRS2alpha), a scaffolding protein previously identified as a mediator of FGF activity in other biological contexts. SNT-1 is widely expressed during early Xenopus development, consistent with a role for this protein in mesoderm formation. Ectopic SNT-1 induces mesoderm in Xenopus ectodermal explants, synergizes with low levels of FGF, and is blocked by inhibition of Ras activity, suggesting that SNT-1 functions to transmit signals from the FGF receptor during mesoderm formation. Furthermore, dominant-inhibitory SNT-1 mutants inhibit mesoderm induction by FGF, suggesting that SNT-1 is required for this process. Expression of dominant-negative SNT-1 in intact embryos blocks mesoderm formation and dramatically disrupts trunk and tail development, indicating a requirement for SNT-1, or a related factor inhibited by the mutant construct, during axis formation in vivo. Finally, we demonstrate that SNT-1 physically associates with the Src-like kinase Laloo, and that SNT-1 activity is required for mesoderm induction by Laloo, suggesting that SNT-1 and Laloo function as components of a signaling complex during mesoderm formation in the vertebrate.     

Dkk3 is required for TGF-beta signaling during Xenopus mesoderm induction

Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.     

Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos

Mesoderm development in Xenopus laevis depends on inductive cell interactions mediated by diffusible molecules. The mesoderm inducer activin is capable of redirecting the development of animal explants both morphologically and biochemically. We have studied the induction of four regulatory genes, Mix. 1, goosecoid (gsc), Xlim-1 and Xbra in such explants by activin, and the influence of other factors on this induction. Activin induction of gsc is strongly enhanced by dorsalization of the embryo by LiCl, while expression of the other genes is only slightly enhanced. The protein synthesis inhibitor cycloheximide (CHX) inhibits the activin-dependent induction of Xbra partially, while induction of Mix. 1 and Xlim- 1 is essentially unaffected. In contrast, gsc shows strong superinduction in the presence of activin and CHX, and can be induced in animal explants by CHX alone. Induction and superinduction by CHX have previously been observed for immediate early genes in a variety of systems, notably for the activation of c-fos expression by serum stimulation, but have not been reported in early amphibian embryos. © 1993Wiley-Liss, Inc.     

Clonal analysis of mesoderm induction in Xenopus laevis

Acidic fibroblast growth factor (aFGF) has been used to induce mesoderm from single animal pole cells of midblastula stage Xenopus embryos. The cells are individually cultured in a completely defined medium and are able to differentiate as small clones in a high proportion of cases. FGF-treated cells can give rise to several mesodermal cell types, while untreated cells show only epidermal or neural differentiation. Mesodermal differentiation can occur in clones of as few as eight cells, indicating that any additional cell-cell interactions required for mesodermal differentiation can be met by the medium used.     

Mesoderm induction by the mesoderm of Xenopus neurulae

Combinations were made between explants of mesoderm from the archenteron roof of early Xenopus neurulae and explants of ectoderm from mid-blastulae. In each combination one component was labeled with the fluorescent lineage label RDA (rhodamine-dextran-amine). Frequent and large mesoderm inductions, consisting mainly of muscle, were found where the presomite plate was used as the inducer. Less frequent and smaller mesoderm inductions were found when notochord was used as the inducer. We conclude that induced mesoderm can itself be active as a mesoderm inducing tissue. If this capability is acquired in the blastula then it follows that mesoderm induction must propagate from cell to cell and its spread be antagonized by some other factor.     

Involvement of the MAP kinase cascade in Xenopus mesoderm induction.

Mitogen-activated protein kinase (MAPK) is activated by MAPK kinase (MAPKK) in a variety of signaling pathways. This kinase cascade has been shown to function in cell proliferation and differentiation, but its role in early vertebrate development remains to be investigated. During early vertebrate embryogenesis, the induction and patterning of mesoderm are thought to be determined by signals from intercellular factors such as members of the fibroblast growth factor (FGF) family and members of the transforming growth factor-beta family. Here we show that the microinjection of either mRNA encoding a constitutively active mutant of MAPKK or mRNA encoding a constitutively active form of STE11, a MAPKK kinase, leads to the induction of mesoderm in ectodermal explants from Xenopus embryos. Moreover, the expression of MAPK phosphatase-1 (MKP-1, also called CL100) blocks the growth factor-stimulated mesoderm induction. Furthermore, injection of CL100 mRNA into two-cell stage embryos causes severe defects in gastrulation and posterior development. The effects induced by CL100 can be rescued by co-injection of wild-type MAPK mRNA. Thus, the MAPK cascade may play a crucial role in early vertebrate embryogenesis, especially during mesoderm induction.     

Nuclear translocation of Xenopus laevis paxillin

Paxillin has been recognized as a focal adhesion adapter protein that participates in the integrin-mediated signaling. An earlier study [Ogawa et al. Biochim. Biophys. Acta 1519 (2001) 235] found that frog paxillin was expressed in the kidney epithelial cell line A6 and localized in the nucleus. Here, in this study, we have found that the expression of frog paxillin is up-regulated in the S phase of cell cycle. The protein became phosphorylated on tyrosine when the cells were grown on vitronectin; the tyrosine phosphorylation was not detectable when the cells were cultured on fibronectin, laminin or poly-D-lysine. On the other hand, MAP kinase was revealed to phosphorylate frog paxillin on serine. Both phosphorylation events, namely on tyrosine and serine, were essential for the nuclear translocation of this protein. Our results suggest that the integrin-mediated signaling pathway and the MAP kinase pathway meet at paxillin.