XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development.
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XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation. As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor. We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor. We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain. By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3. Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development. Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes. (+info)
Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus.
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The co-activation of Wnt signaling and concomitant inhibition of BMP signaling has previously been implicated in vertebrate neural patterning, as evidenced by the combinatorial induction of engrailed-2 and krox-20 in Xenopus. However, screens have not previously been conducted to identify additional potential target genes. Using a PCR-based screening method we determined that XA-1, xCRISP, UVS.2, two UVS.2-related genes, and xONR1 are induced in response to Xwnt-3a and a BMP-antagonist, noggin. Two additional genes, connexin 30 and retinoic acid receptor gamma were induced by Xwnt-3a alone. To determine whether any of the induced genes are direct targets of Wnt signaling, we focussed on engrailed-2. In the present study we show that the Xenopus engrailed-2 promoter contains three consensus binding sites for LEF/TCF, which are HMG box transcription factors which bind to beta-catenin in response to activation of the Wnt- 1 signaling pathway. An engrailed-2 promoter luciferase reporter construct containing these LEF/TCF sites is induced in embryo explant assays by the combination of Xwnt-3a or beta-catenin and noggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as well as expression of the reporter construct. Moreover, mutation of these three LEF/TCF sites abrogates expression of the reporter construct in response to noggin and Xwnt-3a or beta-catenin. We conclude that the engrailed-2 gene is a direct target of the Wnt signaling pathway, and that Wnt signaling works with BMP antagonists to regulate gene expression during patterning of the developing nervous system of Xenopus. (+info)
The modulation of inductive competence is a major theme in embryonic development, but, in most cases, the underlying mechanisms are not well understood. In principle, the capacity of extracellular signals to elicit particular responses could be regulated by changes in cell surface receptors, in intracellular signaling pathways, or in the responsiveness of individual target gene promoters. As an example of regulated competence, we have examined dorsal axis induction in Xenopus embryos by Wnt signaling. Competence of Wnt proteins such as Xwnt-8 to induce an ectopic axis or the dorsal early response genes siamois and Xnr3 is lost by the onset of gastrulation, when these same ligands now produce a distinct set of "late" effects, including anterior truncation and induction of the midbrain/hindbrain marker engrailed-2. Although other Wnts apparently make use of alternative signaling mechanisms, we demonstrate that late-expressed Xwnt-8 continues to employ the canonical Wnt signaling pathway used earlier in dorsal axis induction, stabilizing cytosolic beta-catenin, and activating gene expression through Tcf/Lef transcription factors. Moreover, an activated, hormone-inducible version of XTcf-3 (TVGR) that can reproduce both early and late Wnt responses when activated at appropriate stages becomes unable to induce siamois and secondary axes at the same time as Wnt ligands themselves. Finally, we show that TVGR also loses the ability to induce expression of a reporter construct containing a small fragment of the siamois promoter, implying that this fragment contains sequences governing the loss of Wnt responsiveness before gastrulation. Together, these results argue that the competence of Wnts to induce a dorsal axis is lost in the nucleus, as a result of changes in the responsiveness of target promoters. (+info)
Difference in XTcf-3 dependency accounts for change in response to beta-catenin-mediated Wnt signalling in Xenopus blastula.
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Wnt signalling functions in many tissues and during different stages of animal development to produce very specific responses. In early Xenopus embryos there is a dramatic change in response to Wnt signalling within only a few hours of development. Wnt signalling in very early embryos leads to a dorsalising response, which establishes the endogenous dorsal axis. Only a few hours later in development, almost the opposite happens: Xwnt-8 functions to pattern the embryonic mesoderm by promoting ventral and lateral mesoderm. The specificity of the response could conceivably be carried out by differential use of different signal transduction pathways, many of which have recently been described. We have found, however, that this dramatic shift in response to Wnt signalling in early Xenopus is not brought about by differential use of distinct signal transduction pathways. In fact beta-catenin, a downstream component of the canonical Wnt signal transduction pathway, functions not only in the early dorsalising response but also in the later ventrolateral-promoting response. Interaction of beta-catenin with the XTcf-3 transcription factor is required for the early dorsalising activity. In contrast, our experiments suggest that late Wnt signalling in the ventrolateral mesoderm does not require a similar dependency of beta-catenin function on XTcf-3. Our results highlight the potential versatility of the canonical Wnt pathway to interact with tissue-specific factors downstream of beta-catenin, in order to achieve tissue-specific effects. (+info)
Functional diversity of Xenopus lymphoid enhancer factor/T-cell factor transcription factors relies on combinations of activating and repressing elements.
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Lymphoid enhancer factor/T-cell factor (LEF/TCF) high mobility group box transcription factors are the nuclear transducers of the Wnt/beta-catenin signaling cascade. In Xenopus, three members of the LEF/TCF family, XLEF-1, XTCF-3, and XTCF-4, with distinct but partially overlapping expression patterns have been identified. The individual Xenopus LEF/TCF family members differ extremely in their properties of target gene regulation. We observed that in contrast to LEF-1, neither XTCF-3 nor XTCF-4 can induce secondary axis formation upon ventral overexpression in Xenopus embryos. To identify functional motifs within the LEF/TCF transcription factors responsible for target gene activation or repression, we created various mutants and a set of XLEF-1/XTCF-3 chimeras. In overexpression studies, we asked whether these constructs can mimic an activated Wnt/beta-catenin pathway and lead to the formation of a secondary body axis. In addition, we examined their capacity to rescue a loss-of-function phenotype given by dominant negative LEF-1 expression. We further analyzed their ability to directly activate target genes in reporter gene assays using the LEF/TCF target promoters, siamois and fibronectin. We found that a region homologous to exon IVa of human TCF-1 is an activating element. This is flanked by two small repressing motifs, LVPQ and SXXSS. Our findings implicate that the motifs identified here play an essential role in determining cell type-specific activity of LEF/TCF transcription factors. (+info)
Repression through a distal TCF-3 binding site restricts Xenopus myf-5 expression in gastrula mesoderm.
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The development of skeletal muscle in the vertebrate embryo is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf-5, MyoD, Myogenin, and MRF4. The dynamic expression pattern of myf-5 during myogenesis is thought to be consistent with its role during early determination of the myogenic lineage. To study the factors and mechanisms, which regulate myf-5 transcription in Xenopus, we isolated a genomic DNA clone containing 4858 bp of Xmyf-5 5' flanking region. Using a transgenic reporter assay, we show here that this genomic contig is sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of Xmyf-5 from the gastrula to tail bud stages. For the primary induction of myf-5 transcription, we identify three main regulatory elements, which are responsible for (i) activation in dorsal mesoderm, (ii) activation in ventral mesoderm, and (iii) repression in midline mesoderm, respectively. Their combined activities define the two-winged expression domain of myf-5 in the preinvoluted mesoderm. Repression in midline mesoderm is mediated by a single TCF binding site located in the 5' end of the -4.8 kbp sequence, which binds XTcf-3 protein in vitro. Endogenous Wnt signaling in the lateral mesoderm is required to overcome the long-range repression through this distal TCF site, and to stimulate myf-5 transcription independently from it. The element for ventral mesoderm activation responds to Activin. Together, these results describe a regulatory mosaic of repression and activation, which defines the myf-5 expression profile in the frog gastrula. (+info)
Lef-1 and Tcf-3 transcription factors mediate tissue-specific Wnt signaling during Xenopus development.
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Wnt signaling functions repeatedly during embryonic development to induce different but specific responses. What molecular mechanisms ensure that Wnt signaling triggers the correct tissue-specific response in different tissues? Early Xenopus development is an ideal model for addressing this fundamental question, since there is a dramatic change in the response to Wnt signaling at the onset of zygotic gene transcription: Wnt signaling components encoded by maternal mRNA establish the dorsal embryonic axis; zygotically expressed Xwnt-8 causes almost the opposite, by promoting ventral and lateral and restricting dorsal mesodermal development. Although Wnt signaling can function through different signal transduction cascades, the same beta-catenin-dependent, canonical Wnt signal transduction pathway mediates Wnt signaling at both stages of Xenopus development. Here we show that, while the function of the transcription factor XTcf-3 is required for early Wnt signaling to establish the dorsal embryonic axis, closely related XLef-1 is required for Wnt signaling to pattern the mesoderm after the onset of zygotic transcription. Our results show for the first time that different transcription factors of the Lef/Tcf family function in different tissues to bring about tissue-specific responses downstream of canonical Wnt signaling. (+info)
VegT activation of the early zygotic gene Xnr5 requires lifting of Tcf-mediated repression in the Xenopus blastula.
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Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-beta factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localised throughout the vegetal pole, and beta-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, we have defined a minimal promoter, which mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional beta-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that beta-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a beta-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. We propose a model in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of beta-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of beta-catenin/XTcf-3. (+info)