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D Kimelman

Publications and source records attributed to D Kimelman.

At least 37 records · Page 2Linked to original sources

Transcriptional regulation in Xenopus: a bright and froggy future.

Xenopus has played a key role in defining the general mechanisms that underlie early vertebrate development. Recent studies reveal how the transcriptional regulation of signaling and transcription factors is used to pattern the early dorsal-ventral axis. With the development of new methods for producing transgenic frogs, Xenopus will become a very attractive system for studying transcriptional regulation at all stages of embryogenesis.

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XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development.

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.

Amino Acid Sequence↗

GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis.

Dorsal accumulation of beta-catenin in early Xenopus embryos is required for body axis formation. Recent evidence indicates that beta-catenin is dorsally stabilized by the localized inhibition of the kinase Xgsk-3, utilizing a novel Wnt ligand-independent mechanism. Using a two-hybrid screen, we identified GBP, a maternal Xgsk-3-binding protein that is homologous to a T cell protooncogene in three well-conserved domains. GBP inhibits in vivo phosphorylation by Xgsk-3, and ectopic GBP expression induces an axis by stabilizing beta-catenin within Xenopus embryos. Importantly, antisense oligonucleotide depletion of the maternal GBP mRNA demonstrates that GBP is required for the establishment of the dorsal-ventral axis in Xenopus embryos. Our results define a family of GSK-3-binding proteins with roles in development and cell proliferation.

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From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus.

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal-determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt-1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that beta-catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, beta-catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of beta-catenin and Tcf-3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf-3 likely works in the absence of beta-catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of beta-catenin establishes a range of subsequent dorsoventral asymmetries in the embryo.

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Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes.

Inhibition of fibroblast growth factor (FGF) signaling prevents trunk and tail formation in Xenopus and zebrafish embryos. While the T-box transcription factor Brachyury (called No Tail in zebrafish) is a key mediator of FGF signaling in the notochord and tail, the pathways activated by FGF in non-notochordal trunk mesoderm have been uncertain. Previous studies have shown that the spadetail gene is required for non-notochordal trunk mesoderm formation; spadetail mutant embryos have major trunk mesoderm deficiencies, but relatively normal tail and notochord development. We demonstrate here that spadetail encodes a T-box transcription factor with homologues in Xenopus and chick. Spadetail is likely to be a key mediator of FGF signaling in trunk non-notochordal mesoderm, since spadetail expression is regulated by FGF signaling. Trunk and tail development are therefore dependent upon the complementary actions of two T-box genes, spadetail and no tail. We show that the regulatory hierarchy among spadetail, no tail and a third T-box gene, tbx6, are substantially different during trunk and tail mesoderm formation, and propose a genetic model that accounts for the regional phenotypes of spadetail and no tail mutants.

Amino Acid Sequence↗

A beta-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus.

The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, we show that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos.

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Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway.

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.

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Spatial regulation of floating head expression in the developing notochord.

The zebrafish homeobox gene floating head (flh) is essential for notochord development and is one of the earliest genes to be expressed in notochord precursors. To understand how flh is regulated during notochord development, we compared the wild-type flh expression pattern to that in embryos mutant for flh and no tail (ntl), the zebrafish homologue of Brachyury. In the early gastrula, the pattern of flh expression is not affected in either flh or ntl mutants, implying that the initial establishment of a gastrula notochord domain is independent of the function of these genes. However, flh RNA is expressed at lower levels in flh mutants suggesting that flh positively regulates its own expression. During gastrulation, flh mutants show an abrupt loss of flh expression in cells which have involuted and entered the hypoblast, while the rest of the expression pattern appears normal, thus flh+ function is specifically required to maintain flh expression in hypoblast cells. The anterior-most part of the notochord rudiment differentially maintains flh expression in both wild types and flh mutant embryos, suggesting that there is unique regulation of flh in this region of the developing notochord. In ntl mutants the spatial pattern of flh expression is altered as early as the late gastrula stage, becoming broad and diffuse. We hypothesize that ntl+ is required for the proper convergence movements of flh-expressing cells.

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A role for notochord in axial vascular development revealed by analysis of phenotype and the expression of VEGR-2 in zebrafish flh and ntl mutant embryos.

The notochord is required for the differentiation of nearby tissues, including the neural tube and the floor plate. Because the dorsal aorta and axial vein are midline structures, their development might also be influenced by the notochord. To investigate this possibility, we cloned zebrafish VEGR-2, homologous to the earliest known marker of endothelial cells in mammals. In flh and ntl mutant embryos, which lack a notochord, we found a defect in axial blood vessel formation, and a delay in the fusion of VEGR-2 positive endothelial progenitor cells into the primary vascular plexus and a block in the establishment of mature vessels. Differences in the vascular phenotype between the two mutations correlated with the severity of their axial mesodermal defects. These observations support a role for the notochord in vasculogenesis.

Amino Acid Sequence↗

Activation of Siamois by the Wnt pathway.

The Wnt pathway and the recently described Xenopus homeobox gene Siamois share the ability to mimic Nieuwkoop organizer signaling activity when ectopically expressed, prompting us to ask whether the Wnt pathway can activate Siamois expression. We show that activators of the Wnt pathway, and the dorsalizing substance LiCl, activate Siamois expression in Xenopus animal caps. Furthermore, Siamois expression is shown to be activated by beta-catenin cell-autonomously. Although certain TGF-beta family members, such as Vg1, are potent dorsal mesoderm inducers, the TGF-beta pathway only weakly activated Siamois. Finally, we demonstrate that Siamois expression is eliminated from the marginal zone of UV-irradiated Xenopus embryos, supporting the hypothesis that cortical rotation moves a Wnt-like activity into the dorsal region. These results suggest that formation of the Nieuwkoop organizer is dependent on activation of Siamois by a localized Wnt-like signaling activity, which may act synergistically with a vegetally localized TGF-beta signal.

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The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.

The serine/threonine kinase Xgsk-3 and the intracellular protein beta-catenin are necessary for the establishment of the dorsal-ventral axis in Xenopus. Although genetic evidence from Drosophila indicates that Xgsk-3 is upstream of beta-catenin, direct interactions between these proteins have not been demonstrated. We demonstrate that phosphorylation of beta-catenin in vivo requires an in vitro amino-terminal Xgsk-3 phosphorylation site, which is conserved in the Drosophila protein armadillo. beta-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type beta-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of beta-catenin that acts through the amino-terminal site. Inhibition of endogenous Xgsk-3 function with a dominant-negative mutant leads to an increase in the steady-state levels of ectopic beta-catenin, indicating that Xgsk-3 functions to destabilize beta-catenin and thus decrease the amount of beta-catenin available for signaling. The levels of endogenous beta-catenin in the nucleus increases in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of beta-catenin within specific subcellular compartments. These studies provide a basis for understanding the interaction between Xgsk-3 and beta-catenin in the establishment of the dorsal-ventral axis in early Xenopus embryos.

Amino Acid Sequence↗

Overexpression of Xgsk-3 disrupts anterior ectodermal patterning in Xenopus.

The Xenopus homolog of glycogen synthase kinase-3, Xgsk-3, plays a major role in regulating the formation of the dorsal-ventral axis, most likely through effects on the mesoderm. To determine whether Xgsk-3 is involved in ectodermal patterning, Xgsk-3 was ectopically overexpressed in the presumptive ectoderm. This approach resulted in a dramatically expanded cement gland, which is due to early changes in cement gland specification at the anterior end of the embryo. Explant experiments were used to show that Xgsk-3 overexpression enhances the response of ectoderm to cement-gland-inducing signals from the mesoderm and to the intercellular signaling factor noggin. Expression of two other noggin-inducible genes, Xotx2 and XANF-2, was also expanded in whole embryos, while the expression of the epidermal marker, Xgbx-2, was eliminated. These results suggest that Xgsk-3 may play a role in anterior ectodermal patterning as a component of an intracellular pathway that regulates the ectodermal responsiveness to endogenous inducing signals.

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The Xenopus laevis homeobox gene Xgbx-2 is an early marker of anteroposterior patterning in the ectoderm.

In a search for homeobox genes expressed during early Xenopus development, we have isolated a gene which appears to be the Xenopus cognate of the mouse Gbx-2 gene. Expression of Xgbx-2 is first detectable by in situ hybridization at the midgastrula stage when it is predominantly expressed in the dorsolateral ectoderm, with a gap in expression at the dorsal midline. By the end of gastrulation and during neurulation, Xgbx-2 is expressed dorsolaterally in the neural ectoderm and laterally and ventrally in the epidermis with sharp anterior expression borders in both tissues. The anteriormost expression in the neural ectoderm persists throughout the early stages of development, and was mapped to the region of rhombomere 1, with an anterior expression border in the region of the midbrain-hindbrain boundary. Thus Xgbx-2 is expressed anterior to the Hox genes. Xgbx-2 expression is induced by retinoic acid (RA) in animal caps, and RA treatment of whole embryos expands and enhances Xgbx-2 expression in the ectoderm. We suggest a role for Xgbx-2 in establishing the midbrain-hindbrain boundary, which appears to separate early neurectodermal regions expressing genes that are positively and negatively regulated by RA.

Amino Acid Sequence↗

Regulation of dorsal-ventral patterning: the ventralizing effects of the novel Xenopus homeobox gene Vox.

The formation of the dorsal-ventral axis in Xenopus laevis is elicited by a signaling cascade on the dorsal side of the embryo initiated by cortical rotation. These early developmental events impart an initial axial polarity to the embryo. By the time gastrulation occurs, the embryo has established opposing dorsal and ventral regulatory regions. Through a dynamic process, the embryo acquires a definitive pattern that reflects the distribution of future cell fates. Here we present a novel homeobox gene, Vox, whose expression reflects this dynamic process. Vox is first expressed throughout the embryo and subsequently eliminated from the notochord and neural plate. Ectopic expression of Vox demonstrates that the normal function of this gene may be to suppress dorsal genes such as Xnot and chordin, and induce ventral and paraxial genes such as Bmp-4 and MyoD. Ectopic expression of BMP-4 ventralizes embryos and positively regulates the expression of Vox, suggesting that these genes are components of a reciprocal regulatory network.

Amino Acid Sequence↗

Regulation of dorsal-ventral axis formation in Xenopus by intercellular and intracellular signalling.

The dorsal-ventral axis in the frog, Xenopus laevis, is specified by a microtubule-based process that begins at fertilization. Rotation of the egg cytoplasm leads to the activation of a signalling system on the future dorsal side of the embryo that is responsible for the formation of the Spemann organizer at the dorsal equator. Members of the Wnt family, as well as noggin and activated Vg1, have been shown to mimic this signalling system, but it is not known which factor or factors are used by the embryo. To elucidate this process, we have isolated the Xenopus homologue of the Drosophila zw3/shaggy gene, Xgsk-3, since zw3/shaggy is regulated by the Drosophila Wnt homologue, wingless. We have found that a dominant-negative mutant of Xgsk-3 can induce an ectopic organized when expressed on the ventral side of the Xenopus embryo, leading to embryos with duplicated axes. These results indicate that repression of Xgsk-3 is critical for formation of the dorsal-ventral axis in Xenopus. Inactivation of Xgsk-3 can regulate the fate of neighbouring cells, indicating that the repression of Xgsk-3 may activate a downstream signalling pathway.

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A homeobox gene essential for zebrafish notochord development.

The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. We show that floating head is the zebrafish homologue of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. We propose that flh regulates notochord precursor cell fate.

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BMP-4 regulates the dorsal-ventral differences in FGF/MAPKK-mediated mesoderm induction in Xenopus.

Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. In this study, we establish that fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. We demonstrate that a partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Similar to the results reported earlier with delta FGFR, expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. We tested whether the regional localization of bone morphogenetic protein-4 (BMP-4) could explain why both MAPKKD and delta FGFR eliminate the dorsal and not the ventral expression of Xcad-3 and Xbra. We show that ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR and that expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction.

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