The effect of IQGAP1 on Xenopus embryonic ectoderm requires Cdc42.

IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta-catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, we document here that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-DeltaIQ), Ras-GAP-related (IQGAP1-DeltaGRD), or C-terminal (IQGAP1-DeltaC) domains abrogated the effect of IQGAP1 on the embryos. None of the latter mutants bound Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-DeltaGRD, significantly increased the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocked the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm requires Cdc42 function.

A positive role for the PP2A catalytic subunit in Wnt signal transduction.

Protein phosphatase-2A (PP2A) is a multisubunit serine/threonine phosphatase involved in intracellular signaling, gene regulation, and cell cycle progression. Different subunits of PP2A bind to Axin and Adenomatous Polyposis Coli, components of the Wnt signal transduction pathway. Using early Xenopus embryos, we studied how PP2A functions in Wnt signal transduction. The catalytic subunit of PP2A (PP2A-C) potentiated secondary axis induction and Siamois reporter gene activation by Dishevelled, a component of the Wnt pathway, indicating a positive regulatory role of this enzyme in Wnt signaling. In contrast, small t antigen, an antagonist of PP2A-C, inhibited Dishevelled-mediated signal transduction, as did the regulatory PP2A-B'epsilon subunit, consistent with the requirement of PP2A function in this pathway. Although Wnt signaling is thought to occur via regulation of beta-catenin degradation, PP2A-C did not significantly affect beta-catenin stability. Moreover, the pathway activated by a stabilized form of beta-catenin was sensitive to PP2A-C and its inhibitors, suggesting that PP2A-C acts downstream of beta-catenin. Because previous work has suggested that PP2A can act upstream of beta-catenin, we propose that PP2A regulates the Wnt pathway at multiple levels.

Functional and structural diversity of the human Dickkopf gene family.

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.

Axis determination by inhibition of Wnt signaling in Xenopus.

The Wnt family of secreted polypeptides participate in a variety of developmental processes in which embryonic polarity is established. To study a role for Wnt ligands in vertebrate axis determination, we interfered with Wnt signaling in the embryo using the extracellular domain of Xenopus Frizzled 8 (ECD8), which blocks Wnt-dependent activation of a target gene in Xenopus ectodermal explants. Expression of ECD8 in ventral blastomeres resulted in formation of secondary axes containing abundant notochord and head structures. These results suggest that Wnt signaling is required to maintain ventral cell fates and has to be suppressed for dorsal development to occur.

Wnt signaling and dorso-ventral axis specification in vertebrates.

The dorso-ventral axis is specified in vertebrates through the formation of a dorsal signaling center known as the Spemann organizer. This process depends on signal transduction by beta-catenin that can be regulated by secreted Wnt proteins. Recent discoveries of new players in this signaling pathway have narrowed down the search for the initial cues for axis specification in vertebrate embryos.

Differential expression of frpHE: a novel human stromal protein of the secreted frizzled gene family, during the endometrial cycle and malignancy.

By using the differential display technique to identify genes that are differentially expressed in human endometrial carcinoma compared with normal endometrium, we have cloned frpHE, a novel member of the secreted frizzled gene family. By in situ hybridization, we have determined that frpHE is expressed by mesenchymal cells but not by epithelial cells. The expression of frpHE is modulated during the endometrial cycle: it is expressed in the stroma of proliferative endometrium and not significantly detectable in secretory or menstrual endometrium, suggesting that frpHE is under hormonal regulation. In addition, the expression of frpHE mRNA is markedly up-regulated in the stroma of endometrial hyperplasia and carcinoma and in the stroma of in situ and infiltrating breast carcinomas. Injection of frpHE mRNA in Xenopus embryos inhibited the Wnt-8 mediated dorsal axis duplication. These results indicate that frpHE functions as a regulator of the Wnt-frizzled signaling pathway and is involved in endometrial physiology and carcinogenesis.

The polycystic kidney disease 1 gene product modulates Wnt signaling.

Two distinct signaling pathways, involving Wnt signaling and polycystin, have been found to be critical for normal kidney development. Renal tubulogenesis requires the presence of certain Wnt proteins, whereas mutations in polycystin impede the terminal differentiation of renal tubular epithelial cells, causing the development of large cystic kidneys that characterize autosomal dominant polycystic kidney disease. Polycystin is an integral membrane protein, consisting of several extracellular motifs indicative of cell-cell and cell-matrix interactions, coupled through multiple transmembrane domains to a functionally active cytoplasmic domain. We report here that expression of the C-terminal cytoplasmic domain of polycystin stabilizes soluble endogenous beta-catenin and stimulates TCF-dependent gene transcription in human embryonic kidney cells. Microinjection of the polycystin C-terminal cytoplasmic domain induces dorsalization in zebrafish. Our findings suggest that polycystin has the capacity to modulate Wnt signaling during renal development.

Functional and biochemical interactions of Wnts with FrzA, a secreted Wnt antagonist.

Wnts are highly conserved developmental regulators that mediate inductive signaling between neighboring cells and participate in the determination of embryonic axes. Frizzled proteins constitute a large family of putative transmembrane receptors for Wnt signals. FrzA is a novel protein that shares sequence similarity with the extracellular domain of Frizzled. The Xenopus homologue of FrzA is dynamically regulated during early development. At the neurula stages, XfrzA mRNA is abundant in the somitic mesoderm, but later becomes strongly expressed in developing heart, neural crest derivatives, endoderm, otic vesicle and other sites of organogenesis. To evaluate possible biological functions of FrzA, we analyzed its effect on early Xenopus development. Microinjection of bovine or Xenopus FrzA mRNA into dorsal blastomeres resulted in a shortened body axis, suggesting a block of convergent extension movements. Consistent with this possibility, FrzA blocked elongation of ectodermal explants in response to activin, a potent mesoderm-inducing factor. FrzA inhibited induction of secondary axes by Xwnt8 and human Wnt2, but not by Xdsh, supporting the idea that FrzA interferes with Wnt signaling. Furthermore, FrzA suppressed Wnt-dependent activation of the early response genes in ectodermal explants and in the marginal zone. Finally, immunoprecipitation experiments demonstrate that FrzA binds to the soluble Wingless protein in cell culture supernatants in vitro. Our results indicate that FrzA is a naturally occurring secreted antagonist of Wnt signaling.

Wnt signaling and transcriptional control of Siamois in Xenopus embryos.

The Wnt-inducible homeobox gene Siamois is expressed in Xenopus embryos before gastrulation and is necessary for formation of the Spemann organizer. Here we show that 5'-flanking sequences of the Siamois coding region can specifically activate a heterologous reporter gene in dorsovegetal cells, thus mimicking Siamois's endogenous expression. A 245-bp DNA fragment is sufficient for activation by both Wnts and endogenous inducers. A dominant negative form of Xenopus T cell-specific factor 3 (XTCF-3) inhibited promoter activity, indicating that T cell-specific factor (TCF)/lymphocyte enhancer binding factor 1 (LEF-1) signaling is necessary for regulation of Siamois. Mutagenesis of two individual TCF sites in the -245 promoter revealed that the proximal, but not distal, site is necessary for dorsovegetal activation. These observations suggest that Siamois is directly regulated by TCFs during dorsoventral axis determination. Further deletion analysis identified a positive regulatory region that is required for dorsal activation, but not for Wnt inducibility, of the promoter. We also present evidence for autoregulation of Siamois transcription. Furthermore, the Siamois promoter was activated by Wnt signaling in 293T tissue culture cells, demonstrating that regulation of the promoter is functionally conserved.

Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin.

Signaling by the Wnt family of extracellular proteins is critical in a variety of developmental processes in which cell and tissue polarity are established [1-5]. Wnt signal transduction has been studied mostly by the genetic approach in Drosophila and Caenorhabditis elegans [1,2,5], but the biochemical mechanisms involved remain to be elucidated. The Wnt pathway also operates during axis determination in vertebrates [3,5]. Frizzled receptors transduce a signal to Dishevelled, leading to inactivation of glycogen synthase kinase 3 (GSK3) and regulation of gene expression by the complex of beta-catenin with LEF/TCF (lymphocyte enhancer factor/T-cell factor) transcription factors [3,5]. Axin is a negative regulator of Wnt signaling and dorsal axial development in vertebrates [6]. Here, we demonstrate that axin is associated with GSK3 in the Xenopus embryo and we localize the GSK3-binding domain to a short region of axin. Binding of GSK3 correlates with the ability of axin to inhibit axial development and with the axis-inducing activity of its dominant-negative form (delta RGS). We also find that wild-type axin, but not delta RGS, forms a complex with beta-catenin. Thus, axin may act as a docking station mediating negative regulation of beta-catenin by GSK3 during dorsoventral axis determination in vertebrate embryos.

A role for Siamois in Spemann organizer formation.

The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.

Xwnt-2b is a novel axis-inducing Xenopus Wnt, which is expressed in embryonic brain.

Xwnt-2b is a novel member of the Wnt gene family and is 73-74% similar to human and mouse Wnt-2 proteins. Starting from stage 15, Xwnt-2b transcripts are localized to a non-contiguous stripe in the anterior neural plate of the Xenopus embryo. In the tailbud, Xwnt-2b is expressed along the dorsoanterior side of the prosencephalon-mesencephalon boundary. At the tadpole stages, the brain-specific expression fades, but the total amount of Xwnt-2b mRNA does not decline due to activation of its expression in non-brain areas. Microinjection of Xwnt-2b mRNA into a ventral blastomere of 4-8-cell embryos results in the formation of complete secondary body axes. These results suggest that Xwnt-2b is a member of the axis-inducing Wnts and that it is involved in brain development and in later organogenesis.

Graded amounts of Xenopus dishevelled specify discrete anteroposterior cell fates in prospective ectoderm.

Signals emitted from the prospective dorsal marginal zone (the organizer) are thought to specify neuroectodermal cell fates along the anteroposterior (AP) axis, but the mechanisms underlying this signaling event remain to be elucidated. To assess the effect of Xenopus Dishevelled (Xdsh), a proposed component of the Wnt, Notch and Frizzled signal transduction pathways, on AP axis determination, it was supplied in varying doses to presumptive ectodermal cells. Two-fold increments in levels of microinjected Xdsh mRNA revealed a gradual shift in cell fates along the AP axis. Lower doses of Xdsh mRNA activated anterior neuroectodermal markers, XAG1 and Xotx2, whereas the higher doses induced more posterior neural tissue markers such as En2, Krox20 and HoxB9. At the highest dose of Xdsh mRNA, explants contained maximal amount of HoxB9 transcripts and developed notochord and somites. When compared with Xdsh, Xwnt8 mRNA also activated anterior neuroectodermal markers, but failed to elicit mesoderm formation. Analysis of explants overexpressing Xdsh at the gastrula stage revealed activation of several organizer-specific genes which have been implicated in determination of neural tissue (Xotx2, noggin, chordin and follistatin). Whereas Goosecoid, Xlim1 and Xwnt8 were not induced in these explants, another early marginal zone marker, Xbra, was activated at the highest level of Xdsh mRNA. These observations suggest that the effects of Xdsh on AP axis specification may be mediated by combinatorial action of several early patterning genes. Increasing levels of Xdsh mRNA activate posterior markers, whereas increasing amounts of the organizer stimulate the extent of anterior development (Stewart, R.M. and Gerhart, J.C. (1990) Development 109, 363-372). These findings argue against induction of the entire organizer by Xdsh in ectodermal cells and implicate signal transduction pathways involving Xdsh in AP axis determination. Thus, different levels of a single molecule, Xdsh, can specify distinct cell states along the AP axis.

Analysis of Dishevelled signalling pathways during Xenopus development.

BACKGROUND: Recent studies have demonstrated that the Wnt, Frizzled and Notch proteins are involved in a variety of developmental processes in fly, worm, frog and mouse embryos. The Dishevelled (Dsh) protein is required for Drosophila cells to respond to Wingless, Notch and Frizzled signals, but the molecular mechanisms of its action are not well understood. Using the ability of a mutant form of the Xenopus homologue of Dsh (Xdsh) to block Wnt and Dsh signalling in a model system, this work attempts to clarify the role of the endogenous Xdsh during the early stages of vertebrate development. RESULTS: A mutant Xdsh (Xdd1) with an internal deletion of the conserved PDZ/DHR domain was constructed. Overexpression of Xdd1 mRNA in ventral blastomeres of Xenopus embryos strongly inhibited induction of secondary axes by the wild-type Xdsh and Xwnt8 mRNAs, but did not affect the axis-inducing ability of beta-catenin mRNA. These observations suggest that Xdd1 acts as a dominant-negative mutant. Dorsal expression of Xdd1 caused severe posterior truncations in the injected embryos, whereas wild-type Xdsh suppressed this phenotype. Xdd1 blocked convergent extension movements in ectodermal explants stimulated with mesoderm-inducing factors and in dorsal marginal zone explants, but did not affect mesoderm induction and differentiation. CONCLUSIONS: A vertebrate homologue of Dsh is a necessary component of Wnt signal transduction and functions upstream of beta-catenin. These findings also establish a requirement for the PDZ domain in signal transduction by Xdsh, and suggest that endogenous Xdsh controls morphogenetic movements in the embryo.

Specific modulation of ectodermal cell fates in Xenopus embryos by glycogen synthase kinase.

Shaggy is a downstream component of the wingless and Notch signaling pathways which operate during Drosophila development. To address the role of glycogen synthase kinase 3 beta (GSK3 beta), a mammalian homologue of Shaggy, in vertebrate embryogenesis, it was overexpressed in Xenopus embryos. Microinjection of rat GSK3 beta mRNA into animal ventral blastomeres of 8-cell-stage embryos triggered development of ectopic cement glands with an adjacent anterior neural tissue as evidenced by in situ hybridization with Xotx2, a fore/midbrain marker, and NCAM, a pan-neural marker. In contrast, animal dorsal injection of the same dose of GSK3 beta mRNA caused eye deficiencies, whereas vegetal injections had no pronounced effects on normal development. Using several mutated forms of rat GSK3 beta, we demonstrate that the observed phenotypes are dose-dependent and tightly correlate with GSK3 beta enzymatic activity. Lineage tracing experiments showed that the effects of GSK3 beta are cell autonomous and that ectopic cement glands and eye deficiencies arose directly from cells containing GSK3 beta mRNA. Molecular marker analysis of ectodermal explants overexpressing GSK3 beta has revealed activation of Xotx2 and of cement gland marker XAG-1, but expression of NCAM and XIF-3 was not detected. Phenotypic effects of mRNA encoding a Xenopus homologue of GSK3 beta were identical to those of rat GSK3 beta mRNA. We hypothesize that GSK3 beta mediates the initial steps of neural tissue specification and modulates anteroposterior ectodermal patterning via activation of Otx2 transcription. Our observations implicate GSK3 beta in signaling pathways operating during neural tissue development and during specification of anterior ectodermal cell fates.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.

Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled.

Signaling factors of the Wnt proto-oncogene family are implicated in dorsal axis formation during vertebrate development, but the molecular mechanism of this process is not known. Studies in Drosophila have indicated that the dishevelled gene product is required for wingless (Wnt1 homolog) signal transduction. We demonstrate that injection of mRNA encoding a Xenopus homolog of dishevelled (Xdsh) into prospective ventral mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere contribute to anterior and dorsal structures of the induced axis. In contrast to its effect on mesoderm, overexpression of Xdsh mRNA in prospective ectodermal cells triggers anterior neural tissue differentiation. These studies suggest that Wnt signal transduction pathway is conserved between Drosophila and vertebrates and point to a role for maternal Xdsh product in dorsal axis formation and in neural induction.

The SH2-containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development.

SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. We assayed the effects of injection of wild-type and mutant SH-PTP2 RNAs on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. Our results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase.

The pregastrula establishment of gene expression pattern in Xenopus embryos: requirements for local cell interactions and for protein synthesis.

Although it has been proposed that mesoderm forms in the marginal zone of the amphibian embryo through inductive signaling from vegetal pole cells, the details of this process remain to be clarified. To determine when marginal zone cells become committed to mesodermal fates, cell contact and protein synthesis requirements for early transcriptional responses were analyzed in Xenopus blastulae. Marginal zone explants were isolated from embryos at different stages and either were cultured untreated, or were dissociated in a medium lacking calcium and magnesium ions, or cultured in the presence of cycloheximide. Whereas many mesoderm-specific transcripts are efficiently induced by activin in dissociated animal pole cells, the same markers were not activated in dissociated marginal zone cells, which were isolated from mid or even late blastulae and cultured in the absence of exogenous inducers. These observations suggest that early specification of mesodermal fates requires cell-cell interactions within the marginal zone and that the marginal zone cells are not committed to express early mesodermal markers until the late blastula stage. Specification of mesodermal fates was also assessed by the ability of marginal zone cells to express early mesodermal markers in the absence of protein synthesis. Induction of Xlim1, 1A11, and, partially, Xbrachyury transcripts in the marginal zone was blocked by cycloheximide treatment through late blastula stages, whereas Goosecoid and Xwnt8 mRNAs were expressed in the absence of protein synthesis, indicating that these sets of markers are activated in vivo through different pathways. These observations demonstrate that determination of mesoderm in the marginal zone is a multistep process which occurs during late blastula stages and depends on cell-cell contact and protein synthesis.