Xenopus msx-1 regulates dorso-ventral axis formation by suppressing the expression of organizer genes

We demonstrated previously that Xmsx-1 is involved in mesoderm patterning along the dorso-ventral axis, under the regulation of BMP-4 signaling. When Xmsx-1 RNA was injected into the dorsal blastomeres, a mass of muscle tissue formed instead of notochord. This activity was similar to that of Xwnt-8 reported previously. In this study, we investigated whether the activity of Xmsx-1 is related to the ventralizing signal and myogenesis promoting factor, Xwnt-8. Whole-mount in situ hybridization showed that Xmsx-1, Xwnt-8, and XmyoD were expressed in overlapping areas, including the ventro-lateral marginal zone at mid-gastrula stage. The expression of XmyoD was induced by the ectopic expression of either Xmsx-1 or Xwnt-8 in dorsal blastomeres, and Xwnt-8 was induced by the ectopic expression of Xmsx-1. On the other hand, the expression of Xmsx-1 was not affected by the loading of pCSKA-Xwnt-8 or dominant-negative Xwnt-8 (DN-Xwnt-8) RNA. In addition, Xmsx-1 RNA did not abrogate the formation of notochord if coinjected with DN-Xwnt-8 RNA. These results suggest that Xmsx-1 functions upstream of the Xwnt-8 signal. Furthermore, the antagonistic function of Xmsx-1 to the expression of organizer genes, such as Xlim-1 and goosecoid, was shown by in situ hybridization analysis and luciferase reporter assay using the goosecoid promoter construct. Finally if Xmsx-1/VP-16 fusion RNA, which was expected to function as a dominant-negative Xmsx-1, was injected into ventral blastomeres, a partial secondary axis formed in a significant number of embryos. In such embryos, the activity of luciferase, under the control of goosecoid promoter sequence, was significantly elevated at gastrula stage. These results led us to conclude that Xmsx-1 plays a central role in establishing dorso-ventral axis in gastrulating embryo, by suppressing the expression of organizer genes.     

The maternal Xenopus beta-catenin signaling pathway, activated by frizzled homologs, induces goosecoid in a cell non-autonomous manner

In spite of abundant evidence that Wnts play essential roles in embryonic induction and patterning, little is known about the expression or activities of Wnt receptors during embryogenesis. The isolation and expression of two maternal Xenopus frizzled genes, Xfrizzled-1 and Xfrizzled-7, is described. It is also demonstrated that both can activate the Wnt/beta-catenin signaling pathway as monitored by the induction of specific target genes. Activation of the beta-Catenin pathway has previously been shown to be necessary and sufficient for specifying the dorsal axis of Xenopus. beta-Catenin is thought to work through the cell-autonomous induction of the homeobox genes siamois and twin, that in turn bind to and activate the promoter of another homeobox gene, goosecoid. However, it was found that the beta-catenin pathway regulated the expression of both endogenous goosecoid, and a goosecoid promoter construct, in a cell non-autonomous manner. These data demonstrate that maternal Frizzleds can activate the Wnt/beta-catenin pathway in Xenopus embryos, and that induction of a known downstream gene can occur in a cell non-autonomous manner.     

Xenopus Wnt-5a induces an ectopic larval tail at injured site, suggesting a crucial role for noncanonical Wnt signal in tail regeneration

Amputation of the larval tail of Xenopus injures the notochord, spinal cord, muscle masses, mesenchyme, and epidermis, induces the growth and differentiation of cells in those tissues, and results in tail regeneration. A dorsal incision in the larval tail injures the same tissues and induces cell growth and differentiation, but never results in the formation of any extra appendages. The first sign of tail regeneration is the multilayered wound epidermis and Xwnt-5a expression in the distal region, neither of which is observed in the recovering region after a dorsal incision. To evaluate the role of Xwnt-5a in tail regeneration, Xwnt-5a was overexpressed in the recovering region. When an animal cap injected with Xwnt-5a mRNA was grafted into the dorsal incision, an ectopic protrusion was formed. Morphological and molecular analyses revealed that the protrusion was an ectopic larval tail, which was equivalent to the regenerating tail but different from the tail that develops from the embryonic tail bud. Lineage labeling revealed that the major differentiated structures of the ectopic tail were formed from host cells, suggesting that Xwnt-5a induced host cells to make a complete tail. The ectopic tail was not induced by Xwnt-8 or Xwnt-11, demonstrating the specificity of Xwnt-5a in this process. A pharmacological study showed that JNK signaling is required in tail regeneration. These results support the proposition that Xwnt-5a plays an instructive role in larval tail regeneration via Wnt/JNK signaling.     

Injected Wnt RNA induces a complete body axis in Xenopus embryos.

Studies in Xenopus have shown that growth factors of the TGF beta and Wnt oncogene families can mimic aspects of dorsal axis formation. Here we directly compare the inductive properties of two Wnt proteins by injecting synthetic mRNA into developing embryos. The results show that Wnt-1 and Xwnt-8 can induce a new and complete dorsal axis and can rescue the development of axis-deficient, UV-irradiated embryos. In contrast, activin mRNA injection induces only a partial dorsal axis that lacks anterior structures. These studies demonstrate that the mechanism of Wnt-induced axis duplication results from the creation of an independent Spemann organizer. The relationship between the properties of the endogenous dorsal inducer and the effects of Wnts and activins is discussed.     

Murine Wnt-1 with an internal c-myc tag recombinantly produced in Escherichia coli can induce intracellular signaling of the canonical Wnt pathway in eukaryotic cells

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.     

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.     

Functional specificity of the Xenopus T-domain protein Brachyury is conferred by its ability to interact with Smad1

Members of the T-box gene family play important and diverse roles in development and disease. Here, we study the functional specificities of the Xenopus T-domain proteins Xbra and VegT, which differ in their abilities to induce gene expression in prospective ectodermal tissue. In particular, VegT induces strong expression of goosecoid whereas Xbra cannot. Our results indicate that Xbra is unable to induce goosecoid because it directly activates expression of Xom, a repressor of goosecoid that acts downstream of BMP signaling. We show that the inability of Xbra to induce goosecoid is imposed by an N-terminal domain that interacts with the C-terminal MH2 domain of Smad1, a component of the BMP signal transduction pathway. Interference with this interaction causes ectopic activation of goosecoid and anteriorization of the embryo. These findings suggest a mechanism by which individual T-domain proteins may interact with different partners to elicit a specific response.     

Antisense inhibition of Xbrachyury impairs mesoderm formation in Xenopus embryos

Expression of the Xbrachyury (Xbra) gene was inhibited by antisense RNA synthesized in situ from an expression vector read by RNA polymerase III, injected into the fertilized egg or the 2-cell stage embryo of Xenopus laevis. Antisense-treated embryos had markedly reduced levels of Xbra mRNA and protein, and showed deficiencies in mesodermal derivatives and axis formation. In particular, organization of the posterior axis was affected, but often the anterior axis was also reduced. Some embryos failed to form mesoderm altogether and remained amorphous. The antisense effect is dose-dependent and may be "rescued" by overexpression of Xbra. In Xbra-deficient embryos, expression of several mesodermal genes (Xvent, pintallavis, Xlim, Xwnt-8 and noggin) was reduced to varying degrees, whereas goosecoid levels remained normal. The modified expression levels were partly normalized when Xbra deficiency was rescued. The observation that antisense inhibition yields slightly different phenotypes from dominant-negative inhibition suggests the recommendation of using several surrogate genetic approaches to determine the functional role of a gene in Xenopus development.     

Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells

We have recently reported the chondrogenic effect of bone morphogenetic protein-2 (BMP-2) in high density cultures of the mouse multipotent mesenchymal C3H10T1/2 cell line and have shown the functional requirement of the cell-cell adhesion molecule N-cadherin in BMP-2-induced chondrogenesis in vitro (Denker, A. E., Nicoll, S. B., and Tuan, R. S. (1995) Differentiation 59, 25-34; Haas, A. R., and Tuan, R. S. (1999) Differentiation 64, 77-89). Furthermore, BMP-2 treatment also results in an increased protein level of beta-catenin, a known N-cadherin-associated Wnt signal transducer (Fischer, L., Haas, A., and Tuan, R. S. (2001) Signal Transduction 2, 66-78), suggesting functional cross-talk between the BMP-2 and Wnt signaling pathways. We have observed previously that BMP-2 treatment up-regulates expression of Wnt-3A in high density cultures of C3H10T1/2 cells. To assess the contribution of Wnt-3A to BMP-2-mediated chondrogenesis, we have generated C3H10T1/2 cell lines overexpressing Wnt-3A and various forms of glycogen synthase kinase-3beta (GSK-3beta), an immediate cytosolic component of the Wnt signaling pathway, and examined their response to BMP-2. We show that overexpression of either Wnt-3A or kinase-dead GSK-3beta enhances BMP-2-mediated chondrogenesis. Furthermore, Wnt-3A overexpression results in decreases in both N-cadherin and GSK-3beta protein levels, whereas Wnt-3A as well as kinase-dead GSK-3beta overexpression increase total and nuclear levels of both beta-catenin and LEF-1. Direct cross-talk between Wnts and BMP-2 was also indicated by the up-regulated interaction between beta-catenin and SMAD-4 in response to BMP-2. These results suggest that Wnt-3A acts in a manner opposite to that of other Wnts, such as Wnt-7A, which were previously identified as inhibitory to chondrogenesis, and is the first BMP-2-regulated, chondrogenesis-enhancing member of the Wnt family.     

Functional analysis of the Xenopus frizzled 7 protein domains using chimeric receptors

Seven-transmembrane receptors of the frizzled family can interact with secreted Wnt ligands and transmit Wnt signals into the cell. Dependent on the ligand receptor combination, distinct Wnt pathways are activated. Xenopus frizzled 7 (Xfz7) and Xwnt-8b as well as Human frizzled 5 (Hfz5) and Xwnt-5a can act synergistically in the activation of Wnt/beta-catenin target genes siamois (Xsia) and nodal related 3 (Xnr3) and in the induction of ectopic axes in Xenopus embryos. In order to characterize the role of different protein domains of Xfz7 in Wnt/beta-catenin signaling, chimeric Xfz7/Hfz5 receptors were generated in which the extracellular (N5-TC7) or the intracellular domains (NT7-C5) between Xfz7 and Hfz5 were exchanged. We present evidence that the extracellular domain of Xfz7 can interact with Xwnt-5a and that the intracellular C-terminus can transmit a Wnt/beta-catenin signal. Despite these abilities, Xfz7 and Xwnt-5a do not act synergistically in the activation of Wnt/beta-catenin targets. This implies that the interaction of a frizzled receptor with different ligands can result in distinct cellular responses.     

Spatially regulated translation in embryos: asymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus.

Transition from symmetry to asymmetry is a central theme in cell and developmental biology. In Xenopus embryos, dorsal-ventral asymmetry is initiated by a microtubule-dependent cytoplasmic rotation during the first cell cycle after fertilization. Here we show that the cytoplasmic rotation initiates differential cytoplasmic polyadenylation of maternal Xwnt-11 RNA, encoding a member of the Wnt family of cell-cell signaling factors. Translational regulation of Xwnt-11 mRNA along the dorsal-ventral axis results in asymmetric accumulation of Xwnt-11 protein. These results demonstrate spatially regulated translation of a maternal cell-signaling factor along the vertebrate dorsal-ventral axis and represent a novel mechanism for Wnt gene regulation. Spatial regulation of maternal RNA translation, which has been established in invertebrates, appears to be an evolutionarily conserved mechanism in the generation of intracellular asymmetry and the consequential formation of the multicellular body pattern.     

Wnt-11 and Fz7 reduce cell adhesion in convergent extension by sequestration of PAPC and C-cadherin

Wnt-11/planar cell polarity signaling polarizes mesodermal cells undergoing convergent extension during Xenopus laevis gastrulation. These shape changes associated with lateral intercalation behavior require a dynamic modulation of cell adhesion. In this paper, we report that Wnt-11/frizzled-7 (Fz7) controls cell adhesion by forming separate adhesion-modulating complexes (AMCs) with the paraxial protocadherin (PAPC; denoted as AMCP) and C-cadherin (denoted as AMCC) via distinct Fz7 interaction domains. When PAPC was part of a Wnt-11-Fz7 complex, its Dynamin1- and clathrin-dependent internalization was blocked. This membrane stabilization of AMCP (Fz7/PAPC) by Wnt-11 prevented C-cadherin clustering, resulting in reduced cell adhesion and modified cell sorting activity. Importantly, Wnt-11 did not influence C-cadherin internalization; instead, it promoted the formation of AMCC (Fz7/Cadherin), which competed with cis-dimerization of C-cadherin. Because PAPC and C-cadherin did not directly interact and did not form a joint complex with Fz7, we suggest that Wnt-11 triggers the formation of two distinct complexes, AMCC and AMCP, that act in parallel to reduce cell adhesion by hampering lateral clustering of C-cadherin.