Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.     

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.     

Migration of neuronal cells along the anterior-posterior body axis of C. elegans: Wnts are in control

Migrating neuronal cells are directed to their final positions by an array of guidance cues. It has been shown that guidance molecules such as UNC-6/Netrin and SLT-1/Slit play a major role in controlling cell and axon migrations along the dorsal-ventral body axis. Much less is known, however, about the mechanisms that mediate migration along the anterior-posterior (AP) body axis. Recent research in Caenorhabditis elegans has uncovered an important role of the Wnt family of signalling molecules in controlling AP-directed neuronal cell migration and polarity. A common theme that emerges from these studies is that multiple Wnt proteins function in parallel as instructive cues or permissive signals to control neuronal patterning along this major body axis.     

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.     

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.     

Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development

Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.     

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.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.     

Left-right asymmetry in the alimentary canal of the Drosophila embryo

In many animal groups, left-right (LR) asymmetry within the body is observed. The left and right sides of the body are generally defined with reference to the anterior-posterior (AP) and dorsal-ventral (DV) axes. In this study, we investigated whether LR asymmetry is solely dependent on the AP and DV polarities in Drosophila embryos. We focused on the proventriculus, a posterior part of the foregut, and the hindgut because LR asymmetry in these body parts is highly stable in normal embryos. In embryos with a fully reversed AP polarity, LR asymmetry in both the proventriculus and the hindgut was re-oriented in relation to the reversed AP polarity. This demonstrates that inversion of AP polarity does not affect LR asymmetry of these tissues, and implies that LR asymmetry is specified in relation to the AP and DV polarities. Our findings were not consistent with the alternative hypothesis that LR asymmetry is predetermined by maternal signals that localize asymmetrically along the LR axis in the oocyte and/or early embryo.     

Maternal control of pattern formation in Xenopus laevis

We review the essential role of maternal factors in pattern formation for Xenopus laevis, focusing on VegT, Vg1, and Wnt11. Results from loss of function experiments demonstrate a clear requirement for these genes in germ layer specification, dorsal-ventral axis formation, and convergence extension. We also discuss these genes in the broader context of metazoan development, exploring whether and how their functions in the X. laevis model organism may or may not be conserved in other species. Wnt11 signaling in particular provides a classic example where understanding context in development is crucial to understanding function. Genomic sequencing, gene expression, and functional screening data that are becoming available in more species are providing invaluable aid to decoding and modeling signaling pathways. More work is needed to develop a comprehensive catalog of the Wnt signaling, T-box, and TGF-beta genes in metazoans both near and far in evolutionary distance. We finally discuss some specific experimental and modeling efforts that will be needed to understand the behavior of these signaling networks in vivo so that we can interpret these critical pathways in an evolutionary framework.     

XTsh3 is an essential enhancing factor of canonical Wnt signaling in Xenopus axial determination

In Xenopus, an asymmetric distribution of Wnt activity that follows cortical rotation in the fertilized egg leads to the dorsal-ventral (DV) axis establishment. However, how a clear DV polarity develops from the initial difference in Wnt activity still remains elusive. We report here that the Teashirt-class Zn-finger factor XTsh3 plays an essential role in dorsal determination by enhancing canonical Wnt signaling. Knockdown of the XTsh3 function causes ventralization in the Xenopus embryo. Both in vivo and in vitro studies show that XTsh3 substantially enhances Wnt signaling activity in a beta-catenin-dependent manner. XTsh3 cooperatively promotes the formation of a secondary axis on the ventral side when combined with weak Wnt activity, whereas XTsh3 alone has little axis-inducing ability. Furthermore, Wnt1 requires XTsh3 for its dorsalizing activity in vivo. Immunostaining and protein analyses indicate that XTsh3 is a nuclear protein that physically associates with beta-catenin and efficiently increases the level of beta-catenin in the nucleus. We discuss the role of XTsh3 as an essential amplifying factor of canonical Wnt signaling in embryonic dorsal determination.     

Dorsal downregulation of GSK3beta by a non-Wnt-like mechanism is an early molecular consequence of cortical rotation in early Xenopus embryos

Cortical rotation and concomitant dorsal translocation of cytoplasmic determinants are the earliest events known to be necessary for dorsoventral patterning in Xenopus embryos. The earliest known molecular target is beta-catenin, which is essential for dorsal development and becomes dorsally enriched shortly after cortical rotation. In mammalian cells cytoplasmic accumulation of beta-catenin follows reduction of the specific activity of glycogen synthase kinase 3-beta (GSK3beta). In Xenopus embryos, exogenous GSK3beta) suppresses dorsal development as predicted and GSK3beta dominant negative (kinase dead) mutants cause ectopic axis formation. However, endogenous GSK3beta regulation is poorly characterized. Here we demonstrate two modes of GSK3beta regulation in Xenopus. Endogenous mechanisms cause depletion of GSK3beta protein on the dorsal side of the embryo. The timing, location and magnitude of the depletion correspond to those of endogenous beta-catenin accumulation. UV and D(2)O treatments that abolish and enhance dorsal character of the embryo, respectively, correspondingly abolish and enhance GSK3beta depletion. A candidate regulator of GSK3beta, GSK3-binding protein (GBP), known to be essential for axis formation, also induces depletion of GSK3beta. Depletion of GSK3beta is a previously undescribed mode of regulation of this signal transducer. The other mode of regulation is observed in response to Wnt and dishevelled expression. Neither Wnt nor dishevelled causes depletion but instead they reduce GSK3beta-specific activity. Thus, Wnt/Dsh and GBP appear to effect two biochemically distinct modes of GSK3beta regulation.     

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.     

Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus

Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium (Ca(2+)) and activation of protein kinase C (PKC). Since targets of this Ca(2+) release could play a role in Wnt signaling, we first tested the hypothesis that Ca(2+)/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs. We report that Wnt and Frizzled homologs that activate Ca(2+) release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate beta-catenin function do not. This activation occurs within 10 min after receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, we then tested the hypothesis that CamKII participates in axis formation in the early embryo. Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos revealed elevated activity on the prospective ventral side, which was suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression of CamKII mutants were consistent with this prediction, revealing that CamKII contributes to a ventral cell fate.