RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development

RGS family members are GTPase activating proteins (GAPs) that antagonize signaling by heterotrimeric G proteins. Injection of Xenopus embryos with RNA encoding rat RGS4 (rRGS4), a GAP for G(i) and G(q), resulted in shortened trunks and decreased skeletal muscle. This phenotype is nearly identical to the effect of injection of either frzb or dominant negative Xwnt-8. Injection of human RGS2, which selectively deactivates G(q), had similar effects. rRGS4 inhibited the ability of early Xwnt-8 but not Xdsh misexpression to cause axis duplication. This effect is distinct from axin family members that contain RGS-like domains but act downstream of Xdsh. We identified two Xenopus RGS4 homologs, one of which, Xrgs4a, was expressed as a Spemann organizer component. Injection of Xenopus embryos with Xrgs4a also resulted in shortened trunks and decreased skeletal muscle. These results suggest that RGS proteins modulate Xwnt-8 signaling by attenuating the function of a G protein.     

Ectopic induction of dorsal mesoderm by overexpression of Xwnt-8 elevates the neural competence of Xenopus ectoderm.

The ectoderm of early Xenopus gastrula is competent to become induced to neural tissue, but dorsal ectoderm is more neural competent than ventral ectoderm. It is a tenable, but as yet untested possibility that the higher neural competence of dorsal gastrula ectoderm is dependent on the presence of the dorsal mesoderm. To test this hypothesis we overexpressed Xwnt-8 in order to ectopically induce dorsal mesoderm in the ventral side of the embryo. We found that this elevated the level of neural competence of ventral ectoderm to that of dorsal ectoderm. The effect of Xwnt-8 on neural competence of ventral ectoderm was strictly correlated with its ability to enhance the amount of dorsal structures. The data indicate that the presence of dorsal mesoderm is a prerequisite for establishing the differences in neural competence between gastrula dorsal and ventral ectoderm.     

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.     

Transition of Xwnt-11 mRNA from inactive form to polyribosomes in frogs during early embryogenesis

The distribution of Xwnt-11 mRNA between polysomes and informosomes was studied in Xenopus laevis and Rana temporaria during early embryogenesis. The ratio between polysomes and informosomes suggests their involvement in translation of these mRNAs. In eggs and immediately after fertilization the Xwnt-11 mRNAs are mostly positioned in informosomes. During the cleavage stage, these mRNAs have also been recognized in polysomes. Just before the onset of zygote genome functioning (at the stage of mid blastula), Xwnt-11 mRNA rapidly appears in polysomes of Rana embryos. However, in Xenopus, Xwnt-11 mRNA appears in polysomes only at the end of gastrula. Before this stage, the Xwnt-11 mRNA in Xenopus can be found mostly in informosomes.     

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.     

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.     

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.     

The role of Xmsx-2 in the anterior-posterior patterning of the mesoderm in Xenopus laevis

Many molecules are involved in defining mesodermal patterning of the Xenopus embryo. In this paper, evidence is provided that a member of the msx family of genes, the Xmsx-2 gene, is involved in anterior-posterior patterning of the mesoderm. A comparison of its sequence to another previously cloned msx-2 Xenopus homolog, Xhox-7.1' [45] showed that they are closely related. The Xmsx-2 gene is first expressed at midgastrulation predominantly in the dorsal part of the embryo. It showed a complex pattern of spatial expression, consistent with a role in patterning of the anterior-posterior axis. This inference is confirmed by gain-of-function experiments in which overexpressed msx-2 mRNA in developing Xenopus embryos resulted in embryos lacking anterior structures. Analysis of markers in mutant embryos showed that genes involved in ventral-posterior patterning such as Xhox-3, Xwnt-8, and Xvent-1 were upregulated, confirming the posteriorized nature of the embryos. We believe that the Xmsx-2 gene is involved in refining the patterning of the anterior-posterior part of the dorsal mesoderm after the initial signals determining the dorsal or ventral nature of the mesoderm have been specified.     

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.     

父系HBeAg 阳性流产胚胎绒毛中HBV-DNA 的表达

目的:探讨在父系HBeAg阳性的流产胚胎中,乙型肝炎病毒在绒毛中的表达。方法:募集仅父系感染乙型肝炎病毒组合,即母HBsAg(-)且父HBsAg(+)流产胚胎。按以下组合将入选对象分为4组:组1为父HBeAg(+)母HBsAb(+);组2为父HBeAg(+)母HBsAb(-);组3为父HBeAg(-)母HBsAb(+);组4为父HBeAg(-)母HBsAb(-),采用酶联免疫吸附实验(ELISA)对胎儿父、母亲血清进行乙肝抗原、抗体检测,并使用荧光定量PCR法对胚胎绒毛进行HBV DNA检测。结果:父系感染乙型肝炎病毒的142例胚胎中,仅在父系HBeAg阳性组别(1、2组)84例胚胎中发现3例绒毛HBV-DNA升高,阳性率为3.57%。其中父HBeAg(+)母HBsAb(-)组合中2例,父HBeAg(+)母HBsAb(+)组合中1例。父系HBeAg均阳性,母系HBsAb阳性与阴性组间子代绒毛HBV-DNA升高率差异无显著性(P>0.05)。结论:HBeAg阳性父亲可能更容易导致乙肝父婴垂直传播。