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.     

Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1.

We have identified a novel homeobox gene, Xvent-1, that is differentially expressed in the ventral marginal zone of the early Xenopus gastrula. Evidence is presented from mRNA microinjection experiments for a role for this gene in dorsoventral patterning of mesoderm. First, Xvent-1 is induced by BMP-4, a gene known to be a key regulator of ventral mesoderm development. Second, Xvent-1 and the organizer-specific gene goosecoid are able to interact, directly or indirectly, in a cross-regulatory loop suppressing each other's expression, consistent with their mutually exclusive expression in the marginal zone. Third, microinjection of Xvent-1 mRNA ventralizes dorsal mesoderm. The results suggest that Xvent-1 functions in a ventral signaling pathway that maintains the ventral mesodermal state and antagonizes the Spemann organizer.     

Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos

In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.     

The nodal target gene Xmenf is a component of an FGF-independent pathway of ventral mesoderm induction in Xenopus

The interplay of fibroblast growth factor (FGF) and nodal signaling in the Xenopus gastrula marginal zone specifies distinct populations of presumptive mesodermal cells. Cells in the vegetal marginal zone, making up the presumptive leading edge mesoderm, are exposed to nodal signaling, as evidenced by SMAD2 activation, but do not appear to be exposed to FGF signaling, as evidenced by the lack of MAP kinase (MAPK) activation. However, in the animal marginal zone, activation of both SMAD2 and MAPK occurs. The differential activation of these two signaling pathways in the marginal zone results in the vegetal and animal marginal zones expressing different genes at gastrulation, and subsequently having different fates, with the vegetal marginal zone contributing to ventral mesoderm (e.g. ventral blood island) and the animal marginal zone giving rise to dorsal fates (e.g. notochord and somite). We report here the cloning of a cDNA encoding a novel nuclear protein, Xmenf, that is expressed in the vegetal marginal zone. The expression of Xmenf is induced by nodal signaling and negatively regulated by FGF signaling. Results from animal cap studies indicate that Xmenf plays a role in the pathway of ventral mesoderm induction in the vegetal marginal zone.     

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.     

The head inducer Cerberus in a multivalent extracellular inhibitor

Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants.     

Spatial and temporal properties of ventral blood island induction in Xenopus laevis.

Questions of dorsoventral axis determination and patterning in Xenopus seek to uncover the mechanisms by which particular mesodermal fates, for example somite, are specified in the dorsal pole of the axis while other mesoderm fates, for example, ventral blood island (VBI), are specified at the ventral pole. We report here that the genes Xvent-1, Xvent-2, and Xwnt-8 do not appear to be in the pathway of VBI induction, contrary to previous reports. Results from the selective inhibition of bone morphogenetic protein (BMP) activity, a key regulator of VBI induction, by ectopic Noggin, Chordin, or dominant negative BMP ligands and receptors suggest an alternative route of VBI induction. Injection of noggin or chordin RNA into animal pole blastomeres effectively inhibited VBI development, while marginal zone injection had no effect. Cell autonomous inhibition of BMP activity in epidermis with dominant negative ligand dramatically reduced the amount of (&agr;)T3 globin expression. These results indicate that signaling activity from the Spemann Organizer alone may not be sufficient for dorsoventral patterning in the marginal zone and that an inductive interaction between presumptive VBIs and ectoderm late in gastrulation may be crucial. In agreement with these observations, other results show that in explanted blastula-stage marginal zones a distinct pattern develops with a restricted VBI-forming region at the vegetal pole that is independent of the patterning activity of the Spemann Organizer.     

Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.     

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.