Selective degradation of excess Ldb1 by Rnf12/RLIM confers proper Ldb1 expression levels and Xlim-1/Ldb1 stoichiometry in Xenopus organizer functions

The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and Ldb1. Although XRnf12 functions as a E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, we found that co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1, suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12 throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues.     

RNA interference for the organizer-specific gene Xlim-1 in Xenopus embryos

Double-stranded RNA (dsRNA) interferes with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is also effective in modifying gene expression in Xenopus embryos. First, expression of an exogenous luciferase gene as a reporter in embryos was reduced by coinjection with dsRNA corresponding to the luciferase gene. Next, injection of dsRNA for Xlim-1, a homeobox gene suggested to be involved in Spemann organizer functions, reduced the endogenous level of Xlim-1 mRNA and produced embryos with reduced eyes or anterior truncation at high efficiency. In addition, injection of an antisense expression construct of Xlim-1 elicited phenotypes very similar to those of Xlim-1 dsRNA-injected embryos. These results indicate the effectiveness of RNAi for loss of function studies in Xenopus embryos, and the importance of Xlim-1 in head formation.     

Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.     

Regulation of early expression of Dlx3, a Xenopus anti-neural factor, by beta-catenin signaling

The ectoderm of the pre-gastrula Xenopus embryo has previously been shown to be at least partially patterned along the dorsal-ventral axis. The early expression of the anti-neural homeodomain gene Dlx3 is localized to the ventral ectoderm by a mechanism that occurs prior to gastrulation and is independent of the Spemann organizer. The repression of Dlx3 is mediated by signaling though beta-catenin, but is probably not dependent on the induction of the Xnr3 or chordin genes by beta-catenin. We propose a model in which this early regulation of Dlx3 accounts for the pro-neural bias of dorsal ectoderm.     

The Toll/IL-1 receptor binding protein MyD88 is required for Xenopus axis formation

The Toll/Dorsal pathway regulates dorsoventral axis formation in the Drosophila embryo. We had previously obtained evidence that a homologous pathway exists in Xenopus, however, its role during normal frog development had not been established. Here we report the cloning of Xenopus MyD88 (XMyD88), whose mammalian homologs are adaptor proteins linking Toll/IL-1 receptors and IRAK kinases. We show that in the frog embryo overexpression of a dominant-negative form of XMyD88 blocked Toll receptor activity, specifically inhibited axis formation and reduced expression of pivotal organizer genes. The observed stage-dependency of interference suggests a function for maternal XMyD88 soon after fertilization. We conclude that XMyD88 activity is required for normal Spemann organizer formation, implying an essential role for maternal Toll/IL-1 receptors in Xenopus axis formation.     

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.     

The Spemann-Mangold organizer: the control of fate specification and morphogenetic rearrangements during gastrulation in Xenopus

Vertebrate embryonic development is controlled by sequentially operating signalling centres that organize spatial pattern by inductive interactions. The embryonic body plan is established during gastrulation through the action of the Spemann-Mangold or gastrula organizer, a signalling source discovered 75 years ago by Hans Spemann and Hilde Mangold. Transplantation of the organizer to a heterotopic location in a recipient embryo results in the formation of a secondary embryonic body axis, in which several tissue types, most notably somites and the neural tube, are derived from ventral host cells. Because of these non-cell autonomous recruiting or inducing activities the organizer has become a paradigm for studying intercellular communication in the vertebrate embryo. Here, I review some of the recent advances in understanding 1) the initiation of the Spemann-Mangold organizer, 2) its function in pattern formation along the dorsal-ventral and anterior-posterior axes and 3) the integration of cell fate specification events and downstream execution of morphogenetic movements during gastrulation in Xenopus laevis.     

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.