Calcium-activated potassium channel gene expression in the midgut of Drosophila

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein beta-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic beta-catenin expression. In fact, axis induction by APC required the availability of cytosolic beta-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of beta-catenin, indicating that it has direct positive signaling activity in addition to its role in beta-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/beta-catenin signaling pathway, either upstream of, or in conjunction with, beta-catenin.     

A role for Siamois in Spemann organizer formation

The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.     

Identification of retinal ganglion cells projecting to the lateral hypothalamic area of the rat

Previous gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. We report that expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. We further demonstrate that the ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants is dependent upon a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants was found to be dependent on endogenous Wnt signaling. We conclude that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways.     

Characterization of CMIX, a chicken homeobox gene related to the Xenopus gene mix.1

Members of the TGFbeta, Wnt and FGF families act in concert to induce and pattern the mesoderm of gastrulating embryos. Downstream effectors for these growth factors include homeobox proteins, which also feed back to activate and repress upstream signaling pathways (e.g. Fainsod, A., Steinbeisser, H., De Robertis, E.M. 1994. On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13, 5015-5025; Carnac, G., Kodjabachian, L., Gurdon, J.B., Lemaire, P. 1996. The homeobox gene Siamois is a target of the Wnt dorsalization pathway and triggers organizer activity in the absence of mesoderm. Development 122, 3055-3065). As well as having interwoven upstream and downstream regulatory pathways Mix.1, siamois and goosecoid, all paired-type homeobox genes, may physically interact with each other as heterodimers to regulate dorsal-ventral polarity (Mead, P.E., Brivanlou, I.H., Kelley, C.M., Zon, L.I. 1996. BMP-4 responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature 382, 357-360). We report here a chicken paired-type homeobox gene, CMIX, with a homeodomain having 72% aa identity to its nearest homolog, Xenopus Mix.1. CMIX is expressed in the epiblast of the posterior marginal zone of early chick embryos, and later along the entire anterior-posterior axis of the primitive streak in cells of the medial ectoderm, in nascent mesoderm, but not in endoderm. Coincident with formation of prechordal mesoderm, CMIX mRNA levels rapidly decline throughout the embryo.     

Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.