Doris Wedlich

The Wnt/Wg signal transducer beta-catenin controls fibronectin expression.

ABSTRACT β-Catenin stabilizes the cadherin cell adhesion complex but, as a component of the Wnt/Wg signaling pathway, also controls gene expression by forming a heterodimer with a transcription factor of the LEF-TCF family. We demonstrate that the substrate adhesion molecule fibronectin is a direct target of Wnt/Wg signaling. Nuclear depletion of β-catenin following cadherin transfection in Xenopusfibroblasts resulted in downregulation of fibronectin expression which was restored by activating the Wnt/Wg signaling cascade via LiCl treatment or transfection of either Xwnt-8 or β-catenin. We isolated the Xenopus fibronectin gene (FN) promoter and found four putative LEF-TCF binding sites. By comparing the activities of different fibronectin gene reporter constructs in fibroblasts and cadherin transfectants, the LEF-TCF site at position −368 was identified as a Wnt/Wg response element. LEF-1-related proteins were found in nuclei of the fibroblasts but were absent in a kidney epithelial cell line. Consistent with the lack of these transcription factors, the FN promoter was silent in the epithelial cells but was activated upon transfection of LEF-1. Wild-typeXenopus Tcf-3 (XTcf-3) was unable to activateFN promoter reporter constructs, while a mutant lacking the groucho binding region behaved like LEF-1. In contrast to XTcf-3, LEF-1 does not interact with groucho proteins, which turn TCFs into activators or repressors (J. Roose, M. Molenaar, J. Hurenkamp, J. Peterson, H. Brantjes, P. Moerer, M. van de Wetering, O. Destreé, and H. Clevers, Nature 395:608–612, 1998). Together these data provide evidence that expressing LEF-1 enables fibroblasts, in contrast to epithelial cells, to respond to the Wnt/Wg signal via β-catenin in stimulating fibronectin gene transcription. Our findings further promote the idea that due to its dual function, β-catenin regulates the balance between cell-cell and cell-substrate adhesion.

Xenopus Eya1 demarcates all neurogenic placodes as well as migrating hypaxial muscle precursors

We cloned two isoforms of the Xenopus Eya1 orthologue. They show identical patterns of expression that closely resemble the previously described expression of XSix1, but partly differ from the expression of Eya1 in other vertebrates. XEya1 is expressed in the somites and hypaxial muscle precursors, but not in the pronephros. Moreover, all ectodermal placodes except the lens placode strongly express XEya1. At neural plate stages, ectodermal XEya1 expression starts in two domains, the anterior neural folds and a domain lateral to the neural folds. At tailbud stages, XEya1 expression continues in the adenohypophysis, all neurogenic placodes and placodally-derived structures including cranial ganglia, the otic vesicle and lateral line primordia.

Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules.

Cadherins are discussed not in terms of their adhesive function but rather as morphoregulatory proteins. Changes in gene expression following cadherin transfection of cells in culture or by overexpression in embryos have, until now, not been reported. We established a protocol for stable transfection of Xenopus XTC cells and generated cells bearing high levels of membrane-integrated mouse uvomorulin (E-cadherin) or Xenopus XB-cadherin. These cell lines showed drastically impaired substrate adhesion on fibronectin and laminin. In immunoblot and radioimmunoprecipitation experiments, we found that fibronectin and alpha 3/beta 1 integrin are downregulated. The reduced amounts of proteins result from a decrease of the respective mRNAs as proven by RNase protection assays. Coprecipitations revealed that transfected cadherin molecules are complexed with alpha-catenin and beta-catenin at plasma membranes. However, the alpha-catenin present in the XB-cadherin complex differs immunologically from that found in the uvomorulin complex. When a truncated form of XB-cadherin lacking 38 of the most C-terminal amino acids was expressed in XTC cells, complex formation with endogenous catenins was abolished. In these transfectants, substrate adhesion was not affected. These results prove that complex formation of transfected cadherins in XTC cells with endogenous beta-catenin correlates with altered synthesis of certain substrate adhesion molecules.

Keeping a close eye on Wnt-1/wg signaling in Xenopus.

Nearly two decades have past since description of the ®rst member of the Wnt family by Nusse and Varmus in 1982. Wnt-1 (at that time int-1) was shown to encode a secreted glycoprotein, and misexpression of Wnt-1 was found to promote mammary tumors in mice (Nusse and Varmus, 1982). Tremendous efforts have been undertaken to decipher the intracellular signaling events triggered by this family of extracellular glycoproteins, and now many of the principal mechanisms have been elucidated. Results from different experimental systems have contributed to our present understanding of the Wnt signaling cascade, but two organisms, Drosophila melanogaster and Xenopus laevis have played a critical role in this race. The observation that Wnt-1 is the mammalian homolog of the Drosophila segment polarity gene wingless (wg) (Rijsewijk et al., 1987) was the basis for the hypothesis that both, Wnt-1 and Wg, trigger the same signaling events. Xenopus laevis came into play as a result of the observation by McMahon and Moon (1989) that overexpressing Wnt-1 in ventral blastomeres of early Xenopus embryos elicits a duplication of the embryonic axis. This axis induction assay also showed the central role of b -catenin in Wnt-1/wg signaling (Funayama et al., 1995; Guger and Gumbiner, 1995). Even homologs of pathway genes in other species were found to be suf®cient to trigger axis duplication, e.g. Drosophila armadillo or dishevelled. In the last few years, this cross species axis duplication assay has been widely accepted to allocate newly identi®ed proteins to the Wnt-1/wg signaling cascade (for example see Fig. 2). The family of Wnt proteins is divided into two functional classes based on various activity assays. Only some members of the Wnt family, called Wnt-1/wg class, are able to induce the formation of a secondary axis when injected ventrally into a 4-cell stage Xenopus embryo (review by Moon and Kimelman, 1998). The same subset of Wnt members has been shown to transform C57mg cells (Wong et al., 1994). Both of these effects are thought to be mediated by the Wnt-1/wg signaling cascade which will be described later in this review. The Wnt-5A class of Wnts failed in both assays; i.e. Xwnt-5A, Xwnt-4 and Xwnt-11 do not possess axis inducing capacity, and Wnt-4 and Wnt5A do not transform C57mg cells. In addition, members of this class (Xwnt-4, -5A, -11) are able to antagonize the axis inducing effect of the Wnt-1/wg class in the Xenopus embryo, and Wnt-5A is able to reverse the transforming properties of Wnt-1 in C57 mg cells (Olson and Gibo, 1998). For these reasons, they are discussed as tumor suppressors. In Xenopus assays, the overexpression of the three Wnt-5A class members resulted in disruption of morphogenetic movements. In zebra®sh embryos, Xwnt5A has been shown to signal via intracellular release of calcium ions (Slusarski et al., 1997a,b). The effects of Xwnt-4, Xwnt-5A and Xwnt-11 on cell migration can be Mechanisms of Development 86 (1999) 3±15

Expression of the Armadillo family member p120cas1B in Xenopus embryos affects head differentiation but not axis formation.

Abstractu2002The Armadillo family is formed by proteins which possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat, initially described for the Drosophila segment polarity gene product Armadillo. The Arm domain serves in protein-protein interactions which are required for the family members Armadillo, β-catenin and plakoglobin to mediate cell-cell adhesion and Wnt/Wingless signalling. Similarily, p120cas, the Arm domain containing src substrate, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function of p120cas in adhesion or signalling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos we show by expression of murine p120cas1B (mp120cas1B) in ventral blastomeres that this catenin cannot replace β-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120cas1B into dorsal blastomeres led instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin was altered in the injected embryos we assume that mp120cas1B influences motility or orientation of migrating mesodermal cells.

The HMG-box transcription factor XTcf-4 demarcates the forebrain-midbrain boundary.

A small subfamily of HMG-box transcription factors, the LEF/TCF group, serves as nuclear transducer of the Wnt-1/Wg signaling cascade. Upon Wnt-1/Wg signaling their members interact with beta-catenin and regulate the expression of Xenopus target genes siamois, twin, nodal related-3 or fibronectin. We have isolated a new HMG-box transcription factor in Xenopus that will be addressed XTcf-4 based on its homology to human and murine Tcf-4. Unlike XTcf-3, which is a maternal gene, and XLef-1 that is expressed after mid blastula transition (Molenaar et al., 1998. Mech. Dev. 75, 151-154), XTcf-4 expression starts at late neurula stage and is restricted to the anterior most midbrain demarcating the forebrain-midbrain boundary. The expression partially overlaps with a broad set of Xenopus Wnt family members in distinct patterns. XTcf-4 transcripts were also found partially co-localized with those of Xaxin, an intracellular antagonist of Wnt-1/Wg signaling.