Abstract
Among the Xenopus cadherins the maternal XB/U- and EP/C-cadherin have extraordinary significance. Both are coexpressed in the embryo and are a prerequisite for the integrity of the blastula. Beside their mechanical function in cell-cell adhesion, cadherins are discussed in terms of Wnt dependent signal transduction in the context of dorsoventral specification of the mesoderm. Wether the influence of the cadherin system on Wnt dependent signal transduction observed as it has beenobserved so far, is simply caused by an artifically induced depletion of the common mediator β-catenin from the Wnt-cascade or is additionally influenced by cadherin mediated cell-cell adhesion in vivo, has been controversely discussed.
The constructed extracellularly deleted mutant of XB/U cadherin, XB/UΔe349, lacks all extracellular calcium binding domains, including the disfunction of the HAV domain, but still contains the signal peptide. This mutant was used for functional analysis of maternal cadherins during Xenopus mesoderm specification by dominant negative overexpression of transcripts in a pancadherin manner.
The expression of all cadherin constructs in the membrane of overexpressing cells were shown by immunfluorescent staining of histologically sectioned Xenopus dorsal marginal zones.
The dorsal marginal overexpression of XB/U-cadherin RNA into Xenopus 4 cell stage embryos lead to three different phenotypes in tadpole stages. Two of those, type I and type II, have previously been identified in embryos overexpressing the cytoplasmically deleted mutant XB/UΔc38 after MBT under the control of the CMV-promoter. It was previously shown that those phenotypes developed due to disturbed adhesion processes affecting convergent extension, leading to a misdirection of mesoderm during the gastrulation process. In the work presented here, an additional phenotype III was observed, with strongly reduced or even lacking axial structures. This phenotype was also reported after b-catenin antisense oligodesoxynucleotid knockout experiments and therefore is thought to be a consequence of depleting b-catenin from the Wnt-signaling pathway. However, although overexpressing different cadherin mutants only underline the diverse functions of their wild type domains, the rescue experiments presented here emphasize the necessity of cadherin mediated cell adhesion for signaling events, leading to dorsoventral axis formation in Xenopus. Coexpression of XB/UΔe349 with XB/UWt not only restored cell adhesion, but also dramatically reduced axis deficiency, although providing additional β-catenin binding sites.
Coexpression of XB/UΔe349 with β-catenin or plakoglobin suggest a main function in cell adhesion processes of plakoglobin, rather than being a mediator of the Wnt signaling cascade like its homolog β-catenin. Therefore β-catenin and plakoglobin should not be redundant in the respect of adhesion and signaling functions.
The effects of overexpressing cadherin constructs on mesodermal marker gene expression was investigated by whole mount in situ hybridization. The observed loss of dorsal structures was confirmed on the molecular level. It was shown that a disturbance of the cadherin system at the time and place of the dorsal mesoderm specification interferes with gene expression of diffusible and DNA binding proteins, essential for regulating the dorsal fate of the embryo. This can not sufficiently be explained by β-catenin depletion. Rather, the tissue integrity itself seems to play an important role in recieving and maintainig those signals. Rescue experiments using β-catenin and BMP-4 also confirmed an indirect effect of cadherin mediated cell adhesion of the dorsal marginal zone on the expression of ventral marker genes.
Contrary to the described Wnt-dependent regulation of fibronectin expression, a connection of cadherin expression and fibronectin matrix assembly could not be shown. In animal cap assays the disturbance of the cadherin system lead to a reduced activin dependent recognition of the fibronectin synergy site by integrin receptors. The underlying mechanism remains unclear. However, the results point to a participation of cadherin dependent tissue integrity and the community effect. |
