Functional Analysis of WNT-Signaling in Embryonic Stem Cells

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Nearly one third of all extant animal phyla including the annelids produce their embryonic cell lineages via a stereotypic program, called spiral cleavage. This developmental program generates a number of distinct embryonic stem cells. The size and location of those cells allows the unequivocal identification of homologous embryonic stem cells across phyla. Despite the fact that this widely used mode of cell fate specification has many ancestral features, the underlying molecular mechanisms have not been elucidated.The purpose of this project is to understand the role of wnt-signaling in early cell fate specification and segmentation of the annelid Helobdella triseralis whose embryos are suitable for the proposed experiments. The wnt-pathway has been shown to control early cell fate decisions in vertebrates, insects, nematodes and echinoderms which suggests that it is an ancient patterning mechanisms. In order to investigate the role of wnt-signaling in a spiral cleaving embryo, the sites and function of wnt-signaling will be analyzed. Two downstream components and key mediators of the pathway, a beta-catenin and a LEF-1 ortholog, have been already recovered from Helobdella. Specific antisera against both will be used to localize wnt-responsive cells in early embryos. The function of the wnt-pathway will be determined by the overexpression of dominant negative and dominant active forms of both molecules in distinct embryonic stem cells to inhibit or elicit wnt-responses in a targeted cell lineage. The proposed research will provide information to determine the role of wnt-signaling in an ancestral type of embryogenesis and thereby will give insights into the evolutionary history of a fundamental developmental program that operates during axis formation and segmentation in extant embryos of bilaterian animals.