The overall aims are to understand the roles and activities of the epidermal growth factor receptor (EGFR) in developing embryos in vivo. Known effects of signalling after ligand binding to the EGFR range from changes in membrane motility, cell shape and epithelial polarity to the stimulation of proliferation or to increased differentiation. Over- expression of the EGFR gene in EC cells can lead to differentiation. In contrast, in non-embryonic cells, over-expression can lead to aberrant or transformed growth. Our understanding of how EGFR activities are modulated and how gene expression is regulated is still fragmentary. In the last grant period, we characterized the activities of a truncated, kinase- negative version of EGFR after transfection of the expression plasmid into Pl9 embryonal carcinoma (EC) cells. We showed that truncated EGFR acted as a dominant negative mutation, abrogating the activities of the normal EGF receptor. The result was inhibition of the process of differentiation to nervous tissues after induction by retinoic acid. The present proposal builds on and extends this approach. We propose to perturb the levels of EGFR in EC cells, embryonal stem (ES) cells and in embryos as an approach to understanding the roles of the EGFR in normal and aberrant growth.
The specific aims are:- l. To study the roles of the EGFR in ES cells and in transgenic mice by introducing a dominant negative mutation of the receptor. 2. To study the roles of EGFRs in vitro and in vivo by over- expression of human EGFRs with and without accompanying transforming growth factor alpha (TGFalpha) expression in EC and ES cells and in transgenic mice. 3. To make initial studies of the roles and regulation of EGFRs in ovarian cells and tumors. 4. To determine some of the components in the EGFR signal transduction pathway in preimplantation embryos using antibodies and RT-PCR. A number of hypotheses will be tested. For instance, that autocrine-stimulated EGF-Rs provide a driving force to the continuation of differentiation and its irreversibility and stability such that when aberrant, tumors are initiated or stimulated to progress.
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