The overall goals of the proposed project are first, to develop a detailed characterization of the regulation of fibronectin gene expression and second, to identify the functions specific to the various forms of fibronectin. Fibronectins are a family of adhesive glycoproteins found in the extracellular matrix as well as in plasma. Several different protein variants are produced from a single gene by alternative splicing. Fibronectins are involved in many morphogenetic events during embryonic development, as well as in tissue remodelling events such as wound healing and inflammation-induced fibrosis. The levels and types of fibronectin synthesized vary during these processes in a cell type specific fashion. Understanding the mechanisms by which fibronectin expression is regulated thus complements studies of fibronectin functions. The liver will serve as an ideal model tissue on which to focus for the pursuit of these aims. Hepatocyte-derived fibronectin is a significant component of plasma, and this material can be deposited in a variety of other tissues as well be incorporated into blood clots. Numerous studies have reported changes in fibronectin synthesis in the liver relating to hepatic fibrosis, liver regeneration and malignancy. These goals will be achieved through the production of transgenic mice. The basis for the cell type specific regulation of fibronectin will be evaluated using a marker gene, lacZ, under the control of fragments derived from the cloned rat fibronectin gene. The pattern of expression of this easily assayable indicator gene will be interpreted in light of detailed in situ hybridization studies which will be undertaken in parallel. Particular emphasis will be placed on embryonic liver development. Through analysis of a number of individual trangenics, a picture will emerge regarding the structure of the regulatory regions of the fibronectin gene. Transgenic mice will also provide the means for achieving the other goals related to determining the functions of the various forms of fibronectin. cDNAs encoding specific fibronectin isoforms will be placed under the control of tissue specific promoters; initial experiments will target gene expression to the liver. Lines of mice will be generated which express individual forms, with the expectation that synthesis of inappropriate amounts or types of fibronectin will reveal information regarding their specific functions. More detailed studies involving mutagenesis of specific portions of FN will follow from the initial studies of two FN isoforms.
