Basement membranes are specialized forms of extracellular matrix which are built around a scaffold of collagen IV molecules. Although the precise functions of basement membranes are unclear, they play a critical role in a number of processes, including glomerular filtration, cell migration, cell adhesion, and the regulation of cell proliferation and differentiation. It is significant that the synthesis of collagen IV is one of the earliest observable examples of developmentally controlled gene expression in the early mouse embryo. This expression is observed when uncommitted embryonic stem cells, called primitive endoderm, differentiate to yield parietal endoderm cells, which produce basement membrane components, and visceral endoderm cells. This differentiation can be mimicked in mouse F9 embryonal carcinoma cells by simple manipulation of the culture conditions. This affords us a powerful assay system for factors which affect the synthesis of collagen IV and other basement membrane components. In this research proposal we will examine how the expression of collagen IV is controlled both in mouse embryos and F9 cells. In support of this goal, we have recently isolated the gene for mouse alpha2(IV) collagen, and are beginning to characterize the promoter region. We have cloned and sequenced the entire cDNA for mouse alpha2(IV) collagen, and have cloned, but not yet fully sequenced the entire cDNA for mouse alphal(IV) collagen. Work is in progress to isolate and characterize the promoter region for alphal(IV) collagen. In the first phase of the proposed research we will determine which portions of the gene are required for correct temporal and spatial expression of the collagen IV genes, both in F9 cells and in mouse embryos. In the second phase we will identify, isolate, and characterize DNA-binding and other proteins which are implicated in the regulation of collagen IV gene expression. In the third phase, we will clone and further characterize these proteins. It is our belief that this work will lead to a further understanding of gene regulation during early mouse development and will have important implications for the for the pathobiology of basement membranes.
Showing the most recent 10 out of 13 publications
