Retinoic acid, a vitamin A derivative, is a powerful signalling molecule that controls aspects of vertebrate cell differentiation nd pattern formation during embryonic development. In addition, retinoic acid can inhibit or reverse the process of neoplastic transformation. One of the mechanisms by which retinoic acid (RA) may influence these processes is via the regulation of the expression of genes encoding constituents of the basement membrane such as laminin and collagen IV. Over the past four years under the auspices of HD24319 we have studied the regulation of the murine laminin B1 gene by retinoic acid during the differentiation of cultured murine embryonic stem cells into parietal endoderm, a type of embryonic epithelial cell. We have shown that the laminin B1 gene is transcriptionally activated by 10-fold via an RARE (RA-response element) in its promoter that binds retinoic acid receptor:RA complexes. In addition, we have defined a region in the laminin B1 promoter between - 4.3 and 3.6 kb that enhances transcription. Finally, we have identified new molecular markers for the perietal endoderm cells, including F117, a chondroitin sulfate proteoglycan, and BMP-2. Both of these genes are expressed at much higher levels in parietal endoderm than in undifferentiated stem cells. Over the next five years we want to understand how the spatial and temporal regulation of laminin B1 gene expression during embryogenesis is achieved, and to determine how much of the spatial and temporal regulation of laminin B1 in the embryo is controlled by retinoic acid via the RARE. We will generate various laminin B1 promoter/beta- galactosidase expression vectors and inject these into fertilized 1-cell eggs to create transgenic mice that express beta-gal under the control of various portions of the laminin B1 promoter DNA. These transgenic mice will be analyzed to determine if the spatial and temporal regulation of beta-gal activity (measured by a colorimetric assay) reflects that of the endogenous laminin B1 gene (measured by in situ hybridization). We also plan to generate mice in which the laminin B1 gene is disrupted via homologous recombination to study the function of laminin B1 in early embryogenesis. Finally, we plan to elucidate the mechanism by which RA regulates the other laminin genes, A and B2, and a chondroitin sulfate proteoglycan, F117, during F9 embryonic stem cell differentiation.
This aim will be accomplished by using DNAse I hypersensitivity assays, promoter/chloramphenicol acetyl transferase constructs in transient transfection assays of F9 cells +/- RA, gel retardation assays, and footprinting experiments. Our studies of the regulation of these basement membrane genes by retinoic acid should lead to a better understanding of processes as diverse as cell differentiation, teratogenesis, cancer metastasis, angiogenesis, and diabetic nephropathy.
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