The goal of this proposal is to establish a molecular basis for pattern formation during early vertebrate development. The discovery of the retinoic acid receptor and its homology to the steroid/thyroid receptor superfamily, offers for the first time in a vertebrate system, the hope of analyzing the mechanisms by which positional information are established and thus, how the fate and behavior of embryonic cells are controlled. Utilizing the cloned retinoic acid receptor gene products as models, we will define, in molecular terms, how retinoids influence development and physiology by controlling patterns of gene expression. We will characterize the molecular interactions of the retinoic acid receptor with DNA by identifying putative response elements (RAREs) present in the retinoic acid receptor and osteocalcin genes. In addition to biochemical dissection of the DNA-protein complex, the DNA-binding domain of the RAR and the ligand-binding domain will be overproduced in E. coli to begin NMR and X-ray crystallographic studies. Activation domains of the retinoid receptors will be identified by deletion, truncation, and point mutation analysis. Proteins interacting with the RARs will be sought by chemical approaches to identify the encoding genes. These approaches include chromatography of nuclear extracts to RAR affinity columns and the detection of receptor-DNA complexes with altered electrophoretic mobilities. In situ hybridization and immunohistochemical studies will be used to localize subtype-specific RAR mRNA and protein during mouse embryogenesis. Functional roles of the RARs during early embryonic development will be assessed by creating transgenic animals expressing a variety of mutant RAR derivatives. RAR responsive promoters linked to beta-galactosidase will be used to visualize endogenous RA gradients. In summary, retinoic acid, because of its described role in limb development and its capacity to induce differentiation in embryo carcinoma cells, is an ideal candidate for a vertebrate morphogen. In combination with the recently cloned retinoic acid receptor gene products in several species, we are now poised to examine the mechanism by which RA and the RARs specify pattern formation in vertebrates.
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