THYROID HORMONE RECEPTORS IN DEVELOPMENT

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The long range goal of this research program is to elucidate the mechanisms of action of thyroid hormone (TH) during development using the amphibian Rana catesbeiana (RC) as the animal model. TH is essential for all aspects of development in this species. The proposed research will determine 1) the role that TH receptors (TRs) and deiodinases play in the coordination of metamorphosis and 2) why the TRbeta gene is responsive to TH in the tadpole but not in the adult frog.. The first three SAs will test the following hypothesis: an important feature in the coordination of metamorphosis is the control by TH per se of the expression of four genes whose products are involved in determining the level of TH action in the cell; t he genes for the TRalpha, TRbeta, 5-Deiodinase (5D) and 5'- Deiodinase (5'D). Thus the relationships between the level of expression of these genes and the ability of a tissue to exhibit its TH-dependent developmental characteristics will be investigated. Studies will determine, in two tissues (leg and tail) which metamorphose at different stages of the life-cycle an thus at markedly different plasma TH levels, 1) whether these genes are expressed in tadpoles in the absence of TH and 2) the effects of thyroid status on the expression of these genes during pre- and prometamorphosis and at climax. Next, the role of 5D and 5'D in regulating tissue sensitivity to TH will be examined by inhibiting the activity of these enzymes in tadpoles undergoing TH-induced metamorphosis. Then the cell-specific patterns of expression and regulation of the TR, 5D and 5'D genes will be defined using an RT in situ PCR technique. SA #5 will test a second hypothesis: the loss of TH-responsiveness of the TRbeta gene following metamorphosis is due to either the absence of a factor(s) essential for the response or the presence of a factor)s) that interferes with the response. The presence of such a factor in frog or tadpole nuclear extracts will first be demonstrated using constructs containing the TRbeta promoter and a X. laevis oocyte nuclei expression system. Then, electromobility shift assays, DNA footprinting and Far-western blotting techniques will be used to ascertain if more than one factor is involved, the location of the DNA/protein binding sites and whether binding to DNA is direct or indirect. Time permitting we will clone and characterize the cDNAs for the protein(s). Thyroid dysfunction during human development results in brain damage, the most severe manifestation of which is cretinism. Yet the mechanisms and regulation of TH action during this period have received relatively little attention, due in part to the difficulties of conducting studies in fetal mammals. Thr proposed studies will utilize an eminently suitable animal model to investigate these important biological processes.