Virtually all of the diverse physiological effects of thyroid hormone are thought to be initiated by the binding of triiodothyronine (T3) to nuclear DNA binding proteins, termed T3 receptors (T3R), which regulate gene transcription. There are 2 separate T3R genes, alpha and beta, with differences in tissue distribution, ligand affinity, developmental expression, and regulation by T3. Generalized Thyroid Hormone Resistance (GTHR) is an inherited clinical syndrome, linked to T3R gene deletions and mutations, which is associated with marked phenotypic variation in the degree to which different tissues are affected. This indicates that tissues vary in their ability to compensate for the loss of a specific T3R, although the mechanisms underlying the various phenotypes are not known due to the inaccessibility of human tissue for study. The focus of this application is to develop cellular and animal models to study the function and interrelationship of the alpha and beta T3Rs. We will employ gene targeting techniques to inactivate selectively the T3R genes in mouse embryonic stem (ES) cells and use these cells to create T3R deficient progeny mice. In preliminary studies, we have already cloned and characterized the critical portions of the mouse T3Ralpha and beta genes. We have also constructed a vector and successfully inactivated a T3Ralpha allele in ES cells by homologous recombination. Similar approaches will be used to inactivate the second T3Ralpha allele in these cells and both T3Rbeta alleles in others. These ES cells, expressing either T3Ralpha or beta, will be used in transient transfection studies to determine the specificity of the T3Ralpha and beta for T3 response elements from T3 dependent genes. ES cells with a single null T3Ralpha or beta allele will also be used to breed chimeric mice. Progeny with germ line transmission of the inactivated gene will be identified and bred to produce homozygous alpha or beta T3R deficient mice. These animals will be analyzed for specific nuclear T3 binding in various tissues. Subsequent biochemical and physiologic studies will evaluate the effects of T3R gene inactivation on growth and development, cardiac function, regulation of TSH, growth hormone secretion, and thyroid hormone response liver enzyme activity. The effects of changes in thyroid status on these biochemical and physiological parameters will also be assessed. Cell lines and mice with specific T3R gene inactivation will provide a powerful model to understand the tissue and gene specific roles of alpha and beta T3Rs in thyroid hormone regulated processes.
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