The long range goal of this project is to elucidate the mechanism(s) of action of the thyroid hormones(s) (TH) during development using the amphibian Rana catesbeiana (RC) as the animal model. It is well known that TH is essential for all aspects of development of this species. However, metamorphosis does not take place in all tissues at the same time. For example, hindlimb development is accomplished at the relatively low levels of plasma TH present in prometamorphosis while tail resorption does not begin until climax. Elucidation of the factors and mechanisms which control TH-responsiveness of a tissue is key to the question of how metamorphosis is coordinated. cDNAs for the amphibian iodothyronine 5-deiodinase (5D) have recently been cloned and a potentially highly significant finding is the expression of the 5D gene is rapidly enhanced by TH. Moreover, this response can be obtained in RC tadpoles soon after hatching. The expression of the TH receptor genes (TRalpha and TRbeta) is also enhanced by TH in developing tadpoles. This research project will test the hypothesis that an important feature in the coordination of metamorphosis is the regulation by TH of the expression of the genes which encode the 5-deiodinase and the two TH receptors. The approach will be to first characterize the full-length RC cDNA for the 5D. This cDNA and RC cDNAs for the two receptor gene will then be used to determine the ontogenic profile of the expression of the three genes at the mRNA level. 5D activity will be determined using hybridization and reverse transcription-PCR techniques. Next, the role of TH in the initiation and maintenance of the expression of the genes will be examined. The effects of inhibiting the activity of the 5D enzyme on both TH-induced morphological development and the expression of the three genes will be determined. Finally ireverse transcription in situ PCR techniques will be used to target gene expression to specific cell types within a tissue or organ.
