Thyroid hormone (TH) is critically important for many aspects of vertebrate growth and development. In amphibia, it is essential for all post-embryonic stages of development and metamorphosis, and in this laboratory the Rana catesbeiana (RC) tadpole has proved to be an excellent vertebrate model in which to study the actions of TH in development at the whole system, cellular and molecular levels. To this end, the cDNAs for the RC TH receptors (TRalpha,TRbeta), and the deiodinases, which activate (D2) and degrade (D3) TH, have been cloned and characterized. All four genes are differentially expressed in tadpole tissues, their expression is developmentally regulated and the TRs and D3 genes are upregulated by TH. Furthermore, expression of all four genes is notably high in eye even during premetamorphosis. Recently, using TRs as bait, cDNAs for three novel amphibian TR interactive proteins (ATIP) were isolated from RC tadpole cDNA libaries by yeast two hybrid screening. Preliminary studies suggest a role for these ATIPs in TH action. Of particular interest was the finding that, as with the TRs and deiodinases, eye is major site of expression of mRNA transcripts related to these cDNAs. The tadpole eye has long been used as a model for the study of TH-dependent development of vertebrate vision and specific changes have been reported, several of which can be readily induced by systemic or local TH treatment and documented. Since the eye expresses the TRs and deiodinases, all factors critical for TH-dependent development, and the three proteins postulated to be involved in TH action it provides an ideal model system in which to test the hypothesis that the three ATIP proteins play a role in TH action. In the first specific aim, the interactions between the RC TRs and each of the ATIP proteins in the presence and absence of TH will be determined using a cultured cell line. The effects of each ATIP protein on TR-mediated transcription will also be assessed. In the second specific aim, the temporo-spatial patterns of expression in the RC eye of the TRs, D2, D3 and the ATIP proteins will be determined by in situ hybridization. Those cells which co-express TR and an ATIP will be identified and correlated with the cells undergoing T3-dependent and/or stage specific proliferation. In the third specific aim, the physiological roles of the ATIP proteins in the RC eye will be determined. The effect of disrupting the expression of the endogenous form of each ATIP protein on indicators of T3 action in the intact eye or the appropriate cultured cell line will be examined. In addition the ATIP proteins will be used as bait in the screening of RC eye cDNA libraries to identify additional proteins involved in TR-mediated gene regulation. These studies will provide new and important information concerning the molecular mechanisms involved in TR mediated effects on gene transcription in a developing vertebrate and the role of thyroid hormone action in the developing amphibian eye. In addition they will offer an invaluable basis from which to explore the role of TH in the developing mammalian eye.
