Furlow, J.D.*; Lim, W.; Ermio, D.J.; Chiellini, G.; Scanlan, T.S.: Molecular mechanisms underlying thyroid hormone induced gene expression cascades during amphibian metamorphosis

Amphibian metamorphosis is an excellent system to understand nuclear receptor function during development. Thyroid hormone (TH) induces a wide range of morphological changes by regulating gene expression via a pair of nuclear receptors, TH receptor ALPHA (TRALPHA) and TH receptor BETA (TRBETA). Our laboratory is interested in how this dramatic tissue specific transcriptional control by TH occurs. Previous expression studies in the frog Xenopus laevis implicated TRALPHA in adult tissue formation and TRBETA in larval tissue resorption. We employed the TRBETA selective agonist GC-1 as a pharmacological probe of TR function. In transfection assays, GC-1 activated Xenopus TRBETA 20-fold better than Xenopus TRALPHA, compared to the natural ligand T3. In GC-1 treated tadpoles, tail and gill resorption was efficiently induced with little effect on limb growth. In addition, GC-1 preferentially activated a subset of TH-response genes in the tail, including several protease genes. We are currently screening additional synthetic TH analogs, as well as exploring other means to specifically manipulate TR isotype activity in living cells. Finally, we are characterizing the regulatory control regions of several TH response genes in vivo using transgenesis. We have shown that the TH/bZIP promoter drives green fluorescent protein (GFP) expression in transgenic tadpoles in a proper temporal and spatial pattern. Our initial studies with the fibronectin promoter, a late TH response gene at metamorphosis, showed a high level of GFP expression in gastrulating embryos, as expected. We are now following GFP expression driven by wild type and mutated fibronectin promoter constructs throughout development.