This is a comprehensive study of the mechanisms of differentiation and the action of thyroid hormone (TH) using amphibian metamorphosis as a model system. Particular attention is paid to the initiating mechanisms by which TH trigger the metamorphic process. This includes a study of TH receptors and their effect on genetic expression originating in the cell nuclei. We expect this research to contribute also to our knowledge of comparative biochemistry, embryonic development, endocrinology and biochemical evolution. Our four major objectives are: (1) Characterization of the nuclear receptors of TH in anurans. This includes the study of the effect of temperature, subcellular distribution, the binding of T3 to tadpole chromatin, and the isolation and properties of nuclear-T3 binding proteins. Rationships to the onset and completion of metamorphosis, specific tissue response, and to hormonal states in other amphibian systems will be sought. An unusual opportunity is provided by nucleated erythrocytes in transition which possess typical nuclear receptors for T3. (2) Determine the effects of TH on the nucleus and macromolecular biosynthesis during spontaneous and induced metamorphosis. This includes an analysis of changes in mRNA biosynthesis and processing, particularly the mRNA for albumin and metallothionein. Also we will continue our focus on the enzymic machinery involved in nuclear action on RNA proteins, RNA polymerases and the enzymes involved in protein synthesis and degradation. (3) TH also stimulates membrane responses which may not be mediated by the nucleus. We have reported that cyclic-GMP increased 100% 60 min after T3 treatment. These data need to be reinforced by studies on guanylate cyclase, phosphodiesterase inhibitors, and permeable forms of cGMP (Bu2cGMP) and appropriate prosphokinases. The relation between this cGMP response and nuclear actions will be studied to determine how independent are these membrane effects. (4) These ideas and results will be analyzed for the basis of endocrine control of developmental blocks in other amphibia. The urodeles provide a richer reservoir of biological systems, frequently incomplete or modified in their endocrine mechanisms and developmental expressions. Thus we hope to answer basic questions on the endocrine mechanisms which have evolved to reach the present state of vertebrate development.
