Amphibian metamorphosis is a complex series of changes in gene expression that affect every tissue and organ of the tadpole. The process is controlled entirely by thyroid hormone (TH). The goal of this project is to understand at a molecular level how one hormone can initiate so many different developmental programs and to elucidate the molecular details of a few of these programs. The experimental strategy has three phases: first, the isolation of up and down regulated gene in several programs, second, the identification of regulated genes by sequencing and comparison with the public data base, and third, the determination of the role of these genes in the developmental program. During the past 5 years we have completed the first two phases, and in this proposal we outline functional assays for the many genes that we have identified. In the course of these experiments it has become apparent that amphibian metamorphosis is a superb model system for the study of TH action at the molecular level. These studies will provide information relevant to the various questions addressed by researchers who have used exclusively mammals and chickens as their experimental organisms. The hormone receptors are the same, and the general pattern of their expression is similar. The well known negative feedback loop between the thyroid and pituitary glands in mammals actually develops during metamorphosis. The amphibian system is far easier to manipulate than chickens and mammals as will become evident from the body of this application. Because of the accessibility of the system and our development of a powerful subtractive hybridization method (the """"""""gene expression screen""""""""), large numbers of TH regulated genes have been identified in tadpoles far in excess of all the TH regulated genes combined that have been found in other organisms. This prompts a reexamination of the thyroid response elements (TRE) and some classic questions about the molecular basis of TH action. In addition, TH induced metamorphosis provides a novel way to study vertebrate organogenesis since the characteristic organ changes can be induced at will by the experimenter simply by adding exogenous hormone to a competent tadpole. In Th-induced metamorphosis the expression of a predetermined program is studied. However, we are now analyzing two related complex developmental systems designed to find genes that set up the programs. These two systems are tail regeneration and the homeotic transformation from tail to limb induced by retinoic acid. These systems are examples of important biological events controlled by changes in gene expression that are not subject to genetic analysis but can be studied by this three phase approach. The affect of any hormone on its target tissue, the action of drugs that work by affecting gene expression, and the comparison of genes expressed in a variety of diseased versus normal tissues are candidates for this kind of analysis.
