Amphibian metamorphosis involves the growth and differentiation, cell death and resorption, or remodeling of most tadpole tissue to become a frog. These changes are controlled by thyroid hormone (TH). Metamorphosis is a remarkably tractable developmental system since the diverse changes can be induced precociously with exogenous TH or inhibited pharmacologically with well known """"""""goitrogens"""""""" added to the rearing water. Many features of TH physiology are the same in all vertebrates including the negative feedback loop between the thyroid gland the pituitary, the remarkable similarity in the TH receptors (TR), their RCR partners, and the DNA elements (TRE) to which these receptors bind. We have developed a three phase strategy to study individual programs of metamorphosis. First, we identified genes that are up- and down-regulated by TH in the tail, limb, intestine, and a cultured cell by means of a subtractive hybridization method that has the added power of estimating the number of genes in each program. We have concentrated our attention on the simplest program, tail resorption, in which we isolated about half of the TH-induced up- and down-regulated genes (17 and 4, respectively) clues about their possible roles in the resorption program. This grant application deals with the third phase which is underway, namely assaying genes for functional role in metamorphosis. The recent discovery of a transgenesis method for Xenopus laevis has provided us with such an assay. The function of the TH response gene in metamorphoses can be assessed as well as the various receptors, co-activators, and o-repressors that are presumed to play a role in the ability of TH to alter gene expression. Of special interest is an examination of the role of unliganded thyroid receptor in suppressing metamorphic changes. The participation of various hormones other than TH that have been implicated in amphibian metamorphosis is being assessed. The molecular explanation for many well documented biological features of metamorphosis such as the onset of competence to respond to TH, the different chronology of tissue response to the hormone (heterochrony) can be studied from a new perspective. We have already observed instructive phenotypes by over-expressing genes that inhibit metamorphosis. Strategies are described that can take advantage of these phenotypes and elucidate developmental pathways.
