Donald D. Brown

Amphibian Metamorphosis

The fertilized egg of the frog Xenopus laevis takes about one week to become a feeding tadpole. Three days later the tadpole forms a small thyroid gland. As the tadpole grows, the gland enlarges and synthesizes increasing amounts of thyroid hormone (TH). The concentration of this hormone controls the metamorphosis of tadpoles into frogs. TH is known to function by binding to proteins located in the nucleus of target cells called thyroid receptors, which in turn activate or repress genes. The Brown group has studied metamorphosis as a set of complex genetic programs, which are controlled by this simple hormone.

How can a simple hormone trigger such very diverse responses in most of the tissues of a tadpole? To find out the answer, the scientists began by cloning the two forms of thyroid hormone receptors. One of these is present all of the time while the amount of the other is controlled by the TH. Next they identified many of the genes that are up- or down-regulated by TH in several target tadpole organs. These include the tail, which dies and is resorbed, the limb, which grows and differentiates, and the intestine, which is completely remodeled during metamorphosis. Next the researchers found the exact cell types that express these genes. By sequencing the genes that are regulated by TH, the team identified the protein that each gene encodes. Knowing the identity of the gene and where and when it is expressed provided clues on the possible role of each gene in metamorphosis. One conclusion has been that the extensive thyroid-hormone-induced cell death that occurs consists of multiple genetic programs.

Recently, the Brown group has been aided by the development of a technique called transgenesis, in which any isolated gene can be introduced into the genome of a Xenopus embryo before first cleavage (Kroll, K. L., and E. Amaya, Development 122, 3173-3183,1996). This guarantees that all the cells of the embryo and the tadpole will contain the gene. Using this powerful method, the Brown group has tested many of their genes and developed functional assays for gene function in metamorphosis.

Some of the discoveries that the group has made are as follows:

The hormone prolactin, previously thought to be a juvenile hormone for anuran tadpoles, does not retard metamorphosis. However, it specifically inhibits the tail resorption program, yielding a powerful tool in the search for the basis of cell death in the tail (Fig. 1).

Growth hormone has the same effect on tadpoles and frogs as it does on mammals. It dramatically increases growth (Fig. 2).

The group has identified two independent methods to inhibit functions that are dependent on TH; namely, overexpressing either a gene encoding an enzyme that destroys the hormone, or an altered form of the hormone's receptor called a dominant negative inhibitor. Both kinds of genes are introduced under the control of a specific promoter. The resulting transgenic animals are unable to complete specific sets of functions. These new methods are making possible a new approach to the age-old problem of amphibian metamorphosis.

Fig. 1. This is a picture of frogs with overexpressed prolactin. The result is inhibited tail reabsorption.

Fig. 2. Both control tadpoles and frogs, and those overexpressing growth hormone, are shown.

¹ Kroll, K. L., and E. Amaya, Development 122, 3173-3183,1996.