This laboratory is exploring molecular mechanisms in amphibian metamorphosis. The control of this developmental process by thyroid hormone (TH) offers a unique paradigm in which to study gene function in postembryonic organ development. During metamorphosis, different organs undergo vastly different changes. Some, like the tail, undergoes complete resorption, while others, such as the limb, are developed de novo. The majority of the larval organs persist through metamorphosis but are dramatically remodeled to function in a frog. For example, tadpole intestine in Xenopus laevis is a simple tubular structure consisting of primarily a single layer of primary epithelial cells. During metamorphosis, it is transformed into a multiply folded adult epithelium with elaborate connective tissue and muscles through specific cell death and selective cell proliferation and differentiation. The wealth of knowledge from past research and the ability to manipulate amphibian metamorphosis both in vivo by using transgenesis or hormone treatment of whole animals, and in vitro in organ cultures offer an excellent opportunity to 1) study the developmental function of thyroid hormone receptors (TRs) and the underlying mechanisms in vivo and 2) identify and functionally characterize genes which are critical for postembryonic organ development in vertebrates. FUNCTION OF TR DURING DEVELOPMENT. We have proposed a dual function model based on our earlier studies in the oocyte and developing embryos and tadpoles. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) activate gene expression during metamorphosis when TH is present, while in premetamorphic tadpoles, they repress gene expression to prevent metamorphosis, thus ensuring a tadpole growth period. Our studies since have provided strong support and mechanistic insights for such a model. In our most recent studies, we have shown that corepressors N-CoR and SMRT are recruited to TH-response genes in premetamorphic tadpoles and are released upon treatment of the tadpoles with TH, indicating the unliganded TR recruit these corepressors to repress target genes in tadpoles. In agreement with the ability of these corepressors to form complexes containing histone deacetylases (HDACs), TH treatment leads to increase in local histone acetylation at the TH response genes at least in the intestine and tail, arguing that histone acetylation is an important factor in gene regulation by TR. To investigate the function of TR in vivo, we have adapted sperm-mediated transgenic method to generate transgenic animals expressing a dominant negative TR. Phenotypic analysis indicate that overexpression of the dominant negative TR inhibits TH-induced metamorphosis. More importantly, we have shown that the dominant negative TR specifically blocks the expression of TH response genes that we and others have identified previously. This provides molecular support for the central role of TR in regulating the response genes to mediate the effects of TH on metamorphosis. ROLES OF COFACTORS IN GENE REGULATION BY TR. TR regulates gene transcription by recruiting cofactors to target genes. Many biochemical and molecular studies have been done on such cofactors. Our focus is to investigate how TR utilizes different cofactors in the context of development in various organs. We have recently characterized one coactivator, the Xenopus TRIP7. However, it has relative small effect on TR function based on our studies using the frog oocyte model system and thus its role in developmental gene regulation by TR may be limited. Thus, we have begun to study p300 and SRC, whose involvement in TR function has been supported by in vitro and tissue culture cell studies. We have obtained the cDNA clones for Xenopus p300, SRC1, and SRC2, and have shown that they are expressed during metamorphosis. In the corepressor area, as indicated above, we have shown that the corepressor N-CoR and SMRT are recruited to TH response genes in developing tadpoles and that histone acetylation is involved in gene regulation by TR in tadpoles. Both N-CoR and SMRT are known to interact with other proteins. To investigate how these corepressors participate in the repression by unliganded TR, we have isolated three N-CoR-corepressor complexes. However, due to low abundance, we have yet to determine the identities of the components in the complexes. On the other hand, the TBL1 (transducing-b-like I) protein has been shown to exist in an N-CoR complex in Hela cells that was identified by others. We have cloned the frog homolog and intend to investigate whether TBL1 is present in our N-CoR complexes and whether it participates in N-CoR-dependent gene repression by TR. With both corepressors and coactivators, we intend to use chromatin immunoprecipitation assay to determine their recruitment by TR to target genes in tadpoles. To directly investigate their function in vivo, we will overexpress precociously wild type or dominant negative cofactors and analyze the resulting effects on animal development and target gene expression (and local changes in chromatin structure, when appropriate). INVOLVEMENT OF MATRIX METALLOPROTEINASES DURING TH-INDUCED TISSUE REMODELING. In an effort to identify genes important for postembryonic development, we have isolated many TH response genes during metamorphosis. Expression analyses and other studies have led us to focus on the TH-response genes encoding matrix metalloproteinases (MMPs) for functional investigations. MMPs are extracellular enzymes capable of digesting various ECM components. Our earlier studies have led us to propose that the MMP stomelysin-3 (ST3) is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior. By using a function-blocking antibody against the catalytic domain of ST3, we have demonstrated in organ cultures that blocking ST3 function inhibits TH-induced apoptosis of larval intestinal epithelial cells and the invasion of the proliferating adult epithelial cells into the connective tissue. These effects are accompanied by an inhibition of the remodeling of the basal lamina or basement membrane, the ECM that separate the connective tissue and the epithelium. These results support the argument that ST3 is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior. To directly investigate the roles of MMPs in developing animals, we are employing the transgenic approach to express wild type and mutant MMPs in Xenopus embryos and tadpoles. In our initial study, we over-expressed Xenopus MMPs stromelysin-3 (ST3) and collagenases-4 (Col4) under the control of a ubiquitous promoter and observed that embryos with over-expressed ST3 or Col4, but not the control green fluorescent protein (GFP) or a mutant ST3, died in a dose-dependent manner during late embryogenesis. This lethality in early development prevented us from investigating the roles of MMPs during metamorphosis. Thus, we have developed a double promoter approach to overexpress MMPs under an inducible promoter. In our preliminary study, we generated transgenic tadpoles by using a double promoter construct where ST-3 is under the control of the heat shock inducible promoter. Heat shock at tadpole stages led to overexpression of stromelysin-3 in all organs, although no visible morphological changes of the tadpoles were observed for 4 days. Analysis of the intestine showed that overexpression of stromelysin-3 caused premature apoptosis in the tadpole epithelium, consistent with our organ culture studies earlier. We are currently investigating how the transgenic animals undergo natural and TH-induced metamorphosis.
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