Gene Regulation And Function By Thyroid Hormone During T

Child Health And Human Development

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Abstract

The research in the Unit on Molecular Morphogenesis focuses on the understanding of the molecular mechanism of amphibian metamorphosis. The control of this developmental process by thyroid hormone (TH) offers a unique paradigm in which to study genes that are important for 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 and in vitro in organ culture 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 and Mechanism of Gene Regulation by TR during Frog Development. With regard to TR function, 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. First, chromatin immunoprecipitation (ChIP) assay shows for the first time that TR/RXR are bound to thyroid hormone response elements (TREs) in the target genes in premetamorphic tadpoles. In support of a role of histone acetylation in gene regulation by TR/RXR, blocking deacetylase function with the drug trichostatin A (TSA) can activate TH response genes in the intestine of premetamorphic tadpoles, just like TH. Furthermore, treatment of premetamorphic tadpole with TH leads to increased histone acetylation at the TRE regions as the target genes are being activated. In addition, TH treatment causes a reduction in the association of the histone deacetylase Rpd3 with TH response genes. Together, these studies suggest a role of deacetylase complex in gene repression by unliganded TR in premetamorphic tadpoles. Interestingly, our studies with TSA also showed that histone deacetylases is required at a step(s) downstream of gene activation by TH-bound TR during metamorphosis. Thus, histone deacetylases plays a role at two distinct steps during frog development. To investigate how histone deacetylases participate in development, we have begun to characterize frog histone deacetylase complexes. To do this, we made use of the fact that frog egg contains all necessary components for gene regulation by TR to isolate complexes that contain the TR-interaction corepressor N-CoR. These led to the purification of three N-CoR complexes. Two of them possess histone deacetylase activity, one of which contains Sin3, Rpd3, and RbAp48, the first such complexes purified even though interactions among Sin3, Rpd3, and RbAp48 have been known for some time. The second complex contains a Sin3-independent histone deacetylase. The third complex lacks histone deacetylase activity. Our complexes differ from the apparently identical mammalian complexes reported by a few other laboratories, demonstrating the complexity of gene repression mediated by N-CoR. Immunoprecipitation studies show that N-CoR binds to unliganded TR expressed in the frog oocyte confirming that N-CoR complexes are involved in repression by unliganded TR. The failure to bring down Sin-3 by anti-TR antibody suggest that complex 1 is unlikely involved in gene repression by unliganded TR. Our current studies aim at identifying the components of the complexes and investigate which complex is involved in gene repression by TR during frog development. In addition, we are also employing the recently developed transgenesis technology in Xenopus to analyze TR function directly during metamorphosis. Preliminary data showed over-expressing a dominant negative TR in the tadpole intestine had no effect on tadpole development but interfered with adult epithelial morphogenesis, consistent with the role of TR in mediating the effects of TH during tissue transformation. Roles of Matrix Metalloproteinases during TH-Induced Tissue Remodeling. In the second area, we have been focusing on TH-response genes encoding matrix metalloproteinases (MMPs), which 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 intestinal organ cultures, we have now shown that ST3 expression is associated with ECM remodeling and cell death in TH-induced intestinal remodeling in vitro. Studies with primary cell cultures of the intestine have shown that ECM inhibits TH-induced larval epithelial cell death in vitro. These observations prompted us to investigate whether ST3 is required for epithelial transformation in the organ cultures. By using a function-blocking antibody against the catalytic domain of ST3, we have demonstrated 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 in the remodeling of the basal lamina or basement membrane, the ECM that separate the connective tissue and the epithelium, supporting 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), died in a dose-dependent manner during late embryogenesis. The specificity of this embryonic lethal phenotype was confirmed by the failure of a catalytically inactive mutant of ST3 to affect development. Finally, over-expression of a mammalian membrane type-MMP also led to late embryonic lethality in Xenopus embryos, suggesting that membrane type-MMPs have functions in vivo for ECM remodeling, in addition to being activators of other proMMPs. These data together with the developmental expression of several MMPs during Xenopus development, suggest that MMPs play important roles during mid to late embryogenesis and that proper regulation of MMP genes is critical for tissue morphogenesis and organogenesis. They also suggest that alteration in MMP expression during metamorphosis may alter TH-dependent tissue remodeling process. Thus we have begun to investigate MMP function during metamorphosis by using tissue specific promoters to drive the expression of MMPs during metamorphosis.