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 an organ of a multiply folded adult epithelium surrounded by 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 for TR during frog development. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) activate gene expression during metamorphosis when TH is present. In premetamorphic tadpoles, they repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. By using the sperm-mediated transgenic approach, we have previously generated transgenic animals expressing a dominant negative TR (dnTR), which allowed us to show that gene activation by TR in the presence of TH, in part through the release of corepressors, is necessary for metamorphosis. In addition, by generating transgenic tadpoles expressing a dominant positive TR, which activates TH response genes without a requirement for TH, under a heat shock-inducible promoter, we have shown more recently that heat shock induction of its expression is sufficient to induce all morphological and gene expression changes associated with TH-induced metamorphosis. Such studies have led us to conclude that the metamorphic role of TH is predominantly, if not exclusively, through genomic action of the hormone. Non-genomic action of TH, while exists, plays a minor role, if any, during this postembryonic process. They further provide the first example where TR is shown to mediate directly and sufficiently the developmental effects of TH in individual organs by regulating target gene expression in these organs. Our current research focuses on how TR differentially regulates different genes in various organs/tissues during metamorphosis. ROLES OF COFACTORS IN GENE REGULATION BY TR. TR regulates gene transcription by recruiting cofactors to target genes. In the presence of TH, TR can bind to coactivators while the unliganded TR binds to corepressors. Many in vitro biochemical and molecular studies have been done on such cofactors. On the other hand, much less is known about whether and how they participate in gene regulation by TR in different biological processes in vivo. Our focus is to investigate how TR utilizes different cofactors in the context of development in various organs. Among the corepressors, we have been studying the role of N-CoR and SMRT in gene repression by TR in premetamorphic tadpoles. Both N-CoR and SMRT are known to exist in histone deacetylase-containing complexes in mammals. Among the proteins in the complexes is TBLR1 (transducin beta-like protein 1-related protein). Using chromatin immunoprecipitation (ChIP) assay, we have demonstrated that unliganded TR recruits TBLR1, together with N-CoR and/or SMRT, to its target promoters in chromatin in premetamorphic tadpoles, and that TH treatment of premetamorphic tadpoles leads to the release of TBLR1, together with N-CoR and/or SMRT. These results argue that TBLR1 or related factors are required for transcription repression by unliganded TR in tadpoles and that the release of TBLR1-containing corepressor complexes is one of the mechanisms by which TH response genes are activated during metamorphosis. In addition, our earlier studies have shown that Xenopus coactivator SRC3 is upregulated as a late TH response gene during natural as well as TH-induced metamorphosis in both the tail and intestine. Using ChIP assay, we have found surprisingly that SRC3 is recruited in a gene- and tissue-dependent manner to target genes by TR. Furthermore, we have generated transgenic tadpoles expressing a dominant negative form of SRC3 (F-dnSRC3). We have shown that transgenic expression of F-dnSRC3 inhibits essentially all aspects of metamorphosis and that F-dnSRC3 functions by blocking the recruitment of endogenous coactivators to TH-target genes without affecting corepressor release. Our studies thus demonstrate that coactivator recruitment, aside from corepressor release, is required for TH function in development and further provide the first example where a specific coactivator-dependent gene regulation pathway by a nuclear receptor has been shown to underlie specific developmental events. INVOLVEMENT OF MATRIX METALLOPROTEINASES DURING TH-INDUCED TISSUE REMODELING. We have previously identified several TH-response genes encoding matrix metalloproteinases (MMPs) during intestinal remodeling. Furthermore, earlier studies suggest that the MMP stomelysin-3 (ST3) is involved in ECM (extracellular matrix) remodeling, which in turn influences cell behavior. This has been supported by functional studies in organ cultures. We have now provided in vivo evidence for a role of ST3 in regulating ECM remodeling and cell death. By generating transgenic animals expressing ST3 or a catalytically inactive mutant under a inducible promoter, we have shown that overexpression of wild type but not mutant ST3 causes premature apoptosis in the tadpole epithelium. Furthermore, the apoptosis is accompanied by drastic remodeling of the basal lamina, or the ECM that separates the connective tissue and epithelium in the intestine. These results suggest that ST3 directly or indirectly modifies the ECM, which in turn facilitate cell fate changes and tissue morphogenesis. Toward understanding the mechanism by which ST3 affects tissue remodeling, we have used yeast two-hybrid screen to identify potential substrates. We have thus isolated the 37 kd laminin receptor precursor (LR) as a likely substrate. LR binds to ST3 in vitro and can be cleaved by ST3 at two sites, distinct from where other MMPs cleave. Peptide sequencing revealed that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence, suggesting that LR cleavage by ST3 will alter cell-ECM interaction. Developmentally, LR is expressed in the intestinal epithelium of premetamorphic tadpoles. During intestinal metamorphosis, LR is downregulated in the apoptotic epithelium and concurrently upregulated in the connective tissue but with little expression in the developing adult epithelium. Toward the end of metamorphosis, as adult epithelial cells differentiate, they begin to express LR. Furthermore, LR is cleaved during intestinal remodeling when ST3 is highly expressed or in premetamorphic intestine of transgenic tadpoles overexpressing ST3. Thus, LR is likely a physiological substrate of ST3 and plays a role in cell fate determination and tissue morphogenesis, in part through changes in its spatial expression during development and in part through its cleavage by ST3.

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U.S. National Inst/Child Hlth/Human Dev
United States
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Ishizuya-Oka, Atsuko; Hasebe, Takashi; Shi, Yun-Bo (2010) Apoptosis in amphibian organs during metamorphosis. Apoptosis 15:350-64
Matsuda, Hiroki; Shi, Yun-Bo (2010) An essential and evolutionarily conserved role of protein arginine methyltransferase 1 for adult intestinal stem cells during postembryonic development. Stem Cells 28:2073-83
Mathew, Smita; Fu, Liezhen; Hasebe, Takashi et al. (2010) Tissue-dependent induction of apoptosis by matrix metalloproteinase stromelysin-3 during amphibian metamorphosis. Birth Defects Res C Embryo Today 90:55-66
Matsuda, Hiroki; Paul, Bindu D; Choi, Cheol Young et al. (2009) Novel functions of protein arginine methyltransferase 1 in thyroid hormone receptor-mediated transcription and in the regulation of metamorphic rate in Xenopus laevis. Mol Cell Biol 29:745-57
Heimeier, Rachel A; Das, Biswajit; Buchholz, Daniel R et al. (2009) The xenoestrogen bisphenol A inhibits postembryonic vertebrate development by antagonizing gene regulation by thyroid hormone. Endocrinology 150:2964-73
Shi, Yun-Bo (2009) Dual functions of thyroid hormone receptors in vertebrate development: the roles of histone-modifying cofactor complexes. Thyroid 19:987-99
Fiorentino, Maria; Fu, Liezhen; Shi, Yun-Bo (2009) Mutational analysis of the cleavage of the cancer-associated laminin receptor by stromelysin-3 reveals the contribution of flanking sequences to site recognition and cleavage efficiency. Int J Mol Med 23:389-97
Wang, Di; Guo, Ming-Xiong; Hu, Hai-Ming et al. (2008) Human T-cell leukemia virus type 1 oncoprotein tax represses ZNF268 expression through the cAMP-responsive element-binding protein/activating transcription factor pathway. J Biol Chem 283:16299-308
Heimeier, Rachel A; Hsia, Victor Shaochung; Shi, Yun-Bo (2008) Participation of Brahma-related gene 1 (BRG1)-associated factor 57 and BRG1-containing chromatin remodeling complexes in thyroid hormone-dependent gene activation during vertebrate development. Mol Endocrinol 22:1065-77
Hasebe, Takashi; Kajita, Mitsuko; Shi, Yun-Bo et al. (2008) Thyroid hormone-up-regulated hedgehog interacting protein is involved in larval-to-adult intestinal remodeling by regulating sonic hedgehog signaling pathway in Xenopus laevis. Dev Dyn 237:3006-15

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