Molecular mechanism of thyroid hormone receptor function during metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

ANALYSIS OF THYROID HORMONE RECEPTOR KNOCKOUT TADPOLES REVEALS THAT THE ACTIVATION OF CELL CYCLE PROGRAM IS INVOLVED IN THYROID HORMONE-INDUCED LARVAL EPITHELIAL CELL DEATH AND ADULT INTESTINAL STEM CELL DEVELOPMENT DURING XENOPUS TROPICALIS METAMORPHOSIS. We have recently knocked out the TR genes, TR and TR, individually or both in Xenopus tropicalis and analyzed its effect on tadpole development and metamorphosis of different organs. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We observed that both TR and TR play important roles for intestinal remodeling. To understand the underlying molecular mechanism, we recently studied the function of endogenous TR in the tadpole intestine by using knockout animals and RNA-seq analysis. We observed that removing endogenous TR caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild type and TR knockout tadpoles treated with or without TH for 1 day, prior to any detectable TH-induced cell death and stem cell formation in the tadpole intestine, we identified over 1500 genes regulated by TH treatment of the wild type but not TR knockout tadpoles. Gene ontology and biological pathway analyses revealed that surprisingly, these TR-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Our findings suggest that TR-mediated TH-activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling. A ROLE OF ENDOGENOUS HISTONE ACETYLTRANSFERASE STEROID HORMONE RECEPTOR COACTIVATOR (SRC) 3 IN THYROID HORMONE SIGNALING DURING XENOPUS INTESTINAL METAMORPHOSIS. We have shown previously that during metamorphosis, liganded TR recruits coactivator complexes that include steroid receptor coactivator SRC3, which is a histone acetyltransferase, to TH responsive promoters. To investigate the functions of endogenous coactivators such as SRC3 during metamorphosis, we have generated Xenopus tropicalis animals lacking a functional SRC3 gene and analyzed the resulting phenotype. While removing SRC3 had no apparent effect on external development and animal gross morphology, the SRC3 (-/-) tadpoles displayed a reduction in the acetylation of histone H4 in the intestine comparing to that in wild type animals. Furthermore, the expression of TR target genes was also reduced in SRC3 (-/-) tadpoles during intestinal remodeling. Importantly, SRC3 (-/-) tadpoles had inhibited/delayed intestinal remodeling during natural and TH-induced metamorphosis, including reduced adult intestinal stem cell proliferation and apoptosis of larval epithelial cells. Our results thus demonstrate that SRC3 is a critical component of the TR-signaling pathway in vivo during intestinal remodeling. EVOLUTIONARY DIVERGENCE IN TAIL REGENERATION BETWEEN XENOPUS LAEVIS AND XENOPUS TROPICALIS. Tissue regeneration is of fast-growing importance in the development of biomedicine, particularly organ replacement therapies. Unfortunately, many human organs cannot regenerate. Anuran Xenopus laevis has been used as a model to study regeneration as many tadpole organs can regenerate. In particular, the tail, which consists of many axial and paraxial tissues, such as spinal cord, dorsal aorta and muscle, commonly present in vertebrates, can fully regenerate when amputated at late embryonic stages and most of the tadpole stages. Interestingly, between stage 45 when feeding begins to stage 47, the pseudotetraploid Xenopus laevis tail cannot regenerate after amputation. This period, termed refractory period, has been known for about 20 years. The underlying molecular and genetic bases are unclear in part due to the difficulty to carry out genetic studies in this pseudo-tetraploid species. The availability of the highly related but diploid Xenopus tropicalis offers an opportunity to study the molecular and genetic mechanisms of tail regeneration during this refractory period. We compared tail regeneration between Xenopus laevis and Xenopus tropicalis and found surprisingly that Xenopus tropicalis lacked the refractory period. Further molecular and genetic studies, more feasible in this diploid species, should reveal the basis for this evolutionary divergence in tail regeneration between two related species and facilitate the understanding how tissue regenerative capacity is controlled. In addition, it is well known that many tadpole tissues lose their regenerative capacity during metamorphosis, suggesting a role of TH and TR in regeneration. Making use of our recently generated TR knockout animals, we plan to also investigate if and how TH and TR regulate tissue regeneration. Such studies should have important implications for human regenerative medicine.