Molecular mechanism of thyroid hormone receptor function during metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

COMPLEMENTARY AND ADDITIVE FUNCTIONS OF TRΑ AND TRΒ DURING INTESTINAL REMODELING AS REVEALED BY CHIP-SEQ ANALYSIS ON WILD TYPE AND TR KNOCKOUT ANIMALS. Our earlier studies on TRα and TRβ knockout animals reveals distinct effects of the two TR gene knockouts on limb development, intestinal remodeling, and tail resorption during development. The effects correlate with the relative expression levels of the TRα and TRβ genes during development in these organs as previously reported by us and others for both Xenopus laevis and Xenopus tropicalis. On the other hand, individual TR knockout does not prevent the eventual completion of metamorphosis and the development of reproductive adult frogs, suggesting the two TR genes can completement each other. To investigate this further, we used intestinal remodeling during Xenopus tropicalis metamorphosis, which serves as a model for human postembryonic development, to identify TR-bound genes and determine the relative contribution to target gene binding by TRα and TRβ. We first examined the localization of TRα and TRβ mRNA during metamorphosis in Xenopus tropicalis and found that TRα was broadly expressed in the intestinal tissues from premetamorphosis to the end of metamorphosis, while TRβ was expressed at low levels during premetamorphosis but was upregulated at the climax of metamorphosis when intestinal stem cells are formed and proliferate. Interestingly, both TR genes were co-expressed in different cell types, including stem cells. Chromatin immunoprecipitation (ChIP)-seq analyses of the intestine from wild type, TRα- or TRβ-knockout premetamorphic tadpoles treated with or without TH for 18 hours identified many TR-bound genes and revealed the effects of individual TR knockout on the binding of target genes by TR. We found that individual TR knockout reduced both the number of TR-bound genes and the extent of TR binding to target genes with TRα knockout had a much more dramatic effect than TRβ knockout. On the other hand, the TR-bound genes were largely common among the three genotypes. These findings suggest that both TRα and TRβ contribute to target binding with TRα having a bigger contribution in premetamorphic intestine. STAGE- AND TR-DEPENDENT GENE REGULATION PROGRAMS AFFECT THE PERMISSIVE ENVIRONMENT DURING THE INITIAL PERIOD OF XENOPUS TROPICALIS TAIL REGENERATION. Animal regeneration is the natural process of replacing or restoring damaged or missing cells, tissues, organs, and even entire body to full function. Studies in mammals have revealed that many organs lose regenerative ability soon after birth when TH level is high. This suggests that TH plays an important role in organ regeneration. Intriguingly, plasma TH level peaks during amphibian metamorphosis, which is very similar to postembryonic development in humans. In addition, many organs, such as heart and tail, also lose their regenerative ability during metamorphosis. These make frogs as a good model to address how the organs gradually lose their regenerative ability during development and what roles TH may play in this. Early tail regeneration studies have been done mainly in the tetraploid Xenopus laevis (X. laevis), which is difficult for gene knockout studies. We have used the highly related but diploid anuran X. tropicalis to investigate the role of TH signaling in tail regeneration with gene knockout approaches. We discovered that X. tropicalis tadpoles could regenerate their tail from premetamorphic stages up to the climax stage 59 and then lose regenerative capacity as tail resorption begins, just like what observed for X. laevis. To test the hypothesis that TH-induced metamorphic program inhibits tail regeneration, we used TR double knockout (TRDKO) tadpoles lacking both TR and TR, the only two receptor genes in vertebrates, for tail regeneration studies. Our results showed that TRs were not necessary for tail regeneration at any stages. However, unlike wild type tadpoles, TRDKO tadpoles retained regenerative capacity at the climax stages 60/61, when wild type animals lose their tail regeneration capacity. To further investigate this, we carried out RNA-seq analyses to investigate the gene regulation programs underlying the initiation of tail regeneration, i.e., wound healing and blastema formation. We discovered that GO (gene ontology) terms related to inflammatory response, metabolic process, cell apoptosis, and epithelial cell migration were highly enriched among commonly regulated genes during wound healing at either stage 56 (when both wild type and TRDKO animals can regenerate the tail) or stage 61 (when only TRDKO tadpoles can regenerate the tail), consistent with the morphological changes associated with wound healing occurring in both regenerative (wild type stage 56, TRDKO stage 56, TRDKO stage 61) and nonregenerative (wild type stage 61) animals. Interestingly, ECM (extracellular matrix)-receptor interaction and cytokine-cytokine receptor interaction were significantly enriched among regulated genes in the 3 regenerative groups but not the non-regenerative group at the blastema formation period. In addition, the regulated genes specific to the nonregenerative group were highly enriched with genes involved in cellular senescence, suggesting that TR-mediated, TH-induced gene regulation changed the permissive environment during the initial period of regeneration and affected the patterning/outgrowth period of the regeneration process. Further studies, particularly on the regenerative microenvironment that may depend on ECM-receptor interaction and cytokine-cytokine receptor interaction, should provide important insights on the regulation of regenerative capacity during vertebrate development.