Molecular mechanism of thyroid hormone receptor function during metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

THYROID HORMONE RECEPTOR CONTROLS THE HINDLIMB METAMORPHOSIS BY REGULATING CELL PROLIFERATION AND WNT SIGNALING PATHWAYS IN XENOPUS TROPICALIS. We have been studying Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TR knockout induces precocious hindlimb development. To reveal the molecular pathways regulated by TR during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on hindlimb of premetamorphic wild type and TR knockout tadpoles and identified over 700 TR-bound genes up-regulated by TH treatment in wild type but not TR knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hindlimb of premetamorphic TR knockout tadpoles compared to stage-matched wild type tadpoles, suggesting their derepression upon TR knockout. Bio-informatic analyses revealed that these genes were highly enriched with cell-cycle and WNT signaling related genes. Furthermore, cell cycle and WNT signaling pathways were also highly enriched among genes bound by TR in wild type but not TR knockout hindlimb. These findings suggests that direct binding of TR to target genes related to cell-cycle and WNT pathways is important for limb development: first preventing precocious hindlimb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hindlimb development during metamorphosis by mediating TH activation of these pathways. TRANSCRIPTOME PROFILING REVEALS GENE REGULATION PROGRAMS UNDERLYING TAIL DEVELOPMENT IN THE ORNAMENTED PYGMY FROG MICROHYLA FISSIPES. For comparative studies on the role of TH in anuran metamorphosis, we have previously carried out RNA-Seq analysis of the TH-induced gene expression program during tail resorption in Microhyla fissipes. Parallel to the metamorphic study, we also analyzed the tail at different developmental stages. Tadpole tail develops from the tailbud, an apparently homogenous mass of cells at the posterior of the embryo. While much progress has been made in understanding the origin and the induction of the tailbud, the subsequent outgrowth and differentiation have received much less attention, particularly with regard to global gene expression changes. By using RNA-seq with SMRT and further analyses, we revealed the transcriptome profiles at four key stages of tail development, from a small tailbud to the onset of feeding (S18, S19, S21 and S28) in Microhyla fissipes. We obtained 48826 transcripts and discovered 8807 differentially expressed transcripts (DETs, q < 0.05) among these four developmental stages. We functionally classified these DETs by using GO and KEGG analyses and revealed 110 significantly enriched GO categories and 6 highly enriched KEGG pathways (Protein digestion and absorption; ECM-receptor interaction; Pyruvate metabolism; Fatty acid degradation; Valine, leucine, and isoleucine degradation; and Glyoxylate and dicarboxylate metabolism) that are likely critically involved in developmental changes in the tail. In addition, analyses of DETs between any two individual stages demonstrated the involvement of distinct biological pathways/GO terms at different stages of tail development. Furthermore, the most dramatic changes in gene expression profile are those between S28 and any of the other three stages. The upregulated DETs at S28 are highly enriched in myosin complex and potassium channel activity, which are important for muscle contraction, a critical function of the tail that the animal needs by the end of embryogenesis. Additionally, many DETs and enriched pathways discovered here during tail development, such as HDAC1, Hes1 and Hippo signaling pathway, have also been reported to be vital for the tissue/organ regeneration, suggesting conserved functions between development and regeneration.