Molecular mechanism of thyroid hormone receptor function during metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

ORGAN-SPECIFIC EFFECTS ON TARGET BINDING DUE TO KNOCKING OUT THYROID HORMONE RECEPTOR DURING XENOPUS METAMORPHOSIS. There are two TR genes, TR and TR, in all vertebrates. We have previously generated TR knockout (Xtr.thratmshi) tadpoles and shown that TR is important for TH-dependent intestinal remodeling and hindlimb development, but not tail resorption during metamorphosis. To investigate the underlying molecular basis, we earlier used chromatin immunoprecipitation-sequencing (ChIP-seq) to identify genes bound by TR in the intestine and hindlimbs of premetamorphic wild type and Xtr.thratmshi tadpoles with or without TH treatment. We have now carried out similar analyses on the tail and compared the findings with those in the intestine and hindlimb. We found that the tail had much fewer genes bound by TR or affected by TR knockout. Bioinformatics analyses revealed that among the genes bound by TR in wild type but not Xtr.thratmshi organs, fewer gene-ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TR knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TR has the tissue-specific roles in regulating TH-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis. COMPARATIVE ANALYSIS OF TRANSCRIPTOME PROFILES REVEALS DISTINCT AND ORGAN-DEPENDENT GENOMIC AND NONGENOMIC ACTIONS OF THYROID HORMONE IN XENOPUS TROPICALIS TADPOLES. TH is essential for development and organ metabolism in all vertebrates. TH has both genomic and nongenomic effects on target cells. While much has been learnt on its genomic effects via TRs during vertebrate development, mostly through TR-knockout and knockin studies, little is known about the effects of TH on gene expression in animals in the absence of TR. By using the recently generated TR double knockout (TRDKO) Xenopus tropicalis animals, we have compared the effects of TH on global gene expression in tadpole tissues in the presence or absence of TR. We carried out RNA-seq analyses on gene expression in tadpole tail and intestine of wild-type and TRDKO tadpoles with or without TH treatment. We observed that removing TRs reduced the number of genes regulated by TH in both organs. Gene Ontology (GO) and KEGG pathway analyses revealed that TH affected distinct biological processes and pathways in wild-type and TRDKO tadpoles. Many GO terms and KEGG pathways were enriched among genes regulated in wild-type tissues are likely involved in mediating the effects of TH on metamorphosis, e.g., those related to development, stem cells, apoptosis, and cell cycle/cell proliferation. However, such GO terms and pathways were not enriched among TH-regulated genes in TRDKO tadpoles. Instead, in TRDKO tadpoles, GO terms and pathways related to metabolism and immune response were highly enriched among TH-regulated genes. We further observed strong divergence in the TR-independent, nongenomic effects of TH in the intestine and tail. Our data suggest that TH have distinct and organ-dependent effects on gene expression in developing tadpoles. The TR-mediated effects are consistent with the metamorphic changes, in agreement with the fact that TR is necessary and sufficient to mediate the effects of TH on metamorphosis. TH appears to have a major effect on metabolism and immune response via TR-independent nongenomic processes. THYROID HORMONE RECEPTOR KNOCKOUT PREVENTS THE LOSS OF XENOPUS TAIL REGENERATION CAPACITY AT METAMORPHIC CLIMAX. 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, likely in part by increasing apoptosis at the early regenerative period and enhancing subsequent cell proliferation. In addition, TRDKO animals had higher levels of amputation-induced expression of many genes implicated to be important for tail regeneration, compared to the non-regenerative wild type tadpoles at stage 61. Finally, the high level of apoptosis in the remaining uncut portion of the tail as wild type tadpoles undergo tail resorption after stage 61 appeared to also contribute to the loss of regenerative ability. Our findings for the first time revealed an evolutionary conservation in the loss of tail regeneration capacity at metamorphic climax between X. laevis and X. tropicalis. Our studies with molecular and genetic approaches demonstrated that TR-mediated, TH-induced gene regulation program is responsible not only for tail resorption but also for the loss of tail regeneration capacity. Further studies by using the model should uncover how TH modulates the regenerative outcome and offer potential new avenues for regenerative medicines toward human patients. COMPETITIVE PCR WITH DUAL FLUORESCENT PRIMERS ENHANCES THE SPECIFICITY AND REPRODUCIBILITY OF GENOTYPING ANIMALS GENERATED FROM GENOME EDITING. Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, leading to DNA repair through homologous recombination or non-homologous end joining (NHEJ). DNA repair through NHEJ mechanism is an error-prone process that often results in point mutations or stretches of indels (insertions and deletions) within the targeted region. Such mutations in embryos are germline transmissible, thus providing an easy means to generate organisms with gene mutations. However, point mutations and short indels present difficulty for genotyping, often requiring labor intensive sequencing to obtain reliable results. We have developed a single-tube competitive PCR assay with dual fluorescent primers that allows simple and reliable genotyping. While we used Xenopus tropicalis as a model organism, the approach should be applicable to genotyping of any organisms.