Molecular mechanism of thyroid hormone receptor function during metamorphosis

变态过程中甲状腺激素受体功能的分子机制

• 批准号: 10459124
• 负责人: Yun-Bo Shi
• 金额: $ 79.62万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10459124
• 关键词: Adult; Amphibia; Amputation; Animals; Aorta; Apoptosis; Biological; Biological Metamorphosis; Birth; Cell Cycle; Cell Death; Cell Differentiation process; Cell Proliferation; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Connective Tissue; Defect; Development; Developmental Process; Diploidy; Dorsal; Embryo; Epithelial; Epithelial Cells; Gene Expression; Gene Transfer Techniques; Genes; Genetic Transcription; Genetic study; Goals; Histone Acetylation; Histone H4; Hormone Responsive; Hormones; Human; In Vitro; Individual; Intestines; Knock-out; Knowledge; Ligands; Limb structure; Mammals; Mediating; Modeling; Molecular; Molecular Genetics; Morphogenesis; Morphology; Muscle; NCOA3 gene; Natural regeneration; Ontology; Organ; Organ Culture Techniques; Organ Model; Pathway Analysis; Pathway interactions; Phenotype; Play; Process; RNA; Rana; Receptor Gene; Receptor Signaling; Refractory; Regenerative Medicine; Regenerative capacity; Regenerative research; Replacement Therapy; Research; Role; Signal Pathway; South African; Spinal Cord; Stem Cell Development; Steroid Receptors; Structure; System; Tadpoles; Tail; Thyroid Function Tests; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Tubular formation; Uterus; Vertebrates; Xenopus; Xenopus laevis; adult stem cell; base; chromatin immunoprecipitation; cofactor; epithelial stem cell; feeding; histone acetyltransferase; hormonal signals; in vivo; interest; intestinal maturation; knockout animal; knockout gene; model development; programs; promoter; receptor; receptor function; recruit; regenerative tissue; self-renewal; stem cell proliferation; stem cells; steroid hormone receptor; tissue regeneration; tissue/cell culture; toad; transcription activator-like effector nucleases; transcriptome sequencing

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.

对甲状腺激素受体敲除蝌蚪的分析表明，热带爪蟾变态过程中细胞周期程序的激活参与了甲状腺激素诱导的幼虫上皮细胞死亡和成虫肠干细胞的发育。我们最近在热带爪蟾中单独或同时敲除了TR基因TR和TR，并分析了其对蝌蚪发育和不同器官变态的影响。令人感兴趣的是肠道重塑，这涉及幼虫上皮通过细胞凋亡几乎完全退化。同时，成体肠道干细胞从头形成，随后产生自我更新的成体上皮系统，类似于哺乳动物出生前后的肠道成熟。我们观察到 TR 和 TR 对肠道重塑都起着重要作用。为了了解潜在的分子机制，我们最近通过使用基因敲除动物和 RNA-seq 分析研究了蝌蚪肠道中内源性 TR 的功能。我们观察到去除内源性 TR 会导致肠道重塑缺陷，包括大幅减少幼虫上皮细胞死亡和成体肠道干细胞增殖。在蝌蚪肠道中任何可检测到的 TH 诱导的细胞死亡和干细胞形成之前，使用 RNA-seq 对来自用或不用 TH 处理的变态前野生型和 TR 敲除蝌蚪的肠道 RNA 进行分析，我们鉴定了超过 1500 个受 TH 调节的基因处理野生型蝌蚪，但不处理TR敲除蝌蚪。基因本体和生物通路分析显示，令人惊讶的是，除了与干细胞和凋亡相关的基因外，这些TR调节的基因还高度富集与细胞周期相关的基因。我们的研究结果表明，TR 介导的细胞周期程序 TH 激活涉及肠道重塑过程中幼虫上皮细胞死亡和成体上皮干细胞发育。 内源性组蛋白乙酰转移酶类固醇激素受体 (SRC) 3 在爪蟾肠变态过程中甲状腺激素信号传导中的作用。我们之前已经证明，在变态过程中，配体 TR 会招募共激活子复合物，其中包括类固醇受体共激活子 SRC3（一种组蛋白乙酰转移酶）到 TH 响应启动子。为了研究内源性共激活因子（例如 SRC3）在变态过程中的功能，我们培育了缺乏功能性 SRC3 基因的热带爪蟾动物，并分析了由此产生的表型。虽然去除 SRC3 对外部发育和动物总体形态没有明显影响，但与野生型动物相比，SRC3 (-/-) 蝌蚪表现出肠道中组蛋白 H4 乙酰化的减少。此外，在肠道重塑过程中，SRC3 (-/-)蝌蚪中TR靶基因的表达也有所减少。重要的是，SRC3 (-/-) 蝌蚪在自然和 TH 诱导的变态过程中抑制/延迟了肠道重塑，包括减少成体肠道干细胞增殖和幼虫上皮细胞凋亡。因此，我们的结果表明，SRC3 是肠道重塑过程中体内 TR 信号通路的关键组成部分。 非洲爪蟾和热带爪蟾尾巴再生的进化差异。组织再生在生物医学的发展中，特别是器官替代疗法中，变得越来越重要。不幸的是，许多人体器官无法再生。由于许多蝌蚪器官可以再生，无尾爪蟾已被用作研究再生的模型。特别是尾巴，由许多轴向和近轴组织组成，如脊椎动物中常见的脊髓、背主动脉和肌肉，在胚胎晚期和大多数蝌蚪阶段截肢时可以完全再生。有趣的是，从开始进食的第45阶段到第47阶段，伪四倍体非洲爪蟾尾巴在截肢后无法再生。这个时期被称为不应期，大约已有 20 年的历史。潜在的分子和遗传基础尚不清楚，部分原因是很难在这种伪四倍体物种中进行遗传研究。高度相关的二倍体热带爪蟾的出现为研究不应期尾巴再生的分子和遗传机制提供了机会。我们比较了非洲爪蟾和热带爪蟾的尾部再生，令人惊讶地发现热带爪蟾缺乏不应期。进一步的分子和遗传学研究，在这种二倍体物种中更为可行，应该揭示两个相关物种之间尾部再生的进化差异的基础，并有助于理解组织再生能力是如何控制的。此外，众所周知，许多蝌蚪组织在变态过程中失去了再生能力，这表明 TH 和 TR 在再生中发挥着作用。 利用我们最近生成的 TR 敲除动物，我们还计划研究 TH 和 TR 是否以及如何调节组织再生。此类研究应该对人类再生医学具有重要意义。