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: 8941474
• 负责人: Yun-Bo Shi
• 金额: $ 55.37万
• 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/8941474
• 关键词: Address; Adult; African; Age; Amphibia; Animals; Biological Metamorphosis; Body Size; Categories; Cell Death; Cell Differentiation process; Cell Proliferation; Connective Tissue; Data; Development; Developmental Gene; Developmental Process; Diploidy; Embryo; Embryonic Development; Enzymes; Epigenetic Process; Epithelial Cells; Epithelium; Evolution; Gene Expression; Gene Expression Regulation; Gene Targeting; Gene Transfer Techniques; Genes; Genetic Programming; Genetic Transcription; Goals; Growth; Growth and Development function; Histones; Hormones; Human; Human Development; In Vitro; Intestines; Knock-out; Knowledge; Limb structure; Mammals; Mediating; Methylation; Modeling; Modification; Molecular; Molecular Profiling; Morphogenesis; Mus; Muscle; Mutate; Ontology; Organ; Organ Culture Techniques; Organ Model; Organogenesis; Pathway interactions; Pattern; Phase; Plasma; Process; Rana; Receptor Gene; Regulation; Regulator Genes; Research; Resistance; Role; Somatotropin; Staging; Stem Cell Development; Structure; Study models; System; Tadpoles; Tail; Thyroid Hormone Receptor; Thyroid Hormones; Time; Tissues; Transcription Coactivator; Transgenic Organisms; Tubular formation; Uterus; Vertebrates; Xenopus; Xenopus laevis; adult stem cell; base; comparative; design; egg; feeding; fetal; gene function; genome-wide analysis; histone methyltransferase; human disease; improved; in vivo; interest; model development; molecular marker; mouse development; novel; nuclease; programs; receptor; receptor function; tissue/cell culture; toad; tool; xenopus development; zygote

UNLIGANDED THYROID HORMONE RECEPTOR Α CONTROLS DEVELOPMENTAL TIMING IN XENOPUS TROPICALIS. Earlier studies have shown that during development, of the two TR genes (TRα and TRβ), TRα expression appears earlier than TH synthesis and secretion into the plasma. This and the ability of TRs to regulate gene expression both in the presence and absence of TH have implicated a role of unliganded TR during vertebrate development. On the other hand, it has been difficult to study the role of unliganded TR during development in mammals due to the difficulty to manipulate the uterus-enclosed, late stage embryos. Amphibian development offers an excellent opportunity to address this question. We have designed TALENs (transcriptional activator like effector nucleases) to mutate the TRα gene in Xenopus tropicalis. We show that knockdown of TRα enhances tadpole growth in premetamorphic tadpoles, in part because of increased growth hormone gene expression. More importantly, the knockdown also accelerates animal development, with the knockdown animals initiating metamorphosis at a younger age and with a smaller body size. On the other hand, such tadpoles are resistant to exogenous TH and have delayed natural metamorphosis. Thus, our studies have not only directly demonstrated a critical role of endogenous TRα in mediating the metamorphic effect of TH but also revealed novel functions for unliganded TRα during early development, that is, regulating both the tadpole growth rate and the timing of metamorphosis. TH-INDUCED HISTONE METHYLTRANSFERASE DOT1L IS REQUIRED FOR POSTEMBRYONIC DEVELOPMENT BUT DISPENSABLE FOR XENOPUS EMBRYOGENESIS. In our study of gene regulation by TR during metamorphosis, we discovered that the levels of the methylation of histone H3K79 at TH target genes were upregulated during natural and TH-induced metamorphosis. Interestingly, the histone methyltransferase Dot1L is the only known enzyme capable of methylating histone H3K79, suggesting that Dot1L may function as a TR coactivator. In addition, we have shown that Dot1L is directly activated by TH via TR at the transcription level. To investigate the role of Dot1L during metamorphosis, we have generated a Dot1L-specific TALEN nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We have shown that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis since normal feeding tadpoles were formed. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings and the lack of maternal Dot1L expression suggest that Dot1L and H3K79 methylation are dispensable for embryogenesis but essential for tadpole growth and development prior to metamorphosing into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of two separate phases of vertebrate development with distinct requirements for epigenetic modifications. GLOBAL EXPRESSION PROFILING REVEALS GENETIC PROGRAMS UNDERLYING THE DEVELOPMENTAL DIVERGENCE BETWEEN MOUSE AND HUMAN EMBRYOGENESIS. Through a collaborative effort to study global gene regulation during vertebrate development, we have analyzed the developmental gene expression profiles in mouse. Mouse has served as an excellent model for studying human development and diseases. Advances in transgenic and knockout studies in mouse have dramatically strengthened the use of this model and significantly improved our understanding of gene function during development in the past few decades. On the other hand, little information is known about the gene regulatory networks governing the mouse organogenesis. Importantly, mouse and human development diverges during organogenesis. For instance, the mouse embryo is born around the end of organogenesis while in human the subsequent fetal period of ongoing growth and maturation of most organs spans more than 2/3 of human embryogenesis. To investigate the underlying molecular basis, we carried out a detailed analysis of the global gene expression profiles from egg to the end of organogenesis in mouse (5). Our studies revealed distinct temporal regulation patterns for genes belonging to different functional (Gene Ontology or GO) categories that support their roles during organogenesis. More importantly, comparative analyses identified both conserved and divergent gene regulation programs in mouse and human organogenesis, with the latter likely responsible for the developmental divergence between the two species, and further suggest a novel developmental strategy during vertebrate evolution. Given our earlier observation that genes function in a given process tends to be developmentally co-regulated during organogenesis, our microarray data should help to identify genes associated with mouse development and/or infer the developmental functions of unknown genes. In addition, our study might be useful for investigating the molecular basis of vertebrate evolution.

甲状腺激素受体α无物体控制热带爪蟾的发育时机。较早的研究表明，在开发过程中，在两个TR基因（TRα和TRβ）中，TRα表达出现在血浆中的TH合成和分泌早。在存在和不存在TH的情况下，TRS调节基因表达的能力也暗示了在脊椎动物发育过程中无配合的TR的作用。另一方面，由于难以操纵子宫封闭的后期胚胎，因此很难研究哺乳动物在发育过程中非配体TR的作用。两栖动物的发展提供了一个很好的机会来解决这个问题。 我们已经设计了Talens（转录激活因子（例如效应子核酸酶））以突变Trapus tropicalis中的TRα基因。我们表明，TRα的敲低会增强t骨前t骨的or t，部分原因是生长激素基因的表达增加。更重要的是，敲低还可以加速动物的发育，而击倒动物在年轻时起产生的变形，体积较小。 另一方面，这种t骨对外源性TH具有抵抗力，并且延迟了自然变形。因此，我们的研究不仅直接证明了内源性TRα在介导Th的变质作用中的关键作用，而且还揭示了早期发育过程中无配合的TRα的新功能，也就是说，可以调节t的生长速率和变质的时间。 Th诱导的组蛋白甲基转移酶DOT1L是胚胎后发育所必需的，但对于异爪蟾胚胎发生了。在我们对变态过程中TR调控基因调节的研究中，我们发现在天然和TH诱导的变态过程中，在TH靶基因的组蛋白H3K79的甲基化水平上调。有趣的是，组蛋白甲基转移酶DOT1L是唯一已知的能够甲基化组蛋白H3K79的酶，这表明DOT1L可能充当TR共激活剂。此外，我们已经证明DOT1L在转录级别通过TR直接激活。为了研究DOT1L在变态过程中的作用，我们已经产生了DOT1L特异性的TALEN核酸酶，以敲低内源性DOT1L在Xenopus tropicalis中，这是一种与众所周知的发育模型Laevis高度相关的二倍体物种，二核模型Xenopus laevis，pseudototetraploid amphibian。我们已经表明，在受精卵中表达时Talen在突变DOT1L方面非常有效，从而产生了少量H3K79甲基化的DOT1L敲除胚胎。重要的是，我们观察到DOT1L敲低对胚胎发生没有明显的影响，因为形成了正常的进食t。另一方面，DOT1L敲低严重阻碍了t的生长，并在变形之前导致t刺致命。这些发现以及缺乏母体DOT1L表达表明，DOT1L和H3K79甲基化对于胚胎发生是可分配的，但在变质成青蛙之前，对于t骨生长和发育至关重要。我们的发现进一步揭示了爪蟾和小鼠发育之间有趣的相似性和差异，并表明存在脊椎动物发育的两个独立阶段，对表观遗传修饰有不同的要求。 全球表达谱图揭示了小鼠与人类胚胎发生之间发育差异的基础遗传程序。通过研究脊椎动物发育过程中的全球基因调节的协作努力，我们分析了小鼠中的发育基因表达谱。小鼠是研究人类发育和疾病的绝佳模型。小鼠中转基因和敲除研究的进步已经显着加强了该模型的使用，并显着提高了我们对过去几十年发展过程中基因功能的理解。另一方面，关于管理小鼠器官发生的基因调节网络知之甚少。重要的是，在器官发生过程中，小鼠和人类发育分歧。例如，小鼠胚胎出生于器官发生结束，而在人类中，大多数器官的持续生长和成熟的胎儿时期跨越了人类胚胎的2/3以上。为了研究潜在的分子基础，我们对小鼠中器官发生的整体基因表达谱进行了详细的分析（5）。我们的研究揭示了属于不同功能（基因本体论或GO）类别的基因的独特时间调节模式，这些基因支持其在器官发生过程中的作用。更重要的是，比较分析确定了小鼠和人体器官中的保守基因调节程序和不同的基因调节程序，后者可能导致这两个物种之间的发育差异，并进一步提出了脊椎动物进化过程中新型的发育策略。鉴于我们较早的观察结果是，在器官发生过程中，基因在给定过程中的功能倾向于在发育中共同调节，因此我们的微阵列数据应有助于鉴定与小鼠发育相关的基因和/或推断未知基因的发育功能。此外，我们的研究可能对研究脊椎动物进化的分子基础很有用。