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: 9984107
• 负责人: Yun-Bo Shi
• 金额: $ 73.75万
• 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/9984107
• 关键词: Adult; Amphibia; Animals; Bioinformatics; Biological Metamorphosis; Cell Death; Cell Differentiation process; Cell Proliferation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Connective Tissue; Development; Developmental Process; Diploidy; Embryo; Encyclopedias; Epithelial Cells; Epithelium; Evolution; Extracellular Matrix; Future; Gene Expression; Gene Expression Regulation; Gene Transfer Techniques; Genes; Genetic Transcription; Genetic study; Genome; Goals; Hormones; In Situ Hybridization; In Vitro; Intestines; Investigation; Knock-out; Knowledge; Life; Limb structure; MMP9 gene; Mammals; Matrix Metalloproteinases; Mediating; Modeling; Molecular; Morphogenesis; Muscle; Ontology; Organ; Organ Culture Techniques; Organ Model; Pathway interactions; Pattern; Play; Process; RNA; Rana; Receptor Gene; Regulation; Research; Rest; Role; Sampling; Siblings; South African; Stem Cell Development; Structure; System; Tadpoles; Tail; Technology; Thyroid Function Tests; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Transcript; Tubular formation; Uterus; Vertebrates; Xenopus; Xenopus laevis; adult stem cell; cofactor; developmental genetics; differential expression; genome wide association study; in vivo; insight; knockout gene; model development; notochord; programs; receptor; receptor expression; receptor function; single molecule real time sequencing; tissue/cell culture; toad; transcription activator-like effector nucleases

DISCOVERED A UNIQUE ROLE OF TR IN REGULATING NOTOCHORD RESORPTION DURING XENOPUS METAMORPHOSIS. Tail resorption during anuran metamorphosis is perhaps the most dramatic tissue transformation that occurs during vertebrate development. Like all other process during metamorphosis, tail resorption is controlled by TH. Earlier studies in highly related anuran species Xenopus laevis and Xenopus tropicalis have shown that TR plays a necessary and essential role for metamorphosis. Of the two known TR genes in all vertebrates, TR is highly expressed during both premetamorphosis and metamorphosis while TR expression is low in premetamorphic tadpoles but highly upregulated as a direct target gene of TH during metamorphosis. In addition, the two TR genes have different temporal regulation patterns in different organs during development. These suggest that the two TRs have different functions during metamorphosis. Indeed, gene knockout studies by us and others have shown that TR is not essential for metamorphosis but controls metamorphic timing and rate of metamorphosis progression during early metamorphosis. TR knockout, however, has no effect on metamorphic timing or early metamorphosis, but significantly delays late metamorphosis, particularly tail resorption. Homozygous TR knockout tadpoles become tailed frogs well after sibling wild type ones complete metamorphosis. Most noticeably, in TR-knockout tadpoles, an apparently normal notochord is present in the tail as late as 3 days after the initiation of tail shortening (stage 62), while in wild-type and TR-knockout tadpoles, the tail notochord disappears in about 1 day. We have investigated how tail notochord resorption is regulated by TR. We show that TR is selectively very highly expressed in the notochord compared to TR. We have also discovered differential regulation of several matrix metalloproteinases (MMPs), which are known to be upregulated by TH and implicated to play a role in tissue resorption by degrading the extracellular matrix. In particular, MMP9-TH and MMP13 are extremely highly expressed in the notochord compared to the rest of the tail. In situ hybridization analyses show that these MMPs are expressed in the outer sheath cells and/or the connective tissue sheath surrounding the notochord. Our findings suggest that high levels of TR expression in the notochord specifically upregulate these MMPs, which in turn degrades the ECM, leading to the collapse of the notochord and its subsequent resorption during metamorphosis. REVEALED THE GENE EXPRESSION PROGRAM UNDERLYING TAIL RESORPTION DURING THYROID HORMONE-DEPENDENT METAMORPHOSIS OF THE ORNAMENTED PYGMY FROG MICROHYLA FISSIPES. Studies on amphibian metamorphosis have been largely focused on the two highly related species Xenopus laevis and Xenopus tropicalis. However, adult X. laevis and X. tropicalis animals remain aquatic. This contrasts with most other anurans that truly changes from being aquatic to terrestrial during metamorphosis, which more closely mimics the postembryonic development in mammals. This makes important to study metamorphosis in a truly terrestrial frog species. In this regard, the anuran Microhyla fissipes offers a number of advantages as an alternative model for developmental and genetic studies. We have made use of the advances in sequencing technologies to investigate the gene regulation profiles underlying the tail resorption program during metamorphosis in M. fissipes. We first used single molecule real-time (SMRT) sequencing to obtain 67, 939 expressed transcripts in M. fissipes. We next identified 4,555 differentially expressed transcripts (DETs) during tail resorption by using Illumina sequencing on RNA samples from tails at different metamorphic stages. Bioinformatics analyses revealed that a number of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and Gene Ontology (GO) terms associated with tail resorption were enriched. Our findings suggest that tail resorption during M. fissipes and X. laevis shares many of the same programs. Future investigations on function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian tail resorption and adaptive evolution from aquatic to terrestrial life. Furthermore, analysis of the M. fissipes model, especially, on the changes in other organs associated with the transition from aquatic to terrestrial living, should help to reveal important mechanistic insights governing mammalian postembryonic developments.

发现TR在爪蟾变态过程中调节脊索吸收的独特作用。阿努族变态过程中的尾部吸收可能是脊椎动物发育过程中发生的最戏剧性的组织转化。像变形过程中的所有其他过程一样，尾部吸收受到TH的控制。早期在高度相关的Anuran物种Xenopus laevis和Tropicalis的研究表明，TR起着变态的必要和重要作用。在所有脊椎动物中的两个已知TR基因中，TR在预型和变形过程中都高度表达，而TR表达在themorphic的t骨中较低，但在变质过程中作为TH的直接靶基因高度上调。此外，在发育过程中，两个TR基因在不同器官中具有不同的时间调节模式。这些表明这两个TR在变形过程中具有不同的功能。实际上，我们和其他人的基因敲除研究表明，TR对于变态并不是必不可少的，而是控制早期变态过程中变态时序和变态进展的速率。然而，TR敲除对变态时机或早期变态没有影响，但显着延迟了晚期变质，尤其是尾部吸收。纯合的TR敲除t t端在兄弟式野生型完全变形后变成了尾蛙。最值得注意的是，在Tr-Knockout Tadpoles中，尾巴缩短后3天，尾巴上存在一个正常的脊索（第62阶段），而在野生型和Tr-tr-knockout Tadpoles中，尾部的脊索在大约1天内消失了。我们已经调查了如何通过TR调节尾部脊椎的吸收。我们表明，与TR相比，在脊索中有选择性地表达了TR。我们还发现了几种基质金属蛋白酶（MMP）的差异调节，这些调节被TH上调，并与通过降解细胞外基质来在组织吸收中发挥作用。特别是，与其他尾巴相比，MMP9-TH和MMP13在脊索中非常高度表达。原位杂交分析表明，这些MMP在外鞘细胞和/或脊索周围的结缔组织鞘中表达。我们的发现表明，脊索中高水平的TR表达明确上调了这些MMP，从而使ECM降解，从而导致脊索的崩溃及其在变质过程中的后续吸收。 在甲状腺激素依赖性的变态过程中，揭示了基因表达程序的基因表达程序。关于两栖变态的研究主要集中在两个高度相关的物种Xenopus laevis和xenopus tropicalis上。但是，成年X. laevis和X. tropicalis动物仍然是水生的。这与大多数其他Anurans形成鲜明对比，这些Anurans真正从水生中变成了陆地，它们更紧密地模仿了哺乳动物的胚胎后发育。这对于研究真正的陆蛙物种的变形很重要。在这方面，Anuran Microhyla Fissipes提供了许多优势，作为发展和遗传研究的替代模型。我们利用了测序技术的进步来研究M. fissipes M. fissipes M. tamorphosis期间尾声吸收计划的基因调节概况。我们首先使用单分子实时（SMRT）测序获得67、939在M. fissipes中表达的转录本。接下来，我们通过在不同变质阶段的尾部的RNA样品上使用Illumina测序在尾部吸收期间确定了4,555个差异表达的转录本（DET）。生物信息学分析表明，许多与尾部吸收相关的KEGG（基因和基因组百科全书）和基因本体学（GO）术语富集。我们的发现表明，M. fissipes和X. Laevis期间的尾声有许多相同的程序。对这些基因和途径的功能和调控的未来研究应有助于揭示有关两栖动物尾部吸收和从水生生物的自适应进化的机制。此外，对菲西氏菌模型的分析，尤其是对与从水生生物过渡相关的其他器官的变化，应有助于揭示有关哺乳动物后胚胎后发展的重要机械洞察力。