Regulation of stem cell development during tissue remodeling

组织重塑过程中干细胞发育的调节

• 批准号: 10266510
• 负责人: Yun-Bo Shi
• 金额: $ 103.33万
• 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/10266510
• 关键词: Active Biological Transport; Adult; Affect; Amino Acids; Animals; Apoptosis; Basal lamina; Binding; Biological Metamorphosis; CRISPR/Cas technology; Cell Culture Techniques; Cell Differentiation process; Cell physiology; Cells; Cellular Stress; Cessation of life; ChIP-on-chip; Complement; Connective Tissue; Data; Defect; Development; Embryo; Enterobacteria phage P1 Cre recombinase; Epithelial; Epithelial Cells; Epithelium; Exhibits; Extracellular Matrix; Food Processing; Gene Expression; Genes; Genetic Transcription; Goals; Histones; Intestines; Knock-out; Knockout Mice; Life; Ligands; Light; Limb Bud; LoxP-flanked allele; Mammals; Mediating; Messenger RNA; Metabolic; Metabolic stress; Methylation; Morphogenesis; Mus; Muscle; Mutation; Organ; Organ Culture Techniques; Pathway interactions; Pattern; Phenotype; Phosphorylation; Physiological; Play; Primary Cell Cultures; Process; Proliferating; Promoter Regions; Property; RNA Polymerase II; Rana; Regulation; Role; South African; Stem Cell Development; Stress; Structure; System; Tadpoles; Technology; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Transcription Initiation Site; Transcriptional Activation; Transfer RNA; Translations; Up-Regulation; Vertebrates; WNT Signaling Pathway; Xenopus; Xenopus laevis; adult stem cell; base; biological adaptation to stress; chromatin immunoprecipitation; conditional knockout; epithelial stem cell; hormone response element; in vivo; intestinal epithelium; knock-down; knockout animal; mutant; neonatal period; nutrient absorption; operation; osteoclastogenesis; pathogen; promoter; recruit; relating to nervous system; response; stem cell division; stem cell proliferation; stem cells; tRNA Methyltransferases; tissue regeneration; tissue repair; toad; transcriptomics

DEMONSTRATED THAT TR IS CRITICAL FOR INTESTINAL REMODELING DURING XENOPUS TROPICALIS METAMORPHOSIS. We have analyzed the effect of TR knockout on TH-induced intestinal remodeling by using animals containing an out-frame-mutation of 5 base deletion generated with the CRISPR/Cas9 gene-editing technology. We observed that TR knockout does not affect premetamorphic tadpole development. However, we found that the basal expression of direct TH-inducible genes is increased and their upregulation by TH is reduced in the intestine of premetamorphic homozygous TR knockout animals, accompanied by reduced target binding by TR. More importantly, we have observed reduced adult stem cell proliferation and larval epithelial apoptosis in the intestine during Th-induced metamorphosis. Our data suggest that TR plays a critical role in intestinal remodeling during metamorphosis. DISCOVERED THAT TRNA METHYLTRANSFERASE-LIKE 1 (METTL1) GENE IS DIRECDTLY REGULATED BY THYROID HORMONE RECEPTOR DURING XENOPUS TROPICALIS METAMORPHOSIS, IMPLICATING A ROLE IN ADULT INTESTINAL STEM CELL DEVELOPMENT AND PROLIFERATION. We have previously used ChIP-on-chip assay to identify many putative TR target genes. Among them is the tRNA methyltransferase Mettl1.We studied the regulation of Mettl1 gene by TH during intestinal metamorphosis, a process involves near complete degeneration of the larval epithelial cells via apoptosis and de novo formation of adult epithelial stem cells and their subsequent proliferation and differentiation. We observed that Mettl1 was activated by TH in the intestine during both natural and TH-induced metamorphosis and that its mRNA level peaks at the climax of intestinal remodeling. We further showed that Mettl1 promoter could be activated by liganded TR via a TH response element upstream of the transcription start site in vivo. More importantly, we found that TR binding to the TRE region correlated with the increase in the level of H3K79 methylation, a transcription activation histone mark, and the recruitment of RNA polymerase II by TH during metamorphosis. Our findings suggest that Mettl1 is activated by liganded TR directly at the transcriptional level via the TRE in the promoter region in the intestine during metamorphosis. Mettl1 in turn regulate target tRNAs to affect translation, thus facilitating stem cell formation and/or proliferation during intestinal remodeling. DISCOVERED THAT LAT1 REGULATES OSTEOCLASTOGENESIS AND BONEHOMEOSTASIS THROUGH THE MTORC1 PATHWAY. To regulate cellular processes, TH has to be actively transported into cells and this process is mediated by several different types of transporters. One of our previously identified TH-response genes in the intestine, LAT1, encodes the light chain of a heterodimeric system L type of TH transporter, which also transports several amino acids. Interestingly, LAT1 is highly upregulated at the climax of metamorphosis in the tadpole intestine, coinciding with the formation and rapid proliferation of the adult intestinal stem cells. We also found out that LAT1 was also highly expressed in the mouse intestine during the neonatal period when the mouse intestine matured into the adult form, a process that appears also involves TH-dependent formation and/proliferation of the adult intestinal stem cells. Through a collaborative study, we generated a mouse line with the LAT1 gene floxed, which allows conditional knockout of the LAT1 upon expression of the Cre recombinase. Through a collaborative study, we showed that LAT1 is highly expressed in mouse tissues undergoing morphogenesis and that LAT1-null mouse embryos, generated by crossing the LAT1-floxed mice with a mouse line expressing CRE ubiquitously, have profound neural and limb-bud outgrowth defects. LAT1-null neural tissue exhibited mild proliferation defects and aberrant mTORC1 activity; transcriptomics, protein phosphorylation and apoptosis analyses further indicated induction of the integrated stress response as the likely cause of observed defects. The pattern of stress-response gene expression induced in LAT1-null embryos was also detected at low-level in wildtype embryos and identified stress-vulnerability specifically in tissues undergoing morphogenesis. The LAT1-null phenotype is reminiscent of Wnt-pathway mutants and we show that loss of Wnt or -catenin inhibits LAT1 expression and induces this stress response. Wnt-signaling therefore normally supports the metabolic demands of morphogenesis and constrains cellular stress. Moreover, operation in the embryo of the integrated stress response, which is triggered by pathogen-mediated as well as metabolic stress, may provide a mechanistic explanation for a range of developmental defects.

证明TR对于热带爪蟾爪形成过程中的肠道重塑至关重要。我们通过使用含有CRISPR/CAS9基因编辑技术产生的5个基本缺失的动物，分析了TR敲除对Th诱导的肠道重塑的影响。我们观察到TR敲除不会影响前型t t的发展。但是，我们发现直接TH诱导基因的基础表达增加了，并且在预型纯合纯合TR敲除动物的肠道中，其上调的上调降低，并伴随着TR降低靶标结合。更重要的是，我们已经观察到在TH诱导的变态过程中，肠中成年干细胞增殖和幼虫上皮凋亡的降低。我们的数据表明，TR在变态过程中在肠重塑中起关键作用。 发现在热带Xenopicals tropicalis变质过程中，tRNA甲基转移酶样1（METTL1）基因受甲状腺激素受体受体调节，暗示在成人肠道干细胞发育和增殖中的作用。我们以前已经使用芯片芯片测定法确定了许多推定的TR靶基因。 其中包括tRNA甲基转移酶mettl1。我们研究了肠道变态过程中TH对METTL1基因的调节，这一过程涉及幼虫上皮细胞的完全变性，并通过凋亡和从头形成成人上皮干细胞的从头形成及其后期的扩散和分化。我们观察到，在天然和TH诱导的变态过程中，TH在肠中被Th激活，并且其mRNA水平在肠重塑的高潮处峰值。 我们进一步表明，METTL1启动子可以通过体内转录起始位点上游的TH响应元件来激活METTL1启动子。更重要的是，我们发现与TRE区域的结合与H3K79甲基化水平的增加，转录活化组蛋白标记以及在变质过程中TH募集RNA聚合酶II的水平相关。我们的发现表明，在变态过程中，通过TRE在转录水平上直接在转录水平上直接在转录水平上激活METTL1。 METTL1反过来调节目标TRNA以影响翻译，从而促进干细胞的形成和/或肠重塑期间的增殖。 发现LAT1通过MTORC1途径调节破骨细胞生成和骨homeostasis。为了调节细胞过程，必须将TH积极​​运输到细胞中，并且该过程由几种不同类型的转运蛋白介导。我们先前鉴定出的肠道lat1中的Th响应基因之一，编码了杂二聚体系统l类型的TH转运蛋白的轻链，该系统还传输了几种氨基酸。有趣的是，LAT1在t肠中变态的高潮时高度上调，与成年肠干细胞的形成和快速增殖相吻合。我们还发现，在新生儿时期，当小鼠肠道成熟成成人形式时，LAT1在小鼠肠中也高度表达，这一过程似乎也涉及成人肠干细胞的Th依赖性形成和////增殖。 通过一项协作研究，我们生成了一条用LAT1基因的小鼠线，该系列允许在CRE重组酶表达时将LAT1的有条件敲除。通过一项协作研究，我们表明LAT1在经历形态发生的小鼠组织中高度表达，而Lat1-Null小鼠胚胎是通过将LAT1-氟的小鼠与小鼠线越过无处不在的小鼠线产生的，具有深刻的神经和肢体群可能出生的缺陷。 LAT1无效的神经组织表现出轻度的增殖缺陷和异常的MTORC1活性。转录组学，蛋白质磷酸化和凋亡分析进一步表明综合应力反应可能是观察到的缺陷的原因。还检测到在野生型胚胎中的低水平，在LAT1-NULL胚胎中诱导的应激反应基因表达的模式，并在形态发生的组织中特别鉴定了应力 - 脱硫性。 LAT1-NULL表型让人联想到Wnt-Pathway突变体，我们表明Wnt或-Catenin的丧失会抑制LAT1表达并诱导这种应力反应。因此，WNT信号通常支持形态发生的代谢需求并限制细胞应激。此外，由病原体介导的和代谢应激触发的综合应力反应的胚胎中的操作可能为一系列发育缺陷提供机械解释。