Regulation of stem cell development during tissue remodeling

组织重塑过程中干细胞发育的调节

基本信息

批准号：
10898491
负责人：
Yun-Bo Shi
金额：
$ 116.35万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10898491
关键词：
Active Biological Transport Adult Affect Amino Acid Transporter Amino Acids Amphibia Apoptosis Basal lamina Binding Biological Metamorphosis Cell Culture Techniques Cell Differentiation process Cell Proliferation Cell physiology Cells Cessation of life Characteristics Complement Complex Connective Tissue Data Defect Development Diabetes Mellitus Embryo Embryonic Development Enterobacteria phage P1 Cre recombinase Epithelial Cells Epithelium Erythrocytes Erythropoiesis Exhibits Exons Extracellular Matrix FRAP1 gene Failure Fibroblasts Food Processing Functional disorder Gene Expression Genes Genetic Transcription Glucose Goals Hematopoietic Hepatocyte Homeostasis Hormones Human Hypertrophy Hypothalamic structure In Vitro Intestines Iron deficiency anemia Knock-out Leptin Life Light Lipids Liver Liver Regeneration LoxP-flanked allele Mammals Mediating Mediator Modeling Molecular Molecular Analysis Mus Muscle Mutation Natural regeneration Neurons Nuclear Protein Nutrient Obesity Organ Organ Culture Techniques Pathway interactions Phenotype Physiological Plasma Postembryonic Primary Cell Cultures Process Proliferating Property Proto-Oncogenes RXR Rana Regulation Response Elements Retinoic Acid Receptor Role SKI gene South African Stem Cell Development Structure System Tadpoles Tail Technology Thyroid Hormone Receptor Thyroid Hormones Tissues Up-Regulation Vertebrates WNT Signaling Pathway Xenopus Xenopus laevis adult stem cell bone bone mass chromatin immunoprecipitation conditional knockout hormone regulation hormone response element improved in vivo insight intestinal epithelium knock-down leptin receptor liver development mouse model neonatal period nutrient absorption organ repair promoter receptor receptor binding response selective expression single-cell RNA sequencing stem cell division stem cells tissue repair toad transcriptome sequencing uptake urea cycle villin

项目摘要

UPREGULATION OF PROTOONCOGENE SKI BY THYROID HORMONE IN THE INTESTINE AND TAIL DURING XENOPUS METAMORPHOSIS. TH affects frog metamorphosis through TH receptor (TR)-mediated regulation of TH response genes, where TR forms a heterodimer with RXR (9-cis retinoic acid receptor) and binds to TH response elements (TREs) in TH response genes to regulate their transcription. To study how TH regulates intestinal stem cell development and/or proliferation, we have previously identified many putative direct TH response genes in Xenopus tropicalis tadpole intestine by using ChIP (chromatin immunoprecipitation)-on-chip assays. Among them is the proto-oncogene Ski, which encodes a nuclear protein with complex functions in regulating cell fate. We have now shown that Ski is upregulated in the intestine and tail of premetamorphic tadpoles upon TH treatment and its expression peaks at stage 62, the climax of metamorphosis. We have further discovered a TRE in the first exon that can bind to TR/RXR in vitro and mediate TH regulation of the promoter in vivo. These data demonstrate that Ski is activated by TH through TR binding to a TRE in the first exon during Xenopus tropicalis metamorphosis, implicating a role of Ski in regulating cell fate in this process. LIVER DEVELOPMENT DURING XENOPUS TROPICALIS METAMORPHOSIS IS CONTROLLED BY TH-ACTIVATION OF WNT SIGNALING. Many mammalian organs and tissues including erythrocytes mature into their adult forms during postembryonic development when plasma TH level peaks, resembling amphibian metamorphosis. TR mutations/deletions can cause hematopoietic dysfunction, suggesting that TH plays a role in erythropoiesis during development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis as a model to study TH function during postembryonic development. Our analyses of TRDKO tadpoles during metamorphosis revealed that TRDKO tadpoles exhibited characteristics similar to human iron deficiency anemia. As the liver is the hematopoietic organ, our finding suggests a defect in liver development in TRDKO tadpoles. We analyzed liver metamorphosis in wild type and TRDKO tadpoles and found that wild type liver metamorphosis involved increased cell proliferation, hepatocyte hypertrophy, and activation of urea cycle gene expression, a key feature of adult/mature liver in vertebrates. Interestingly, TRDKO liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate the expression of urea cycle genes. To reveal the molecular pathways regulated by TH during liver remodeling, we performed RNA-seq analysis and found that TH activated canonical Wnt pathway in the liver. Particularly, Wnt11 was activated in both fibroblasts and hepatic cells, and in turn, likely acted to promote stem cell development and/or proliferation and maturation of hepatocytes. Our findings also resemble those from studies on liver regeneration in mammals. Thus, analyses of liver metamorphosis have the potential to bring new insights on not only how TH regulates liver development but also potential means to improve liver regeneration. L-TYPE AMINO ACID TRANSPORTER 1 (LAT1) IN HYPOTHALAMIC NEURONS IN MICE MAINTAINS ENERGY AND BONE HOMEOSTASIS. 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 Xenopus 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 tadpole intestine, coinciding with the formation and rapid proliferation of adult intestinal stem cells. We also found out that LAT1 was also highly expressed in the mouse intestine during the neonatal period when mouse intestine matured into the adult form, a process that appears also to involve TH-dependent formation and/proliferation of adult intestinal stem cells. Through a collaborative study, we generated a mouse line with the LAT1 gene floxed, which allows conditional knockout of LAT1 upon expression of the Cre recombinase. We are currently analyzing the effect of LAT1 knockout specifically in the mouse intestine by expressing Cre under the control of the intestinal epithelial specific villin promoter. In addition, through another collaborative study, we discovered LAT1 expression in hypothalamic neurons, which regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). Importantly, we found that LAT1 in hypothalamic leptin receptor (LepR)-expressing neurons was important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.

在异爪蟾变态过程中，甲状腺激素在肠和尾部上的质量雪质上调。 TH会通过TH受体（TR）介导的TH反应基因的调节影响青蛙的变形，其中TR形成具有RXR（9- cis视黄酸受体）的异二聚体，并与TH反应基因中的TH反应元素（TRE）结合以调节其转录。为了研究TH如何调节肠道干细胞的发育和/或增殖，我们以前已经通过使用ChIP（染色质免疫沉淀） - 芯片分析方法确定了热带Xenopicalis tadpole肠中许多推定的直接TH反应基因。其中的是原始癌基因滑雪，它在调节细胞命运中编码具有复杂功能的核蛋白。现在，我们已经表明，在治疗后的肠道前肠道和尾部的肠道和尾部被上调，其表达峰在第62阶段，即变态的高潮。我们进一步在第一个外显子中发现了可以在体外与TR/RXR结合并介导体内启动子的调节的TRE。这些数据表明，在热带异常变态过程中，通过TR结合与第一个外显子中的TRE通过TR结合激活滑雪，这暗示了滑雪在此过程中调节细胞命运中的作用。 thnopicalis变态过程中的肝脏发育受到Wnt信号传导的控制。当血浆TH水平峰值时，许多哺乳动物器官和组织在胚胎后发育过程中成人形式成熟，类似于两栖动物的变形。 TR突变/缺失会引起造血功能障碍，这表明TH在发育过程中在促红细胞生成中起作用。我们最近生成了TR双基因敲除（TRDKO）Tropicalis，作为在胚胎后发育过程中研究TH功能的模型。我们对变态过程中Trdko t的分析表明，trdko t的特征表现出类似于人类铁缺乏症贫血的特征。由于肝脏是造血器官，我们的发现表明trdko t的肝发育缺陷。我们分析了野生型和trdko t的肝脏变形，发现野生型肝脏变形涉及增加细胞增殖，肝细胞肥大和尿素循环基因表达的激活，这是脊椎动物中成人/成熟的肝脏的关键特征。有趣的是，Trdko肝脏具有发育缺陷，例如细胞增殖减少和无法接受肝细胞肥大或激活尿素循环基因的表达。为了揭示肝脏重塑过程中TH调节的分子途径，我们进行了RNA-Seq分析，发现TH激活了肝脏中的规范WNT途径。特别是，Wnt11在成纤维细胞和肝细胞中都被激活，进而可以促进干细胞发育和/或肝细胞的增殖和成熟。我们的发现也类似于哺乳动物肝脏再生的研究。因此，对肝脏变形的分析有可能对TH调节肝脏发育的方式一些新的见解，而且还具有改善肝脏再生的潜在手段。小鼠下丘脑神经元中的L型氨基酸转运蛋白1（LAT1）保持能量和骨稳态。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Xenopus肠LAT1中的TH响应基因之一编码了TH转运蛋白类型的杂二聚体系统的轻链，该型TH转运蛋白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，小鼠肠道成年形式时，LAT1在小鼠肠中也高度表达，这一过程似乎也涉及TH依赖性的形成和///////////////////livient。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。我们目前正在通过在肠上皮特异性villin启动子的控制下表达CRE来分析LAT1敲除在小鼠肠中的效果。此外，通过另一项协作研究，我们发现了下丘脑神经元中的LAT1表达，这些神经元通过感应和整合关键激素和原发性营养素（氨基酸，葡萄糖和脂质）的水平来调节身体稳态。重要的是，我们发现在下丘脑瘦素受体（LEPR）表达神经元中的LAT1对于全身能量和骨稳态很重要。我们观察到下丘脑中LAT1依赖性氨基酸的摄取，该氨基酸在肥胖和糖尿病的小鼠模型中受到损害。在表达LEPR的神经元中缺乏LAT1（由SLC7A5编码）的小鼠表现出与肥胖相关的表型和较高的骨骼质量。 SLC7A5缺乏在肥胖发作之前引起表达LEPR的神经元的交感神经功能障碍和瘦素不敏感性。重要的是，在表达LEPR的腹侧下丘脑神经元中选择性地恢复SLC7A5表达，从而在表达LEPR表达细胞中缺乏SLC7A5的小鼠中挽救了能量和骨稳态。发现雷帕霉素复合物1（MTORC1）的机械靶标是能量和骨稳态调节LAT1依赖性调节的关键介体。这些结果表明，表达LEPR的神经元中的LAT1MTORC1轴通过微调交感神经流出来控制能量和骨稳态，从而在体内提供了在体内稳态中通过下丘脑神经元传感氨基酸对氨基酸传感的影响的证据。