Regulation of Satellite Cell Development, Programming and Differentiation by Myogenic Factors

成肌因子对卫星细胞发育、编程和分化的调节

• 批准号: 10670113
• 负责人: DAVID J GOLDHAMER
• 金额: $ 45.38万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670113
• 关键词: Address; Adopted; Adult; Alleles; Anatomy; Bioinformatics; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Cell Lineage; Cell Therapy; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data; Defect; Degenerative Disorder; Development; Differentiation and Growth; Distal; Elements; Embryo; Embryonic Development; Enhancers; Environment; Exhibits; Fiber; Gene Targeting; Genes; Genetic; Genetic Enhancer Element; Genetic Recombination; Genetic Transcription; Heterogeneity; Immunofluorescence Immunologic; Injury; Knock-out; Mediating; Messenger RNA; Methodology; MicroRNAs; Molecular; Mus; Muscle; Muscle satellite cell; Mutagenesis; MyoD Protein; Myoblasts; Myogenic Regulatory Factors; Natural regeneration; Neonatal; Nucleic Acid Regulatory Sequences; Pathway interactions; Phenotype; Polymerase; Population; Positioning Attribute; Property; Proteins; Publishing; Regenerative capacity; Regulation; Regulator Genes; Regulatory Element; Regulatory Pathway; Reporter Genes; Research; Running; Skeletal Muscle; Skeletal muscle injury; Testing; Tissues; Transcriptional Regulation; Transfection; Transgenic Mice; Transgenic Organisms; blastomere structure; candidate identification; cell type; conditional knockout; dosage; embryo cell; experimental study; fetal; genetic regulatory protein; genome-wide; in vivo; injured; insight; muscle regeneration; mutant; novel; prenatal; programs; promoter; response; satellite cell; self-renewal; single-cell RNA sequencing; skeletal muscle growth; stem cell function; stem cells; tibialis anterior muscle; tool; transcriptome; transcriptome sequencing

Project Summary Satellite cells are muscle-specific stem cells that are responsible for skeletal muscle growth and regeneration. The myogenic regulatory factors (MRFs) MYOD and MYF5 are essential for muscle lineage determination in the embryo and are induced in activated satellite cells as an early response to muscle injury. Recent gene- targeting studies using a new MyoD conditional knockout allele (MyoDcKO) showed that either MyoD or Myf5 is essential for muscle regeneration; satellite cells lacking both genes (dKO) accumulate in injured muscle but are unable to undergo myogenic differentiation. In this proposal, new genetic tools and strategies are used to determine the functions of MyoD and Myf5 in satellite cell development, lineage determination, differentiation and self-renewal. In addition, transcriptional control mechanisms that regulate MyoD expression in satellite cells and during embryogenesis are interrogated. In Aim 1, cell type identification by immunofluorescence and single-cell RNA sequencing (scRNA-seq) will establish whether dKO satellite cells adopt non-myogenic cell fates, the extent to which they retain myogenic programming, and their capacity for self-renewal. Experiments will also distinguish cell-autonomous and non-autonomous effects of MRF deficiency. Aim 2 will utilize RNA- seq to define the transcriptome of mutant satellite cells in uninjured and injured skeletal muscle, which will identify direct and indirect transcriptional targets of MYOD and MYF5 as well as regulatory pathways and cellular processes impacted by the loss of these MRFs. In addition, Pro-seq (genome-wide Precision Run-On) analyses will quantify changes in active gene transcription, will identify candidate genes regulated by promoter- proximal polymerase pausing, and will identify potential enhancer targets of MYOD and MYF5. Aim 3 will determine whether the function of MyoD or Myf5 is required for satellite cell development by producing dKO satellite cell precursors at embryonic, fetal and neonatal stages and testing their capacity to generate adult satellite cells, as assessed by molecular and anatomical criteria. Recent data demonstrate that the only enhancer elements known to regulate MyoD expression (the core enhancer and distal regulatory region) are not necessary for MyoD transcription during embryogenesis or in satellite cells. Aim 4 will utilize transfection, transgenic and CRISPR-based knockout methodologies to define the regulatory functions of novel putative enhancer elements identified by PRO-seq and bioinformatic analyses. The proposed research will contribute significantly to an understanding of fundamental gene regulatory mechanisms that control satellite cell stem cell functions and may lead to the development of new cell types and strategies for cell-based therapies for muscle degenerative diseases.

项目摘要 卫星细胞是负责骨骼肌生长和再生的肌肉特异性干细胞。 肌源性调节因素（MRF）Myod和MyF5对于确定肌肉谱系至关重要 胚胎并在活化的卫星细胞中诱导，作为对肌肉损伤的早期反应。最近的基因 使用新的Myod有条件淘汰等位基因（Myodcko）进行靶向研究，表明Myod或MyF5是 对于肌肉再生至关重要；缺乏两个基因（DKO）的卫星细胞积聚在受伤的肌肉中，但是 无法经历肌源性分化。在此提案中，新的遗传工具和策略用于 确定MYOD和MYF5在卫星细胞开发，谱系测定，分化中的功能 和自我更新。此外，调节卫星中MYOD表达的转录控制机制 细胞和胚胎发生期间受到询问。在AIM 1中，通过免疫荧光鉴定细胞类型和 单细胞RNA测序（SCRNA-SEQ）将确定DKO卫星细胞是否采用非肌生成细胞 命运，保留肌源性编程的程度以及自我更新的能力。实验 还将区分MRF缺乏症的细胞自主和非自治作用。 AIM 2将利用RNA- SEQ在未受伤和受伤的骨骼肌中定义突变体卫星细胞的转录组，这将 确定MYOD和MYF5的直接和间接转录目标以及监管途径以及 这些MRF丢失影响的细胞过程。此外，Pro-Seq（全基因组精度跑步） 分析将量化活性基因转录的变化，将确定受启动子调节的候选基因 近端聚合酶暂停，并将识别MYOD和MYF5的潜在增强靶标。目标3意志 通过产生DKO来确定MYOD或MYF5是否需要卫星细胞开发的功能 胚胎，胎儿和新生儿阶段的卫星细胞前体，并测试其产生成人的能力 通过分子和解剖标准评估的卫星细胞。最近的数据表明唯一 增强子元素已知用于调节MYOD表达的元素（核心增强子和远端调节区域）是 在胚胎发生或卫星细胞中，Myod转录不需要。 AIM 4将利用转染， 基于转基因和基于CRISPR的敲除方法，以定义新型推定的调节功能 通过Pro-Seq和生物信息学分析确定的增强子元素。拟议的研究将做出贡献 显着了解控制卫星细胞茎的基本基因调节机制 细胞功能，可能导致开发新的细胞类型和基于细胞的疗法的策略 肌肉退行性疾病。