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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要卫星细胞是肌肉特异性干细胞，负责骨骼肌的生长和再生。肌源性调节因子 (MRF) MYOD 和 MYF5 对于肌肉谱系的确定至关重要胚胎并在激活的卫星细胞中被诱导，作为对肌肉损伤的早期反应。最近的基因- 使用新的 MyoD 条件敲除等位基因 (MyoDcKO) 的靶向研究表明，MyoD 或 Myf5 是对于肌肉再生至关重要；缺乏这两种基因（dKO）的卫星细胞在受伤的肌肉中积聚，但不能进行肌原性分化。在该提案中，新的遗传工具和策略被用于确定 MyoD 和 Myf5 在卫星细胞发育、谱系确定、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过程中受到询问。在目标 1 中，通过免疫荧光和单细胞 RNA 测序 (scRNA-seq) 将确定 dKO 卫星细胞是否采用非肌源性细胞命运、它们保留生肌编程的程度以及它们自我更新的能力。实验还将区分 MRF 缺陷的细胞自主和非自主效应。目标 2 将利用 RNA- seq 来定义未受伤和受伤骨骼肌中突变卫星细胞的转录组，这将识别 MYOD 和 MYF5 的直接和间接转录靶点以及调控途径细胞过程受到这些 MRF 丢失的影响。此外，Pro-seq（全基因组精确运行）分析将量化活性基因转录的变化，将识别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 的潜在增强子靶点。目标3将通过产生 dKO 来确定卫星细胞发育是否需要 MyoD 或 Myf5 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卫星细胞，根据分子和解剖学标准进行评估。最近的数据表明，唯一已知调节 MyoD 表达的增强子元件（核心增强子和远端调节区）是在胚胎发生期间或卫星细胞中，MyoD 转录不是必需的。目标 4 将利用转染，转基因和基于 CRISPR 的敲除方法来定义新假定的调控功能通过 PRO-seq 和生物信息学分析鉴定的增强子元件。拟议的研究将有助于对理解控制卫星细胞干的基本基因调控机制具有重要意义细胞功能，并可能导致新细胞类型和细胞疗法策略的开发肌肉退行性疾病。