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

项目摘要 卫星细胞是肌肉特有的干细胞，负责骨骼肌的生长和再生。 生肌调节因子(MRF)MYOD和MYF5对于确定骨骼肌的谱系是必不可少的 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条件基因敲除等位基因(MyoDcKO)进行的靶向研究表明，MyoD或Myf5是 对肌肉再生是必不可少的；缺乏这两种基因的卫星细胞(DKO)在受伤的肌肉中积累，但 不能进行肌源性分化。在这项建议中，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MyoD表达的转录调控机制 细胞和胚胎发育过程中被询问。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，它们在多大程度上保留了肌源性编程，以及它们自我更新的能力。实验 也将区分MRF缺乏的细胞自主和非自主影响。AIM 2将利用RNA- SEQ来定义未损伤和损伤的骨骼肌中突变卫星细胞的转录组，这将 确定MYOD和MYF5的直接和间接转录靶点以及调控途径和 这些MRF的丢失影响了细胞过程。此外，Pro-seq(全基因组精确运行) 分析将量化活跃基因转录的变化，将确定受启动子调控的候选基因- 近端聚合酶暂停，并将确定MYOD和MYF5的潜在增强子靶点。目标3将 通过产生dKO确定卫星细胞发育是否需要MyoD或Myf5的功能 胚胎、胎儿和新生儿时期卫星细胞前体细胞及其成体生成能力的检测 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，只有 已知的调节MyoD表达的增强子元件(核心增强子和末端调节区)是 在胚胎发育或卫星细胞中，MyoD的转录不是必需的。AIM 4将利用转染法， 基于转基因和CRISPR的基因敲除方法来确定新的推定的调控功能 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 对于理解控制卫星细胞干细胞的基本基因调控机制具有重要意义 细胞功能，并可能导致开发新的细胞类型和基于细胞的治疗策略 肌肉退行性疾病。

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.