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

项目摘要 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MYF 5是确定肌肉谱系所必需的， 胚胎和诱导激活的卫星细胞作为肌肉损伤的早期反应。最近的基因- 使用新的MyoD条件性敲除等位基因（MyoDcKO）的靶向研究表明，MyoD或Myf 5是 对于肌肉再生至关重要;缺乏这两种基因的卫星细胞（dKO）在受伤的肌肉中积累， 不能进行肌原性分化。在这项建议中，新的遗传工具和战略被用来 确定MyoD和Myf 5在卫星细胞发育、谱系确定、分化 和自我更新此外，调控MyoD在卫星细胞中表达的转录控制机制， 询问细胞和胚胎发生过程中的细胞。在目的1中，通过免疫荧光和免疫荧光技术鉴定细胞类型。 单细胞RNA测序（scRNA-seq）将确定dKO卫星细胞是否采用非肌原性细胞 命运，他们保留肌原性编程的程度，以及他们自我更新的能力。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， 鉴定MYOD和MYF 5的直接和间接转录靶标以及调控途径， 细胞过程受到这些MRF损失的影响。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，将识别受启动子调控的候选基因， 在一个实施方案中，该方法将使邻近聚合酶暂停，并将鉴定MYOD和MYF 5的潜在增强子靶标。目标3将 通过产生dKO来确定MyoD或Myf 5的功能是否是卫星细胞发育所需的 胚胎、胎儿和新生儿阶段的卫星细胞前体，并测试它们产生成年 卫星细胞，如通过分子和解剖学标准评估的。最近的数据显示， 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 在胚胎发生或卫星细胞中，MyoD转录不是必需的。Aim 4将利用转染， 转基因和基于CRISPR的敲除方法来定义新的推定的 通过PRO-seq和生物信息学分析鉴定的增强子元件。这项研究将有助于 这对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， 肌肉退化性疾病

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.