Molecular mechanisms of muscle stem cells transitioning intoquiescence

描述(申请人提供)：哺乳动物的肌肉系统为运动提供机械力。肌肉显示出从肌肉干细胞储存库再生的非凡能力。肌肉干细胞如何从发育过程中活跃的增殖群体转变为成年后用于肌肉修复/再生的储备群体，目前尚不清楚。我获得的证据表明，在小鼠模型中，这一过程依赖于Pax7基因的功能，该基因编码一种转录因子。我计划的目标如下：1)Pax7的确切作用；2)转录组在发育到成人的转变过程中的变化；3)调节这一转变的其他基本基因(与Pax7平行或下游)。目的1：检测突变型Pax7卫星细胞的细胞和分子缺陷。Pax7突变细胞何时以及如何在发育到成体的转变过程中变得有缺陷？我将结合过多的测试来确定几种肌肉干细胞的特性，包括肌肉分化、细胞增殖、细胞死亡、壁龛占有率和细胞极性。这些检测将通过从新生儿到成人的时间进程研究进行。目的2：对Pax7对照和突变型卫星细胞进行mRNA序列分析。在这一转变过程中，转录组的变化是什么？Pax7控制的具体过程是什么？对照细胞和Pax7突变细胞从发育到成体的整个过渡期的转录本的比较时间进程将使用mRNA-seq进行，随后将进行生物信息学分析。目的3：筛选卫星细胞功能的候选基因。除了Pax7外，哪些基因对肌肉干细胞从发育到成体的转变至关重要？从目标2中，我将选择多达50个基因来测试它们在这方面的功能；它们可能独立于Pax7发挥作用，也可能在Pax7下游发挥作用。我将开发一种新的方法，通过结合基因敲除和肌肉干细胞肌肉内移植来快速筛选这些基因的功能。 公共卫生相关性：肌肉干细胞为损伤后的肌肉再生提供补充来源。这项提议旨在以小鼠为模型，确定控制肌肉干细胞生成的基因。这项研究的结果将对基于干细胞的肌肉营养不良和因战斗或事故造成的急性肌创伤的治疗具有临床意义。

DESCRIPTION (provided by applicant): The mammalian musculature provides mechanical force for locomotion. Muscles display remarkable ability to regenerate from reservoirs of muscle stem cells. How muscle stem cells transition from an actively proliferating population during development into a reserved population in adulthood for muscle repair/regeneration throughout lifetime is not known. I have obtained evidence that, in the mouse model, this process depends on the function of the Pax7 gene, which encodes a transcription factor. I plan the following aims to define 1) the precise role of Pax7, 2) the transcriptome changes during the development-to-adult transition, and 3) other essential genes (acting in parallel or downstream of Pax7) that mediate this transition. Aim 1: Determining the cellular and molecular defects of Pax7 mutant satellite cells. When and how do Pax7 mutant cells become defective in the development-to-adult transition? I will combine a plethora of assays for determining several muscle stem cell properties, including muscle differentiation, cell proliferation, cell death, niche occupancy, and cell polarity. These assays will be conducted with time course studies from newborn to adult. Aim 2: Performing mRNA-seq of Pax7 control and Pax7 mutant satellite cells. What are the transcriptome changes during this transition and what are the specific processes governed by Pax7? A comparative time course study of transcriptomes between control and Pax7 mutant cells throughout the development-to-adult transitional period will be performed using mRNA-seq, followed by bioinformatic analysis. Aim 3: Screening candidate genes for their function in satellite cells. What are the genes (in addition to Pax7) critical for the development-to-adult muscle stem cell transition? From Aim 2, I will select up to 50 genes to test for their function in this aspect; they may either act independently or downstream of Pax7. I will develop a novel assay for rapid screening of these genes' functions by combining gene knock-down and intramuscular transplantation of muscle stem cells. PUBLIC HEALTH RELEVANCE: Muscle stem cells provide the replenishing source for muscle regeneration after injury. This proposal aims to determine the genes that control the genesis of muscle stem cells, using the mouse as a model. Results from this study will have clinical implications for stem cell based therapies of muscular dystrophies and of acute myotrauma from combat or accident.