Role of AP-1 in Skeletal Myogenesis

骨骼肌的发育、维持和再生是真生动物（除海绵动物和placozoans外的所有动物）的原始特征。肌肉作为专门用于收缩的组织，是神经系统运动、逃跑、进食和防御等活动的基本效应器。在之前的研究中，我们认为AP-1转录复合体在肌源性分化的基本过程中参与了骨骼肌基因调控的控制。最近，我们报道了AP-1不仅在胚胎成肌细胞中表达，也在成人骨骼肌的常驻干细胞（卫星细胞）中表达。我们还记录了ERK信号通路稳定AP-1复合体的Fra2亚基。在所有哺乳动物中，卫星细胞对骨骼肌的产后维护和修复至关重要。在拟议的研究中，我们计划进一步评估骨骼肌发育和再生对AP-1的需求。为了做到这一点，我们将解决以下特定的假设驱动目标：******1)AP-1是肌母细胞和卫星细胞中的Notch信号所必需的。***2)小鼠条件性cjun/fosl2缺失会破坏胚胎肌肉发生和/或出生后肌肉再生。***3)生长因子信号控制Fra2转录复合物的组成和肌肉发生。******实验策略：***目的1：先前的工作发现AP-1转录靶向肌肉中Notch信号通路的许多组分。下一步是利用AP1的功能增益和损失分析来评估它如何影响原代成肌细胞和单个肌纤维培养中Notch信号的组成和下游靶点。我们将利用传统的生物化学和最先进的共聚焦活细胞成像技术来解决这个问题。***目的2：条件Cre-lox介导的小鼠Floxed cJun和Fra2切除是一项雄心勃勃的目标，旨在评估AP-1在胚胎肌肉形成和产后肌肉维持和再生中的作用。***目的3：表征Fra2复合物组成和稳定性的信号通路调控将使用我们开发的最先进的相互作用分析管道进行，该管道使用亲和纯化-质谱，以及抗体介导的Fra2翻译后修饰检测。******可行性和影响：***该提案基于NSERC几个资助周期产生的大量数据，将解决AP-1的基本调控及其在骨骼肌中的作用。人员，试剂和技术到位，以高度可行性解决这些目标。这些研究将产生有影响力的科学，与我们在该领域的其他贡献一致。

Development, maintenance and regeneration of skeletal muscle is a primal feature of eumetazoans (all animals except sponges and placozoans). Muscles, as tissues specialized for contraction, are essential effectors of the nervous system underlying activities such as locomotion, escape, feeding, and defence. In previous work we implicated the AP-1 transcription complex in the control of skeletal muscle gene regulation during the fundamental process of myogenic differentiation. Recently, we reported that AP-1 is expressed not only in embryonic myoblasts but also in the resident stem cells (satellite cells) in adult skeletal muscle, We also documented that ERK signalling stabilizes the Fra2 subunit of the AP-1 complex. Satellite cells are crucial for the vital post-natal maintenance and repair of skeletal muscle in all mammals. In the proposed studies we plan to further assess the requirement for AP-1 in skeletal muscle development and regeneration. To do this we will address the following specific hypothesis driven objectives:******1) AP-1 is required for Notch signalling in myoblasts and satellite cells.***2) Conditional cjun/fosl2 deletion in mice will disrupt embryonic myogenesis and/or post-natal muscle regeneration.***3) Growth factor signaling controls Fra2 transcription complex composition and myogenesis.******Experimental Strategy:***Objective 1: Previous work identified that AP-1 transcriptionally targets a number of components of the Notch signaling pathway in muscle. The next step is to employ gain and loss of function analysis of AP1 to assess how this influences components and downstream targets of Notch signalling in primary myoblast and single muscle fiber cultures. We will utilize traditional biochemistry and state of the art confocal live cell imaging technology to address this aim.***Objective 2: Conditional Cre-lox mediated excision of Floxed cJun and Fra2 in mice is an ambitious goal to assess the role of AP-1 in embryonic muscle formation and also post-natal muscle maintenance and regeneration.***Objective 3: Characterization of the signalling pathway regulated control of Fra2 complex composition and stability will be carried out using an innovative state of the art interactome analysis pipeline that we have developed using affinity purification-mass spectrometry, along with antibody mediated detection of post-translational modification of Fra2.******Feasibility and Impact: ***This proposal, based on extensive data generated over several NSERC funding cycles, will address the fundamental regulation of AP-1 and its role in skeletal muscle. Personnel, reagents and technology are in place to address these objectives with high feasibility. These studies will result in impactful science in line with our other contributions in this field.