Grant: Discovery Grants Program - Individual

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.