PROJECT SUMMARY Myoblast fusion, the process in which mononucleate myoblasts fuse to form multinucleate, contractile muscle fibers, is essential for skeletal muscle development, maintenance and regeneration. Insights into the molecular and cellular mechanisms of myoblast fusion to date have mainly come from studies of a genetic system, the fruit fly Drosophila. Studies in Drosophila have uncovered a handful of evolutionarily conserved regulators of myoblast fusion, ranging from cell adhesion molecules to actin polymerization regulators to mechanical sensors. More importantly, Drosophila studies have identified a novel cellular mechanism underlying myoblast fusion at the site of fusion – an attacking cell aggressively invades its fusion partner using actin-propelled membrane protrusions, whereas the receiving cell increases mechanical tension to resist the invasion, leading to cell membrane juxtaposition, fusogen engagement and plasma membrane fusion. Besides evolutionarily conserved fusion-promoting proteins, recent studies in zebrafish and mouse have identified a pair of vertebrate-specific fusogenic proteins, Myomaker and Myomixer (also known as Myomerger and Minion). However, how and where these proteins facilitate myoblast fusion is largely unknown. Compared to Drosophila studies, a major issue that hinders the study of the mechanisms underlying vertebrate myoblast fusion is the lack of knowledge of the precise sites of fusion. While myoblast fusion appears to occur at undefined location(s) along a broad cell-cell contact zone in cultured mammalian myoblasts, the sites of myoblast fusion in an intact animal remain completely unknown. Thus, it is imperative to identify the sites of fusion in vivo and provide a cellular framework upon which future studies can be built. Zebrafish is an excellent vertebrate model to study myoblast fusion in vivo, due to the large number of small and transparent zebrafish embryos and their rapid ex-utero development. In this proposal, we will use zebrafish as an in vivo model to define the sites of myoblast fusion in an intact vertebrate animal with molecular markers. In addition, we will study the localization and potential interaction between the fusogens, Myomaker and Myomixer. Furthermore, we will explore the interaction between the fusogens and the cell adhesion molecules and the actin cytoskeleton. Insights from the proposed studies will have a broad impact on understanding the fundamental principles of muscle development and regeneration, and ultimately may be exploited for the development of therapeutic strategies to optimize satellite cell-mediated muscle regeneration in patients with muscle degenerative diseases.