RecrutCell

Muscle fibers consist of multi-nucleated cells that are the basic cellular building block of muscle. In adults, muscle growth and regeneration mainly occur through the activation, proliferation and differentiation of a resident population of muscle stem cells called satellite cells. Once activated, (known as myoblast) these cells differentiate and either fuse to existing myofibers (hypertrophy) or fuse together to form new myofibers (hyperplasia). Contrary to muscle growth in terrestrial livestock animals, post-larval muscle growth in large and rapidly growing fish, such as trout, results from both hypertrophy and hyperplasia. Thus, in fish, satellite cells have the choice to fuse with an existing myofiber or with another satellite cell. The factors that control the fusion with either a myofiber or another myoblast to enhance the growth of muscle are of great interest from an agronomic and therapeutic standpoint. Our project aims to identify new molecular factors involved in the commitment of a satellite cell to fuse with a myoblast or with a myotube. We are particularly interested in the identification of the differences between the genetic programs underlying myoblast-myoblast fusion and myoblast-myotube fusion. To achieve this objective, we propose two complementary parts: 1) a double functional screen using siRNA to identify genes that are potentially involved in myoblast commitment and 2) the functional analysis of the identified genes using in vivo and in vitro studies in fish. The siRNA screen will be performed on the C2C12 myogenic cell line. This cell line is the most commonly used line for the study of myoblast fusion because it recapitulates both myoblast-myoblast fusion as well as myoblast-myotube fusion in vitro. The siRNA screens are based on a primary and a secondary functional screen. To automate and simplify the analysis, we will use three fluorescent C2C12 cell lines that we have already generated. The primary screen, which will use the MCK-GFP C2C12 cell line, will aim to identify genes that induce a fusion default upon knockdown. The second screen, which will use two stable C2C12 cell lines that constitutively express GFP or mCherry, will aim to identify genes that are involved exclusively in either myoblast-myoblast or myoblast-myotube fusion. For the newly identified genes, we will study their expression (task 3) during the embryonic development of zebrafish, as well as during the fusion of satellite cells in vitro. We will determine the function of the identified genes in vivo during embryonic myogenesis using a morpholino. Finally, we will determine the involvement of the identified genes in the balance between hyperplasia and hypertrophy. We will carry out gain-of-function and loss-of-function experiments for the candidate genes in trout muscle, which exhibits a high level of hyperplasia.