Identification of the crucial molecular events during the large-scale myoblast fusion in sheep

It is well known that in sheep most myofibers are formed before birth; however, the crucial myogenic stage and the cellular and molecular mechanisms underpinning phenotypic variation of fetal muscle development remain to be ascertained. We used histological, microarray, and quantitative real-time PCR (qPCR) methods to examine the developmental characteristics of fetal muscle at 70, 85, 100, 120, and 135 days of gestation in sheep. We show that day 100 is an important checkpoint for change in muscle transcriptome and histomorphology in fetal sheep and that the period of 85-100 days is the vital developmental stage for large-scale myoblast fusion. Furthermore, we identified the cis-regulatory motifs for E2F1 or MEF2A in a list of decreasingly or increasingly expressed genes between 85 and 100 days, respectively. Further analysis demonstrated that the mRNA and phosphorylated protein levels of E2F1 and MEF2A significantly declined with myogenic progression in vivo and in vitro. qRT-PCR analysis indicated that PI3K and FST, as targets of E2F1, may be involved in myoblast differentiation and fusion and that downregulation of MEF2A contributes to transition of myofiber types by differential regulation of the target genes involved at the stage of 85-100 days. We clarify for the first time the timing of myofiber proliferation and development during gestation in sheep, which would be beneficial to meat sheep production. Our findings present a repertoire of gene expression in muscle during large-scale myoblast fusion at transcriptome-wide level, which contributes to elucidate the regulatory network of myogenic differentiation.