SRY-box Transcription Factor 6 and Folliculin-Interacting Protein 1 as Key Regulators of Skeletal Muscle Fiber Formation in Chicken

SRY-box transcription factor 6 (Sox6) and folliculin-interacting protein 1 (Fnip1) play essential roles in muscle fiber specification during muscle development. However, their involvement in chicken muscle development remains largely unexplored. In this study, CRISPR-Cas9 was used to knock out (KO) Sox6 and Fnip1 in a chicken myoblast cell line (pCM cells). The functional significance and regulatory mechanisms of these genes were then examined during cell proliferation and muscle fiber differentiation. The loss of Sox6 and Fnip1 led to increased expression of Type 1 muscle-specific genes, including myosin light chain 2 (MYL2), myosin heavy chains (MYH1B, MYH1E, and MYH7B), and ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 2 (ATP2A2). In KO pCM cells, the expression of muscle Type 2-specific genes, including MYL1 and Troponin C (TnnC2), was significantly reduced. Moreover, mitochondrial abundance increased following gene deletion. Simultaneously, genes associated with the tricarboxylic acid (TCA) cycle and oxidative phosphorylation exhibited substantially elevated expression in KO pCM cells. Notably, the loss of Sox6 and Fnip1 not only suppressed cell proliferation but also impaired muscle fiber differentiation. Overall, these findings indicate that Sox6 and Fnip1 play a crucial role in regulating muscle fiber specification and differentiation in chickens.