Single-Nucleus RNA Sequencing Reveals Cellular Heterogeneity and Trajectories of Lineage Differentiation during Chicken Skeletal Muscle Development

Understanding chicken skeletal muscle development is crucial for meat production, yet its cellular and molecular mechanisms are not fully understood. To address this, we built a single-nucleus RNA sequencing atlas of chicken skeletal muscle across key developmental stages (E4-P30) using 111,328 nuclei. We identified 22 cell populations, including nine myogenic ones, revealing extensive heterogeneity and transcriptional diversity. Myonuclei were classified into three fiber-type-associated subpopulations: <i>FGF13</i>⁺ type IIA fast-twitch, <i>MYH1F</i>⁺ type IIB fast-twitch, and <i>MYH7B</i>⁺ type I slow-twitch. Pseudotime analysis reconstructed the developmental trajectory from myogenic progenitors to mature myofibers and highlighted RBM24 as a pivotal regulator. Functional validation confirmed that RBM24 promotes myoblast differentiation while inhibiting proliferation. We further identified putative fiber-type-specific regulators (e.g., <i>TBX15</i> for IIA, <i>TNNT3</i> for IIB, and <i>FGFRL1</i> for I). This study delineates myogenic lineage development and identifies key regulatory genes, offering valuable insights into chicken skeletal muscle biology.