Integrative single-cell RNA-seq and ATAC-seq profiling identifies NTN5⁺LSAMP⁺ myoblasts as mediators of impaired embryonic myogenesis

Prenatal myogenesis establishes the cellular and regulatory framework that determines postnatal muscle mass and contractile performance, and represents a critical period for elucidating mechanisms underlying developmental myopathies and impaired fetal growth. In this study, integrated single-cell transcriptomic and chromatin accessibility profiling was applied to compare the regulatory landscapes between growth-restricted and normal porcine embryonic somites. This analysis uncovered a distinct pathogenic myoblast population characterized by co-expression of netrin-5 (NTN5) and limbic system-associated membrane protein (LSAMP), markedly enriched in growth-restricted embryos. These NTN5⁺LSAMP⁺ myoblasts exhibited a cell-autonomous differentiation blockade accompanied by coordinated epigenetic and metabolic dysregulation. Chromatin landscapes revealed sustained accessibility at the cytoskeletal regulator tropomyosin-3 (TPM3), suppressed accessibility at the muscle-specific actin depolymerization factor cofilin-2 (CFL2), and transcriptional programs consistent with impaired glycolytic flux. Perturbation of the Hippo/TGF-β signaling pathway, together with disrupted ligand–receptor interactions, exacerbated the differentiation arrest. Reconstruction of a multi-omics regulatory network delineated the mechanisms underlying aberrant embryonic myogenesis, identifying NTN5 and LSAMP as key regulators of porcine skeletal muscle development. Collectively, these findings define mechanistic determinants of impaired fetal myogenesis and provide candidate targets for therapeutic intervention in growth restriction as well as molecular strategies for optimizing muscle accretion in livestock.