Integrated Transcriptomic and Proteomic Analysis Reveals Runx2b-Associated Molecular Regulation of IB Development in...

Intermuscular bones (IBs) are ossified structures within the myoseptal connective tissues of cyprinid fishes and negatively affect eating quality and processing suitability. Although runx2b has been shown to be essential for IB formation, the molecular changes associated with runx2b-mediated IB reduction or loss remain poorly understood. In this study, runx2b-edited Culter alburnus F1 individuals were generated and classified into wild-type, heterozygous mutant, and homozygous mutant groups. Skeletal staining revealed a clear genotype-associated phenotypic gradient, with normal IBs in wild-type individuals, markedly reduced IBs in heterozygous mutants, and complete IB absence in homozygous mutants. Integrated transcriptomic and proteomic analyses were then performed using matched muscle/myoseptal tissues to characterize molecular changes associated with this phenotypic gradient. Global mRNA-protein correlations were weak and DEG-DEP overlaps were limited, suggesting that transcriptomic changes alone cannot fully explain protein-level remodeling after runx2b mutation. Functional enrichment and integrated omics analysis highlighted coordinated changes in cytoskeletal organization, extracellular matrix remodeling, cell adhesion, calcium-related signaling, muscle-associated processes, and ossification-related pathways. Representative candidate molecules, including MYH1s, CDH26, PLIN5, PAH, HBA/HBB, GPX4, KRT1, GSN, LGALS9, and ANXA3, were mainly associated with structural, metabolic, and microenvironmental remodeling rather than being interpreted as direct downstream osteogenic targets of runx2b. Overall, these findings suggest that runx2b-associated IB reduction and loss may involve coordinated remodeling of the myoseptal osteogenic microenvironment rather than disruption of a single ossification pathway. This study provides a discovery-oriented multi-omics resource and a hypothesis-generating molecular model for future functional validation of IB development in cyprinid fishes.