Single-nucleus RNA sequencing unveils the sequential fate transitions of myogenic cells during sheep skeletal muscle development

Description

Background Skeletal muscle development is a critical biological process that dictates meat yield and quality in sheep; however, its underlying molecular regulatory mechanisms remain largely unexplored. Methods We profiled the longissimus dorsi muscle of Sunit sheep at three developmental stages, fetal (gestational day 100), neonatal (postnatal week 1), and adult (2 years of age), using single-nucleus RNA sequencing (snRNA-seq). A comprehensive single-cell transcriptomic atlas spanning these stages was constructed, enabling the systematic dissection of cellular composition, myogenic differentiation trajectories, and stage-specific regulatory networks via unsupervised clustering, pseudotime analysis, hdWGCNA, and SCENIC.. Results Our analysis yielded 81,811 high-quality nuclei, which were grouped into 21 clusters representing the major skeletal muscle cell types, including myocytes, fibro/adipogenic progenitors (FAPs), endothelial cells, macrophages, T/NK cells, and adipocytes. Re-clustering of myocyte subsets followed by pseudotime trajectory analysis revealed a continuous differentiation trajectory from fetal to adult stages, characterized by four dynamic gene expression modules. hdWGCNA uncovered co-expression modules significantly associated with distinct developmental stages and pinpointed their respective hub genes. SCENIC analysis further identified the stage-specific, sequential activation of transcription factors, including BACH1, ZNF639, and RXRA. Immunofluorescence and western blotting confirmed RXRA protein localization in satellite cells and its progressive downregulation with advancing development. In silico knockout of RXRA predicted a substantial remodeling of the transcriptional regulatory network, affecting pathways vital to skeletal muscle development, such as the MAPK cascade. Conclusions This study provides a single-cell-resolution transcriptional atlas of sheep skeletal muscle development, revealing the sequential order of myogenic cell fate transitions from proliferation to maturation and the stage-specific molecular regulatory networks. Our findings offer important theoretical resources and candidate genes for molecular breeding aimed at improving sheep muscle growth and meat production traits.

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Xingdong Wang

Institutions

• Inner Mongolia Normal University

  Inner Mongolia, Hohhot

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