Single-nucleus developmental atlas of the sheep adipose-skeletal muscle system reveals stem cell differentiation programs driven by core regulatory genes

Abstract Introduction: Adipose and skeletal muscle are interdependent for metabolism homeostasis, with their stem cells maintaining self-renewal and tissue formation through precise fate determination. However, the cellular developmental heterogeneity and stem cell differentiation regulation mechanisms of the sheep adipose-skeletal muscle system remain unclear. Methods: Single nucleus RNA sequencing was performed on 24 samples (four tissues from six female Hu sheep, three at 1 month and three at 5 months). Average depth >10,000 reads/cell, with median genes 755-2,122 per cell. Core regulatory genes were identified using combined machine learning and virtual knockout. Results: This study constructed a single-nucleus developmental atlas of the sheep adipose-skeletal muscle system (155,012 cells, 19467 features). Based on ensemble machine learning, we identified core regulatory genes (Subcutaneous adipose: COL1A1, FBN1, MFAP5; Perirenal adipose: FABP4, ADIPOQ, PLIN1; Biceps femoris muscle: ACTA1, MYH1, TTN; Longissimus dorsi muscle: ACACB, AUTS2, FOXP1) during the differentiation process of each tissue. Combining trajectory inference and virtual knockout, we uncovered key perturbation nodes and downstream effector pathways in the differentiation process driven by these regulatory genes. Furthermore, this study developed the SheepCellMap (https://sheepcellmap.lwzlab.com/) platform with marker search and visualization to support data for subsequent breeding practices. Conclusion: This study creatively explains cross‑tissue developmental logic of the adipose‑skeletal muscle system at single-nucleus resolution and reveals stem cell differentiation programs driven by core regulatory genes. It provides single‑cell portal and targets for adipose deposition and myofiber maturation. Despite sample and time limitations, future multi omics studies are needed for breeding application.