Single‐Cell RNA‐Sequencing Provides Insight into Skeletal Muscle Evolution during the Selection of Muscle Characteristics

Abstract Skeletal muscle comprises a large, heterogeneous assortment of cell populations that interact to maintain muscle homeostasis, but little is known about the mechanism that controls myogenic development in response to artificial selection. Different pig (Sus scrofa) breeds exhibit distinct muscle phenotypes resulting from domestication and selective breeding. Using unbiased single‐cell transcriptomic sequencing analysis (scRNA‐seq), the impact of artificial selection on cell profiles is investigated in neonatal skeletal muscle of pigs. This work provides panoramic muscle‐resident cell profiles and identifies novel and breed‐specific cells, mapping them on pseudotime trajectories. Artificial selection has elicited significant changes in muscle‐resident cell profiles, while conserving signs of generational environmental challenges. These results suggest that fibro‐adipogenic progenitors serve as a cellular interaction hub and that specific transcription factors identified here may serve as candidate target regulons for the pursuit of a specific muscle phenotype. Furthermore, a cross‐species comparison of humans, mice, and pigs illustrates the conservation and divergence of mammalian muscle ontology. The findings of this study reveal shifts in cellular heterogeneity, novel cell subpopulations, and their interactions that may greatly facilitate the understanding of the mechanism underlying divergent muscle phenotypes arising from artificial selection.


Figure S1 .
Figure S1.Related to Figure 1.Cell populations present in skeletal muscle tissues for each pig breed.t-SNE plot revealed cellular heterogeneity with nine major populations and violin plots showed the expression levels and distribution of representative marker genes within each breed.

Figure S2 .
Figure S2.Related to Figure 2 and 3. Unbiased dissection of FAP subpopulations among pig breeds.(A-B) FAPs from Laiwu pig (A) and Duroc pig (B) skeletal muscle were selected and reanalyzed.
(E) Representative confocal images of skeletal muscle stained for FAP marker PDGFRa (red), DAPI (blue) and MT-rich FAPs marker TOM20 (Green).White arrowheads denote FAPs that are positive for the marker (scale, 10 μm).Yellow arrowheads mark FAPs that are negative for expression of the marker.(F) Expressions of CD142 within wide boars, Laiwu pigs and Duroc pigs.(G) Heatmap illustrating the TF of differentially expressed genes (DEGs) dynamics towards myocyte-like FAPs and tenocytes/adipocytes fate along pseudotime.

Figure S4 .
Figure S4.Related to Figure 4 and 5. Myogenic lineage subpopulations from wild boars, Laiwu and Duroc pigs (A, B) Myogenic lineages (satellite cells, myoblasts and myocytes) from Laiwu (A) and Duroc pigs (B) were selected and re-analyzed.t-SNE plot colored by myogenic lineages (left) and manual classified cell subpopulations (right).
(C) t-SNE maps showing the expression levels and distribution of markers for mesenchymal stem cell (CD34), satellite cell (PAX7), myoblast (MYOG) and myocyte (ENO3) of wild boars (top), Laiwu pigs (middle), and Duroc pigs (bottom).(D) t-SNE maps showing the distribution in the expression of selected markers of indicated satellite cell subpopulations of wild boars (top), Laiwu pigs (middle), and Duroc pigs (bottom).
(E-F) t-SNE maps showing the expression levels of genes related to muscle maturation and Ca 2+ -binding capacity in two myoblast subpopulations of Laiwu (E) and Duroc pigs (F).(G-H)Fast and slow myocytes recognized by myofiber type-specific markers (MYL1 and MYL2 respectively) in Laiwu (G) and Duroc pigs (H).

Figure S7 .
Figure S7.Related to Figure 6.CellChat analysis of the communications across skeletal cell type (A) The alluvial plot showing outgoing communication patterns of secreting cells.(B, D, E) Circle plot showing the number of statistically significant intercellular interactions for the selected pathway family of molecules.Each circle (color) represents one cell type; edges connecting circles represent significant intercellular signaling inferred between those cell types.Circles and edges are normalized to the number of cells for a given cell type and inferred strength of signaling, respectively.(C) Incoming communication patterns of target cells.

Figure S8 .
Figure S8.Related to Figure6.Differential intercellular signaling from immune cells among pig breeds (A) Hierarchical plot shows the inferred intercellular communication network for FGF (top) and IL1 (bottom) signaling within FAPs subsets of Duroc pigs.Solid and open circles represent

Figure S10 .
Figure S10.Related to Figure 9.Comparison of human satellite cells and porcine endothelial cells (A) Comparison of expression levels between human satellite cells and porcine endothelial cells.Satellite cell markers were shown in green, while endothelial cell markers in red.

Table Captions for Table S1-4 Table S1: The quality information of sequencing data Table S2: Enriched GSEA terms of biological processes on up-regulated genes of each homologous cell subpopulation among breeds Upload seperately Table S3: Shared markers and species-specific markers of homologous cells among pigs, mice, and humans Upload seperately Table S4: Reagent and resource used in this paperTable S1 .
The quality information of sequencing data

Table S4 .
Reagent and resource used in this paper