MNC clusters with differential expression of myogenesis and muscle function genes identified by MERFISH. (A) Two-dimensional UMAP projection of gene expression profiles of all MNCs. (Left) UMAP overlaid with the corresponding cluster identities; (right) Composition of each cluster in each genotype. (B) Heatmap illustrating DUX4 target gene expression level of the MNC clusters. The expression profile for each MNC cluster is the averaged, normalized, log-transformed expression across different samples and genotypes. (C) Volcano plot visualizes differential expression between the two identified MNC clusters across all samples. Myoblast and myotube marker genes (TTN, MYH3, MYH8, and NEB) show upregulation in cluster B MNCs (Log2FC threshold: 0.5; P-value threshold: 0.001). (D) Comparison of expression profiles of the two MNC clusters and myotube populations. (Top) UMAP calculated from the expressions of both MNCs and myotubes from all samples. The myotube population mostly overlaps with cluster 1 MNCs in UMAP space. Each dot represents a single MNC or a myotube. (Bottom) UMAP colored by genotype. Each dot represents a single MNC or myotube. (E) UMAP as in D, but overlaid with the normalized expression level of the myotube target genes, as well as two example DUX4 target genes (CCNA1, ZSCAN4). Cells with higher expression levels of TTN, MYH3, MYH8, and NEB mainly colocalize with cluster B MNCs, further indicating that cluster B MNCs are at a more mature differentiation state. CCNA1 and ZSCAN4 are highly expressed in the area occupied by FSHD and DEL5 myotubes.
