Fig. 1: EDB(+) but not EDA(+) FN is enriched in the MuSC niche.

a Uniform manifold approximation and projection (UMAP) analysis of single cell RNA sequencing data from uninjured adult mouse skeletal muscle and at 5 days post injury (5 dpi) overlaid with the gene expression density of total FN²². FAPs, fibro-adipogenic progenitors; MuSC, muscle stem cells; ICs, immune cells; ECs, endothelial cells; MNs, myonuclei b Graphical representation of the mouse FN protein structure and location of the EDA, EDB, and IIICS variable domains, which undergo alternative splicing. Arrows indicate the location of primers used for detection of the respective FN splice variants. c,d PCR detection of alternative EDB and EDA splicing of FN in uninjured mouse tibialis anterior (TA) muscles (0 dpi) and at 5 dpi (c), and in early passage FAPs, freshly isolated MuSC-derived myoblasts in culture, and differentiated myotubes (d) using primers shown in Fig. 1b. The percent spliced in index (PSI) was determined using the formula: in/[in+ex] x 100 (in=exon inclusion, ex=exon exclusion). Mouse 36b4 (Rplp0) is shown as a housekeeping (HK) gene. Bars represent means ± SEM from n = 4 (c) and n = 3 (d) biological replicates per condition, each from tissues or cells from separate young mice. P-values were calculated using a one-tailed Student’s t-test (c) and one-way ANOVA with Dunnett’s post-hoc test (d). ****p < 0.0001, ***p < 0.001, **p < 0.01. e–h Immunostaining of EDB(+) and EDA(+) FN (green) in combination with the MuSC membrane marker m-cadherin (M-Cad, red) or total FN (panFN, red) in TA muscle sections of young mice 24 hours (h) post inury. DNA (blue) was counterstained using DAPI. Scale bar = 200 µm (large images) and 20 µm (inserts). i, j Immunostaining of EDB(+) and EDA(+) FN (green) in combination with M-Cad (red) or panFN (red) on single extensor digitorum longus (EDL) muscle fibers of young mice after 0, 42, and 72 h of culture. DNA (blue) was counterstained using DAPI. Scale bar = 10 µm. Source data are provided as a Source Data file.