Adult regenerative myogenic cells express the transgene ScxGFP. A. Experimental scheme for data in (B-D). Tg-ScxGFP (ScxGFP) mice were injured by CTX to the TA muscle, followed by daily EdU administration for 5 days (5d), and their TA muscles were harvested for analysis at 5d post-injury (dpi). B. Muscle samples obtained in (A) were sectioned and stained with Pax7 and GFP (for ScxGFP expression) antibody. Arrows indicate Pax7 and ScxGFP double positive cells; 97.77% Pax7+ SCs were ScxGFP+ (N = 4 mice; n = 1274 cells). C. Muscle samples obtained in (A) were sectioned and stained in pairs of GFP/Pax7, GFP/MyoD1, GFP/MyoG (N = 4 mice). Asterisks indicate cells double-positive for ScxGFP and each respective myogenic marker. D. Muscle samples obtained in (A) were sectioned and stained for Pax7 and GFP, followed by EdU reaction (N = 4 mice). Arrows indicate Pax7, ScxGFP, and EdU triple-positive SCs. E. Experimental scheme of SC isolation from Tg-ScxGFP hindlimb muscles using four surface markers (CD31-, CD45-, Sca1-, Vcam1+) by FACS. Isolated SCs were assayed immediately after isolation (D0; data in (F)), after culture in growth media for 2d (D2(GM); data in (G)), or after cultured for 4d in GM followed by 2d in differentiation media (DM) (D6(DM); data in (H)). F, G. D0 (F) and D2 cultured (G) SCs obtained in (E) were stained with for GFP (i.e. ScxGFP), Pax7, and MyoD. At D0, no Pax7+ cells were GFP+ or MyoD+. At D2, 95.3% of Pax7+ cells were GFP+, whereas 99.2% of MyoD+ were GFP+. (N = 3 mice; n = 1805 cells at D0; n = 1332 cells at D2). H. D6(DM) cells obtained in (E) were stained for GFP (i.e. ScxGFP) and MHC. 94.58% MHC+ were GFP+. (N= 3 mice; n = 1539 nuclei in MHC+ domain examined). Nuclei were stained with DAPI (blue); Scale bars = 20 μm.

Endogenous Scx is expressed by activated but not quiescent SCs. A. Experimental design for ScxCreERT2 mediated inducible lineage tracing with the RtdT reporter. The two experimental groups are: 1) TMX administrated before injury for 5 days (TMX before injury) and 2) TMX administrated after injury for 5 days (TMX after injury). TA muscles in both groups were harvested at 14d post injury. B. TA muscles from experiment groups in (A) were stained with Pax7 (green) and Laminin (white) and visualized with tdT (no staining). Open arrowheads indicate Pax7+ SCs; arrows, Pax7+tdT+ SCs. C. Percentages of Pax7+tdT+ SCs in Pax7+ SCs examined, from data in B. (N = 4 mice per group; n = 191 (Before, TMX before injury) and 256 (After, TMX after injury) Pax7+ cells). D. Percentages of tdT+ myofibers in regenerated muscle fibers (with centrally located nuclei), from data in B. (N = 4 mice; n = 1956 (Before, TMX before injury) and n = 2024 (After, TMX after injury) regenerated myofibers). E. UMAP plot of Scx expression in SCs isolated from uninjured muscles (left, freshly sorted) and from muscles at 2.5 dpi in a published dataset(Dell’Orso, Juan et al. 2019). Nuclei were stained with DAPI; Scale bar = 20 μm. Data are presented with mean ± s.d.; P value are indicated. C-D, Unpaired two-tailed Student’s t-test were applied.

Efficient muscle regeneration requires Scx function. A. Experimental designs to compare phenotypes of control (Ctrl) and ScxcKO mice. The RtdT reporter was included (tdT lineage; see Figure S3A for genotypes). TMX was administered before and after the CTX induced injury to maximize gene inactivation. TA muscles were harvested at 5d or 14d after injury. B-C. (B) Ctrl and ScxcKO TA muscles at 5d post-injury were sectioned and stained with hematoxylin and eosin (H&E) at low (top) and high (bottom) magnifications. (C) Histogram of regenerated muscle fiber cross-sectional area from data in (B). (N = 5 mice per group). D-E. (D) Ctrl and ScxcKO TA muscles at 14d post-injury were sectioned and stained with H&E. (E) Histogram of regenerated muscle fiber cross-sectional area from data in (D). (N = 5 mice per group) F. Histogram of average Pax7+ SCs number per imaged field (0.08mm2) of TA muscle sections from 5 dpi Ctrl and ScxcKO mice (N = 5 mice per group; n = 2807 Ctrl and n = 442 ScxcKO Pax7+ SCs). G. After EdU administration, Ctrl and ScxcKO TA muscles at 5d post-injury were sectioned and stained for Pax7, followed by EdU reaction. Arrows indicate Pax7+EdU+cells, whereas asterisk, Pax7+EdU- cells. Nuclei were stained with DAPI; Scale bar = 20 μm. H. Percentages of EdU+ cells within the Pax7+ cell population of Ctrl and ScxcKO, from data in G (N = 5 mice per group; n = 858 Ctrl and n = 325 ScxcKO Pax7+ SCs). I. Averaged Pax7+ SC number per image field (0.4mm2) in Ctrl and ScxcKO TA muscle sections from 14d post-injury samples (N = 5 mice per group; n = 350 Ctrl and n = 186 ScxcKO Pax7+ SCs). Data are presented with the mean ± s.d.; P values are indicated. (C, E-F, H-I) Unpaired two-tailed Student’s t-test were applied.

ScxcKO SCs display a proliferation defect. A. Experimental design to obtain YFP lineage marked Pax7+ SCs for in vitro analyses in (B-F). B. Box plot of percentages of FACS-isolated tdT and YFP marked cells expressing Pax7 by staining immediately after isolation (as D0); each dot represents one image data, 10 image per group, totally n > 1000 cells for each group. C. YFP lineage marked cells were cultured in GM and assayed at days 2, 3, and 4 (D2-D4) intervals. 10 μM EdU was added for 6 h prior to harvesting for EdU detection. D. Box plot of percentages of EdU+ cells from data in (C), N=2 mice, each dot represents one image data, 3 well per group, 8 images per well. E. Box plot of ratios of total cell numbers from data in (C); normalized to the average control cell number at D2 as 1. F. Box plot of ratios of program cell death assayed by staining for cleaved Caspase3; n > 300 cells for each group. Nuclei were stained with DAPI; Scale bar = 20 μm. Data are presented with the mean ± s.d.; adjusted P values are shown. (B) Two-way ANOVA; (D-F) Unpaired two-tailed Student’s t-test.

scRNA-seq helps identify the role of Scx in myogenic differentiation and fusion.

A. SC scRNA-seq scheme for YFP lineage marked SCs. YFP+ cells were FACS-isolated from 2.5 dpi BaCl2 injured TA and the gastrocnemius (GA) muscles. B. Trajectory analysis of the 7 myogenic clusters (complete cell cluster analysis in Fig. S5) indicated to the right. Arrow indicates the direction of pseudotime trajectory. C. Cell densities of Ctrl and ScxcKO cells along the trajectory in (B). Cell in Peaks 2-4 were used for the DEG analysis; The asterisk indicates Peak 2 as our main focus. D. In vitro differentiation assay scheme. SC-derived myoblasts were cultured in GM for 12 h (D0), switched into DM, and harvested daily for analysis over 3 days (D1 - D3). E. Myoblasts subjected the scheme in (D) were stained for MyoG (for differentiation index in F) and for MHC (for fusion index in G). Nuclei were stained with DAPI; Scale bar = 20 μm. F, G. Box plot of differentiation index (F) and fusion index (G) from data in (E). Each dot represents data from one image. Unpaired two-tailed Student’s t-tests were applied and adjusted P values are shown. (N = 3 mice; 3 wells per group per time point; 10 images per well; in total, 3342 control and 2561 ScxcKO cells examined). A. Volcano plot of relative gene expression (Log2 fold change) in Ctrl versus ScxKO cells in Peak 2 (in C). B. GO term enrichment of muscle development related processes from DGEs in (H).

CUT&RUN and scRNA-seq identify direct targets of Scx. A. Experimental scheme for CUT&RUN profiling of the Scx binding in the genome of ScxTy1/Ty1 and ScxGFP myoblasts. Primary myoblasts derived from SCs of ScxTy1/Ty1 (experimental group) and ScxGFP (control group) mice were used. They were cultured in GM for 12 h (D0), and switched to DM for 12 h for use. 500,000 (500K) cells per group were subjected to CUT&RUN using an anti-Ty1 antibody or an IgG control antibody, in duplicate. B. Pie chart for distribution of Scx CUT&RUN peaks in various regions of the genome. C. Motif enrichment analysis with SEA from MEME suite (v. 5.5.0) identified bHLH protein binding motif (i.e. E-box) in all Scx CUT&RUN binding peaks. D. Venn diagram of intersecting genes (207 genes) between Scx CUT&RUN target genes (861) and DEGs (3749) in Peak 2 of Fig. 5C. E. Motif enrichment analysis (as in C) of the 207 genes in (D) also showed enrichment of bHLH protein binding motifs, the E-box. F. GO term analysis of the 207 genes in (D). GO terms with P < 0.0001 were plotted. G. Genomic snapshots of Scx CUT&RUN peaks on 4 select genes related to muscle differentiation. Expression levels (CPM, counts per million UMI) of the 4 select genes in (G) along the pseudotime trajectory (same trajectory as Fig. 5C).

Addition data for Figure 1. A. TA muscle from uninjured ScxGFP mice were sectioned and stained with Pax7 and GFP (N = 4 mice; n = 203 Pax7+ cells examined and no Pax7+GFP+ cells found). The arrow indicates the Pax7+ SC; asterisk, ScxGFP+ intramuscular CT; dashed line, outline of a muscle fiber. B-D. Split channel images of data in Figure 1C; asterisks indicate the same cells. E, F. FACS strategy (E) and profiles (F) to support Fig. 1E. (F) FACS plot and population hierarchy of SC from 4 surface markers sorting in pseudocolor plots. G. Freshly isolated SCs from ScxGFP mice were cyto-spun and stained with Pax7 and DAPI. H. Percentage of Pax7+ cells in freshly isolated ScxGFP SCs by FACS procedures in (E, F). (N = 3 mice; n =1805 cells). J. Scale bar = 20 μm. Data are presented with mean ± s.d.

Additional information for Figure 2. C. Averaged Pax7+ SC number per field (0.06mm2) from data in B. Mouse and cell numbers are the same as in Figure 2C. D. Gene expression levels (in FPKM) of Scx and Pax7 in SCs isolated from wild type (WT) and mdx mice using bulk-RNA-seq. Data are extracted from published data sets(Li, Rozo et al. 2019, Madaro, Torcinaro et al. 2019). Data are presented with the mean ± s.d.; the P value is indicated. Unpaired two-tailed Student’s t-test was applied.

Addition data for Figure 3. A. Detailed description for the genotypes used as Ctrl and ScxcKO mice in Fig. 3. For tdT lineage, RtdT reporter was included. For YFP lineage, RYFP reporter was included. B. Depiction of Scx gene inactivation by TMX-induced recombination of loxP sites flanking the exon 1 of Scx using the Pax7CE allele. PCR primer sets P1, P2, and P3 were used to detect exon 1 (E1), intron 1 (I1) and E2 of the Scx gene, respectively. Primer sequences are in Table S10. C. Experimental scheme to determine the recombination efficiency using FACS-isolated Ctrl and ScxcKO SCs. Freshly sorted SCs were plated down and cultured in growth medium for 3 days and harvested for genomic DNA extraction and qPCR for data in (D). D. Relative levels of E1, I1, and E2 in control and ScxcKO myoblasts determined by qPCR, followed by 2-ΔΔCt analysis. E. Experimental scheme as Fig. 3A for 5 dpi data in F-G. F, G. TA muscle sections (from E) were sectioned and stained with Pax7 and Laminin in (F), and MHC and laminin in (G). Arrows indicate Pax7+ SCs in (F), whereas arrows indicate Laminin+MHC- ghost fibers (G) (N = 5 mice in each group; n = 1748 control and n = 442 ScxcKO Pax7+ SCs). H. Experimental scheme as Fig. 3A for 14 dpi data in (I). I. TA muscle section from (H) from were sectioned and stained with Pax7 and Laminin (N = 5 mice in each group; n = 350 control and n = 186 ScxcKO Pax7+ SCs examined). J. Same experimental scheme as in (E), except that RYFP reporter (YFP lineage), instead of RtdT reporter, was included. K. Histogram of the fiber CSA from 5 dpi TA muscle sections from (J). (N = 6 mice for control, and N = 5 mice for ScxcKO group) Nuclei were stained with DAPI. Scale bar = 20 μm. Data are presented with the mean ± s.d.; adjusted P values are shown. (D, K) Unpaired two-tailed Student’s t-test.

Additional data for Figure 4. A, B. FACS profiles for isolating tdT (A) and YFP (B) linage marked SCs. E. Similar experimental scheme as in Fig. 4 A for refence to live imaging data in (D-F). F. Line plot of relative cell number per frame; normalized to cell number of the Ctrl group at the beginning as 1. G. Box plot of relative dividing cell ratio. Relative dividing ratio is defined as the divided cell number of each day per group / the beginning cell number of that day. There were no dividing cells detected at D1, each dot represents one image data, 3 well per group. H. Box plot of cell migration speed based on tracking cell displacement by pixel (0.33 μm / pixel), each dot represents a cell data. I. Still frames from time lapse show examples of necrotic cells at D3 of the ScxcKO group. Frames 1 - 6 are sequential time lapse images (covering 500 mins). Two cells of focus are indicated by red and black arrows to track their appearance from a healthy state (earlier frames) to a necrotic state (later frames). Scale bar = 20 μm. H, Box plot of necrotic cell ratio. Necrotic cell was manually identified by morphology in each frame on each day, and normalized to the beginning cell number of that day, each dot represents one image data. Data are presented with mean ± s.d.; adjusted P values are shown. Unpaired two-tailed J. Student’s t-test were applied to data collected in each day.

Additional analyses of data in Figure 5. B. Detailed scRNA-seq procedures diagram as in Fig. 5A for reference to analyses in (B-G). C. UMAP plot of combined scRNA-seq of Ctrl and ScxcKO cells. A total of 11,388 control cells and 12,844 ScxcKO cells were included for analysis, and 18 cell clusters were delineated (shown by different colors and with assigned cell types indicated to the right). Asterisks denote myogenic clusters subjected to further analyses in Figure 5B, C, H, I. D. Cell density distribution of each cell cluster in Ctrl and ScxcKO samples. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. E. Expression of select marker genes in each cell cluster. Dot size represents the percentage of expressed cells within each cluster, whereas color intensity represents relative expression level (keys at bottom). F. Pax7 expression levels and cell numbers in each cluster; each dot represents one cell. G. Scx expression levels and cell numbers in each cluster. Relative expression levels (CPM, counts per million UMI) of 4 select cell cycle genes along the pseudotime depicted in Figure 5C.

Additional data to support Figure 6. A. Diagram of the genomic structure of ScxTy1 allele. Three (3X) Ty1 tags were inserted just before the TGA codon of the Scx gene. B. Neonatal patellar tendons of ScxTy1/Ty1 and wild type (WT) mice were fixed, sectioned, and stained for Ty1. C. In vitro myogenic differentiation scheme using ScxTy1/Ty1 SC-derived myoblasts. Myoblasts were cultured in GM for 12 h (D0), and switched to DM for 3 days. Cells in each day were stained for Ty1 (D0-3). D. ScxTy1/Ty1 and WT myoblasts at D0 were stained for Ty1 as an example for data in (E). E. Boxplot of percentages of cells with detectable Ty1 signal assayed at different time points listed in (C). Each dot represents one image data, 3 wells per time point, 10 images per well, and totally 2785 cells examined. N = 3 mice. Data are present as the mean ± s.d.; adjusted P values are shown. Unpaired two-tailed Student’s t-test were applied, D0 sample as the refence group. F, Genomic snapshots of Scx CUT&RUN signals across a 10 Kb region of each of the 4 select genes in Figure 6G. The green lines represent CUT&RUN signal from anti-Ty1 on ScxGFP myoblasts; the magenta and blue lines represent the IgG control and anti-Ty1 antibody CUT&RUN signals detected in ScxTy1/Ty1 primary myoblasts, respectively. Nuclei were stained with DAPI; Scale bar = 20 μm. Dell’Orso, S., A. H. Juan, K. D. Ko, F. Naz, J. Perovanovic, G. Gutierrez-Cruz, X. Feng and V. Sartorelli (2019). “Single cell analysis of adult mouse skeletal muscle stem cells in homeostatic and regenerative conditions.” Development 146(12). Li, L., M. Rozo, S. Yue, X. Zheng, J. T. F, C. Lepper and C. M. Fan (2019). “Muscle stem cell renewal suppressed by Gas1 can be reversed by GDNF in mice.” Nat Metab 1(10): 985-995. Madaro, L., A. Torcinaro, M. De Bardi, F. F. Contino, M. Pelizzola, G. R. Diaferia, G. Imeneo, M. Bouche, P. L. Puri and F. De Santa (2019). “Macrophages fine tune satellite cell fate in dystrophic skeletal muscle of mdx mice.” PLoS Genet 15(10): e1008408.