Mouse skeletal muscle satellite cells co-opt the tenogenic gene Scleraxis to instruct regeneration
- Department of Embryology, Carnegie Institution for Science, United States
- College of Life Sciences, Zhejiang University, China
Figures
Figure 1 with 1 supplement
Adult regenerative myogenic cells express the transgene ScxGFP.
(A) Experimental scheme for data in (B–D). Tg-ScxGFP (ScxGFP) mice were injured by cardiotoxin (CTX) to the tibialis anterior (TA) muscle, followed by daily 5-ethynyl-2′-deoxyuridine (EdU) administration for 5 days (5d), and their TA muscles were harvested for analysis at 5 days 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, GFP/MYH (N=4 mice). Asterisks indicate cells double-positive for ScxGFP and each respective myogenic marker. All myofibers are GFP and myosin heavy chain (MHC) double positive, thus without additional labeling. (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 satellite cells (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 2 days (D2(GM); data in (G)), or after cultured for 4 days in GM followed by 2 days in differentiation media (DM) (D6(DM); data in H). F-G. D0 (F) and D2 cultured (G) SCs obtained in (E) were stained 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.
Figure 1—figure supplement 1
ScxGFP transgene is expressed in tendon, regenerative muscle, and cultured myoblast.
(A) Tibialis anterior (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 connective tissue (CT). (B–D) Split channel images of data in Figure 1C; asterisks indicate the same cells. (E, F) Fluorescent activated cell sorting (FACS) strategy (E) and profiles (F) to support Figure 1E. (F) FACS plot and population hierarchy of SC from four surface markers sorting in pseudocolor plots. (G) Freshly isolated satellite cells (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). Scale bar = 20 µm. Data are presented with mean ± s.d.
Figure 2 with 1 supplement
Endogenous Scx is expressed by activated but not quiescent satellite cells (SCs).
(A) Experimental design for ScxCreERT2-mediated inducible lineage tracing with the RtdT reporter. The two experimental groups are: (1) Tamoxifen (TMX) administered before injury for 5 days (TMX before injury) and (2) TMX administered after injury for 5 days (TMX after injury). Tibialis anterior (TA) muscles in both groups were harvested at 14 days 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) Re-analyses for Scx expression in two published scRNA-seq data sets of activated myogenic cells at 2 dpi and 2.5 dpi (De Micheli et al., 2020; Dell’Orso et al., 2019), displayed by UMAP; colored keys to expression levels are included correspondingly. Nuclei were stained with DAPI; Scale bar = 20 µm. Data are presented with mean ± s.d.; p-values are indicated. (C, D) Unpaired two-tailed Student’s t-test were applied.
Figure 2—figure supplement 1
Pax7+ and Scx expression in published data set.
(A) Averaged Pax7+ satellite cell (SC) number per field (0.06 mm2) from data in B. Mouse and cell numbers are the same as in Figure 2C. (B) 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 et al., 2019; Madaro et al., 2019). (C) Uniform manifold approximation and projection (UMAP) plot of re-analyzed scRNA-seq for Scx expression in SCs isolated from uninjured muscles (freshly sorted) using published data in Dell’Orso et al., 2019. Data are presented with the mean ± s.d.; the p-value is indicated. An unpaired two-tailed Student’s t-test was applied.
Figure 3 with 1 supplement
Efficient muscle regeneration requires Scx function.
(A) Experimental designs to compare phenotypes of control (Ctrl) and Scx cKO mice. The RtdT reporter was included (tdT lineage; see Figure 3—figure supplement 1A for genotypes). Tamoxifen (TMX) was administered before and after the cardiotoxin (CTX)-induced injury to maximize gene inactivation. Tibialis anterior (TA) muscles were harvested at 5 days or 14 days after injury. (B, C) (B) Ctrl and Scx cKO TA muscles at 5 days 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 Scx cKO TA muscles at 14 days 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+ SC number per imaged field (0.08 mm2) of TA muscle sections from 5 dpi Ctrl and Scx cKO mice (N=5 mice per group; n=2,807 Ctrl and n=442 Scx cKO Pax7+ SCs). (G) After 5-ethynyl-2′-deoxyuridine (EdU) administration, Ctrl and Scx cKO TA muscles at 5 days post-injury were sectioned and stained for Pax7, followed by EdU reaction. Arrows indicate Pax7+EdU+ cells, whereas asterisks indicate Pax7+EdU- cells. Nuclei were stained with DAPI. (H) Percentages of EdU+ cells within the Pax7+ cell population of Ctrl and Scx cKO, from data in G (N=5 mice per group; n=858 Ctrl and n=325 Scx cKO Pax7+ SCs). (I) Averaged Pax7+ SC number per image field (0.4 mm2) in Ctrl and Scx cKO TA muscle sections from 14d post-injury samples (N=5 mice per group; n=350 Ctrl and n=186 Scx cKO 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 was applied, Scale bars = 20 μm in (B, D, G).
Figure 3—figure supplement 1
Experimental design for recombination efficiency and staining of 5 dpi and 14 dpi muscle sections in control and Scx cKO group.
(A) Detailed description for the genotypes used as Ctrl and Scx cKO mice in Figure 3. For the tdT lineage, RtdT reporter was included. For the YFP lineage, RYFP reporter was included. (B) Depiction of Scx gene inactivation by tamoxifen (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 Supplementary file 1j. (C) Experimental scheme to determine the recombination efficiency using FACS-isolated Ctrl and Scx cKO satellite cells (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 Scx cKO myoblasts determined by qPCR, followed by 2-ΔΔCt analysis. (E) Experimental scheme as Figure 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 myosin heavy chain (MHC) and laminin in (G). Arrows indicate Pax7+ SCs in (F), whereas arrows indicate Laminin+MHC- ghost fibers (G) (N=5 mice per group; n=1748 control and n=442 Scx cKO Pax7+ SCs). (H) Experimental scheme as Figure 3A for 14 dpi data in (I). (I) Tibialis anterior (TA) muscle section from (H) were sectioned and stained with Pax and Laminin (N=5 mice per group; n=350 control and n=186 Scx cKO 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 Scx cKO 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.
Figure 4 with 1 supplement
Scx cKO satellite cells (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 images 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 5-ethynyl-2′-deoxyuridine (EdU) was added for 6 hr 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, three wells per group, eight 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, G) (F) FACS-isolated Ctrl and Scx cKO SCs were cultured in growth medium for 4 days, harvested, and immuno-stained for Pax7 and cleaved Caspase 3; actin cytoskeleton (to identify cell body) was stained by Phalloidin. (G) Box plot of percentages of cell death (i.e. cleaved Caspase 3+ cells) from data in (F); N=2 mice; each dot represents one image data; two wells per group, >10 images per well; 240 Ctrl and 353 Scx cKO cells examined. 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.
Figure 4—figure supplement 1
Experimental design for live imaging, live-imaging results, and programmed cell death analyses in control and Scx cKO mice.
(A, B) FACS profiles for isolating tdT (A) and YFP (B) linage marked satellite cells (SCs). (C) Similar experimental scheme as in Figure 4A for reference to live imaging data in (D–F). (D) Line plot of relative cell number per frame; normalized to cell number of the Ctrl group at the beginning as 1. (E) 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, three wells per group. (F) Box plot of cell migration speed based on tracking cell displacement by pixel (0.33 µm/pixel), each dot represents a cell data. (G) Still frames from time lapse show examples of necrotic cells at D3 of the Scx cKO group. Frames 1–6 are sequential time-lapse images (covering 500 min). Two cells of focus are indicated by red and black arrowheads to track their appearance from a healthy state (earlier frames) to a necrotic state (later frames). (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. (I) For positive control, 5 dpi tibialis anterior (TA) muscle from a Pax7 CE/+;ScxTy1/Ty1;RYFP/YFP mouse was treated with 200 μgml–1 DNase I for 10 min, followed by TUNEL assay and YFP staining (top panel; four sections examined and five images taken). Lower two panels are Ctrl and Scx cKO 3 dpi TA muscle sections subjected to TUNEL assay and YFP staining (N=4 mice per group, nine sections per slides per group, and 3–7 images per section analyzed). No appreciable number of TUNEL+YFP+ cells were found in Ctrl and Scx cKO; hence, quantification omitted. (J) FACS-isolated Ctrl and Scx cKO SCs were cultured in growth medium for 4 days. For positive control, 1 µM of staurosporine was added for 6 hr prior to harvesting. Cells were then stained for cleaved Caspase-3 and Pax7; Phalloidin was used to identify cells by actin cytoskeleton. Nuclei were stained with DAPI; Scale bar = 20 µm. Data are presented with mean ± s.d.; adjusted p-values are shown. Unpaired two-tailed Student’s t-tests were applied to data collected on each day.
Figure 5 with 2 supplements
Single-cell RNA-sequencing (scRNA-seq) helps identify the role of Scx in myogenic differentiation and fusion.
(A) Satellite cell (SC) scRNA-seq scheme for YFP lineage-marked SCs. YFP+ cells were FACS-isolated from 2.5 dpi BaCl2 injured tibialis anterior (TA) and the gastrocnemius (GA) muscles. (B) Trajectory analysis of the seven myogenic clusters (complete cell cluster analysis in Figure 5—figure supplement 1) indicated to the right. Arrowindicates the direction of pseudotime trajectory. (C) Cell densities of Ctrl and Scx cKO cells along the trajectory in (B). Cell in Peaks 2–4 were used for the differentially expressed gene (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 hr (D0), switched into differentiation media (DM), and harvested daily for analysis over 3 days (D1-D3). (E) Myoblasts subjected to the scheme in (D) were stained for MyoG (for differentiation index in F) and for myosin heavy chain (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 one image data. Unpaired two-tailed Student’s t-tests were applied and adjusted p-values are shown. (N=3 mice; three wells per group per time point; 10 images per well; in total, 3342 control, and 2561 Scx cKO cells examined). (H) Volcano plot of relative gene expression (Log2 fold change) in Ctrl versus ScxKO cells in Peak 2 (in C). (I) Gene Ontology (GO) term enrichment of muscle development-related processes from DGEs in (H).
Figure 5—figure supplement 1
Assembly and curation of single-cell RNA-sequencing (scRNA-seq) atlas of muscle stem cell.
(A) Detailed scRNA-seq procedures diagram as in Figure 5A for reference to analyses in (B–G). (B) Uniform manifold approximation and projection (UMAP) plot of combined scRNA-seq of Ctrl and Scx cKO cells. A total of 11,388 control cells and 12,844 Scx cKO 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 and I. (C) Cell density distribution of each cell cluster in Ctrl and Scx cKO samples. NS, not significant; *p<0.05; **p<0.01; ***p<0.001. (D) 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). (E) Pax7 expression levels and cell numbers in each cluster; each dot represents one cell. (F) Scx expression levels and cell numbers in each cluster.
Figure 5—figure supplement 2
Relative expression levels of four cell cycle genes.
(A) Relative expression levels (CPM, counts per million UMI) of four select cell cycle genes along the pseudotime depicted in Figure 5C.
Figure 6 with 1 supplement
CUT&RUN and single-cell RNA-sequencing (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 hr (D0) and switched to differentiation media (DM) for 12 hr 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 (3956) in Peak 2 of Figure 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). Gene Ontology (GO) terms with p<0.0001 were plotted. (G) Genomic snapshots of Scx CUT&RUN peaks on four select genes related to muscle differentiation. (H) Expression levels (CPM, counts per million UMI) of the four select genes in (G) along the pseudotime trajectory (same trajectory as Figure 5C).
Figure 6—figure supplement 1
Ty1 expression in tendon and myoblasts, genomic snapshots of four select genes related to muscle differentiation.
(A) Diagram of the genomic structure of ScxTy1 allele. Three (3 X) 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, Nuclei were stained with DAPI; Scale bar = 20 µm. (C) In vitro myogenic differentiation scheme using ScxTy1/Ty1 SC-derived myoblasts. Myoblasts were cultured in GM for 12 hr (D0) and switched to differentiation media (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, three wells per time point, 10 images per well, and a total of 2785 cells examined. N=3 mice. Data are presented as the mean ± s.d.; adjusted p-values are shown. Unpaired two-tailed Student’s t-tests were applied, D0 sample as the reference group. (F) Genomic snapshots of Scx CUT&RUN signals across a 10 Kb region of each of the four 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.
Additional files
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Supplementary file 1
Supplementary tables.
(a) Different gene expression (DEGs) between control and Scx cKO group in peak 2. 3956 genes differently expressed between control and Scx cKO cells in peak 2 along the trajectory in Figure 5C. (b) Muscle development-related GO term enrichment from DGEs in peak 2. 16 GO term enrichments of muscle development-related processes from DGEs in Ctrl versus ScxKO cells in Peak 2 along the trajectory in Figure 5C. (c) Distribution of Scx CUT&RUN peaks. A total of 1003 binding peaks were identified in 861 gene loci, with 33.4%, 38.88%, and 22.44% located in intergenic regions, introns, and promoters, respectively, alongside other genomic regions, respectively. (d) Common Gene list between Scx CUT&RUN target genes and DEGs in Peak 2. 207 intersecting genes between Scx CUT&RUN target genes (861) and DEGs in Peak 2. (e) GO term enrichment of the 207 common genes. 67 GO term enrichment analysis of the 207 common genes. (f) Different gene expression (DEGs) between control and Scx cKO group in peak 3. 1942 genes were differently expressed between control and Scx cKO cells in peak 3 along the trajectory in Figure 5C. (g) DEGs between control and Scx cKO group in peak 3. 755 genes were differently expressed between control and Scx cKO cells in peak 4 along the trajectory in Figure 5C. (h) GO term enrichment from DGEs in peak 3. Go term analysis of the DGEs in peak 3, muscle-related processes were marked in red. (i) GO term enrichment from DGEs in peak 4. Go term analysis of the DGEs in peak 4, muscle-related processes were marked in red. (j) Primer pairs used in this study. PCR primer sets P1, P2, and P3 were used to detect exon 1 (E1), intron 1 (I1) and E2 of the Scx gene, respectively, PCR primer set used for CUT&RUN library preparation. (k) Antibodies information. Sources and dilution for primary and secondary antibodies are provided.
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Mouse skeletal muscle satellite cells co-opt the tenogenic gene Scleraxis to instruct regeneration
eLife 13:RP95854.
https://doi.org/10.7554/eLife.95854.3
