Muscle denervation promotes functional interactions between glial and mesenchymal cells through NGFR and NGF

Figures

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  Figure 1. scRNA-seq-mediated identification of denervation-responsive muscle-resident cell types (A) Experimental design. Single-cell suspensions were generated from whole TA and GA muscles from non-denervated mice and from three time points following denervation through sciatic nerve transection (namely, 0-, 2-, 5-, or 15-day post-denervation, abbreviated as CTR (Non-DEN), DEN2d, DEN5d, and DEN15d in figures and tables). (B and C) UMAP embedding of all the single cells comprising the dataset colored either by experimental condition (B) or by meta-cluster (C). (D) Violin plots of the expression profile of marker genes for each meta-cluster, color legend as in 1C. (E–G) UMAP embedding of the integration analysis of denervation and injury (Oprescu et al., 2020)10 datasets (E), with Oprescu’s meta-cluster identities (F), or with the meta-cluster identities from our dataset (G)—color legend as in 1C.

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  Figure 2. Dynamic changes in cell number and gene expression in muscle-resident cells following denervation (A) Barplot representing the proportion of single cells belonging to each meta-cluster for each experimental condition. (B) Venn diagram of upregulated genes (upper) and downregulated genes (bottom) in all single cells at DEN2d, DEN5d, and DEN15d compared with CTR. DE genes with p-value-adj ≤ 0.01 are counted. (C) Heatmap of top 20 upregulated genes in all cells at DEN2d, DEN5d, and DEN15d respectively compared with CTR, clustered by condition and meta-cluster identity. Gene expression values visualized on a scale from low (purple) to high (yellow), with black for zero expression value. (D) IPA comparison of DE genes in all cells at DEN2d, DEN5d, and DEN15d respectively compared with CTR. Pathways significantly enriched in either time point with absolute Z score > 1 were shown. Pathways with p value ≥ 0.05 were indicated with gray dots. Pathways activation status (Z-scores) visualized on a scale from repressed (blue) to activated (orange), with neutral status (Z-Score = 0) set to white. (E) Violin plots in DEN5d cells of genes involved in hepatic fibrosis signaling pathways.

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  Figure 3. Response of muscle-resident glial cells to denervation (A) Venn diagram of upregulated genes (left) and downregulated genes (right) in glial cells at DEN2d, DEN5d, and DEN15d compared with CTR. Genes with p-value-adj ≤ 0.01 are counted. (B) Heatmap of top 20 upregulated genes in glial cells at DEN2d, DEN5d, and DEN15d respectively compared with CTR. Gene expression values visualized on a scale from low (purple) to high (yellow), with black for zero expression value. (C) IPA comparison of DE genes in glial cells at DEN2d, DEN5d, and DEN15d respectively compared with CTR. Selected pathways related to neurogenic processes are shown. Pathways with p value ≥ 0.05 were indicated with gray dots. Pathways activation status (Z-scores) visualized on a scale from repressed (blue) to activated (orange), with neutral status (Z-Score = 0) set to white. (D) UMAP embedding subset from the whole dataset of glial cells colored by time point. (E) UMAP embedding of glial cells after re-clustering, with superimposition of pseudo-time trajectories from Monocle3. Glial cells are colored by time point. (F) Same as (E), but glial cells are colored by pseudo-time. Pseudotime visualized on a scale from…

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  Figure 4. Response of muscle-resident mesenchymal cells to denervation (A) Venn diagram of upregulated genes (left) and downregulated genes (right) in activated fibroblasts at DEN2d, DEN5d, and DEN15d compared with CTR. Genes with p-value-adj ≤ 0.01 are counted. (B) Heatmap of top 20 upregulated genes in activated fibroblasts at DEN2d, DEN5d, and DEN15d respectively compared with CTR. Gene expression values visualized on a scale from low (purple) to high (yellow), with black for zero expression value. (C) IPA comparison of DE genes in activated fibroblasts at DEN2d, DEN5d, and DEN15d respectively compared with CTR. Pathways significantly enriched in either time point with absolute Z score > 1 were shown. Pathways with p value ≥ 0.05 were indicated with gray dots. Pathways activation status (Z-scores) visualized on a scale from repressed (blue) to activated (orange), with neutral status (Z-Score = 0) set to white. (D) UMAP embedding subset from the whole dataset of activated fibroblasts colored by time point. (E) UMAP embedding of activated fibroblasts after re-clustering, with superimposition of pseudo-time trajectories from Monocle3. Activated fibroblasts are colored by time point. (F)…

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  Figure 5. Chromatin accessibility at single nucleus level of muscle-resident cells upon denervation (A) UMAP embedding of snATAC-seq data after integration, colored according to our scRNA-seq data. (B) Table illustrating the number of nuclei assigned to each muscle-resident cell population and the number of differential accessibility regions (DARs) between DEN5d and CTR. (C) Histogram of the number of DARs overlapping promoters (red, UP) or non-promoter (enhancer, in blue, DOWN) regions, per cell type. (D–F) UMAP embedding of snATAC-seq data, showing the predicted gene expression profile for Thy1 (CD90) (D), Ngf (E) ,and Ngfr (F), either dim (blue) or bright (red).

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  Figure 6. Ligand-receptor interaction analysis (A) Heatmap plot from CellPhoneDB showing the total number of interactions (log count) between cell types in our scRNA-seq data. Interaction log count visualized on a scale from low (blue) to high (dark red). (B) Venn diagram of the intersection between molecular pathways downstream to receptors on glial cells predicted to interact with ligands expressed by activated fibroblasts (Predicted), and molecular pathways predicted to be activated in glial cells based on gene expression (Activated). (C) Heatmap containing the molecular pathways connected to a specific receptor-ligand pair (only downstream of NGF-NGFR; complete information can be found in Figure S9) for the interactions between glial cells and activated fibroblasts (Interactome). On the right each pathway activation status calculated by Ingenuity Pathway Analysis (IPA; red for activation, blue for repression, and gray or white for activation status info missing) at DEN2d, DEN5d, and DEN15d, respectively compared to the control. EMT: epithelial-mesenchymal transition; NO: nitric oxide; ROS: reactive oxygen species. Pathways activation status (Z-scores) visualized on a scale from…

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  Figure 7. Recombinant NGF promotes expansion of NGFR-positive glail cells ex vivo (A) FACS plot showing the sorting strategy for CD90posSCA1pos activated fibroblasts (abbreviated as CD90pos cells), CD59AposNGFRneg cells (abbreviated as CD59Apos cells), and CD59AposNGFRpos Glial cells (abbreviated as NGFRpos cells) simultaneously from control (upper, CTR) and DEN5d (bottom) muscles. (B) Percentage of 3 populations (gated in A) by FACS at CTR and DEN5d conditions. N = 8 mice. (C) Sorted NGFRpos glial cells were treated with 0/5/10/20 ng/mL beta-NGF for 2 days in culture. Hoechst and propidium iodide (PI) were added into the medium to help detect total cell number and dead cell number, respectively. Images were captured at day 0 and day 2. Quantified live cell number at day 2 was normalized to that at day 0. N = 5 mice. In B and C, data are represented as mean ± SD. Statistical significance was analyzed by two-way ANOVA in (B) and one-way ANOVA in (C) with p values shown as ∗p < 0.05 and ns for not significant.

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  Figure 8. Co-culture with NGF-expressing mesenchymal cells promotes expansion of NGFR-positive glial cells ex vivo (A) Graphic illustration of co-culture experiment with NGFRpos cells and CTR- or DEN5d- CD90pos cells (abbreviated as CTR group or DEN5d group, respectively). Cells were fixed after 3 days culture for EdU assay (EdU was added for 4 h pulsing before fixation) and TUNEL assay, respectively. (B) Representative cell staining images of SOX10 (green) and EdU (magenta), counterstained with Hoechst (blue). Arrow heads indicate SOX10posEdUpos Glial cells. Scale bar: 100μm. (C) Representative cell staining images of SOX10 (green) and TUNEL (red), counterstained with Hoechst (blue). Arrows indicate SOX10posTUNELpos Glial cells. Scale bar: 100μm. (D) SOX10pos Glial cell number per field was quantified in two co-culture groups. By normalization to the CTR group, relative Glial cell number was shown. N = 6 mice. (E) EdUpos percentage of SOX10pos Glial cells in two co-culture groups. N = 6 mice. (F) TUNELpos percentage of SOX10pos Glial cells in two co-culture groups. N = 3 mice. Statistical significance was analyzed by paired Student’s t test in (D-F) with p values shown as ∗∗p < 0.01 and…