Distinct response profiles of FAPs upon nerve crush injury versus denervation

(A) Single-cell transcriptome data of nerve injury-affected FAPs displayed separately by samples on UMAP plots. (B–E) Volcano plots showing different numbers of DEGs identified from comparing SNC-vs DEN-affected FAPs at (B) 3, (C) 7, (D) 14, (E) 28 days post injury (dpi). (F–H) Pathway terms enriched from gene set overrepresentation analyses using g:Profiler. DEGs used as input were (F) DEN-28dpi-upregulated versus SNC-28dpi, (G) SNC-28dpi-upregulated versus DEN-28dpi, and (H) DEGs upregulated commonly in SNC-3dpi, SNC-7dpi, DEN-3dpi, and DEN-7dpi versus uninjured control.

Nerve injury-responsive subsets within FAPs

(A) Seven clusters identified by unsupervised clustering using all nine scRNA-seq samples obtained in this study displayed on the UMAP plot. (B) Violin plots showing expressions of unique marker genes identified in each cluster. (C) Dotplot showing the expression levels and percentages of top 10 DEGs enriched in each cluster. (D) UMAP plots of clustered scRNA-seq data displayed separately by samples. (E) Barplots showing the proportions of the seven clusters that comprise each scRNA-seq sample of nerve injury-affected FAPs. For 0 dpi, data from the same uninjured control sample is displayed for both SNC and DEN.

GDNF signaling pathway in the nerve injury-sensing mechanism by FAPs

(A–B) UMAP plots showing the expressions of (A) Ret and (B) Gfra1 in the merged scRNA-seq data. (C–D) RT-qPCR results showing the expressions of (C) Ret and (D) Gfra1 in mononuclear cells isolated from uninjured muscles by FACS. MuSC, muscle stem cells; Lin+, lineage-positive cells; DN, Vcam1/Sca1 double-negative cells. n = 4; one-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant. (E) Shared pathway terms commonly identified from gene set overrepresentation analyses using DEGs specifically upregulated in clusters 1, 2 or 3. (F) Venn diagram showing the results from TRRUST analyses using DEGs enriched in clusters 2 and 3. Transcription factors predicted to regulate genes upregulated in each cluster are listed. (G) Simplified diagram of the GDNF signaling pathway. Blue: GDNF ligand; orange: GDNF receptor Ret expressed in cluster 1; pink: downstream cascade genes expressed in clusters 1-3; red: transcription factors commonly predicted to regulate upregulated genes in clusters 2 and 3.

Bdnf expression in FAPs by both endogenous and exogenous GDNF

(A) Scheme for identification of candidate genes expressed in FAPs in response to GDNF that may contribute to nerve regeneration. Number of genes that fit into each criterion is indicated. (B) Violin plot displaying the expression levels of Bdnf in the seven clusters. (C) Expression of Bdnf in each scRNA-seq sample shown on UMAP plots. (D) Experimental scheme for sampling Schwann cells and FAPs at different time points post SNC for gene expression analyses. (E) RT-qPCR results showing the expression levels of Gdnf from Schwann cells (orange dot and line, left y-axis) and Bdnf from FAPs (blue dot and line, right y-axis) at different time points post SNC displayed on the same plot. n = 4, except for 0 and 2 dpi, where n = 3. One-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, *** p < 0.001, n.s., not significant. (F) Experimental scheme for intramuscular injection of either PBS or recombinant mouse GDNF protein, with the time point for FAP isolation post injection indicated. (G) RT-qPCR results showing the expression level of Bdnf in FAPs 48 hours post intramuscular injection of either PBS (n = 4) or GDNF (n = 5). Unpaired t-test with Welch’s correction. ** p < 0.01.

Remyelination by FAP-derived BDNF during peripheral nerve regeneration

(A) Experimental scheme displaying mice used and the time points selected for EMG measurements and sciatic nerve dissection. wpi, weeks post injury. (B) Representative EMG measurement results of both injured and uninjured GA muscles from Ctrl or cKO mice at the indicated time points post SNC. (C–D) Quantified results of EMG measurement showing (C) CMAP amplitude and (D) CMAP latency. n = 5. One-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant. (E) Representative images showing toluidine blue-stained, semi-thin cross-sections of sciatic nerves dissected from Ctrl or cKO mice at 6 wpi. Scale bars, 10 μm. (F–G) Quantification of (F) calculated G-ratio values and (G) axon diameters from analyzing toluidine blue-stained sciatic nerve sections dissected from Ctrl or cKO mice at 6 wpi. 50 axons were randomly selected from each sciatic nerve for quantification. n = 5. Mann-Whitney U test. *** p < 0.001, n.s., not significant.

Implication of FAP-derived BDNF in the age-related delay in nerve regeneration

(A) Experimental scheme indicating ages of mice used and the time point for FAP isolation to compare the expression level of Bdnf post SNC. (B) RT-qPCR results showing the expression level of Bdnf in FAPs isolated from either adult (5-6 months) or aged (24 months) mice at 7 dpi. n = 5. Unpaired t-test. ** p < 0.01. (C) Graphical summary of this study.

Establishment and validation of nerve injury-affected FAPs’ scRNA-seq data

(A) Experimental scheme depicting the procedures for sample collection and scRNA-seq. The types of nerve injuries and time points for FAP isolation for each sample are specified. (B) Gating strategies used for FACS isolation of FAPs. (C) Integration of scRNA-seq data obtained in this study and data from Nicoletti et al. (2023) visualized on UMAP plots. Left: integrated data labeled with cell types; middle: cells from Nicoletti et al. (2023) only; right: data from this study overlaid on top of data from Nicoletti et al. (2023). (D) Expressions of marker genes that distinguish cell types within skeletal muscle in the integrated scRNA-seq data visualized on UMAP plots.

DEG analyses reveal similarities and differences between FAPs affected by SNC or DEN at different time points

(A) Number of DEGs identified from pairwise comparisons of all nine scRNA-seq samples visualized as a heatmap. (B) DEGs identified from comparing nerve injury-affected FAPs versus uninjured control shown on volcano plots. (C) Hierarchical clustering of the nine scRNA-seq samples using DEGs identified in (A) displayed as a heatmap. (D and F) UMAP plots showing the expressions of (D) Il6 and (F) Stat3. (E and G) Violin plots showing the expressions of (E) Il6 and (G) Stat3, with p values calculated from comparing each sample to uninjured control. Wilcoxon rank sum test.

Gene set overrepresentation analyses using DEGs from pairwise comparisons of the nine scRNA-seq samples

(A–B) Results from g:Profiler showing pathways enriched using DEGs upregulated in (A) DEN-28dpi versus SNC-28dpi and (B) vice versa. (C) Venn diagram showing the number of overlapping genes identified as DEGs by comparing each indicated sample to uninjured control. (D) Results from g:Profiler showing pathways enriched using DEGs shared in all four samples compared to uninjured control, as shown in (C).

Identification of FAP clusters that respond to nerve injury

(A) UMAP plots showing expressions of cluster-specific marker genes identified in this study. (B) Results from RNA velocity analysis visualized on UMAP plots. Arrows indicate the predicted direction of cellular movement in the near future on the UMAP plots. (C) Hierarchical clustering of the seven FAP clusters displayed with a heatmap. (D) Violin plots showing the expressions of marker genes previously reported by Leinroth et al. (2022) in the seven clusters identified in this study. (E–F) Expressions of (E) Hsd11b1 and (F) Mme shown on UMAP plots.

Involvement of GDNF signaling pathway in the nerve injury-sensing mechanism by cluster 1 FAPs

(A) Top 10 genes specifically enriched in cluster 1 FAPs. P values were drawn from Wilcoxon rank sum test. (B) Results from g:Profiler showing pathways enriched using genes specifically enriched in cluster 1. (C) MAPK signaling pathway retrieved from KEGG, with color-coding to highlight relevant genes. Blue: GDNF ligand; orange: GDNF receptor Ret expressed in cluster 1; pink: downstream cascade genes expressed in clusters 1-3; red: transcription factors (TFs) commonly predicted to regulate upregulated genes in clusters 2 and 3; green: TFs predicted to regulate genes upregulated in cluster 2; gold: TFs predicted to regulate genes upregulated in cluster 3.

Pathways enriched in the two activated clusters within nerve injury-affected FAPs

(A–B) Results from g:Profiler showing pathways enriched using genes specifically enriched in (A) cluster 2 or (B) cluster 3.

Validation of cKO mice used in this study and methods used for analysis

(A) Genomic loci and structure of Prrx1Cre and Bdnffl alleles labeled with primers used for genotyping and genomic DNA recombination validation. (B) Results from genotyping (left and middle) and genomic DNA recombination PCR (right). Genomic DNA recombination of the LoxP flanking sites in the cKO mice were confirmed using primers P4 and P6 shown in (A). (C) RT-qPCR results of Bdnf expression using FAPs isolated from either Ctrl or cKO mice at 3 or 7 days post SNC. n = 2; Two-way ANOVA. ** p < 0.01. (D) Scheme for CMAP measurement showing the positions of electrodes used. (E) Diagram of a typical CMAP graph along with amplitude and latency used for analysis. (F) Diagram depicting calculation of G-ratio. (G) Scatter plots with linear regressions displaying G-ratios (y-axis) in relation to axon diameters (x-axis). Solid lines are linear regressions and dotted lines represent error in 95% confidence level. ANCOVA, *** p < 0.001.

Expression of GDNF receptor genes Ret and Gfra1 in nerve-resident cells

(A–C) scRNA-seq data from Carr et al. (2019) and Toma et al. (2020) (accession numbers: GSM3408137, GSM3408139, GSM4423509, GSM4423506) were merged into a single Seurat object and visualized on UMAP plots. Cell types identified using markers listed by Toma et al. (2020) are shown in (A), and expression levels of (B) Ret and (C) Gfra1 are displayed. epi.MES: epineurial mesenchymal cells; peri.MES: perineurial mesenchymal cells; endo.MES: endoneurial mesenchymal cells; diff.MES: differentiating mesenchymal cells; prol.MES: proliferating mesenchymal cells; NM.SC: non-myelinating Schwann cells; MY.SC: myelinating Schwann cells; Mac/Mo: macrophage/monocyte. (D–F) scRNA-seq data from Zhao et al. (2022) (accession number: GSE198582) were merged into a single Seurat object and visualized on UMAP plots. Cell types annotated by Zhao et al. (2022) are shown in (D), and expression levels of (E) Ret and (F) Gfra1 are displayed. cDC: conventional dendritic cells; DCx: dendritic cells destined for homing; dMES: differentiating mesenchymal cells; EC: endothelial cells; eMES: endoneurial mesenchymal cells; Fb: fibroblasts; GC: granulocytes; Mac: macrophages; Mast: mast cells; Mo: monocytes; MoDC: monocyte-derived dendritic cells; NK: natural killer cells; pDC: plasmocytoid dendritic cells; pMES: perineurial mesenchymal cells; prol.MES: proliferating mesenchymal cells; SC: Schwann cells; TC: T cells; vSMC_PC: vascular smooth muscle cells/pericytes; NA: not applicable.

Expression of nerve injury-induced, cluster-specific genes in FAPs

(A–B) Expressions of (A) Rspo1 and (B) Csmd1 shown on UMAP plots, separated by samples. scRNA-seq data obtained in this study were used.

Expression of Bdnf in muscle-resident mononuclear cells affected by denervation

(A) UMAP plot showing data from Nicoletti et al. (2023) labeled by cell types identified. (B) Expression pattern of Bdnf in data from Nicoletti et al. (2023) shown on UMAP plots, separately by days post denervation. (C) Expression of Bdnf in each cell type on different days post denervation displayed in violin plots. P values were calculated by comparing each injury-affected cells’ expression levels versus its uninjured state (0 dpi). Only significant p values are shown. Wilcoxon rank sum test.

Results from TRRUST showing transcription factors predicted to regulate genes specifically enriched in cluster 2

Genes known to be regulated by each transcription factor is listed.

Results from TRRUST showing transcription factors predicted to regulate genes specifically enriched in cluster 3

Genes known to be regulated by each transcription factor is listed.

Categorization of genes predicted to be regulated by transcription factors that act downstream of the GDNF signaling pathway

Note that only Bdnf fits into all three criteria.

Primers used in this study for genotyping PCR or RT-qPCR