The injured sciatic nerve atlas (iSNAT), insights into the cellular and molecular basis of neural tissue degeneration and regeneration

  1. Xiao-Feng Zhao
  2. Lucas D Huffman
  3. Hannah Hafner
  4. Mitre Athaiya
  5. Matthew C Finneran
  6. Ashley L Kalinski
  7. Rafi Kohen
  8. Corey Flynn
  9. Ryan Passino
  10. Craig N Johnson
  11. David Kohrman
  12. Riki Kawaguchi
  13. Lynda JS Yang
  14. Jeffery L Twiss
  15. Daniel H Geschwind
  16. Gabriel Corfas
  17. Roman J Giger  Is a corresponding author
  1. Department of Cell and Developmental Biology, University of Michigan-Ann Arbor, United States
  2. Neuroscience Graduate Program, University of Michigan–Ann Arbor, United States
  3. Kresge Hearing Institute, University of Michigan–Ann Arbor, United States
  4. Departments of Psychiatry and Neurology, University of California, Los Angeles, United States
  5. Department of Neurosurgery, University of Michigan-Ann Arbor, United States
  6. Department of Biological Sciences, University of South Carolina, United States
  7. Department of Neurology, Program in Neurogenetics, David Geffen School of Medicine, University of California, Los Angeles, United States
  8. Department of Human Genetics,David Geffen School of Medicine, University of California, Los Angeles, United States
  9. Institute of Precision Health, University of California, Los Angeles, United States
  10. Department of Neurology, University of Michigan–Ann Arbor, United States
10 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
The cellular and molecular landscape of naïve mouse sciatic nerve.

(A) Schematic of workflow for peripheral nervous tissue analysis. Cartoon of a mouse spinal cord, lumbar dorsal root ganglia (DRGs), and sciatic nerve trunk with main branches. The nerve segment marked with a red bracket was harvested for further analysis. Immune cells were captured with anti-CD45 conjugated magnetic beads. The flow through, containing non-immune (CD45-) cells, was collected as well. In separate scRNAseq runs, CD45+ and CD45- single-cell transcriptomes were determined. A total of 21,973 high-quality transcriptomes, including 4539 CD45+ cells and 17,434 CD45- cells were generated and used for downstream analysis. (B) UMAP embedding of naïve sciatic nerve cells. Unsupervised Seurat-based clustering identified 24 clusters. (C) List of cell types identified in the naïve sciatic nerve, grouped into immune cells (clusters 0–7), structural/stromal cells (clusters 8–16), cells associated with the nerve vasculature (clusters 17–22), and Schwann cells (clusters 23 and 24). (D–L) Feature plots of scRNAseq data showing expression of canonical markers used for assignment of major cell types, including epineurial fibroblasts (Pcolce2/procollagen C-endopeptidase enhancer 2), endoneurial MES (Cdkn2a/cyclin dependent kinase inhibitor 2A), perineurial MES (Cldn1/claudin-1), endothelial cells (Pecam1/CD31), vascular smooth muscle cells and pericytes (Notch3/notch receptor 3), Schwann cells (Sox10/SRY-box transcription factor 10), epineurial Mac (Ccl8/C-C motif chemokine ligand 8), endoneurial Mac (Cx3cr1/C-X3-C motif chemokine receptor 1), and dendritic cells (Napsa/napsin A, aspartic peptidase). Expression levels are color coded and calibrated to average gene expression.

Figure 1—figure supplement 1
Cell-type-enriched gene products in the naïve mouse sciatic nerve.

Heatmaps of top cluster-enriched gene products. (A) Nonimmune cells (CD45 negative) include Fb, fibroblasts; eMES, endoneurial mesenchymal cells; pMES, perineurial mesenchymal cells; prol.MES, proliferating mesenchymal cells; EC, endothelial cells; LyEC, lymphatic endothelial cells; PC, pericytes; nmSC, non-myelinating Schwann cells; mSC, myelinating Schwann cells. (B) Nerve resident immune cells (CD45 positive) include epiMac, epineurial macrophages; endoMac, endoneurial macrophages; Mac0, macrophages expressing high levels of fibronectin (Fn1); DC, dendritic cells; MAST, mast cells; NK, natural killer cells; TC, T cells; and UI, unidentified cells. Expression levels are calibrated to median gene expression.

Figure 1—figure supplement 1—source data 1

X-ray films of cytokine ELISA membranes probed with serum or sicatic nerve lysate of naive mice.

https://cdn.elifesciences.org/articles/80881/elife-80881-fig1-figsupp1-data1-v3.zip
Figure 1—figure supplement 2
Markers for nerve resident macrophages.

(A–F) Feature plots of naïve mouse sciatic nerve resident macrophages. (A) Cd163 (scavenger receptor cysteine-rich 1 protein M130) is expressed by epi-Mac and endo-Mac. (B) A small subpopulation of Mac (Mac0) expresses high levels of Fn1 (fibronectin). (C) Fcna (Ficolin A) is selectively expressed by epi-Mac. (D–F) Expression of the Mac markers Trem2 (triggering receptor expressed on myeloid cells 2), Lilra5 (leukocyte immunoglobulin like receptor A5), and Unc93b1 (Unc-93 homolog B1) a Toll-like receptor signaling regulator. Expression levels are projected onto UMAP with a minimum expression cutoff of 1. (G, G’) Longitudinal sections of naïve mouse sciatic nerve. In situ hybridization of Ccl8 transcripts with RNAscope revealed preferential staining of epi-Mac, labeled with arrows. (H, H’) Longitudinal sciatic nerve section of Cx3cr1-GFP reporter mice revealed preferential labeling of endo-Mac, labeled with arrows. Scale bar (G, H), 500 µm; (G’,H’), 75 µm.

Figure 1—figure supplement 3
Validation of naïve nerve scRNAseq dataset by ELISA.

(A) ELISA membranes probed with naïve sciatic nerve lysate, n = 2 biological replicates. (B) List of abundantly detected proteins. For quantification, ELISA signals were averaged and normalized to reference spots shown at coordinates (A1, A2), (A23, A24), and (J1, J2). (C) Dotplot analysis of the corresponding gene products, using scRNAseq datasets of naïve sciatic nerve. Relative expression levels, normalized to average gene expression (color coded) are shown. For each cell cluster, the percentile of cells expressing a specific gene product is indicated by the dot size. Gene products marked with a red asterisk are detected at the protein level but not at the transcriptional level, n = 1 biological replicate (D) ELISA membrane probed with serum from naïve mice. (E) List of top serum proteins detected by ELISA. For quantification, ELISA signals were normalized to reference spots shown at coordinates (A1, A2), (A23, A24), and (J1, J2). Coordinates of proteins detected by the ELISA: (A1, A2) Reference spots, (A3, A4) Adiponectin [Adipoq], (A5, A6) Amphiregulin [Areg], (A7, A8) Angiopoientin-1 [Angpt1], (A9, A10) Angiopoientin-2 [Angpt2], (A11, A12) Angiopoientin-like 3 [Angptl3], (A13, A14) BAFF [Tnfrsf13b], (A15, A16) C1qR1 [Cd93], (A17, A18) CCL2 [Ccl2], (A19, A20) CCL3 [Ccl3], (A21, A22) CCL5 [Ccl5], (A23, A24) Reference spots, (B3, B4) CCL6 [Ccl6], (B5, B6) CCL11 [Ccl11], (B7, B8) CCL12 [Ccl12], (B9, B10) CCL17 [Ccl17], (B11, B12) CCL19 [Ccl19], (B13, B14) CCL20 [Ccl20], (B15, B16) CCL21 [Ccl21], (B17, B18) CCL22 [Ccl22], (B19, B20) CD14 [Cd14], (B21, B22) CD40 [Cd40], (C3, C4) CD160 [Cd160], (C5, C6) Chemerin [Rarres2], (C7, C8) Chitinase 3-like 1 [Chil3], (C9, C10) Coagulation Factor III [F3], (C11, C12) Complement Component C5 [C5], (C13, C14) Complement Factor D [Cfd], (C15, C16) C-Reactive Protein [Crp], (C17, C18) CX3CL1 [Cx3cl1], (C19, C20) CXCL1 [Cxcl1], (C21, C22) CXCL2 [Cxcl2], (D1, D2) CXCL9 [Cxcl9], (D3, D4) CXCL10 [Cxcl10], (D5, D6) CXCL11 [Cxcl11], (D7, D8) CXCL13 [Cxcl13], (D9, D10) CXCL16 [Cxcl16], (D11, D12) Cystatin C [Cst3], (D13, D14) DKK-1 [Dkk1], (D15, D16) DPPIV [Dpp4], (D17, D18) EGF [Egf], (D19, D20) Endoglin [Eng], (D21, D22) Endostatin [Col18a1], (D23, D24) Fetuin A [Ahsg], (E1, E2) FGF acidic [Fgf1], (E3, E4) FGF-21 [Fgf21], (E5, E6) Flt-3 Ligand [Flt3l], (E7, E8) Gas 6 [Gas6], (E9, E10) G-CSF [Csf3], (E11, E12) GDF-15 [Gdf15], (E13, E14) GM-CSF [Csf2], (E15, E16) HGF [Hgf], (E17, E18) ICAM-1 [Icam1], (E19, E20) IFN-gamma [Ifng], (E21, E22) IGFBP-1 [Igfbp1], (E23, E24) IGFBP-2 [Igfbp2], (F1, F2) IGFBP-3 [Igfbp3], (F3, F4) IGFBP-5 [Igfbp5], (F5, F6) IGFBP-6 [Igfbp6], (F7, F8) IL-1alpha [Il1a], (F9, F10) IL-1Beta [Il1b], (F11, F12) IL-1ra [Il1rn], (F13, F14) IL-2 [Il2], (F15, F16) IL-3 [Il3], (F17, F18) IL-4 [Il4], (F19, F20) IL-5 [Il5], (F21, F22) IL-6 [Il6], (F23, F24) IL-7 [Il7], (G1, G2) IL-10 [Il10], (G3, G4) IL-11 [Il11], (G5, G6) IL-12 p40 [Il12], (G7, G8) IL-13 [Il13], (G9, G10) IL-15 [Il15], (G11, G12) IL-17A [Il17a], (G13, G14) IL-22 [Il22], (G15, G16) IL-23 [Il23], (G17, G18) IL-27 p28 [Il27], (G19, G20) IL-28 [Ifnl3], (G21, G22) IL-33 [Il33], (G23, G24) LDL R [Ldlr], (H1, H2) Leptin [Lep], (H3, H4) LIF [Lif], (H5, H6) Lipocalin-2 [Lcn2], (H7, H8) LIX [Cxcl5], (H9, H10) M-CSF [Csf1], (H11, H12) MMP-2 [Mmp2], (H13, H14) MMP-3 [Mmp3], (H15, H16) MMP-9 [Mmp9], (H17, H18) Myeloperoxidase [Mpo], (H19, H20) Osteopontin [Spp1], (H21, H22) Osteoprotegerin [Tnfrsf11b], (H23, H24) PD-ECGF [Tymp], (I1, I2) PDGF-BB [Pdgfb], (I3, I4) Pentraxin 2 [Nptx2], (I5, I6) Pentraxin 3 [Ptx3], (I7, I8) Periostin [Postn], (I9, I10) Pref-1 [Dlk1], (I11, I12) Proliferin [Prl2c2], (I13, I14) Proprotein Convertase 9 [Pcsk9], (I15, I16) RAGE [Ager], (I17, I18) RBP4 [Rbp4], (I19, I20) Reg3G [Reg3g], (I21, I22) Resistin [Retn], (J1, J2) Reference spots, (J3, J4) E-Selectin [Sele], (J5, J6) P-Selectin [Selp], (J7, J8) Serpin E1 [Serpine1], (J9, J10) Serpin F1 [Serpinf1], (J11, J12) Thrombopoietin [Thpo], (J13, J14) TIM-1 [Havcr1], (J15, J16) TNF-alpha [Tnf], (J17, J18) VCAM-1 [Vcam1], (J19, J20) VEGF [Vegf], (J21, J22) WISP-1 [Ccn4], (J23, J24) negative control.

Figure 2 with 3 supplements
Longitudinal analysis of single-cell transcriptomes of injured peripheral nervous system (PNS).

(A) Schematic of workflow for single-cell analysis of injured mouse sciatic nerve trunk. Cartoon of lumbar spinal cord with dorsal root ganglia (DRGs) and sciatic nerve. The nerve injury site is shown, and the segment marked with the red bracket harvested at different post-injury time points for analysis by scRNAseq. (B) UMAP plot embedding of sciatic nerve cells at 1dpc. A total of 29,070 high-quality cells (n = 5 replates) were subjected to unsupervised Seurat-based clustering resulting in 22 clusters. (C) List of cell types identified, grouped into immune cells (clusters 0–14), structural cells (clusters 15–19), cells associated with the nerve vasculature (clusters 20 and 21), and Schwann cells (cluster 22). (D–I) Feature plots of canonical immune cell markers reveals clusters that harbor GC (Cxcr2), including a subset of eosinophils (Siglecf/sialic acid binding Ig-like lectin F), Mo (Ly6c2/Ly6C), Mac (Adgre1/F4/80), MoDC (Cd209a/DC-SIGN), and antigen-presenting myeloid cells (H2-Aa/histocompatibility 2, class II antigen A, alpha). (J) UMAP plot embedding of sciatic nerve cells at 3dpc. A total of 24,672 high-quality cells (n = 6 replates) were subjected to unsupervised Seurat-based clustering, resulting in 24 cell clusters. (K) List of cell types in the 3-day injured nerve, grouped into immune cells (clusters 0–12), structural cells (clusters 13–17), cells associated with the nerve vasculature (clusters 18–21), and Schwann cells (clusters 22–24). (L–Q) Feature plots of canonical markers for immune cells to identify clusters with GC (Cxcr2), Mo (Ly6c2), Mo/Mac (Ccr2), Mac (Adgre1), MoDC (Cd209a), and antigen-presenting cells (H2–Aa). (R) UMAP plot embedding of sciatic nerve cells at 7dpc. A total of 32,976 high-quality cells (n = 8 replates) were subjected to unsupervised Seurat-based clustering resulting in 34 cell clusters. (S) List of cell types in the nerve at 7dpc, grouped into immune cells (clusters 0–17), structural cells (clusters 18–24), cells associated with the nerve vasculature (clusters 25–29), and Schwann cells (clusters 30–34). (T–Y) Feature plots of canonical markers for immune cells revealed GC (Cxcr2), Mo (Ly6c2), Mo/Mac (Ccr2), Mac (Adgre1), MoDC (Cd209a), and antigen-presenting cells (H2–Aa). Expression levels are projected onto UMAP with a minimum expression cutoff of 1. Abbreviations: Mo, monocytes; Mac, macrophages; prol.Mac, proliferating macrophages; MoDC, monocyte-derived dendritic cells; cDC, conventional dendritic cells; DCx mature/migrating dendritic cells; pDC, plasmacytoid dendritic cells; MAST, mast cells; GC, granulocytes (including neutrophils and eosinophils), GC-L, granule cell-like; TC, T cells; NK, natural killer cells. Abbreviations for stromal cells: Fb, fibroblast; dMES, differentiating mesenchymal cells; prol.MES, proliferating mesenchymal cells; eMES, endoneurial mesenchymal cells; pMES, perineurial mesenchymal cells. Abbreviations for vascular cells: EC, endothelial cells, vSMC, vascular smooth muscle cells; PC, pericytes; vPC, venous pericytes; aPC, arterial pericytes; prol.PC, proliferating pericytes. Abbreviations for Schwann cells, nmSC, non-myelinating Schwann cells; mSC myelinating Schwann cells; rSC, repair Schwann cells; prol.SC, proliferating Schwann cells. UI, unidentified cells.

Figure 2—figure supplement 1
Identification of marker genes for cell type identification in the naïve and injured peripheral nervous system (PNS).

Dotplot analysis of scRNAseq datasets of (A) naïve nerve, (B) 1dpc nerve, (C) 3dpc nerve, and (D) 7dpc nerve. Expression levels are normalized to average gene expression (color coded). For each cell cluster, the percentile of cells expressing a specific gene product is indicated by the dot size.

Figure 2—figure supplement 2
Quality test of scRNAseq datasets and identification of proliferating cells in the naïve and injured sciatic nerve.

Feature plots for Mki67 reveals proliferating cells in (A) naïve nerve, (B) 1dpc, (C) 3dpc, and (D) 7dpc. The expression levels are projected onto the UMAP with a minimum expression cutoff of 1. (E–H) Feature plot showing the number of unique transcripts detected in naïve and injured sciatic nerve cells. Color-coded calibration is shown. Note, cells with less than 500 unique features or more than 7500 were excluded from the study. (I–L) Feature plots showing the mitochondrial content of cells in naïve and injured nerves. Color-coded calibration is shown. Cells with higher than 15% mitochondrial content were excluded from the analysis.

Figure 2—figure supplement 3
Cell composition on naïve and injured sciatic nerve.

Sciatic nerve scRNAseq datasets generated as part of a longitudinal study were used to assess changes in cell proportion before and after nerve crush injury.

Figure 3 with 4 supplements
Top injury-regulated Mac gene products.

(A) Heatmap of top 107 injury-upregulated gene products from a list of significant genes, filtered for upregulation in Mac at 1dpc and expression in at least 25% of cells. Shown are z-scores for average expression levels in naïve nerve Mac, and Mac at 1dpc, 3dpc, and 7dpc. (B–G) Examples of injury-induced gene products, violin plots are shown for naïve and injured nerves. (H) Ingenuity pathway analysis for injury-induced gene products in Mac utilizing the top 107 differentially expressed genes determined by Seurat’s FindAllMarkers function and the Wilcoxon rank-sum test. The top-scoring enriched canonical pathways are represented through activation z-scores and p-values.

Figure 3—figure supplement 1
Structural cells in the injured nerve shape the local immune microenvironment.

Dotplots of structural cells, including Fb (fibroblasts), eMES (endoneurial mesenchymal cells), pMES (perineurial mesenchymal cells), and dMES (differentiating mesenchymal cells). Expression of extracellular immune molecules, as detected by scRNAseq of naïve nerve, and at 1-, 3-, and 7dpc is shown. Color-coded gene expression levels, normalized to average gene expression. The dot size reflects the percentage of cells that express the gene.

Figure 3—figure supplement 2
Top injury-regulated gene products in endoneurial mesenchymal cell (eMES).

(A) Heatmap of top 78 injury-upregulated gene products, showing z-scores for average expression levels in naïve nerve eMES, and at 1dpc, 3dpc, and 7dpc. (B–G) Examples of injury-induced gene products, violin plots are shown for naïve and injured nerves. (H) Ingenuity pathway analysis for injury-induced gene products in eMES utilizing the top 78 differentially expressed genes determined by Seurat’s FindAllMarkers function and the Wilcoxon rank-sum test. The top-scoring enriched canonical pathways are represented through activation z-scores and p-values.

Figure 3—figure supplement 3
Top injury-regulated gene products in perineurial mesenchymal cells (pMES).

(A) Heatmap of top 75 injury-upregulated gene products, showing z-scores for average expression levels in naïve nerve pMES, and at 1dpc, 3dpc, and 7dpc. (B–G) Examples of injury-induced gene products, violin plots are shown for naïve and injured nerves. (H) Ingenuity pathway analysis for injury-induced gene products in pMES utilizing the top 75 differentially expressed genes determined by Seurat’s FindAllMarkers function and the Wilcoxon rank-sum test. The top-scoring enriched canonical pathways are represented through activation z-scores and p-values.

Figure 3—figure supplement 4
Top injury-regulated gene products in fibroblasts/differentiating mesenchymal cells (Fb/dMES).

(A) Heatmap of top 104 injury-upregulated gene products, showing calibrated expression levels in naïve nerve Fb, and Fb/dMES at 1dpc, 3dpc, and 7dpc. (B–G) Examples of injury-induced gene products, violin plots are shown for naïve and injured nerves. (H) Ingenuity pathway analysis for injury-induced gene products in Fb/dMES utilizing the top 104 differentially expressed genes determined by Seurat’s FindAllMarkers function and the Wilcoxon rank-sum test. The top-scoring enriched canonical pathways are represented through activation z-scores and p-values.

Figure 4 with 4 supplements
CellChat reveals chemotactic cell–cell communication networks in the injured peripheral nervous system (PNS).

Hierarchical plots of CellChat analysis showing the inferred intercellular communication networks for (A–H) CXCL-chemokines and (I–P) CCL-cytokines in naïve nerve and during the first week following injury. The sender cells (ligand sources) are shown on the y-axis and receiving cells (receptor expression) on the x-axis. The probabilities for cells to communicate with each other are indicated. (B, D ,F, H) The bar graphs show the contributions of CXCL ligand–receptor pairs for each time point. (J, L, N, P) The bar graphs show the relative contributions of CCL ligand–receptor pairs for each time point. (Q–S) Volcano plot of extracellular proteins detected by ELISA compared to naïve nerve. The most abundant and strongly upregulated proteins in the 1dpc nerve (Q), the 3dpc nerve (R), and the 7dpc nerve (S) are labeled in red. The normalized signal on the x-axis shows the log2 fold-change and the y-axis shows the -log(p-value), normalized to naïve nerve.

Figure 4—figure supplement 1
Identification of extracellular proteins in the injured sciatic nerve by ELISA.

(A–C) Representative ELISA membranes probed with injured sciatic nerve lysates prepared at (A) 1dpc (n = 2), (B) 3dpc (n = 2), and (C) 7dpc ( n = 1). (D–F) List of top proteins detected at each time point. For quantification, ELISA signals were normalized to reference spots shown at coordinates (A1, A2), (A23, A24), and (J1, J2). (G, H) Dotplots generated from scRNAseq data obtained from 1dpc, 3dpc, and 7dpc nerves, showing transcripts for top 25 proteins detected by ELISA. Relative RNA expression levels, normalized to average gene expression (color coded). For each cell cluster, the percentile of cells expressing a specific gene product is indicated by the dot size. Gene products marked with a red star are detected by ELISA but not by scRNAseq.

Figure 4—figure supplement 1—source data 1

X-Ray films of ELISA membranes probed with injured sciatic nerve lysates.

https://cdn.elifesciences.org/articles/80881/elife-80881-fig4-figsupp1-data1-v3.zip
Figure 4—figure supplement 2
Identification of extracellular proteins in the injured sciatic nerve.

Heatmap shows proteins detected by ELISA in serum, sciatic nerve trunk of naïve mice (n = 2), 1dpc (n = 2), 3dpc (n = 2), and 7dpc (n = 1) nerves. Relative protein levels were averaged and normalized to internal controls on ELISA membranes shown at coordinates (A1, A2), (A23, A24), (J1, J2). Coordinates of proteins that can be detected by the ELISA: (A3, A4) Adiponectin [Adipoq], (A5, A6) Amphiregulin [Areg], (A7, A8) Angiopoientin-1 [Angpt1], (A9, A10) Angiopoientin-2 [Angpt2], (A11, A12) Angiopoientin-like 3 [Angptl3], (A13, A14) BAFF [Tnfrsf13b], (A15, A16) C1qR1 [Cd93], (A17, A18) CCL2 [Ccl2], (A19, A20) CCL3 [Ccl3], (A21, A22) CCL5 [Ccl5], (A23, A24) Reference spots, (B3, B4) CCL6 [Ccl6], (B5, B6) CCL11 [Ccl11], (B7, B8) CCL12 [Ccl12], (B9, B10) CCL17 [Ccl17], (B11, B12) CCL19 [Ccl19], (B13, B14) CCL20 [Ccl20], (B15, B16) CCL21 [Ccl21], (B17, B18) CCL22 [Ccl22], (B19, B20) CD14 [Cd14], (B21, B22) CD40 [Cd40], (C3, C4) CD160 [Cd160], (C5, C6) Chemerin [Rarres2], (C7, C8) Chitinase 3-like 1 [Chil3], (C9, C10) Coagulation Factor III [F3], (C11, C12) Complement Component C5 [C5], (C13, C14) Complement Factor D [Cfd], (C15, C16) C-Reactive Protein [Crp], (C17, C18) CX3CL1 [Cx3cl1], (C19, C20) CXCL1 [Cxcl1], (C21, C22) CXCL2 [Cxcl2], (D1, D2) CXCL9 [Cxcl9], (D3, D4) CXCL10 [Cxcl10], (D5, D6) CXCL11 [Cxcl11], (D7, D8) CXCL13 [Cxcl13], (D9, D10) CXCL16 [Cxcl16], (D11, D12) Cystatin C [Cst3], (D13, D14) DKK-1 [Dkk1], (D15, D16) DPPIV [Dpp4], (D17, D18) EGF [Egf], (D19, D20) Endoglin [Eng], (D21, D22) Endostatin [Col18a1], (D23, D24) Fetuin A [Ahsg], (E1, E2) FGF acidic [Fgf1], (E3, E4) FGF-21 [Fgf21], (E5, E6) Flt-3 Ligand [Flt3l], (E7, E8) Gas 6 [Gas6], (E9, E10) G-CSF [Csf3], (E11, E12) GDF-15 [Gdf15], (E13, E14) GM-CSF [Csf2], (E15, E16) HGF [Hgf], (E17, E18) ICAM-1 [Icam1], (E19, E20) IFN-gamma [Ifng], (E21, E22) IGFBP-1 [Igfbp1], (E23, E24) IGFBP-2 [Igfbp2], (F1, F2) IGFBP-3 [Igfbp3], (F3, F4) IGFBP-5 [Igfbp5], (F5, F6) IGFBP-6 [Igfbp6], (F7, F8) IL-1alpha [Il1a], (F9, F10) IL-1Beta [Il1b], (F11, F12) IL-1ra [Il1rn], (F13, F14) IL-2 [Il2], (F15, F16) IL-3 [Il3], (F17, F18) IL-4 [Il4], (F19, F20) IL-5 [Il5], (F21, F22) IL-6 [Il6], (F23, F24) IL-7 [Il7], (G1, G2) IL-10 [Il10], (G3, G4) IL-11 [Il11], (G5, G6) IL-12 p40 [Il12], (G7, G8) IL-13 [Il13], (G9, G10) IL-15 [Il15], (G11, G12) IL-17A [Il17a], (G13, G14) IL-22 [Il22], (G15, G16) IL-23 [Il23], (G17, G18) IL-27 p28 [Il27], (G19, G20) IL-28 [Ifnl3], (G21, G22) IL-33 [Il33], (G23, G24) LDL R [Ldlr], (H1, H2) Leptin [Lep], (H3, H4) LIF [Lif], (H5, H6) Lipocalin-2 [Lcn2], (H7, H8) LIX [Cxcl5], (H9, H10) M-CSF [Csf1], (H11, H12) MMP-2 [Mmp2], (H13, H14) MMP-3 [Mmp3], (H15, H16) MMP-9 [Mmp9], (H17, H18) Myeloperoxidase [Mpo], (H19, H20) Osteopontin [Spp1], (H21, H22) Osteoprotegerin [Tnfrsf11b], (H23, H24) PD-ECGF [Tymp], (I1, I2) PDGF-BB [Pdgfb], (I3, I4) Pentraxin 2 [Nptx2], (I5, I6) Pentraxin 3 [Ptx3], (I7, I8) Periostin [Postn], (I9, I10) Pref-1 [Dlk1], (I11, I12) Proliferin [Prl2c2], (I13, I14) Proprotein Convertase 9 [Pcsk9], (I15, I16) RAGE [Ager], (I17, I18) RBP4 [Rbp4], (I19, I20) Reg3G [Reg3g], (I21, I22) Resistin [Retn], (J1, J2) Reference spots, (J3, J4) E-Selectin [Sele], (J5, J6) P-Selectin [Selp], (J7, J8) Serpin E1 [Serpine1], (J9, J10) Serpin F1 [Serpinf1], (J11, J12) Thrombopoietin [Thpo], (J13, J14) TIM-1 [Havcr1], (J15, J16) TNF-alpha [Tnf], (J17, J18) VCAM-1 [Vcam1], (J19, J20) VEGF [Vegf], (J21, J22) WISP-1 [Ccn4], (J23, J24) negative control.

Figure 4—figure supplement 3
Identification of multiple dendritic cell (DC) populations in the injured peripheral nervous system (PNS).

(A) Dotplot for DC marker genes at 7dpc identifies MoDC (monocyte-derived dendritic cells), cDC (conventional dendritic cells), pDC (plasmacytoid dendritic cells), and DCx (nerve exiting dendritic cells, destined for homing to draining lymph nodes). Relative RNA expression levels, normalized to average gene expression (color coded) are shown. For each cell cluster, the percentile of cells expressing a specific gene product is indicated by the dot size. Ingenuity pathway analysis at 7dpc for (B) MoDC, (C) cDC, (D) pDC, and (E) DCx. Top-scoring enriched canonical pathways in each cell type are represented by activation z-scores and p-values.

Figure 4—figure supplement 4
Identification of specific T cell (TC) and natural killer cell (NK) populations in the injured peripheral nervous system (PNS).

Longitudinal analysis of TC and NK populations during the first week following injury. Feature plots for (A–D) Trac/T cell receptor alpha constant, (E–H) Cd3g/CD3 gamma subunit of T cell receptor complex, (I–L) Cd4/T cell surface glycoprotein CD4, and (M–P) Ncr1/Natural cytotoxicity triggering receptor-1 of naïve, 1, 3, and 7dpc nerve. Expression levels are projected onto the UMAP with a minimum expression cutoff of 1. (Q, R) Ingenuity pathway analysis (IPA) for TC and NK at 7dpc. Top-scoring enriched canonical pathways are represented by activation z-scores and p-values.

Figure 5 with 2 supplements
Application of injured sciatic nerve atlas (iSNAT) reveals metabolic reprogramming of immune cells.

Metabolic pathways regulated by peripheral nervous system (PNS) injury. (A) Glycolysis: metabolites and enzymes for the catabolism of glucose into pyruvate and pyruvate to lactate. Glucose-6-phospate is required for nucleotide synthesis through the pentose phosphate pathway. Glycolysis and the pentose phosphate pathway occur in the cytosol. Pyruvate can be metabolized into Acetyl-CoA and enter the tricarboxylic acid (TCA) cycle. The TCA takes place in mitochondria. The Warburg effect allows for rapid ATP production through aerobic glycolysis and fermentation of pyruvate to lactate. The Warburg effect is characterized by limited mitochondrial ATP production because of TCA cycle fragmentation at the conversion of aconitate to α-ketoglutarate and succinate to fumerate, steps marked with dotted arrows. (B) Injury-regulated gene products associated with glycolysis, as inferred by scRNAseq data. Log2 average expression of genes for cells classified as Mac. (C) Quantification of gene expression by qRT-PCR in the 1dpc nerve relative to naïve nerve. Log2-fold changes relative to naïve nerve are shown. Per gene product, n = 4 biological replicates. p-values, *<0.05, **<0.001, ***<0.0001, Student’s t test. ns, not significant. (D, E) PNS injury-regulated gene products associated with inhibition of mitochondrial energy synthesis (D) and inflammation resolution (E), as inferred by scRNAseq data. Log2 average expression of genes for cells classified as Mac. (F) Longitudinal section of 3dpc injured sciatic nerve stained for Mo/Mac (anti-F4/80, green) and LDHA (white), proximal is to the left. Dotted boxes mark regions at the injury site and the distal nerve, respectively. Scale bar, 100 µm. (G–I) Higher magnification view of the dotted box at the injury site. Some F4/80 and LDHA double-positive cells are labeled with arrows (yellow). (J–L) Higher magnification view of distal nerve, dotted box showin in F. box in. Some F4/80 and LDHA double-positive cells are labeled with arrows (yellow). Scale bar (G–L), 50 µm. (M) Quantification of Mo/Mac (F4/80+) and SC (Sox10+) per field of view (FOV) at the 3dpc injury site (red dots) and distal nerve (blue dots). (N–P) Quantification of double labeled cells per FOV at the injury site and distal nerve for (N) LDHA+ Mac (O) LDHA+ SC, and (P) PKM+ Mac. Biological replicates n = 3 with two technical replicates each. Nonparametric independent Student's t tests. *<0.05, **<0.01, GraphPad Prism 9.

Figure 5—figure supplement 1
Longitudinal analysis of gene products that regulate glycolysis.

Feature plots showing gene expression in the naïve and crushed sciatic nerve during the first week post injury (1dpc, 3dpc, and 7dpc). (A–D) Hif1a (hypoxia inducible factor 1 subunit alpha), (E–H) Slc2a1 (glucose transporter), (I–L) Hk2 (hexokinase 2), (M–P) Pgk1 (phosphoglycerate kinase 1), and (Q–T) Pkm (pyruvate kinase muscle). Gene product expression, as inferred by scRNAseq data. Expression levels are projected onto the UMAP plot with a minimum expression cutoff of 1.

Figure 5—figure supplement 2
Peripheral nervous system (PNS) injury induces glycolytic macrophages and Schwann cells.

(A–H) Longitudinal sections of naïve and injured sciatic nerves stained for Mo/Mac (anti-F4/80, red). Nerve sections were stained with (A–D) anti-PKM2 (green) and (E–H) anti-LDHA (green). Proximal is to the left. Scale bar, 100 µm. (I–N) Longitudinal nerve sections at 3dpc stained for SC (anti-Sox10, purple) and LDHA (green). The nerve injury site and distal nerve as shown. Scale bar, 100 µm. Representative examples of n = 3 biological replicates.

Figure 6 with 2 supplements
Catalog of peripheral blood mononuclear cells (PBMCs).

(A) Blood was collected by cardiac puncture of naïve mice. UMAP plot embedding of PBMC revealed 20 cell clusters, including B cells (BC1-BC4), Platelets/megakaryocytes (P/M), T cells (TC1-TC5), natural killer cells (NK), immature and mature granulocytes (iGC and mGC), monocytes (Mo_1 and Mo_2), macrophages (Mac_0), dendritic cells (DC), plasma blasts (PB), proliferating leukocytes (prol.Lk), mast cells (MAST), and one cluster with unidentified cells (UI1). A total of 34,386 high-quality PBMC were analyzed (n = 2 replicates). (B) Dotplot with marker genes enriched in PBMC subpopulations. Color-coded expression levels are shown. The dot size reflects the percentage of cells that express the gene. (C–Q) Feature plots of marker gene expression in PBMC used for cell-type identification. Cd79a, CD79A antigen (immunoglobulin-associated alpha); Gp5, glycoprotein V platelet; Cd3e, T cell receptor complex CD3 epsilon subunit; Cd8b1, CD8 antigen beta chain 1; Cd4, CD4 antigen; Nkg7, natural killer cell group 7; Ly6g, lymphocyte antigen 6 complex locus G; Cxcr2, chemokine (C-X-C motif) receptor 2; Ly6c2, lymphocyte antigen 6 complex locus 2, Chil3, chitinase-like 3 (Ym1); Ccr2, chemokine (C-C motif) receptor 2; Csf1r, colony stimulating factor 1 receptor; Ear2, eosinophil-associated ribonuclease A family member 2; Prg4, proteoglycan 4; Cd209a, DC-SIGN (C-type lectin). (R–V) Assessment of PBMC metabolic state. In feature plots classical monocytes (Mo) are labeled with an arrow and immature granulocytes (iGC) with a black arrowhead. Feature plots for Hif1a (hypoxia inducible factor 1 alpha), Pfkl (phosphofructokinase liver type), Pgk1 (phosphoglycerate kinase 1), Ldha (lactate dehydrogenase alpha), and Slc16a3 (lactate exporter) are shown. Expression levels are projected onto the UMAP with a minimum expression cutoff of 1.

Figure 6—figure supplement 1
Differential gene expression between patrolling and classical Mo in blood.

A heatmap showing differential gene expression between Mo1 and Mo2 identified by scRNAseq of peripheral blood mononuclear cells (PBMC) obtained from naïve mouse cardiac blood. Hierarchical clustering, expression levels are calibrated to median gene expression. Note: Mo2 show elevated expression of Ly6c2, Chil3, and Hp, markers for classical Mo.

Figure 6—figure supplement 2
Activation of circulating immune cells upon nerve entry.

Feature plots for the glycolytic enzyme Pfkl (A–E), the monocarboxylate transporter Slc16a3 (F–J), the arginine degrading enzyme Arg1 (K–O), and the NO-producing enzyme Nos2 (P–T) in peripheral blood mononuclear cells (PBMC), naïve nerve, 1dpc, 3dpc, and 7dpc nerve. Color-coded expression levels are shown. Expression levels are projected onto the UMAP with a minimum expression cutoff of 1.

Figure 7 with 4 supplements
Integrated analysis of single-cell transcriptomes generated from peripheral blood mononuclear cells (PBMC), naïve nerve, and injured nerve.

(A–E) UMAP plots of integrated myeloid cells split into (A) PBMC, (B) naïve sciatic nerve trunk, (C), 1dpc nerve, (D) 3dpc nerve, and (E) 7dpc nerve. (F–J) Integrated analysis of Chil3+ in (F) PBMC, (G) naïve nerve, (H) 1dpc nerve, (I) 3dpc nerve, and (J) 7dpc nerve. (K–D) Integrated analysis of Gpnmb+ Mac in (K) PBMC, (L) naïve nerve, (M) 1dpc nerve, (N) 3dpc nerve, and (O) 7dpc nerve. For feature plots (F–O), expression values are projected onto the integrated UMAP with a minimum expression cutoff of 1. Abbreviations: iGC, immature granulocytes; mGC, mature granulocytes; Mo, monocytes; Mac, macrophages: DC, dendritic cells (MoDC, monocyte-derived DC; cDC, conventional DC; prol.DC, proliferating DC; pDC, plasmocytoid DC; and DCx, homing DC); MAST, mast cells; T_NK, T cells and natural killer cells; TC, T cells; NK, natural killer cells; UI, unidentified cells.

Figure 7—figure supplement 1
Transient upregulation of proinflammatory gene products in the injured sciatic nerve.

(A–E) UMAP plots of integrated immune cells split into (A) peripheral blood mononuclear cells (PBMC) (of naïve mice), (B) naïve sciatic nerve trunk, (C) 1dpc nerve, (D) 3dpc nerve, and (E) 7dpc nerve. (F–D’) Tracking of temporal changes in Cxcr2+ (F–J), Il1b+ (K–O), Hifa+ (P–T), Ccl2+ (U–Y), and Ccl7+ (Z–D’) expression in immune cells. For all feature plots shown (–-D’), expression values are projected onto the integrated UMAP with a minimum expression cutoff of 1. Abbreviations: GC, granulocytes; Mo, monocytes; Mac, macrophages; DC, dendritic cells (MoDC, monocyte-derived DC; cDC, conventional DC; prol.DC, proliferating DC; pDC, plasmocytoid DC; and DCx, homing DC); MAST, mast cells; TC, T cells and NK, natural killer cells; UI, unidentified cells.

Figure 7—figure supplement 2
Tracking of myeloid cells before and after entering the injured nerve.

(A–C) UMAP embedding of integrated immune cells with simplified cluster labels. (D) Principal component analysis (PCA) of integrated immune cells using the same cluster labels as in (A). The first four principal components were used as input to SlingShot pseudotime analysis. (B) SlingShot pseudotime, for Mo to Mac trajectory, projected onto the UMAP. (E) Pseudotime projected onto the PCA showing the predicted trajectory, starting from Mo and differentiating toward Mac. (C) A separate trajectory, starting from Mo shows differentiation toward MoDC; pseudotime projected on UMAP. (F) Mo to MoDC pseudotime projected on PCA. (G) Integrated immune cell UMAP embedding with all cluster labels from each time poin analyzed. (H) Integrated immune cells PCA with all cluster labels from each time point analyzed. Abbreviations: GC, granulocytes [iGC and mGC (in blood), GC (1dpc, 3dpc, and 7dpc nerve)], Mo, monocytes [Mo_1 and Mo_2 (blood), Mo (1dpc, 3dpc, and 7dpc nerve)], Mac, macrophages [endo-Mac and epi-Mac (naïve nerve), (Mac-I, Mac-II, Mac-III, Mac-IV), Mac-V (1dpc nerve), Mac1, Mac2, Mac3, Mac4 (3dpc nerve), Mac-a, Mac-b, Mac-c, Mac-d, Mac-e (7dpc nerve)], MoDC (monocyte-derived dendritic cells), cDC (conventional dendritic cells), pDC, plasmacytoid dendritic cells, DCx (mature dendritic cells), DC (dendritic cells in blood), Mast (mast cells), TC (T cells), NK (natural killer cells), UI (unidentified).

Figure 7—figure supplement 3
Heatmap showing the top 30 genes by importance in predicting pseudotime (Figure 7—figure supplement 2E) as determined by random forest analysis.

Colors represent the z-score calculated across genes.

Figure 7—figure supplement 4
Heatmap showing the top 30 genes by importance in predicting pseudotime (Figure 7—figure supplement 2F) as determined by random forest analysis.

Colors represent the Z-score calculated across genes.

Figure 8 with 1 supplement
Spatial differences in the immune landscape of the injured sciatic nerve.

(A) Schematic of workflow for analysis of wound tissue at the site of nerve injury and distal nerve tissue. Cartoon of a mouse lumbar spinal cord, dorsal root ganglia (DRGs), and major branches of the sciatic nerve. Nerve segments ~3 mm in length (injury or distal), marked with blue brackets, were harvested separately. Innate immune cells were captured with anti-CD11b magnetic beads and analyzed by scRNAseq. (B) UMAP plot of sciatic nerve myeloid cells captured at the injury site (left) and the distal nerve (right) at 3dpc. A total of 17,404 high-quality cells were subjected to unsupervised Seurat-based clustering, resulting in 13 cell clusters. (C) Bar graph of population size at the injury site versus distal nerve for 3d injured nerve immune cells. (D, E) Feature plots for Arg1 at 3dpc showing injury site and distal nerve (F, G). Feature plots for Cd38 at 3dpc showing injury site and distal nerve. Expression levels are color coded and calibrated to average gene expression. (H, I) Projection of the 3dpc 'whole nerve' reference data onto cells at the 3dpc injury site (H) and 3dpc distal nerve (I) onto 3dpc 'whole nerve' reference data. The y-axis shows the prediction score for each cell’s top predicted cell population. The number of cells assigned to each population is shown on top. (J, K) Quantification of gene expression by qRT-PCR in the 3d injured nerve injury site versus distal nerve (n = 3). p-values, *<0.05, **<0.001, ***<0.0001, Student’s t test. ns, not significant. (L) Longitudinal sections of naïve sciatic nerve stained for Gpnmb expression by RNAscope. (M) Longitudinal sections of 3d injured nerve stained for Gpnmb expression by RNAscope, proximal is to the left. Sections were counterstained with Hoechst. High power images of injury site (M’) and distal nerve (M’’) are shown. Scale bar: 200 μm (L, M’’).

Figure 8—figure supplement 1
Location-specific distribution of Mac subpopulations in the injured sciatic nerve.

(A) Longitudinal section through 3dpc sciatic nerve trunk of Arg1-YFP (YARG) reporter mice, co-labeled for Gpnmb mRNA expression by RNAscope. Proximal is to the left. Boxes marked with dotted lines show regions at the injury site and within the distal nerve. Scale bar, 150 µm. (B–D) Higher magnification view of box at the injury site shown in (A). (B) Cells labeled green express the YFP under the Arg1 promoter, (C) cells labeled red are stained for Gpnmb transcript by RNAscope, and (D) cells labeled yellow show co-expression of Arg1 and Gpnmb. (E–G) Higher magnification view of dotted box in the distal nerve shown in (A). (E) Cells labeled green express the YFP under the Arg1 promoter, (F) cells labeled red are stained for Gpnmb transcript by RNAscope, and (G) cells labeled yellow show co-expression of Arg1 and Gpnmb. Scale bar, 50 µm. (H–K) Longitudinal section through 7dpc sciatic nerve trunk. MoDC are visualized by RNAscope with a probe specific for Cd209a. Nuclei are stained with Hoechst dye. (H, I) show nerve injury site. (J, K) show distal nerve. Scale bar, 50 µm.

Figure 9 with 1 supplement
Nerve trauma causes Wallerian degeneration (WD)-independent nerve inflammation.

(A) Cartoon of a mouse lumbar spinal cord, dorsal root ganglia (DRGs), and major branches of the sciatic nerve. A nerve injury divides the nerve trunk into a proximal segment, the injury site, and distal segment, each marked with blue brackets. Nerve segments were harvested and subjected to analysis. (B–I) Longitudinal sciatic nerve sections from WT and Sarm1-/- mice, stained with anti-F4/80 for identification of Mac. Representative examples of (B, C) naïve nerve, (D, E), 1dpc (F, G), 3dpc, and at (H, I) 7dpc. Injury site is marked with a dashed red line, proximal is to the left. Scale bar, 500 µm. (J–O) Longitudinal sciatic nerve sections from WT and Sarm1-/- mice at 7dpc, stained with fluoromyelin. Representative images of proximal nerve, the injury site and distal nerve are shown. Scale bar, 200 µm. (P) Western blots of sciatic nerve segments collected at 3dpc and 7dpc from WT and Sarm1-/- mice. Nerves were divided into proximal, injury site, and distal segments and blots probed with anti-CD11b, and anti-ERK1/2. (Q–V) Flow cytometry dotplots for Mo/Mac of sham-operated WT and Sarm1-/- sciatic nerve trunks, the 3dpc nerve injury site and distal nerve. (W) Quantification of Mo/Mac (Ly6Chi + Ly6Cint + Ly6C-) in sham-operated mice, the 3dpc injury site, and 3dpc distal nerve of WT and Sarm1-/- mice. N = 3, with 3–5 mice per genotype per replica. Flow data are represented as mean ± SEM. Statistical analysis was performed in GraphPad Prism (v9) using two-way, paired t-test. **p<0.01.

Figure 9—source data 1

Western blots (LiCOR) of sciatic nerve segments probed with anti-CD11b and anti-Erk1/2.

https://cdn.elifesciences.org/articles/80881/elife-80881-fig9-data1-v3.zip
Figure 9—figure supplement 1
Gating strategy for flow cytometry.

(A) Cells were first gated with forward scatter (FSC-A) and side scatter (SSC-A) to exclude debris. (B) Cells were then gated with forward scatter height (FSC-H) and FSC-A to find single cells and to exclude doublets. (C) Live cells were isolated by negative staining for fixed viability dye (Fix Via). (D–G) Leukocytes (D) were analyzed as follows: lymphocytes were isolated as CD45+, CD11b-. Myeloid cells (CD45+, CD11b+) were further separated into Ly6G+ granulocytes (E). The remaining cells (CD45+, CD11b+, Ly6G-) were characterized as DC (F) (CD45+, CD11b+, CD11c+, Ly6G-), and Mo/Mac (G) (CD45+, CD11b+, CD11c-, Ly6G-) and further analyzed for Ly6C surface expression.

Figure 10 with 1 supplement
Evidence for Wallerian degeneration (WD)-dependent and WD-independent nerve inflammation.

(A) Timeline for parabiosis experiments. After a 2-week co-housing period, 10-week-old WT or Sarm1-/- and tdTomato mice were surgically paired. (B) To assess chimerism, blood was harvested and analyzed by flow cytometry. Dotplot of tdT+ myeloid cells (CD45+CD11b+) is shown. (C) Quantification of tdT+ myeloid cells in host parabionts (n = 6), revealed chimerism of 28 ± 2%. (D, E) Bilateral SNC was performed 3 weeks after pairing and tissue harvested at 7dpc. Longitudinal sciatic nerve sections from (D) WT and (E) Sarm1-/- parabionts showing infiltrating tdT+ leukocytes (magenta). Nuclei (blue) were labeled with Hoechst dye. The nerve crush site is marked by the white dashed line, proximal is to the left. Scale bar, 500 μm. (F) Quantification of tdT+ cells per high power field (HPF, 500 μm × 250 μm) at the injury site (0 μm) and at 1000, 2000, and 3000 μm distal to the injury site. The average cell number ± SEM is shown, n = 3 mice per genotype, average of four HPF per two nerves. Student’s t test, *p<0.05. (G) HPF of sciatic nerves from WT and Sarm1-/- parabionts 7dpc taken from the injury site, 1000, 2000, and 3000 μm distal to the injury site showing infiltrating tdT+ leukocytes (magenta), F4/80+ macrophages (green), and nuclei (blue). Scale bar, 100 μm. (H) Quantification of tdT+F4/80+ cells per HPF ± SEM at indicated distances distal to the injury site, n = 3 mice, average of four HPF per two nerves. Student’s t test, *p<0.05. (I, J) Flow cytometric analysis of sciatic nerves from single (not part of parabiosis complex) WT and Sarm1-/- mice 7dpc. Sciatic nerve trunks were microdissected and separated in 3 mm injury site and distal nerve segments. Dotplots showing Mo/Mac maturation assessed by Ly6C surface staining, Mo (Ly6Chi), Mo/Mac (Ly6Cint), Mac (Ly6C-), previously gated as CD45+CD11b+Ly6G-CD11c- cells. (K) Quantification of Mo/Mac shown in panels (I) and (J) as a percentage of single cells ± SEM, n = 3, injury and distal sites were pooled from 5 mice per genotype per biological replicate. Two-way ANOVA with Tukey’s post-hoc test for multiple comparisons, *p<0.05, **p<0.01.

Figure 10—figure supplement 1
Neutrophils are reduced in the Sarm1-/- distal nerve.

Quantification of tdT, Ly6G double-positive neutrophils in the 7dpc nerve of host parabionts (n = 6). (A) High power field (HPF) of longitudinal sections of a WT and Sarm1-/- parabionts at 7dpc. Representative images taken from the injury site, 1000, 2000, and 3000 μm distal to the injury site. Infiltrating tdT+ leukocytes (magenta), Ly6G+ neutrophils (green), and nuclei (Hoechst) are labeled. Scale bar, 100 μm. (B). Quantification of tdT+Ly6G+ cells per (HPF) ± SEM at indicated distances distal to the injury site, n = 3 mice, an average of four HPF per two nerves. Student’s t test.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyNeurofilament heavy chain
(chicken polyclonal)
Aves LabNFH1:750
AntibodyAnti-chicken Cy3
(donkey polyclonal)
Jackson Immunoresearch703-165-1551:200
AntibodyIba1
(rabbit polyclonal)
Wako Chemicals019-197411:500
AntibodyF4/80
(rat IgG2b monoclonal)
Thermo Fisher Scientificma1-911241:500–1:1000
AntibodyCD68
(rabbit polyclonal)
Abcamab1252121:500
AntibodySCG10
(rabbit polyclonal)
Novus BiologicalsNBP1494611:500–1:1000
AntibodyCD11b
(rabbit monoclonal)
Abcamab1333571:200–1:1000
AntibodyERK1/2
(rabbit polyclonal)
Cell Signaling91021:5000
AntibodyAnti-rabbit HRP
(donkey polyclonal)
EMD MilliporeAP182P1:2000–1:10,000
AntibodyCD16/32
(rat IgG2a monoclonal)
BD Pharmingen5531411 µg/1 million cells/25 µl
AntibodyCD11b-PE-Cy7
(rat IgG2b monoclonal)
Thermo Fisher Scientific25-0112-821:200
AntibodyIsotype Control-PE-Cy7
(rat-IgG2b monoclonal)
Thermo Fisher Scientific25-4031-821:100
AntibodyCD45-e450
(rat-IgG2b monoclonal)
Thermo Fisher Scientific48-0451-821:100
AntibodyCD45.1-e450
(mouse-IgG2a monoclonal)
BioLegend1107211:100
AntibodyIsotype Control-e450
(mouse-IgG2a monoclonal)
BioLegend4002351:100
AntibodyCD45.2-APC
(mouse-IgG2a monoclonal)
BioLegend1098131:100
AntibodyIsotype Control-APC
(mouse-IgG2a monoclonal)
BioLegend4002211:100
AntibodyLy6G-APC-Cy7
(rat-IgG2a monoclonal)
BD Biosciences5606001:100
AntibodyCD11c-PerCP-Cy5.5
(ArmHam-IgG monoclonal)
Thermo Fisher Scientific45-0114-821:100
AntibodyIsotype Control-PerCP-Cy5.5
(ArmHam-IgG monoclonal)
Thermo Fisher Scientific45-4888-801:100
AntibodyLy6C-FITC
(rat-IgM monoclonal)
BD Biosciences5531041:100
AntibodyIsotype Control-FITC
(rat-IgM monoclonal)
BD Biosciences5539421:100
AntibodyIba1 (goat polyclonal)Novus BiologicalsNB100-10281:200
AntibodyAnti-goat Alexa Fluor
488 (donkey polyclonal)
Jackson Immunoresearch705-545-1471:200
AntibodyLDHA
(rabbit polyclonal)
Cell Signaling Technology25581:300
Chemical compound, drugTOPRO pan-nuclear stainThermo Fisher ScientificT36051:2000
Chemical compound, drugFixable Viability DyeThermo Fisher Scientific650866141:500
Chemical compound, drugProteinase KNew England BiolabsP8107S
Chemical compound, drug10 mM dNTP mixPromegaC1141
Chemical compound, drug5X Green GoTaq BufferPromegaM791A
Chemical compound, drugGoTaq DNA polymerasePromegaM3005
Chemical compound, drugBuprenorphinePar PharmaceuticalNDC12496-0757-1
Chemical compound, drugKetaminePar PharmaceuticalNDC42023-115-10
Chemical compound, drugXylazineAkornNDC59399-110-20
Chemical compound, drugFluriso (Isoflurane, USP)Vet One501017
Chemical compound, drugRhodamine-conjugated
dextran MW 3000 (Microruby)
Life TechnologiesD-7162
Chemical compound, drugCholera toxin B (CTB)Life TechnologiesC34775
Chemical compound, drugPuralube Eye ointmentDechraNDC-17033-211-38
Chemical compound, drugN2 media supplementGibco17502048Cell culture
Chemical compound, drugN1 media supplementSigmaN6530Cell culture
Chemical compound, drugLeibovitz-15 (L-15)Gibco21083-027Cell culture
Chemical compound, drugPenicillin/StreptomycinLife Technologies15140-122Cell culture
Chemical compound, drugDMEM Ham’s F-12Gibco10565-018Cell culture
Chemical compound, drugFetal bovine serumAtlanta BiologicalsS11550Cell culture
Chemical compound, drugCytosine arabinosideSigma-AldrichC1768Cell culture
Chemical compound, drugCollagenase type 2Worthington BiochemicalLS004176Tissue digestion
Chemical compound, drugPBS without
calcium, magnesium
Gibco10010023Cell culture
Chemical compound, drugPoly-L-lysine MW
70,000–150,000
Sigma-AldrichP4707Cell culture
Chemical compound, drugLamininSigma-AldrichL2020Cell culture
Chemical compound, drugParaformaldehydeSigma-Aldrich158127-500G
Chemical compound, drugTriton-X100Sigma-AldrichT8787
Chemical compound, drugBovine serum albumin
(BSA) heat shock fraction V
Fisher ScientificBP1600
Chemical compound, drugHoechst 33342InvitrogenH3570Nuclear dye
Chemical compound, drugTissue-Tek O.C.T. CompoundElectron Microscopy Sciences62550-01
Chemical compound, drugβ-GlycerophosphateSigma-AldrichG9422-100G
Chemical compound, drugSodium orthovanadate
(Na3VO4)
Sigma-AldrichS6508-10G
Chemical compound, drugProtease inhibitor cocktailSigma-AldrichP8340-5ML
Chemical compound, drugDC Protein Assay KitBio-Rad5000111
Chemical compound, drug2× Laemmli sample bufferBio-Rad1610737
Chemical compound, drugβ-MercaptoethanolEMD Millipore6010
Chemical compound, drugBlotting-grade blockerBio-Rad1706404
Chemical compound, drugSuperSignal West Pico PLUS Chemiluminescent SubstrateThermo Fisher Scientific34580
Chemical compound, drugSuperSignal West Femto
Maximum Sensitivity Substrate
Thermo Fisher Scientific34095
Chemical compound, drugWesternSure PREMIUM
Chemi Substrate
LI-COR Biosciences926-95000
Chemical compound, drugFixable Viability Dye eF506Thermo Fisher Scientific65-0866-14
Chemical compound, drugTRIzolThermo Fisher Scientific15596026
Chemical compound, drugDispaseSigma-AldrichD4693
Chemical compound, drugActinomycin DSigma-AldrichA1410
Chemical compound, drugPercollSigma-AldrichP4937
Chemical compound, drugMACS bufferMiltenyi130-091-376
Chemical compound, drugCD45 MicroBeadsMiltenyi130-052-301
Chemical compound, drugCD11b MicroBeadsMiltenyi130-049-601
Chemical compound, drugMyelin removal BeadsMiltenyi130-096-733
Chemical compound, drugLS ColumnsMiltenyi130-042-401
Chemical compound, drugHanks balanced salt solutionGibco14025092
Chemical compound, drugSucroseFisher ScientificS5-500
OtherSuperfrost Plus Microscope SlidesFisher Scientific12-550-15For histology
OtherZeiss Axio Observer Z1Zeiss491912-0049-000Microscope
OtherZeiss Axiocam 503 mono cameraZeiss426559-0000-000Microscope camera
OtherEC PlnN ×10 objectiveZeiss420341-9911-000Objective for microscope
OtherMotorized tissue homogenizerRPI299200Homogenization of nerve tissue
OtherFisher Scientific Sonic DismembratorFisher ScientificModel 500Western blot equipment
Otherphoto spectrometerMolecular DevicesSpectraMax M5eMeasurement of protein concentration
OtherLI-COR C-DigitLI-COR BiosciencesCDG-001313Scanning of Western blot membranes
Other40 µm filterBD Falcon352340Cell isolation
Other70 µm cell strainerCorning352350Cell isolation
Chemical compound, drugPVDF membraneEMD MilliporeIPVH00010
Chemical compound, drugAmmonium-chloride-potassium (ACK) Lysing BufferGibcoA1049201Removal of erythrocytes
Commercial assay or kitMyelin Removal BeadsMiltenyi130-096-731
Commercial assay or kitMidiMACS separatorMiltenyi130-042-302
OtherLS ColumnsMiltenyi130-042-401Cell sorting
OtherHemacytometerMilliporeSigmaZ359629Cell counting
Commercial assay or kitChromium Next GEM Chip G10X Genomics, IncNC1000127
Other10X Genomic Chromium Controller10X Genomics, IncGCG-SR-1Barcoding of cells for scRNA-sequencing
Commercial assay or kitChromium Next GEM Single
Cell 3′ Kit v3.1
10X Genomics, Inc1000268
Commercial assay or kitChromium Next GEM Chip
G Single Cell Kit
10X Genomics, Inc1000127
Commercial assay or kitDual Index Kit TT Set A10X Genomics, Inc1000125
Chemical compound, drugDynabeads10X Genomics, Inc2000048
Chemical compound, drugSPRIselectBeckman CoulterB23318
OtherNovaSeq Illumina 6000IlluminaN/ADNA library sequencing
OtherCryostatLeica BiosystemsCM3050STissue sectioning
OtherConfocal MicroscopeNikonC1Imaging of tissue sections
OtherConfocal MicroscopeLeica BiosystemsSP8Imaging of tissue sections
Commercial assay or kitProteome Profiler, Mouse XL
Cytokine membranes (ELISA)
R&D Systems, Minneapolis, MN, USAARY028
Strain, strain background (Mus musculus)Sarm1-/-
C57BL/6
Jackson LaboratoriesStock# 018069PMID:22678360
Strain, strain background (M. musculus)ROSA26-mTdt/mGFP
C57BL/6
Jackson LaboratoriesStock# 007576PMID:17868096; MGI: J:124702
Strain, strain background (M. musculus)Arg1-eYFP
C57BL/6
Jackson LaboratoriesStock# 015857PMID:17450126; MGI: J:122735;
PMID:33263277
Strain, strain background (M. musculus)CD45.1
C57BL/6
Jackson LaboratoriesStock# 002014PMID:11698303; MGI: J:109863;
PMID:11994430; MGI: J:109854;
PMID:12004082; MGI: J:109853
Strain, strain background (M. musculus)Wildtype, WT
C57BL/6
TaconicsB6NTac
Sequence-based reagentNeomycin ForwardIntegrated DNA TechnologiesN/A5’-CTTGGGTGGAGAGGCTATTC-3’
Sequence-based reagentNeomycin ReverseIntegrated DNA TechnologiesN/A5’-AGGTGAGATGACAGGAGATC-3’
Software, algorithmWIS-NeuromathWeizmann Institute of ScienceVersion 3.4.8PMID:23055261
Software, algorithmImage Studio SoftwareLI-COR BiosciencesVersion 5.2.5
Software, algorithmNovaSeq control softwareIlluminaVersion 1.6
Software, algorithmReal Time Analysis (RTA) softwareIlluminaVersion 3.4.4
Software, algorithmCellRanger10X Genomics, IncVersion 3.1.0
Software, algorithmSeuratSatija Lab–New York Genome CenterVersion 4.0.5
Software, algorithmSeurathttps://github.com/satijalab/seurat; Srivastava and Hoffman, 2022; Hao et al., 2021Version 4.1.1.9006
Software, algorithmRr-project.orgVersion 4.1.2
Software, algorithmSlingShotbioconductor.orgVersion 2.2.1
Software, algorithmRangerComprehensive R Archive NetworkVersion 0.13.1
Software, algorithmCellChathttps://github.com/sqjin/CellChat; Jin and CaoWei-UM, 2022; Jin et al., 2021Version 1.1.3
Software, algorithmshinyRstudio.comVersion 1.7.1
Software, algorithmPrismGraphPadVersions 7 and 8
Software, algorithmIngenuity pathway analysisQIAGENVersion
81348237
Software, algorithmImarisBitplane
Software, algorithmLeica Application Suite (LAS X)Leica
Software, algorithmZen Application SoftwareZeissPro 3.8
OtherSomnoSuiteKent ScientificSS-01Anesthesia
OtherPovidone-Iodine Prep PadPDI HealthcareB40600Disinfection
OtherAlcohol Prep, Sterile, Md, 2 PlyCovidien6818Disinfection
OtherFine Forceps Dumont #55 DumoxelRoboz Surgical InstrumentRS-5063Surgical tool
Other7 mm Reflex Wound ClipsCell Point Scientific203-1000Surgical tool
OtherMicro Friedman RongeurRoboz Surgical InstrumentRS-8306Surgical tool
OtherMcPherson-Vannas Micro Dissecting Spring scissorsRoboz Surgical InstrumentRS-5600Surgical tool
OtherCOATED VICRYL (polyglactin 910) SutureEthiconJ463GSurgical suture
OtherDumont #7 curved forcepsFine Science Tools11271-30Surgical tool
OtherMiltex Halsted mosquito forcepsIntegra LifeSciences724Surgical tool
OtherNanofil 10 µl syringeWorld Precision InstrumentsNANOFILSmall syringe
Other36g beveled nanofil needleWorld Precision InstrumentsNF36BV-2Perfusion
OtherNon-absorbable suturesEthicon640GSurgical sutures for parabiosis
OtherAbsorbable suturesEthiconJ463GSurgical sutures
Sequence-based reagentRNAscope Probe- Mm-Gpnmb-C3-
Mus musculus glycoprotein (transmembrane) Gpnmb
(Gpnmb) mRNA
ACD Bio489511-C31:50
Sequence-based reagentRNAscope Probe- Mm-Ccl8-C2-Mus musculus chemokine
(C-C motif) ligand 8 (Ccl8) mRNA
ACD Bio546211-C21:50
Sequence-based reagentRNAscope Probe- Mm-Cd209a-C2- musculus CD209a antigen (Cd209a) mRNAACD Bio480311-C21:50
Commercial assay or kitRNAscope Multiplex
Fluorescent Reagent Kit v2
ACD Bio323100
OtherModel 1525 incubatorVWR Scientific1525RNAscope equipment
OtherACD hybridization ovenACD Bio321710RNAscope equipment
Chemical compound, drugHydrogen peroxide solutionACD BioPN 322381
Chemical compound, drug10× antigen retrieval solutionACD Bo322000
Chemical compound, drugProtease Plus solutionACD Bio322331
Chemical compound, drugCy3AKOYA BiosciencesNEL744001KT1:2000
Chemical compound, drugCy5AKOYA BiosciencesNEL745001KT1:2000
Chemical compound, drugDAPI mounting mediaSouthern Biotech0100-20
Chemical compound, drugRNAscope wash bufferACD Bio310091
Chemical compound, drugFormalin (1:10)Fisherbrand427-098
AntibodyPFKfb3
(rabbit monoclonal)
Cell Signaling Technology131231:300
AntibodyPKM
(rabbit polyclonal)
Abcamab1377911:300
AntibodySox10
(goat polyclonal)
R&D SystemsAF28641:300
Sequence-based reagentSpp1 ForwardThis paperPCR primerAAGTCTAGGAGTTTCCAGGTTTC
Sequence-based reagentSpp1 ReverseThis paperPCR primerGCTCTTCATGTGAGAGGTGAG
Sequence-based reagentGapdh ForwardThis paperPCR primerAACTTTGGCATTGTGGAAGG
Sequence-based reagentGapdh ReverseThis paperPCR primerGGATGCAGGGATGATGTTCT
Sequence-based reagentChil3 ForwardThis paperPCR primerAGCCCTCCTAAGGACAAAC
Sequence-based reagentChil3 ReverseThis paperPCR primerGGAATGTCTTTCTCCACAGATTC
Sequence-based reagentRlp13a ForwardThis paperPCR primerGCTGCTCTCAAGGTTGTTC
Sequence-based reagentRlp13a ReverseThis paperPCR primerGTACTTCCACCCGACCTC
Sequence-based reagentHif1a ForwardThis paperPCR primerCTGATGGAAGCACTAGACAAAG
Sequence-based reagentHif1a ReverseThis paperPCR primerCAATATTCACTGGGACTGTTAGG
Sequence-based reagentAcod1 ForwardThis paperPCR primerGGCACAGAAGTGTTCCATAAAG
Sequence-based reagentAcod1 ReverseThis paperPCR primerGTGGGAGCCTGAAGTCTG
Sequence-based reagentIl1b ForwardThis paperPCR primerCTTCCAGGATGAGGACATGAG
Sequence-based reagentIl1b ReverseThis paperPCR primerTCACACACCAGCAGGTTATC
Sequence-based reagentSlc16a3 ForwardThis paperPCR primerGCAGAAGCATTATCCAGATCTAC
Sequence-based reagentSlc16a3 ReverseThis paperPCR primerGATTGAGCATGATGAGGGAAG
Sequence-based reagentLdha ForwardThis paperPCR primerCATTGTCAAGTACAGTCCACAC
Sequence-based reagentLdha ReverseThis paperPCR primerTTCCAAGCCACGTAGGTC
Sequence-based reagentPkm ForwardThis paperPCR primerCTGGATACAAAGGGACCTGAG
Sequence-based reagentPkm ReverseThis paperPCR primerCAGAGTGGCTCCCTTCTTC
Sequence-based reagentPgk1 ForwardThis paperPCR primerGTGGAATGGCCTTTACCTTC
Sequence-based reagentPgk1 ReverseThis paperPCR primerGACAATCTTGGCTCCTTCTTC
Sequence-based reagentCd38 ForwardThis paperPCR primerACTGTCCCAACAACCCTATTAC
Sequence-based reagentCd38 ReverseThis paperPCR primerATCACTTGGACCACACCAC
Sequence-based reagentPfkl ForwardThis paperPCR primerCTGCTGAGCTACACAGAGG
Sequence-based reagentPfkl ReverseThis paperPCR primerCGTGTCCCTTGGTGAGAAG
Sequence-based reagentGatm ForwardThis paperPCR primerTTGCTTTGATGCTGCTGAC
Sequence-based reagentGatm ReverseThis paperPCR primerCACTCGATGCCCAGGTAG
Chemical compound, drugSYBR Green Fluorescein Master MixThermo Scientific4364344
OtherQuantStudio 3 real-time PCR systemApplied BiosystemsA28567Genotyping
OtherPestle motor mixerRPI299200Tissue homogenization
OtherNanodropThermo ScientificMeasurement of nucleic
acid concentration
Commercial assay or kitSuperScript III First-Strand
Synthesis System kit
Invitrogen18080051
Chemical compound, drugRIPA bufferSigmaR0278Supplemented with 50 mM BGP,
1 mM Na3VO4, and 1:100 PIC
Chemical compound, drugDC protein assayBio-Rad5000111
Chemical compound, drugβ-MercaptoethanolSigma60242
Chemical compound, drug2X Laemmli bufferBio-Rad1610737
OtherImmune-Blot PVDF membraneBioRad1620260Western blotting
Chemical compound, drugTBST blocking bufferThis paperBuffer48.4 g Tris Base, 351.2 g NaCl,
ddH2O 2 L, pH 7.4, 0.5% Tween-
20 with BSA 200 mg
Chemical compound, drugBovine serum albuminFisherBP1600-100
Chemical compound, drugSuperSignal West Pico PLUS Chemiluminescent SubstrateThermo Fisher Scientific34580

Additional files

Supplementary file 1

Datasets included in injured sciatic nerve atlas (iSNAT).

The table shows newly generated and existing scRNAseq datasets used in this study. Columns show cell numbers before and after applying exclusion criteria, replicates, statistics on reading depth and sequence saturation. A total of 157,409 high-quality single-cell transcriptomes were analyzed from naïve mouse sciatic nerve, injured sciatic nerves, and peripheral blood mononuclear cells (PBMC). Some of the 3 day (3d) injured nerves were divided into injury site and distal nerve and sequenced separately. SN, sciatic nerve; UMI, unique molecular identifier.

https://cdn.elifesciences.org/articles/80881/elife-80881-supp1-v3.docx
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https://cdn.elifesciences.org/articles/80881/elife-80881-mdarchecklist1-v3.docx

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  1. Xiao-Feng Zhao
  2. Lucas D Huffman
  3. Hannah Hafner
  4. Mitre Athaiya
  5. Matthew C Finneran
  6. Ashley L Kalinski
  7. Rafi Kohen
  8. Corey Flynn
  9. Ryan Passino
  10. Craig N Johnson
  11. David Kohrman
  12. Riki Kawaguchi
  13. Lynda JS Yang
  14. Jeffery L Twiss
  15. Daniel H Geschwind
  16. Gabriel Corfas
  17. Roman J Giger
(2022)
The injured sciatic nerve atlas (iSNAT), insights into the cellular and molecular basis of neural tissue degeneration and regeneration
eLife 11:e80881.
https://doi.org/10.7554/eLife.80881