Figure 8. Identification and characterization of ligands expressed in nerve mesenchymal cells that locally promote sympathetic axon growth. A, tSNE gene expression overlays of Angpt1 and Vegfc on the combined 3 and 9 DPI nerve dataset shown in Figure 1E. Cells that detectably express the ligand are colored blue, and the numbers correspond to the clusters. B, Heatmap showing expression of Ccl11 mRNA in single cells within clusters of the combined injured nerve scRNA-seq dataset shown in Figure 1E. Each column line represents the level of expression in a single cell. Gene expression represents scaled expression values using Seurat’s scaling function and is color coded as per the adjacent color key, where yellow indicates the highest expression. Cluster numbers are on the bottom, and the cell types in that cluster are annotated on the top. Mes. = mesenchymal, Endoth. = endothelial, Endo Mes. = endoneurial mesenchymal, SC = Schwann cell. C–E, Neonatal (P4) rat sciatic nerve mesenchymal cells were sorted for cell-surface PDGFRα using FACS, cultured in defined growth medium, and medium was collected after 1–4 d of conditioning. C, Schematic of the experiments. D, Representative image of the mesenchymal cell cultures immunostained for PDGFRα and for the Schwann cell protein S100β to indicate the relative purity of the cultures. Scale bar = 50 μm. E, Quantitative ELISA analysis of the nerve mesenchymal cell conditioned medium for ANGPT1, CCL11, and VEGFC (CM). Control growth medium was used as a negative control in each experiment (Con). Shown are the mean ± SEM from three independent experiments; **p < 0.01 (CCL11, p = 0.0055; VEGFC, p = 0.0045) and *p < 0.05 (ANGPT1, p = 0.031), two-tailed unpaired Student’s t test. F, Images of the 9-d injured distal sciatic nerve of an adult PdgfraEgfp/+ mouse analyzed by FISH for Angpt1, Ccl11, and Vegfc mRNAs. Hatched white lines outline EGFP-positive cells (green nuclei) that were also positive for the mRNA of interest (red dots). Also shown is Hoechst 33258 counterstaining (white/gray) to highlight cell nuclei. The arrows indicate cells that are shown at higher magnification in the insets. Scale bars = 20 μm. Scale bars in insets = 8.75 μm. G, Schematic of the compartmented culture axon outgrowth experiments. Neonatal SCG sympathetic neurons were established in compartmented cultures in the presence of 10 ng/ml NGF. When their axons had crossed into the side compartments, the side compartment medium was replaced with medium containing 0.5 ng/ml NGF plus 100 ng/ml of ANGPT1, CCL11, or VEGFC for three additional days. As a positive control, 50 ng/ml NGF was added into side compartments and as a baseline control, axons were maintained in 0.5 ng/ml NGF alone. H, Brightfield images of sympathetic axons in side compartments grown as described in G, located within 1 mm of the furthest extent of axonal outgrowth. Positive control (Max NGF) = 50 ng/ml, negative control (Min NGF) = 0.5 ng/ml. Scale bar = 100 μm. I, Scatter plots showing the density of outgrowth in response to the different ligands. A vertical line was drawn within the farthest 1 mm of axonal outgrowth, and the number of axons crossing the line was quantified. A maximum of eight separate lanes was scored per technical replicate, with three to four technical replicate cultures (i.e., cultures generated from sympathetic ganglia harvested from the same litter) scored per biological replicate (n values in plot). Individual points show mean ± SEM of individual biological replicates; *p < 0.05 (p = 0.041), ****p < 0.0001, one-way ANOVA with Holm–Sidak’s multiple comparison’s test (n = 5 for all treatments except VEGFC, where n = 3).