Fig. 3

Pseudotime analyses reveal putative fibroblast differentiation trajectories. a Pseudotime ordering on wound fibroblasts arranged them into a major trajectory, with two minor bifurcations (top panel). Stacked bar chart shows relative abundance of fibroblasts from distinct subclusters across the pseudotime, which was divided into ten equal bins (bottom panel). Pdgfra^low sC3/9/11 cells primarily occupy right half of the trajectory, with the remaining part consisting of Pdgfra^high cells. All cells on the pseudotime and the bar chart are color-coded to match the colors in Fig. 2a. b RNA velocity analysis distinguished three sets of velocity vectors across the pseudotime: Path1 (red), Path2 (green) and Path3 (blue). Stacked bar charts show relative abundance of fibroblast subclusters across individual RNA velocity paths (middle panel). Bottom panel defines fibroblast subcluster colors and matches the colors in Fig. 2a. c scEpath analysis performed on pseudotime identified five gene clusters (pC1 through pC5) of pseudotime-dependent genes. Heatmap of the expression levels for all differentially expressed genes in all analyzed wound fibroblasts is shown on the left. Average gene expression dynamics and gene counts in all five clusters are shown on the right. d Feature plots of expression distribution for Col14a1 and Mest across pseudotime. Both genes are part of cluster pC1 and mark the beginning points of RNA velocity Path1 and Path2. Expression levels for each cell are color-coded with the highest expression level colored black. e Feature plots of expression distribution for contractile markers Acta2 and Tagln across pseudotime. Cells with the highest expression level are colored black. Numbers of both Acta2⁺ and Tagln⁺ cells peak around minor bifurcations and along Path3. f, g Expression level heatmaps for selected signaling factors (f) and transcription factors (g) identified as differentially expressed on c. Each heatmap is accompanied by color-coded pseudotime feature plots for two selected genes. Numbers of Ogn⁺ and Klf4⁺ cells peak at the beginning points of RNA velocity Path1 and Path2, while numbers of Pdgfa⁺ and Ebf1⁺ cells increase along Path3. Cells with the highest expression level are colored black