Extended Data Fig. 5: Prox1a⁺Pou2f3⁺ macrophages (cluster 6) identified by scRNA-seq are a dwelling macrophage subset independent from Mmp9⁺ macrophages (cluster 2).

a, After appropriate filtering, 1,309 macrophages were analysed by scRNA-seq. These cells express antigen processing and presentation genes, confirming their macrophage character. Feature plots highlighting antigen-presenting genes expressed by the majority of cells (cd83, cd81a and cd40) as well as genes differentially expressed by individual subsets. The percentage of cells expressing a gene of interest is also recorded (maroon, information extracted from Supplementary Tables 1, 3). b, Graphical representation of UMAP scatter plot clusters. Uninjured macrophages cluster together (cluster 3). Macrophages isolated from a ‘transient’ time point (1 dpi) also predominantly cluster together (cluster 1). The remaining six clusters (0, 2, 4, 5, 6, 7) are mainly composed of macrophages isolated from ‘dwelling’ time points (2–3 dpi). c, Lineage analysis using PAGA visualizes gene expression changes leading to the definition of an mmp9⁺ dwelling macrophage subset along path 3-0-4-2 of the PAGA cluster and a prox1a⁺pou2f3⁺ dwelling macrophage subset along path 3-5-1-6 of the PAGA cluster. The numerical scale (right) expresses normalized gene expression from Seurat, whereas clusters (bottom) indicate subsets along the PAGA path with length being proportional to the cell numbers in each cluster. d, Violin plots show the expression pattern of mmp9 and nampta based on cluster identity and isolation time point. The percentage of cells expressing the markers at 2 dpi are documented. e–j, Violin plots (percentage of cells in cluster 6 expressing a specific marker recorded as percentage (e, h)) and antibody staining for a subset of the cluster-6 markers (Pou2f3 (f) and Prox1a (i)) after needle-stab muscle injury (white dotted line), showing a specific, late-injury-dwelling macrophage population (Supplementary Table 3 (worksheet 7)). Antibody staining against mCherry was used to identify all mpeg1⁺ macrophages in the Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry) (yellow) line. After injury, at 1 dpi, almost no Pou2f3⁺mpeg1⁺ (f; scale bar, 50 μm) or Prox1a⁺mpeg1⁺ (i; scale bar, 50 μm) macrophages are present in the wound site; their numbers start to increase slightly at 2 dpi with the highest percentage being present at 3 dpi, identifying this subset as a late-injury-dwelling macrophage subset. g, j, Quantification (n = 10 at 2 and 3 dpi in g and n = 7, n = 11 and n = 7 at 1, 2 and 3 dpi, respectively, in j). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t₁₇ = 4.574, P = 0.0003 (g) or one-way ANOVA with Dunnett’s post hoc test for multiple comparisons (j). k, l, Cluster-2 macrophages display a metabolic shift towards glycolysis at the gene expression level. KEGG pathway analysis of cluster-2 differentially expressed genes (Supplementary Table 3) identified genes associated with glycolysis (term: dre00010) and oxidative phosphorylation (term: dre00190) pathways. Genes associated with glycolysis were upregulated (k), whereas the majority of genes associated with oxidative phosphorylation were downregulated (l), highlighting a metabolic shift towards glycolysis in cluster-2 mmp9⁺ dwelling macrophages.

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