Extended Data Fig. 5: Additional characterization of injury-responsive profibrotic CNS myeloid cells, related to Fig. 2.

a-b, Quantification of Fig. 2o showing number of myeloid cells (IBA1^hi, macrophages and reactive microglia) per slice (a) and median myeloid cell distance from the nearest fibroblast-associated ECM (ER-TR7⁺) surface (b). n = 3(2dpi), n = 4(7dpi), or n = 5(14/21/60dpi) mice (2–4 slices/mouse). c, Schematic showing myeloid cell distribution by ontogeny. d-f, Immunofluorescent imaging from microglia lineage tracer mice (d, P2ry12^creER; Rosa26^TdT; tamoxifen days ⁻14–⁻12 before injury), with quantification of lineage traced cell density (e) and proportion (f) in inner core, fibrotic border, or outer glial regions at late timepoints (14/21dpi). n = 6 mice. g-i, Imaging from monocyte lineage tracer mice (g, Ccr2^creER; Rosa26^TdT; tamoxifen 0–2dpi), with quantification of density (h) and proportion (i) by region, as above. n = 5 mice. j-k, Time course showing Ccr2-lineage traced cells differentiating into both lesional macrophages and dendritic cells, shown as normalized counts (j) or as a proportion of the indicated population (k). Tamoxifen 0–2dpi. n = 4(0/2dpi), n = 3(7/21dpi), or n = 5(14dpi) mice. l-n, Imaging from BAM/PVM lineage tracer mice (l, PF4^cre; Rosa26^TdT), with quantification of density (m) and proportion (n) by region, as above. n = 4 mice. o-q, UMAP of reclustered myeloid cells (o, colored by timepoint; major resting populations labelled), dot plot showing myeloid subcluster marker genes (p), and violin plot of Disease Associated Microglia (“DAM”) score (q, an aggregate of 30 published DAM-specific markers). n = 1267(DAM), n = 816(SAM), n = 305(PVM/BAM), n = 173(ECM_Macs), n = 116(Mono/DC), n = 20(DCs), n = 110(Microglia), or n = 19(Osteoclasts) nuclei. r, DAM/SAM snRNAseq identities mapped onto spatial transcriptomic data. s-u, Imaging of FABP5⁺ cells near Cthrc1^creER; R26^TdT+ myofibroblasts at 7dpi (s), and surfaced images of FABP5⁺ DAM (t, microglia-derived) or SAM (u, monocyte-derived) in perilesional/border regions at 7dpi (with inset) or 21dpi. v-w, Density plots showing expression of SAM score among myeloid cells after stroke in marmosets (v) or TBI in humans (w). x, Ligand-receptor interactions between SAM or DAM and myofibroblasts (CellPhoneDB). Left half: ligand (first/purple molecule in labelled pair) expressed by SAM/DAM, receptor (grey) expressed by myofibroblast. Right half: ligand expressed by myofibroblast, receptor expressed by SAM/DAM. Dotted lines (left) and schematic (right) highlight modules with macrophage-expressed ligands (blue) or myofibroblast-expressed ligands (orange). y, Expression of DAM markers (CD9/CD63) on sorted homeostatic microglia cultured for 72 h alone or with 7/21dpi lesions. n = 7 wells/condition. One-way ANOVA, Tukey post-test (a,b,y); one-way repeated-measures ANOVA, Tukey post-test (e,f,h,i,m,n); Kruskall-Wallis test, Dunn’s multiple comparisons correction (q, relevant comparisons shown). 14μm slices; images represent 2 or more mice.

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