Extended Data Fig. 9: Additional molecular characterization of injury response after fibroblast impairment, related to Fig. 4.

a, snRNAseq schematic. Control (WT), cKO (Col1a2^creER; Tgfbr2^flox), and α_vβ₈-blocking antibody (ADWA11)-treated mice were harvested at 7 and 21dpi (2 mice per timepoint/condition), lesions were micro-dissected, and nuclei were sorted (DAPI⁺) and sequenced (final library 60,070 nuclei). b-c, Global UMAPs from snRNAseq data showing indicated cell types (b) or Col1a2 expression (c). d-e, UMAPs (18,455 fibroblasts and 496 mural cells) with fibroblast subclusters (d) or timepoint (e). f, Violin plots mapping “CNS fibroblast signatures” (derived from Fig. 2, x-axis) onto clusters identified in d (y-axis). g-i, Characterization of “Altered dural/lymphocyte-interactive” fibroblasts, shown as highlighted cluster in Cxcl12 feature plot (g), UMAP with subclusters (h), and violin plots showing subcluster expression of “Lymphocyte-interactive” and “Altered-dural” signatures (i, derived from Fig. 2), highlighting 3 lymphocyte-interactive-like and 1 altered-dural-like subcluster(s). j, Fibroblast UMAP separated by condition, with pan-fibroblast reduction in cKO mice and late-inner fibroblast reduction in α_vβ₈-blocked (ADWA11) mice. k-p, Images (top) and quantification (bottom) of fibroblast subset marker expression among Col1a2^creER R26^TdT fibroblasts in control or cKO mice. 7dpi cKOs show a decrease in αSMA⁺ myofibroblasts (k, quantified as myofibroblast density [left] or proportion [right]). 21dpi cKOs show a trending decrease in FGF13⁺ late inner fibroblasts (l) and decreases in CD80⁺ lymphocyte interactive fibroblasts (m), ALPL⁺ altered dural fibroblasts (n), and LAMA1⁺ pial fibroblasts (p), though ALDH1A2⁺ leptomeningeal/arachnoid fibroblasts are preserved (o). Blue dotted lines show lesion border. n = 3(control) or n = 4(cKO) mice. q, Violin plots showing expression of myofibroblast-related genes among the myofibroblast cluster. r, Gene set enrichment among the myofibroblast cluster, with controls showing ECM-process enrichment. s-t, UMAPs showing myeloid cell subclusters (s) or timepoint (t). u, Violin plots mapping expression of “CNS myeloid cell signatures” (derived from Fig. 2, x-axis) onto clusters identified in s (y-axis). v-w, Pseudotime (v, Monocle3) and UMAP plots separated by condition (w) showing potential myeloid differentiation trajectories, including monocyte-to-SAM (top) and microglia-to-DAM (bottom), with monocytes and microglia as root states. x, Schematic showing effect of TGFβ-activated myofibroblasts on SAM/DAM formation. y-z, Violin plots (y) or feature plots (z) showing “dysmature” transcriptional signature across conditions. n = 1841/1281(Ctrl/ADWA11 DAM), n = 573/5551(Ctrl/ADWA11 SAM), or n = 1724/3181(Ctrl/ADWA11 PVM/BAM) nuclei (y). aa, Surfaced T cells (CD3ε⁺; CD45⁺) in control and cKO mice, with fibroblast-rich (ER-TR7⁺) lesion. ab-ac, UMAP showing lymphocyte subclusters (ab) or genotype (ac, Ctrl and cKO). Dotted black lines highlight CD8 and γδT cells. ad, Violin plot of lymphocyte Itgae expression. n = 495(WT) or n = 336(cKO) nuclei. Two-way Student’s T-test (k-p); over-representation test (one-sided Fisher’s exact test, FDR-adjusted, r); two-way Mann-Whitney test (y [Bonferroni correction], ad). 14μm slices; images represent two or more mice.

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