Fig. 1: Identification of a dynamic and persistent fibroblast response to brain injury.

a, Homeostatic brain fibroblasts are labelled in Col1a2^creER; Rosa26^tdT+ mice. Pial/perivascular fibroblasts (top right), periventricular/choroid plexus fibroblasts (bottom right). Mice were treated with tamoxifen from day −9 to day −7 before mice were euthanized for imaging. Scale bars: 2,000 μm (left), 200 μm (right). b, Lesional fibroblasts are labelled in Col1a1^GFP+; Col1a2^creER; Rosa26^tdT+ mice after PT cortical brain injury (image of GFAP⁺ gliosis (far right) is from a separate mouse). Mice were treated with tamoxifen from day −16 to day −14 before injury. Scale bars, 200 μm. c, A fibrotic lesion labelled for collagen 1 (COL1) and ER-TR7 (a fibroblast marker monoclonal antibody) within a glial scar (GFAP⁺) in the controlled cortical impact (CCI) model of TBI. Scale bars: 500 μm (left), 100 μm (right). d, Lesional fibroblasts after tMCAO stroke. Mice were treated with tamoxifen on days −7 to −5 before injury to track ontogeny. Scale bar, 250 μm. e, Time course showing collagen 6 (COL6) expression and persistence after endothelin-1-induced (ET-1) ischaemic stroke in adult marmosets. Scale bars, 250 μm. f,g, Time course in the PT injury model, showing fibroblasts (f; Col1a1-GFP⁺) and/or associated ECM (g; ER-TR7). Scale bars: 500 μm (f), 200 μm (g). h, Immunofluorescence showing astrocyte-dense (GFAP⁺) and fibroblast-dense regions (ER-TR7⁺) (top) and spatial plots (bottom, from same tissue) with 55-μm spot-based clusters. Early necrosis-associated autofluorescence is prominent; white dotted lines denote lesion borders (10X Genomics, Visium). i,j, Uniform manifold approximation and projection (UMAP) plots showing spot-based clusters (i) or fibroblast-enriched spots (j; Col1a1⁺). Four main fibroblast-enriched clusters are highlighted (black dashed line). k, Time course showing fibroblast-enriched spots in UMAP (top) or spatial (bottom) plots. l,m, UMAP and spatial plots (l) and volcano plot (m) comparing 7 dpi (early) fibroblast-enriched signature (cluster 10_0, red) and 21 dpi (late) fibroblast-enriched signature (cluster 8, blue). n,o, TGFβ score (n; genes upregulated in lung adventitial fibroblasts cultured with TGFβ) and proliferation score (o; 23-gene signature) across early and late fibroblast signatures. n = 118 (early) and n = 160 (late) spots. MAST (model-based analysis of single-cell transcriptomics) test (hurdle model with likelihood ratio test, false discovery rate (FDR)-adjusted; m); two-way Mann–Whitney test (n,o). Slice thickness: 200 μm (a), 14 μm (b,f,g), 30 μm (c), 50 μm (d), 40 μm (e) and 10 μm (h). Images represent two or more mice.