Extended Data Fig. 1: Additional characterization of the CNS fibroblast response to PT injury, related to Fig. 1.

a, Homeostatic dural meningeal fibroblasts (Col1a2^creER; Rosa26^TdT+; whole-mounted dura, tamoxifen days ⁻11–⁻7 before harvest). b-c, fibroblast infiltration (Col1a1^GFP+, b, or Col1a2^creER; Rosa26^TdT+, c) after CCI model of TBI (tamoxifen days ⁻18–⁻16). d, Fibroblast expansion near lesion borders (white dotted line) by 4dpi (tamoxifen days ⁻14–⁻12,⁻7,0–2dpi). e-f, Flow plot (e) and quantification (f) of fibroblast nuclei in Pdgfrα^GFP mice (marking fibroblasts and oligodendrocyte precursor cells [OPC]). n = 7(resting cortex), n = 11(ipsilateral cortex/lesion, 14dpi), or n = 8(dura, resting or 14dpi) mice. g-h, Expanded perilesional vasculature and associated ECM after PT injury, with Pdgfrα^GFP+ nuclei (g) or fibroblasts (h) visualized in lesional perivascular spaces. i-l, Resting fibroblasts (Col1a1^GFP+) partially recombined by Col1a2^creER (i.e., TdT⁺; i), quantification of GFP expression within recombined cells (j), 21dpi lesional fibroblasts (Col1a1^GFP+) with a fibroblast origin (TdT⁺) (k), and quantification of recombination efficiency before and after injury (l). Tamoxifen days ⁻16–⁻14 (before injury, “uninjured”/“lineage-traced”) or 7–9dpi (“post-injury induction”). n = 6(uninjured), n = 5(lineage traced), or n = 3(post-injury) mice (1 40μm slice [uninjured] or 2 14μm slices/mouse). m, Lineage-traced fibroblasts in Gli1^creER; Rosa26^TdT mice (preferentially recombining fibroblast subsets in multiple tissues). Tamoxifen days ⁻16–⁻14 before injury. n, Time course showing stromal/fibroblast (Twist2^cre; Rosa26^TdT+) expression of fibroblast markers (Dcn, Col6a1, Postn) and pericyte marker (Desmin). Serial sections; images representative of n = 2–3 (2dpi), n = 2–6 (7dpi), or n = 2–7 (21dpi) mice. o-q, Lack of lineage-traced lesional cells (with extra-lesional accumulation) in Ng2^creER; Rosa26^TdT mice (o), images of Ng2-lineage⁺ pericytes (Desmin⁺) or oligodendrocyte-lineage cells (SOX10⁺; p), and quantification of recombination efficiency within each lineage (q). n = 4 mice. Tamoxifen days ⁻16–⁻14 before injury. r, Lack of lineage-traced lesional cells (with sparse smooth muscle visible) in Acta2^creER; Rosa26^TdT mice. Tamoxifen days ⁻16–⁻14 before injury. s, Quantification of lesional recombined cells in Acta2^creER, Ng2^creER, Gli1^creER, and Col1a2^creER mice. n = 3(Acta2^creER, Gli1^creER, Col1a2^creER) or n = 4(Ng2^creER) mice. t-u, Lineage traced fibroblasts (t, Col1a2^creER; Rosa26^TdT) but not pericytes (u, Atp13a5^creER; Rosa26^TdT) accumulating within fibrotic lesions (Col1⁺) after distal Middle Cerebral Artery Occlusion (dMCAO), 14dpi. Tamoxifen given for 3 days, 6–8 weeks prior to injury. v-w, Images of Atp13a5-lineage⁺ pericytes (v, PDGFRβ⁺, NG2⁺) and quantification of recombination efficiency (w). n = 3 mice (3 slices/mouse). x, Quantification of lesional or contralateral recombined pericytes or fibroblasts (TdT⁺ area in Atp13a5^creER or Col1a2^creER; Rosa26^TdT mice) after dMCAO, 14dpi. n = 6(Atp13a5^creER) or n = 5(Col1a2^creER) mice (2–3 slices/mouse). y-ac, Schematic (y) showing transcranial 4-hydroxy-tamoxifen (4-OHT) induction (top) and intraperitoneal tamoxifen induction (bottom). Transcranial 4-OHT sparsely recombines dural (z) but not perivascular or leptomeningeal (pial or arachnoid) fibroblasts (aa), highlighted with arachnoid markers ALDH1A2 and E-Cadherin (ab, top and bottom) and quantified in ac. Shown 2–10 days after induction; n = 3(intraperitoneal), n = 5(transcranial-perivascular/leptomeningeal), or n = 6(transcranial-dural) mice. ad-ae, Image (ad) and quantification (ae) of lesional fibroblasts recombined via transcranial 4-OHT. n = 6(lesion [2 slices/mouse] and dura-transcranial) or n = 3(dura-intraperitoneal) mice. One-way ANOVA, Tukey post-test (f,l,s); two-way ANOVA, Sidak’s post-test (x [repeated measures], ae); multiple two-way T-tests, Holm-Sidak correction (ac). 14μm (b-d,h,k,m-p,r,ad), 200μm (g), 40μm (i,n [desmin],aa,ab), or 10μm (t,u,v) slices; images represent two or more mice.

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