Extended Data Fig. 6: No contribution of bone-marrow-derived cells or microglia to the pool of perivascular macrophages during development, but dependence on the transcription factors Irf8 and Mafb.

(a) Scheme of the experimental set-up. (b) Flow cytometry-based quantification of tdT-labelled Ly6C^hi or Ly6c^lo blood monocytes in Cxcr4^CreERT2/+R26^tdT/+ mice (n = 3). Symbols represent individual mice. Data are shown as mean ± s.e.m. (c) Left, representative immunofluorescence images of tdT signals (red) in vessel cells, but not in pvMΦ (CD206, green) and microglia (IBA1, cyan), combined with immunofluorescence for Col IV (blue), from Cxcr4^CreERT2/+R26^tdT/tdT mice at P14. Blank white, and blank yellow arrowheads indicate single positive microglia or single positive pvMΦ, respectively. Representative images from three mice are depicted. Scale bars: 50 µm. Right, quantification of tdT-expressing microglia (MG, IBA1), pvMΦ (CD206) and mMΦ (CD206) in Cxcr4^CreERT2/+R26^tdT/tdT mice. Each symbol represents one mouse (n = 3). Three sections per mouse were quantified (means ± s.e.m.). (d) Scheme of experimental set-up. (e) Flow cytometry-based quantification of YFP-labelled Ly6C^hi or Ly6c^lo blood monocytes in Hexb^{CreERT2/CreERT2}R26^YFP/YFP mice (n = 7). Each symbol represents one mouse. Data are shown as mean ± s.e.m. (f) Left, representative immunofluorescence images of YFP signals (green) in microglia (IBA1, purple), but not in pvMΦ (CD206, red) and mMΦ (CD206, red), combined with immunofluorescence for Col IV (blue), from Hexb^{CreERT2/CreERT2}R26^YFP/YFP mice at P14. Filled white and blank yellow arrowheads indicate double positive microglia or single positive pvMΦ/mMΦ, respectively. Representative images from seven mice are depicted. Scale bars: 50 µm. Right, quantification of YFP-expressing microglia (MG, IBA1⁺), pvMΦ (CD206⁺) and mMΦ (CD206⁺) in Hexb^{CreERT2/CreERT2}R26^YFP/YFP mice. Each symbol represents one mouse (n = 7). Three sections per mouse were quantified. Means ± s.e.m. (g) Scheme of experimental set-up. (h) Quantitative PCR of Irf8 mRNA levels in sorted microglia and CAMs from Cx3cr1^CreERT2/+Irf8^fl/fl and controls at P6. Each symbol represents one mouse (n = 6 for control, n = 10 for Cx3cr1^CreERT2/+Irf8^fl/fl). Two-sided unpaired t-test. (i) Representative immunofluorescence images from the cortex of Cx3cr1^CreERT2/+Irf8^fl/fl and control mice for CD11b (red), CD206 (green) and laminin (blue) depicting microglia (blank arrowheads) and pvMΦ (filled arrowheads). Scale bars: 50 µm. (j) Quantification of microglia (MG) and pvMΦ. Each symbol represents one mouse (Irf8^fl/fl, n = 6; Cx3cr1^CreERT2/+Irf8^fl/fl, n = 7). Three sections per mouse were quantified. Means ± s.e.m. Two-sided unpaired t-test. (k) Histological pictures from the meninges of Cx3cr1^CreERT2/+Irf8^fl/fl and control mice for CD11b (red), CD206 (green) and laminin (blue) showing microglia (blank arrowheads) and mMΦ (filled arrowheads). Scale bars: 50 µm. (l) Quantification thereof. Each symbol represents one mouse (Irf8^fl/fl, n = 6; Cx3cr1^CreERT2/+Irf8^fl/fl, n = 7). Three sections per mouse were quantified. Means ± s.e.m. Two-sided unpaired t-test. (m) Scheme of experimental set-up. (n) Quantitative PCR of Mafb mRNA levels in sorted microglia and CAMs from Cx3cr1^CreERT2/+Mafb^fl/fl (n = 9 mice) and controls (n = 10 mice) at P6. Each symbol represents one mouse. Two-sided unpaired t-test. (o) Typical histological images from the cortex of Cx3cr1^CreERT2/+Mafb^fl/fl and control mice for CD11b (red), CD206 (green) and laminin (blue) depicting pvMΦ (filled arrowheads). Scale bars: 50 µm. (p) Quantification of microglia (MG) and pvMΦ. Each symbol represents one mouse (n = 7 per genotype). Three sections per mouse were quantified. Means ± s.e.m. Two-sided unpaired t-test. (q) Histological pictures from the meninges of Cx3cr1^CreERT2/+Mafb^fl/fl mice for CD11b (red), CD206 (green) and laminin (blue) showing mMΦ (filled arrowheads). Scale bars: 50 µm. (r) Quantification thereof. Each symbol represents one mouse (n = 7 per genotype). Three sections per mouse were examined. Means ± s.e.m. Two-sided unpaired t-test.

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