Extended Data Fig. 3: Repopulation dynamics of CNS macrophages throughout the brain.

(a) Experimental scheme. HSC: hematopoietic stem cell; MG: microglia, Mono: monocyte, TAM: Tamoxifen. (b-d) Flow cytometric quantification of reporter gene expression in Ly6C^hi and Ly6C^lo blood monocytes from Hexb^CreERT2R26^YFP (b), Mrc1^CreERT2R26^tdT (c) and Cxcr4^CreERT2R26^tdT (d). Symbols represent individual mice, Hexb^CreERT2R26^YFP: n = 3 (2w CTL), n = 4 (2w BLZ945, 8w, 26w), Mrc1^CreERT2R26^tdT: n = 3 (2w CTL, 26w CTL), n = 4 (2w BLZ945, 26w BLZ945), n = 6 (8w), Cxcr4^CreERT2R26^tdT: n = 3 (2w CTL, 8w CTL, 12w CTL, 26w CTL), n = 4 (8w BLZ945, 12w BLZ945), n = 5 (26w BLZ945), n = 6 (5d CTL), n = 8 (5d BLZ945), mean ± SEM. Ordinary one-way ANOVA with Šidák-adjusted multiple-comparisons p values. (e-f) Representative images (e) and quantifications (f) of labeled MG, leptomeningeal (lmMΦ) and perivascular (pvMΦ) macrophages. Symbols represent individual mice, Hexb^CreERT2R26^YFP: n = 4; Mrc1^CreERT2R26^tdT: n = 4 (olfactory tubercle BLZ945), n = 5 (all other groups), mean ± SEM. Unpaired two-tailed t-test with Welch’s correction. Arrowheads: magenta = YFP⁺ MG; red = YPF⁻ MG; blue = YFP⁻ leptomeningeal (lmMΦ) and perivascular (pvMΦ) macrophages; open = tdT⁺ lmMΦ and pvMΦ; orange = tdT⁻ lmMΦ and pvM. (g) Representative images from Cxcr4^CreERT2R26^tdT mice. Arrowheads: open = tdT⁻ lmMΦ and pvMΦ; orange = tdT⁺ lmMΦ and pvMΦ. (h-k) Flow cytometric analysis. Representative histograms (h) and quantification of reporter gene expression in MG and sdCAMs from Hexb^CreERT2R26^YFP (i), Mrc1^CreERT2R26^tdT (j) and Cxcr4^CreERT2R26^tdT (k). Symbols represent individual mice, Hexb^CreERT2R26^YFP: n = 3 (2w CTL), n = 4 (2w BLZ945, 8w, 26w); Mrc1^CreERT2R26^tdT: n = 3 (2w CTL, 26w CTL), n = 4 (2w BLZ945, 26w BLZ945), n = 6 (8w); Cxcr4^CreERT2R26^tdT: n = 3 (5d CTL, 2w CTL, 8w CTL, 12w CTL, 26w CTL), n = 4 (8w BLZ945, 12w BLZ945), n = 5 (5d BLZ945, 26w BLZ945), n = 6 (2w BLZ945), mean ± SEM. Ordinary one-way ANOVA with Šidák-adjusted multiple-comparisons p values. (l) Representative images showing Ki-67 immunofluorescence in the cortex of Cxcr4^CreERT2R26^tdT mice 5d post CTL or BLZ945 treatment, respectively and quantification. Arrowheads: blue = Ki-67⁻ lmMΦ and pvMΦ; red = Ki-67⁺ lmMΦ and pvMΦ. Symbols represent individual mice, n = 3 per group, mean ± SEM. Unpaired two-tailed t-test with Welch’s correction (MG) or ordinary one-way ANOVA with Šidák’s multiple comparison test, multiplicity adjusted p values (lmMΦ and pvMΦ).

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