Fig. 2.

Repopulating microglia have dual origins. a Analysis of chimerism in F4/80^low and F4/80^hi populations in CD45.1 chimeras. Top panel gated on CD11b⁺F4/80⁺Ly6C^–Ly6G^–. Bottom panels gated on F4/80^low and F4/80^hi as indicated. Percentages are mean ± s.d. of n = 5 mice/group. The experiment was performed twice. b Analysis of chimerism in Cx3cr1^GFP/+Ccr2^RFP/+ → Cx3cr1^CreER/+R26^DTA/+ chimeras that had received busulfan chemotherapeutic for myeloablation. Successful separation of GFP⁺ and YFP⁺ populations is demonstrated in Supplementary Fig. 3. Blood is gated on CD11b⁺Ly6G^–CD115⁺Ly6C^hi and CNS is gated on CD11b + Ly6C^–. Values in plots are mean ± s.d of n = 4 and six mice. The experiment was performed once. c Quantification of CX3CR1-YFP⁺Ki67⁺ proliferating microglia in the indicated brain regions. Representative images are from the hippocampus. n = 4–5 mice/group. Lines represent mean values. ***p < 0.001, *p < 0.05 by one-way ANOVA with Dunnett’s Multiple Comparison test. Scale bar 20 μm. d Proliferation in repopulating F4/80^low microglia assessed by EdU. EdU was administered in the drinking water for 14 days during the indicated time periods after TAM. Control mice were given EdU days 0–14 after TAM. Gated on CD11b⁺Ly6C^–Ly6G^–CX3CR1⁺F4/80^low. n = 3–4 mice/group, the experiment was performed twice. Lines represent mean values. ***p < 0.001 by one-way ANOVA with Dunnett’s Multiple Comparison test