Extended Data Fig. 2: Kinetics of microglia, meningeal and perivascular macrophages as a HSC-independent cell population during development.

(a) Left: representative images showing Ki-67⁺ CX3CR1–GFP⁺ cells in the parenchyma of Cx3cr1^GFP/+ mice at P10. Arrowheads indicate double positive cells. Representative pictures out of four mice investigated are shown. Scale bar. 50 μm. Right: density (cells / mm²) and frequency (ratio of Ki-67⁺ cells out of all CX3CR1–GFP⁺ cells) of Ki-67⁺ CD206⁻ CX3CR1–GFP⁺ cells in the parenchyma of Cx3cr1^GFP/+ mice during development (n=3 mice for E16.5, P3, P5, P21, P28; n = 4 mice for E12.5, E14.5, P0, P7, P10, P14). Three sections per mouse were quantified (means ± s.e.m.). (b) Left: representative images showing Ki-67⁺ cells in the Col IV⁺ perivascular compartment of Cx3cr1^GFP/+ mice at P10. Filled arrowheads: CD206⁺ CX3CR1–GFP⁺ Ki-67⁺ cells, blank arrowheads: single Ki-67⁺ cell. Images representative of four mice investigated. Scale bar: 50 μm. Right: density and frequency of Ki-67⁺ CD206⁺ CX3CR1–GFP⁺ or Ki-67⁺CD206⁻CX3CR1–GFP⁺ cells in the perivascular compartment of Cx3cr1^GFP/+ mice (n=3 mice for E16.5, P3, P5, P21, P28; n = 4 mice for E12.5, E14.5, P0, P7, P10, P14). Three sections/mouse were quantified (means ± s.e.m.). (c) Left, representative images showing Ki-67⁺ or Ki-67⁻ cells in the Col IV⁺ brain surfaces of Cx3cr1^GFP/+ mice at E14.5, P3, and P21. Filled arrowhead indicate CD206⁺CX3CR1–GFP⁺Ki-67⁺ cells, and blank arrowhead indicate CD206⁺CX3CR1–GFP⁺Ki-67⁻ cells. Representative pictures out of four mice investigated are shown. Scale bar: 50 μm. Right, density and frequency of CD206⁺CX3CR1–GFP⁺Ki-67⁺ or CD206⁻ CX3CR1–GFP⁺Ki-67⁺ cells in the brain surfaces of Cx3cr1^GFP/+ mice during development (n=3 mice for E16.5, P5, P14, P21, P28; n = 4 mice for E12.5, E14.5, P0, P3, P7, P10, P56). Three sections per mouse were quantified (means ± s.e.m.). (d) Upper, representative images showing active-caspase 3 (Cas3)⁻ CX3CR1–GFP (GFP)⁺ cells in the perivascular compartment of Cx3cr1^GFP/+ mice at E14.5. Filled arrowhead indicates GFP⁺Cas3⁻ cell, and blank arrowhead indicates single Cas3⁺ cell. Representative pictures out of four mice investigated are shown. Scale bar: 50 μm. Bottom, quantification of Cas3⁺ CX3CR1–GFP⁺ cells in the perivascular compartment of Cx3cr1^GFP/+ mice during embryogenesis (n = 4 for all timepoints). N.d., not detected. Three sections per mouse were quantified. (e) Scheme of experimental set-up. Pregnant Cx3cr1^CreERT2/+R26^YFP/YFP females were injected with 4OH-TAM at E9.5, and the embryos or the offspring were then analysed at E14.5, P0, or P14, respectively. (f) Representative immunofluorescence images showing YFP-expressing IBA1⁺ cells, in combination with Col IV⁺ basal membrane, in the parenchyma, perivascular compartment, and brain surface of Cx3cr1^CreERT2/+R26^YFP/YFP embryos (E14.5) or offspring (P0, P14). Filled yellow, filled white, blank white arrowheads indicate double positive cells in perivascular compartment, double positive cells in the parenchyma or meninges, or single positive cells, respectively. Representative pictures out of five mice investigated are shown. Scale bar: 50 μm. (g) Quantification thereof. Each symbol represents one mouse (n = 5). Means ± s.e.m. are shown. (h) Illustration of the experimental procedure for tamoxifen injection (TAM) and subsequent analysis of adult Cxcr4^CreERT2/+R26^tdT/+ mice. (i) Frequency of tdTomato (tdT)⁺ Ly6C^lo and Ly6C^hi monocytes in the blood of Cxcr4^CreERT2/+R26^tdT/+ mice after tamoxifen injection (n = 5 mice for all timepoints tested). Data are depicted as mean ± s.e.m. (j) Typical images showing that tdT (filled white arrowheads) is not expressed in IBA1⁺ microglia (green), CD206⁺ pvMΦ (green) or CD206⁺ meningeal macrophages (mMΦ, green), but in IBA1⁺ choroid plexus macrophages (cpMΦ, green) and in the lumen of CD31⁺ endothelial cells (green) in the brains of Cxcr4^CreERT2/+R26^tdT/+ mice 8 weeks after TAM injection. Yellow and blank arrowheads indicate double positive or single positive cells, respectively. EC, endothelial cell. Representative images out of 5 mice investigated are shown. Scale bars: 50 μm. (k) Frequency of tdT positive microglia, pvMΦ, and mMΦ in the cortex, and cpMΦ of Cxcr4^CreERT2/+R26^tdT/+ mice after TAM treatment (n = 5 mice for all timepoints tested). Data are depicted as mean ± s.e.m.

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