Extended Data Fig. 1: Preferential localization of perivascular macrophages in the arterial perivascular space during development in mice and humans.

(a) Electron microscopy (EM) showing the establishment of a narrow PVS at P3 in the brains of wildtype mice. Blue: outer vascular and glial basal laminas. Representative images out of 6 mice investigated are shown. SAS: subarachnoid space; E: endothelium; SM: smooth muscle cell; L: vascular lumen. (b, c) P2RY12 (blue) and LYVE1 (magenta) and Col IV (red) immunofluorescence in the cortex of Cx3cr1^GFP/+ (green) mice. Arrowheads indicate double positive (filled) or single positive (blank) cells. Pictures representative out of at least three investigated mice per time point are shown. Scale bars: 50 μm. (d) Quantification thereof. Symbols represent individual mice. Bars indicate means ± s.e.m. (n = 4: E14.5 and P10 P2RY12, n = 3: E14.5 LYVE1, n = 5: P10 LYVE1). (e) Top: Immunofluorescence of CD206 (white) and laminin (blue, basement membranes) in the cortex of adult mice. Bottom: Most of the CD206⁺ perivascular macrophages (pvMΦ) are located in alpha smooth muscle actin (aSMA, red)⁺ transferrin receptor (TfR, green)^– arteries/arterioles, with a minor population in aSMA^-TfR⁺ veins/venules. Typical pictures out of 6 mice (3 male and 3 female) investigated are shown. A, artery/arteriole. Cap, capillary. V, vein/venule. Scale bar: 200 μm. (f) High-magnification images of dashed frames shown in d. Arrowheads indicate CD206⁺ pvMΦ. Scale bar: 50 μm. (g) Distribution of CD206⁺ pvMΦ in different vessels types (mean ± s.e.m., n = 3 mice for both genders). (h) Left: Immunofluorescence of CD206 (magenta or green), SOX17 (blue) and aSMA (red) in the cortex of adult Cx3cr1^GFP/+ (green) mice. Pictures representative of three mice investigated. Scale bar: 50 μm. Right: Frequency of CD206⁺ CX3CR1–GFP⁺ pvMΦ within aSMA⁺ vessels (n = 3 mice). Three sections per mouse were quantified (means ± s.e.m.). (i) Left: Immunofluorescence of CD206 (green), endomucin (blue) CD31 (red) in the cortex of adult Cx3cr1^GFP/+ (green) mice. Pictures representative of three mice investigated. Scale bar. 50 μm. Right: quantification of CD206⁺ CX3CR1–GFP⁺ pvMΦ within endomucin^- CD31⁺ vessels (n = 3 mice). Three sections per mouse were quantified (means ± s.e.m.). (j) Representative images showing CX3CR1–GFP⁺ pvMΦ in endomucin^- Col IV⁺ vessels at different developmental time points. Arrowheads indicate CX3CR1–GFP⁺ pvMΦ located in the endomucin^- vessels. Images representative of 3 mice for each timepoint investigated. Scale bar: 50 μm. (k) EM illustrating that pvMΦ (yellow) are preferentially localized in PVS of arterial vessels of wild-type mice at P21. Vascular smooth muscle cells (blue) are lacking in venous vessels. Images representative of 6 mice investigated. L: lumen, N: neuropil, E: endothelial cell. Arrows: glial basement membrane. Asterisks: processes of fibroblasts. (l) Distribution of pvMΦ around arteries and veins based on quantification in EM images in cortical sections of P21 mouse brains. Multiple non-consecutive sections per mouse were quantified. Symbols represent individual mice (n = 6, mean ± s.e.m.). (m) Immunohistochemistry for CD206 (brown) combined with Elastica-Van-Gieson (EvG) staining in human cortical brain. Asterisks: pvMΦ, arrowheads: EvG-positive arteries. Images representative of one sample per time point.

Source data