Extended Data Fig. 7: Different roles of integrin signalling in the development of microglia, meningeal and perivascular macrophages.

(a) Illustrative electron microscopy images depicting the intersection of a brain-penetrating arterial blood vessel with the leptomeninges. Left: overview, 1: higher magnification of the area indicated in the overview image and a consecutive section (1*) of the same area. Arrowheads depicting a gap (in 1) and a direct contact (in 1*) between processes of pial cells that cover the PVS. Yellow: pia mater, Blue: arachnoid mater, Asterisk: PVS, L: vessel lumen, N: neuropil. Representative images out of 6 mice investigated are shown. (b) Representative images showing IBA1⁺ or CD206⁺ cells in the parenchyma, the perivascular compartment, or the brain surface of either control Tln^fl/fl or Cx3cr1^CreTln^fl/fl embryos at E14.5. Col IV staining (red) was also included. Representative images from five (Tln^fl/fl) or six mice (Cx3cr1^CreTln^fl/fl) are depicted. Scale bars: 50 µm. (c) Quantification thereof. Each symbol represents one embryo (n = 5 for Tln^fl/fl; n = 6 for Cx3cr1^CreTln^fl/fl). Three sections/mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. Ns: not significant. (d) Representative images showing claudin 5 (Cldn5, cyan), fibrinogen (red) and laminin (grey) immunoreactivity in the cortex of Cx3cr1^Cre/+Tln1^fl/fl and Tln1^fl/fl controls. Arrowheads indicate fibrinogen immunofluorescence within the vessel lumen. Representative images from four (Tln^fl/fl) or six mice (Cx3cr1^CreTln^fl/fl) are depicted. Scale bar: 50 µm. (e) Quantification of the area covered by Cldn5 or fibrinogen immunofluorescence, respectively. Each symbol represents one mouse (n = 4 for Tln^fl/fl; n = 6 for Cx3cr1^CreTln^fl/fl). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. Ns: not significant. (f) Left, representative immunofluorescence images of IBA1⁺ microglia (red) and CD206⁺ pvMΦ (green), combined with immunofluorescence for Col IV (blue), in control Itgb1^fl/fl and Vav^CreItgb1^fl/fl mice at P14. Representative images from four (Itgb1^fl/fl) or seven (Vav^CreItgb1^fl/fl) mice are depicted. Scale bar: 50 μm. Right, quantification thereof (n = 4 for Itgb1^fl/fl, n = 7 for Vav^CreItgb1^fl/fl). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. (g) Left, representative immunofluorescence images of CD206⁺ mMΦ green) with Col IV⁺ basement membranes (blue) in control Itgb1^fl/fl and Vav^Cre Itgb1^fl/fl mice at P14. Representative images from four (Itgb1^fl/fl) or seven (Vav^CreItgb1^fl/fl) mice are depicted. Scale bar: 50 μm. Right, quantification thereof (n = 4 for Itgb1^fl/fl, n = 7 for Vav^CreItgb1^fl/fl). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test.

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