Supplementary Figure 1: Lack of dural leakage and characterization of hot spots of the meningeal lymphatic vessels.

a, Representative images of the lymphatic vessels immunostained by both an i.c.v. injected anti-Lyve-1^A488 and exogenously applied anti-Lyve-1^A660 at different time points after i.c.v. injection. Inset at 5 and 15 min illustrate the initial points where the i.c.v. injected anti-Lyve-1^A488 labelled the meningeal lymphatics. Inset at 60 min illustrate the lack of staining of the middle meningeal artery lymphatics. Scale bar = 1000 µm, 75 µm (insets). Representative of 2-3 independent mice per group. b, Representative images of the meningeal beads around the meningeal lymphatics of the transverse sinuses after i.c.m., i.c.v. or i.n. injection of 2µl of fluorescently conjugated beads. Scale bar = 300 µm, 75 µm (insets). Arrowheads in the i.n. injection inset points towards individual beads. Representative of 3 independent mice. c, Representative images of whole mount meninges immunostained for macrophages (Iba1 – green) one hour after i.c.m. injection or ex-vivo exposure to fluorescent ovalbumin. Scale bar = 500 µm, 120 µm (insets). Representative of 2-3 independent mice per group. d, Quantification of the ovalbumin uptake by meningeal macrophages in different meningeal regions one hour after i.c.m. injection or ex-vivo exposure to ovalbumin (mean ± s.e.m.) e_i, Representative images of Ova diffusion in the skullcap meninges (dura) and at the surface of the brain (pia). Scale bar = 1000 µm. e_ii, Representative images of the diffusion of Ova (both perivascular diffusion and macrophage uptake) on different region of the cerebellum 10 min after i.c.m. injection. Scale bar = 300 or 200 µm. Images in e, are representative of 3 independent mice. f, Representative images of VE-Cadherin and Claudin-5 expression on lymphatic sprouts of the meninges (upper panel) and diaphragm (lower panel) of adult wild-type mice. Scale bar = 20 µm (meninges) and 30 µm (Diaphragm). Representative images of sprouts from 2 mice. g, Representative side view of the meningeal lymphatics along the upper region of the superior sagittal sinus by multiphoton microscopy. The meningeal lymphatics (Prox1^GFP – green) are detected below the skull and the dura matter (secondary harmonic – blue). Yellow arrow points to sub-arachnoid lymphatic extensions. Scale bar = 40 µm. Representative of 2 independent animals. h, Prox1^GFP mice were injected intravenously (i.v.) with 5µl of Qdot⁶⁵⁵ (diluted in 95µl of saline). The transverse sinus was imaged through a thinned skull ~ 5min after injection. Representative images of the complex meningeal lymphatics associated with the transverse sinuses at the lymphatic extension-rich area (arrows point to lymphatic extensions). Arrow on the 3D reconstruction points to a descending lymphatic extension directed towards the SAS. Scale bar = 60 µm. Representative of 2 independent animals.