Supplementary Figure 4: Drainage in the Prox1^het mice, characterization of the cribriform plate lymphatics, and lethality of i.c.m. injection of tamoxifen and diphtheria toxin.

a, Representative images of exogenously injected T cells (CFSE – green) in the dCLN of Prox1-WT and Prox1-Het mice 12h after i.c.m. injection. Scale bar = 200 µm, 100 µm (insets). b, Quantification of the density of T cells per mm² of dCLN of Prox1-WT and Prox1-Het mice (mean ± s.e.m.; t = 10.72 df = 5; two-tailed unpaired t-test). c, Representative images of exogenously injected fluorescent microbeads (0.5 µm in diameter – red, 1 µm in diameter - green) in the dCLN of Prox1-WT and Prox1-Het mice 2h after i.c.m. injection. Scale bar = 200 µm, 80 µm (insets). d, Quantification of the percentage of beads coverage in the dCLN of Prox1-WT and Prox1-Het mice ((0.25µlof beads in a total of 2µl were injected) mean ± s.e.m.; F(1,9) = 3.038; two-way ANOVA with Sidak’s multiple comparisons test). e, Quantification of the average dCLN section size in Prox1-WT and Prox1-Het mice (mean ± s.e.m.; pooled from 2 independent experiments). f, Representative contour plots of T cells in the meninges of Prox1-WT and Prox1-Het mice. g, Quantification of the number of T cells (TCRb + ) in the meninges of Prox1-WT and Prox1-Het mice (mean ± s.e.m.; pooled from 2 independent experiment; Two-tailed unpaired t-test). h, Representative images of exogenously i.c.m. injected DCs (CFSE – green) in the dCLN of sham and ligated mice. Scale bar = 100 µm. i, Quantification of the density (per mm²) of exogenously i.c.m. injected DCs in the dCLN of sham and ligated mice (mean ± s.e.m.; t = 2.627 df = 7; two-tailed unpaired t test). j, Representative photographs of the mouse cribriform plate. k, Representative photograph of cribriform lymphatics (i) and representative images (Lyve-1 – red) on different sections of the mouse nose (ii). White arrowhead points to a cribriform lymphatic crossing the cribriform plate along the olfactory nerve (laminin – green). Laminin stains the vasculature but also the meninges surrounding the olfactory bulbs and the olfactory nerves. Scale bar = 300 µm. Note that the large majority of the cribriform plate lymphatics are localized on the CNS side of the bone, with some examples of crossing into the nasal side. Representative of 3 independent mice. l, Representative images of VEGFR3 (grey) expression by the cribriform plate lymphatics (Prox1^GFP – green, Lyve-1 – red). Arrowheads point to area of colocalization of Prox1, Lyve-1 and VEGFR3. Scale bar = 100 µm. Representative of 2 independent mice. m, Representative images of podoplanin expression (PDPN – grey) by the cribriform plate lymphatics (Prox1^GFP – green, Lyve-1 – red). Arrowheads point to area of colocalization of Prox1, Lyve-1 and PDPN). Scale bar = 100 µm. Representative of 2 independent mice. n, Representative images of CCL21 (green) expression by the cribriform lymphatics (Lyve-1 – red). Arrowheads point to CCL21⁺ vesicles inside the cribriform LEC. Scale bar = 100 µm. Representative of 2 independent mice. o, Representative images of a sagittal section of Prox1GFP (green) nose region immunostained with Lyve-1 (red). Inset illustrates the large lymphatic bundle localized at the base of the nose draining the lymphatic vasculature of the nasal region. Scale bar = 1000 µm. Representative of 2 independent mice. p, Representative images of the endogenous T cells (CD3e – red) around the lymphatics (Lyve-1 – green) of the cribriform plate of naïve mice. Arrowheads point to T cells. Scale bar = 100 µm. q, Quantification of the number of T cells (CD3e + ) on the CNS, nasal side or within the cribriform plate per section of wild-type mouse cribriform region (mean ± s.e.m.; F(2,12) = 57.22; one way ANOVA with Tukey’s multiple comparison test.) r, Representative images of fluorescently labeled T cells (CFSE – red) around the cribriform plate 12h after low (0.5µl/min) or high (4µl/min) speed of injection. Scale bar = 100 µm. s, Quantification of the number of T cells on the CNS and the nasal mucosa sides of the cribriform plate 12h after i.c.m. injection at 0.5, 1 or 4 µl/min (mean ± s.e.m.; F(2,8) = 11.98; two-way ANOVA with Sidak’s multiple comparison test). t-x, Adult Prox1^CreERT2::DTA^fl/fl and Prox1^CreERT2 mice were injected i.c.m. with 2µl of 4-hydroxi-tamoxifen (TAM - 0.1mg/ml). t, Survival curve of Prox1^CreERT2::DTA^fl/fl and Prox1^CreERT2 mice after i.c.m. injection of TAM (representative of 2 independent experiments). u, Representative images of the lymphatics (Lyve-1 – green) of the superior sagittal sinus of Prox1^CreERT2::DTA^fl/fl after injection of low-dose (100µg) of TAM. Scale bar = 150 µm. Representative of 2 independent mice. v, Survival curve of Prox1^CreERT2::DTR^fl/fl and Prox1^CreERT2 mice after repeated i.c.m. injection of diphtheria toxin (DT) at different concentrations (250ng to 0.25ng; representative of 2 independent experiments). w, Representative images of the meningeal lymphatics (Lyve-1 – green) in Prox1^CreERT2::DTR^fl/fl 10 days after repeated i.c.m. injection of PBS or low dose of DT (25ng). Scale bar = 1000 µm. x, Quantification of the ratio of total lymphatic length over the length of the intracranial sinuses in mice injected i.c.m. with PBS or low dose of DT (mean ± s.e.m.).