Absence of lymphatic vessels in skull bone marrow and cortical bones. (A) Representative images of coronal sections from the parietal bones of adult mice stained with PLVAP (green) and VEGFR3 (red). The white box highlights the zoomed region shown below scale bars: 500 and 100 µm (inset). (B) Schematic showing the experimental procedure in which mice were injected via i.c.m. (intra-cisterna magna) with fluorescent OVA-488 and retro-orbital injection with fluorescently conjugated CD31-PE antibodies to label CSF and blood vessels, respectively. Skulls were collected for clearing and imaging after 30 min. The lower panel diagram indicates OVA-488 labeling of the perivascular space (representing CSF flow) and CD31-PE labeling of blood vessels within the channels. (C and D) Representative images showing bone marrow channels to meninges and periosteum in parietal bones of adult mice, which were injected with OVA-488 (i.c.m.) and CD31-PE (i.v.), followed by staining with VEGFR3 antibodies (white). White dashed lines indicate the boundary between the bone marrow and the channel region, whereas purple dashed lines outline the periosteum and meninges. Scale bars: 20 µm. (E) Diagram outlining the procedure for skull sample collection from Prox1-CreERT2;Ai14 mice, followed by staining of Lyve1 and VEGFR3. (F and G) Representative images showing channels connecting the bone marrow to the periosteum and meninges. Purple dashed lines outline the bone marrow and channel regions, while white dashed lines delineate the periosteum and meninges. Scale bars: 50 µm. (H) Diagram summarizing the marker-positive regions in skull sections from Prox1-CreERT2;Ai14 (red) mice. P: periosteum; BM: bone marrow; M: meninges. All data are represented with n = 4–5 individual mice per experiment.