Figure 4.

Coincidence of subpleural lymphatic plexuses and leakage sites. Image orientation: caudal (left), rostral (right). ADN–VEGF-C mice were analyzed after 0.075 mg/mL doxycycline for 7 days. A: Left panel: Whole mount of dorsal surface of thorax of Prox1-GFP control mouse after horseradish peroxidase (HRP) staining for GFP. Right panel: Drawing of whole mount where green lines show GFP⁺ lymphatic vessels [thoracic duct (TD), paravertebral lymphatic vessel (PVL), and lymphatic plexus (LP)]. Gray lines mark the location of ribs. B: Paravertebral lymphatic vessel (arrows indicate valves) and connecting lymphatic (arrowheads indicate valves) to subpleural lymphatic plexuses. C: Fluorescence stereomicroscopic image of the pleural surface of the chest wall of a 3-week-old Prox1-GFP mouse showing lymphatic vessels (green; arrowheads) that connect the TD to PVLs. Some regions of the connecting lymphatics (arrow) are obscured by overlying tissue. D: Images of a region of chest wall showing sites of tracer leakage (red; left panel), LYVE-1–HRP staining of lymphatic plexuses (brown; middle panel), and an overlay of the two (white; LYVE-1–HRP; right panel). E: Enlarged images of lymphatic plexuses (LYVE-1–HRP) in chest wall of single transgenic control mouse and ADN–VEGF-C mouse, the latter showing bud-like outgrowths (arrowheads). F: Regional area per lymphatic plexus in single-transgenic and ADN–VEGF-C mice. Each dot shows the area of one plexus. Red lines indicate plexus area means. n = 7 mice (D); n = 4 mice per group (E and F). ^∗∗P < 0.01 (t-test). Scale bars: 4 mm (A); 1 mm (B–E). RCA I, Ricinus communis agglutinin I.