Retrograde fluorescent tracer flow into lymphatic plexuses and thoracic cavity. Image orientation: caudal (left), rostral (right). A: Bright-field image of region captured by time-lapse microscopic imaging. B: First frame at 3 seconds (left panel) after start of injection and last frame at 120 seconds (right panel) of a time-lapse video showing fluorescent Ricinus communis agglutinin I (RCA I) lectin (white) confined to thoracic duct of control mouse. C: Time-lapse video frames of ADN–VEGF-C mouse showing rapid movement of tracer out of thoracic duct (9 seconds), into adjacent lymphatics (22 seconds), and leakage into the thoracic cavity (45 and 120 seconds). Arrowheads mark diffuse extravasated tracer. D: Fluorescent lectin was confined to thoracic duct (blue), and not present in paravertebral lymphatics, in 100% of six single transgenic control mice, but was found in paravertebral lymphatics (red) within 6 seconds (two frames) after reaching the thoracic duct of 26 (90%) of 29 ADN–VEGF-C mice. E: Images of fluorescent tracer leakage before (left panel, 120 seconds after injection) and after (right panel) PBS wash of pleural surface showing that some tracer was removed (arrowheads), consistent with entry into the thoracic cavity. n = 6 mice (E). Scale bar = 2 mm (A–C and E).