Autocrine or paracrine S1PR1 signaling regulates LV development and TLO formation. (A and B) Mesenteric (A) and dermal (B) lymphatic vessels of Sphk1/2^ΔLEC mice, in which S1P synthesis in LECs was ablated, were analyzed. (A) Stitched images of mesenteric lymphatic vessels revealed fewer LVs in P10 Sphk1/2^ΔLEC pups. The remaining LVs appeared immature (bottom row). Representative valves are within dotted boxes, and their enlarged images are shown below. (B) Dermal lymphatic vessels of 3-mo-old Sphk1/2^ΔLEC mice had fewer LVs (yellow arrows) and more branches per field. (C) The mesenteries of 1-year-old S1PR1-GS and S1PR1-GS;Sphk1/2^ΔLEC mice were analyzed. GFP autofluorescence was observed in the lymphatic vessels of S1PR1-GS mice (arrows). Weaker GFP expression was observed in blood vessels. In contrast, lymphatic vessels could not be identified based on GFP autofluorescence in S1PR1-GS;Sphk1/2^ΔLEC mice. However, GFP expression was observed in blood vessels and in nodule-like structures (arrows). (D) IF for GFP and VEGFR3 revealed a dramatic downregulation of GFP expression in the lymphatic vessels of S1PR1-GS;Sphk1/2^ΔLEC mice. (E) IF for LYVE1 and B220 revealed significant number of TLOs in 3-, 6- and 12-mo-old Sphk1/2^ΔLEC mice. The picture shows a representative TLO from a 12-mo-old Sphk1/2^ΔLEC mouse. Statistics: (A) n = 3 control and n = 4 Sphk1/2^ΔLEC pups. Total number of LVs in the entire mesentery were counted. Each dot in the graph represents an individual animal. Three proximal vessels and three distal vessels from each mesentery were analyzed to quantify the valve density. Each dot in the graph represents a vessel; (B) n = 4 controls and n = 3 Sphk1/2^ΔLEC mice. (C) n = 7 S1PR1-GS and n = 8 S1PR1-GS;Sphk1/2^ΔLEC mice; (D) n = 3 6-mo-old S1PR1-GS and n = 3 S1PR1-GS;Sphk1/2^ΔLEC littermates; (E) 3-mo-old mice: n = 4 control and n = 4 Sphk1/2^ΔLEC; 6-mo-old mice: n = 6 control and n = 8 Sphk1/2^ΔLEC; and 1-year-old mice: n = 5 control and n = 5 Sphk1/2^ΔLEC. Graphs were plotted as mean ± SD. Unpaired t tests (A), Welch’s t tests (D), and Mann–Whitney tests (B and E) were performed for the statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001.