S1PR1 signaling is active in LVs and is necessary for LV development. (A) The mesenteries of P10 S1PR1-GFP and H2B-GFP littermates where analyzed. Collecting lymphatic vessels of S1PR1-GFP pups were GFP⁺ and GFP expression was stronger in the LVs (arrowheads). GFP expression was not observed in H2B-GFP pups. (B) The mesenteries of P10 S1pr1^f/f;Prox1-tdTomato and S1pr1^iΔLEC;Prox1-tdTomato pups that were administered TM from P1–7 were analyzed. Mesenteric tissue that was connected to the duodenum and jejunum was considered “proximal,” and that which is connected to the ileum and cecum was considered “distal,” LVs (puncta) in the proximal and distal vessels were counted and quantified. LVs were significantly reduced in S1pr1^iΔLEC;Prox1-tdTomato pups. The reduction appeared to be more severe in the posterior section of the gut. (C) The mesenteric lymphatic vessels and LVs of P10 pups that were administered TM from P1–7 were analyzed using the indicated antibodies. CX37 expression appeared to be reduced in the remaining LVs of S1pr1^iΔLEC pups. Statistics: (A) n = 9 S1PR1-GS and n = 3 H2B-GFP pups; (B) n = 5 control and n = 8 S1pr1^iΔLEC pups. Each dot represents an individual animal on the graph for the total number of LVs. Three proximal vessels and three distal vessels from each mesentery were analyzed to quantify the valve density. Each dot represents a vessel on the graph; (C) n = 3 per genotype. The graphs were plotted as mean ± SD. Unpaired t test was performed for the statistical analysis. ****P < 0.0001.