LVs are reduced in the numbers and defective in S1pr1 ^iΔLEC mice. (A and B) 3-mo-old S1pr1^iΔLEC mice that were treated with TM either from P1–P7 (A) or at 8 wk of age (B) were studied. (A) Dermal lymphatic vessels in the ears of S1pr1^iΔLEC (TM@P1–P7) mice had more branches and fewer claudin-5^hi LVs (arrowheads) when compared with control littermates. (B)S1pr1^iΔLEC (TM@8w) mice had elevated number of branch points but did not have any obvious reduction in LVs. (C) Representative stitched image of a terminal ileum-draining mesenteric lymphatic vessel of a control mouse with LVs (arrows) is shown. A corresponding stitched image of a lymphatic vessel from an S1pr1^iΔLEC (TM@P1–7) mouse lacking LVs is also shown (red arrowhead indicates where small nubs remain from a valve). The graph shows that the LV density is significantly reduced in S1pr1^iΔLEC (TM@P1–7) but not in S1pr1^iΔLEC (TM@8w) mice. (D) Ex vivo analysis of LVs in the ileum-draining lymphatic vessels. LVs of control, S1pr1^iΔLEC (TM@P1–7), and S1pr1^iΔLEC (TM@8w) mice were analyzed for back leak. The graph shows that the LVs in the terminal ileum-draining lymphatic vessels of mutant mice were significantly leaky irrespective of the time of gene deletion. (E) Following the back leak analysis, some leaky valves fixed for subsequent immunofluorescence to assess the specific LV structural component(s). IHC was performed on isolated vessel for the indicated markers, imaged by confocal microscopy, and 3D reconstructed. The 3D images were rotated to visualize the LVs on their side (left) or en face (right). LVs with two symmetrical leaflets were observed in control mice. However, in 1 of 6 S1pr1^iΔLEC (TM@P1–7) LVs imaged, only one partial leaflet (arrows, Prox1^Hi-ITGA9⁺) was observed at the LV site, which resulted in complete back leak. (F) We performed live confocal imaging on 3 S1pr1^iΔLEC (TM@P1–7) LVs that exhibited various levels of back leak and two control LVs without back leak using the Prox1-tdTomato reporter under various levels of Pin and Pout. Both control and mutant LVs remained open when Pin and Pout were equal and closed when Pout was slightly elevated. The control LV remained closed when Pout was increased to 8 cm H₂O. In contrast, a gap remained in a mutant LV (arrow), resulting in back leak. Symmetrically located commissures that extend in the downstream direction can be observed in the same control LV visualized from the side (yellow arrows). In contrast, the S1pr1^iΔLEC (TM@P1–7) LV had asymmetrical commissures that extended in both upstream and downstream directions (yellow arrows). Statistics: (A and B) Each dot represents an individual mouse. The graphs were plotted as mean ± SD. Mann–Whitney test and unpaired t test were performed for the statistical analysis. *P < 0.05. (C) LV density was measured in ileum-draining lymphatic vessels harvested from n = 10 3-mo S1pr1^f/f (TM@P1–P7), n = 5 10-mo S1pr1^f/f (TM@P1–P7), n = 11 3-mo S1pr1^iΔLEC (TM@P1–P7), and n = 3 10-mo S1pr1^iΔLEC (TM@8w) mice. One-way ANOVA with Tukey’s post hoc test was performed to determine significance. ***P < 0.001. (D) Each dot represents an individual LV harvested from n = 10 3-mo S1pr1^f/f (TM@P1–P7), n = 2 10-mo S1pr1^f/f (TM@P1–P7), n = 11 3-mo S1pr1^iΔLEC (TM@P1–P7), and n = 3 10-mo S1pr1^iΔLEC (TM@8w) mice. A nonparametric Kruskal–Wallis test with Dunn’s post hoc test was performed to determine significance. *P < 0.05; **P < 0.01. (E and F) Live imaging followed by fixation and whole-mount IHC was performed using n = 2 LVs from 3-mo S1pr1^f/f;Prox1-tdTomato (TM@P1–P7) and n = 3 LVs from 3-mo S1pr1^iΔLEC;Prox1-tdTomato (TM@P1–P7) mice. Additionally, n = 3 LVs that were not imaged live from 3-mo S1pr1^iΔLEC (TM@P1–P7) mice were directly fixed and imaged by whole-mount IHC.