Figure 1.
S1PR1 signaling is active during and is necessary for LVV and venous valve morphogenesis. (A) E16.5 S1PR1-GS and H2B-GFP (control) littermates were frontally sectioned and analyzed. GFP+ cells were observed in the LVVs (arrows) and venous valves (arrowheads) of S1PR1-GS embryos, indicating that S1PR1 signaling is active in these structures. Only a few GFP+ cells were observed in H2B-GFP embryos due to leaky expression of this transgene. (B) 500-μm–thick transverse section of E16.5 Prox1-tdTomato and Lyve1-Cre;S1pr1−/f; Prox1-tdTomato embryos were prepared using a vibratome, and whole-mount IHC and confocal imaging were performed to visualize the LVVs and venous valves (bottom row). Subsequently, the same samples were processed and analyzed by SEM (top row). Normal-looking LVVs (arrows or pseudo colored in magenta) and venous valves (arrowheads or pseudo colored in green) were seen in control embryos. Mutant embryos appeared to have substantially fewer valvular endothelial cells. When present the mutant cells appeared disorganized and arrested at the periphery of the vessels. (C and D) E16.5 control, Lyve1-Cre;S1pr1−/f, and Lyve1-Cre;S1pr1−/f;Vegfr3+/− embryos were frontally sectioned (12-μm thick) and analyzed by IHC. LVVs (arrows) and venous valves (arrowheads) of control embryos had invaginated into the veins. Higher magnification images of control LVVs (boxed areas and the panels below in C) revealed that VEGFR3hi;Prox1hi LECs (asterisks) were located in between two layers of VEGFR3Low;Prox1hi valvular endothelial cells (yellow arrowheads). In contrast, invagination was substantially reduced in the LVVs and venous valves of mutant embryos, and the organization of the two cell types was defective. Blood cells were observed within the lymph sacs of mutant embryos (yellow arrows in C). (D) The invagination defect of Lyve1-Cre;S1pr1−/f embryos was not rescued by Vegfr3 heterozygosity. Statistics: (A, C, and D) n = 5 embryos per genotype; (B) n = 5 controls and n = 7 Lyve1-Cre;S1pr1−/f LVV complexes. LS, lymph sacs; T, thymus; EJV, external jugular vein; SVC, superior vena cava; SCV, subclavian vein; IJV, internal jugular vein.

S1PR1 signaling is active during and is necessary for LVV and venous valve morphogenesis. (A) E16.5 S1PR1-GS and H2B-GFP (control) littermates were frontally sectioned and analyzed. GFP+ cells were observed in the LVVs (arrows) and venous valves (arrowheads) of S1PR1-GS embryos, indicating that S1PR1 signaling is active in these structures. Only a few GFP+ cells were observed in H2B-GFP embryos due to leaky expression of this transgene. (B) 500-μm–thick transverse section of E16.5 Prox1-tdTomato and Lyve1-Cre;S1pr1−/f; Prox1-tdTomato embryos were prepared using a vibratome, and whole-mount IHC and confocal imaging were performed to visualize the LVVs and venous valves (bottom row). Subsequently, the same samples were processed and analyzed by SEM (top row). Normal-looking LVVs (arrows or pseudo colored in magenta) and venous valves (arrowheads or pseudo colored in green) were seen in control embryos. Mutant embryos appeared to have substantially fewer valvular endothelial cells. When present the mutant cells appeared disorganized and arrested at the periphery of the vessels. (C and D) E16.5 control, Lyve1-Cre;S1pr1−/f, and Lyve1-Cre;S1pr1−/f;Vegfr3+/− embryos were frontally sectioned (12-μm thick) and analyzed by IHC. LVVs (arrows) and venous valves (arrowheads) of control embryos had invaginated into the veins. Higher magnification images of control LVVs (boxed areas and the panels below in C) revealed that VEGFR3hi;Prox1hi LECs (asterisks) were located in between two layers of VEGFR3Low;Prox1hi valvular endothelial cells (yellow arrowheads). In contrast, invagination was substantially reduced in the LVVs and venous valves of mutant embryos, and the organization of the two cell types was defective. Blood cells were observed within the lymph sacs of mutant embryos (yellow arrows in C). (D) The invagination defect of Lyve1-Cre;S1pr1−/f embryos was not rescued by Vegfr3 heterozygosity. Statistics: (A, C, and D) n = 5 embryos per genotype; (B) n = 5 controls and n = 7 Lyve1-Cre;S1pr1−/f LVV complexes. LS, lymph sacs; T, thymus; EJV, external jugular vein; SVC, superior vena cava; SCV, subclavian vein; IJV, internal jugular vein.

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