Abnormal and dilated lymphatic vessels with edema in LPA₄-deficient embryos. (A-C) Transverse sections of the jugular area, including typical lymphatic sacs that surround the subclavian arteries, were immunostained with antibodies against PECAM1 (green), LYVE1 (red), and TOTO-3, a nuclear marker, (blue) in WT (A), apparently normal LPA₄-deficient (B), and edematous LPA₄-deficient (C) embryos at E14.5. SV, subclavian vein; SA, subclavian artery; CA, carotid artery; JV, jugular vein; LS, lymph sac. Scale bars, 100 μm. (D) Quantification of the area of lymph sacs in WT, apparently normal LPA₄-deficient, and edematous LPA₄-deficient embryos. Data represent the mean ± SEM (n = 13, 10, and 9, respectively). *P < .05 and **P < .001, using Kruskal-Wallis test followed by Dunn post-hoc test. KO, LPA₄-deficient. (E-J) The ileum (E-G) and the skin (H-J) were immunostained with antibodies against PECAM1 (green), LYVE1 (red), and TOTO-3 (blue) in WT (E,H), apparently normal LPA₄-deficient (F,I), and edematous LPA₄-deficient (G,J) embryos at E18.5. Arrowheads, submucosal lymphatic vessels; arrows, subserosal lymphatic capillaries (lacteals); double arrowheads, severely dilated lymphatic vessels. Scale bars, 50 μm. (K-M) Quantification of area, number, and size ( = area/number) of the skin lymphatic vessels, respectively, in WT and apparently normal LPA₄-deficient embryos. Data represent the mean ± SEM (n = 9 and 8, respectively). *P < .005 and **P < .0001, using unpaired 2-tailed t test.