Figure 2. LEC- and Vascular Smooth Muscle Cell-Derived Wnt Ligands Play Complementary Roles during Lymphatic Vascular Development.
(A) HLECs were cultured under OSS with or without the Wnt agonist BIO. Subsequently, cells were lysed, and western blot was performed for the indicated molecules. Both OSS and BIO enhance FOXC2 expression. BIO enhances FOXC2 expression to a greater extent compared with OSS. Additionally, a much stronger expression of FOXC2 is observed in HLECs cultured with both OSS and BIO.
(B–I) W/s was simultaneously deleted from LECs and vascular smooth muscle cells by Lyve1-Cre and SM22-Cre. The control and mutant embryos were harvested at the indicated developmental time points and analyzed by immunohistochemistry on frontal cryosections (B and C) or by whole-mount immunohistochemistry(D–I).
(B and C) LVVs are observed at the junction of lymph sacs (LSs), IJV, and subclavian vein (SCV) in control (B, arrows) but not in SM22-Cre;Lyve1-Cre;W/sf/f embryos (C).
(D–G) LVs are observed in the mesenteric lymphatic vessels of control embryos (D, arrows, and F). LVs are mostly absent from the mesenteric lymphatic vessels of SM22-Cre;Lyve1-Cre;W/sf/f embryos (E and G).
(H–I´) (H) and (I) are higher-magnification images of the boxed areas in (H´) and (I´), respectively. The lymphatic vessels of the dorsal skin are dilated in mutant embryos. The distance between the opposing migrating fronts is higher in SM22-Cre;Lyve1-Cre;W/sf/f embryos (magenta bars in I’). Additionally, fewer branchpoints are observed in the lymphatic vessels of mutant embryos.
(J–M) The number of mesenteric LVs per lymphatic vessel is quantified in (J). The lymphatic vessels of the dorsal skin were analyzed, and the distance between the migrating fronts (K), diameters of lymphatic vessels (L), and the number of branchpoints (M) were quantified.
Scale bars, 100 mM (B, C, and F–I), 200 mM (H’ and I’), and 250 mM (D and E). Statistics: (A), n = 3; (B)–(M), n = 4 per genotype; ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.