NC-specific loss of Foxc1 induces angiogenesis and lymphangiogenesis in the mouse cornea during development. (A) Neovascularization was evaluated in CD31-stained corneas from control (Foxc1F/F), NC-Foxc1+/−, and NC-Foxc1−/− mouse embryos (E14.5, E15.5, and E17.5) and newborn mice (P0), and from adult (7-wk-old and 43-wk-old) control (Foxc1F/F) and NC-Foxc1+/− mice. The corneas of control (Foxc1F/F) embryos and mice were avascular with developing long ciliary arteries (long arrows) and limbal blood vessels (short arrows). The corneas of NC-Foxc1+/− embryos and mice remained avascular through adulthood, but the long ciliary arteries (long arrows) and limbal blood vessels (Inset; short arrow) of embryos and newborn mice were disrupted. Increased sprouting of limbal blood vessels (arrowheads) was observed in adult NC-Foxc1+/− mice. In NC-Foxc1−/− embryos, corneal vascularization (red arrows) was observed by E14.5 and extended throughout the cornea at later time points; the disruption of limbal blood vessels was apparent on E17.5 and P0. (B) NC-specific loss of Foxc1 increases the growth of corneal lymphatic vessels in mice. Lymphangiogenesis was evaluated in corneas from control (Foxc1F/F), NC-Foxc1+/−, and NC-Foxc1−/− embryos (E17.5) and newborn mice (P0), and also from adult (7-wk-old and 43-wk-old) Foxc1F/F and NC-Foxc1+/− mice. Embryonic corneas were stained with the lymphatic vessel marker Prox1, corneas from newborn mice were stained with Prox1 or the lymphatic vessel marker Lyve-1, and corneas from adult mice were stained with Lyve-1. Growth of lymphatic vessels from the limbus (red arrow) into the cornea was observed in NC-Foxc1−/− embryo at E17.5 and in newborn mice (arrows). The corneas of adult NC-Foxc1+/− mice remained free of lymphatic vessels, but showed increased sprouting of lymphatic vessels (arrowheads). (C) Quantification of blood and lymphatic vessel sprouting in 7-wk-old adult Foxc1F/F and NC-Foxc1+/− mice. The sprouting of both blood and lymphatic vessels was significantly greater in NC-Foxc1+/− mice compared with control Foxc1F/F mice. Values are mean ± SEM. Compared with control mice: blood vessel sprouting in NC-Foxc1+/− mice, P = 0.0000006; lymphatic vessel sprouting in NC-Foxc1+/− mice, P = 0.00003. *P < 0.05, Student t test. lca, long ciliary artery. (Scale bars: 100 μm.)