Nrp2 inhibition reduces lymphatic sprouting in developing intestinal and epicardial lymphatics. (A–D) Developmental time course of intestinal lymphangiogenesis by LYVE-1 immunohistochemistry. (A) At P0, lymphatics are restricted to the submucosa (arrows). (B) By P2, lymphatic branches have extended into a portion of some villi, and newly formed sprouts (arrows) can be seen to form on the submucosal lymphatic vessel adjacent to other villi. Tip cells with filopodia (inset) are present on the growing lacteals. (C and D) By P4 (C), most villi have a developing lacteal, which extends to the villus tip by P8 (D). Tip cells (inset) can still be observed at the ends of the vessels. (E) Scheme of the time course of lymphatic sprouting into intestinal villi and representation of experiments shown in G, I, and J. Anti-Nrp2^B was injected i.p. at P1, 3, and 5 (red dots), animals were sacrificed at P8, and tissues were analyzed. (F) Schematic representation of experiment shown in H, K, and L. Anti-Nrp2^B was injected i.p. at P3, 5, and 7 (red dots), animals were sacrificed at P10, and tissues were analyzed. (G–J) Analysis of intestines from the experiment depicted in E. (G and H) Control-treated intestines (G) have a normal-appearing lymphatic pattern in contrast to anti-Nrp2^B–treated intestines (H) in which a larger portion of villi lack lacteals. (I and J) Quantification of the percent of villi that have lacteals and villi length. (I–L) 50 villi per animal for six animals per treatment condition were analyzed. (M–P) Control (M and N) and anti-Nrp2^B (O and P)–treated hearts showing reduced branching and increased vessel thickness in portions of the anti-Nrp2^B–treated hearts. Error bars indicate SEM. Bars: (A–D) 250 µm; (G and H) 125 µm; (M–P) 400 µm.