Abnormal structure of lymphatic mesenteric collecting vessels in Bmp9-KO neonates at P0. Representative macroscopic views of mesenteric vessels in WT (A) and Bmp9-KO (B) pups at P0. The lymphatic vessels are filled with white chyle and constrictions along the vessels reflecting the presence of valves are shown with white arrows. Whole-mount fluorescence immunostainings of WT (C) and Bmp9-KO (D) mesenteries with CD31 (red) and Prox1 (green); arrows point to Prox1-overexpressing cells in valve areas. Higher magnification views of the mid-part of mesenteric collecting vessels in WT (E) or Bmp9-KO (F). Enlarged vessels, as outlined by the red lines, showing vessel sections are observed in mutants. (G) Quantification of the mean collecting vessel section in WT and Bmp9-KO pups. The mean diameter of mesenteric lymphatic collecting vessels was calculated as the average of 4 different measurements performed using Axiovision 4.8 software throughout the length of 4 different collecting vessels per mesentery. (H-I) Quantification of LEC number per millimeter of collecting vessel length and by square millimeter of collecting vessel surface unit in WT and Bmp9-KO pups. Prox1-positive nuclei were counted per vessel unit length and per vessel surface unit on at least 3 collecting vessel per mesentery using Axiovision 4.8 software. Values are the mean (± SE) from 8 individuals per genotype. ns, not significant; ***P ≤ .001, significantly different from WT pups by unpaired Student t test.