Flt-1 ^−/− embryos have increased mitoses in endothelial cells.

Transverse sections of day 8.5 embryos were processed for immunohistochemistry by using antiphosphohistone H3 to detect mitotic nuclei (A-C), and overlays of adjacent sections were processed individually (see “Materials and methods” section) for immunohistochemistry with antiphospohistone H3 (red), anti-PECAM (green), and DAPI (blue) (D-F). Visualization of yolk sacs offlt-1^+/− (A) or WT (^+/+) (D) embryos that were phenotypically normal showed few mitotic nuclei in vascular areas (arrow in D). In contrast,flt-1^−/− embryos (B,C,E,F) exhibited vascular overgrowth and numerous mitotic nuclei (red; E,F) in PECAM⁺regions (green; E,F) of the yolk sac and allantois (F, left part of panel). (A-C) Asterisks denote the lumina of blood islands in the yolk sac, and arrows point to mitotic nuclei abutting the endoderm with the long axis perpendicular to the long axis of the endoderm cells, a characteristic of dividing endothelial cells. In contrast, the arrowhead in C points to a mitotic nucleus in the endoderm with the long axis parallel to the long axis of the endoderm cells, a characteristic of dividing endoderm. The arrowhead in B points to a mitotic nucleus of unknown cell type. (D-F) Arrows point to mitotic nuclei of PECAM⁺ cells. En, visceral endoderm of the yolk sac.