Anatomic locations of primitive hematopoiesis and vasculogenesis. (A) Transverse section of an E8.5 (6sp) embryo. Flk1 (red) and ϵyglobin (green) are expressed exclusively. Note that Flk1 marks endothelial cells, whereas ϵyglobin marks hematopoietic cells. The domain of the vascular plexus and the blood island region are clearly regionalized. (B-E) High-magnification image of the blood island region stained with a nuclear staining dye, Hoechst 33342 (B), anti-ϵyglobin antibody (C), anti-Flk1 antibody (D), and the merged image (E). (F) Merged image of PECAM-1 (red), ϵyglobin (green), and Hoechst dye staining (blue) of a transverse section of an E9.5 embryo proper. Scale bar represents 100 μm. (G-J) Higher magnification of the boxed area in panel F. (G) Hoechst 33342 staining. (H) Anti–ϵyglobin antibody staining. (I) Anti–PECAM-1 antibody staining. (J) Merged image of panels G-I. (K-O) Merged image of anti-ϵyglobin antibody staining and Hoechst 33342 staining in combination with staining for VE-cadherin (K), Tie2 (L), CD34 (M), endoglin (N), or Flk1 (O). Endothelial markers are expressed in endothelial but not in hematopoietic cells. (P) Merged image of CD34 (red), ϵyglobin (green), and Hoechst dye staining (blue) of a transverse section of an E9.5 embryo proper. (Q-T) Higher magnification of the boxed area in panel P. (Q) Hoechst 33342 staining. (R) Anti-ϵyglobin antibody staining. (S) Anti-CD34 antibody staining. (T) Merged image of panels Q-T. (arrow) Clump of cells at the ventral side of OA. ex indicates extra-embryonic region; em, embryonic region; DA, dorsal aorta; VE, visceral endoderm; EPC, ectoplacental cone; HF, head fold; NT, neural tube; OA, omphalomesenteric artery. All images were captured by a Leica DC500 CCD camera with IM50 Imaging Manager through a Leica DMRXA microscope using a 5×/0.15 NA objective (panel A) or a 20×/0.50 NA objective (panels B-T).