PECAM-1 expression in globin-producing cells and in the extraembryonic region as early as the streak stage. (A) Whole mount anti–PECAM-1 antibody staining of E8.5 (4sp) embryo. Note that most blood vessels, including arteries and veins, are well stained with the anti–PECAM-1 antibody and that there are densely stained cells within the blood island region. (B) Transverse section showing that blood island cells are PECAM-1 positive. Nuclei are visualized by methyl green staining. (C) High magnification of the boxed area in panel B. (D-G) High magnification of the blood island region stained with Hoechst 33342 (D), anti-ϵyglobin antibody (E), anti–PECAM-1 antibody (F), and the merged image (G). PECAM-1 is expressed in globin-positive cells. (H-L) Whole mount PECAM-1 antibody staining of embryo at late-streak stage (H), no-bud (OB) stage (I), early-bud (EB) stage (J), early–head fold (EHF) stage (K), and late–head fold (LHF) stage (L). PECAM-1 is expressed in the extraembryonic region, presumably corresponding to the mesodermal layer as early as the late-streak stage, and is expressed continuously in the extraembryonic regions including the blood island region until E8.5. (H, inset) High magnification of PECAM-1–positive cells in the extraembryonic region. (arrow) Forming dorsal aortae. hc indicates heart crescent; HF, head fold; ec, endocardium; VE, visceral endoderm. Images in panels A and H-L were captured by Leica DC500 CCD camera with IM50 Imaging Manager through Leica MZ FLIII microscope a PLAN APO lens. A Leica DC500 CCD camera with IM50 Imaging Manager through Leica DMRXA microscope was used to capture the images using a PLAN APO 1.0/0.125 NA lens (panel A; total magnification, ×32), a 5×/0.15 NA objective (panel B) or a 20×/0.50 NA objective (panels C-G).