GATA1-positive cell population exists as a subset of EC marker–positive cell. (A) GFP expression in GATA1-GFP embryos at no-bud (OB) stage. Line shows a transverse section used for immunohistochemistry (PLAN APO lens, 1.0×/0.125; final magnification, ×63). (B) Immunohistochemical analysis of a transverse section as shown in panel A using anti-GFP antibody shown in green and anti–PECAM-1 antibody shown in red. Merged image is shown. (C) High magnification of the blood island region shown in panel B, Hoechst 33342 staining, anti–PECAM-1 antibody, anti-GFP antibody, and the merged image. (white arrows) Cells expressing GFP but not PECAM-1. (green arrows) Cells expressing PECAM-1 but not GFP. Note that GFP (GATA1) is expressed in a subset of extraembryonic mesoderm cells, in contrast to the broader spectrum of PECAM-1 expression. (D) Flow cytometric analysis of GATA1-GFP embryos (LS-OB) with GFP and cell surface markers VE-cadherin, PECAM-1, or Tie2. Note that GATA1 (GFP)–positive cells coexpress VE-cadherin and Tie2. Images were captured by a Leica DC500 CCD camera with IM50 Imaging Manager. Images in panel A were captured through a Leica MZ FLIII microscope using a PLAN APO 1.0 lens. Images in panels B and C were taken with a Leica DMRXA microscope using a 5×/0.15 NA objective (B) or 20×/0.50 NA objective (C).