Reduced expression of GATA-1 and EKLF in Runx1^−/− primitive erythrocytes. (A) Relative intensity levels were measured by real-time PCR and normalized to GAPDH. The Student t test was applied for statistical analysis. Significant differences in Runx1^−/− mice from wild-type were observed (**P = .009, *P = .013). (B) Structure of the GATA-1 promoter-LacZ (IE3.9-LacZ) transgene. IE indicates exon IE. (C) Representative pictures of Runx1^+/−::IE3.9-LacZ (n = 7 E8.5, n = 12 E10.5) and Runx1^−/−::IE3.9-LacZ (n = 8 E8.5, n = 4 E10.5) embryos. Expression of β-galactosidase was reduced in Runx1^−/− embryo (C, right panels) compared with Runx1^+/− embryo (C, left panels). (D) Cytospin preparation of blood cells from embryos shown in panel C (E10.5). Intensity of β-galactosidase staining was reduced in Runx1^−/− cells () compared with Runx1^+/− cells (). (E) FACS analysis of GATA-1 (β-galactosidase) expression in E10.5 embryos. FDG was used as a fluorescent substrate for β-galactosidase. Black line, gray bold line, or red bold line represents β-galactosidase expression in Runx1^+/+, Runx1^+/−, or Runx1^−/−, respectively. (F) MFI values of FDG for Runx1^+/+ (n = 2), Runx1^+/− (n = 5), and Runx1^−/− (n = 2) embryos. Significant difference in Runx1^−/− mice from Runx1^+/− mice was observed (*P = .032 by Student t test). Original magnifications: C (top panels), ×16.0; C (bottom panels), ×7.2; D, ×400. Images in panel C were captured with a Leica MZ FLIII microscope and a Leica DC500 CCD camera with IM50 Imaging Manager, with a PLAN APO 1.0 lens (1.0×/0.125 NA). A Leica DC500 CCD camera with IM50 Imaging Manager through Leica DMRXA microscope was used to capture the images using a 20×/0.50 NA objective for the bottom panel and 5×/0.15 NA objective for the top panels.