Definitive erythropoiesis in fetal liver is impaired in c-Maf^−/− embryos but c-Maf^−/− fetal liver cells can form erythroid colonies. (A) Flow cytometric analysis of fetal liver cells isolated from E13.5 embryos labeled with a FITC-conjugated anti–TER-119 mAb and an APC-conjugated anti-CD44 mAb. Regions I to V are defined by a characteristic staining pattern and forward scatter (FSC) intensity of cells as indicated. (B) Representative images of erythroblast structure on stained cytospins from the 5 distinct regions shown in Figure 3A of wild-type fetal liver. Images were acquired by a Biorevo BZ microscope (Plan Apo 20×0.75 DIC N2) at room temperature and processed with the Adobe Photoshop CS4 software. Scale bar represents 20 μm. (C) Comparison of c-Maf^+/+ and c-Maf^−/− fetal livers in region I to region V. □ represents c-Maf^+/+; ■, c-Maf^−/−; n = 8∼10 per group; *P < .05. (D) The ratio of annexin V⁺ cells from region I to region V was compared in c-Maf^+/+ and c-Maf^−/− fetal livers. □ represents c-Maf^+/+; ■, c-Maf^−/−; n = 3 per group; *P < .05. (E) In vitro colony assay with the use of fetal liver cells from c-Maf^+/+ (□) and c-Maf^−/− (■) embryos at E13.5. A total of 20 000 fetal liver cells were plated and cultured with methylcellulose media. The numbers of CFU-E–, BFU-E–, and CFU-GEMM–derived colonies per fetal liver are shown. No significant difference (n.s.) was found in the number of CFU-E–, BFU-E–, or CFU-GEMM–derived colonies per fetal liver in c-Maf^+/+ embryos and c-Maf^−/− embryos; n = 6∼7 per group; data are presented as mean ± SEM. FL indicates fetal liver.