Increased differentiation and apoptosis of PU.1G/G FL erythroid precursors. (A) Differentiation of erythroid cells. FL cells were analyzed for CD71 and TER119 expression after a 4-day culture. The cells normally amplified in these cultures are CD71⁺TER119^– (red gate). Mature erythroid cells are TER119⁺ (black gate). Photos show the morphology of cells from day-4 cultures, as evaluated by May-Grünwald-Giemsa staining. Proerythroblasts and erythroblasts are large cells with big nuclei and dark blue cytoplasm; differentiated erythroid cells are small with light cytoplasm. Original magnification, × 400. (B) Increased differentiation of sorted CD71⁺TER119^– PU.1G/G cells. After 3 days of culture, CD71⁺TER119^– cells were purified and reseeded in erythroid medium. Cells were counted after 48 hours and stained for TER119 and CD71 expression. Bar graphs display the total number of cells versus the number of TER119⁺ cells in each culture. (C) Genes expressed by PU.1G/G erythroid precursors. RNA was extracted from sorted CD71⁺TER119^– cells after 5 days of culture. Expression of the indicated genes was assessed by RT-PCR. “+” or “–” RT indicates the presence or not of reverse transcriptase in the assay. All experiments were performed in erythroid medium. All are representative of more than 3 experiments. (D) Increased apoptosis of PU.1G/G erythroid precursors. The 13.5-dpc FL cells were cultured for 3 days in erythroid medium. Cells were then stained for annexin V positivity. The intensities of annexin V staining are shown for immature CD71⁺TER119^– and mature TER119⁺ cells. The figure is representative of 4 experiments, analyzing cells from 7 PU.1G/G, 4 PU.1^+/G, and 4 WT fetuses.