Maturational defect in erythropoiesis and reduction of globin transcription in Hemgn^−/− mice.Hemgn^−/− mouse E14.5 embryos (A) and livers (B) are smaller in size compared with control littermates. Scale bars, 1 cm. (C) Immunofluorescence indicating that hemogen protein is downregulated during fetal development (E12.5-E16.5) (white arrows). Quantification of hemogen fluorescence intensity in 25 cells (right panel). (D) Flow cytometry with Ter119 (glycophorin A, GYPA) and CD71 (transferrin receptor) staining to separate maturational stages S0-S5 fetal liver cells of Hemgn^−/− mice and WT littermates. (E) Quantification of cell numbers in populations S0-S5 in (D). (F) Relative expression level of hemogen to actin in the S0-S5 population of E14.5 fetal liver (FL) and E1-E4 population of bone marrow (BM) cells. Reverse transcription quantitative PCR (RT-qPCR)showing expression of embryonic Hbb-y and adult Hbb-bs in fetal liver cell populations S3 and S4/5 at E12.5, E14.5, and E16.5. Error bars represent standard deviation. N = 3 biological replicates. *P < .05, **P < .01, 2-tailed Student t test. au, arbitrary units.