Sox6^–/– mice feature abnormal RBCs and are anemic. (A) Blood smears of E14.5 and E16.5 Sox6^–/– and control littermates. Arrowheads indicate large and nucleated primitive RBCs; arrows, small, enucleated, definitive RBCs; and double arrows, Sox6^–/– nucleated and misshapen definitive RBCs. (B) Scanning electron microscopy (SEM) of E18.5 Sox6^–/– and control RBCs. D indicates normal-looking discoid RBCs; *, SEM artifacts; and #, misshapen RBCs. (C) Transmission electron microscopy of RBCs in E18.5 Sox6^–/– and control littermates. Nucleated and abnormally shaped RBCs are seen in the Sox6^–/– fetus. (D) Globin chain analysis in E14.5 Sox6^–/– and control fetuses. The migration level of the primitive Eγ globin chains, definitive β-globin haplotypes, and α-globin chains is indicated. The Sox6^–/– mutant shows the same proportions of primitive and definitive globin chains as its control littermates. (E) TO/CD71 FACS profiles of blood cells in control and Sox6^–/– mice from E18.5 to P13. Thiazole orange, which binds nucleic acids, distinguishes nucleated cells (TO⁺⁺) from reticulocytes (TO⁺) and mature RBCs (TO^–), and CD71 antibodies distinguish reticulocytes (CD71⁺) from mature RBCs (CD71^–). Arrows indicate the trend of reticulocyte (CD71⁺/TO⁺) differentiation into mature RBCs (CD71^–/TO^–).