Altered fetal erythropoiesis in TRα^−/− mice. (A) Day-12.5 fetuses of TRα^+/+ (+/+) and TRα^−/− (−/−) mice. (B) Neutral benzidine/Giemsa-stained fetal liver cells from day-12.5 TRα^+/+ and TRα^−/− embryos, with percentage of nucleated red blood cells (RBCs, arrowheads) enumerated (n = 3). (C) Erythroid colony assays (BFU-Es and CFU-Es) of fetal liver cells from day-12.5 TRα^+/+ and TRα^−/− embryos (n = 3). (D) Flow cytometric analysis of fetal liver cells from day-12.5 TRα^+/+ and TRα^−/− embryos, stained with CD71 and TER119. Erythroid populations corresponding to different stages of maturation (R1, R2, R3) were enumerated (n = 5). (E) Morphology of Giemsa-stained immortalized erythroblasts from day-12.5 TRα^+/+ (J2+), TRα^−/− (Jα−), and TRβ^−/− (Jβ−) fetal livers. (F) Proliferation of J2+ and J2α− cells was measured by a colorimetric (OD: 490 nm) MTT-based assay (n = 6). (G) Clonogenicity of immortalized erythroid cells was determined using methylcellulose cultures (n = 3). (H) Viability of immortalized erythroid cells was determined by eosin dye exclusion (n = 6). (I) Apoptosis was assessed by annexin V staining of immortalized erythroid cells (n = 4). (J) Hemoglobin production was identified by benzidine staining in response to Epo treatment (48 hours). Means (± SD) are shown. Statistically significant (2-way ANOVA) differences are indicated (*P ≤ .05; **P ≤ .01).