Requirement of the N terminus of GATA1 in fetal erythropoiesis. (A) Representative pictures of E14.5 embryos. The FL of ERG/Gata1s males is smaller and paler (arrow). A total of 1 × 107 FL cells were resuspended in PBS. The light color of the ERG/Gata1s FL cells points to the decrease number in mature erythrocytes in those embryos. (B) ERG/Gata1s males die between E12.5 and E14.5. Females and males generated from crossing heterozygous TgERG males with homozygous Gata1s KI females were tested for the presence of ERG transgene on E12.5, E14.5, and adults (n = 85, 98, and 137 for E12.5, E14.5, and adults, respectively). A significant difference in the TgERG male population was found between E12.5 and E14.5 (t test: P < .014). (C-E) ERG and Gata1s inhibit erythroid colony formation. FL cells were isolated from (C) E12.5 TgERG embryos and Wt littermates and (D-E) E12.5 and E14.5 embryos of ERG/Gata1s embryos and Wt/Gata1s littermates and were plated on methylcellulose supplemented with EPO to promote growth of erythroid colonies (BFU-E). Erythroid colonies (BFU-E) were counted 7 to 10 days after plating. Each bar graph represents the average of at least 3 independent experiments. Statistical significance was tested using the t test. (F) Representative figures of flow cytometric analysis of male E12.5 FL cells using the erythroid markers Ter119 and CD71. (G) Expression of Gata1s impairs erythropoiesis. Expression of Ter119 erythroid marker as measured by flow cytometry analysis in FL cells generated from E12.5 females and males of Wt, TgERG, Gata1s, and ERG/Gata1s animals. (H) Increase apoptosis in ERG/Gata1s males. Annexin V levels were measured by flow cytometry in Ter119-positive FL cells for the detection of apoptosis. FL cells were isolated from E12.5 Wt, TgERG, Wt/Gata1s, and ERG/Gata1s males. Significant increase in apoptosis in ERG/Gata1s males compared with Wt/Gata1s males was measured using the t test (P = .017). (I) Decreased expression of erythroid and antiapoptotic genes in ERG/Gata1s males. Expression level of the different genes was obtained from the Affymetrix mouse gene 1.0 ST chip array and confirmed by real-time PCR on at least 2 additional samples for each genotype.
