Defective hematopoiesis in Eng^−/− embryos. (A) Whole E7.5 embryos derived from Eng^+/− intercross were plated in M3434 and cultured in a low-oxygen incubator. The number of EryPs was reduced drastically in Eng^−/− embryos (43.00 ± 18.2; n = 3) compared with Eng^+/− (128.3 ± 31.52; n = 3) embryos (P = .0395 by 1-tailed Student t test). Definitive hematopoietic colonies do not show significant differences at this early time point. (B) YS from WT E9.5 embryos show normal vasculature, full of erythrocytes, whereas YS from Eng^−/− embryos present primitive vasculature containing very few RBCs. Severe anemia was also observed in the AGM region of Eng^−/− embryos (arrows in bottom panels). (C) The frequency of CD71⁺Ter119⁺ cells (erythroblasts) is severely decreased in Eng^−/− YS compared with Eng^+/− or Eng^+/+ YS. **P < .05 and ***P < .01 by ANOVA. (D) qPCR gene-expression analyses in E9.5 YS from WT (+/+; n = 9), heterozygotes (+/−; n = 18), and knockout embryos (−/−; n = 15). Transcripts are normalized to GAPDH. Bars represent average gene expression for each genotype. Error bars indicate the SEM for each genotype. *P < .05 and **P < .01 by ANOVA. (E) E9.5 YS cells from Eng^+/− intercrosses were assayed for hematopoietic colony formation. The frequency of GEMM and BFU-E progenitors was decreased significantly in the YS of knockout embryos (−/−; n = 7) compared with heterozygous (+/−; n = 25) or WT (+/+; n = 12). Horizontal bar represents the mean. *P < .05 and **P < .01 by ANOVA. (F) Representative GM, GEMM, and BFU-E colonies are shown at similar magnification (10× objective).