Effect of K-Ras deficiency on fetal liver erythropoiesis. (A) Representative fetal liver touch preps from WT and K-Ras^-/- day-13.5 embryos. Touch preparations were stained with Wright Giemsa. Numerous erythropoietic cells in all stages of differentiation are observed in WT fetal livers and are represented as follows: proerythroblasts by a blue arrow; basophilic erythroblasts by a black arrow; polychromatophilic erythroblasts by a green arrow; orthochromatophilic erythroblasts by a red arrow; and mature enucleated erythrocytes by a purple arrow. In contrast, a marked increase in proerythroblasts (blue arrow) and basophilic erythroblasts (black arrow) was observed in K-Ras^-/- fetal livers with a concomitant decrease in more mature polychromatophilic and orthochromatophilic erythroblasts and a paucity of enucleated erythrocytes. Slides were visualized under an Olympus BX51 microscope (Olympus, Melville, NY) equipped with a UPlan FI 40 ×/0.75 objective lens. Photomicrographs were acquired with an Olympus DP11 camera using MicroSuite imaging software (Olympus). Five other experiments showed similar results. (B) Representative flow cytometric analysis of erythroid differentiation in vivo. Freshly isolated day-13.5 WT or K-Ras^-/- fetal livers were doubly stained with anti-CD71–FITC and anti–Ter-119–PE. Regions R1 to R5 define distinct populations of erythroid progenitor cells at different stages of differentiation as described previously.¹⁸  Primitive progenitor cells (including mature BFU-Es and CFU-Es) are shown in R1, proerythroblasts and early basophilic erythroblasts in R2, early and late basophilic erythroblasts in R3, chromatophilic and orthochromatophilic erythroblasts in R4, and late orthochromatophilic and reticulocytes in R5, as previously described.¹⁸  Numbers represent the percentage of cells within that region. Day-13.5 wild-type fetal livers display normal distribution of erythroid cells at different stages of differentiation. K-Ras^-/- fetal livers demonstrate a delay in differentiation at the basophilic erythroblast level: R2 to R3. Five other experiments showed similar results. (C) Percentage of freshly isolated K-Ras^-/- or WT day-13.5 fetal liver cells expressing c-kit in regions R1 to R5, which define distinct populations of erythroid progenitor cells at different stages of differentiation. Freshly isolated day-13.5 WT or K-Ras^-/- fetal livers were stained with anti-CD71–FITC, anti–Ter-119–PE, and anti–c-kit–APC antibodies as described in “Apoptosis, proliferation, and differentiation assays.” The majority of c-kit⁺ cells were identified in the more immature erythroid progenitor populations in R1 and R2 and no significant difference in the percentage of c-kit⁺ cells was observed in the different gated populations (R1-R5) between the 2 genotypes. Results represent the mean percentage of c-kit⁺ cells and error bars represent the standard error of the mean of 5 parallel independent experiments from embryos isolated from the same litter.