Effect of p85α deficiency on fetal liver erythropoiesis in day-14.5 embryos. (A) Phenotypic appearance of wild-type and p85α^-/- embryos at day 14.5 of gestation. p85α^-/- embryos are pale compared with wild-type controls. Original magnification, × 10. (B) Representative peripheral blood smears from day-14.5 wild-type and p85α^-/- embryos. Blood smears were stained with Wright-Giemsa, and the total number of mature erythrocytes was quantified (“Results and discussion”). Mature erythrocytes (macrocytes) are indicated by black arrows, yolk sac–derived megaloblasts are indicated by short open arrows, and fetal liver-derived erythroid precursors are indicated by red arrows. p85α^-/- peripheral blood contains fewer nonnucleated red cells and a large number of nucleated erythroblasts. Original magnification, × 40. (C) Representative fetal liver touch preps from wild-type and day-14.5 p85α^-/- embryos. Touch preps were stained with Wright-Giemsa. Numerous erythropoietic cells in all stages of differentiation are observed in wild-type fetal livers. More megaloblasts are observed in the fetal livers of p85α^-/- embryos (short open arrows). Original magnification, × 40. (D) Total number of BFU-Es and CFU-Es in day-14.5 wild-type (▦) and p85α^-/- (▨) fetal livers. Cells were plated for growth of BFU-Es in methylcellulose medium containing various concentrations of KitL as indicated combined with a single concentration of Epo (5 U/mL). Cells were plated for growth of CFU-Es in methylcellulose medium containing various concentrations of Epo alone as indicated. CFU-Es and BFU-Es were counted by indirect microscopy after 2 and 7 days of culture, respectively. Results represent the mean number of colonies per fetal liver ± SEM of 5 independent experiments. *P < .05 by Student paired t test. (E) Frequency of erythroid progenitors in c-kit⁺–sorted fetal liver cells isolated from day-14.5 p85α^-/- (▨) and wild-type (▦) fetal livers. c-kit⁺ cells were isolated by immunomagnetic bead enrichment and sorted as previously described.¹⁵  More than 90% of the cells were c-kit⁺ after sorting as tested by fluorescence cytometry (data not shown). c-kit⁺ cells/mL (2000) were plated in methylcellulose medium containing KitL (10 ng/mL) and Epo (5 U/mL), and the numbers of CFU-Es and BFU-Es were quantified after 2 and 7 days of culture, respectively. In addition, 2000 c-kit⁺ cells/mL were plated in methylcellulose medium containing Epo (5 U/mL) alone, and the number of CFU-Es was counted after 2 days of culture. Results represent the mean number of colonies per 2000 c-kit⁺ cells ± SEM of 3 independent experiments. *P < .05 by Student paired t test.