Megakaryocyte, granulocyte, and T-cell development in the fetus are sensitive to CBFβ dosage. (A) Morphology and acetylcholinesterase staining (brown) of megakaryocyte colonies from CFU-Mk assays. Note the reduced intensity of acetylcholinesterase staining in Cbfb^rss/−; megakaryocyte colonies, indicating impaired differentiation. (B) Megakaryocytes (large cells, arrows) were obtained following in vitro differentiation of 13.5-dpc fetal liver cells in liquid cultures in the presence of thrombopoietin. Note the absence of large cells in the Cbfb^rss/−; culture. (C) Histogram of CD41 expression in platelets isolated from in vitro megakaryocyte cultures. (D) Average percentage of CD41⁺ platelets from megakaryocyte cultures. n_+/+ = 4, n_rss/− = 4. Error bars represent 95% confidence intervals. The difference between Cbfb^+/+ and Cbfb^rss/−; cultures was significant at P < .03. (D) (E) Pictures of spleens (left panel) and thymic lobes (right panel) from 17.5-dpc fetuses. (F) Morphology of Gr-1⁺ cells enriched from 17.5-dpc fetal livers. Arrows in Cbfb^+/+ panel point to mature and band neutrophils. Red arrows in the Cbfb^rss/−; sample indicate immature monocytoid cells, and the black arrow points to a rare band. (G) Real-time PCR for myeloid-specific gene expression in Gr-1⁺ cells enriched from Cbfb^+/+ and Cbfb^rss/−; 17.5-dpc fetal livers. Gene-expression data are presented as the fold change in Cbfb^rss/−; cells relative to Cbfb^+/+ cells (normalized to a value of 1) and represent averages from 3 independent experiments. Error bars indicate 95% confidence intervals. For each sample, expression values were normalized to Gapdh. GCSFR indicates granulocyte colony-stimulating factor receptor; MCSFR, macrophage colony-stimulating factor receptor; MPO, myeloperoxidase; NE, neutrophil elastase; LF, lactoferrin; GelB, gelatinase B; and I°, II°, III°, primary, secondary, and tertiary, respectively, granule proteins. The asterisks indicate expression differences significant at P < .001. (H) Total number of CD4⁺CD8⁺ (DP) thymocytes in 17.5-dpc fetuses. Error bars represent standard errors. The differences between Cbfb^+/+ and all other genotypes were significant at P < .001. (I) CD4 expression is derepressed, and the percentage of TCRβ and γδ cells decreased in 17.5-dpc Cbfb^rss/rss thymocytes. The 2 histograms on the left show the percentage of CD4⁺CD8⁺ (DP) cells and CD4⁺CD8⁻ thymocytes that are TCRβ⁺. The histogram on the far right shows the percentage of total thymocytes that are TCRγδ⁺. The average percentages of TCRβ⁺ DP cells (± SD) are as follows: Cbfb^+/+, 20.1 (2.9), Cbfb^rss/rss, 5.6 (2.6); for percentages of TCRβ⁺ CD4⁺ cells: Cbfb^+/+, 19.8 (10.5), Cbfb^rss/rss, 5.1 (2.2); and for percentages of TCRδγ⁺ cells: Cbfb^+/+, 1.9 (0.2), Cbfb^rss/rss, 0.4 (0.1).