RhoA^Δ/Δ fetal liver cells exhibit increased cell-cycle arrest and cell death. (A) Fetal liver sections stained for the cell-proliferation marker Ki67 show that the RhoA^Δ/Δ binucleated cells in the fetal liver (arrows) are not actively proliferating, implying an abnormal arrest in the binucleated state and failure to complete cytokinesis. The scale bar represents 10 µm. (B) Graphic representation of the gating used to separate fetal liver cell populations in Figure 4C-E, defining the erythroid progenitors S0 (CD71^–;Ter119^– cells) and S1 (CD71⁺;Ter119^– cells) based on CD71 level, and the erythroid precursors E1-E4 based on CD71 and Ter119 levels and size (forward scatter), as detailed in supplemental Figure 2. (C-D) Cell-cycle analysis of WT and RhoA^Δ/Δ fetal liver cells from E14.5 embryos using in vivo BrdU assay shows a significant increase of the polyploid cells (>4N) and the G2/M population in the RhoA^Δ/Δ late erythroblasts (E2 and E3 populations) along with a decrease in the S phase. Flow cytometry dot plots are representative of 5 different biological repeats (C). Bar graphs of mean ± SEM of the percentage of each cell-cycle stage per erythroid population of 5 fetal livers for each genotype (D). *P < .05 of RhoA^Δ/Δ vs WT. (E) No significant difference was found in early apoptosis of the RhoA^Δ/Δ erythroblasts (left panel); however, late apoptosis was increased in E2, E3, and E4 RhoA^Δ/Δ fetal liver cells, as evidenced by an increased number of cells in those populations positive for both annexin V and 7-AAD (middle panel). Clearly necrotic cells (negative for annexin V but positive for 7AAD) were also found to be increased in RhoA^Δ/Δ fetal liver erythroblasts (right panel). Data are shown as mean ± SEM from 6 WT and 4 RhoA^Δ/Δ fetal livers. *P < .05 of RhoA^Δ/Δ vs WT.