EpoR-Cre^Tg/+; RhoA^flox/flox (RhoA^Δ/Δ) mice with erythroid-specific RhoA deficiency die in utero by E16.5 because of failure of definitive erythropoiesis. (A) Representative results of embryo genotyping by PCR (somatic DNA) show bands for EpoR-Cre (top, WT:431bp, Cre⁺:679bp) and RhoA (bottom, WT:482bp, flox:633bp) for all possible genotypes of the offspring of an EpoR-Cre^Tg/+;RhoA^WT/flox × EpoR-Cre^−/−;RhoA^flox/flox timed pregnancy. (B) Immunodetection of RhoA protein in peripheral WT and RhoA^Δ/Δ blood cells from E11.5 embryos (top panel) and in CD71⁺ cells from WT and RhoA^Δ/Δ E13.5 fetal livers (bottom panel), using a size-based capillary electrophoresis instrument, shows significant reduction of RhoA in the RhoA^Δ/Δ embryos. (C-D) Stereoscopic images of E13.5 embryos showing severe anemia in RhoA^Δ/Δ yolk sac (C), as well as pallor and small fetal liver in RhoA^Δ/Δ embryos (D). The scale bar represents 1 mm. (E-F) Light microscopy images of peripheral blood cytospins showing poikilocytosis with large and frequently multinucleated primitive RhoA^Δ/Δ erythroid cells in contrast to the homogeneous population of WT primitive red cells (E); hematoxylin and eosin–stained yolk sacs show the paucity of primitive erythroid cells in the RhoA^Δ/Δ yolk sac vessels (F). The scale bar represents 10 µm. (G) The offspring of EpoR-Cre^Tg/+;RhoA^WT/flox × EpoR-Cre^−/−;RhoA^flox/flox follow Mendelian ratios up to E14.5. By E15.5, only 6% of live embryos are RhoA^Δ/Δ, with none being alive by E16.5. Data based on genotyping of >60 embryos per each time point. (H) RhoA^Δ/Δ embryos exhibit anemia already by E11.5, worsening significantly by E14.5. Data are represented as mean ± SEM of blood counts for at least 3 embryos per genotype per each time point. *P < .05 of RhoA^Δ/Δ vs WT.