Block in erythroid maturation at the yolk sac and fetal liver stages in scl^RER/RER embryos. (A) Yolk sac erythropoiesis. Day E9.5 scl^RER/RER yolk sacs (iii) are paler than wild-type and heterozygotes (i,ii). (iv-vi) MGG staining of yolk sac blood shows asynchronous maturation of primitive erythroid cells in mutant homozygous E9.5 embryos. Arrows show immature erythroid cells absent from wild-type and heterozygous control samples (original magnification, ×400). (vii-ix) MGG/benzidine staining of primitive erythroid colonies obtained on plating of day E8.5 scl^Wt/Wt, scl^Wt/RER, and scl^RER/RER yolk sacs. Arrows point to immature erythroid cells in the mutant homozygous population that are absent from wild-type and heterozygous control samples (original magnification, ×400). (B) Fetal liver erythropoiesis. (i-iii) Day E12.5 embryos. Note the pale scl^RER/RER embryo (iii). (iv-vi) MGG-stained E12.5 blood (original magnification, ×400). The inset shows the presence of immature cells in blood derived from scl^RER/RER embryos (arrows); (vii-ix) representative CFU-E colonies from wild-type, scl^Wt/RER, and scl^RER/RER E12.5 fetal liver replating (magnification, ×200). Note the poor hemoglobinization of the mutant CFU-E colony (ix); (x-xii) MGG/benzidine staining of CFU-E colonies. Note the lack of mature benzidine positive scl^RER/RER erythrocytes (xii) (original magnification, ×400); (xii-xv) day E14.5 embryos. Note the pale scl^RER/RER embryo in (xv). (xvi-xviii) Benzidine staining of E14.5 fetal blood (original magnification, × 400). The arrows show the presence of less mature, benzidine-negative erythroid cells in the mutant homozygous sample (xviii) that are absent from the wild-type and heterozygous samples (xvi,xvii). (C) Characterization of fetal liver hematopoietic cells. (Top) Schematic representation of the progressive maturation of erythroid cells and pattern of expression of the cell surface markers c-kit, CD71 and Ter119.³⁷  ProE indicates proerythroblasts; basoE, polyChE, orthoChE, basophilic, polychromatic, and orthochromatic erythroid cells; retic, reticulocytes; RBC, red blood cells. (Bottom) Fetal liver cells derived from wild-type, heterozygous, and homozygous E12.5 embryos were analyzed by fluorescence-activated cell sorting (FACS). The percentages of fetal liver populations characterized by expression of various combinations of cell surface markers are indicated for each genotype. Error bars indicate plus or minus 1 SD from at least 3 independent experiments; *P < .01. (D) Cell-cycle analysis. (Top left) FACS analysis of PI-stained fetal liver cells representative of both wild-type and scl^RER/RER E12.5 fetal liver samples. (Top right) The 3 cell cycle phases and the sub-G₁ phase are visualized according to PI intensity and pulse width. (Bottom) The graph shows the proportion of fetal liver cells isolated from wild-type and scl^RER/RER mice in sub-G₁, G₁, S, and G₂/M phases. Error bars indicate plus or minus 1 SD from at least 3 independent experiments. (E) Apoptosis analysis. Cryosections of wild-type and scl^RER/RER E12.5 fetal livers were analyzed by TUNEL assay. Arrowheads point to TUNEL-positive apoptotic cells shown at higher magnification in insets (upper right of both panels; original magnification, ×400).