Figure 3.
Definitive erythropoiesis is impaired in the GPS2-deficient fetal liver. (A) Viability of Gps2−/− embryos on the C57BL/6N:129S2/Sv background. Embryos from Gps2+/− intercrosses were harvested at stages E9.5 to E13.5. The percentages reflect the numbers of live Gps2−/− embryos with respect to all embryos harvested in litters at each gestational stage. (B) Representative pictures of E12.5 Gps2+/+ and Gps2−/− embryos (bottom) and fetal livers (top). Scale bars, 1 mm. (C-E) Total cell numbers (C), Ter119+ erythroid cell numbers (D), and CD45+ hematopoietic cell numbers (E) in fetal livers from Gps2+/+ (n = 17), Gps2+/− (n = 21), and Gps2−/− (n = 7) embryos at E12.5. Data are presented as mean ± SEM; Mann-Whitney test. (F) Wright-Giemsa staining of E12.5 Gps2+/+ and Gps2−/− fetal liver cytospin preparations. Scale bars, 10 μm. (G) Wright-Giemsa staining of peripheral blood cytospins from Gps2+/+ and Gps2−/− embryos at E12.5. Scale bars, 20 μm. (H) The percentage of enucleated red blood cells in peripheral blood cytospins from Gps2+/+ and Gps2−/− embryos at E12.5 (n = 3/group, mean ± SEM). Two-tailed unpaired t test. (I) Representative flow cytometry profiles of R1 to R5 erythroblast populations labeled with CD71 and Ter119 in fetal liver from Gps2+/+ embryos at E12.5. (J) The frequency of R1 to R5 cells in Gps2+/+ (n = 5), Gps2+/− (n = 8), and Gps2−/− (n = 4) E12.5 fetal livers. Data are presented as mean ± SEM; 2-tailed unpaired t test. (K) Representative flow cytograms of Gps2+/+ Ter119hi fetal liver cells (E12.5) separated into 3 populations (S1, S2, and S3) based on the forward light scatter (FSC) profile. (L) The frequency of S1 to S3 cells in Ter119hi fetal liver cells from each embryo at E12.5 (Gps2+/+, n = 5; Gps2+/−, n = 8; Gps2−/−, n = 4). Data are presented as mean ± SEM; 2-tailed unpaired t test. (M) Representative flow cytometry of enucleated cells in the S1 to S3 populations using Cyto-16 for nuclei and 7-aminoactinomycin D (7-AAD) for cell viability. (N) Percentages of enucleated cells in S1, S2, and S3 populations (Gps2+/+, n = 6; Gps2+/−, n = 11; Gps2−/−, n = 6). Data are presented as mean ± SEM; 2-tailed unpaired t test. (O) Real-time PCR analysis of the indicated erythroid genes in FACS-sorted CD71+Ter119+ cells from Gps2+/+ and Gps2−/− E12.5 fetal liver. Values are mean ± SEM (n = 3 replicates); 2-tailed unpaired t test. (P) Quantification of BFU-E, CFU-GM, and CFU-GEMM colonies from Methocult cultures of 4 × 104Gps2+/+, Gps2+/−, and Gps2−/− E12.5 fetal liver cells (n = 4/group). Values are mean ± SEM. *P < .05, **P < .01, ***P < .001; ns, not significant.

Definitive erythropoiesis is impaired in the GPS2-deficient fetal liver. (A) Viability of Gps2−/− embryos on the C57BL/6N:129S2/Sv background. Embryos from Gps2+/− intercrosses were harvested at stages E9.5 to E13.5. The percentages reflect the numbers of live Gps2−/− embryos with respect to all embryos harvested in litters at each gestational stage. (B) Representative pictures of E12.5 Gps2+/+ and Gps2−/− embryos (bottom) and fetal livers (top). Scale bars, 1 mm. (C-E) Total cell numbers (C), Ter119+ erythroid cell numbers (D), and CD45+ hematopoietic cell numbers (E) in fetal livers from Gps2+/+ (n = 17), Gps2+/− (n = 21), and Gps2−/− (n = 7) embryos at E12.5. Data are presented as mean ± SEM; Mann-Whitney test. (F) Wright-Giemsa staining of E12.5 Gps2+/+ and Gps2−/− fetal liver cytospin preparations. Scale bars, 10 μm. (G) Wright-Giemsa staining of peripheral blood cytospins from Gps2+/+ and Gps2−/− embryos at E12.5. Scale bars, 20 μm. (H) The percentage of enucleated red blood cells in peripheral blood cytospins from Gps2+/+ and Gps2−/− embryos at E12.5 (n = 3/group, mean ± SEM). Two-tailed unpaired t test. (I) Representative flow cytometry profiles of R1 to R5 erythroblast populations labeled with CD71 and Ter119 in fetal liver from Gps2+/+ embryos at E12.5. (J) The frequency of R1 to R5 cells in Gps2+/+ (n = 5), Gps2+/− (n = 8), and Gps2−/− (n = 4) E12.5 fetal livers. Data are presented as mean ± SEM; 2-tailed unpaired t test. (K) Representative flow cytograms of Gps2+/+ Ter119hi fetal liver cells (E12.5) separated into 3 populations (S1, S2, and S3) based on the forward light scatter (FSC) profile. (L) The frequency of S1 to S3 cells in Ter119hi fetal liver cells from each embryo at E12.5 (Gps2+/+, n = 5; Gps2+/−, n = 8; Gps2−/−, n = 4). Data are presented as mean ± SEM; 2-tailed unpaired t test. (M) Representative flow cytometry of enucleated cells in the S1 to S3 populations using Cyto-16 for nuclei and 7-aminoactinomycin D (7-AAD) for cell viability. (N) Percentages of enucleated cells in S1, S2, and S3 populations (Gps2+/+, n = 6; Gps2+/−, n = 11; Gps2−/−, n = 6). Data are presented as mean ± SEM; 2-tailed unpaired t test. (O) Real-time PCR analysis of the indicated erythroid genes in FACS-sorted CD71+Ter119+ cells from Gps2+/+ and Gps2−/− E12.5 fetal liver. Values are mean ± SEM (n = 3 replicates); 2-tailed unpaired t test. (P) Quantification of BFU-E, CFU-GM, and CFU-GEMM colonies from Methocult cultures of 4 × 104Gps2+/+, Gps2+/−, and Gps2−/− E12.5 fetal liver cells (n = 4/group). Values are mean ± SEM. *P < .05, **P < .01, ***P < .001; ns, not significant.

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