bcl-x prevents apoptotic cell death of both primitive and definitive erythrocytes at the end of maturation

Abstract

bcl-x is a member of the bcl-2 gene family, which regulates apoptotic cell death in various cell lineages. There is circumstantial evidence suggesting that bcl-x might play a role in the apoptosis of erythroid lineage cells, although there is no direct evidence. In this study, we used Bcl-X null mouse embryonic stem (ES) cells, and showed that Bcl-X is indispensable for the production of both embryonic primitive erythrocytes (EryP) and adult definitive erythrocytes (EryD) at the end of their maturation. In vivo, bcl-x-/- ES cells did not contribute to circulating EryD in adult chimeric mice that were produced by blastocyst microinjection of the bcl-x-/- ES cells. bcl-x-/- EryP and EryD were produced by in vitro differentiation induction of ES cells on macrophage colony-stimulating factor-deficient stromal cell line OP9, and further analysis was carried out. The emergence of immature EryP and EryD from bcl-x-/- ES cells was similar to that from bcl-x+/+ ES cells. However, prominent cell death of bcl-x-/- EryP and EryD occurred when the cells matured. The data show that the antiapoptotic function of bcl-x acts at the very end of erythroid maturation.

Figure 3

Percentage of viable erythroid lineage cells in the presence or absence of EPO. Purified day 6.5 EryP (A and B) and purified day 11.5 EryP (C and D) derived from bcl-x ^+/+ and bcl-x ^−/− (clone 18) ES cells were cultured in the presence or absence of EPO until the indicated day. The percentage of viable cells is shown as the mean ± SD of six dishes. The data shown are representative of three independent experiments. bcl-x ^−/− (clone 3a) ES cells gave results similar to bcl-x ^−/− (clone 18) ES cells.

Figure 1

Contribution of ES-derived cells to mature adult definitive erythrocytes of chimeras. Hemoglobin was analyzed in the peripheral blood of C57BL/6 mice (lane 1) and 129/Ola mice (lane 2). The Hbb^s haplotype (single) is specific for host blastocysts of strain C57BL/6, and the Hbb^d haplotype (diffuse; major and minor) is specific for strain 129/ Ola from which the ES cell line used in this study was established. Peripheral blood samples of chimeras made with bcl-x ^+/+ ES cells (lanes 3 and 4), bcl-x ^+/− ES cells (lanes 5 and 6), bcl-x ^−/− ES cells, clone 18 (lanes 7–9), and bcl-x ^−/− ES cells, clone 3a (lanes 10–12) were analyzed to examine the contribution of ES cells.

Figure 2

Gel electrophoresis of low molecular weight DNA extracted from the cells after culture of purified day 6 differentiation– induced EryP and day 12 differentiation–induced EryD in the presence of EPO for 48 h. DNA extracted from the cells from the day 6 induced bcl-x ^+/+ EryP (lane 1), the day 6 induced bcl-x ^−/− EryP (lane 2), the day 12 induced bcl-x ^+/+ EryD (lane 3), and the day 12 induced bcl-x ^−/− EryD (lane 4).