Novel Insights into the Maturation and Enucleation of Primitive Erythroblasts.

Primitive erythroblasts (EryP), the first blood cell type to form during mammalian embryogenesis, arise in the yolk sac (YS) as a synchronous cohort before the establishment of the embryonic circulation. The large, nucleated EryP begin to circulate around midgestation, when connections between YS and embryo mature. Two to three days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryP within the circulation. Cells of the two erythroid lineages differ not only in size but also in their expression of globin genes. Recently it has been shown that EryP begin to enucleate around E12.5 in the mouse. We have begun to characterize EryP maturation during mouse embryogenesis, with a focus on circulating cells. Analysis of embryonic blood at different stages revealed a stepwise developmental progression within the primitive erythroid lineage: loss of nucleoli (E9.5–10.5); decrease in cell diameter and cross-sectional area (E10.5–11.5); progressive nuclear condensation (E10.5 onwards); and enucleation (E12.5 onwards). Thereafter, EryD are also present in the bloodstream. A major obstacle to the study of primitive erythroid development is the current inability to easily distinguish EryP from EryD and to cleanly separate these populations, particularly given that enucleation can no longer be considered an exclusively definitive erythroid characteristic. No EryP-specific surface antigens have been identified that would permit the detection and isolation of cells of this lineage. We have exploited the restricted expression of the human embryonic beta-like globin gene epsilon to generate EryP lineage-specific transgenic mouse and embryonic stem (ES) cell models in which green fluorescent protein (GFP) reporter expression is driven exclusively in EryP and have used this line to track quantitatively the differentiation and maturation of circulating EryP during embryogenesis. The maturation of primitive erythroblasts was reflected in their dynamic expression of Ter119 and CD71. Up-regulation of a number of nonerythroid-specific cell surface markers, including integrins and other adhesion molecules, was apparent beginning around E12.5, reflecting a previously unrecognized EryP maturation process. Interestingly, enucleation of EryP is accompanied by changes in surface antigen expression. Thus, the phenotype of primitive reticulocytes is distinct from that of their nucleated precursors. In contrast with the prevailing view that EryP are present only transiently in the embryo, we find that they comprise a stable cell population that persists through the end of gestation. The human epsilon-globin::GFP transgenic mouse model provides a novel system for defining, at the cellular and molecular level, the mechanisms involved in development of the primitive erythroid lineage.