Blood formation in development is characterized by two distinct stages. Primitive hematopoiesis, which is initiated by a population of primitive hematopoietic stem cells, is progressively replaced by definitive hematopoiesis, which is derived from definitive hematopoietic stem cells that maintain hematopoiesis throughout adulthood. During murine embryogenesis, primitive erythroid cells (EryP) develop in the yolk sac blood islands on embryonic day 7.5 (E7.5), enter the circulation by E9.5, and remain nucleated until E12.5. In contrast, definitive erythroid cells (EryD), which appear later in fetal development, are enucleated prior to entering the blood stream.
In order to better understand how enucleation of EryP is regulated, the investigators designed an elegant system to track EryP. In this system, the promoter elements of the ε-globin gene drive expression of green fluorescent protein (GFP), such that GFP expression is restricted to EryP. This allows tracking of EryP throughout development even during E12 through E16 when progressively greater numbers of EryD appear. The investigators followed maturation and expansion of EryP as well as surface markers on the cells before, during, and after enucleation. They discovered a transient upregulation of particular integrins from E12.5 to E14.5, with little expression of these integrins on EryP before or after this time frame. The investigators hypothesized that upregulation of these integrins promotes adhesion of EryP to macrophages in the developing fetal liver forming so-called erythroid blood islands in which developing erythroid cells form rosettes around central macrophages, which then phagocytose the nuclei as the erythroid cells enucleate. To test this hypothesis, the investigators engineered erythroid blood islands in vitro using isolated fetal liver-derived macrophages from one specific stage of fetal liver development and EryP from multiple different stages of development. They found that the EryP from days E12.5 through E14.5 adhered to the macrophages, while those from earlier and later stages did not. The two-day window during which EryP adhere corresponds to the time during embryogenesis when EryP upregulate integrins and enucleate. By E15.5, enucleation of EryP is complete.
As an erythrocyte enucleates, the nucleus is not just expelled from the cell, but remains surrounded by a thin rim of plasma and plasma membrane and is pinched off from the remaining erythroblast by formation of an actin ring. This results in two cells. One is the young enucleated RBC, which can re-enter the circulation, and the other contains the membrane-bound nucleus. A key feature of this process is the asymmetric distribution of plasma membrane components between the resulting RBC versus the expelled nucleus. To test the hypothesis that integrins are enriched on the plasma membrane surrounding the expelled nucleus may be responsible for the retention of the nuclei near the macrophages, while allowing the RBC to re-enter the circulation, the investigators took their model a step further. They targeted the GFP protein expressed under the control of the ε-globin gene to enter the nucleus, which allowed them to specifically track the "pinched-off" nucleus. As predicted, expression of the integrins was much higher on the nuclei than on the enucleated RBCs, and the nuclei were retained in the fetal liver.
In Brief
The findings presented in this work not only represent an astute observation of the distinct development of primitive versus definitive erythropoiesis, but also employ an elegant model to elucidate the mechanisms underlying these differences. In an era when exploration into stem-cell therapies employing more and more primitive cell types is turning into a realistic hope, a deeper understanding of developmental mechanisms will certainly contribute to the field.
Competing Interests
Drs. Krause and Halene indicated no relevant conflicts of interest.
