Characterization of Circulating Primitive Erythroid Cells during Embryogenesis Using a Human ε-Globin-GFP Transgenic Mouse Model.
Authors: Fraser Stuart T. , Isern Joan , Baron Margaret H. 
Published: April 2020

Abstract
Primitive erythroid cells (EryP) are the first hematopoietic cell type to mature during embryonic development. EryP are characterized by expression of “embryonic” ε- and βh1-globin genes that are not expressed in fetal liver-derived “adult type” or definitive erythroid cells. Although EryP constitute the majority of the blood cells in mid-gestation embryos, EryP maturation remains poorly defined. Here, we utilize a transgenic mouse line in which green fluorescence protein (GFP) expression is driven by the human ε-globin minimal promoter to characterize the development of circulating EryP throughout embryogenesis. ε-globin(+)/GFP(+) EryP appear from 7.5 dpc within blood islands of the yolk sac. As expected, at 9.5 dpc, essentially all circulating blood cells express GFP. By 14.5 dpc, only 50% of the circulating cells express GFP. This sharp decrease in the numbers of circulating ε-globin(+)/GFP(+) cells continues until birth, by which stage fewer than 1% express the reporter gene. To further characterize the maturation of EryP, we analyzed the expression of surface antigens on ε-globin(+)/GFP(+) cells at various stages of embryonic development. From 9.5 dpc onwards, ε-globin(+)/GFP(+) cells express increasing levels of the erythroid marker Ter-119. The transferrin receptor CD71 is also expressed from early stages of development but is down-regulated as ε-globin(+)/GFP(+) cells mature. Increasing Ter-119 expression with concomitant loss of CD71 have previously been reported as hallmarks of maturing fetal liver erythroid cells (EryD) and we now report that ε-globin(+)/GFP(+) EryP demonstrate a similar developmental progression. Therefore, EryP contribute to the Ter119 and CD71 expression reported by others for total embryonic peripheral blood cells. The GPI-anchored surface marker CD24, present on EryD, is also found on ε-globin(+)/GFP(+) EryP. Interestingly, expression of the adhesion molecules CD44 and α4-integrin was upregulated on maturing ε-globin(+)/GFP(+) EryP, perhaps reflecting a requirement for interaction of EryP with other cells. ε-globin(+)/GFP(+) EryP lack expression of surface antigens typical of endothelial cells (Flk1, VCAM1), hematopoietic stem cells (c-kit, Sca1, CXCR4), myeloid (Gr1, Mac1) and lymphoid cells (CD19, CD3). Together, these data help to define the maturation pathway of the primitive erythroid lineage. It was recently shown by immunohistology that murine EryP enucleate, similar to their definitive counterparts. We used the cell permeable DNA-binding dye Draq5 to quantify enucleation in the circulating ε-globin(+)/GFP(+) population by FACS. We show that enucleated ε-globin(+)/GFP(+) cells are Draq5low/neg whereas those bearing nuclei are Draq5high. At 9.5 dpc, ε-globin(+)/GFP(+) cells are Draq5high. The frequency of nucleated EryPs decreases rapidly such that by 14.5 dpc, half of the circulating e-globin(+)/GFP(+) cells are Draq5low/neg. Shortly before birth, almost all EryP have enucleated. This system will allow us to separate nucleated from enucleated EryP by cell sorting and will help us assess changes in surface antigen expression during EryP maturation and enucleation. The human ε-globin-GFP transgenic mouse model is therefore a useful system for defining, at the cellular and molecular level, the developmental pathways of the primitive erythroid lineage.