“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Abstract 2539

Poster Board II-516

A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis.