A Novel Nr4a1^GFP Reporter System Reveals That Nr4a1 Expression Identifies Long-Term Hematopoietic Stem Cells

Abstract 2339

Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1^GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1^GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1^GFP. Interestingly, circulating LSK's in the spleen express Nr4a1^GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1^GFP, 2) Nr4a1^GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1^GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1^GFP expression.