Flt3 fate mapping reveals that most erythroid and megakaryocyte progenitors are derived from Flt3-expressing progenitors. (A) EYFP expression in early thymic progenitors (LIN⁻CD25⁻KIT⁺; bottom left) and BM B220⁺ B-cell progenitors (bottom right) of Flt3-Cre^tg/+R26R^EYFP/+ mice. (B-C) Representative FACS profiles of EYFP expression in myeloid progenitor subsets (B) or LSKCD150⁺CD48⁻ HSCs (also gated as FLT3⁻; C) in the BM of Flt3-Cre^tg/+R26R^EYFP/+ mice. Numbers in histograms are mean percentages from analysis of 3-6 mice. GMP indicates GM progenitor. (D) Quantitative RT-PCR analysis of Flt3 mRNA expression in EYFP⁻ LSKFLT3⁻CD150⁺ CD48⁻ and EYFP⁺ LSKFLT3⁻CD150⁺CD48⁻ HSCs and LSKFLT3^hi LMPPs isolated from 3-week-old Flt3-Cre^tg/+R26R^EYFP/+ mice. Mean (SEM) expression relative to Hprt, 50-100 cells/ well, 2-3 wells/mouse, 4 separate mice in total. (E) Single EYFP⁺ LSKFLT3⁻CD150⁺CD48⁻ HSCs and EYFP⁺ Lin⁻SCA1⁺KIT⁻CD41⁻FcgR⁻CD150⁻CD105⁺ erythroid progenitors isolated from 4-week-old Flt3-Cre^tg/+R26R^EYFP/+ mice were analyzed for Flt3 mRNA expression. Data are expressed as mean (SEM) percentage of single Kit⁺ cells. A total of 64 single EYFP⁺ HSCs were sorted in total, of which 63 cells were Kit⁺, and 24 single EYFP⁺ erythroid progenitors were sorted, of which 24 were Kit⁺. (F) Quantitative PCR analysis of VWF, Gfi1b, and Runx3 expression in EYFP⁺ LSKFLT3⁻ CD150⁺CD48⁻, EYFP⁻ LSKFLT3⁻CD150⁺CD48⁻ HSCs, and LSKFLT3^hi LMPPs isolated from 3-week-old Flt3-Cre^tg/+R26R^EYFP/+ mice. Mean (SEM) expression relative to Hprt, 50-100 cells/well, 2-3 wells/mouse, 4 separate mice in total. (G) Two million BM cells from Flt3-Cre^tg/+R26R^EYFP/+ CD45.2 mice were transplanted into lethally irradiated CD45.1 recipients. At 8 weeks after transplantation, donor-derived (CD45.2) LSKEYFP⁺ and LSKEYFP⁻ cells were sorted for secondary transplantation. FACS profiles show representative distribution of LSKEYFP⁻ and LSKEYFP⁺ cells in primary recipients and purity analysis after sorting. (H) Five thousand purified LSKEYFP⁻ and LSKEYFP⁺ cells from primary recipients of Flt3-Cre^tg/+R26R^EYFP/+ CD45.2⁺ BM cells were transplanted in competition with 300 000 WT CD45.1 BM cells into secondary recipients. At 6 months after transplantation, BM from recipients who underwent transplantation was analyzed for contribution of CD45.2⁺ cells toward the B-cell and myeloid cell lineages. To the left FACS profiles from representative mice. Percentages are mean values relative to total BM cells. To the right mean (SD) contribution in a total of 4-6 recipients each of transplanted LSKEYFP⁺ and LSKEYFP⁻ cells toward total (CD45.2⁺), B (CD19⁺) and myeloid (Gr1⁺Mac1⁺) lineages at 6 months after transplantation.