Hoxb4 is expressed in adult BM HSCs. Flow cytometric analysis of BM cells from WT and Hoxb4^YFP/+ animals. (A) Gating of the LSK population is shown by 4 gates representing the major populations, marked R1 to R4. (B) Comparison of gated populations R1 to R4 indicated in panel A, showing YFP expression. In the histograms dashed lines indicate WT and solid lines indicate Hoxb4^YFP/+ cells. (C) The FACS strategy is outlined, indicating Lin⁻, Sca1⁺, and YFP sorting gates. (D) Lin⁻Sca1⁺Hoxb4/YFP⁺ cells in contrast to Lin⁻Sca1⁺Hoxb4/YFP⁻ cells effectively reconstitute irradiated recipients. Each symbol indicates one recipient animal and reflects the level of engraftment of donor-derived cells (combined data from 2 separate experiments). (E) BM extracted from WT and Hoxb4^YFP/+ mice at 1 week, 4 weeks, 8 months, and 16 months of age were stained with the LSK markers (lacking Mac1 for mice 1 and 4 weeks old). LSK cells were gated (as in panel A) and YFP fluorescence in experimental Hoxb4^YFP/+ mice (2 animals at each time point, red- and blue-outlined histograms) compared with LSK cells from WT (thick black-lined histograms) and 8-week-old Hoxb4^YFP/+ mice (solid gray histogram). Note that, although the peak of Hoxb4/YFP fluorescence in the red histogram is shifted less than in the blue histogram at 16 months and is similar to that in 8-month-old mice, the entire peak volume is shifted significantly higher (arrowhead).