Developmental dichotomy of CLP1 and presumptive CLP2 progenitor cells evidenced by analysis of Gfi1−/− and Gfi1GFP/+ mice.
A) FACS plots indicating relative decrease of CLP1 (upper panels) and normal numbers of presumptive CLP2 (middle panels) in Gfi1−/− bone marrow cells. Cells were pregated on Lin−IL7Rα+ cells (upper panels) and Lin−IL7Rα+Sca1+CD19− cells (middle panels), respectively. Bar diagrams indicating absolute numbers of CLP1 and CLP2 cells of Gfi1−/− and Gfi1+/+ mice (lower panel, n = 3 mice). Lineage markers included CD4, CD8, CD11c, CD11b and B220. Flow-cytometric analysis was performed on pooled BM cells from 5 mice. Shown is a representative experiment of 3. B) FACS plots indicating relative increase of ETP in Gfi1−/− thymus. Lin−CD25−CD44+ cells (upper panels) were gated (G1) and analysed for expression of c-kit and IL7Rα (lower panels). Experiments were performed on pooled thymi (n = 5 mice). Bar diagrams indicating absolute numbers of ETP of Gfi1−/− and Gfi1+/+ mice (lower panel, n = 3 mice). The total number of thymocytes was decreased by ∼6 fold in Gfi1−/− mice. Shown is a representative experiment of 3. C) Transcriptional activity of Gfi1 locus in hematopoietic progenitor cells and thymic B cells. Pooled bone marrow cells or thymocytes (n = 5) were analyzed for GFP expression in the following populations: LSK (HSC), Lin−IL7Rα+Sca1lowc-kitlow (CLP1), Lin−IL7Rα+Sca1+c-kit−B220+CD19− (CLP2) and Lin−CD25−IL7Rα−CD44+c-kit+ (ETP). Shaded histograms represent GFP fluorescence in Gfi1+/GFP cells, open histograms represent autofluorescence of Gfi1+/+ cells. The GMFI (Geo Mean Fluorescence Intensity) of Gfi1+/+ (above) and Gfi1+/GFP (below) cells is indicated. Data are representative of 3 independent experiments.