Cxcr4¹⁰¹³-bearing mice display a cell-intrinsic constraint in multipotent and lymphoid-biased progenitors in the BM. (A) Absolute numbers of MPPs (Lin⁻c-Kit⁺Sca-1⁺ [LSK] CD48⁻CD150⁻), LMPPs (LSK Flt3^highCD34⁺), and CLPs (Lin⁻c-Kit^lowSca-1^lowFlt3⁺CD127⁺) in the BM of WT and mutant mice. Data are from at least four independent experiments with 13–17 mice per group. (B, left) Absolute numbers of CMPs (Lin⁻c-Kit⁺Sca-1⁻CD34⁺CD16/32⁻), GMPs (Lin⁻c-Kit⁺Sca-1⁻CD34⁺CD16/32⁺), and MEPs (Lin⁻c-Kit⁺Sca-1⁻CD34⁻CD16/32⁻). (right) Number of colonies formed from WT, +/1013, or 1013/1013 BM in the myeloid lineage in CFU-C assays. Data are from at least four independent experiments with 7–10 mice per group. (C) Absolute numbers of donor CD45.2⁺ (left, WT, +/1013, or 1013/1013) or CD45.1⁺ (right, WT) MPPs, CLPs, CMPs, GMPs, and MEPs recovered from the BM of chimeras in CD45.1⁺ (left, WT) or CD45.2⁺ (right, WT, +/1013 or 1013/1013) recipients. Data are from at least three independent experiments with 8–15 recipient mice per group. (D) Frequency of CD150^high in LSK CD34⁻ cells in the BM of young WT and mutant mice. Data are from at least four independent experiments with 6–10 mice per group. (E and G) Equal numbers of purified LSK SLAM (CD48⁻CD150⁺; E) or MPPs (G) were cultured for 4 d in a cytokine-supplemented feeder-free media. (left) Representative flow cytometric analyses comparing the frequencies of LSK SLAM and MPPs (E) or LMPPs (G) at day 4. (right) Absolute numbers of LSK SLAM and MPPs (E) and of MPPs and LMPPs (G) after 2 and 4 d of culture. Data are from three independent experiments with six mice per group. (F and H) Representative histograms for the expression of Ki-67 in MPPs (F) and LMPPs (H) after 4 d of culture. (I, left) Membrane expression of Cxcr4 was determined by flow cytometry on gated BM CLPs from WT, +/1013, and 1013/1013 mice. Background fluorescence is shown (isotype, gray histograms). (right) The percentages of LSK SLAM, MPPs, LMPPs, CLPs, pro–B cells, and ETPs expressing Cxcr4 are shown. Data are from three independent experiments with five to seven mice per group. (J) Cell surface expression of Cxcr4 on BM Lin⁻ cells or B220⁺ B cells upon exposure to 10 nM Cxcl12 or 100 nM Ccl2 at 37°C for 45 min. No Cxcr4 internalization was found when cells were incubated at 4°C in the presence of Cxcl12. Cxcr4 expression on BM cells incubated in medium alone was set at 100% (dotted horizontal line). Data are from three independent experiments with five mice per group. (K) Migration of Lin⁻ BM cells from WT, +/1013, and 1013/1013 mice in response to 1 or 10 nM of Cxcl12 was enumerated by CFU-C assays. Data are from at least three independent experiments with five to eight mice per group. (L) The proportions of apoptotic (Annexin V⁺ DAPI⁻) LMPPs and CLPs were determined by flow cytometry in the BM of WT and mutant mice. Data are from three independent experiments with eight to nine mice per group. All displayed results are represented as means ± SEM. Kruskal–Wallis H test–associated p-values (#) are indicated. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 compared with WT or CD45.2⁺ donor WT cells; ^§, P < 0.05; and ^§§, P < 0.005 compared with +/1013 or CD45.2⁺ donor +/1013 cells (as determined using the two-tailed Student’s t test).