Loss of Miz-1 POZ domain affects frequencies of bone marrow ELPs, ALPs, and BLPs and in vitro differentiation of ETPs and ELPs. (A) ELPs were gated on Lin⁻ no CD4, CD25⁻ Sca1⁺CD44⁺CD117⁺ (top panel) and further analyzed for CD135 and CCR9 expression (bottom panel). The plots are composed of an overlay of the CD135 or CCR9 staining in black (WT) or gray (Miz-1^ΔPOZ) with the matching isotype control antibodies staining (dotted black; n = 4). (B) Bone marrow Lin⁻CD127⁺Sca1^medCD117^med CLPs were examined for the expression of Ly6D dividing the progenitor population into Ly6D⁻ ALPs and Ly6D⁺ BLPs. (C) Percentages of positive cells in panels A and B are calculated relative to the total live cells and expressed as absolute cell count (n = 4 for ELPs and n = 2 for ALPs and BLPs). (D) Quantitative real-time PCR analysis of target genes involved in ELP development. RNA was extracted from 5000 sorted bone marrow ELPs from WT and Miz-1^ΔPOZ mice. All values are presented as fold induction relative to values obtained with the respective wild-type control. Average of triplicate values and SD are shown (n = 3). FACS analysis indicating the development of sorted Lin⁻ no CD4, CD25⁻ Sca1⁺CD44⁺CD117⁺ ELPs (E) and ETPs (F) from the bone marrow and thymus of WT or Miz-1^ΔPOZ mice. Fifty sorted ETPs or ELPs were cocultured on OP9DL1 stroma cells for 15 or 20 days in T-cell media. The developmental progression of the cells is evaluated by flow cytometry using T-cell markers CD44, CD25, CD4, and CD8 (n = 4).