(A) Targeting strategy for Tcf7l1 gene locus. Yellow boxes denote exons, with exon numbers marked on top. Exon six is flanked with two LoxP sites (marked with red triangles). The LacZ/Neo cassette flanked by Frt Sites (cyan wedges) was removed with Flippase in germline-transmitted mice. (B) In vivo ablation of Tcf/Lef proteins. CreER+ WT, Tcf1/Lef1 dKO, and Tcf3/4 dKO mice were treated with tamoxifen for four consecutive days. Three days later, total thymocytes were intracellularly stained for Tcf1 and Lef1 in Tcf1/Lef1 dKO and control mice, and values denote geometric mean fluorescent intensity in representative half-stacked histograms (left panels). Total thymocytes were immunoblotted for Tcf4 protein in Tcf3/4 and control mice (right panel). (C) Validation of efficient deletion of targeted Tcf7l1 and Tcf7l2 exons in hematopoietic stem/progenitor cells. CreER+Tcf-qKO and WT mice were treated with tamoxifen for four consecutive days, and two days later, Flt3–LSK cells, which were enriched in both long-term and short-term HSCs, were sort-purified and analyzed by quantitative RT-PCR. Relative expression of Tcf7l1 and Tcf7l2 was determined by normalizing to Hprt, and shown as means ± s.d. (n = 5). NRD, not reliably detected. (D) Thymic cellularity. CreER+ WT, Tcf1/Lef1 dKO, Tcf3/4 dKO, and Tcf-qKO mice were treated with tamoxifen as in B), and thymocytes were enumerated. (E) Detection of thymic maturation stages. Thymocytes were surface-stained with biotinylated lineage markers (minus CD3ε) to exclude non-T cells, and with CD4 and CD8 to identify DN, DP, CD4+ and CD8+ subsets. (F) Detection of DN subsets. DN thymocytes were surface-stained with CD44 and CD25 to identify DN1 to DN4 subsets. Note that deletion of Tcf1 and Lef1, as in Tcf1/Lef1 dKO and Tcf-qKO mice, caused premature, modest upregulation of CD25 in a portion of DN1 cells, and the gate was adjusted accordingly to demarcate DN1 and DN2 subsets. Data in D–F are means ±s.d. from ≥2 experiments. Statistical significance for multiple groups was first assessed by one-way ANOVA, and that for indicated pair comparison was determined with Tukey’s correction. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not statistically significant. In F), n = 2 for Tcf1/Lef1 dKO, and thus no p values are shown. (G) Kaplan-Meier survival curves of 1o and 2o recipients of WT or Tcf-qKO LSCs. Data are pooled from two independent experiments. ns, not statistically significant as determined by log-rank test. (H) Longitudinal tracking of CD45.2+GFP+Mac1+ AML leukemic cells in PBCs of 2o recipients. For week 8, the surviving recipients were analyzed. Data from two independent experiments were displayed separately because modest differences were observed in kinetics of leukemic cell expansion at week 4. These differences did not affect recipient survival (see G). ns, not statistically significant as determined by Student’s t-test.