Thymocyte development in CD4Cre-Ctnnb^Δex3 versus LckCre-Ctnnb^Δex3 mice. (A) β-Catenin protein levels in total thymocyte extracts from LckCre control (lanes 1-2), LckCre-Ctnnb^Δex3 (lane 3), and CD4Cre-Ctnnb^Δex3 (lane 4) mice revealed by Western blot analysis. Arrows depict wild-type and mutated β-catenin. Numbers indicate the predicted molecular weight in kilodaltons. (B) Course of β-catenin stabilization during thymocyte development. Thymocytes from the indicated mice were surface stained for expression of CD4 and CD8, or with a cocktail of lineage-specific antibodies (“Materials and methods”) combined with staining for surface expression of CD44 and CD25. Cells were then permeabilized and stained with anti–β-catenin FITC. Histogram overlays of electronically gated cells in the indicated subsets are representative of 4 independent experiments. (C) Intracellular TCRβ expression of DN3- and DN4-stage thymocytes. Thymocytes from the indicated mouse strains were surface stained as in panel B prior to permeabilization and staining for intracellular TCRβ expression (TCRβ-PE). Histograms depict intracellular TCRβ expression in the indicated electronically gated subsets. Numbers in the histograms indicate the fraction of TCRβ-positive cells and are representative of 5 independent experiments. (D) Fraction of DN3- and DN4-stage thymocytes in S/G₂/M phases of the cell cycle. Thymocytes were surface stained as in panel B prior to permeabilization and intracellular staining with the DNA binding dye 7AAD to estimate their DNA content. Histogram bars represent the average values of 5 independent experiments; error bars represent standard deviation. (E, upper panels) CD4/CD8 profiles of thymocytes from the indicated mouse strains; the numbers in the quadrants indicate the percentage of cells in each. Profiles are representative of 6 independent experiments. (E, lower panels) Thymic cellularity was estimated in 4- to 8-week-old mice. Percent distribution of the different thymocyte subsets was determined after surface staining for CD4 and CD8. Data represent average values from 6 LckCre, 6 CD4Cre-Ctnnb^Δex3, and 5 LckCre-Ctnnb^Δex3. Standard errors are included. While LckCre-Ctnnb^Δex3 thymi have reduced cellularity,^22,23  the cellularity of CD4Cre-Ctnnb^Δex3 thymi (P = .455) and the number of DPs (P = .2348) were comparable to LckCre controls in contrast to the absolute number of SPs, which was reduced in CD4Cre-Ctnnb^Δex3 mice (P = .013 for CD4 and P = .007 for CD8). The fraction of DPs significantly increased in CD4Cre-Ctnnb^Δex3 (P = .001) and LckCre-Ctnnb^Δex3 (P = .001) compared to LckCre mice. The fraction of SPs was significantly reduced in CD4Cre-Ctnnb^Δex3 (P = .001 for CD4⁺ and CD8⁺) and LckCre-Ctnnb^Δex3 (P = .001 for CD4⁺ and CD8⁺). Reduction in the fraction of SPs was comparable between CD4Cre-Ctnnb^Δex3 and LckCre-Ctnnb^Δex3 mice (P = .14 for CD4⁺ and P = .34 for CD8⁺).