Gli2 expression in thymocytes on E16.5, and function in E13.5 and E14.5 thymus. (A) Gli2 expression in sorted C57BL/6 E16.5 thymocytes. Error bars represent SD. (B) Gli2 affects the transition from DN1 to DN2 in E13.5 embryos (WT, n = 5; Gli2^+/−, n = 12; Gli2^−/−, n = 8). Relative number of thymocytes on E13.5. To allow comparison between litters, the number of cells in each thymus was divided by the mean of WT littermates to give a relative cell number for each thymus. Dot plots show DN subset composition of E13.5 thymi. Cells were gated for CD45.2⁺, and analyzed by CD25 and CD44 expression. Bar chart shows the relative percentage of DN1 cells in E13.5 thymi. The difference between WT and Gli2^−/− is statistically significant (P = .02). (C) Thymocyte development in E14.5 Gli2^−/− and littermate embryos (WT, n = 17; Gli2^+/−, n = 27; Gli2^−/−, n = 15). Bar chart shows the relative thymocyte number on E14.5. The difference between WT and Gli2^−/− is significant (P = .04). Top dot plot shows E14.5 thymocytes stained with anti-CD45.2. Cells positive for CD45.2⁺ were analyzed for CD25 and CD44 expression. Righthand bar charts show the relative percentage of DN1 and DN3 cells in E14.5 thymi. Differences in mean relative percentages of DN1 and DN3 populations were statistically significant between WT and Gli2^−/− (P = .04 and P = .002, respectively). Histograms show Draq5 staining of E14.5 thymocytes. The percentage of cells in G2+S/M is given.