Thymocyte development in E16.5 Ihh^−/− and Ihh^+/− thymi. (A) Thymocyte number on E16.5. The mean relative cell number (± SE) in WT, Ihh^+/−, and Ihh^−/− thymi on E16.5. To allow comparison between litters, the number of cells recovered from each thymus was divided by the mean number of cells recovered from WT thymi from the same litter, to give relative cell number from different E16.5 litters. The differences in mean cell number between Ihh^−/− and WT thymi (P < .001), Ihh^+/− and WT thymi (P = .018), and Ihh^−/− and Ihh^+/− (P < .001) were statistically significant by Student t test. (B) Photographs of E16.5 WT, Ihh^+/−, and Ihh^−/− littermate thymus lobes. (C) The mean relative cell number (■) and mean relative percentages (□) of DP cells (± SE) in WT, Ihh^+/−, and Ihh^−/− thymi on E16.5. Differences in mean cell number and percentages between WT and Ihh^+/− (P = .007, P = .02), WT and Ihh^−/− (P < .001 for both), and Ihh^+/− and Ihh^−/− (P < .001 for both) thymi were statistically significant by Student t test. (D) Flow cytometry of E16.5 WT, +/−, and −/− littermate thymi stained with anti-CD4 and anti-CD8. Thymus size: WT 4.56 × 10⁴, Ihh^+/− 1.144 × 10⁵, and Ihh^−/− 1.68 × 10⁴. (E) Transcription of Ihh in DP thymocytes from embryonic Ihh^+/+ and Ihh^+/− thymi. Relative levels of Ihh transcription were measured as described in Figure 1A and were normalized for HPRT mRNA content. (F) (Left panel) Mean percentages of DN thymocytes in WT and Ihh^−/− thymi, relative to mean of WT littermates. The percentage of DN cells in the Ihh^−/− thymus is significantly higher than in the Ihh^+/+ thymi by Student t test (P = .002). (Right panel) Mean percentages of DN4 thymocytes in the DN subset relative to mean of WT littermates are shown for WT and Ihh^−/− thymi. Differences between Ihh^+/+ and Ihh^−/− were statistically significant by Student t test (P < .001). (G) Mean percentages of icTCR-β⁺ cells in the DN3 subset are shown relative to mean of WT littermates for WT and Ihh^−/− thymi. Differences between mean percentage icTCR-β⁺ in DN3 subset of Ihh^+/+ and Ihh^−/− were statistically significant by Student t test (P = .03). (H) Representative histograms of icTCR-β expression in the DN3 subsets of WT littermate (top) and Ihh^−/− (bottom) thymi. (I) TCR-β locus rearrangement was measured in E15.5 DN thymocytes from WT and Ihh^−/− littermates, according to the method of Gounari et al.²⁹  DNA was amplified using primers 5′ to Vβ8.2 or Vβ5.1 and 3′ to Jβ2.7, and products were measured by quantitative PCR on an iCycler (Bio-Rad) using iQ-SYBR Green Supermix (Bio-Rad). DNA content was normalized relative to Thy1. Differences between Ihh^−/− and WT were not significant by Student t test. (J) The bar chart shows the percentage of cells in S + G₂/M, measured by PI staining of FACS-sorted E16.5 CD25⁺ DN cells from WT and Ihh^−/− littermates, relative to the mean percentage in WT littermate populations. Differences in mean percentage between −/− and WT were not statistically significant.