Thymocyte development in Shh/Ihh double mutants and in FTOCs treated with exogenous recombinant Hedgehog protein. (A) Histograms show the mean relative cell number (± SE) in WT (n = 11), Ihh^+/+Shh^+/− (n = 5), Ihh^+/−Shh^+/+ (n = 8), Ihh^+/− Shh^+/− (n = 3), Ihh^−/−Shh^+/+ (n = 4), Ihh^−/−Shh^+/− (n = 3) (left), and in WT (n = 11), Ihh^+/+Shh^−/− (n = 3), and Ihh^+/−Shh^−/− (n = 3) thymi (right) on E16.5. (B) Histogram to show the mean relative percentage ( ± SE) of DP cells in the litters shown in panel A. (C) Expression of CD4 and CD8 on Shh^+/− Ihh^+/+, Shh^+/+ Ihh^−/−, and Shh^+/− Ihh^−/− thymi. Thymus sizes were: Shh^+/− Ihh^+/+: 2.6 × 10⁵; Shh^+/+ Ihh^−/−: 1.28 × 10⁵; and Shh^+/− Ihh^−/−: 5.2 × 10⁴. (D) Bar chart shows the fold change in number of DP thymocytes recovered from WT E15 FTOCs treated for 3 days with different concentrations of r-mShh-N (R&D Systems), compared with the number of DP cells recovered from the untreated thymus lobe from the same embryos; 0.25 μg/mL r-mShh-N decreased the production of DP cells by approximately 2-fold. (E) Bar chart shows the number of cells from DN, CD8⁺ ISP, and DP thymocyte populations recovered from Ihh^+/− and WT littermate E15 FTOCs cultured for 3 days, with or without treatment with 0.25 mg/mL r-mShh-N. Mean cell recovery per thymus lobe: Ihh^+/− control 3.3 × 10⁵; Ihh^+/− + r-mShh-N 6.3 × 10⁴; Ihh^+/+ littermate control 7.0 × 10⁴. In each experiment, the treated thymus lobe was compared with the untreated lobe from the same embryo. (F) Dot plots show anti-CD4 and anti-CD8 staining of individual thymus lobes from the experiment in panel E, in which treated thymus lobes were compared with the untreated lobe form the same embryo, or to a WT littermate lobe. (G) Bar charts show the number of cells from DN, CD8⁺ ISP, and DP thymocyte populations recovered from Ihh^−/− and littermate (LM) E15 FTOC cultured for 5 days, with or without treatment with 0.25 μg/mL r-mShh-N. The treated lobe from one embryo was compared with the untreated lobe from the same embryo.