Increased differentiation to CD4SP in the Shh^{\#8722;/\#8722;} thymus. (A) CD4 and CD8 staining of E14.5 Shh^{\#8722;/\#8722;}, Shh^+/\#8722;, and Shh^+/+ FTOC littermates, cultured for 6 days. Mean thymus sizes were 2.3 × 10⁴ for Shh^{\#8722;/\#8722;}, 1.6 × 10⁵ for Shh^+/\#8722;, and 1.8 × 10⁵ for Shh^+/+. (B-C) Bar charts to show the relative proportion of CD4SP thymocytes in E14.5 Shh^{\#8722;/\#8722;} and littermate FTOC after 3 (B) and 6 (C) days in culture. For each thymus, the proportion of CD4SP cells was calculated relative to the mean proportion in WT littermates. The difference in the relative proportion of CD4SP cells between Shh^{\#8722;/\#8722;} and Shh^+/+ thymi was statistically significant: P = .006 for 3-day cultures (WT [n = 7], Shh^{\#8722;/\#8722;} [n = 5]), P = .022 for 6-day cultures (WT [n = 7], Shh^{\#8722;/\#8722;} [n = 6]). (D) CD4 and CD8 staining of E17.5 Shh^{\#8722;/\#8722;}, Shh^+/\#8722;, and Shh^+/+ FTOC littermates, cultured for 7 days. The average percentage of CD4SPs in the Shh^{\#8722;/\#8722;} was increased 1.4 times compared with littermates (P = .046). The ratio of CD4/CD8 SP cells was 2.6 in Shh^+/+ FTOC and 3.4 in Shh^{\#8722;/\#8722;} FTOC. Bar charts to show CD3 and CD69 expression on CD4 and CD8 SP thymocytes in the Shh^{\#8722;/\#8722;} (\#9633;, n = 3) and littermates (\#9641;, n = 14). Mean thymus sizes were 9 × 10⁴ for Shh^{\#8722;/\#8722;}, 1.3 × 10⁵ for Shh^+/\#8722;, and 1.9 × 10⁵ for Shh^+/+. (E) Bar chart to show mean CD4/CD8 ratio for the different culture conditions. (F) Bar chart to show mean SP/DP ratio for the different culture conditions. Error bars show SE. Data are representative of at least 3 experiments.