Ihh regulates thymus homeostasis. (A) Scatter plots show the percentage of cells in S + G₂/M, measured by PI staining of FACS-sorted E16.5 CD25⁺ DN (left plot), CD8ISP (middle plot), and DP (right plot) populations from WT and Ihh^+/− littermates, relative to the mean percentage in WT littermate populations. Differences in mean percentage between +/− and WT were statistically significant for CD25⁺ DN (P = .042) and CD8ISP (P = .040) populations, but not for DP cells (P = .981). (B) Scatter plots show relative thymocyte number from Ihh^+/+Rag1^−/− and Ihh^+/−Rag1^−/− thymi (relative to mean of Ihh^+/+Rag1^−/− littermates). Differences are significant by Student t test (P < .001). Mean thymocyte number: litter 1 Ihh^+/+Rag1^−/− 1.65 × 10⁶, Ihh^+/−Rag1^−/− 1.33 × 10⁶; litter 2 Ihh^+/+Rag1^−/− 1.7 × 10⁶, Ihh^+/−Rag1^−/− 1.25 × 10⁶; litter 3 Ihh^+/+Rag1^−/− 1.75 × 10⁶, Ihh^+/−Rag1^−/− 1.31 × 10⁶. (C) Bar chart shows fold increase in thymocyte number on induction of differentiation by treatment with 1 μg/mL anti-CD3 after 5 days of FTOCs for Rag1^−/−Ihh^+/+ and Rag1^−/− Ihh^+/− littermates. For each thymus, one lobe was cultured with anti-CD3 and the fold increase in cell number was calculated relative to the number of cells in the other untreated lobe. The fold increase in the Rag1^−/−Ihh^+/− was significantly different from that in the Rag1^−/−Ihh^+/+ by Student t test (P = .03). Mean thymocyte number: litter 1 Ihh^+/+Rag1^−/− untreated 1.65 × 10⁶, Ihh^+/+Rag1^−/− + anti-CD3 1.425 × 10⁷, Ihh^+/−Rag1^−/− untreated 1.33 × 10⁶, Ihh^+/−Rag1^−/− + anti-CD3 1.725 × 10⁷. Litter 2 Ihh^+/+Rag1^−/− untreated 1.7 × 10⁶, Ihh^+/+Rag1^−/− + anti-CD3 1.49 × 10⁷, Ihh^+/−Rag1^−/− untreated 1.245 × 10⁶, Ihh^+/+Rag1^−/− + anti-CD3 1.689 × 10⁷. (D) Dot plots show E17.5 WT, Ihh^+/−, and Ihh^−/− thymi stained with anti-CD4 and anti-CD8. Mean thymocyte number: litter 1 WT 2.05 × 10⁶, Ihh^+/− 3.3 × 10⁶, Ihh^−/− 2 × 10⁶; litter 2 WT 3.5 × 10⁶, Ihh^+/− 2.865 × 10⁶, Ihh^−/− 8.5 × 10⁵; litter 3 WT 3.35 × 10⁶, Ihh^+/− 2.63 × 10⁶ Ihh^−/− 1.675 × 10⁶; litter 4 WT 3.35 × 10⁶, Ihh^+/− 2.336 × 10⁶; litter 5 WT 3.868 × 10⁶, Ihh^+/− 3.69 × 10⁶; litter 6 WT 4.8 × 10⁶, Ihh^+/− 4.756, Ihh^−/− 3.8 × 10⁶. (E) Scatter plot shows cell numbers (relative to mean of WT) from thymi from E17.5 WT, Ihh^+/−, and Ihh^−/− embryos. The difference in mean between WT and Ihh^−/− is significant (P = .002, Student t test), and the difference in SD between WT and Ihh^+/− is also significant (P < .001, F test). (F) Scatter plot shows : thymocyte number (relative to mean of WT) from E17.5 WT littermate and Ihh^+/− embryos. Ihh^+/− thymi are grouped by relative size into 3 sets: I, more than 1.3; II, 0.9 to 1.1; and III, less than 0.9; ○: the percentage of cells in S + G₂/M (relative to mean of WT), measured by PI staining of FACS-sorted E17.5 CD25⁺ from WT and Ihh^+/− mice grouped into 3 groups I, II, and III as detailed in this figure legend.