Early thymocyte development in Ihh^−/− thymi. (A,B) Transcription of Ihh (A) and Gli1 (B) in sorted E16.5 fetal thymocyte populations and thymus stroma from C57BL/6 mice. Levels of Ihh and Gli1 transcription were normalized for HPRT mRNA content and are shown relative to HPRT-normalized transcription in the DN3 (Ihh) or DN1 (Gli1) subsets. cDNA samples were analyzed in triplicate by quantitative PCR on an iCycler (Bio-Rad, Hercules, CA) using iQ-SYBR Green Supermix (Bio-Rad). Thymocytes were sorted on a Modular Flow Cytometer (MoFlo; Dako North America, Carpinteria, CA), and purity was more than 98%. (C-F) Flow cytometry of E13.5 Ihh^−/− and WT littermate thymi. (C) Dot plot of anti-CD45.2 staining versus forward scatter (FSC), showing the gate used for analysis of lymphocyte precursors. (D) The composition of DN subsets gated on CD45.2⁺ and stained with anti-CD44 and anti-CD25. (E) Dot plot of anti-CD117 staining versus anti-CD44 staining, gated on CD45.2⁺. (F) Histogram of anti-B220 staining, gated on CD45.2⁺CD44⁺ cells. Thymus size: control: 6.4 × 10³, Ihh^−/−: 4.8 × 10³. (Gi) Photographs of E14.5 Shh^−/− and WT littermate thymus lobes. (Gii) Photographs of E14.5 Ihh^−/− and WT littermate thymus lobes. (H) The mean (± SE) relative cell number in WT (n = 7), Ihh^+/− (n = 17), and Ihh^−/− (n = 5) thymi on E14.5. 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 4 E14.5 litters. Differences between WT, Ihh^+/−, and Ihh^−/− were not significant by Student t test.