Figure 6
Figure 6. Thymocyte Ihh expression is required for adult thymocyte development. (A,B) Expression of Ihh (A) and Gli1 (B) was assessed by quantitative RT-PCR in FACS-sorted populations of adult and fetal murine tissue. Levels of Ihh and Gli1 transcription were normalized for HPRT mRNA content and are shown relative to HPRT normalized transcription in the CD25+ DN population (Ihh) and DN1 population (Gli1). Thymocytes were sorted on a Modular Flow Cytometer (MoFlo; Dako North America), and purity was more than 98%. (C) Relative thymocyte number (relative to the mean of WT littermates) of 3-week-old WT (n = 8) and Ihh+/− (n = 8) littermates revealed a significant difference in SD as judged by F test (P < .001). Mean thymocyte number: litter 1 WT 1.2 × 108, Ihh+/− 1.77 × 108; litter 2 WT 1.44 × 108, Ihh+/− 1.76 × 108; litter 3 WT 1.35 × 108, Ihh+/− 1.81 × 108. (D) Transcription of Ihh in DP thymocytes from Ihh+/+ and Ihh+/− thymi. Relative levels of Ihh transcription were measured as described in Figure 1A and were normalized for HPRT mRNA content. (E) Excision of the Ihh gene as assessed by PCR from vavCre− (lane1) and vavCre+ Ihhfl/fl mice (lane 2). (F) Relative cell number (calculated relative to the mean of the WT littermates) of the thymus of 4- to 6-week-old mice of littermates (all Cre−: nonknockout mice, n = 9) compared with knockout mice (vavCre+Ihhflnull, n = 4) showed a significant decrease in thymocyte cell number (P = .003). Mean thymocyte number: litter 1 littermate (LM) 1.7 × 108, vavCre+Ihhflnull 0.75 × 108; litter 2 LM 1.95 × 108, vavCre+Ihhflnull 0.47 × 108; litter 3 LM 1.48 × 108, vavCre+Ihhflnull 1.28 × 108; litter 4 LM 1.2 × 108, vavCre+Ihhflnull 9.2 × 107. (G) Relative cell number (calculated as in panel D) of the thymus isolated from 4- to 6-week-old mice of littermate vavCre−Ihhfl/fl mice (WT, n = 15) compared with vavCre+Ihhfl/fl mice (knockout mice, n = 5) showed a significant decrease in thymocyte number (P = .016). Mean thymocyte numbers: litter 1 vavCre−Ihhfl/fl 3 × 108, vavCre+Ihhfl/fl 1.925 × 108; litter 2 vavCre−Ihhfl/fl 3.24 × 108, vavCre+Ihhfl/fl 2.59 × 108; litter 3 vavCre−Ihhfl/fl 2.115 × 108, vavCre+Ihhfl/fl 1.3 × 108; litter 4 vavCre−Ihhfl/fl 2.8 × 108, vavCre+Ihhfl/fl 1.75 × 108. (H) FACS analysis of CD44 and CD25 expression on CD4−8−3− thymocytes from vavCre−Ihhfl/fl and vavCre+Ihhfl/fl mice revealed reduced transition to the DN4 stage in development. (I) Histogram shows the percentage of DN4 cells in the DN population, relative to the mean percentage in Cre− littermates for vavCre−Ihhfl/fl (n = 13) and vavCre+Ihhfl/fl knockout mice (n = 6). *Significant difference by Student t test (P = .028). (J) Intracellular TCR-β expression was analyzed in DN3 thymocytes from littermate vavCre−Ihhfl/fl WT (n = 14) and vavCre+Ihhfl/fl knockout mice (n = 6). (K) The bar chart shows the percentage of icTCR-β+ cells in the DN3 population, relative to the mean percentage of icTCR-β+ cells in the DN3 population in WT littermates, for vavCre−Ihhfl/fl WT (n = 14) and vavCre+ihh fl/fl knockout mice (n = 6). A significant increase in icTCR-β expression was observed in vavCre+Ihhfl/fl knockout DN3 population, as determined by Student t test (P = .012).

Thymocyte Ihh expression is required for adult thymocyte development. (A,B) Expression of Ihh (A) and Gli1 (B) was assessed by quantitative RT-PCR in FACS-sorted populations of adult and fetal murine tissue. Levels of Ihh and Gli1 transcription were normalized for HPRT mRNA content and are shown relative to HPRT normalized transcription in the CD25+ DN population (Ihh) and DN1 population (Gli1). Thymocytes were sorted on a Modular Flow Cytometer (MoFlo; Dako North America), and purity was more than 98%. (C) Relative thymocyte number (relative to the mean of WT littermates) of 3-week-old WT (n = 8) and Ihh+/− (n = 8) littermates revealed a significant difference in SD as judged by F test (P < .001). Mean thymocyte number: litter 1 WT 1.2 × 108, Ihh+/− 1.77 × 108; litter 2 WT 1.44 × 108, Ihh+/− 1.76 × 108; litter 3 WT 1.35 × 108, Ihh+/− 1.81 × 108. (D) Transcription of Ihh in DP thymocytes from Ihh+/+ and Ihh+/− thymi. Relative levels of Ihh transcription were measured as described in Figure 1A and were normalized for HPRT mRNA content. (E) Excision of the Ihh gene as assessed by PCR from vavCre− (lane1) and vavCre+ Ihhfl/fl mice (lane 2). (F) Relative cell number (calculated relative to the mean of the WT littermates) of the thymus of 4- to 6-week-old mice of littermates (all Cre−: nonknockout mice, n = 9) compared with knockout mice (vavCre+Ihhflnull, n = 4) showed a significant decrease in thymocyte cell number (P = .003). Mean thymocyte number: litter 1 littermate (LM) 1.7 × 108, vavCre+Ihhflnull 0.75 × 108; litter 2 LM 1.95 × 108, vavCre+Ihhflnull 0.47 × 108; litter 3 LM 1.48 × 108, vavCre+Ihhflnull 1.28 × 108; litter 4 LM 1.2 × 108, vavCre+Ihhflnull 9.2 × 107. (G) Relative cell number (calculated as in panel D) of the thymus isolated from 4- to 6-week-old mice of littermate vavCre−Ihhfl/fl mice (WT, n = 15) compared with vavCre+Ihhfl/fl mice (knockout mice, n = 5) showed a significant decrease in thymocyte number (P = .016). Mean thymocyte numbers: litter 1 vavCre−Ihhfl/fl 3 × 108, vavCre+Ihhfl/fl 1.925 × 108; litter 2 vavCre−Ihhfl/fl 3.24 × 108, vavCre+Ihhfl/fl 2.59 × 108; litter 3 vavCre−Ihhfl/fl 2.115 × 108, vavCre+Ihhfl/fl 1.3 × 108; litter 4 vavCre−Ihhfl/fl 2.8 × 108, vavCre+Ihhfl/fl 1.75 × 108. (H) FACS analysis of CD44 and CD25 expression on CD4−8−3− thymocytes from vavCre−Ihhfl/fl and vavCre+Ihhfl/fl mice revealed reduced transition to the DN4 stage in development. (I) Histogram shows the percentage of DN4 cells in the DN population, relative to the mean percentage in Cre− littermates for vavCre−Ihhfl/fl (n = 13) and vavCre+Ihhfl/fl knockout mice (n = 6). *Significant difference by Student t test (P = .028). (J) Intracellular TCR-β expression was analyzed in DN3 thymocytes from littermate vavCre−Ihhfl/fl WT (n = 14) and vavCre+Ihhfl/fl knockout mice (n = 6). (K) The bar chart shows the percentage of icTCR-β+ cells in the DN3 population, relative to the mean percentage of icTCR-β+ cells in the DN3 population in WT littermates, for vavCre−Ihhfl/fl WT (n = 14) and vavCre+ihh fl/fl knockout mice (n = 6). A significant increase in icTCR-β expression was observed in vavCre+Ihhfl/fl knockout DN3 population, as determined by Student t test (P = .012).

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