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. 2017 Aug 9;292(38):15814–15825. doi: 10.1074/jbc.M117.777532

Figure 7.

Figure 7.

Compound mutation of Sufu and Gli2 in CNC rescues the developmental defects of calvarial bones in the Sufufx/fx;Wnt1-Cre mutant. A–C, skeletal staining with Alcian blue/Alizarin red showing the complete restoration of calvarial bone formation in Sufufx/fx;Gli2fx/fx;Wnt1-Cre mice (n = 5). D–F, von Kossa staining showing the restoration of frontal bone mineralization in Sufufx/fx;Gli2fx/fx;Wnt1-Cre at E14.5 (n = 2). G–I, immunofluorescence showing the restored expression of cyclin D1 and osteogenic marker Runx2 in the frontal primordium of Sufufx/fx;Gli2fx/fx;Wnt1-Cre at E14.5 (n = 3). J–L, in situ hybridization analysis reveals that expression of Gli1 in the frontal primordium of Sufufx/fx;Gli2fx/fx;Wnt1-Cre is comparable with wild type control at E12.5 (n = 3). Dashed lines outline the presumptive frontal primordium. Scale bars, 1 mm (A–C) and 200 μm (E–L). M, Western blot showing substantial removal of Sufu and Gli2 in calvarial mesenchyme of compound mutant Sufufx/fx;Gli2fx/fx;Wnt1-Cre at E12.5 (n = 3). N, qRT-PCR shows that the expression of the target and downstream genes of the Hh signaling pathway in Sufufx/fx;Gli2fx/fx;Wnt1-Cre at E12.5 is comparable with wild-type control (n = 3). The significance level of differential expression between wild type control and mutants was evaluated with Student's t test. p values are indicated above each pair compared. ns, non-significant. Error bars, S.D. Ctrl, wild-type control; M1, Sufufx/fx;Wnt1-Cre; M2, Sufufx/fx;Gli2fx/fx;Wnt1-Cre.