Temporal profile of Gli activity in neural cells exposed to different concentrations of Shh.

(A) Experimental scheme. The reporter plasmid, which carries the coding sequence of the firefly luciferase gene (FF-luciferase) driven by octameric Gli-binding sites (8xGBS), was introduced with a normalization plasmid, containing Renilla luciferase, into HH st.10 chick embryos by in ovo electroporation. [i] Explants were removed and cultured ex vivo in the presence of different concentrations of recombinant Shh. Dual luciferase assays were performed at appropriate time points. (B) Gli activity, measured with GBS-Luc, in neural cells exposed to the indicated concentrations of Shh (nM) for 6 h. Graph shows the fold induction of Gli activity in response to Shh compared to the level of Gli activity in untreated explants (relative Gli activity ± s.e.m.). Between 0.1 nM and 1 nM Shh the level of Gli activity at 6 h is a function of Shh concentration. (C) Temporal dynamics of Gli activity in explants exposed to Shh. The fold increase in Gli activity (relative Gli activity ± s.e.m.) measured with GBS-Luc in [i] explants treated with either 0.25 nM Shh (blue) or 2 nM Shh (green) for the indicated times. Data for 1 nM Shh (red dashed line) taken from Dessaud et al. (2007) [24] is included for comparison. Cells become adapted to these concentrations of Shh; moreover the time taken for Gli activity to return to less than 4-fold above pre-stimulus levels (inset) when exposed to the indicated concentrations of Shh is proportional to Shh concentration.