Figure 8. Effect of different Hh variants on en, dpp-LacZ and ptc-LacZ expression in the wing disc (a,b) Dpp-LacZ (a) and ptc-LacZ (b) reporter gene expression at the a/p border in wild-type third-instar discs.
Nuclear β-galactosidase is immunofluorescently labeled (red). Overexpression of CD8-GFP under en-control labels the posterior compartment. Fly larvae developed at 25°C. The left image is a merge. (c,d) Hh overexpression expands dpp-LacZ and ptc-LacZ expression anteriorly. 4D9 α-engrailed/invected (inv) antibodies label the posterior compartment in this and the following panels (green). Fly larvae developed at 25°C. (e,f) En-controlled HAHh overexpression reduced dpp-LacZ expression in the anterior wing disc. Ptc-LacZ expression was always completely abolished, and en/inv expression was restricted to most posterior wing disc regions (arrow). Fly larvae for this and all subsequent analyses developed at 18°C because wing disc growth arrested at 25°C, preventing further analysis. (g,h) Additional deletion of the palmitate membrane anchor increased dpp-LacZ expression and also restored a stripe of weak yet expanded ptc-LacZ expression. The expansion of ptc-LacZ and dpp-LacZ domains beyond wild-type levels may be linked to reduced inv/en expression in anterior target cells (note the unchanged posterior restriction of inv/en-expression). (i,j) En-controlled HhC85S expression leads to comparable dpp-LacZ expression. Ptc-LacZ reporter expression was reverted into more intense and restricted staining, indicating an additional inhibitory effect of the HA-tag. (k,l) Restored wild-type pattern of dpp-LacZ expression and ptc-LacZ expression as a consequence of en-controlled HhC85S;Δ86-100 expression shows that expanded dpp-LacZ expression and reduced ptc-LacZ expression in en >HhC85S and en >HAHhC85S discs were caused by the unprocessed N-terminal peptide. Wing discs are oriented such that anterior is right and dorsal is up; all magnification, camera and processor settings were kept identical. Scale bar: 100 μm.