The silencing of CG5964, CG3305, or CG12693 abolishes HhM1-induced anterior outgrowth and alters Hh distribution in producing cells.

<p>All panels show confocal images of wing imaginal discs immunostained for Hh (green), Ptc (blue) and phalloidin (red). (<b>A–F″</b>) Confocal x/y sections at low (A–F) and high magnifications (A′–F″). Anterior is to the left and dorsal down. The overexpression of Hh from producing cells using <i>hh-Gal4</i> generates an anterior outgrowth (arrow in A). Note the broadening of Ptc expression domain (A–A″). The expression of dsRNA against GFP (B–B″), or dsRNA against CG8441 (C–C″) together with HhM1 have no effect on HhM1-induced disc morphology and Hh signalling in receiving cells. In contrast, the overexpression of dsRNAs targeting CG5964, CG3305, or CG12693 prevents HhM1-induced anterior outgrowth (D, E, F). Note that Ptc expression is restored to normal levels in 2 rows of anterior cells (D′–F″ compared to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033665#pone.0033665.s007" target="_blank">Figure S7A</a>–A′). (<b>G–I′</b>) Confocal Z sections showing Hh subcellular distribution when HhM1 is expressed alone (G–G′) or together with dsRNA against GFP (H–H′), or with dsRNA against CG8441 (I–I′). Hh is membranous and localizes mainly to the apical membrane. Note also the high accumulation of Hh at the apical plane of receiving cells. (<b>J–L′</b>) The silencing of CG5964 (J–J′), CG3305 (K–K′), and CG12693 (L–L′) alters Hh distribution. Hh is patchy and appears to be distributed over the entire apical/basal axis. Note also that Hh neither accumulates at the apical membrane of producing cells nor at that of receiving cells. Dashed lines mark the position of the compartment boundary (G–L) or position of the Z section within the disc (A′–F′). Apical is up and basolateral is down in panels G–L′.