Imaging

Third instar larval whole-mount specimen were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, rinsed phosphate-buffered saline with 0.1% (v/v) Triton X100, mounted on a slide using Vectashield H1000 (Vector Laboratories, Burlingame, USA) and a coverslip, whose edges were sealed with nail varnish. Confocal images were obtained with a Leica SP8 system. Z-stacks were collected at 4 µm intervals while capturing the entire larvae in XY dimensions. Individual Z-planes and maximum projections of the Z-stacks were inspected for expression pattern analysis.