Figure 1. Membrane potential is patterned in the third‐instar wing imaginal disc.

• A, B
  Live third‐instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A–A′) and basal (B–B′) optical sections. Increased fluorescence is observed in the centre of the pouch (white arrow, B′) and at the dorsoventral (D‐V) compartment boundary in the anterior compartment (yellow arrow, (B′), and inset, (D)).
• C–E
  Comparison of DiBAC with the voltage‐insensitive dye FM4‐64. Incubation of live discs in FM4‐64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N = 7 discs for each treatment, data compared using an unpaired t‐test, ** indicates P < 0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D‐V compartment boundary.
• F, G
  Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.
• H–H″
  Live wing discs expressing UAS‐RFP anterior to the compartment boundary, under the control of ptc‐Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.
• I, I′
  Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).

Data information: Scale bars are 100 µm in all panels, except for (A′ and B′), where scale bars are 50 µm.