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For developmental analysis of individual ORN axon targeting, wild type single-cell clones were generated using the Flybow (FB) construct (41) in combination with a heat-induced FLPm5 on second chromosome. Flies expressing FB1.1B transgene under the control of R86G11-Gal4 (19) were exposed to single heat shock for 90 min at 37°C to induce transient mFLP5 (41) activity between 0 and 5 hours after pupa formation (APF). Confocal images were processed and analyzed using ImageJ and Imaris 9.2 (Bitplane).

To characterize the neuronal morphology of cPIN, wild type single-cell clones were generated using the hs-FLP and FRT/FLP system (42). Wild type cPINs were labeled with R19H07-Gal4 (19), driving the expression of UAS-mCD8::GFP (42). Second instar larvae were heat-shocked for 20 to 30 min at 37°C. Pupae at the desired stage were dissected. The developmental pattern of pioneer ORNs, their synaptogenesis and LNs, in wild type and Nrg mutant background was analyzed in confocal image stacks of stained pupal and adult brains. R86G11-Gal4 (19) and R20F11-Gal4 (19) driver lines for ORNs and LNs were used, respectively. Nrg mutant hemizygous males and heterozygous females (control) were preselected at third instar and separated in different food vials for both clonal and expression analysis. White pupae (defined as 0 hours APF) were collected for staging at 25°C. Pupae at desired age and adult flies were dissected after eclosion. For developmental analysis, 5 to 10 brains were analyzed at each time point.

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