Fly husbandry and stocks
Flies were reared on a standard cornmeal medium at 25 °C under a 12-h–12-h light–dark cycle. To enhance transgene expression levels, flies from all genetic perturbation experiments, including control groups, were shifted to 29 °C shortly before puparium formation. Detailed genotypes for each experiment are listed in Supplementary Table 1.
Molecular cloning and generation of transgenic flies
To generate QF2 lines, we used pENTR/D-TOPO vectors with various enhancer insertions (gifts from the laboratory of G. Rubin) as entry vectors for Gateway cloning into the pBPQF2Uw vector using LR Clonase II Enzyme mix (Invitrogen, 11791020). pBPQF2Uw was made using NEBuilder HiFi DNA assembly master mix (New England Biolabs) to replace the GAL4 on the pBPGAL4.2Uw-2 vector (Addgene, 26227) with QF2 from pBPGUw-HACK-QF2 (Addgene, 80276). The resulting constructs were sequence-verified and inserted into JK22C landing sites by Bestgene. pGP-5XQUAS-IVS-Syn21-jGCaMP8m-p10 was made using NEBuilder HiFi DNA assembly master mix (New England Biolabs) to replace the 20XUAS on the pGP-20XUAS-IVS-Syn21-jGCaMP8m-p10 vector (Addgene, 162387) with 5XQUAS from pQUAST (Addgene, 24349). Plasmids were injected to embryos at BestGene. Genetic labelling with these drivers is unlikely to disrupt normal development; a previous study showed that drivers with improved translation efficiency could increase GFP expression by 20-fold with no apparent effect on neuronal morphology37.
Immunostaining
The procedures used for fly dissection, brain fixation and immunostaining were described previously10. For primary antibodies, we used rat anti-DNcad (1:30, DSHB, RRID AB_528121), chicken anti-GFP (1:1,000, Aves Labs, RRID AB_10000240), rabbit anti-DsRed (1:500, Takara Bio, RRID AB_10013483) and mouse anti-rat CD2 (1:200, Bio-Rad, OX-34).
Confocal imaging
Immunostained brains were imaged using a laser-scanning confocal microscope (Zeiss LSM 780). Images of antennal lobes were taken as confocal stacks with 1-mm-thick sections. Representative single sections were shown to illustrate the arborization features of ORN axons and PN dendrites, with brightness adjustment, contrast adjustment and image cropping done in ImageJ.
Calculating the percentage of ORN axons matching with PN dendrites
PN dendritic pixels and ORN axonal pixels were defined by first smoothening the image using ‘gaussian blur’ (radius = 2 pixels) and then thresholding the image based on the algorithm ‘Otsu’ in Fiji. We found that this algorithm could efficiently separate the neurons of interest from the background. Irrelevant signals (such as the PN axons, cell bodies or autofluorescence) that still persisted after these operations were manually masked out in the analysis. A portion of ORN axons were considered as matching with PN dendrites if they had overlapping pixels on a single z-plane in the image. Note that the definition of glomerulus becomes vague as ORN axons and PN dendrites innervate more and more outside the original glomerulus.
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