201y gal4

Flies were reared on cornmeal/molasses/agar medium under standard culture conditions (29°C, 25°C or 18°C depending on the presence of tubP-Gal80^ts, 12 h:12 h light/dark cycle). CO₂ was used as an anesthetic. slgA^NP4104, UAS-mCD8-gfp, UAS-mito-tomato, UAS-RFP, CkIIα^JF01436, CkIIα ^GL0003, slgA^GL01514, TubP-Gal80^ts, TubP-Gal4, OK107-Gal4 and 201y-Gal4 were obtained from the Bloomington Drosophila Stock Center, Bloomington, IN, USA. P{cry-Gal4.E39} and P{pdf-Gal4.P2.4} were a gift of Dr Bassem Hassan (ICM, Paris, France). All fly stocks were isogenized by mating females to Canton-S males for ten generations to exclude effects due to differences in genetic background. The RNAi lines used were predicted to have no off-target effects (Ni et al., 2009 (link)).

All Drosophila stocks and crosses were fed with malt and semolina-based food with 12 h light and dark daily rhythms at 25°C with 60% humidity, unless mentioned otherwise. Drosophila strains used in this study were UAS-da^RNAi GD51297 and UAS-da^RNAi KK105258 from the Vienna Drosophila Resource Center, CantonS (a gift from Dr Bertram Gerber, Leibniz Institute for Neurobiology, Magdeburg, Germany), da^G32-Gal4 (a gift from Riitta Lindström, University of Helsinki, Helsinki, Finland), UAS-TCF4B (Tamberg et al., 2015 (link)), 201Y-Gal4 [Bloomington Drosophila Stock Center (BDSC, 4440)], 30Y-Gal4 (BDSC, 30818) (Yao Yang et al., 1995 (link)) and OK107-Gal4 (BDSC, 854) were gifts from Mark Fortini, Thomas Jefferson University, Philadelphia, PA, USA. R12B08-Gal4 (BDSC, 48489) (Pfeiffer et al., 2008 (link), flweb.janelia.org/cgi-bin/view_flew_imagery.cgi?line=R12B08), elav-Gal4 (BDSC, 8760) (Luo et al., 1994 (link)), UAS-Dcr2;Pin¹/CyO (BDSC, 24644) (Dietzl et al., 2007 (link)), UAS-nls-GFP (BDSC, 4776), UAS-da^G (BDSC, 37291), UAS-mCD8-GFP; Pin¹/CyO (BDSC, 5136) and da mutant line da¹⁰ (BDSC, 5531) (Caudy et al., 1988 (link)) were obtained from the BDSC. The following transgenic lines were generated in this study: 3xFLAG-da^2M4 and sfGFP-da^4M1.