(a–c) Immunolabeled repo>GFP wing (glia in green: anti-GFP; neurons in red: anti-22c10) at different stages. (a) Initiation of migration, (b) reaching the level of the costa, and (c) migration completion. (d) Schematic of a 18 hAPF developing Drosophila wing, insets indicate the regions shown in panels (e–f’’’’’) and panels (i–k’’’). L1 and L3 indicate L1 and L3 nerves. (e–e’’’’’) gcm>GFP/+ 18 hAPF wing immunolabeled with anti-22c10 (red), anti-Fra (gray) and anti-GFP (green). mCD8-GFP was used to label the membrane. (f–g’’) 18 hAPF repo>GFP/+ wing, immunolabeled with anti-22c10 (neurons in red), anti-Fra (gray) and anti-GFP (glia in green). (e–g’’) The presence of Fra in the glial soma (white arrowheads) at the front of migration. The position of the high-magnification panels (g–g”) is highlighted by the white rectangle in (f). Maximum confocal projections are shown in all figures, unless otherwise specified. White arrowheads indicate the glial cells that are expressing Fra. (h) Schematic representations of the coinFLP technique (modified from Bosch et al. (2015); and the phenotypes of the different cells. (i–k’’’) Immunolabeled Fra KD/WT-coinFLP wing at 18 hAPF. The WT clones display GFP labeling at the membranes (anti-GFP), the fra KD clones display RFP labeling at the membranes (anti-RFP); glial nuclei are labelled with anti-Repo in red and anti-Fra is in gray. (i) A projection of 10 confocal sections from a 18 hAPF wing. The arrowheads indicate glial cells and the white lines outline the L1 nerve. (j–k’’’) Individual sections: (j–j’’’) represents section 2/10; (k–k’’’) represents section 5/10, which corresponds to a deeper layer than section 2/10. (j, k) The overlay of the three channels (anti-RFP/Repo, anti-GFP and anti-Fra), (j’, k’) show anti-GFP alone, (j’’,k’’) anti-Fra and (j’’’, k’’’) the overlay of anti-RFP/Repo and anti-Fra. Glial cells are indicated by white arrowheads, the dashed lines indicate the fra KD clones and the continuous lines indicate the WT clones. For technical reasons, RFP (membrane labeling) and Repo (nuclear labeling) are shown in the same channel. Note the decrease in Fra levels in the fra KD clones. The scale bar in (a–c) represents 80 µm, in (e–f) 10 µm and in (i–k) 5 µm.