(A) A third instar larval brain of nSyb-GAL4/+; Trim9MI12525EGFP-T2A-mCherry/Trim9Df, raised at 29°C stained for EGFP (green, a, d and f), mCherry (red, b, d and g) and Elav (blue, c, d and h). In the absence of UAS-FLP, only GFP signal is detected, reflecting the Trim9-EGFP-Trim9 expression pattern. In contrast, mCherry is not expressed. (b, d and g). Magnification of the region indicated by a white square in panel d (e–h). Third instar larval brains of UAS-FLP/nSyb-GAL4; Trim9MI12525EGFP-T2A-mCherry/Trim9Df, expressing FLP under the control of nSyb-GAL4 show cassette inversion that leads to the loss of EGFP and gain of mCherry expression in neurons (a’–h’). Magnification of the region indicated by a white square in panel (d’) shows the existence of three populations of neurons (e’–h’): neurons that express mCherry and have completely lost GFP expression (1), neurons that have not undergone cassette inversion and thus still express GFP but lack mCherry expression (2) and neurons that express both tags, suggesting they have recently undergone cassette flip (as indicated by mCherry expression), and still contain some remaining Trim9-EGFP-Trim9 protein that perdures following the cassette flip, scale bar = 50 µm (a, b, c, d, a’, b’, c’ and d’), scale bar = 5 µm (e, f, g, h, e’, f’, g’ and h’). (B) Adult brain of tubP-GAL80ts; Trim9MI12525EGFP-T2A-mCherry/Trim9Df; UAS-FLP/nSyb-GAL4 animals stained for EGFP (green), mCherry (red) and Elav (blue). The temperature-sensitive GAL80 (GAL80ts), expressed under a tubulin promoter (tubP) was used to prevent nSyb-GAL4 from driving expression of UAS-FLP during development. (a–d) Adult brain of tubP-GAL80ts; Trim9MI12525EGFP-T2A-mCherry/Trim9Df; UAS-FLP/nSyb-GAL4 control animals, raised at 18°C display Trim9-EGFP-Trim9 (green, a and d) and do not show any mCherry expression (red, b and d). (a’–d’) Adult brains of tubP-GAL80ts; Trim9MI12525EGFP-T2A-mCherry/Trim9Df; UAS-FLP/nSyb-GAL4 animals, raised at 18°C until 4 days after eclosion, and then shifted to 29°C to induce FLP-expression, show mCherry-expressing neuronal cells in which the cassette inversion has taken place (red, b’ and d’), scale bar = 50 µm.