Neuropeptide T2A-GAL4 knock-ins illustrate practical considerations for interpreting GAL4 reporter patterns in Drosophila

Transgenic GAL4 lines are widely used in Drosophila to label and manipulate specific cell types, but the resulting reporter expression patterns do not always represent ongoing driver activity. We generated a collection of neuropeptide T2A-GAL4 knock-ins and characterized their expression by native-fluorescence imaging using UAS-CD8::GFP and a brp-miRFP680 knock-in, eliminating the need for immunostaining. During characterization, we observed both timing-dependent changes in adult GFP labeling and GAL4-associated cellular abnormalities. Using temperature-sensitive GAL80, we found that adult GFP labeling in certain neurons driven by CCHa2-T2A-GAL4 and Dsk-T2A-GAL4 depended on GAL4 activity before eclosion and was not reproduced by adult-restricted induction. Several GAL4 lines also showed dominant developmental phenotypes. Heterozygous Burs-T2A-GAL4 animals, for example, displayed wing expansion defects that were fully suppressed by GAL80. Severe wing defects were not accompanied by a reduction in the number of reporter-labeled neurons before eclosion, but the morphology of these neurons was affected. These observations illustrate how developmental driver history and GAL4-associated cellular effects can influence reporter patterns in the adult nervous system.