Approaches for conditional and inducible synaptic transgene expression in Drosophila. a) The GAL4/UAS, QF/QUAS, and LexA/LexAop binary expression systems are each comprised of 2 components: a transcription factor (Gal4, QF, or LexA) and its cognate promoter (UAS, QUAS, or LexAop, respectively). Specific promoters (left) regulate the expression of Gal4, QF, and LexA, which in turn drive expression of their responder transgenes in specific cells or tissues. a’) Example application of multiple binary expression systems in the CNS. The synaptic compartment is labeled in the presynaptic neuron via expression of a fluorescent protein- or epitope-tagged AZ protein under Gal4/UAS control; expression of a postsynaptic compartment marker (e.g. a fluorescently labeled NT receptor) in the postsynaptic neuron is under QUAS control; pan-neuronal expression of a short hairpin RNA, under LexA/LexAop control, knockdown expression of the gene of interest in all neurons. b, c) Cell-type-specific strategies that use FLP recombinase to label a protein only in specific cells (GRE, gene regulatory elements; UTR, untranslated region; FP, fluorescent protein). Flippase recombinase (FLP) induces site-specific recombination between matching FRTs. When tandem FRT sites are arranged in the same orientation, FLP recombination excises the intervening sequences, eliminating one of the FRT sites as shown in (b) and (c). When tandem FRT sites are arranged in opposing orientations, FLP recombination inverts the orientation of the intervening sequences as shown in (c). Each of these FLP/FRT approaches has been leveraged to generate inducible systems for synaptic labeling: (b) In the STaR method, conditional FLP expression in presynaptic neurons leads to FLP-mediated excision of the brp transcriptional terminator allowing transcription of an engineered cassette containing a fluorescent protein or epitope tag, thus producing a tagged version of Bruchpilot (Brp), which labels presynaptic AZs (Chen et al. 2014). This cassette also features a ribosomal skipping sequence, T2A, followed by LexA ORF (Daniels et al. 2014), which effectively couples inducible synaptic labeling with activation of the LexA/LexAoP binary expression system. The LexA/LexAoP system can be used to drive expression of additional transgenes only in FLP-expressing cells, for example, a membrane marker as diagrammed in (b’). c) In the FlpTag method, conditional FLP expression in a postsynaptic neuron inverts the orientation of an artificial exon inserted into, for example, an NT receptor gene. The artificial exon encodes GFP, which is only spliced into the mature mRNA when the donor and acceptor sites are in the correct orientation. The resultant NT receptor::GFP fusion protein labels the postsynaptic compartment in FLP expressing cells. Similar approaches have also been used to generate inducible Rab3-, vGAT-, and vGlut-based synaptic vesicle markers (Williams et al. 2019; Certel, McCabe, et al. 2022; Certel, Ruchti, et al. 2022).