For the analysis of neural circuits and behavior, neuroscientists use transgenic techniques to isolate neuronal groups with precision. Neurogeneticists working with the vinegar fly
Drosophila melanogaster have developed a sophisticated, versatile toolkit that includes a foundational transcriptional system for mapping and manipulating neural circuits: Gal4–UASG [
5]. This system typically uses two fusion transgenes: endogenous fly enhancer sequences are placed upstream of the yeast transcription factor Gal4; effector transgenes are fused to Gal4’s upstream activation sequence (UASG). This arrangement places the effector under the
in trans transcriptional control of the enhancer [
5]. The Gal4–UASG method has been used for cell-specific genetic rescue, gene overexpression, reporter expression, RNA-interference screens, optogenetic physiology, and many other applications [
3,
18]. While this tool is vitally useful, one challenge to dissecting neuron–behavior relationships has been that Gal4-linked enhancers often capture more cells than are functionally relevant. To improve the precision of transgene expression, neural circuit analysis uses a variety of molecular strategies to produc AND and NOT genetic logic, producing expression refinements by intersection. Intersectional methods use either a repressor of Gal4, a targeted recombinase system, a leucine-zipped split-Gal4, or a combination. The native Gal4 repressor, Gal80, is used as a genetic NOT operator to exclude expression from a subset of cells captured by a driver [
25]. The flippase (Flp) recombinase specifically excises genomic sequences flanked by flippase recognition target (FRT) sites. In the Flp-out method, Flp is transiently expressed under the control of a heat shock promoter to both generate AND and NOT operations [
26]. Stochastic single-cell specificity can be achieved with the ‘mosaic analysis with repressible cell marker’ (MARCM) technique [
16]. Flp-FRT is also used in the ‘Flippase-induced intersectional Gal80/Gal4 repression’ (FINGR) intersectional method [
4], wherein stable, elevated levels of Flp are expressed from an enhancer to add or remove Gal80 expression from a subset of Gal4 driver cells with some stochasticity [
24]. The split-Gal4 method uses a bipartite Gal4 variant, in which a heterodimerization leucine zipper joins the DNA-binding and activation domains; it is active as a transcription factor when both components are expressed in the same cell, producing AND logic between the two half-drivers [
17]. A non-intersectional approach to improving cell set specificity uses driver lines constructed with small enhancer fragments instead of large upstream regions [
12,
13,
21]. Such genomic fragments contain fewer enhancer modules, so they tend to express in more restricted anatomical ranges: an estimated 4- to 10-fold greater specificity compared with enhancer traps [
21].
In light of the extensive Gal4 resources currently available, we aimed to develop an tool that would refine existing Gal4 lines. The DNA-binding domain of Gal4 is a zinc finger that can be substituted with another domain, conferring novel DNA-binding affinity in vitro [
22]. We implemented and tested a zinc finger variant of Gal4 that works both as a standalone binary transcription system and as a genetic AND operator in combination with existing Gal4 lines. Using several enhancer sequences associated with particular neurotransmitter systems, we demonstrated that the variant transcription factor -termed
Zinc finger-replaced G
al4 (Zal1) can drive expression from a corresponding upstream activating sequence, termed UASZ. When co-expressed in the same cells, Gal4 and Zal1 were active in the presence of a hybrid upstream activation sequence that contained asymmetric binding sites (UASGZ) for the Gal4-Zal1 heterodimer. This method allowed targeting of expression to neurons in which both transcription factor types are expressed. The Zal1-Gal4-UASGZ system will enable the refinement of existing Gal4 lines to isolate precise neuronal types.