Facilitating Neuron-Specific Genetic Manipulations in Drosophila melanogaster Using a Split GAL4 Repressor

Abstract

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter’s ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely-used technique for refined cell type-specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS (Upstream Activating Sequence) system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the “Killer Zipper” (KZip+), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip+ acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip+ technology, we have developed a versatile set of LexAop-KZip+ fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. KZip+ significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.

A genetic, genomic, and computational resource for exploring neural circuit function

“…In some cases, available driver lines, including some used in this study, may label some additional cell types. While drivers with even higher specificity could be obtained through testing of additional split-GAL4 intersections or perhaps triple intersections (Dolan et al, 2017), we did not find the contributions of small numbers of 'offtarget' cells to be a major limitation for many applications of expression data. In general, the transcriptomes support the high specificity of the intersectional lines we used to access visual system cells (Figure 1).…”

Circuits for integrating learnt and innate valences in the fly brain

“…SS02108 drives expression in two neurons with similar morphology in each hemisphere, CN-33 and MB2ON-86, and weakly in segmentally repeated local interneurons in the nerve cord downstream of aversive mechanosensory neurons. As a control, we removed SS02108-driven nerve cord expression using the Split-GAL4 repressor Killer Zipper (Dolan et al, 2017) under the control of teashirt-LexA (J.-M. Knapp and J. Simpson, unpublished data) promoter (Extended Data Fig. 12a-b).…”

“…a. We removed SS02108-driven neural expression in the nerve cord by expressing the Split-GAL4 repressor Killer Zipper (LexAop-KZip + ::3xHA , Dolan et al, 2017) under the control of teashirt-LexA driver (SS02108>CsChrimson; tsh>KZip + ). With this genetic manipulation we verified that the approach behavior of larvae observed in Fig.4b was due to the brain neurons present in the SS02108 expression pattern.…”

An Interscholastic Network to Generate LexA Enhancer Trap Lines inDrosophila

“…Experimental approaches in biology benefit from temporal-or cell-specific control of gene expression, like that possible with binary expression strategies pioneered in the Drosophila GAL4-UAS system (Brand and Perrimon, 1993). Intersectional approaches, like simultaneous use of the LexA-lexAop and GAL4-UAS systems, have also greatly enhanced experimental and interpretive power in fly biology, particularly studies of neuroscience and intercellular communication (Simpson, 2016, Martin et al, 2017, Dolan et al, 2017. For example, enhancer traps generated here (Suppl.…”