Evaluation of Ligand-Inducible Expression Systems for Conditional Neuronal Manipulations of Sleep inDrosophila

Abstract

Drosophila melanogaster is a powerful model organism for dissecting the molecular mechanisms that regulate sleep, and numerous studies in the fly have identified genes that impact sleep–wake cycles. Conditional genetic analysis is essential to distinguish the mechanisms by which these genes impact sleep: some genes might exert their effects developmentally, for instance by directing the assembly of neuronal circuits that regulate sleep; other genes may regulate sleep in adulthood; and yet other genes might influence sleep by both developmental and adult mechanisms. Here we have assessed two ligand-inducible expression systems, Geneswitch and the Q-system, for conditional and neuronally restricted manipulations of sleep in Drosophila. While adult-specific induction of a neuronally expressed Geneswitch transgene (elav-GS) is compatible with studies of sleep as shown previously, developmental induction of elav-GS strongly and nonspecifically perturbs sleep in adults. The alterations of sleep in elav-GS animals occur at low doses of Geneswitch agonist and in the presence of transgenes unrelated to sleep, such as UAS-CD8-GFP. Furthermore, developmental elav-GS induction is toxic and reduces brood size, indicating multiple adverse effects of neuronal Geneswitch activation. In contrast, the transgenes and ligand of the Q-system do not significantly impact sleep–wake cycles when used for constitutive, developmental, or adult-specific neuronal induction. The nonspecific effects of developmental elav-GS activation on sleep indicate that such manipulations require cautious interpretation, and suggest that the Q-system or other strategies may be more suitable for conditional genetic analysis of sleep and other behaviors in Drosophila.

Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function

“…Shaker ( Sh ) and Hyperkinetic ( Hk ) mutations decrease sleep in adults [ 26 , 57 ] but increase both excitability and synaptic growth at the NMJ [ 58 – 60 ], suggesting that synaptic functions of Inc may affect sleep by a mechanism different than broad neuronal hyperexcitability. While a parsimonious model is that Inc directs the ubiquitination of a target critical for synaptic transmission both at the larval NMJ and in neuronal populations that promote sleep, this hypothesis awaits the elucidation of Inc targets, definition of the temporal requirements of Inc activity, and further mapping of the neuronal populations through which Inc impacts sleep [ 11 , 12 , 61 ]. Finally, determining the localization of endogenous Inc within neurons is essential to distinguish possible presynaptic and postsynaptic functions of Inc and whether Inc engages local synaptic proteins or extrasynaptic targets that ultimately influence synaptic function.…”

A versatile genetic tool for post-translational control of gene expression in Drosophila melanogaster

“…Third, application of the current chemical-dependent tools requires the generation of new transgenic stocks, such as new promoter-GAL4 lines. Additionally, RU486, a chemical used to induce gene expression in the Geneswitch system, has been reported to cause developmental lethality in flies with pan-neuronal expression of the RU486-sensitive GAL4 ( Li and Stavropoulos, 2016 ).…”

An auxin-inducible, GAL4-compatible, gene expression system forDrosophila

“…GENESWITCH (Osterwalder et al, 2001) Non-toxic for adult flies Not compatible with existing GAL4 lines Leaky expression (Poirier et al, 2008;Scialo et al, 2016) Larval exposure impacts on adult sleep patterns (Li and Stavropoulos, 2016) Drug is unsafe to handle for female researchers Drug is expensive QF SYSTEM (Potter et al, 2010) Non-toxic for flies Not compatible with existing GAL4 lines…”