Genome-Wide Screen for Genes Involved inCaenorhabditis elegansDevelopmentally Timed Sleep

Abstract

In Caenorhabditis elegans, Notch signaling regulates developmentally timed sleep during the transition from L4 larval stage to adulthood (L4/A) . To identify core sleep pathways and to find genes acting downstream of Notch signaling, we undertook the first genome-wide, classical genetic screen focused on C. elegans developmentally timed sleep. To increase screen efficiency, we first looked for mutations that suppressed inappropriate anachronistic sleep in adult hsp::osm-11 animals overexpressing the Notch coligand OSM-11 after heat shock. We retained suppressor lines that also had defects in L4/A developmentally timed sleep, without heat shock overexpression of the Notch coligand. Sixteen suppressor lines with defects in developmentally timed sleep were identified. One line carried a new allele of goa-1; loss of GOA-1 Gαo decreased C. elegans sleep. Another line carried a new allele of gpb-2, encoding a Gβ5 protein; Gβ5 proteins have not been previously implicated in sleep. In other scenarios, Gβ5 GPB-2 acts with regulators of G protein signaling (RGS proteins) EAT-16 and EGL-10 to terminate either EGL-30 Gαq signaling or GOA-1 Gαo signaling, respectively. We found that loss of Gβ5 GPB-2 or RGS EAT-16 decreased L4/A sleep. By contrast, EGL-10 loss had no impact. Instead, loss of RGS-1 and RGS-2 increased sleep. Combined, our results suggest that, in the context of L4/A sleep, GPB-2 predominantly acts with EAT-16 RGS to inhibit EGL-30 Gαq signaling. These results confirm the importance of G protein signaling in sleep and demonstrate that these core sleep pathways function genetically downstream of the Notch signaling events promoting sleep.

Gap Junctions and NCA Cation Channels Are Critical for Developmentally Timed Sleep and Arousal in Caenorhabditis elegans

“…To corroborate the sleep defects observed based on motion detection, we also examined another important aspect of sleep: altered arousal thresholds. During C. elegans L4/A lethargus sleep bouts, response to a variety of external stimuli is diminished (Raizen et al 2008;Singh et al 2011;Huang et al 2017b); animals carrying mutations that perturb sleep quantity usually also have perturbed arousal thresholds during sleep bouts (Singh et al 2014). Consistent with this general rubric, we found arousal defects in unc-7(lf) and unc-9(lf) animals.…”

“…Arousal threshold assays with blue light stimulation were conducted as previously described (Huang et al 2017b). Briefly, L4/A lethargus animals were picked to freshly made assay plates with food and allowed to recover for 15 min.…”

“…To identify conserved genes required for C. elegans developmentally timed sleep during the transition to adulthood (L4/ A lethargus), we previously undertook a forward genetic screen (Huang et al 2017b). Subsequently, we determined that the unc-7(rt212) mutant allele, a premature stop mutation (W365Opal, isoform a), caused decreased total time in sleep bouts during L4/A lethargus (total sleep time, Figure S1A).…”

“…There is deep conservation of genes and pathways regulating sleep and sleep homeostasis between C. elegans and other species (Singh et al 2014;. Studies in C. elegans have contributed to a growing list of genes affecting sleep, including egl-4/cyclic-GMP (cGMP)-dependent kinase, lin-12/glp-1/Notch receptors, aptf-1/AP2-family transcription factor, and gpb-2/G b protein (Raizen et al 2008;Singh et al 2011;Huang et al 2017b).…”

Epidermal Growth Factor signaling acts directly and through a sedation neuron to depolarizes a sleep-active neuron following cellular stress

“…We obtained seven genes whose mutation changed sleep amount significantly and more than 50% from wild type (Figure 2A). The four genes that reduced sleep had already been described to control sleep ( aptf-1 , flp-11 , goa-1 , frpr-3 )[31, 32, 51]. We obtained three genes whose mutation caused increased sleep.…”