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insomniac links the development and function of a sleep-regulatory circuit

  1. Qiuling Li
  2. Hyunsoo Jang
  3. Kayla Y Lim
  4. Alexie Lessing
  5. Nicholas Stavropoulos  Is a corresponding author
  1. Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, United States
  2. Waksman Institute, Rutgers University, United States
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Cite this article as: eLife 2021;10:e65437 doi: 10.7554/eLife.65437

Abstract

Although many genes are known to influence sleep, when and how they impact sleep-regulatory circuits remain ill-defined. Here, we show that insomniac (inc), a conserved adaptor for the autism-associated Cul3 ubiquitin ligase, acts in a restricted period of neuronal development to impact sleep in adult Drosophila. The loss of inc causes structural and functional alterations within the mushroom body (MB), a center for sensory integration, associative learning, and sleep regulation. In inc mutants, MB neurons are produced in excess, develop anatomical defects that impede circuit assembly, and are unable to promote sleep when activated in adulthood. Our findings link neurogenesis and postmitotic development of sleep-regulatory neurons to their adult function and suggest that developmental perturbations of circuits that couple sensory inputs and sleep may underlie sleep dysfunction in neurodevelopmental disorders.

Editor's evaluation

This is an interesting study showing that the short sleep phenotype of inc mutants in Drosophila depends on the loss of the gene at a specific developmental time, and in a specific region, the mushroom bodies (MB). There are very few studies assessing the effects of sleep during development, in any animal species, and thus this paper is a very welcomed addition. The experiments are carefully done, and the conclusions are warranted.

https://doi.org/10.7554/eLife.65437.sa0

Introduction

A central goal of sleep research has been elucidating the mechanisms by which genes shape normal sleep patterns and cause sleep disorders. While numerous genes that strongly impact sleep have been identified in humans and in animals ranging from mammals to invertebrates (Chemelli et al., 1999; Chiu et al., 2016; Cirelli et al., 2005; Funato et al., 2016; He et al., 2009; Lin et al., 1999; Raizen et al., 2008), when these genes act to influence sleep is in many cases unresolved. Genes that act in the adult brain to modulate the activity of sleep-regulatory circuits in an ongoing manner have been intensively investigated (e.g. Chemelli et al., 1999; Lin et al., 1999), including with conditional gain-of-function, loss-of-function, and rescue in adult animals (Chiu et al., 2016; Clasadonte et al., 2017; Foltenyi et al., 2007; Guo et al., 2011; Ishimoto and Kitamoto, 2010; Joiner et al., 2006; Van Buskirk and Sternberg, 2007). In contrast, despite great progress in understanding neuronal development (Doe, 2008; Jessell and Sanes, 2000; Sanes and Zipursky, 2020; Tessier-Lavigne and Goodman, 1996; Weinstein and Hemmati-Brivanlou, 1999), developmental mechanisms by which genes influence sleep remain poorly explored, despite the likely relevance of such mechanisms to sleep disturbances in autism and other neurodevelopmental disorders (Angriman et al., 2015; Souders et al., 2017). Notably, the temporal contributions of genes that impact sleep are rarely assessed in a comprehensive manner, and a further challenge has been linking particular genes to developmental processes that control the structure and function of discrete sleep-regulatory circuits.

Here, we assess the temporal contributions of insomniac (inc), a gene whose mutation sharply curtails sleep in Drosophila (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). Pan-neuronal depletion of inc causes short sleep, while restoring inc solely to neurons is largely sufficient to rescue the sleep deficits of inc mutants, indicating that inc impacts sleep chiefly through neurons (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). inc is expressed in the larval, pupal, and adult brain (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011), but when inc acts to influence sleep remains uncertain (Li and Stavropoulos, 2016; Pfeiffenberger and Allada, 2012). inc encodes an adaptor for the Cul3 ubiquitin ligase (Li et al., 2019), which, like inc, is required in neurons for normal sleep (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). Both inc and Cul3 are highly conserved, and mammalian inc orthologs restore sleep to inc mutants (Li et al., 2017), suggesting that functions and substrates of inc are conserved in mammals. Human Cul3 mutations are implicated as a cause of autism and its associated sleep dysfunction (Codina-Solà et al., 2015; Kong et al., 2012; O’Roak et al., 2012), but the underlying mechanisms are unknown. Studies of inc may thus reveal fundamental and conserved mechanisms underlying sleep regulation which are altered in sleep disorders.

Using conditional genetic manipulations of inc, we show that inc acts transiently in developing neurons to impact sleep in adulthood. We furthermore identify developmental defects in inc mutants within the mushroom body (MB), a brain structure that integrates sensory stimuli and regulates sleep. Loss of inc alters MB neurogenesis, causing the overproduction of late-born neurons and changes in postmitotic development that impair the assembly of MB circuits. These developmental alterations persist into adulthood and are associated with specific deficits in the ability of MB neurons to promote sleep in inc adults, in contrast to the anatomy and function of other sleep-regulatory circuits which remain intact. Together, these results elucidate an unexpected mechanism by which inc shapes the development and function of sleep-regulatory neurons to exert a lasting impact on sleep–wake behavior. Our findings additionally suggest that developmental alterations of neurogenesis and within brain centers that integrate sensory inputs may contribute to sleep dysfunction in autism and other neurodevelopmental disorders.

Results

inc acts transiently during a restricted developmental period to impact sleep in adulthood

inc impacts sleep through neurons and is expressed in the developing and adult brain (Figure 1; Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). To assess the temporal mechanisms by which inc impacts sleep, we manipulated inc expression in neurons using the ligand-inducible Q-system (Potter et al., 2010; Riabinina et al., 2015). The Q-system circumvents nonspecific perturbations of sleep caused by other inducible systems and allows constitutive, developmental, and adult manipulations of sleep (Li and Stavropoulos, 2016). We performed a series of conditional rescue experiments in short-sleeping inc1 null mutants bearing a UAS-inc-HA transgene whose expression is induced in neurons by the Q-system upon exposure to quinic acid (Figure 2A). Animals exposed to vehicle throughout development and adulthood slept indistinguishably from inc1 mutants, while animals exposed constitutively to quinic acid exhibited strongly rescued sleep (Figure 2B, C; Figure 2—figure supplement 1), consistent with the rescue conferred by constitutive neuronal expression of inc (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). Anti-HA staining of brains confirmed that the Q-system controlled inc expression as expected: vehicle-fed animals lacked inc-HA signal, while those exposed constitutively to quinic acid expressed inc-HA in the larval, pupal, and adult brain (Figure 2D). We next asked whether inc influences sleep through adult-specific or developmental mechanisms. Animals fed quinic acid in adulthood expressed inc-HA in the adult brain but exhibited no rescue of their sleep deficits (Figure 2B–D; Figure 2—figure supplement 1). In stark contrast, developmental induction of inc-HA from embryonic through pupal stages restored sleep to near wild-type levels (Figure 2B–D; Figure 2—figure supplement 1). These findings indicate that inc is dispensable in adult neurons and acts instead during neuronal development to ultimately impact sleep–wake behavior.

Expression of 3×FLAG-inc driven by inc-Gal4 in the larval, pupal, and adult brain.

Maximal projections are shown for male inc-Gal4; UAS-3×FLAG-inc/+ brains stained with anti-FLAG. For larval brain, projection from a partial z-stack is shown to allow visualization of signal in mushroom body projections (arrowheads). In pupae and adults, signal is prominent in the mushroom body, pars intercerebralis, fan-shaped body, and ellipsoid body. Scale bars, 100 μm.

Figure 2 with 1 supplement see all
inc acts in a restricted period of neuronal development to impact sleep in adulthood.

(A) Conditional rescue of inc1 mutants using the ligand-inducible Q-system. Quinic acid relieves QS suppression of the pan-neuronally expressed Gal4QF transcriptional activator, inducing UAS-inc-HA in neurons. (B) Total sleep duration of controls (gray) and inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ animals exposed to quinic acid (+) or vehicle (−) at indicated life stages; embryos (E), larval stages (1–3), pupae (P), and adults (A). Bars represent mean ± standard error of the mean (SEM). n = 11–86. One-way analysis of variance (ANOVA) (F(7,397) = 86.73, p < 0.0001) and Tukey post hoc tests, *p < 0.01 for comparisons to inc1; UAS-inc-HA/+. (C) Average sleep profiles of flies in (B), with induction regimens indicated below. Shading indicates ± SEM. (D) Anti-HA staining of inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ brains from indicated induction regimens. Scale bars, 100 μm.

We further defined the developmental period in which inc functions, using more precise temporal manipulations. Neuronal induction of inc-HA from the late third instar larval stage through adulthood strongly rescued the inc sleep phenotype (Figure 2B–D; Figure 2—figure supplement 1), indicating that inc is dispensable in embryonic and early larval neurons. Induction of inc activity solely in late third instar larval and pupal neurons, using a pulse of quinic acid exposure (Figure 2D), restored sleep indistinguishably from constitutive neuronal induction (Figure 2B, C; Figure 2—figure supplement 1). The sleep deficits of inc2 animals, which bear an independent inc null allele that can be reverted by Gal4 (Stavropoulos and Young, 2011), were similarly rescued by this pulse of quinic acid (Figure 3A–C; Figure 3—figure supplement 1), confirming that inc activity in this developmental period is sufficient to restore sleep to inc mutants. We next assessed whether inc is required in late third instar larval and pupal neurons for normal sleep in adulthood, by using the Q-system to induce a pulse of inc RNAi. This manipulation markedly decreased sleep (Figure 3D, E; Figure 3—figure supplement 2). Together, these findings indicate that inc acts transiently in neurons of late third instar larvae and pupae to influence adult sleep–wake behavior. During these developmental stages, many neurons of the adult brain are born and assemble into circuits (Truman and Bate, 1988; White and Kankel, 1978).

Figure 3 with 2 supplements see all
Conditional rescue of inc2 mutants and conditional inc RNAi in larval and pupal neurons.

(A) Conditional neuronal rescue of inc2 mutants using the ligand-inducible Q-system. inc2 mutants contain a transposon insertion in the inc 5′UTR immediately upstream of the endogenous start codon. A UAS/TATA element within the transposon terminus permits Gal4-dependent restoration of inc expression (Stavropoulos and Young, 2011). (B) Total sleep duration in inc2; tub-QS/+; nysb-Gal4QF/+ animals exposed to vehicle or quinic acid at the late third instar larval and pupal stages. n = 20–83. One-way analysis of variance (ANOVA) (F(3, 170) = 70.66, p > 0.0001) and Tukey post hoc tests, *p < 0.01 for comparisons to inc2. (C) Average sleep profiles of indicated genotypes from (B). (D) Total sleep duration in tub-QS/UAS-inc-RNAi; nsyb-Gal4QF/UAS-dcr2 animals exposed to vehicle or quinic acid at the late third instar larval and pupal stages. n = 16–24. Student’s t-test, *p < 0.01 for comparison to vehicle-treated control. (E) Average sleep profiles of animals from (D). For (B) and (D), bars represent mean ± SEM. For (C) and (E), shading represents ± SEM.

inc has a critical function in the MB that impacts sleep

To identify neurons that might underlie the developmental impact of inc on sleep, we performed a rescue screen in inc2 mutants. We screened 277 Gal4 lines expressed in sleep-regulatory circuits or randomly selected populations of cells in the brain and identified two drivers, c253-Gal4 and c309-Gal4, that rescued sleep similarly to the pan-neuronal nsyb-Gal4 driver (Figure 4A). After backcrossing to an isogenic background, both drivers retained their ability to rescue most of the sleep phenotypes of inc2 mutants (Figure 4B, C; Figure 4—figure supplement 1). In late third instar larvae and adults, c253-Gal4 and c309-Gal4 are strongly expressed in the MB (Figure 4D), a structure important for sensory integration, associative learning, and sleep regulation (Heisenberg, 2003; Joiner et al., 2006; Pitman et al., 2006). Because c253-Gal4 and c309-Gal4 are also expressed outside of the MB, we used independent genetic manipulations to confirm that inc acts in the MB to influence sleep. inc-Gal4, a driver that bears inc regulatory sequences and fully rescues inc mutants when used to restore inc activity (Li et al., 2017; Stavropoulos and Young, 2011), is expressed in the larval, pupal, and adult MB (Figure 1). We tested whether the rescue conferred by inc-Gal4 was altered by MB-Gal80, a Gal4 suppressor expressed in MB neurons during development and adulthood (Krashes et al., 2007; Pauls et al., 2010). MB-Gal80 partially suppressed the ability of inc-Gal4 to restore sleep to inc1 mutants, indicating that while inc does not influence sleep solely through the MB, inc is required in MB neurons for normal sleep regulation (Figure 4E, F; Figure 4—figure supplement 2).

Figure 4 with 2 supplements see all
The mushroom body is a critical brain region through which inc impacts sleep.

(A) Mean sleep is plotted for each line in a Gal4 rescue screen of inc2 animals. n ≥ 5 per genotype. (B) c253-Gal4 and c309-Gal4 rescue sleep in inc2 mutants. n = 14–78. One-way analysis of variance (ANOVA) (F(4, 172) = 20.36, p < 0.0001) and Tukey post hoc test, *p < 0.01 for comparisons to inc2. (C) Average sleep profiles of flies in (B). (D) Anti-GFP immunostaining of indicated genotypes. Scale bars, 100 μm. (E) MB-Gal80 suppresses sleep rescue in inc1 inc-Gal4; UAS-inc-HA/+ animals. n = 30–69. One-way ANOVA (F(3, 161) = 121.4, p < 0.0001) and Tukey post hoc tests, *p < 0.01. (F) Average sleep profiles of indicated genotypes from (E). For (B) and (E), bars represent mean ± SEM. For (C) and (F), shading represents ± SEM.

Loss of inc abolishes the sleep-promoting functions of MB neurons but spares the functions of other sleep-regulatory circuits

While different circuits within the MB can promote or inhibit sleep upon activation (Joiner et al., 2006; Pitman et al., 2006; Sitaraman et al., 2015a), ablation of the MB strongly reduces sleep (Joiner et al., 2006; Pitman et al., 2006), suggesting that the integrated activity of the MB is sleep-promoting. To assess whether the sleep-regulatory functions of the MB are altered in inc mutants, we activated MB neurons in adult wild-type and inc1 flies using the dTrpA1 heat-activated cation channel (Hamada et al., 2008). Wild-type control flies lacking Gal4 drivers exhibited no change in total sleep when shifted to 28.5°C for 24 hr, while inc1 flies lacking Gal4 drivers exhibited decreased sleep at this temperature (Figure 5A, B), suggesting that inc mutants are hyperarousable by thermal stimuli, as for mechanical stimuli (Pfeiffenberger and Allada, 2012). Activation of neurons expressing TrpA1 under the control of c253-Gal4 or c309-Gal4 strongly increased sleep in wild-type animals (Figure 5A, B; Figure 5—figure supplement 1), consistent with observations that inactivating synaptic output using the same drivers promotes wakefulness (Pitman et al., 2006). Because c253-Gal4 and c309-Gal4 are expressed in some cells outside of the MB, we also assessed a split-Gal4 driver expressed specifically in MB neurons (Figure 5—figure supplement 2). Using this driver to express TrpA1 and activate MB neurons increased sleep in wild-type animals (Figure 5A, B, ‘pan-MB’). Strikingly, using the same three drivers to activate neurons in inc1 mutants elicited no significant changes in sleep compared to inc1; UAS-TrpA1/+ controls (Figure 5A, B; Figure 5—figure supplement 1), indicating that the sleep-promoting effects of MB activation are abolished in inc mutants.

Figure 5 with 2 supplements see all
Sleep-promoting functions of the mushroom body are impaired in inc mutants.

(A) Thermogenetic activation of neuronal populations expressing TrpA1 in control and inc1 animals. Percent change in sleep (mean ± SEM) elicited by activation is shown. n = 31–144. Control and inc1 animals expressing dTrpA1 are compared to no-Gal4 controls (UAS-dTrpA1/+ and inc1; UAS-dTrpA1/+, respectively). *p < 0.01 for Dunnet’s post hoc comparisons after one-way analysis of variance (ANOVA) for control animals (F(8, 528) = 92.12, p < 0.0001) or inc1 mutants (F(8, 452) = 50.01, p < 0.0001). Green and pink bars indicate drivers that significantly promote or inhibit sleep, respectively; gray bars indicate no significant change with respect to controls. (B) Average sleep profiles of animals from (A) on the baseline day and during thermogenetic activation. Shading represents ± SEM.

To test whether the loss of inc specifically impairs the sleep-regulatory functions of MB neurons or causes more general deficits in sleep regulation, we assessed other neuronal populations that influence sleep. Activation of sleep-promoting populations that include ellipsoid body R5 (EB) (Liu et al., 2016) or Dorsal Paired Medial (DPM) neurons (Haynes et al., 2015) increased sleep similarly in wild-type and inc1 animals (Figure 5A, B; Figure 5—figure supplement 1). Conversely, activation of sleep-inhibiting populations that include Helicon (Donlea et al., 2018), l-LNv (Sheeba et al., 2008), or pars intercerebralis and dopaminergic PPM3 neurons (PI, PPM3) (Dubowy et al., 2016) strongly decreased sleep in wild-type and inc1 animals (Figure 5A, B; Figure 5—figure supplement 1). The functions of these populations thus appear to be intact in inc mutants, suggesting that the loss of inc specifically impairs the sleep-regulatory functions of MB neurons. These findings, together with the developmental time-of-action of inc and its requirement within the MB for normal sleep, suggest that inc acts developmentally in MB neurons to have a lasting impact on their sleep-regulatory functions in adulthood.

inc regulates the production and anatomy of late-born MB neurons

During the critical developmental period through which inc impacts sleep, MB neurons are born and assemble into adult circuits (Ito and Hotta, 1992; Lee et al., 1999). In each brain hemisphere, four MB neuroblasts proliferate to yield ~2000 neurons comprising seven sequentially born subtypes (γd, γm, α´/β´ap, α´/β´m, α/βp, α/βs, and α/βc) that project axons into distinct lobes (γ, α´/β´, and α/β) (Aso et al., 2014a; Ito et al., 1997; Ito and Hotta, 1992; Kurusu et al., 2002; Lee and Luo, 1999; Tanaka et al., 2008; Truman and Bate, 1988; Zhu et al., 2003). Chemical ablation of the MB by exposing first instar larvae to hydroxyurea, an inhibitor of DNA replication, causes sleep deficits in adulthood (Joiner et al., 2006; Pitman et al., 2006). The sleep deficits caused by MB ablation are similar to but less severe than those of inc mutants, including reductions in sleep across the day and decreased sleep consolidation (Figure 6A–F). These findings and the partial suppression of inc rescue by MB-Gal80 (Figure 4E, F; Figure 4—figure supplement 2) support the notion that reduced sleep in inc mutants results from impairments in the MB, alongside effects in additional neuronal populations.

Sleep phenotypes for mushroom body ablation and inc mutants.

Sleep parameters for inc2 mutants and animals exposed to vehicle or hydroxyurea (HU). For all panels, n = 37–49; *p < 0.01 for post hoc tests. (A) Total sleep. One-way analysis of variance (ANOVA) (F(2, 122) = 132.9, p < 0.0001) and Tukey post hoc tests. (B) Average daily sleep profiles. Shading represents ± SEM. (C) Nighttime sleep. One-way ANOVA (F(2, 122) = 126.6, p < 0.0001) and Tukey post hoc tests. (D) Daytime sleep. One-way ANOVA (F(2, 122) = 74.32, p < 0.0001) and Tukey post hoc tests. (E) Sleep bout length. Kruskal–Wallis (p < 0.0001) and Dunn’s post hoc tests. (F) Sleep bout number. One-way ANOVA (F(2, 122) = 24.89, p < 0.0001) and Tukey post hoc tests. For (A) and (C–F), bars represent mean ± SEM.

To determine whether inc mutants have anatomical changes in the adult MB that might disrupt its sleep-regulatory functions, we examined MB neurons expressing UAS-Myr-GFP-2A-RedStinger, a bicistronic reporter that marks projections and nuclei (Daniels et al., 2014). Specifically, we used split-Gal4 drivers that label MB neuron subtypes born in embryos (γd), late larval stages (α´/β´), and in pupae (α/βc) (Aso et al., 2014a), to assess whether the loss of inc might preferentially alter subtypes whose birth and development coincides with the critical period through which inc impacts sleep. Consistent with this notion, we observed prominent changes in the number and anatomy of larval- and pupal-born MB neurons in inc mutants. While embryonic-born γd neurons were present in similar numbers in adult brains of controls and inc1 mutants (control, 102 ± 4; inc1, 94 ± 2) (Figure 7A, B), the number of larval-born α´/β´ neurons was increased 58% in inc1 animals (control, 141 ± 13; inc1, 223 ± 24), and the number of pupal-born α/βc neurons was doubled (control, 223 ± 11; inc1, 458 ± 45). The surplus of α´/β´ and α/βc neurons varied between left and right hemispheres in individual inc1 brains and this variation was greatest for α/βc neurons, the last-born in the MB (Figure 7A, C; Figure 7—figure supplement 1), indicating that inc mutants have a stochastic and cumulative defect in MB neurogenesis. Four clusters of α/βc neurons were present in control animals, reflecting their birth from four MB neuroblasts (Ito et al., 1997; Ito and Hotta, 1992; Truman and Bate, 1988), whereas inc1 mutants exhibited an average of nearly seven clusters (control, 3.7 ± 0.2; inc1, 6.8 ± 0.6) (Figure 7A, D; Figure 7—figure supplement 1), suggesting an origin from aberrant or excess neuroblasts. The numbers of other sleep-regulatory neurons, including those of the dorsal fan-shaped body (dFB) and DH44+ neurons, were unchanged in inc mutants (Figure 7A, I), indicating that neuronal overproduction in inc mutants is specific to the MB or manifests preferentially within this neuronal lineage. These findings indicate that inc regulates neurogenesis, a fundamental process regulated by proteins conserved from flies to mammals (Doe, 2008; Knoblich, 2008), and suggest that alterations in early nervous system development can exert a lasting impact on sleep.

Figure 7 with 2 supplements see all
inc regulates neurogenesis and anatomy of late-born mushroom body (MB) neurons.

(A) Adult control and inc1 brains expressing UAS-MyrGFP-2A-RedStinger in indicated MB neuron subtypes, stained with anti-GFP (cyan) and anti-dsRed (yellow). (B) MB neuron number per hemisphere. γd, n = 10–11; α ́/β ́, n = 7–10; α/βc, n = 16–18. *p < 0.01, Welch’s t-test. (C) Absolute difference in MB neuron number between left and right brain hemispheres; γd, n = 5–6; α ́/β ́, n = 3–5; α/βc, n = 8–9. *p < 0.01, Welch’s t-test. (D) Number of α/βc neuron clusters per hemisphere. n = 16–18. *p < 0.01, Welch’s t-test. (E) Numbers of dorsal fan-shaped body (dFB) and DH44+ neurons. dFB, n = 26; DH44+, n = 6–8. ns, p > 0.01, Welch’s t-test. (F) Adult control and inc1 brains expressing UAS-DenMark-smGdP-V5 in indicated MB neuron subtypes, stained with anti-GFP. (G) Dendrite volume per hemisphere. γd, n = 16–17; α ́/β ́, n = 19; α/βc, n = 14–16. *p < 0.01, Welch’s t-test. (H) Quantification of axonal projection defects for MB neuron subtypes. Colored bars represent the number of MB lobes in each brain entirely lacking axonal myr-GFP signal. See also panel (A). n = 10–25. (I) Adult control and inc1 brains expressing UAS-MyrGFP-2A-RedStinger in dFB neurons. All scale bars represent 100 μm. For (B–E) and (G), bars represent mean ± SEM.

To further assess MB anatomy in inc mutants, we examined axons marked with myr-GFP and separately examined dendrites by expressing DenMark (Nicolaï et al., 2010). Axons of embryonic-born γd neurons exhibited no obvious changes in inc1 mutants (Figure 7A, H). In contrast, axons of larval- and pupal-born MB neurons exhibited morphological defects whose severity correlated with neuronal overproduction and birth order (Figure 7A, H). While α´/β´ axons were absent from MB lobes in a minority (10%) of inc1 brains, axons of α/βs neurons, the penultimate to be born, were missing from MB lobes in 53% of inc1 brains (1.07 ± 0.33 missing lobes per brain) (Figure 7H). Axons of last-born α/βc neurons showed the most severe defects; they failed to project into lobes in 86% of inc brains (2.23 ± 0.3 missing lobes per brain), fasciculated from ectopic neuronal clusters, and often aggregated near the peduncle (Figure 7A, H; Figure 7—figure supplement 1). The dendrites of γd, α´/β´, and α/βc neurons occupied enlarged territories in inc mutants but otherwise appeared normal (Figure 7F, G). Expansions in dendritic volume for α´/β´ and α/βc subtypes paralleled increases in the numbers of these neurons (Figure 7A, B), while increases for γd dendrites occurred independently of neuron number, consistent with functions of inc in postmitotic γd neurons or non-cell autonomous mechanisms. Axons and dendrites of other sleep-regulatory circuits, including those of the dFB, CRZ+ neurons, and PDF+ circadian pacemaker neurons, exhibited no obvious changes in inc mutants (Figure 7I; Figure 7—figure supplement 2), suggesting that alterations of neuronal anatomy in inc mutants are specific to the MB. These findings indicate that increases in the numbers of late-born MB neurons in inc mutants are associated with changes in postmitotic development expected to perturb circuit assembly and function. In particular, the altered axons of multiple MB neuron subtypes are unlikely to form normal circuits with their targets that influence sleep, including dopaminergic neurons, MB output neurons, and recurrent connections to the MB (Aso et al., 2014b; Sitaraman et al., 2015a; Sitaraman et al., 2015b).

Discussion

Here, we have used temporally restricted genetic manipulations to show that inc acts during neuronal development to ultimately impact sleep in adulthood. While many genes are known to act in adults to impact sleep, developmental mechanisms underlying sleep regulation have only recently gained attention (Chakravarti Dilley et al., 2020; Gong et al., 2021; Iwasaki et al., 2021; Xie et al., 2019). Our results underscore the importance of unbiased temporal genetic manipulations to define critical periods through which genes impact sleep, and suggest that genes may influence sleep through unappreciated developmental mechanisms. A clear implication of these findings is that variations in human sleep patterns, including pathological disruptions of sleep, may have a developmental origin.

Reciprocal conditional manipulations have been critical in revealing surprising developmental and adult contributions of genes to neuronal function and behavior. In one notable example, anxiety-like behaviors in mice caused by mutations of the 5-HT1A serotonin receptor were found to be rescued by developmental expression of the receptor (Gross et al., 2002). Withdrawal of receptor expression in adulthood had no measurable consequences on anxiety-like behavior, and adult-specific receptor expression failed to provide rescue, indicating the necessity and sufficiency of the receptor during development (Gross et al., 2002). A second noteworthy example is provided by a mouse model of Rett syndrome, a neurodevelopmental disorder caused by mutation of MECP2, a transcriptional regulator. Conditional MeCP2 expression solely in adulthood was found to be sufficient to rescue mutant phenotypes, indicating a critical period for MeCP2 function in adults rather than during brain development (Guy et al., 2007; Guy et al., 2012). Inactivation of MECP2 specifically in adulthood causes MECP2 mutant phenotypes (McGraw et al., 2011), confirming its adult requirement. By analogy, various genes that influence sleep might act developmentally or in adulthood in a manner that cannot be anticipated in the absence of conditional manipulations.

inc activity is required in neurons for normal sleep, and conversely, restoring inc solely to neurons is largely sufficient to rescue the short sleep of inc mutants (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). Our conditional neuronal manipulations of inc span embryonic development through adulthood and indicate that inc expression in neurons of late third instar larvae and pupae is sufficient to rescue sleep in inc mutants to near wild-type levels, indistinguishable from the rescue provided by constitutive neuronal inc expression (Pfeiffenberger and Allada, 2012; Stavropoulos and Young, 2011). Extending this developmental pulse of neuronal inc expression into adulthood does not augment the rescue of inc sleep phenotypes, nor does expressing inc only in adult neurons restore sleep to inc animals. inc expression in embryonic, early larval, and adult neurons thus appears dispensable for normal sleep. Instead, inc is required at a time coincident with the birth and development of many adult neurons, including those of the MB (Ito and Hotta, 1992; Lee et al., 1999; White and Kankel, 1978). While our findings suggest that the MB is not the sole brain structure through which inc impacts sleep, they establish a vital role for inc in regulating MB development and its sleep-regulatory functions.

Our findings reveal that inc governs neurogenesis, a fundamental process regulated by genes and pathways conserved from flies to mammals (Doe, 2008; Knoblich, 2008), and suggest that alterations of neurogenesis can cause lasting changes in sleep–wake behavior. The cellular and molecular mechanisms underlying altered neurogenesis in inc mutants, including the stochastic nature of these phenotypes and their apparent restriction to the MB, are of particular interest. inc null mutations are viable (Stavropoulos and Young, 2011), in contrast to the lethality of mutations that globally alter neurogenesis (Betschinger et al., 2006; Lee et al., 2006a; Lee et al., 2006b; Rolls et al., 2003; Vaessin et al., 1991), consistent with the notion that altered neurogenesis in inc mutants manifests preferentially or specifically within the MB. The stochastic nature of neurogenic defects in inc mutants and the overproduction of neurons with projection defects are reminiscent of phenotypes of mushroom body defect (mud) mutants (Guan et al., 2000; Hovhanyan and Raabe, 2009; Prokop and Technau, 1994). In mud mutants, infrequent errors in asymmetric neuroblast division give rise to excess neuroblasts and MB neurons (Bowman et al., 2006; Siller et al., 2006). Similar alterations in neuroblast proliferation in inc mutants may account for the stochastic and cumulative defects in the production of late-born MB neurons; a subtle defect in neuroblast proliferation would be expected to manifest particularly in the MB lineage, the longest in the fly brain. Our results do not yet distinguish the cellular populations through which inc regulates neurogenesis. One possibility is that inc acts in neurons to promote their differentiation, analogous to lola and midlife crisis, genes whose absence causes neurons to dedifferentiate and acquire the proliferative character of neuroblasts (Carney et al., 2013; Southall et al., 2014). Another possibility is that inc functions in neuroblasts, like mud, to govern their asymmetric division.

Our studies and recent findings (Gong et al., 2021) suggest that proper regulation of neurogenesis is essential for normal sleep and that altered neurogenesis in discrete circuits can cause lifelong sleep dysfunction. Intriguing but fragmentary evidence suggests that other genes whose mutation impacts sleep might similarly alter neurogenesis. wide awake (wake), whose mutation causes short sleep in Drosophila (Liu et al., 2014; Zhang et al., 2015), was characterized in an independent study as banderuola (bnd) and shown to regulate the asymmetric division of neuroblasts (Mauri et al., 2014). An interesting possibility yet to be assessed is whether sleep phenotypes of wake/bnd mutants might arise developmentally or through neuroblasts. Similarly, while short sleep phenotypes caused by mutations in the potassium channel subunits encoded by Shaker and Hyperkinetic (Bushey et al., 2007; Cirelli et al., 2005) are thought to reflect their role in regulating excitability in specific adult neurons (Kempf et al., 2019; Pimentel et al., 2016), developmental functions that could contribute to their impact on sleep remain unexplored. Notably, mutations in the Shaker ortholog Kv1.1 analogous to those that strongly reduce sleep in Drosophila (Cirelli et al., 2005; Gisselmann et al., 1989) cause megencephaly and neuronal overproduction in mammals, implicating Kv1.1 in regulating neurogenesis (Chou et al., 2021; Donahue et al., 1996; Petersson et al., 2003; Yang et al., 2012). Explicit tests of whether wake/bnd and Shaker impact sleep through adult or developmental mechanisms, or through a combination of the two, await conditional temporal analysis.

While further manipulations of inc are required to elucidate the precise developmental mechanisms by which it impacts sleep, Cul3 is known to regulate various aspects of neuronal development. Clonal analysis of Cul3 mutations in Drosophila indicates that Cul3 is required for normal axonal arborization and dendritic elaboration within the MB, as well as axonal fasciculation (Zhu et al., 2005). These phenotypes overlap those of inc mutants, although direct comparisons are complicated by the pleiotropic nature of Cul3 mutations, which dysregulate multiple adaptor and substrate pathways. Mosaic analysis of inc is required to discern its developmental functions in postmitotic neurons, to compare its phenotypes with Cul3, and to distinguish cell autonomous and non-cell autonomous mechanisms. In mammals, Cul3 mutations alter neurogenesis, cortical lamination, neuronal migration, synaptic development, and cause behavioral deficits (Amar et al., 2021; Dong et al., 2020; Fischer et al., 2020; Rapanelli et al., 2021). inc and Cul3 are present at synapses in flies and mammals (Kikuma et al., 2019; Li et al., 2017) and are required at the Drosophila larval neuromuscular junction for synaptic homeostasis (Kikuma et al., 2019), a process proposed to be a core function of sleep (Tononi and Cirelli, 2003). The impact of inc on the development and function of central synapses has yet to be assessed, and whether such functions contribute to inc sleep phenotypes remains unknown. As a Cul3 adaptor, inc may engage multiple molecular targets and cellular pathways. Identifying and manipulating inc substrates are thus important goals in elucidating the mechanisms through which inc impacts neuronal development and sleep–wake behavior.

The loss of inc causes enduring developmental and functional impairments in the MB, a structure important for sensory integration, learning, and sleep regulation. The MB integrates olfactory (de Belle and Heisenberg, 1994; Heisenberg et al., 1985), gustatory (Keene and Masek, 2012; Masek and Scott, 2010), visual (Li et al., 2020; Vogt et al., 2016), and thermal inputs (Frank et al., 2015; Hong et al., 2008; Shih et al., 2015), and its activity is altered by sleep pressure (Bushey et al., 2015; Sitaraman et al., 2015a). The MB may thus integrate and filter sensory stimuli to promote sleep in appropriate environmental conditions, in a manner modulated by learning and sleep history. The anatomical defects in inc mutants may render the MB hypersensitive to sensory stimuli, alter functions of the MB that link learning and sleep (Berry et al., 2015; Cervantes-Sandoval et al., 2017; Haynes et al., 2015; Seugnet et al., 2011; Seugnet et al., 2008), or impair the relay of sensory input from MB neurons to downstream sleep-promoting circuits (Aso et al., 2014b; Sitaraman et al., 2015a). While MB circuits and genetic pathways that act in the MB to influence sleep have been manipulated with increasing precision (Aso et al., 2014b; Cavanaugh et al., 2016; Guo et al., 2011; Joiner et al., 2006; Pitman et al., 2006; Sitaraman et al., 2015a; Sitaraman et al., 2015b; Yi et al., 2013), much remains unknown about the function of the MB in sleep regulation, and additional analysis is required to elucidate how inc lesions might alter discrete circuits within the MB and signaling to their targets.

While sensory hypersensitivity and sleep dysfunction are hallmarks of autism and other neurodevelopmental disorders, the underlying mechanisms remain obscure. Given the conserved functions of Cul3–inc complexes and the associations of Cul3 lesions with autism (Kong et al., 2012; Li et al., 2017; O’Roak et al., 2012), elucidating inc substrates and their contributions to neurogenesis and neuronal anatomy may provide insights into brain development, tumorigenesis, and sleep disorders.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Antibodyα-HA (rat monoclonal)RocheCat# 11867431001, RRID:AB_390919(1:100)
Antibodyα-Brp (mouse monoclonal)DSHBCat# nc82, RRID:AB_2314866(1:20 and 1:50)
Antibodyα-FLAG (mouse monoclonal)Sigma-AldrichCat# F1804, RRID:AB_262044(1:100)
Antibodyα-GFP (mouse monoclonal)DSHBCat# GFP-G1, RRID:AB_2619561(1:1000)
Antibodyα-GFP (rabbit polyclonal)Thermo Fisher ScientificCat# A11122, RRID:AB_221569(1:2000)
Antibodyα-dsRed (rabbit polyclonal)Takara BioCat# 632496, RRID:AB_10013483(1:1000)
Antibodyα-FasII (mouse monoclonal)DSHBCat# 8 C6, RRID:AB_2314391(1:50)
Antibodyα-mouse Alexa Fluor 488 (donkey polyclonal)Thermo Fisher ScientificCat# A21202, RRID:AB_141607(1:1000)
Antibodyα-rabbit Alexa Fluor 488 (donkey polyclonal)Thermo Fisher ScientificCat# A21206, RRID:AB_2535792(1:1000)
Antibodyα-rat Alexa Fluor 488 (donkey polyclonal)Thermo Fisher ScientificCat# A21208, RRID:AB_2535794(1:1000)
Antibodyα-rabbit Alexa Fluor 568 (donkey polyclonal)Thermo Fisher ScientificCat# A10042, RRID:AB_2534017(1:1000)
Antibodyα-mouse Alexa Fluor 647 (donkey polyclonal)Thermo Fisher ScientificCat# A31571, RRID:AB_162542(1:1000)
Chemical compound, drugHydroxyureaSigma-AldrichH8627
Genetic reagent (D. melanogaster)w1118Bloomington Drosophila Stock CenterRRID:BDSC_5905Ryder et al., 2004
Genetic reagent (D. melanogaster)inc1Stavropoulos labFLYB:FBal0266013Stavropoulos and Young, 2011; BDSC #5,905 background
Genetic reagent (D. melanogaster)inc2Stavropoulos labFLYB:FBal0162225Stavropoulos and Young, 2011; BDSC #5,905 background
Genetic reagent (D. melanogaster)tub-QS; nsyb-Gal4QFChristopher PotterRiabinina et al., 2015; Li and Stavropoulos, 2016; BDSC #5,905 background
Genetic reagent (D. melanogaster)inc-Gal4Stavropoulos labStavropoulos and Young, 2011; BDSC #5,905 background
Genetic reagent (D. melanogaster)inc1inc-Gal4Stavropoulos labLi et al., 2017; BDSC #5,905 background
Genetic reagent (D. melanogaster)nsyb-Gal4Julie SimpsonSimpson, 2016; BDSC #5,905 background
Genetic reagent (D. melanogaster)c253-Gal4 (MB)Bloomington Drosophila Stock CenterRRID:BDSC_6980Pitman et al., 2006; BDSC #5,905 background; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)c309-Gal4 (MB)Bloomington Drosophila Stock CenterRRID:BDSC_6906Connolly et al., 1996; Pitman et al., 2006 Joiner et al., 2006; Aso et al., 2009; BDSC #5,905 background; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)c929-Gal4 (l-LNv)Amita SehgalHewes et al., 2000; Hewes et al., 2003; Sheeba et al., 2008; Parisky et al., 2008; Shang et al., 2008; iso31 background; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)c584-Gal4 (PI, PPM3)Amita SehgalMartin et al., 1999; Dubowy et al., 2016; iso31 background; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)R69F08-Gal4 (EB)Mark WuLiu et al., 2016; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)R24B11-Gal4 (Helicon)Bloomington Drosophila Stock CenterRRID:BDSC_49070Donlea et al., 2018
Genetic reagent (D. melanogaster)R23E10-Gal4 (dFB)Bloomington Drosophila Stock CenterRRID:BDSC_49032Donlea et al., 2014
Genetic reagent (D. melanogaster)NP2721-Gal4 (DPM)Leslie GriffithWu et al., 2011; Haynes et al., 2015; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)DH44-Gal4Bloomington Drosophila Stock CenterRRID:BDSC_39347Cavanaugh et al., 2014
Genetic reagent (D. melanogaster)pdf-Gal4Stavropoulos labRenn et al., 1999
Genetic reagent (D. melanogaster)crz-Gal4Stavropoulos labTayler et al., 2012
Genetic reagent (D. melanogaster)MB004B (pan-MB)Yoshinori AsoSitaraman et al., 2015a
Genetic reagent (D. melanogaster)MB607B (ɣd)Yoshinori AsoSitaraman et al., 2015a
Genetic reagent (D. melanogaster)MB370B (α'β'm + α'β'ap)Yoshinori AsoSitaraman et al., 2015a
Genetic reagent (D. melanogaster)MB185B (αβs)Yoshinori AsoSitaraman et al., 2015a
Genetic reagent (D. melanogaster)MB594B (αβc)Yoshinori AsoSitaraman et al., 2015a
Genetic reagent (D. melanogaster)MB-Gal80Michael YoungKrashes et al., 2007
Genetic reagent (D. melanogaster)UAS-3xFLAG-IncStavropoulos labLi et al., 2017; BDSC #5,905 background
Genetic reagent (D. melanogaster)UAS-inc-HAStavropoulos labLi et al., 2017; BDSC #5,905 background
Genetic reagent (D. melanogaster)UAS-inc-RNAiVienna Drosophila Resource CenterFLYB:FBst0453067Dietzl et al., 2007; Stavropoulos and Young, 2011
Genetic reagent (D. melanogaster)UAS-dcr2Bloomington Drosophila Stock CenterRRID:BDSC_24651Dietzl et al., 2007; BDSC #5,905 background
Genetic reagent (D. melanogaster)UAS-TrpA1Stavropoulos labHamada et al., 2008; BDSC #5,905 background
Genetic reagent (D. melanogaster)UAS-MyrGFP-2A-RedStingerBarry GanetzkyDaniels et al., 2014
Genetic reagent (D. melanogaster)5xUAS-DenMark::smGdP-V5Bloomington Drosophila Stock CenterRRID:BDSC_62138Nern et al., 2015
Genetic reagent (D. melanogaster)5xUAS-IVS-Syt1::smGdP-HABloomington Drosophila Stock CenterRRID:BDSC_62142Nern et al., 2015
Genetic reagent (D. melanogaster)20xUAS-IVS-CD8-GFPBloomington Drosophila Stock CenterRRID:BDSC_32194Pfeiffer et al., 2010
Genetic reagent (D. melanogaster)NP1227-Gal4Kathy NagelOkada et al., 2009; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)R2-Split Gal4Greg SuhLiu et al., 2016; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)R72G06-Gal4Mark Wuused in inc[2] rescue screen
Genetic reagent (D. melanogaster)VT64246-Gal4Leslie Griffithused in inc[2] rescue screen
Genetic reagent (D. melanogaster)c305a-Gal4Leslie Griffithused in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR49E09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38692Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR49F01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38694Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR49F02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38695Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR49G06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38707Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR51G05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38797Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR53B06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38863Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR53C04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_38871Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR54F06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39081Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55A03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39095Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55B12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39103Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55D01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39110Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55D05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39112Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55F07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39128Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55G11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39132Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR56H02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39164Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR56H09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39166Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR58E10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39184Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR58H05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39198Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59B10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39209Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59E09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39220Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59H05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39229Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR60C01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39240Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR60D05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39247Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR60H12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39268Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR64A11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39289Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR64F03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39309Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR64G05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39316Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65B04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39336Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65D06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39352Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65D07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39353Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR67A04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39396Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR69C02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39483Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR71D01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39579Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR72H03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39799Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR74H01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39872Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR76F06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39937Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR77H03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39976Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR78A01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_39985Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR78G06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40013Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR79A01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40021Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR79B08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40029Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR83H01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40368Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR85C07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40422Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR87A08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40473Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR92G09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40629Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93C06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40647Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93G05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40662Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93H07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40669Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR94D04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40681Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR94E07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40688Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR94F06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40694Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR95E08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40710Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR95F11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_40714Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR40B09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41235Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR40E08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41238Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR41G11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41244Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR42F06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41253Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR60D10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41284Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65C03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41290Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR74B11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41301Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR87B02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41316Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65B09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_41353Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR34C12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45219Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR45D10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45323Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR60G12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45360Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR23G07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45493Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26C01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45518Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48D06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45774Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR20E01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45837Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25G01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_45851Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR53G07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46041Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR55G02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46070Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR35H03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46205Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR46H09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46275Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR58G05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46410Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59H01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46423Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR64D08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46539Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65C05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46554Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65H08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46566Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR69H02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46620Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR70G11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46641Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR71E04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46658Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR72A04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46665Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR73D06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46692Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR56F05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46714Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR77A04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_46976Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR80C12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47059Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR81C04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47087Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR81D04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47094Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR91A08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47148Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR91G01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47175Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR92H11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47211Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93B04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47215Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93D01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47221Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93D06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47224Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93G11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47238Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR94H10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47268Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR16D12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47325Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR16H05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47327Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10E03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47447Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR42E09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47589Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR52A01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47634Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR70A09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47720Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR72F10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47731Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR74G04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47742Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10A11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47839Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10A12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47840Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR13C06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47860Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR19G10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47887Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR21C11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47898Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR30F07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47911Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR44G12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47933Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR52F09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_47943Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR28F06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48083Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR33H11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48119Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR50A07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48179Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR51B08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48183Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR52C05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48190Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR54H12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48205Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR58F01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48213Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59B11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48215Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59C12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48219Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59E04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48221Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10D10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48261Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10H09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48277Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR67B06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48294Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR73H09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48318Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR87C01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48389Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR89C02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48404Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR92A08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48414Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93C08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48417Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR93F02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48422Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR95F03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48433Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10E07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48440Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR11C07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48448Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR12B10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48490Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR12D12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48506Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR12G09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48525Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR13B10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48548Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR13D09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48561Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR13E04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48565Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR13E06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48566Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR13F04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48573Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14C08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48606Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR20F01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48610Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14E05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48642Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14E06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48643Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14E09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48645Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14E12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48647Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14F11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48653Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14G08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48661Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14H02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48664Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR15B07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48678Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR15D11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48690Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR15E09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48696Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR16E03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48727Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR17B12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48759Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR17D02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48764Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR17G05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48782Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR18D04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48811Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR18D07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48813Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR18F04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48820Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR18G06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48826Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR19F05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48855Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR20F04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48904Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR21C09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48936Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR21D02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48939Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR21D06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48942Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR22C12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48978Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR22E06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_48986Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR22H10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49005Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR23B04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49016Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR23C06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49023Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR23E10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49032Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR23F05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49035Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24A08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49058Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24B11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49070Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24C06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49073Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24C07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49074Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24C10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49075Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24E05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49081Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24F03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49086Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR24H03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49098Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25A01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49102Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25A06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49105Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25C01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49115Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25C03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49117Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25E04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49125Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR25H06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49144Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26B04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49158Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26B11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49164Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26B12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49165Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26C11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49171Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26E02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49179Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26E07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49182Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR26F09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49194Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR27A02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49207Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10E06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49236Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR14B11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49255Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR15B03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49261Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR18G02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49278Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR32D08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49357Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR35F09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49371Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR60F05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49405Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR28E01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49457Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR29A12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49478Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR30B10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49522Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR43D09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49553Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR47E07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49568Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48D07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49572Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR52F11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49579Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR59A05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49593Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR65H10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49614Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR66A03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49615Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR30G03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49646Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR31F06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49684Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR31G04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49686Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR31H05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49692Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR32E04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49717Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR33H07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49760Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR34B11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49774Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR34C08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49780Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR35B08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49818Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR10G02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49825Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR11E05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49827Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR19C10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49831Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR19E12-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49835Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR20D07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49848Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR20E08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49851Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR21H06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49866Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR22F03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49875Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR35D07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49908Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR37E08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49958Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR37F05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49961Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR38A11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49980Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR38B06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_49986Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR38E08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50008Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR39C07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50039Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR39E10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50053Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR39G09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50064Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR40C07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50080Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR42D11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50156Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR44B03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50200Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR44B10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50202Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR44D02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50205Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR45D05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50227Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR45G01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50241Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR45G05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50243Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR45H11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50248Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR46B05-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50253Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR47D07-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50304Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR47F04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50319Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR47G08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50328Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR47H01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50330Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48A03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50339Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48A08-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50341Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48B10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50352Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48C06-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50357Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48E02-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50367Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48G01-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50381Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48G04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50383Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48H04-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50392Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48H10-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50395Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR48H11-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50396Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR49A09-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50403Jenett et al., 2012; used in inc[2] rescue screen
Genetic reagent (D. melanogaster)P{GMR49C03-GAL4}attP2Bloomington Drosophila Stock CenterRRID:BDSC_50414Jenett et al., 2012; used in inc[2] rescue screen

Fly food and culture

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Fly food was prepared in batches containing the following ingredients: 1800 g cornmeal (Labscientific, FLY-8010-20), 1800 ml molasses (Labscientific, FLY-8008-16), 744 g yeast (Labscientific, FLY-8040-20F), 266 g agar (Mooragar, 41084), 56 g methyl paraben (Sigma, H3647), 560 ml alcohol (Fisher, A962P4), 190 ml propionic acid (Fisher, A258500), and 47 l of water. Unless indicated otherwise, crosses were performed with five females and three males in vials (28.5 mm diameter × 95 mm height) containing standard fly food supplemented with dry yeast (Fleischmann, B000LRFVHE). Crosses were cultured at 25°C in 12 hr light–dark (LD) cycles.

To prepare food for conditional induction of the Q-system, solid fly food was melted in a microwave oven and allowed to cool before addition of quinic acid or vehicle. Quinic acid solution was freshly prepared essentially as described (Riabinina et al., 2015). 10 g of quinic acid (Sigma, 138622) was dissolved in 30 ml of water and the pH was adjusted to 6.5 with 10 mM NaOH. A volume of quinic acid solution containing the equivalent of 0.66 g of quinic acid (~2.4 ml) was added for each 10 ml of melted fly food and mixed well; ~12.4 ml was distributed to each empty vial. Food was allowed to cool and subsequently stored at 4°C prior to use. Vehicle food was prepared similarly, substituting an equal volume of water.

Conditional Q-system induction

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Three sets of conditional induction experiments were performed. The first set contained vehicle treatment and constitutive, developmental-specific, and adult-specific induction regimens. The second set included vehicle, constitutive induction, and induction from the late third instar larval stage through adulthood. The third set included vehicle, constitutive induction, and a pulse of induction from the late third instar larval stage through pupal stages. Initiation, maintenance, or termination of induction at desired developmental stages was achieved by transferring larvae, pupae, and/or adults to food containing quinic acid or vehicle as described below. Within each set of experiments, w1118 and inc1 controls were exposed to vehicle and quinic acid induction regimens, and all animals underwent the same physical transfers in parallel. Sleep of w1118 and inc1 animals was not altered by exposure to vehicle or quinic acid, as described previously (Li and Stavropoulos, 2016), nor by physical transfer at larval, pupal, or adult stages. Vehicle-treated w1118 and inc1 animals, pooled across all three sets of experiments, are shown in Figure 2B. Two to three independent biological replications were performed for all induction experiments.

In the first set of experiments, developmental-specific induction was achieved by setting crosses on food containing quinic acid, allowing animals to develop and pupate in the same vials, and transferring adult males within 2–3 hr of eclosion to fresh vials with vehicle-containing food to terminate Q-system induction. Adult animals were maintained in these vials for 3–4 days, anesthetized with CO2, and transferred to DAM tubes with vehicle-containing food for measurement of sleep. For adult-specific induction, crosses were set on vehicle food and animals developed in the same vials. Adult males eclosing from these cultures were transferred within 2–3 hr of eclosion to fresh vials with food containing quinic acid, maintained on this food for 3–4 days, and transferred to DAM tubes containing food with quinic acid for measurement of sleep. For constitutive induction and vehicle treatment, food containing quinic acid or vehicle, respectively, was used throughout, along with the same transfer procedure.

In the second set of experiments, induction from the late third instar larval stage through adulthood was achieved as follows: crosses were set on vehicle-containing food and wandering third instar larvae from these cultures were gently collected with blunt forceps and examined under brief phosphate-buffered saline (PBS) immersion to select males by visual identification of gonads as described (Kerkis, 1931). Larvae were transferred to recipient vials containing isogenic w1118 larvae and pre-churned quinic acid food; these recipient cultures were initiated in parallel with experimental crosses to allow food consistency to be maintained during Q-system induction. Adult animals bearing mini-white-marked transgenes were transferred within 2–3 hr of eclosion to fresh vials containing quinic acid food to maintain Q-system induction. Three- to four-day-old adults were subsequently transferred to DAM tubes with food containing quinic acid for measurement of sleep. Constitutive induction and vehicle treatment were performed similarly, using appropriate food and the same transfer procedure.

In the third set of experiments, a pulse of Q-system induction specific to late third instar larval and pupal stages was achieved as follows: crosses were set on vehicle food and male wandering third instar larval progeny were selected and transferred to w1118 recipient vials containing pre-churned quinic acid food as described above. To prevent adult exposure to quinic acid, pupae bearing mini-white-marked transgenes were identified at approximately the P13–P14 stage by pigmented eyes and black wings (Ashburner et al., 2005; Bainbridge and Bownes, 1981) and gently dislodged from vial walls with a paintbrush and transferred to the walls of fresh vials containing vehicle food. Three- to four-day-old adults eclosing from these vials were transferred to DAM tubes containing vehicle food for measurements of sleep. Constitutive induction and vehicle treatment were performed similarly, using appropriate food and the same transfer procedure.

inc2 rescue screen

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inc2 virgins were crossed to male flies carrying Gal4 transgenes and a minimum of five male progeny were screened for each genotype. A total of 277 Gal4 lines were screened, including 266 randomly selected drivers and 11 drivers previously characterized for expression in sleep-regulatory circuits. To select random lines, 4088 lines from the FlyLight collection available from the Bloomington Drosophila Stock Center were assigned sample numbers. Using the randperm command in Matlab, 300 lines were randomly selected. Expression patterns for these lines in the Janelia Flylight database were examined; 84 lines were excluded due to very low levels of expression, very broad expression patterns unlikely to be useful for functional mapping, or because expression data were unavailable. Expression patterns for the remaining 216 lines ranged from broad to sparse. This procedure for random selection was applied iteratively to yield 266 lines. Top-ranking hits from the initial screen were rescreened in independent crosses. Rescreening of c253-Gal4 and c309-Gal4 was performed after backcrossing each line six generations to an isogenic w1118 stock (BDSC #5905) (Ryder et al., 2004).

MB ablation

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MB ablation was performed essentially as described previously (de Belle and Heisenberg, 1994). Egg collection was performed on grape juice agar plates containing a spot of rehydrated dry yeast. w1118 larvae at the first instar stage were transferred to a well of a 24-well plate bearing a spot of rehydrated dry yeast paste, containing water vehicle or 50 mg/ml hydroxyurea (Sigma, H8627). After 4–5 hr, larvae were collected and washed briefly with distilled water on a Nitex mesh filter (Genesee Scientific, 57–102) to remove yeast and subsequently transferred to vials containing standard food. Vials were cultured at 25°C in LD cycles and adult animals eclosing from these cultures were assayed for sleep as described below. MB ablation was verified in adult brains in a separate cohort of animals by staining with anti-FasII primary antibody (1:50, DSHB) and Alexa 488-conjugated donkey anti-mouse secondary as described below. Vehicle-treated animals exhibited MB lobes demarcated with FasII signal (100%, n = 9), while hydroxyurea-treated animals exhibited complete MB ablation as indicated by the lack of residual FasII staining (100%, n = 12); FasII signal within the EB was observed in all brains, providing a control for staining of the MB.

Immunohistochemistry

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All fixing, washing, and incubation steps for immunohistochemistry were performed on a nutator. To assess conditional induction of inc-HA using the Q-system, larval, pupal, and adult brains were dissected from inc1; UAS-inc-HA/tub-QS; nsyb-Gal4QF/+ males. Wandering third instar male larvae were selected by visual identification of gonads as described above. Larval brains were dissected in ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, and washed 3× 15 min in PBS containing 0.2% Triton X-100 (PBST). Male pupae at stage P13–P14 were identified by the staging criteria described above and the presence of sex combs. Pupal brains were dissected in ice-cold PBST, fixed with 4% paraformaldehyde in PBST for 30 min at room temperature, and washed 3× 15 min in PBST. To prepare adult brains, 2- or 4-day-old whole male adults were fixed with 4% paraformaldehyde in PBST for 3 hr at 4°C and washed 3× 15 min in PBST at room temperature prior to brain dissection in PBST. After dissection, all brains were blocked with 5% normal donkey serum (NDS) (Lampire Biological, 7332500) in PBST at room temperature for 30–60 min. Samples were incubated overnight at 4°C in rat anti-HA (1:100; Sigma, 11867431001) and mouse anti-Brp (1:20, DSHB, nc82) antibodies prepared in 5% NDS in PBST. Brains were subsequently washed 3× 15 min in PBST at room temperature, incubated overnight at 4°C in Alexa 488 donkey anti-rat (1:1000; Life Technologies, A21208) and Alexa 647 donkey anti-mouse (1:1000, Life Technologies A31571) antibodies prepared in 5% NDS in PBST, washed 3× 15 min at room temperature in PBST, and mounted on microscope slides (Fisher, 1255015) in Vectashield (Vector Labs, H-1000).

For all other immunohistochemistry, adult brains of 4-day-old males were dissected in PBST, fixed with 4% paraformaldehyde in PBST for 30 min at room temperature, and washed 3× 20 min in PBST at room temperature. Brains of male wandering third instar larvae and stage P13–P14 pupae were dissected, fixed, and stained as described above for Q-system experiments. Primary antibodies were mouse anti-FLAG (1:100; Sigma, F1804), rabbit anti-GFP (1:2000; Fisher, A11122), mouse anti-GFP (1:1000; DSHB, GFP-G1), rabbit anti-dsRed (1:1000; Takara, 632496), and mouse anti-Brp (1:50, DSHB, nc82). Secondary antibodies were Alexa 488 donkey anti-rabbit (1:1000; Life Technologies, A21206), Alexa 488 donkey anti-mouse (1:1000; Life Technologies, A21202), and Alexa 568 donkey anti-rabbit (1:1000; Life Technologies, A10042).

Imaging and quantitation of neuron number and cluster number

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All imaging was performed on a Zeiss LSM800 confocal microscope, using a 10X air objective to capture z-stacks at 512 × 512 pixel resolution with 1 μM z-slices, unless indicated otherwise. All imaging settings were identical for each experiment comprising control and experimental brains stained in parallel.

To quantify MB neuron numbers, wild-type and inc1 brains expressing UAS-MyrGFP-2A-RedStinger under the control of split-Gal4 drivers were imaged as described above. For each neuron subtype, wild-type and inc1 brains were assigned sample numbers and a subset, randomly selected using the randperm command in Matlab, was imaged at higher resolution with a 63X oil objective. Both hemispheres of brains were imaged, capturing dsRed and myr-GFP channels separately. Only a single hemisphere could be imaged for two wild-type brains, one each in the γd and α´/β´ groups, due to sample compression by the objective. z-stacks encompassing nuclei were captured at 512 × 512 resolution for γd neurons and at 1024 × 1024 resolution for α´/β´ and α/βc neurons; 2 μM z-slices were used to ensure that all nuclei (diameter ~3 μM) were segmented in at least one optical section.

High resolution z-stacks were assigned a random letter code and neurons were counted in a single-blind manner by two independent experimenters. Nuclei of γd and α´/β´ neurons exhibited minimal overlap along the z-axis, allowing nuclei to be counted in maximum intensity z-projections using the Cell Counter plug-in in ImageJ; visual inspection of z-stacks in parallel allowed overlapping nuclei to be differentiated. Dense distribution of α/βc neurons prohibited accurate counting in single maximum intensity z-projections; maximum intensity z-projections were generated for every 10 z-slices, yielding three to four maximum intensity z-projections representing 20 μM each. To improve visualization of densely clustered α/βc nuclei, background was subtracted using a rolling ball/sliding paraboloid algorithm (radius set to the size of the largest nucleus: 50 pixels) and image intensity display range was adjusted (minimum: 5; maximum: 175). Processed maximum intensity z-projections representing 20 μM each were then merged into a single z-stack for manual counting using the Cell Counter plug-in in ImageJ; to avoid double-counting of nuclei segmented in adjacent z-projections, the original unprocessed z-stack was examined in parallel. The variation in MB neuron counts between experimenters, calculated as the absolute difference between the two counts divided by their mean, was (mean ± SEM) 2.0% ± 0.2% for γd; 1.5% ± 0.3% for α´/β´; and 2.6% ± 0.3% for α/βc. Where neuron counts were different for a given hemisphere, the average was plotted. Numbers of γd neurons in wild-type animals were intermediate between those reported in prior studies (Aso et al., 2014a; Shih et al., 2019), while numbers of α´/β´ and α/βc neurons were lower, likely reflecting conservative assignment of nuclei in our study and the use of different antibodies and reporters (Aso et al., 2014a; Shih et al., 2019). α´/β´ counts obtained using the MB370B driver were similar to previously reported numbers of α´/β´m neurons; because MB370B labels α´/β´m neurons strongly and α´/β´ap neurons weakly, the lower absolute numbers of α´/β´ neurons in our studies may reflect detection sensitivity and correspond chiefly to α´/β´m neurons.

To count the number of α/βc neuron clusters in wild-type and inc1 brains, the same randomly selected samples used to quantify neuron numbers were assessed in a single-blind manner by two independent experimenters. Each z-stack was analyzed using a combination of visual inspection of z-sections and rotating the image stack in three dimensions using the 3D Viewer plug-in in ImageJ (threshold: 0; resampling factor: 2). A group of nuclei distributed continuously along all axes was classified as a cluster; a continuous gap at least one nuclear diameter in width across all axes was used to define cluster edges and discrete clusters. Cluster counts were identical for wild-type brains; total cluster counts for inc1 brains differed by 8.2% ± 0.2% (mean ± SEM) between experimenters. Where cluster counts were different for a given hemisphere, the average was plotted.

DH44 and dFB somata were counted in a single-blind manner by two independent experimenters as described for γd and α´/β´ neurons, using DH44-Gal4 to drive UAS-MyrGFP-2A-RedStinger and 23E10-Gal4 to drive 5× UAS-IVS-Syt1::smGdP-HA. Numbers of dFB and DH44 neurons were identical between two independent experimenters.

Analysis of axonal projections and dendritic volume

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To analyze axonal projections and dendritic volume, image stacks were captured using a 10X objective at 512 × 512 resolution with 1 μM z-slices. Axonal projection defects were assessed in maximum intensity z-projections. The number of horizontal and/or vertical lobes missing myr-GFP signal entirely was counted for each brain. To quantify dendritic volume, the Threshold command in ImageJ was applied to z-stacks to select dendrites based on DenMark immunofluorescence; high signal to noise allowed unambiguous demarcation of dendrites and clear separation from background. The same minimum and maximum threshold values were applied to all wild-type and inc1 brains stained in parallel in an experiment and captured the entirety of dendritic signal for all samples. A single rectangular region of interest of minimal area encompassing dendritic signals from both brain hemispheres across all z-slices was drawn for each z-stack. Dendritic volume was quantified using the Voxel Counter plug-in in ImageJ.

Sleep analysis

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Three- to four-day-old male flies eclosing from LD-entrained cultures raised at 25°C were loaded in glass tubes (5 mm diameter × 65 mm length) containing standard food or appropriate food for Q-system experiments as described above. Animals were monitored for 5–7 days at 25°C in LD cycles using DAM2 monitors (Trikinetics). Locomotor activity data were collected in 1 min bins. Inactive periods of 5 min or longer were classified as sleep. The first 36–48 hr of data were discarded to allow acclimation of animals to tubes, and 3–5 integral days of data were analyzed beginning with ZT0. Dead animals were excluded from analysis by a combination of automated filtering and visual inspection of locomotor traces. Matlab code used to analyze sleep is available in Source code 1.

Thermogenetic activation

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Crosses were set on standard fly food as described above and cultured at 21.5°C. One- to four-day-old male flies eclosing from these cultures were assayed for 5 days in LD cycles. Animals were maintained at 21.5°C for the first 60–72 hr of the assay, including 36–48 hr of acclimation and the subsequent baseline day beginning at ZT0. Temperature was increased to 28.5°C for 24 hr to activate dTrpA1, followed by 24 hr of recovery at 21.5°C. The percent change in sleep was calculated for each animal by subtracting the amount of sleep on the baseline day from the amount of sleep on the activation day and dividing this difference by the amount of sleep on the baseline day. The percent change in sleep for individual animals was averaged for each genotype.

Gal4 drivers used to express TrpA1 were as follows: pan-MB, MB004B split-Gal4; MB, c253-Gal4 and c309-Gal4; EB, R69F08-Gal4; DPM, NP2721-Gal4; Helicon, R24B11-Gal4; l-LNV, c929-Gal4; PI, PPM3, c584-Gal4.

Statistics

One-way analysis of variance (ANOVA) and Tukey post hoc tests were used for comparisons between more than two groups of animals for total sleep, daytime sleep, nighttime sleep, and sleep bout number; for comparisons of these sleep parameters between two groups, unpaired two-sided Student’s t-tests were used. Kruskal–Wallis tests and Dunn’s post hoc tests were used for comparisons of sleep bout length between more than two groups of animals; for comparison between two groups, Mann–Whitney tests were used. One-way ANOVA and Dunnett’s post hoc tests were used for comparisons of percent change in sleep. Unpaired two-sided Welch’s t-tests were used for pairwise comparisons of neuron number, cluster number, and dendrite volume.

Data availability

Data for all figures and code used to analyze sleep are included in the supporting files.

References

  1. Book
    1. Ashburner M
    2. Golic KG
    3. Hawley RS
    (2005)
    Drosophila: A Laboratory Handbook (2nd ed)
    New York: Cold Spring Harbor Laboratory Press.

Decision letter

  1. Ronald L Calabrese
    Senior and Reviewing Editor; Emory University, United States
  2. Chiara Cirelli
    Reviewer; University of Wisconsin, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "insomniac links the development and function of a sleep regulatory circuit" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Chiara Cirelli (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The authors should briefly discuss inc's previously described role in homeostatic plasticity, which may be relevant to this manuscript. Perhaps MBs require homeostatic plasticity to promote sleep but they cannot undergo it without inc In that case, multiplication and rewiring of MB neurons may not be cell-autonomous direct effects of reducing inc's molecular function but instead may be compensatory responses (i.e. expansion and rewiring of sleep-promoting neurons) to make up for loss of sleep that begins to manifest in larvae. Hypofunctionality of MB neurons in this context might thus be expected to reduce sleep just like loss of MB neurons following ablation with hydroxyurea. Previous studies indicate that hydroxyurea ablation of the MBs phenocopies inc mutants' reduction in sleep. A comparison between these two short-sleeping phenotypes should be made and can be accomplished using ablation data already published.

2) The authors should add at least a few lines of commentary to the banderuola (bnd) gene. Bnd controls asymmetric cell division of sensory organ neural precursors. 5' wake alleles are derived from bnd, and these alleles reduce sleep, like LOF mutants of inc (Mauri et al., Curr Biol 2014; Liu et al., Neuron 2014; see also Zhang et al., PLoS Genet 2015). Those findings bolster the contention in the current study that the function of many sleep-regulating genes may be developmental in origin.

3) There were concerns related to how the data is presented, how some conclusions are made and why some critically important data is left out. Several suggesting for improvement in these regards are in the reviewers' "Recommendations for the authors." Some important points to address:

a. In Figures 1-5, sleep data is only represented as a sleep profile or total sleep. Sleep is a complex behavior and when we manipulate genes several parameters are affected. Regarding sleep parameters it would be great if authors can be consistent. For example, figure 4-supplement 1 has all important sleep parameters for the Mb sleep rescue experiment but the same parameters are not shown for other experiments (e.g. quinic acid). If there is a specific reason for presenting additional parameters for one set of experiments and not the others, then it should be made explicit. It appears that authors think these parameters are important because they show them for this one set of experiments. Also, these experiments are different from the published Neuron 72:964-976. doi:10.1016/j.neuron.2011.12.003 paper showing inc mutants and these manipulations could affect sleep parameters differently.

b. There is a screen of 283 Gal4 lines in Figure 4A. The lines are listed by Bloomington id but the genotype information is absent from material and methods (copy and paste genotype information from Bloomington). We are interested in information like 49E09-Gal4 instead of BL38692 (Bloomington id) in the Materials and methods section. The text says sleep regulatory circuits or randomly selected without any additional details. How many were random, how many were sleep regulatory and where, were they clean or mixed population?

c. Further, the figures define several transgenic lines as pan-MB gal4, DPM-gal4 etc. (Figure 5). Several gal4 lines could have a DPM neuron and many other neurons so it potentially confusing to call each a DPM Gal4. Please make it clear to the reader what Gal4 was used and where its expressed. This can be done simply in the figure legend and in a supplementary table with appropriate references to the literature so people can see for themselves where a certain line is expressed by going to the appropriate reference. This would also help people doing similar RNAi screens.

Reviewer #1:

The submitted manuscript explores an interesting area in neuroscience as to how genes control development of circuits that regulate behavior. What do these genes do in these circuits and at what stage do they become critical. Authors focus on 3 main things:

1) Where inc is expressed and when its required for circuit development that underlies inc dependent sleep regulation.

2) inc expression in MB and how that controls sleep.

3) How inc expression regulates development of MB-KCs and assemble into functional circuits.

These findings are important for the field and experiments are appropriate and figures are generally clear. My major concerns are related to how the data is presented, how some conclusions are made and why some critically important data is left out. Figure 1-5, sleep data is only represented as a sleep profile or total sleep. Sleep is a complex behavior and when we manipulate genes several parameters are affected. These sleep parameters should be shown for all genetic and other (quinic acid) manipulations.

The screening of 283 GAL4 drivers for inc expression and its affect on sleep is shown but that data is barely discussed. If the intention is not to discuss the data then its positioning in main figures should be reconsidered as it is not clear what that screen is all about.

The effect of inc expression on KC cell number is intriguing but there is no direct evidence that reduction in the percentage of KC population affects sleep. Although, inc is expressed in MBs and inc is involved in KC neurogenesis but how do these flies with defective KCs behave. Is it the number of KCs that regulate sleep or molecular role of inc in MB functioning/ physiological activity?

Since this paper focusses on the role of a single gene, so KCs with inc knockdown could be physiologically altered suggesting that while inc is involved in neurogenesis it is not necessarily the reason flies sleep differently. What this gene does to circuits and connectivity is a key focus of the paper but the experiments do directly address this. what is the significance of KC number to sleep and how are specific KCs related to the inc phenotypes? This needs to be addressed thoroughly.

Specific Comments

Introduction: Since gene expression is the big focus of this paper the authors need to elaborate on what specific genes have been identified and how they control sleep circuits. While, the focus is Drosophila sleep it might be worthwhile mentioning some genes and how they shape circuits in other animals. This is an important area in sleep research that has not received much focus but the central problem and specifically role of inc and Cul3 gene need to be significantly elaborated.

Results section: Lines 72-80 detail main experiments of Figure 1 and 2. The information seems to be highly intermingled and its confusing what the approach was and what was the result. Rather than jumping between experiments it might be worthwhile to focus on Figure 1 and what it shows about inc expression. Figure 1 images are small and don't look very high resolution and it appears that the expression is widespread and not restricted to MB and EB as claimed by authors in the figure legend.

Figure 1 needs to be larger and if the researchers used a counter stain like nc82 that stain should also be shown as it allows neuropil detection. Nc82 stain MB and EB very clearly so the staining overlap will strengthen the authors argument.

Line 86-92: "inc influences sleep through adult-specific or developmental mechanisms". This is an elegant approach to address a developmental role of the gene and the schematic in Figure 2 is helpful but the rational for choose specific development stages should be expanded on as its central to the paper's conclusions. For example, why was quinic acid was not applied during individual larvae stages or two stages at a time etc. How were the time points chosen and tightly controlled between flies that could be developing at slightly different rates?

In Figure 2-5 there is extensive sleep data but only total sleep is shown. The sleep analysis involves several other features like daytime sleep, nightime sleep, sleep bout structure, activity and latency. These sleep parameters are also important and inc could be involved in these as well. These data should be added for all sleep experiments. This would require some data mining but no additional experiments but would be extremely useful to readers as these genes could have more specific effects on these features.

In almost all figures range of n's is provided. For example, in Figure 2 of conditional rescue N=11-86 this is quite the range so how was statistical power considered in these data sets. If there is a specific reason for why some samples were low (n-11) and so as high as 86.

Line 106-108: the authors results show that inc mediated circuit development is happening around third instar and this stage is critical. Why is this stage critical? What regions develop during this time and what does molecularly inc really do to these circuits. This needs to be discussed as it is a key conclusion of Figures 1-3.

Line 113: 283 Gal4 drivers were chosen based on what criteria? The criteria for screening are important here as there is no description of the 283 lines in methods and the data is represented in a way that not much can be concluded.

Line 124-128: Authors use inc-Gal4; mb-Gal80. MB is a structure with over 3000 neurons (KCs, ONS and DANS). The authors should make it explicit and clear as what mb-gal80 really blocks (KCS?).

In all figures, sleep profiles should show error bars (mean and SEM).

Figure 5: Please show all sleep parameters. While total sleep data is used here to reach conclusions other features that change for manipulating different cell types in inc + and inc – background will be very interesting.

Figure 6 data is extremely interesting and really ties in the developmental role with behavior. I am surprised why they chose dfb and not EB even though inc expression is high in eb. Dfb is sleep-promoting but not sure how it relates to inc function.

The MB Gal4 drivers used in behavioral experiments are distinct from neurogenesis experiments. Reasons for doing so should be made explicit.

In all figures number of samples and statistical measures (F value, type of statistic etc.) should be stated.

Rather than using pan-MB gal4, DPM-gal4 etc. please state the name of driver in the figures and results. Several gal4 lines could have a DPM neuron and many other neurons so it won't be right to call that a DPM Gal4. This is potentially misleading. The gal4 names should be consistent and specific as they can alter the conclusions of the study.

Reviewer #2:

The results are very well presented and discussed. I have only 2 suggestions.

First, in the end, it seems that the authors suggest that while the inc mutation results in an increased number of MB neurons, these cells are hypofunctional because their axons are underdeveloped. In the limit, they may be not functional at all. If so, however, the inc mutation and the short sleep phenotype due to the ablation of MB – after larvae are fed with hydroxyurea – may not be very different. Is this the case? A comparison between these two short-sleeping phenotypes should be done, even just using ablation data already published.

Second, previous studies from the same authors have shown a very interesting role for inc in presynaptic homeostatic potentiation. I understand that this may be very difficult to study outside the neuromuscular junction, but the authors should at least try to comment on whether they think that given the postsynaptic role of inc in this phenomenon, the lack of this in the inc mutants may underlie the structural defects seen in the MB.

Reviewer #3:

Li et al., investigate the relationship between the insomniac (inc) gene's role in neural circuit development and sleep regulation in adult Drosophila using a combination of sophisticated genetics, behavioral analyses, neural circuit mapping and immunohistochemical-based measurements of neuronal structural changes. The authors find that adult sleep depends on inc facilitating proper wiring of the mushroom body (MB), but not other established arousal-controlling loci, during a critical window during late development that excludes adulthood. This study stands out for addressing a possibility that is often ignored in behavioral genetics (especially the field of sleep research) – namely that a gene's function may be more related to establishment of neural circuitry that controls a given behavior than acute regulation of neuronal function while the behavior is being performed. As the authors point out, their findings may also be relevant to poorly understood neurodevelopmental disorders. For example, the wiring deficiencies of inc mutants are consistent with inc's molecular function as an adaptor of Cul3, for which human mutations have been implicated in autism.

This is a very straightforward, clear manuscript. The experiments are well-defined, rigorously performed with appropriate controls, and support the authors' conclusions quite well.

1) Lines 223-225: "Our results… suggest that many genes influence sleep through unappreciated developmental mechanisms." This statement struck me as overly bold and should probably be toned down. The findings in this study suggest that the research community should more rigorously test whether identified sleep-regulating genes function developmentally rather than assuming those functions are restricted to adult behavior. However, this study does not provide evidence that many genes have actually been misassigned adult-specific roles in regulating sleep.

2) Line line 241: "While our findings suggest that the MB is not the sole brain structure through which inc impacts sleep…" Presumably this statement relates to only partial suppression by MB-Gal80 of inc1 rescue by inc-Gal4/UAS-inc Please clarify.

3) The present study cites Pfeiffenberger and Allada (Plos Genetics, 2012). However, it does not state that those authors previously showed that inc and Cul3 are required during development. That omission should be corrected.

4) The authors may not want to mention this point, but I thought it was interesting that stimulation of all tested arousal circuits except MBs elicited normal (expected) changes in sleep in inc mutants. That suggests to me that MBs function either presynaptically or in parallel to those other sleep-regulating circuits.

https://doi.org/10.7554/eLife.65437.sa1

Author response

Essential Revisions:

1) The authors should briefly discuss inc's previously described role in homeostatic plasticity, which may be relevant to this manuscript. Perhaps MBs require homeostatic plasticity to promote sleep but they cannot undergo it without inc. In that case, multiplication and rewiring of MB neurons may not be cell-autonomous direct effects of reducing inc's molecular function but instead may be compensatory responses (i.e. expansion and rewiring of sleep-promoting neurons) to make up for loss of sleep that begins to manifest in larvae. Hypofunctionality of MB neurons in this context might thus be expected to reduce sleep just like loss of MB neurons following ablation with hydroxyurea. Previous studies indicate that hydroxyurea ablation of the MBs phenocopies inc mutants' reduction in sleep. A comparison between these two short-sleeping phenotypes should be made and can be accomplished using ablation data already published.

In the revised Discussion, we cite the role of inc and Cul3 in regulating synaptic homeostasis at the larval neuromuscular junction. While the simplest hypothesis supported by our current findings is that the effects that inc exerts on sleep through the MB reflect alterations in MB neurogenesis and anatomy, our findings do not exclude functions of inc at synapses that may contribute to its developmental impact on sleep. We note in the Discussion that inc may engage multiple substrates as a Cul3 adaptor and impact sleep by more than one mechanism.

We have added a figure comparing the short sleep phenotypes caused by inc mutations and MB ablation. We reproduce short sleep phenotypes elicited by MB ablation (Pitman et al., 2006; Joiner et al., 2006) and find them to be less severe than those of inc mutants. Because the number and anatomy of early-born MB neurons appears unaffected by the loss of inc, MB function in inc mutants is likely to be at least partially intact. Together, these data are consistent with the idea that MB function is impaired in inc mutants and contributes to their sleep phenotypes.

2) The authors should add at least a few lines of commentary to the banderuola (bnd) gene. Bnd controls asymmetric cell division of sensory organ neural precursors. 5' wake alleles are derived from bnd, and these alleles reduce sleep, like LOF mutants of inc (Mauri et al., Curr Biol 2014; Liu et al., Neuron 2014; see also Zhang et al., PLoS Genet 2015). Those findings bolster the contention in the current study that the function of many sleep-regulating genes may be developmental in origin.

We thank the reviewer for pointing out that developmental functions of the wake/bnd gene may underlie or contribute to its sleep phenotypes. The revised Discussion considers this relevant and interesting possibility for wake/bnd and other sleep mutants.

3) There were concerns related to how the data is presented, how some conclusions are made and why some critically important data is left out. Several suggesting for improvement in these regards are in the reviewers' "Recommendations for the authors." Some important points to address:

a. In Figures 1-5, sleep data is only represented as a sleep profile or total sleep. Sleep is a complex behavior and when we manipulate genes several parameters are affected. Regarding sleep parameters it would be great if authors can be consistent. For example, figure 4-supplement 1 has all important sleep parameters for the Mb sleep rescue experiment but the same parameters are not shown for other experiments (e.g. quinic acid). If there is a specific reason for presenting additional parameters for one set of experiments and not the others, then it should be made explicit. It appears that authors think these parameters are important because they show them for this one set of experiments. Also, these experiments are different from the published Neuron 72:964-976. doi:10.1016/j.neuron.2011.12.003 paper showing inc mutants and these manipulations could affect sleep parameters differently.

We have added supplemental figures with absolute sleep parameters for animals assessed under LD cycles at constant temperature (Figures 2-4 and new Figure 6; note Figure 1 contains only immunostaining). We have also added a supplemental figure for thermogenetic activation experiments (Figure 5) containing daily sleep profiles for genotypes not included initially. For thermogenetic activation experiments, we present (1) percent change in total sleep for the entire activation day versus the baseline day and (2) daily sleep profiles, to focus on relative changes in sleep for each genotype. This enables comparison of TrpA1 activation in different neuronal populations while controlling for variation in baseline sleep across many genotypes and the differential effects of temperature alone in wild-type animals and inc mutants. We believe this presentation strikes a balance between plotting an overwhelming number of absolute sleep parameters for 18 genotypes over 3 days and conveying the main result: the effect on sleep elicited by neuronal activation is altered specifically for the MB in inc mutants.

b. There is a screen of 283 Gal4 lines in Figure 4A. The lines are listed by Bloomington id but the genotype information is absent from material and methods (copy and paste genotype information from Bloomington). We are interested in information like 49E09-Gal4 instead of BL38692 (Bloomington id) in the Materials and methods section. The text says sleep regulatory circuits or randomly selected without any additional details. How many were random, how many were sleep regulatory and where, were they clean or mixed population?

The revised Methods and Key Resources Table contain information for screened lines, fragment identity for Gal4s from the Janelia collection, additional references for Gal4 driver expression patterns, and procedures for selecting random lines. The revised manuscript contains 277 unique lines; six lines screened in duplicate were inadvertently listed as unique in the earlier revision. For the rescue screen, all but eleven lines were randomly selected.

c. Further, the figures define several transgenic lines as pan-MB gal4, DPM-gal4 etc. (Figure 5). Several gal4 lines could have a DPM neuron and many other neurons so it potentially confusing to call each a DPM Gal4. Please make it clear to the reader what Gal4 was used and where its expressed. This can be done simply in the figure legend and in a supplementary table with appropriate references to the literature so people can see for themselves where a certain line is expressed by going to the appropriate reference. This would also help people doing similar RNAi screens.

We agree that this shorthand may overstate the specificity of Gal4 lines. The revised text clarifies that expression patterns of Gal4 drivers include neurons of interest but are not limited to them. We use short labels within Figure 5A for spatial clarity; explicit identifications of Gal4s (e.g. R69F08) are present in Figure 5B, Figure 5–supplement 1, in the relevant Methods subsection, and in the Key Resources Table with relevant references.

Reviewer #1:

The submitted manuscript explores an interesting area in neuroscience as to how genes control development of circuits that regulate behavior. What do these genes do in these circuits and at what stage do they become critical. Authors focus on 3 main things:

1) Where inc is expressed and when its required for circuit development that underlies inc dependent sleep regulation.

2) inc expression in MB and how that controls sleep.

3) How inc expression regulates development of MB-KCs and assemble into functional circuits.

These findings are important for the field and experiments are appropriate and figures are generally clear. My major concerns are related to how the data is presented, how some conclusions are made and why some critically important data is left out. Figure 1-5, sleep data is only represented as a sleep profile or total sleep. Sleep is a complex behavior and when we manipulate genes several parameters are affected. These sleep parameters should be shown for all genetic and other (quinic acid) manipulations.

As noted above, we have added supplemental figures with additional sleep parameters.

The screening of 283 GAL4 drivers for inc expression and its affect on sleep is shown but that data is barely discussed. If the intention is not to discuss the data then its positioning in main figures should be reconsidered as it is not clear what that screen is all about.

We focus on the identification of c253 and c309 as the main result of the inc2 rescue screen (Figure 2A) and include screen data in the main figures to convey the scale of the screen, the distribution of screened lines, and the rank order of rescuing hits. We have refrained from discussing Gal4 drivers we did not carefully characterize or pursue; backcrossing and additional analysis of spatiotemporal expression patterns would be required to interpret possible rescue of inc mutants or lack thereof.

The effect of inc expression on KC cell number is intriguing but there is no direct evidence that reduction in the percentage of KC population affects sleep. Although, inc is expressed in MBs and inc is involved in KC neurogenesis but how do these flies with defective KCs behave. Is it the number of KCs that regulate sleep or molecular role of inc in MB functioning/ physiological activity?

Since this paper focusses on the role of a single gene, so KCs with inc knockdown could be physiologically altered suggesting that while inc is involved in neurogenesis it is not necessarily the reason flies sleep differently. What this gene does to circuits and connectivity is a key focus of the paper but the experiments do directly address this. what is the significance of KC number to sleep and how are specific KCs related to the inc phenotypes? This needs to be addressed thoroughly.

The simplest model consistent with our findings is that developmental changes in the MB in inc mutants, including altered neurogenesis and circuit formation, impair its effects on sleep when activated in adulthood. We acknowledge that our present data do not distinguish whether changes in KC number, anatomy, physiology, or a combination of these attributes contribute to the sleep phenotypes of inc mutants. Resolving these mechanistic questions is non-trivial, as experimental manipulations of one process, such as MB neurogenesis, may alter the production and postmitotic development of multiple KC subtypes. We hope to pursue the underlying mechanisms, including the role of neurogenesis and MB neuron subtypes, in future studies.

Specific Comments

Introduction: Since gene expression is the big focus of this paper the authors need to elaborate on what specific genes have been identified and how they control sleep circuits. While, the focus is Drosophila sleep it might be worthwhile mentioning some genes and how they shape circuits in other animals. This is an important area in sleep research that has not received much focus but the central problem and specifically role of inc and Cul3 gene need to be significantly elaborated.

Results section: Lines 72-80 detail main experiments of Figure 1 and 2. The information seems to be highly intermingled and its confusing what the approach was and what was the result. Rather than jumping between experiments it might be worthwhile to focus on Figure 1 and what it shows about inc expression. Figure 1 images are small and don't look very high resolution and it appears that the expression is widespread and not restricted to MB and EB as claimed by authors in the figure legend.

Figure 1 needs to be larger and if the researchers used a counter stain like nc82 that stain should also be shown as it allows neuropil detection. Nc82 stain MB and EB very clearly so the staining overlap will strengthen the authors argument.

The revised Introduction and Discussion add context and elaborate on the roles of inc and Cul3 in neuronal development. We have edited the text describing Figures 1 and 2 and hope that the changes improve clarity. We increased the size of Figure 1, allowing better resolution of staining in the EB and MB. We note that the Figure 1 legend and prior studies (Stavropoulos and Young, 2011; Pfeiffenberger and Allada, 2012) describe prominent (but not exclusive) inc expression in the mushroom body, ellipsoid body, fan-shaped body, and pars intercerebralis.

Line 86-92: "inc influences sleep through adult-specific or developmental mechanisms". This is an elegant approach to address a developmental role of the gene and the schematic in Figure 2 is helpful but the rational for choose specific development stages should be expanded on as its central to the paper's conclusions. For example, why was quinic acid was not applied during individual larvae stages or two stages at a time etc. How were the time points chosen and tightly controlled between flies that could be developing at slightly different rates?

We first validated the Q-system in constitutive rescue and then assessed developmental- and adult-specific induction regimens. We chose the wandering third instar larval stage as the first timepoint for analysis, as animals at this stage are easily selected, sexed, and transferred to different food for induction regimens. Because experiments with third instar larvae revealed that inc has no measurable contribution in neurons of embryos and first and second instar larvae that impacts sleep in adulthood, we did not attempt to analyze these earlier stages separately. We focused further analysis on the restricted pulse condition to map the developmental sufficiency of inc. While our protocol does not exclude variation in the precise developmental age of wandering third instar larvae, any such differences are accounted for in scatterplots of individual animals in Figure 2B.

In Figure 2-5 there is extensive sleep data but only total sleep is shown. The sleep analysis involves several other features like daytime sleep, nightime sleep, sleep bout structure, activity and latency. These sleep parameters are also important and inc could be involved in these as well. These data should be added for all sleep experiments. This would require some data mining but no additional experiments but would be extremely useful to readers as these genes could have more specific effects on these features.

We have added additional sleep parameters in supplemental figures, as described above.

In almost all figures range of n's is provided. For example, in Figure 2 of conditional rescue N=11-86 this is quite the range so how was statistical power considered in these data sets. If there is a specific reason for why some samples were low (n-11) and so as high as 86.

inc mutants exhibit reduced viability in many but not all genetic backgrounds in the absence of rescue (Stavropoulos and Young, 2011). The low number of animals (n=11) in the adult-specific induction regimen reflects a low number of animals obtained in this non-rescuing condition; the high number of animals (n=86) in the vehicle condition reflects the inclusion of this induction regimen in all experiments that were pooled in Figure 2. One-way ANOVA and post-hoc tests account for statistical significance between populations of different sizes.

Line 106-108: the authors results show that inc mediated circuit development is happening around third instar and this stage is critical. Why is this stage critical? What regions develop during this time and what does molecularly inc really do to these circuits. This needs to be discussed as it is a key conclusion of Figures 1-3.

As noted in the text, proliferating neuroblasts in third instar larvae and pupae give rise to adult neurons in various regions of the nervous system including the optic lobe, mushroom bodies, other brain regions, and the ventral nerve cord (White and Kankel, 1978; Truman and Bate, 1988). Prior to adulthood, these neurons elaborate projections and form synapses with their targets; many embryonic-born neurons and circuits are simultaneously remodeled and/or incorporate newly born neurons. The revised Results and Discussion note that the developmental impact of inc on sleep may involve processes spanning neurogenesis and postmitotic neuronal development in multiple brain regions including the mushroom body.

Line 113: 283 Gal4 drivers were chosen based on what criteria? The criteria for screening are important here as there is no description of the 283 lines in methods and the data is represented in a way that not much can be concluded.

Almost all Gal4 lines were selected randomly; eleven represent previously characterized populations whose manipulation was shown to impact sleep. The revised Methods section adds details on the identity of screened lines and their selection.

Line 124-128: Authors use inc-Gal4; mb-Gal80. MB is a structure with over 3000 neurons (KCs, ONS and DANS). The authors should make it explicit and clear as what mb-gal80 really blocks (KCS?).

We have edited the text to clarify that MB-gal80 is expressed chiefly in intrinsic MB neurons (Kenyon cells) during development and adulthood (Krashes et al., 2007; Pauls et al., 2010).

In all figures, sleep profiles should show error bars (mean and SEM).

All sleep profiles now include shading to represent SEM.

Figure 5: Please show all sleep parameters. While total sleep data is used here to reach conclusions other features that change for manipulating different cell types in inc + and inc – background will be very interesting.

Supplemental figure panels include additional sleep parameters, as described above.

Figure 6 data is extremely interesting and really ties in the developmental role with behavior. I am surprised why they chose dfb and not EB even though inc expression is high in eb. Dfb is sleep-promoting but not sure how it relates to inc function.

inc is expressed in CRZ, dFB, EB, MB, and PDF neurons, among other populations (Figure 1 of this study; Stavropoulos and Young, 2011; Pfeiffenberger and Allada, 2012; Li et al., 2017); we chose CRZ, dFB, and PDF neurons for controls for anatomical analysis because their somata are easily counted and their projections are well defined. We note in the Discussion that our findings do not exclude the possibility that inc regulates the development of circuits outside of the MB.

The MB Gal4 drivers used in behavioral experiments are distinct from neurogenesis experiments. Reasons for doing so should be made explicit.

Split-Gal4 drivers used for anatomical analysis of specific MB neuron subtypes do not rescue inc sleep phenotypes (data not shown). The lack of rescue for sparsely expressed MB drivers may reflect limited strength, breadth, spatiotemporal patterns of expression, and/or a broad requirement for inc within the MB; we note that the anatomical defects of inc mutants are not limited to one subtype of MB neurons. As noted in the revised Discussion, additional manipulations will likely be required to distinguish the precise cellular populations that underlie the sleep phenotypes of inc mutants.

In all figures number of samples and statistical measures (F value, type of statistic etc.) should be stated.

We have added information to figure legends including F-statistics. Statistical tests described in Methods are now also included in figure legends.

Rather than using pan-MB gal4, DPM-gal4 etc. please state the name of driver in the figures and results. Several gal4 lines could have a DPM neuron and many other neurons so it won't be right to call that a DPM Gal4. This is potentially misleading. The gal4 names should be consistent and specific as they can alter the conclusions of the study.

We have added identifications for drivers as noted above.

Reviewer #2:

The results are very well presented and discussed. I have only 2 suggestions.

First, in the end, it seems that the authors suggest that while the inc mutation results in an increased number of MB neurons, these cells are hypofunctional because their axons are underdeveloped. In the limit, they may be not functional at all. If so, however, the inc mutation and the short sleep phenotype due to the ablation of MB – after larvae are fed with hydroxyurea – may not be very different. Is this the case? A comparison between these two short-sleeping phenotypes should be done, even just using ablation data already published.

We thank the reviewer for these comments and helpful suggestions. The revised manuscript includes comparative analysis of the sleep phenotypes caused by inc mutations and MB ablation.

Second, previous studies from the same authors have shown a very interesting role for inc in presynaptic homeostatic potentiation. I understand that this may be very difficult to study outside the neuromuscular junction, but the authors should at least try to comment on whether they think that given the postsynaptic role of inc in this phenomenon, the lack of this in the inc mutants may underlie the structural defects seen in the MB.

The revised discussion cites the role of inc and Cul3 in synaptic homeostasis at the larval neuromuscular junction and notes that developmental functions of inc at synapses may contribute to inc sleep phenotypes. Although we are unaware of instances in which defects in synaptic homeostasis elicit compensatory alterations in neurogenesis or neuronal projections in a cell autonomous or non-cell autonomous manner, we cannot at present exclude this possibility for inc. As noted in the revised Discussion, the identification of Inc substrates that impact sleep may provide insight into the downstream cellular and molecular pathways.

Reviewer #3:

Li et al., investigate the relationship between the insomniac (inc) gene's role in neural circuit development and sleep regulation in adult Drosophila using a combination of sophisticated genetics, behavioral analyses, neural circuit mapping and immunohistochemical-based measurements of neuronal structural changes. The authors find that adult sleep depends on inc facilitating proper wiring of the mushroom body (MB), but not other established arousal-controlling loci, during a critical window during late development that excludes adulthood. This study stands out for addressing a possibility that is often ignored in behavioral genetics (especially the field of sleep research) – namely that a gene's function may be more related to establishment of neural circuitry that controls a given behavior than acute regulation of neuronal function while the behavior is being performed. As the authors point out, their findings may also be relevant to poorly understood neurodevelopmental disorders. For example, the wiring deficiencies of inc mutants are consistent with inc's molecular function as an adaptor of Cul3, for which human mutations have been implicated in autism.

This is a very straightforward, clear manuscript. The experiments are well-defined, rigorously performed with appropriate controls, and support the authors' conclusions quite well.

1) Lines 223-225: "Our results… suggest that many genes influence sleep through unappreciated developmental mechanisms." This statement struck me as overly bold and should probably be toned down. The findings in this study suggest that the research community should more rigorously test whether identified sleep-regulating genes function developmentally rather than assuming those functions are restricted to adult behavior. However, this study does not provide evidence that many genes have actually been misassigned adult-specific roles in regulating sleep.

This point is well taken and we have adjusted the language of this section.

2) Line line 241: "While our findings suggest that the MB is not the sole brain structure through which inc impacts sleep…" Presumably this statement relates to only partial suppression by MB-Gal80 of inc1 rescue by inc-Gal4/UAS-inc. Please clarify.

This is correct. The ability of MB-gal80 to partially suppress the rescue conferred by inc-Gal4 suggests that the mushroom body is a critical region mediating the effects of inc on sleep, among other neuronal populations. We have edited the text to clarify this point.

3) The present study cites Pfeiffenberger and Allada (Plos Genetics, 2012). However, it does not state that those authors previously showed that inc and Cul3 are required during development. That omission should be corrected.

The Introduction references earlier publications (Pfeiffenberger and Allada, 2012 and Li and Stavropoulos, 2016) relevant to the temporal requirements of inc with regard to sleep regulation. Pfeiffenberger and Allada (2012) reported developmental manipulations of inc using the Geneswitch system, but we were unable to reproduce their findings (Li and Stavropoulos, 2016). In particular, we found that developmental induction of Geneswitch in neurons causes lethality and strong nonspecific changes in sleep in the absence of effector transgenes or with UAS-GFP (Li and Stavropoulos, 2016). We believe these experimental artifacts preclude use of the Geneswitch system for developmental manipulations of sleep, and thus we regard the temporal requirements of inc as unresolved prior to the current study. In contrast, Q-system transgenes and their induction with quinic acid have no measurable effect on sleep in the absence of effector transgenes in wild-type animals or inc mutants, as shown in the present study and as reported previously (Li and Stavropoulos, 2016).

4) The authors may not want to mention this point, but I thought it was interesting that stimulation of all tested arousal circuits except MBs elicited normal (expected) changes in sleep in inc mutants. That suggests to me that MBs function either presynaptically or in parallel to those other sleep-regulating circuits.

Recent connectome data for the mushroom body provide information on its direct and indirect synaptic connectivity (Li et al., 2020, eLife; Schlegel et al., 2021, eLife), including to fan-shaped body neurons in the central complex. MB output neurons (MBONs) provide direct and indirect presynaptic input to many neurons of the fan-shaped body (Li et al., 2020, eLife). While no direct inputs from fan-shaped body neurons to the MB are apparent from connectome data, some fan-shaped body neurons provide direct input to dopaminergic neurons (DANs), which can modulate synapses between MB neurons and MBONs. Thus, FB neurons may indirectly modulate MB output. The functional relevance of these synaptic connections in the context of sleep remains unknown and awaits combinatorial manipulations of circuit elements; we note that residual MB function in inc mutants might not fully block potential presynaptic input from neuronal populations in our analysis.

https://doi.org/10.7554/eLife.65437.sa2

Article and author information

Author details

  1. Qiuling Li

    Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, United States
    Contribution
    Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6101-8779
  2. Hyunsoo Jang

    Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, United States
    Present address
    Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9191-3697
  3. Kayla Y Lim

    Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, United States
    Present address
    Graduate Program in Molecular, Cellular, & Integrative Physiology, University of California, Los Angeles, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7168-5877
  4. Alexie Lessing

    Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2044-7822
  5. Nicholas Stavropoulos

    1. Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, United States
    2. Waksman Institute, Rutgers University, Piscataway, United States
    Contribution
    Conceptualization, Funding acquisition, Project administration, Software, Supervision, Writing – original draft, Writing – review and editing
    For correspondence
    stavropoulos@waksman.rutgers.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5915-2760

Funding

Howard Hughes Medical Institute (International Student Research Fellowship)

  • Qiuling Li

National Institute of Neurological Disorders and Stroke (R01NS112844)

  • Nicholas Stavropoulos

National Institute of Neurological Disorders and Stroke (R21NS111304)

  • Nicholas Stavropoulos

G. Harold and Leila Y. Mathers Foundation

  • Nicholas Stavropoulos

Whitehall Foundation (2013-05-78)

  • Nicholas Stavropoulos

Alfred P. Sloan Foundation

  • Nicholas Stavropoulos

Leon Levy Foundation

  • Nicholas Stavropoulos

Brain and Behavior Research Foundation (NARSAD Young Investigator)

  • Nicholas Stavropoulos

Sleep Research Society Foundation (J. Christian Gillin, M.D. Research Grant)

  • Nicholas Stavropoulos

Irma T. Hirschl Trust (Career Scientist Award)

  • Nicholas Stavropoulos

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank C Desplan, N Ringstad, M Shirasu-Hiza, and members of the Stavropoulos lab for comments on the manuscript; Y Aso, B Ganetzky, L Griffith, W Joiner, W Li, K Nagel, C Potter, A Sehgal, J Simpson, G Suh, M Wu, M Young, the Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Center, and the Janelia Flylight collection for fly stocks; and DSHB for antibodies. This work was supported by an International Student Research Fellowship from the Howard Hughes Medical Institute (HHMI) to QL and by grants from the National Institutes of Health (R01NS112844 and R21NS111304), the Mathers Foundation, Whitehall Foundation grant 2013-05-78, fellowships from the Alfred P Sloan and Leon Levy Foundations, a NARSAD Young Investigator Award from the Brain and Behavior Foundation, the J Christian Gillin, M.D. Research Award from the Sleep Research Society Foundation, and a Career Scientist Award from the Irma T Hirschl/Weill-Caulier Trust to NS.

Senior and Reviewing Editor

  1. Ronald L Calabrese, Emory University, United States

Reviewer

  1. Chiara Cirelli, University of Wisconsin, United States

Publication history

  1. Received: December 4, 2020
  2. Accepted: October 15, 2021
  3. Accepted Manuscript published: December 15, 2021 (version 1)
  4. Accepted Manuscript updated: December 16, 2021 (version 2)
  5. Version of Record published: January 13, 2022 (version 3)

Copyright

© 2021, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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