EEC-derived Unpaired signaling regulates sleep.

a, upd3 expression levels in midguts RNAi-mediated knockdown of upd3 in EECs using voilá-GAL4 in combination with Tubulin-GAL80ts (voilá>) (N=7). b, upd2 and upd3 expression levels in midguts in animals with EEC knockdown of upd2 or upd3 using voilá-GAL4 in combination with Tubulin-GAL80ts and R57C10-GAL80 (EEC>) (N=4-6). c, Confocal imaging of upd3-GAL4 driven UAS-GFP expression in EECs, co-stained with Prospero (Pros) as an EEC marker (Scale bar, 20 µm). d, Twenty-four-hour sleep profiles in animals with EEC-specific upd2 and upd3 knockdown (N=29-34). e, Total daytime sleep and f, nighttime sleep durations in animals with EEC-specific upd2 and upd3 knockdown flies (N=29-34). g, Motion bout length for control and EEC-specific upd2 and upd3 knockdown flies (N=23-27). h, Motion bout activity across experimental groups (N=23-30). i, Twenty-four-hour sleep profiles for global upd2/3 mutants (N=19-32). j, Daytime and k, nighttime sleep durations in global upd2/3 mutants (N=19-32). l, Sleep profiles following EEC-specific CRISPR-mediated upd2 and upd3 knockout (N=24-31). m, Daytime and n, night sleep durations in animals with EEC-specific CRISPR-mediated upd2 and upd3 activation (N=25-31). Statistical analyses performed using Mann-Whitney tests for panels a and b; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test for panels e, g, j, and k; Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels f, h, m, and n. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

EEC-derived Unpaired signaling regulates glial JAK-STAT activity that modulates sleep.

a, Day sleep measurements for flies with knockdown of IL-6 related Unpaired cytokine receptor domeless (dome), which activates JAK-STAT, in neurons (R57C10-GAL4, R57C10>) and glial cells (repo-GAL4, repo>) (N=25-39). b, Twenty-four-hour sleep profiles for controls and animals with glial-specific dome knockdown (N=25-32). c, Total day sleep duration and d, total night sleep duration for animals with glial-specific dome knockdown and control flies (N=25-32). e, Motion bout length and f, motion bout activity in animals with glial-specific dome knockdown and controls (N=25-32). g, Images of brain from controls and animals with EEC knockdown upd2 or upd3 using voilá>10xSTAT-GFP. STAT-GFP expression (green) with Repo labeling (red) indicates glial cells (Scale bar, 50 µm). Insets show zoomed views of STAT-GFP+ and Repo+ glial cells (Scale bar, 15 µm). h, Quantitative analysis of GFP intensity in the layer of glial cells located at the surface of the brain in animals with EEC knockdown of upd2 or upd3 and controls (N=8). Statistical analyses were conducted using parametric t-tests for panel a; Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels c, d, e and f; and Ordinary one-way ANOVA with Dunnett’s multiple comparisons test for panel h. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

EEC-derived Unpaired signaling modulates sleep in response to intestinal oxidative stress.

a, Measurement of upd2 and upd3 expression in the midguts upon 20-hour treatment with 1% H2O2-laced food or overexpression of upd3 (upd3-OE) using EEC> (N=4). b and c Assessment of sleep duration over consecutive days during daytime (ZT0-ZT12) and nighttime (ZT12-ZT24) in animals exposed to food containing (b) 0.1% H2O2 (N=26-31) or (c) 1% H2O2 (N=23-29). d, Daily sleep and e, nightly sleep amounts measured over one day under standard food conditions followed by two consecutive days on 1% H2O2-containing food in animals with EEC-specific upd2 or upd3 knockdown and controls (N=23-30). Experiments measuring sleep levels in controls and animals lacking EEC-derived upd2 or upd3 were performed concurrently and share the control but are presented in separate figures (b, c, d, and e) for clarity. f, Survival rates under a 1% H2O2-induced oxidative stress diet in controls and animals with EEC-specific upd2 or upd3 knockdown (N=23-30). g, A 48-hour sleep profile comparison between global upd2/3 mutants and w1118 controls under one day of standard food conditions followed by one day of 1% H2O2-induced stress (N=18-63). h and i, Quantification of daytime and nighttime sleep durations in upd2/3 mutants versus w1118 controls under normal food conditions and the following day exposed to food containing 1% H2O2 (N=18-63). j, Observation of sleep patterns and k, measurements of daytime sleep across a three-day period, encompassing a day on standard diet, subsequent day on 1% H2O2-laced food to induce oxidative stress, and a final day back on standard diet to monitor recovery, in flies with EEC-specific overexpression of upd3 (upd3-OE) compared to control (N=29-32). Statistical analyses were performed using Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels a, b, c, d, e, f, and k; Mann-Whitney test for panels h and i. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

Activation of glial JAK-STAT by EEC-derived Unpaired cytokines in response to oxidative stress.

a, Representative images of brains from flies expressing STAT::dGFP:2A::RFP reporter, with green representing destabilized GFP (dGFP) and purple indicating stable RFP. Left panels show brains under normal light conditions (ZT0), middle panels show brains with lights off (ZT12), and right panels depict brains after 20 hours of oxidative stress induced by 1% H2O2-containing food, imaged at light-on time ZT0. White dotted lines outline the brain perimeter. Scale bar, 50 µm. b, Ratio of dGFP to RFP fluorescence intensity under normal light conditions at ZT0, at ZT12, and after oxidative stress (ZT0), as depicted in panel a, to show dynamic changes in JAK-STAT activity (N=108-461, indicating the number of cells counted). c, Representative images of brains displaying 10xSTAT::GFP expression under homeostatic conditions and after oxidative stress in control flies (Ctrl) and flies with EEC-specific upd2 or upd3 knockdown. Scale bar, 50 µm. Inset ’Zoom’ panels provide magnified views of glial cells labeled by Repo. Scale bar, 15 µm. d, Quantification of 10xSTAT::GFP intensity in glial cells under homeostatic and oxidative stress conditions, demonstrating the impact of EEC-specific cytokine knockdown (N=8, indicating the number of brains). e, qPCR analysis of dome expression in the brains of flies with EEC-specific upd3 knockdown in comparison to voilà> controls (N=5). Statistical analyses were conducted using Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panel b; ordinary one-way ANOVA with Tukey’s multiple comparison test for panel d; and t-tests for panel e. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

EEC-derived Unpaired and glial Domeless signaling modulate sleep during intestinal oxidative stress.

a, Daytime sleep duration in flies with glial-specific dome knockdown under standard and oxidative stress conditions induced by 1% H2O2 in food (N=25-32). b, Daytime sleep duration in flies with repo-driven dome knockdown restricted to the adult stage using Tub-GAL80ts(repoTS>) under normal conditions and during exposure to 1% H2O2-containing food (N=29-32). c, Daytime sleep during a three-day period, encompassing a day on standard diet, subsequent day on 1% H2O2-laced food to induce oxidative stress, and a final day back on standard diet to monitor recovery, in controls and animals with glial-specific dome knockdown (N=24-32). d, Survival rates of controls and flies with glial-specific dome knockdown after exposure to oxidative stress by 1% H2O2-laced food (N=31). e, Sleep profiles and f, daytime sleep duration for animals with EEC-specific upd3 knockdown compared to control flies across a 36-hour period encompassing 24 hours on standard diet followed by 12 hours on oxidative stress conditions, induced by 4% H2O2-containing food (N=15-30). g, Sleep profiles and h, daytime sleep duration for animals with glial-specific dome knockdown compared to control flies across a 36-hour period encompassing 24 hours on standard diet followed by 12 hours on oxidative stress conditions, induced by 4% H2O2-containing food (N=20-32). i, Nighttime sleep durations for animals under 4% H2O2 oxidative stress with adult restricted knockdown of dome in glial cells and control (N=31-32). Statistical tests: Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels a, c and d; Unpaired t-tests for panels b, f, and h; and Mann-Whitney test for panel i. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

BBB glia drives Domeless-mediated sleep responses to intestinal oxidative stress.

a, Representative images showing GFP expression driven by 10xSTAT::GFP in controls and animals with voilà-GAL4 (voilà>) driven upd3 knockdown in EECs under intestinal ROS induced by 20 hours exposure to 1% H2O2-containing food. The top panels depict overall brain and ventral nerve cord (VNC) structure with views of surface or deeper layers; the bottom panels provide zoomed-in views, highlighting the BBB glia at the interface between the brain and external environment. Dotted lines indicate brain and VNC perimeters. Scale bars, 50 µm (top) and 15 µm (bottom). b, Quantification of GFP intensity in the brain and VNC in BBB glial controls and animals with EEC knockdown of upd3 under ROS stress, induced by exposure to 1% H2O2-laced food (N=7). c, Daytime and d, nighttime sleep durations in flies with BBB glial-specific knockdown of dome or overexpression of hopTum under normal conditions, during exposure to 1% H2O2-containing food, and subsequent recovery on normal diet (N=23-32). e, Daytime and f, nighttime sleep durations in flies with BBB glial-specific knockdown of dome or overexpression of hopTum under normal conditions, during exposure to 4% H2O2-containing food, and subsequent recovery on normal diet (N=23-32). Statistical tests used: Unpaired t-tests for panel b; Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels c, d and f; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test for panel e. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

Gut Unpaired cytokine signaling inhibits wake-promoting AstA signaling.

a, Images of brains from animals with AstA-R1-GAL4 and AstA-R2-GAL4 driving nuclear dsRed (nRFP, magenta) and co-stained with anti-Repo antibodies (green) show glial cells. The yellow dashed line indicates the interface between the brain and the external space, with the area below housing the (BBB glial cells (Scale bar, 10 µm). The yellow demarcation accentuates the separation between the cerebral interior and the external milieu, identifying the location of BBB glial cells underneath this partition (Scale bar is 10 µm). b, Relative expression of AstA-R1 and AstA-R2 in heads from animals with upd3 knockdown in EECs driven by voilà-GAL4 (voilà>) compared to the control group after 20 hours on 1% H2O2-laced food to induce oxidative stress (N=5-6). c, Relative expression of AstA-R1 and AstA-R2 in brains from animals with dome knockdown in glial cells driven by repo-GAL4 (repo>) compared to the control group after 20 hours on 1% H2O2-laced food to induce oxidative stress (N=6).d Sleep patterns and e, daytime sleep across a three-day period, encompassing a day on a standard diet, a subsequent day on 1% H2O2-laced food to induce oxidative stress, and a final day back on the standard diet to monitor recovery, in flies with BBB glial-specific knockdown of AstA-R2 compared to control (N=28-32). f, AstA transcript levels in brains and midguts from controls and animals with knockdown of AstA in AstA+ EECs using AstA-GAL4 (AstA>) in combination with R57C10-GAL80 to suppress neuronal GAL4 activity, referred to as AstAGUT> (N=5-6). g Sleep profiles and h, daytime sleep on standard food of animals with AstA knockdown in AstA+ EECs using AstAGUT> and controls (N=30-32). i Sleep profiles, j daytime sleep on standard food of controls and animals with TrpA1-mediated activation of AstA+ EECs, and animals with TrpA1-mediated activation of AstA+ EECs with simultaneous knockdown of AstA (N=30-32). k, Diagram illustrating the role of enteroendocrine cells (EECs) in regulating wakefulness and sleep through Unpaired cytokine signaling under homeostatic and disease conditions. Left: Under homeostatic conditions, EECs release baseline levels of Unpaired, which interacts with the blood-brain barrier (BBB) to maintain normal JAK-STAT signaling, promoting healthy wakefulness. Right: In response to disease, reactive oxygen species (ROS) increase in EECs, leading to elevated release of Unpaired. This surge in Unpaired upregulates JAK-STAT signaling in BBB glia, which inhibits wake-promoting AstA signaling by suppressing AstA receptors, thus resulting in increased sleep, a state termed ’sickness-sleep,’ to promote recovery. The diagrams depict the gut lining with EECs highlighted, the interface with the BBB, and the resulting systemic effects on the organism’s sleep-wake states. EC; enterocyte. Statistical tests used: Unpaired t-tests for panel b, c and f; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test for panel e, h and j. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

Effects on sleep, feeding, and metabolic parameters of Unpaired cytokine manipulation in EECs.

a, Twenty-four-hour sleep profile for animals with EEC knockdown of upd2 and upd3 using voilà-GAL4 (voilà>). b, Day and c, night sleep durations for EEC upd2 and upd3 knockdown and control groups (N=50-61). d, Sleep profiles for EEC-specific upd3 knockdown using R57C10-GAL80, Tub-GAL80ts, voilà> (ECC>) and UAS-upd3-RNAi controls (N=23-31). e, Day and f, night sleep durations for UAS-RNAi controls and EEC-specific upd2 and upd3 knockdown (N=23-31). g, Motion bout length and h, motion bout activity for EEC upd2 and upd3 knockdown using voilà> and control flies (N=49-60). i, Motion bout length and j, motion bout activity for EEC upd2 and upd3 knockdown and UAS-RNAi control flies (N=21-32). k, Total feeding time for EEC-specific upd2 and upd3 knockdown and control flies using FLIC (N=9-36). l, Food intake over 1 hour for animals with EEC-specific upd3 overexpression (upd3-OE) and controls (N=13-20). m, Relative triacylglyceride (TAG) levels in EEC-specific upd3 knockdown and control flies (N=5-8). Statistical analyses were performed using t-tests for panel e, f, and m; Mann Whitney tests for panels i,j, k, and l; Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels b, c, g, and h. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

Characterization of sleep patterns, activity, and metabolic impact of dome knockdown in glial cells.

a-c, Sleep profiles over a 24-hour period for animals with glial-specific dome knockdown and UAS-RNAi controls (N=25-32). d, Total daytime sleep and e, nighttime sleep for UAS-RNAi controls and animals with glial-specific dome knockdown (N=25-32). f, Duration of motion bouts and g, motion bout activity for the same groups as in a-d (N=25-32). h, Food intake measured over a 1-hour period for animals with dome knockdown in glial cells and UAS-RNAi control flies (N=23-31). i, Triacylglyceride (TAG) levels relative to controls in glial-specific dome knockdown flies. Statistical analyses were performed using Mann Whitney tests for panels d,e, f, g, h, and i, except t-tests, were performed for dome-iTRiP versus control in panel d and g, and for dome-iGD versus control in panel d. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

Sleep duration influenced by EEC-specific Unpaired cytokine disruption and dietary changes.

a-d, Daytime sleep duration in flies with upd2 or upd3 knockdown in EECs and UAS-RNAi controls measured over one day under standard food conditions followed by two consecutive days on 1% H2O2-containing food (N=21-32). e, Sleep profiles over a 48-hour period for flies with EEC-specific upd3 knockdown under a transition from standard to 1% agar starvation diet compared to control flies (N=27-32). f and g, Daytime and nighttime sleep durations for flies with EEC-specific upd3 knockdown under standard diet and 1% agar starvation diet conditions (N=27-32). Statistical tests: Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test was used for panels a, b, c, d; two-way ANOVA with Sidak’s multiple comparisons test was used for panels f and g. Data are presented as mean ± SEM. ns, non-significant (p>0.05).

Expression of upd2 and upd3.

a upd2 and upd3, b expression levels in heads of animals with RNAi-mediated knockdown in EECs using voilá-GAL4 in combination with Tubulin-GAL80ts (voilá>) (N=6). Statistical analyses were performed using t-tests for panel a and b. Data are presented as mean ± SEM.

Impact of glial-specific dome knockdown on sleep patterns post oxidative stress and after sleep deprivation.

a-c, Daytime sleep measured over three days, starting with a normal diet, followed by a day with 1% H2O2-supplemented food to induce oxidative stress, and concluding with a return to a normal diet to assess recovery, in flies with UAS-RNAi constructs without dome knockdown (UAS-RNAi controls) and those with glial-specific dome knockdown (N=25-31). d, Sleep profiles during a night that included a 6-hour period of sleep deprivation, followed by a recovery phase, in control flies and those with glial-specific dome knockdown (N=17-24). e, Sleep quantity measured during the first 2 hours of the recovery period following sleep deprivation in control flies and those with glial-specific dome knockdown (N=17-24). Statistical tests: Kruskal-Wallis One-Way ANOVA with Dunn’s multiple comparison test for panels a, b, and e; Ordinary one-way ANOVA with Dunnett’s multiple comparison test for panel c. Data are presented as mean ± SEM. ns, non-significant (p>0.05)

AstA signaling from EECs promotes wakefullness.

a, AstA-R2 expression in heads of flies with knockdown of AstA-R2 in BBB glia using moody-GAL4 (moody>) compared to the control group (N=5). b, Sleep profiles over two consecutive days, encompassing one day under standard diet conditions followed by one day on oxidative stress conditions, induced by 1% H2O2-containing food, in animals with BBB glial-specific knockdown of AstA-R1 or AstA-R2 and controls (N=30-32). c, Daytime sleep durations over two consecutive days, encompassing one day under standard diet conditions followed by one day on oxidative stress conditions, induced by 1% H2O2-containing food, in animals with BBB glial-specific knockdown of AstA-R1 or AstA-R2 and controls (N=30-32). d, Sleep profiles and e, daytime sleep on standard food in animals with AstA knockdown in AstA+ EECs and UAS-RNAi controls (N=30-31). Statistical tests used: Unpaired t-tests for panel a; two-way ANOVA with Sidak’s multiple comparisons test was used for panel b; Unpaired t-tests for panel d. Data are presented as mean ± SEM.

Oligos used for cloning the upd2 and upd3 CRISPR constructs, with gRNA sequences indicated in bold.

List of qPCR primers