Ecdysone acts through cortex glia to regulate sleep in Drosophila
- Howard Hughes Medical Institute and Chronobiology and Sleep Institute, Perelman School of Medicine at the University of Pennsylvania, United States
- Department of Biology, University of Pennsylvania, United States
- Department of Pharmacology, Perelman School of Medicine at the University of Pennsylvania, United States
Figures
Figure 1
A screen of all nuclear hormone receptors (NHRs) in Drosophila identifies sleep-regulating functions of ecdysone receptor (EcR) and its downstream target, NHR E75.
(A) Flies carrying a pan-neuronal driver nSyb-GS or pan-glial driver Repo-GS were crossed with different UAS lines carrying RNAi constructs against NHRs, and their 5- to 7-day-old F1 female progeny …
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Figure 1—source data 1
Detailed sleep phenotypes of nuclear hormone receptor (NHR) RNAi screening lines.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig1-data1-v2.xlsx
Figure 2 with 4 supplements
Baseline sleep phenotypes resulting from pan-neuronal or pan-glial knockdown of ecdysone receptor (EcR).
(A, B) Show representative sleep traces of nSyb-GS>EcR RNAi #1 and Repo-GS>EcR RNAi #1. N = 14–16 per genotype. Data are based on at least three independent experiments. Representative sleep traces …
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Figure 2—source data 1
Sleep and circadian phenotypes resulting from pan-neuronal or pan-glial knockdown of ecdysone receptor (EcR).
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig2-data1-v2.xlsx
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Figure 2—source data 2
Sleep phenotypes resulting from pan-neuronal or pan-glial knockdown of E75.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig2-data2-v2.xlsx
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Figure 2—source data 3
Sleep phenotypes resulting from pan-neuronal or pan-glial knockdown of Hr51 and sleep phenotypes of adult-specific neuronal and glial knockdown of ecdysone receptor (EcR).
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig2-data3-v2.xlsx
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Figure 2—source data 4
Sleep phenotypes resulting from pan-glial knockdown of ecdysone receptor (EcR) by temporal and regional gene expression targeting (TARGET) system and different EcR RNAi lines.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig2-data4-v2.xlsx
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Figure 2—source data 5
Sleep phenotypes resulting from pan-neuronal or pan-glial knockdown of EcI and overexpression of ecdysone receptor (EcR) common isoforms.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig2-data5-v2.xlsx
Figure 2—figure supplement 1
Baseline sleep phenotypes resulting from pan-neuronal and pan-glial knockdown of E75, the direct downstream target of ecdysone receptor (EcR).
(A, B) show representative sleep traces of nSyb-GS>E75 RNAi #1 and Repo-GS>E75 RNAi #1. N = 12–16 per genotype per experiment. Experiments were independently repeated at least two times, and one …
Figure 2—figure supplement 2
Adult-specific neuronal and glial knockdown of ecdysone receptor (EcR) reduces sleep.
(A, B) show representative sleep traces of nSyb-GS>Hr51 RNAi and Repo-GS>Hr51 RNAi. N = 26–32 per genotype. Experiments were independently repeated two times, and one experiment is shown here. (C, D)…
Figure 2—figure supplement 3
Adult-specific knockdown of ecdysone receptor (EcR) by using the temporal and regional gene expression targeting (TARGET) system reduces sleep.
(A) Sleep traces resulting from pan-glial adult-specific EcR knockdown by using Repo-Gal4; TubulinGal80ts crossed with UAS-EcR RNAi #1. Five-day-old F1 progeny flies were maintained at 18 degrees …
Figure 2—figure supplement 4
Baseline sleep phenotypes resulting from pan-neuronal and pan-glial overexpression of ecdysone receptor (EcR) common isoforms and knockdown of EcI, the membrane importer of ecdysone.
(A, B) show representative sleep traces of nSyb-GS>EcI RNAi #1 and Repo-GS>EcI RNAi #1 flies, (C, D) Total sleep quantification of the nSyb-GS>EcI RNAi #1 and Repo-GS>EcI RNAi #1 flies and their …
Figure 3 with 2 supplements
Ecdysone feeding prevents sleep loss in response to starvation, while ecdysone receptor (EcR) KD exacerbates starvation-induced sleep loss.
Sleep was monitored for 3 days either under continuous feeding with and without 0.2 mM ecdysone (A) or under each of the following three conditions—continuous feeding for 3 days or with an …
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Figure 3—source data 1
Sleep phenotypes of starvation on wild-types and ecdysone receptor (EcR) disrupted flies.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig3-data1-v2.xlsx
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Figure 3—source data 2
Sleep phenotypes of ecdysone treatment and heat shock when ecdysone receptor (EcR) was disrupted in neurons and glial cells.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig3-data2-v2.xlsx
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Figure 3—source data 3
Ecdysone levels in both fly brain and periphery, and the expression levels of ecdysone responsive genes.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig3-data3-v2.xlsx
Figure 3—figure supplement 1
Ecdysone receptor (EcR) disruption in neurons or glia does not affect the response to heat shock.
(A) Total sleep of EcR RNAi #1 knockdown flies in both neurons and glia and their genetic control flies after 0.1 mM 20E feeding, N = 29–46, and experiments were repeated two or three times …
Figure 3—figure supplement 2
Ecdysone levels are higher in the fly periphery at ZT14 compared to ZT2.
(A) Ecdysone level was measured in the fly’s whole body or brain using an ELISA (enzyme-linked immunosorbent assay) kit. 20E levels are higher at ZT14 than ZT2 in the periphery. (B) A and B isoforms …
Figure 4
Ecdysone receptor (EcR) is expressed in cortex glia.
(A) EcR antibody DDA2.7 staining overlaps with reporter expression driven by cortex glia drivers (GMR77A03>mCD8 RFP and Np2222>mCD8 RFP), indicating that EcR is expressed in the cortex glia. EcR …
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Figure 4—source data 1
Quantification of Anti-EcR staining in the cortex glia layer.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Ecdysone receptor (EcR) functions in cortex glia to affect sleep.
(A) Multiple constitutive Gal4 drivers labeling different subglial populations were crossed to EcR RNAi flies. Only GMR77A03 for cortex glia (CG), Eaat-1 for astrocyte-like glia (ALG), and GMR56F03 …
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Figure 5—source data 1
Sleep phenotypes resulting from subglial knockdown of ecdysone receptor (EcR).
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig5-data1-v2.xlsx
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Figure 5—source data 2
Sleep phenotypes of adult-specific ecdysone receptor (EcR) disruption in different subglial populations.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
Adult-specific disruption of ecdysone receptor (EcR) in most subglial populations does not affect sleep.
Temperature-sensitive tubulin-Gal80ts were used to restrict the expression of Gal4 to adults. Sleep was monitored for 1 day at the permissive temperature of 18 degrees, then 2 days at the …
Figure 6 with 3 supplements
Lipid metabolism mediates the effects of 20E on sleep.
(A, B) Lipid droplet (LD) staining of representative brains from flies treated with vehicle or 0.5 mM 20E. A z-stack slice that shows the maximal structure of the cortex glia was selected, and …
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Figure 6—source data 1
The effects of ecdysone on lipid droplets and sleep.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig6-data1-v2.xlsx
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Figure 6—source data 2
Lipid droplets changes resulting from glial ecdysone receptor (EcR) knockdown.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig6-data2-v2.xlsx
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Figure 6—source data 3
Sleep phenotypes of gaboxadol treatment in lsd-2 mutant flies.
- https://cdn.elifesciences.org/articles/81723/elife-81723-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
EcR knockdown in glial cells resulted in more LDs.
(A, B) Lipid droplet (LD) staining of representative brains from UAS-EcR RNAi #1 control flies and Repo-GS>EcR RNAi #1 flies treated with vehicle or RU486. A z-stack slice that shows the maximal …
Figure 6—figure supplement 2
Gaboxadol promotes sleep in lsd-2 mutant flies.
A representative sleep trace of lsd-2 mutant flies with vehicle control or 0.1 mg/ml gaboxadol is shown in the left panel and quantification of all data points are shown on the right. N = 37–38 per …
Figure 6—figure supplement 3
Model for sleep function of ecdysone in cortex glia.
This schematic shows how ecdysone acts as a long-distance signal to affect sleep in cortex glia. Ecdysone synthesis genes are not expressed in the fly brain, suggesting that glial ecdysone comes …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w[*]; P{w[+mW.hs]=Switch2}GSG2326 | Bloomington Stock Center | BDSC:40990 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS-Eip78C.miRNA}attP16/CyO | Bloomington Stock Center | BDSC:44390 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS Hr3.miRNA}attP16 | Bloomington Stock Center | BDSC:44399 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS Hr96.miRNA}attP16 | Bloomington Stock Center | BDSC:44395 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS-Hnf4.miRNA}attP16/CyO | Bloomington Stock Center | BDSC:44398 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02546}attP2 | Bloomington Stock Center | BDSC:27258 | |
| Genetic reagent (D. melanogaster) | y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01620}attP2 | Bloomington Stock Center | BDSC:36729 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS Hr78.miRNA}attP16 | Bloomington Stock Center | BDSC:44393 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02545}attP2 | Bloomington Stock Center | BDSC:27242 | |
| Genetic reagent (D. melanogaster) | y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01316}attP2 | Bloomington Stock Center | BDSC:34329 | |
| Genetic reagent (D. melanogaster) | y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01951}attP2 | Bloomington Stock Center | BDSC:39032 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02537}attP2 | Bloomington Stock Center | BDSC:29373 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS02272}attP40 | Bloomington Stock Center | BDSC:41707 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS Hr83.miRNA}attP16 | Bloomington Stock Center | BDSC:44397 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS svp.miRNA}attP16 | Bloomington Stock Center | BDSC:44394 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS ERR.miRNA}attP16/CyO | Bloomington Stock Center | BDSC:44391 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS Hr38.miRNA}attP16/CyO | Bloomington Stock Center | BDSC:44396 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02738}attP2 | Bloomington Stock Center | BDSC:27659 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00019}attP2/TM3, Sb[1] | Bloomington Stock Center | BDSC:33625 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00018}attP2/TM3, Sb[1] | Bloomington Stock Center | BDSC:33624 | |
| Genetic reagent (D. melanogaster) | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02432}attP2 | Bloomington Stock Center | BDSC:27086 | |
| Genetic reagent (D. melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS Hr4.miRNA}attP16 | Bloomington Stock Center | BDSC:44392 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; PBac{y[+mDint2] w[+mC]=I-SceI(FRT.Rab9-GAL4.ATG(loxP.3xP3-RFP))}VK00033/TM3, Sb[1] | Bloomington Stock Center | BDSC:51587 | |
| Genetic reagent (D. melanogaster) | w;; Repo-Gal4, 6 x crossed to Iso | Bloomington Stock Center | BDSC:7415 | |
| Genetic reagent (D. melanogaster) | w[1118]; UAS-EcR.A.dsRNA/TM3 | Bloomington Stock Center | BDSC:9328 | |
| Genetic reagent (D. melanogaster) | w[1118]; P{w[+mC]=UAS EcR.B1.dsRNA}168 | Bloomington Stock Center | BDSC:9329 | |
| Genetic reagent (D. melanogaster) | w[*]; P{w[+mC]=UAS EcR.A.F645A}TP2 | Bloomington Stock Center | BDSC:9452 | |
| Genetic reagent (D. melanogaster) | y1 v1; P{TRiP.JF02257}attP2 | Bloomington Stock Center | BDSC:26717 | |
| Genetic reagent (D. melanogaster) | w[1118]; P{w[+mC]=hs-GAL4-EcR.LBD}SBM | Bloomington Stock Center | BDSC:23656 | |
| Genetic reagent (D. melanogaster) | w; UAS-GFP-Lsd2 | Michael Welte | ||
| Genetic reagent (D. melanogaster) | w;; UAS-EcI RNAi | Naoki Yamanaka | BDSC:37295 | |
| Genetic reagent (D. melanogaster) | w1118; P{GD1434}v44851 | VDRC Stock Center | VDRC:44851 | |
| Genetic reagent (D. melanogaster) | w1118; P{GD1428}v37058 | VDRC Stock Center | VDRC:37058 | |
| Genetic reagent (D. melanogaster) | w1118; P{GD1428}v37059 | VDRC Stock Center | VDRC:37059 | |
| Genetic reagent (D. melanogaster) | UAS-E75 RNAi GD | VDRC Stock Center | VDRC:44851 | |
| Genetic reagent (D. melanogaster) | UAS-E75 RNAi KK | VDRC Stock Center | VDRC:108399 | |
| Genetic reagent (D. melanogaster) | white Canton-S | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | nSyb-GS74 | Laboratory Stocks | PMID: 29590612 | |
| Genetic reagent (D. melanogaster) | Repo-GS11 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | Actin-GS | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w[*]; P{w[+mW.hs]=Switch2}GSG5961 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; 9–137 Gal4; TubGal80ts | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; MZ0708-Gal4; TubGal80ts | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; Eaat1-Gal4; TubGal80ts | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; TubGal80ts; NP222-Gal4 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; NP2222-Gal4 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; GMR85601-Gal4 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; Eaat1-Gal4;;, 6 x crossed to Iso | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; GMR56F03-Gal4 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; NP6293-Gal4, 6 x crossed to Iso | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; Alarm-Gal4 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; 9–137 Gal4; 6 x crossed to Iso | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; MZ0709-Gal4 | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; moody-Gal4, 6 x crossed to Iso | Laboratory Stocks | ||
| Genetic reagent (D. melanogaster) | w; TublinGal80ts/CYO; Repo-Gal4/TM6 | Laboratory Stocks | ||
| Antibody | EcR Antibody DDA2.7 (mouse monoclonal) | Developmental Studies Hybridoma Bank (DSHB) | DDA2.7 (EcR common) | 2 ug/ml |
| Sequence-based reagent | E74_FOR | This paper, Integrated DNA Technologies | PCR primers | TGA GAC GCG AGG AAT ACC CTG GAC |
| Sequence-based reagent | E74_REV | This paper, Integrated DNA Technologies | PCR primers | AAC TGC CAG CGT GTA GCC GTT TCC |
| Sequence-based reagent | E75A_FOR | This paper, Integrated DNA Technologies | PCR primers | TCA GCA GGC CAA TCT GCA CCA CTC |
| Sequence-based reagent | E75A_REV | This paper, Integrated DNA Technologies | PCR primers | TGA TGT ACT CGG GAG TCT GGG GAC |
| Sequence-based reagent | E75B_FOR | This paper, Integrated DNA Technologies | PCR primers | AGC AGC ACC AGC ACC AGC AAC AAC |
| Sequence-based reagent | E75B_REV | This paper, Integrated DNA Technologies | PCR primers | ATT GCC CGC ACT GGA GTT GCT CGA |
| Sequence-based reagent | EcI_FOR | This paper, Integrated DNA Technologies | PCR primers | TGC AGT GCC GCT CTC AAC TGT ACC |
| Sequence-based reagent | EcI_REV | This paper, Integrated DNA Technologies | PCR primers | TCA CAG TAA CCG TTG ACC GCC TCC |
| Sequence-based reagent | EcR_C_FOR | This paper, Integrated DNA Technologies | PCR primers | TCA ACC ACA GCC ACA GCT CCT TCC |
| Sequence-based reagent | EcR_C_REV | This paper, Integrated DNA Technologies | PCR primers | TGA TGG GTC CTA TGG CCG CAC TTC |
| Sequence-based reagent | EcR_1_FOR | This paper, Integrated DNA Technologies | PCR primers | GCG GCC AAG ACT TTG TTA AG |
| Sequence-based reagent | EcR_1_REV | This paper, Integrated DNA Technologies | PCR primers | GGC CAA CTG ATT GTA CGT TAA G |
| Sequence-based reagent | EcR_2_FOR | This paper, Integrated DNA Technologies | PCR primers | GCC ATC TGA AGA GGA TCT CAG |
| Sequence-based reagent | EcR_2_REV | This paper, Integrated DNA Technologies | PCR primers | AAC GCT GGT AGA CCT TTA GC |
| Commercial assay or kit | 20-Hydroxyecdysone EIA kit | Cayman | Item No. 501390 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | Item No. 74134 | |
| Chemical compound, drug | BODIPY 493/503 | Fisher | D3922 | 1 ug/ml |
| Chemical compound, drug | 20-Hydroxyecdysone | Sigma | H5142 | 0.5 mM |
| Chemical compound, drug | Gaboxadol | Sigma-Aldrich | T101 | 0.1 mg/ml |
| Software, algorithm | Clocklab | Actimetrics | https://actimetrics.com/ | |
| Software, algorithm | Adobe Illustrator 2020 | Adobe | https://www.adobe.com/ | |
| Software, algorithm | BioRender | BioRender | App.biorender.com | |
| Software, algorithm | Pysolo | Gilestro and Cirelli, 2009 | https://www.pysolo.net/about/ | |
| Software, algorithm | GraphPad Prism v9 | GraphPad Software | https://www.graphpad.com/ | |
| Software, algorithm | JTK_CYCLE | Hughes et al., 2010 | Hughes et al., 2010 | |
| Software, algorithm | DAMFileScan113 | Trikinetics | https://trikinetics.com/ |
