Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet‐Polycomb‐Octβ2R cascade
2021; Springer Nature; Volume: 22; Issue: 2 Linguagem: Inglês
10.15252/embr.201947910
ISSN1469-3178
AutoresZhangwu Zhao, Xianguo Zhao, Tao He, Xiaoyu Wu, Pengfei Lv, Alan Jian Zhu, Juan Du,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoArticle7 January 2021free access Source Data Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octβ2R cascade Zhangwu Zhao Zhangwu Zhao orcid.org/0000-0002-3006-7770 Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Xianguo Zhao Xianguo Zhao Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Tao He Tao He MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Xiaoyu Wu Xiaoyu Wu Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Pengfei Lv Pengfei Lv Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Alan J Zhu Alan J Zhu MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Juan Du Corresponding Author Juan Du [email protected] orcid.org/0000-0002-1850-3613 Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Zhangwu Zhao Zhangwu Zhao orcid.org/0000-0002-3006-7770 Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Xianguo Zhao Xianguo Zhao Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Tao He Tao He MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Xiaoyu Wu Xiaoyu Wu Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Pengfei Lv Pengfei Lv Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Alan J Zhu Alan J Zhu MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Juan Du Corresponding Author Juan Du [email protected] orcid.org/0000-0002-1850-3613 Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Author Information Zhangwu Zhao1, Xianguo Zhao1, Tao He2, Xiaoyu Wu1, Pengfei Lv1, Alan J Zhu2 and Juan Du *,1 1Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China 2MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China *Corresponding author. Tel: +86 15120098776; E-mail: [email protected] EMBO Reports (2021)22:e47910https://doi.org/10.15252/embr.201947910 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. In this study, we identify an epigenetic regulatory cascade (Stuxnet-Polycomb-Octβ2R) that modulates the octopamine receptor in Drosophila. We demonstrate that stuxnet positively regulates Octβ2R through repression of Polycomb in the ellipsoid body of the adult fly brain and that Octβ2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octβ2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octβ2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octβ2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis. SYNOPSIS An epigenetic regulatory mechanism regulates sleep homeostasis through an octopamine-responsive Stuxnet-Pc-Octβ2R cascade reducing Octβ2R receptor levels, thus avoiding unfavorable effects of octopamine on sleep. Octopamine-responsive Stuxnet-Polycomb-Octβ2R cascade regulates sleep through modulating octopamine reception in the ellipsoid body of Drosophila. The study clarifies diverse functions of multiple Octβ receptors in mediating octopamine signaling in different aspects of sleep. Octopamine-activated Octβ1R induces the downregulation of stuxnet, which reduces Octβ2R transcription in the presence of octopamine. Introduction Drosophila has been used as a model system to study mechanisms of sleep regulation. The first studies on sleep in Drosophila revealed that they periodically enter a quiescence state that meets a set of criteria for sleep (Hendricks et al, 2000; Shaw et al, 2000). Drosophila sleep is monitored normally by a Drosophila activity monitoring system (DAMS) and is defined as immobility for 5 min or longer which is a sleep bout. Drosophila sleep mainly happens at night, while a period of siesta is in the mid-day. For example, total sleep time is around 380 min (male) and 250 min (female) during the day time, and 480 min (male) and 490 min (female) during the night time in w1118. In Drosophila, central complex structures, especially the ellipsoid body (EB) and fan-shaped body (FSB), are important for sleep homeostasis regulation. Activation of dorsal FSB neurons is sufficient to induce sleep (Donlea et al, 2011). The dorsal FSB also integrates some sleep inhibiting signals (Liu et al, 2012). Both dorsal FSB and EB ring 2 are important in sleep homeostasis (Donlea et al, 2014; Liu et al, 2016; Pimentel et al, 2016). Recently, the helicon cells were found to connect the dorsal FSB and EB Ring 2 (Donlea et al, 2018), indicating that these EB and FSB are connected. Multiple studies indicate that the epigenetic mechanisms are involved in circadian regulation (Etchegaray et al, 2003; Doi et al, 2006; Nakahata et al, 2008; Valekunja et al, 2012; Aguilar-Arnal & Sassone-Corsi, 2015; Tamayo et al, 2015; Xu et al, 2016). However, a direct link between epigenetic regulation and sleep homeostasis is not yet established. Octopamine (OA) in Drosophila is a counterpart of vertebrate noradrenaline. Previous studies in Drosophila showed that OA is a wake-promoting neurotransmitter and plays an important role in regulating both sleep amount and sleep homeostasis. The mutants of the OA synthesis pathway show an increased total sleep (Crocker & Sehgal, 2008). Activation of OA signaling inhibits sleep homeostasis (Seidner et al, 2015), while in OA synthesis pathway mutants, an enhanced sleep homeostasis is observed (Crocker & Sehgal, 2008). Study of the neural circuit responsible for the sleep/wake effect of OA showed that octopaminergic ASM neurons (Busch et al, 2009) project to the pars intercerebralis (PI), where OAMB (one of the OA receptors)-expressing insulin-like peptide (ILP)-secreting neurons act as downstream mediators of OA signaling (Crocker et al, 2010). However, the effects of manipulating ASM neurons or ILP-secreting neurons are much weaker than those observed by manipulating all OA secreting neurons (Dubowy & Sehgal, 2017). Moreover, the effect of octopamine is not completely suppressed in the OAMB286 mutant (Crocker et al, 2010), arguing that another receptor or circuit may participate in this process. Eight OA receptors are identified to date: OAMB, Octβ1R, Octβ2R, Octβ3R, TAR1, TAR2, TAR3, and Octα2R (Qi et al, 2017). Although the expression pattern of OA is identified (Busch et al, 2009), the endogenous expression profile of these receptors is lacking (El-Kholy et al, 2015). A previous study demonstrated that the mushroom body-expressed OAMB mediates the sleep:wake effect of OA (Crocker et al, 2010). Recently, Octβ2R was shown to be important for the OA effect on endurance exercise adaptation (Sujkowski et al, 2017). How the versatility of OA function is mediated by the diverse array of its receptors needs further study. Moreover, the upstream regulatory mechanisms of OA receptors are still unknown. In a previous study, we showed that Stuxnet (Stx) is important in mediating Polycomb (Pc) protein degradation in the proteasome (Du et al, 2016). Stx, which is an ubiquitin like protein, mediates Polycomb (Pc) protein degradation through binding to the proteasome with a UBL domain at its N terminus and to Polycomb through a Pc-binding domain. Stx level changes result in a series of homeotic transformation phenotypes. Pc is an epigenetic regulator functioning in Polycomb Group (PcG) Complexes. Although it is reported that PcG component E(Z) is involved in circadian regulation (Etchegaray et al, 2006), the role of stx in adult physiological process is unknown. In this study, we identified the role of the epigenetic regulator Stx in sleep regulation. We found that Stx positively regulates Octβ2R through regulation of Polycomb in the EB of the adult fly brain. Further study demonstrated that the Stuxnet-Polycomb-Octβ2R cascade plays an important role in sleep regulation. In order to elucidate the role of this Stuxnet-Polycomb-Octβ2R cascade in sleep regulation, we systematically identified the role of various Octβ receptors in sleep regulation. We found that Octβ2R was one of the receptors that mediates OA function in sleep homeostasis. More interestingly, we found that stx was OA-responsive depending on the Octβ1R. Based on our data, we propose that the Stuxnet-Polycomb-Octβ2R cascade provides a feedback mechanism for OA signals to the EB to regulate sleep homeostasis and sleep amount. Results Stuxnet (stx) is involved in sleep regulation in adult Drosophila In a Drosophila genetic screen for sleep regulators, we found that mutation of stx leads to increased sleep. We tested this with two different alleles—stxd77, which was generated by imprecise P element excision (Du et al, 2016) (Fig EV1A), and stx34, which was made by CRISPR-Cas9-mediated deletion of exon 3 and exon 4 in the stx gene region (Fig EV1B). The hemizygous males of both stx34 and stxd77 show increased total daytime sleep, caused by increases of sleep bout duration and decreases of sleep bout number (Fig 1A–D and F–I). Although the total sleep at nighttime is not significantly different compared with control, the sleep quality is significantly improved with increases of sleep bout duration and decreases of sleep bout number (Fig 1A–D and F–I). These results indicate that Stx is a negative regulator of sleep. As stx has a role in development, we used the Gal80ts system (McGuire et al, 2003) to dissect whether the sleep defect is due to a stx effect on development or on adult fly neurons. After induction of stx RNAi before or after eclosion, we found that knockdown of stx in the adult fly is sufficient to cause the sleep phenotype (Fig EV1C and D), while knockdown of stx only before eclosion does not (Fig EV1E and F). These results show that the stx function on sleep is due to its effects on adult fly neurons. Click here to expand this figure. Figure EV1. Verification of stx mutant phenotypes A, B. RT–PCR shows that the RNA level of stx is downregulated in stxd77 and stx34 alleles (For A, mean ± SEM: w1118, 0.8117 ± 0.110, N = 3; stxd77/Y, 0.0183 ± 0.004, N = 3; For B, mean ± SEM: canton-S, 1.073 ± 0.0730, N = 3; stx34/Y, 0.1213 ± 0.0715, N = 3). C–F. Tub-Gal80ts is introduced to stx-Gal4;; stx RNAi to conditionally knockdown stx. (C, D) Knockdown of stx at adult stage only. Fly cross is kept at 18°C until the behavior test at 29°C. (C) 24-h sleep curve, dark bar and white bar indicate the nighttime and daytime, respectively. n = 16 for each group. The y-axis is average sleep time in every 30 min. (D) Total sleep is shown significantly increased in knocking down of stx during the daytime (From left to right: mean ± SEM: 410.4 ± 10.90, n = 92; 393.7 ± 11.19, n = 132; 359.6 ± 11.26, n = 104; 507.7 ± 8.986, n = 116; 503.1 ± 8.174, n = 92; 406.9 ± 12.66, n = 132; 500.6 ± 10.88, n = 104; 431.8 ± 10.48, n = 116). (E-F) Knockdown of stx at developmental stage only. Fly cross is kept at 29°C until the behavior test at 18°C. (E) 24-h sleep curve, dark bar and white bar indicate nighttime and daytime, respectively. n = 16 for each group. The y-axis is average sleep time in every 30 min. (F) Total sleep is not significantly changed by knockdown of stx (From left to right: mean ± SEM: 528.5 ± 10.57, n = 89; 495.9 ± 10.76, n = 75; 460.5 ± 20.35, n = 50; 510.9 ± 14.55, n = 53; 562.1 ± 8.145, n = 89; 616.9 ± 6.692, n = 75; 594.4 ± 10.79, n = 50; 591.0 ± 8.757, n = 53). G–J. Sleep deprivation test of stx mutants. (G, H) 12-h mechanical day sleep deprivation is performed in stxd77 and w1118 control (For G, from left to right: mean ± SEM: 18.05 ± 10.05, n = 63; 300.5 ± 19.28, n = 63; 351.9 ± 16.48, n = 63; 14.96 ± 5.13, n = 76; 379.0 ± 16.87, n = 76; 469.3 ± 15.87, n = 76; For H, from left to right: mean ± SEM: 47.69 ± 11.42, n = 68; 84.73 ± 11.24, n = 81). (I, J) 12-h mechanical day sleep deprivation was performed in stx34 and canton-S control (For I, from left to right: mean ± SEM: 86.31 ± 20.29, n = 35; 484.8 ± 19.75, n = 35; 533.8 ± 12.94, n = 35; 128.1 ± 22.33, n = 53; 473.2 ± 12.31, n = 53; 564.4 ± 11.14, n = 53; For J, from left to right: mean ± SEM: 46.14 ± 19.26, n = 35; 91.15 ± 10.69, n = 53). (G, I) Increased sleep amount in the recovery sleep comparing to the baseline. The black bar indicates the day sleep amount of the sleep deprivation day, the blue bar indicates the night sleep amount of previous day, and the green bar indicates the night sleep amount after the deprivation. (H, J) Value of changed sleep recovery which is calculated by the subtraction of the day sleep amount after the deprivation (Recovery) by the day sleep amount of previous day (Baseline). K, L. Group activity and the wake activity of stxd77 (K) and stx34 (L) (For K, group activity samples size: For K, group activity samples size: w1118, n = 16; stxd77/Y, n = 16; waking activity mean ± SEM: w1118, 1.543 ± 0.101, N = 3; stxd77/Y, 1.680 ± 0.253, N = 3. For L, group activity samples size: canton-S, n = 16; stx34/Y, n = 16; waking activity mean ± SEM: canton-S, 1.146 ± 0.127, N = 3; stx34/Y, 0.737 ± 0.100, N = 3). M, N. Climbing test of stxd77 and stx34. The climbing speed of control and stx mutants flies show no significant differences (From left to right mean ± SEM: 1.250 ± 0.070, N = 6; 1.133 ± 0.101, N = 6; 1.820 ± 0.053, N = 6; 1.847 ± 0.068, N = 6). (N) Percentage of control and stx mutants flies that climb 6 cm in 5 s show no significant differences (From left to right mean ± SEM: 91.05 ± 3.421, N = 4; 87.48 ± 4.507, N = 4; 75.25 ± 4.661, N = 4; 75.00 ± 5.401, N = 4). O. In stx-Gal4 line, stx is up-regulated (mean ± SEM: w1118, 0.953 ± 0.047, N = 3; stx-Gal4/Y, 1.327 ± 0.074, N = 3). Data information: Bar graphs are presented as mean ± SEM. (A–B, H, J–O) Statistical differences were measured using unpaired Student's t-test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, and ***P < 0.001. (D, F) Statistical differences were measured using one-way ANOVA, Tukey's multiple comparison test, n.s. indicates no significant difference, **P < 0.01, and ***P < 0.001. (G, I) Statistical differences were measured using non-parametric test with two-tailed Mann–Whitney test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, and ***P < 0.001. n indicates the number of tested flies; N indicates the number of biological repetitions (For A, B, and O, each repeat with a sample size of 30 individual fly heads). All the P-values are listed in Table EV3. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Stuxnet (stx) is involved in sleep regulation in adult Drosophila A–E. Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. (A) 24-h sleep curve (y-axis was average sleep time in every 30 min), n = 16. (B) Total sleep (From left to right mean ± SEM: 275.8 ± 17.08, n = 47; 358.6 ± 23.47, n = 39; 517.0 ± 16.49, n = 47; 519.7 ± 15.89, n = 39). (C) Sleep bout duration (From left to right mean ± SEM: 13.04 ± 1.287, n = 47; 18.01 ± 2.212, n = 39; 66.70 ± 9.569, n = 47; 59.41 ± 6.909, n = 39). (D) Number of sleep bouts (From left to right mean ± SEM: 23.61.5 ± 0.882, n = 47; 25.95 ± 1.353, n = 39; 12.64 ± 0.713, n = 47; 12.44 ± 0.888, n = 39). (E) sleep latency (From left to right mean ± SEM: 49.00 ± 2.041, N = 4; 33.09 ± 5.100, N = 4). F–J. Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. (F) 24-h-sleep curve, n = 16. (G) Total sleep (From left to right mean ± SEM: 359.0 ± 12.91, n = 64; 444.5 ± 10.98, n = 74; 478.9 ± 10.76, n = 64; 458.1 ± 13.98, n = 74). (H) Sleep bout duration (From left to right mean ± SEM: 21.01 ± 1.312, n = 64; 37.63 ± 2.988, n = 74; 58.81 ± 5.202, n = 64; 61.0 ± 6.555, n = 74). (I) Number of sleep bouts (From left to right mean ± SEM: 20.23 ± 0.627, n = 64; 16.38 ± 0.693, n = 74; 11.66 ± 0.605, n = 64; 11.93 ± 0.556, n = 74). (J) Sleep latency (From left to right mean ± SEM: 52.20 ± 5.496, N = 7; 22.76 ± 2.621, N = 9). K–N. Sleep deprivation test of stx mutants. (K, L) 12-h mechanical night sleep deprivation was performed in stx34 and Canton-S control. (For K, from left to right mean ± SEM: 34.53 ± 9.85, n = 53; 334.2 ± 14.34, n = 53; 378.8 ± 14.20, n = 53; 108.8 ± 20.73, n = 47; 361.1 ± 16.71, n = 47; 450.8 ± 14.84, n = 47; for L, from left to right mean ± SEM: 45.60 ± 13.43, n = 53; 104.9 ± 17.70, n = 47). (M, N) 12-h mechanical night sleep deprivation was performed in stxd77 and w1118 control. Sleep amount calculation of deprived night sleep (Deprivation), the day sleep amount of previous day (Baseline), and the day sleep amount after the deprivation (Recovery; For M, from left to right mean ± SEM: 35.06 ± 10.76, n = 50; 370.8 ± 16.07, n = 50; 434.5 ± 17.42, n = 50; 59.36 ± 11.59, n = 53; 420.3 ± 15.60, n = 53; 523.9 ± 15.12, n = 53; For N, from left to right mean ± SEM: 61.86 ± 13.35, n = 50; 104.0 ± 11.86, n = 53) (K, M). Increased sleep amount in the recovery sleep comparing to the Baseline (L, N), which was calculated by the subtraction of the day sleep amount after the deprivation (Recovery) by the day sleep amount of previous day (Baseline). Data information: Bar graphs are presented as mean ± SEM. (B–D, G–I, K, M) Statistical differences were measured using non-parametric test with two-tailed Mann–Whitney test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001. (E, J, L, N) Statistical differences were measured using unpaired Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. n indicates the number of tested flies, N indicates the number of biological repetitions. Horizontal white and black boxes along the x-axis indicate light and dark periods under LD, respectively. All the P-values are listed in Table EV3. Source data are available online for this figure. Source Data for Figure 1 [embr201947910-sup-0008-SDataFig1.xls] Download figure Download PowerPoint In addition, we found that the sleep latency is reduced in stx mutants (Fig 1E and J), indicating an increased sleep pressure in the mutants. In the sleep deprivation test, stx mutants still have a significant sleep recovery. Further analysis showed that both of the stx mutants have significant increases on the sleep recovery after sleep deprivation during the nighttime (Fig 1K–N) and during the day time (Fig EV1G–J), indicating an increased sleep pressure when stx function is defective. These results suggest that stx plays a critical role on sleep homeostasis. Both of the stx mutants are verified to have normal activity by the group activity profile, wake activity (Fig EV1K and L), and climbing test (Fig EV1M and N). In order to identify the expression pattern of stx gene, we did antibody staining in adult fly brains. The validity of the antibody was proven by the detection of the endogenous stx in hh-Gal4, stx RNAi fly wing disk. We found that Stx staining is significantly down in the RNAi compartment (Fig EV2A–D). In addition, Stx is colocalized at least partially with DAPI staining (Fig EV2E–H). In the adult fly brain, stx is expressed in the central complex neurons in the EB (Fig 2A–D′) and in most of the neuronal perikarya (Fig 2A′–D′ and E′–H′). The specificity of the brain staining is verified by loss of most of the signal in stx mutants (Fig EV2I–K). Click here to expand this figure. Figure EV2. Stx antibody verification and rescue experiment A–D. The specificity of the Stx antibody is tested in Drosophila wing disk. The genotypes used is hh-Gal4, UAS-rfp/UAS-stx RNAi. Scale bar: 50 μm. E–H. The nuclear localization is shown in the wing disk by colocalization with DAPI. Scale bar: 25 μm. I. Western blot shows that the stxd77 still has a shorter form of Stx which could be detected by the antibody. Tubulin was blotted on separate gel using the same sets of samples and loading to show the quality of the samples. J, K. The specificity of Stx antibody is tested in adult fly brain by staining in stx34 mutants and controls. Data present are merged images of multiple optical sections that have signals. Scale bar: 100 μm. L. stx overexpression results in sleep phenotype. Sleep phenotypes are verified in female flies (For total sleep, from left to right mean ± SEM: 211.0 ± 14.34, n = 52; 293.0 ± 14.36, n = 52; 146.8 ± 16.20, n = 54; 437.9 ± 15.24, n = 52; 493.9 ± 16.00, n = 52; 446.9 ± 19.35, n = 54. For sleep bout duration, from left to right mean ± SEM: 15.39 ± 2.760, n = 52; 19.16 ± 4.531, n = 52; 7.505 ± 0.756, n = 54; 70.23 ± 10.080, n = 52; 83.87 ± 15.420, n = 52; and 94.05 ± 12.450, n = 54. For number of sleep bouts, from left to right mean ± SEM: 18.13 ± 0.991, n = 52; 21.15 ± 0.763, n = 52; 18.27 ± 1.149, n = 54; 12.30 ± 0.893, n = 52; 14.66 ± 1.135, n = 52; 11.35 ± 1.022, n = 54). Data information: Bar graphs are presented as mean ± SEM. (L) Statistical differences were measured using one-way ANOVA, Tukey's multiple comparison test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, and ***P < 0.001. n indicates the number of tested flies. Horizontal white and black boxes along the x-axis indicate light and dark periods under LD, respectively. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. Characterization of stx-expressing neurons A–D′. Colocalization of Stx represented by antibody staining with EB1-Gal4. Scale bar: 10 µm. E–H′. Colocalization of stx antibody staining with stx-Gal4 expression pattern. I. stx-Gal4 expression pattern shown by crossing with UAS-mCD8GFP. J–O. Dendrite (RFP) and axon (GFP) pattern of stx-expressing neurons. The dendrite is highlighted by Denmark (RFP), and the axons are highlighted by syt-eGFP. P, Q. Rescue experiment of stx mutant. The stx mutant phenotype can be rescued by stx overexpression driven by stx-Gal4 (in female flies, P) or EB1-Gal4 (Q). For P, from left to right females mean ± SEM: 226.0 ± 11.76, n = 49; 336.6 ± 14.10, n = 86; 382.7 ± 16.43, n = 50; 357.8 ± 16.55, n = 72; 296.2 ± 16.04, n = 68; 484.9 ± 12.97, n = 49; 507.6 ± 13.83, n = 86; 539.5 ± 12.99, n = 50. 577.0 ± 11.34, n = 72; 520.7 ± 14.96, n = 68; For Q, from left to right mean ± SEM; 398.3 ± 9.535, n = 63; 378.4 ± 23.78, n = 32; 324.4 ± 12.33, n = 45; 495.8 ± 16.43, n = 28; 478.5 ± 14.65, n = 53; 328.7 ± 16.99, n = 34; 540.7 ± 7.690, n = 63; 489.9 ± 21.39, n = 32; 490.9 ± 13.65, n = 45; 494.7 ± 11.99, n = 28; 537.8 ± 12.07, n = 53; 450.3 ± 15.47, n = 34. R. Colocalization of stx-Gal4 with EB1-Gal4. Scale bar showed was 10 µm. Data information: Bar graphs are presented as mean ± SEM. (P, Q) Statistical differences were measured using one-way ANOVA, Tukey's multiple comparison test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, and ***P < 0.001. n indicates the number of tested flies. Horizontal white and black boxes along the x-axis indicate light and dark periods under LD, respectively. Source data are available online for this figure. Source Data for Figure 2 [embr201947910-sup-0009-SDataFig2.rar] Download figure Download PowerPoint The expression pattern of stx-Gal4-driven fluorescent proteins indicates that stx-Gal4 (BL:62766) (Gohl et al, 2011) recapitulates the central complex part of stx expression patterns. With nuclear-localized RFP driven by stx-Gal4, we identified a portion of Stx-positive neurons that colocalized with RFP (Fig 2E–H′), and the position of these neurons indicates that they are colocalized in the cell body of EB neurons (Fig 2A–D′). Consistently, strong signals are detected in EB by stx-Gal4 driven UAS-mCD8GFP (Fig 2I). This evidence indicates that stx-Gal4 drives expression in a part of the EB neurons. In order to further characterize stx-expressing neurons, we used stx-Gal4 to drive expression of Denmark (Nicolaï et al, 2010) and Syb (Zhang et al, 2002) to mark axons and dendrites of these neurons, respectively. Results show that the EB and olfactory lobe are filled with axons and dendrites of these neurons (Fig 2J–L). The FB is mostly composed of axons (Fig 2M–O), while the dendrites are found outside of the EB (Fig 2J–L). In order to validate the function of stx in sleep regulation, we performed overexpression of stx and rescue experiments. stx expression shows an increase in the stx-Gal4 line (Fig EV1O). Overexpression of stx driven by stx-Gal4 results in sleep decrease (Fig EV2L). Over regulation of stx driven by EB1-Gal4 results in sleep decrease (Fig 2Q). Overexpression of stx driven by both stx-Gal4 and EB1-Gal4 driving expression in the EB Ring 2 (R2) (Young & Armstrong, 2010) rescues the stx mutant phenotype (rescue percentage was 36.5% in stx-Gal4, 135.5% in EB1-Gal4, Fig 2P and Q). By labeling the nucleus of the EB1-Gal4-expressing neurons using the nuclear-localized UAS-Red stinger, we found that a portion of Stx-positive neurons colocalized with RFP (Fig 2A–D′). Consistently, in the colocalization experiment by crossing stx-lexA;; EB1-Gal4 with LexAOP-FLP; UAS > stop > GFP, we found that stx-LexA and EB1-Gal4 colocalized in the EB R2 (Fig 2R). This evidence demonstrates that stx mainly functions in the EB R2. Stx regulates sleep through stx-Polycomb-Octβ2R cascade Stx was previously shown as a Pc stability control factor in developmental processes of various tissues (Du et al, 2016). In this study, we found that Pc is also a sleep regulator. In the PcXT109 mutant, total daytime sleep amount and daytime sleep duration are downregulated, while daytime sleep number is up-regulated (Fig 3A–C). In order to determine the molecular mechanisms of stx function on sleep regulation, we tested whether stx regulation on sleep is dependent on Pc. Results showed that removing one copy of Pc could rescue the sleep phenotype of stxd77 hemizygous mutants (percentage of rescue for stxd77 was 128.6% for total day sleep, 109.6% for day sleep bout duration, 140.4% for day sleep bout number, 29.86% for sleep latency, Fig 3A–D), indicating that Pc is downstream of stx in the sleep regulation pathway. This is consistent with the previous finding that Stx stabilize Pc through proteasome-dependent pathway (Du et al, 2016). Figure 3. Stx regulates sleep through regulating Pc downstream targets Octβ2R A–E. Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. (A) Total sleep (Fr
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