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. 2023 Jan 31;12:e81723. doi: 10.7554/eLife.81723

Figure 5. 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 for ensheathing glia (EG) drivers produced viable adult progeny flies, and only GMR77A03>EcR RNAi #1 flies showed reduced total sleep compared with control flies. Green chart columns are experimental groups, and neighboring black and gray columns are Gal4 and UAS flies controls, respectively. N = 16–20 per genotype. (B) Overexpression of EcR by subglial Gal4 drivers—9-137 Gal4 to drive expression in the surface glia (SG), Moody-Gal4 for subperineurial glia (SPG), GMR85G01 for perineurial glia (PG), MZ008-Gal4 for ensheathing glia (EG), GMR77A03 for cortex glia (CG), and Eaat-1 for astrocyte-like glia (ALG). Only overexpression of EcR in the surface glia promotes sleep. N = 16–24 per genotype. (C) Representative sleep traces of the cortex glia GMR77A03>EcR RNAi #1 flies. (D) Gal4/Tubulin-gal80ts was used to achieve adult-specific knockdown of EcR in different subglial populations. Under permissive temperature, Gal80ts inhibits Gal4 activation of UAS, but under restrictive temperature, Gal80ts is inactivated, and genes under the regulation of UAS are expressed. (E, F) Sleep traces resulting from EcR knockdown in the cortex glia using NP2222-Gal4/tubulinGal80ts and surface glia using 9-137 Gal4/tubulinGal80ts. F1 progeny flies were kept at 18 degrees for 1 day, and then the temperature was switched to 31 degrees to inactivate the Gal80ts and thus achieve knockdown of EcR over the following 2 days. Subsequently, temperatures were decreased back to 18 degrees. (G, H) show quantification of total sleep of all EcR knockdown flies in the cortex glia using NP2222-Gal4/tubulinGal80ts and surface glia using 9-137 Gal4/tubulinGal80ts, N = 34–77 per genotype. Total sleep of each genotype was calculated and compared to controls for the above 4 days. Bar graphs show mean ± standard error of the mean (SEM), ns = not significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p values for each comparison were calculated by one-way analysis of variance (ANOVA) with Tukey post hoc test. See also Figure 5—source data 1.

Figure 5—source data 1. Sleep phenotypes resulting from subglial knockdown of ecdysone receptor (EcR).
Figure 5—source data 2. Sleep phenotypes of adult-specific ecdysone receptor (EcR) disruption in different subglial populations.

Figure 5.

Figure 5—figure supplement 1. Adult-specific disruption of ecdysone receptor (EcR) in most subglial populations does not affect sleep.

Figure 5—figure supplement 1.

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 restrictive temperature of 31 degrees data, and then for another day at 18 degrees. EcR knockdown (RNAi) and overexpression (OE) were driven by: (A, B) a cortex glia driver (NP2222>EcR RNAi #1 and NP2222>EcR OE); (C, D) a surface glia driver (9-137>EcR RNAi #1 and 9-137>EcR OE). (E, F) An astrocyte-like glia driver (Eaat-1>EcR RNAi #1 and Eaat-1>EcR OE). (G, H) An ensheathing glia driver (MZ0709>EcR RNAi #1 and MZ0709>EcR OE). Bar graphs show mean + standard error of the mean (SEM); ns = not significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p values for each comparison were calculated by one-way analysis of variance (ANOVA) with the Tukey post hoc test. See also Figure 5—source data 2.