Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP–PKA signaling

Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain’s neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. Here, we address the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. We show that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep.

Supplementary Table 4 Supplementary Table 5 Supplementary Table 6 Supplementary Figure 1. RNAi screening of the MB LD cyclers for sleep a. Boxplot of the daily total sleep of female flies expressing an RNAi transgene with the OK107 driver. The center lines indicate the median, box boundaries are 25th and 27th percentiles, and the whiskers extend to the minimum and maximum values. The total amount of sleep per day was compared with OK107/+ by The Kruskal-Wallis one-way ANOVA with Dunnett's multiple comparisons test (*p < 0.05, * * p < 0.01, * * * * p < 0.0001; n = 21-46 flies per group). b. Results of the screening of the MB LD cyclers for sleep. The boxplot represents the total sleep amount as in a. Each gene was knocked down with the OK107 driver. No significant change in total sleep amount was detected by ANOVA with Dunnett's multiple comparisons test. Source data are provided as a Source Data file.

Supplementary Figure 3. Inhibition of Nf1
or Pka-C1 in the MB does not affect locomotor activity a. Double-plotted actograms of flies of indicated genotypes. Group average activities of virgin female flies (n= 28-32 per genotype) during 5 days in DD and 2 days in DD are shown. White and black boxes indicate light (white) and dark (black) period in 12h:12h-LD cycles, and gray shades indicate the period of darkness. b. Mean activity counts during wake period in LD in the virgin female flies of indicated genotypes. The center lines of the box plots indicate the median, box boundaries are 25th and 27th percentiles, and the whiskers represent the minimum and maximum values. n=28-32 per group. *p < 0.05, ***p < 0.001, ****p < 0.0001 by the Kruskal-Wallis one-way ANOVA with Dunn's multiple comparisons test. Source data are provided as a Source Data file.

Supplementary Figure 4. Ras hyperactivation in the adult MB
Sleep profiles, amount, and structure of MB-GS > Ras v12 and MB-GS > mCD8:GFP flies fed with RU486 (+RU486) or those without RU486 (-RU486) during adulthood (n = 30 per group). MB-GS > Ras v12 (+RU486) was compared with other groups by the two-way ANOVA with Dunnett's multiple comparisons test. *p < 0.05, * * p <0.01, * * * p < 0.001, and * * * * p < 0.0001). The center lines of the box plots indicate the median, box boundaries are 25th and 27th percentiles, and the whiskers represent the minimum and maximum values. a. Sleep per 30-min bin averaged over 5 days of LD. b. Total sleep duration. c. Number of sleep episodes. Data are mean ± SEM. d. Maximum sleep episode duration. e. Mean sleep episode duration. Source data are provided as a Source Data file.

Supplementary Figure 5. NF1 and PKA-C1 mediate calcium rhythms in the MB
Relative calcium levels in the MB lobes are monitored by expressing CaLexA-GFP with 30Y-GAL4 together with UAS-Nf1 KK or UAS-Pka-C1 KK . Quantification of GFP intensity across all MB lobes in LD with Nf1 RNAi (a), in LD with Pka-C1 RNAi (b), in DD with Nf1 RNAi (c) and in DD with Pka-C1 RNAi (d). n= 40-50. The center lines of the box plots indicate the median, box boundaries represent the 25th and 75th percentiles, and the whiskers represent the minimum and maximum values. Stars indicate statistical significance levels comparing values between timepoints using the non-parametric Kruskal-Wallis one-way ANOVA with Dunn's multiple comparison's test (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). arb. units, arbitrary units. Source data are provided as a Source Data file.

Supplementary Table 1. Gene Ontology analysis of the MB LD-DD cyclers
A total of 117 MB LD-DD cyclers were subjected to the GO term enrichment analysis using the GOrilla tool. GO

Supplementary Table 2. Enriched transcription factor binding sites in the promoters of the MB LD-DD cyclers
Top 30 transcription factor (TF) binding sites over-represented in the promoter regions of the MB LD-DD cycling genes, sorted by the Z-score.
Significantly enriched TF binding sites that fulfil both a Z-score > 5.5 and a Fisher score > 0.5 are marked in bold.

Supplementary Table 3. Predicted targets of circadian-relevant miRNAs among the MB LD-DD cyclers
miRNAs known to be expressed rhythmically in the fly brain are marked in bold.