PK2-R1 is required for larval sleep control

(A) Sleep amounts in PK2-R1 or Oamb knock-out mutants. In this and the following panels, ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. *** p < 0.001 (Mann–Whitney U-test with Bonferroni correction). (B) Sleep amounts in larvae in which PK2-R1 neurons were silenced. *** p < 0.001 (Mann– Whitney U-test with Bonferroni correction). (C) The expression pattern of PK2-R12A GAL4 > UAS-Kir2.1::EGFP in the larval CNS. Similar results were obtained across three independent samples.

IPCs and Dilps negatively regulate larval sleep

(A) Triple labeling of PK2-R1 neurons expressing nuclear-localized RFP (magenta), Dilp3 neurons expressing nuclear-localized GFP (green), and anti-Dilp2 positive cells (blue). Top panels show signals in the larval brain and the VNC. The bottom panels show magnified images of the white-squared area in the top panels, where the cell bodies of the IPCs are located. Note that all IPCs labeled by Dilp3-GAL4 are also labeled by PK2-R12A-LexA. Similar results were obtained across five independent samples. (B) Triple labeling of PK2-R1 neurons expressing nuclear-localized RFP (magenta), Dilp5 neurons expressing nuclear-localized GFP (green), and anti-Dilp2 positive cells (blue). Similar results were obtained across five independent samples. (C) Effect of IPCs silencing on larval sleep. In this and the following panels, ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. *** p < 0.001, ** p < 0.01, * p < 0.05 (Mann–Whitney U-test with Bonferroni correction). (D) Sleep amounts in Dilp3 or Dilp5 null mutant larvae. *** p < 0.001, ** p < 0.01 (Mann–Whitney U-test with Bonferroni correction).

Hug neurons negatively regulate larval sleep

(A) Sleep amounts in Hug CRISPR-knock-out mutant larvae. In this and the following panels, ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. **** p < 0.0001 (Mann–Whitney U-test). (B) Effect of silencing Hug neurons on larval sleep amount. *** p < 0.001 (Mann–Whitney U-test with Bonferroni correction). (C) Larval sleep changes induced by optogenetic activation of Hug neurons. For each genotype, sleep duration in the 1-h light-ON period was normalized to that in the 1-h light-OFF phase. * p < 0.05 (Mann–Whitney U-test). (D) Larval sleep during thermogenetic activation of Hug neurons. *** p < 0.001 (Mann–Whitney U-test with Bonferroni correction). (E) Images of HugPC neurons and IPCs labeled by rCD2::RFP (magenta) and mCD8::GFP (green), respectively. The bottom panels show magnified images of the white-squared region in the top panels, in which HugPC neurons project their axons close to the cell bodies of the IPCs. While the top panels are z-stacks of 122 image slices (1 µm interval) covering the entire brain, the bottom panels are projections of 60 slices around the cell bodies of IPCs. Similar results were obtained across five independent samples. (F) Effect of silencing HugPC neurons on larval sleep. *** p < 0.001, ** p < 0.01 (Mann–Whitney U-test with Bonferroni correction).

Activation of Hug neurons triggers Ca2+ responses in larval IPCs

(A) Schematic flow of assessing intracellular Ca2+ levels in IPCs while performing the thermogenetic activation of Hug neurons. (B–C) The level of nuclear-localized CRTC::GFP signal, calculated as NLI (see the Methods section for a detailed explanation of NLI), in IPCs during Hug neurons stimulation ex vivo (B) or in vivo (C). In these panels, ‘N’ indicates the number of cells used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. **** p < 0.0001 (Mann–Whitney U-test). (D) The proposed neuronal connection that regulates larval sleep.

Hug peptides induce Ca2+ responses in larval IPCs via PK2-R1

(A) Schematic flow of assessing intracellular Ca2+ levels in IPCs and bath application of peptides. (B–C) Ca2+ responses in IPCs to ex vivo bath application of either glucose (B) or Hug peptides (C). In this and the following panels, ‘N’ indicates the number of cells used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. **** p < 0.0001, *** p < 0.001 (Mann–Whitney U-test with Bonferroni correction). (D) Representative images of larval IPCs after peptide application. Scale bars, 2 μm. (E) Anti-Dilp3 signal intensity measured within the cytosolic areas of IPCs. *** p < 0.001, * p < 0.05 (Mann–Whitney U-test with Bonferroni correction). (F–G) Ca2+ responses in IPCs of PK2-R1 knock-out mutants, measured following either glucose (F) or Hug peptide application (G). ** p < 0.01, NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction). (H) A model where Hug peptides, but not glucose, activate IPCs via the PK2-R1 receptor.

Distinct impacts of Hugin/PK2-R1 axis on wake/sleep control in larvae and adults

(A) Total sleep amounts in Hug knock-out mutant adults. NS: p ≥ 0.05 (Mann–Whitney U-test). In these and the following panels, ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. NS: p ≥ 0.05 (Mann–Whitney U-test). (B) Sleep amounts of adults in which HugPC neurons were silenced. *** p < 0.001, NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction). (C) Sleep amounts in Dilp3 or Dilp5 null mutant adults. *** p < 0.001 (Mann–Whitney U-test with Bonferroni correction). (D) Schematic flow of peptide application experiments using adult brains. (E–F) Ca2+ responses (E) and anti-Dilp3 signal intensity (F) in adult IPCs after Hug peptide application. ** p < 0.01, NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction).

A schematic model of the Hugin/PK2-R1/Dilps axis in larval and adult sleep regulation

Detailed experimental setup for analyzing larval sleep

(A) Schematic flow of automated larval sleep quantification. A second instar larva was placed in each of 24 wells, and all 24 wells were videotaped from above. For each well and frame, the image of larva was blurred and binarized to automatically locate the larva. Consecutive frames were then compared so that changes in pixel values were summed to estimate larval motion. This motion estimate was then used to classify every consecutive pair of frames as representing “active” or “inactive.” We found that inactivity across 12 consecutive frames meets the sleep criteria, as shown in Figure 1—figure supplement 2. (B) Schematic representation of the 24-well chamber used for larval sleep assays. (C) Flow of automated processes of larval motion detection and active/inactive state determination. For each well and frame, the image of larva was blurred and binarized. Consecutive frames were then compared so that changes in pixel values were summed to estimate larval motion. This motion estimate was then used to classify every consecutive pair of frames as representing “active” or “inactive.”

Larval “sleep state” in this study is consistent with behavioral criteria for sleep

(A) Relationship between “inactive” state duration and spontaneous arousal probability. Sustained quiescence for ≥ 12 frames (open circles) significantly reduced arousal probability compared to 1-frame inactivity. * p < 0.05, NS: p ≥ 0.05 (chi-squared test). (B) The percentage of larvae that showed a response within the next 10 frames after high-intensity blue light. The number of events analyzed was 36 and 60 with and without blue light irradiation, respectively. * p < 0.05 (Fisher’s exact test). (C) Sleep amounts before, during, and after blue light irradiation. The number of larvae tested was 21 and 21 for the control and experimental groups, respectively. ** p < 0.01, NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction). (D) Percentage of larvae that showed a response in the next 1 or 12 frames after blue light stimulation. Number of events analyzed: 1960 or 111 for 1- or 12-frame continuous inactivity prior to blue light illumination, respectively. **** p < 0.0001 (chi-squared test).

CRISPR-knock-out screen for genes that regulate larval sleep.

Total sleep amounts in CRISPR-knock-out mutants were measured for 18 h from the beginning of the second instar. The data for Control, PK2-R1, and Oamb mutants are identical to those shown in Figure 1. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. *** p < 0.005 (Kruskal–Wallis one-way ANOVA and post hoc Mann–Whitney U-test with Bonferroni correction). Number of larvae tested: 475 for the control and 10–52 for each mutant.

Effects of neuronal manipulation on larval sleep

Sleep amounts in larvae in which distinct neuronal populations were silenced. Kir2.1 expression was driven by each enhancer-GAL4 for “hit” genes identified in the CRISPR-knock-out screen. ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. *** p < 0.001, ** p < 0.01, * p < 0.05, NS: ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction).

Locomotion speeds are not consistently affected by genetical manipulations of PK2-R1, IPCs, or Dilps (A–B) Effects of silencing PK2-R1 neurons (A) and IPCs (B) on larval locomotion speed. (C) Larval locomotion speed in null mutants of Dilp3 or Dilp5. The average locomotion speed during the wake periods was measured from the data presented in Figures 1B, 2C, and 2D. In this and the following panels, ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. *** p < 0.0001, * p < 0.05, NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction).

Neither Hug knock-out mutation nor Hug neuron silencing significantly affects larval locomotion speed (A) Average locomotion speed of Hug knock-out mutants during waking periods. The data presented in Figures 3A and 3B were reanalyzed. In this and the following panels, ‘N’ indicates the number of biologically independent animals used for each group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. NS: p ≥ 0.05 (Mann–Whitney U-test). (B) Locomotion speed of larvae in which Hug neurons were silenced. NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction).

Generation of HugPC-LexA transgenic lines

(A–D) Labeling patterns of four HugPC-LexA strains in larval CNS visualized by rCD2::RFP. These transgenic lines were generated by injecting the identical plasmid into the following landing sites, respectively: su(Hw)attP5, VK00005, attP2, and attP40. Arrowheads indicate cell bodies of non-HugPC neurons. Filled and unfilled arrowheads indicate cell bodies within the VNC and near the protocerebrum, respectively.

D-glucose but not Hug peptide cause Dilp2 reduction in IPCs

(A–B) Anti-Dilp2 signal intensity measured in IPCs after glucose (A) or Hug-γ application (B). In this and the following panels, ‘N’ indicates the number of cells used for each group. Box plots are generated so that center line indicates median, box limits indicate upper and lower quartiles, and whiskers indicate the minimum-to-maximum range. **** p < 0.0001, * p < 0.05 (Mann–Whitney U-test). (C) Anti-Dilp3 signal intensity measured within the cytosolic areas of IPCs. NS: ≥ 0.05 (Mann–Whitney U-test).

Sleep patterns of adult flies following manipulations of Hugin and insulin pathways

(A) Experimental setup for monitoring adult sleep using the Drosophila Activity Monitor (DAM). (B) Sleep duration in adult CRISPR-knock-out mutants. These mutants showed significant changes in the sleep amounts in larvae (Figure 1—figure supplement 3). In panels B, C, and E, sleep amounts were calculated as the ratio relative to the median sleep amount of the control group. Box plots are generated so that the center line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers indicate the minimum-to-maximum range. *** p < 0.001, ** p < 0.01 (Kruskal–Wallis one-way ANOVA and post hoc Mann–Whitney U-test with Bonferroni correction). (C, E) Sleep amounts relative to the median value of the control group. Data presented in Figures 6B and 6C were reanalyzed. *** p < 0.001, * p < 0.05, NS: p ≥ 0.05 (Mann–Whitney U-test with Bonferroni correction). (D, F) Temporal sleep patterns over two consecutive days.

Morphology of PK2-R1 neurons, HugPC neurons, and IPCs in the adult brain

(A) Dual labeling of PK2-R1 neurons and IPCs expressing nuclear-localized RFP (stinger; magenta) and GFP (stinger; green), respectively. The top panels show signals in the entire adult brain. The bottom panels show magnified images of the white-squared area in each corresponding top panel, where the cell bodies of IPCs are located. Similar results were obtained across five independent samples. (B) Visualization of HugPC neurons and IPCs labeled by mCD8::GFP (magenta) and the anti-Dilp3 antibody (green), respectively. Similar results were obtained across four independent samples.