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. 2021 Dec 15:10:e65437.
doi: 10.7554/eLife.65437.

insomniac links the development and function of a sleep-regulatory circuit

Qiuling Li  1 , Hyunsoo Jang  1 , Kayla Y Lim  1 , Alexie Lessing  1 , Nicholas Stavropoulos  1   2
Affiliations

insomniac links the development and function of a sleep-regulatory circuit

Qiuling Li et al. Elife. .

Abstract

Although many genes are known to influence sleep, when and how they impact sleep-regulatory circuits remain ill-defined. Here, we show that insomniac (inc), a conserved adaptor for the autism-associated Cul3 ubiquitin ligase, acts in a restricted period of neuronal development to impact sleep in adult Drosophila. The loss of inc causes structural and functional alterations within the mushroom body (MB), a center for sensory integration, associative learning, and sleep regulation. In inc mutants, MB neurons are produced in excess, develop anatomical defects that impede circuit assembly, and are unable to promote sleep when activated in adulthood. Our findings link neurogenesis and postmitotic development of sleep-regulatory neurons to their adult function and suggest that developmental perturbations of circuits that couple sensory inputs and sleep may underlie sleep dysfunction in neurodevelopmental disorders.

Keywords: Cul3; D. melanogaster; autism; developmental biology; neuroblast; neurodevelopmental disorders; neurogenesis; neuroscience; sleep disorders.

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Conflict of interest statement

QL, HJ, KL, AL, NS No competing interests declared

Figures

Figure 1.
Figure 1.. Expression of 3×FLAG-inc driven by inc-Gal4 in the larval, pupal, and adult brain.
Maximal projections are shown for male inc-Gal4; UAS-3×FLAG-inc/+ brains stained with anti-FLAG. For larval brain, projection from a partial z-stack is shown to allow visualization of signal in mushroom body projections (arrowheads). In pupae and adults, signal is prominent in the mushroom body, pars intercerebralis, fan-shaped body, and ellipsoid body. Scale bars, 100 μm.
Figure 2.
Figure 2.. inc acts in a restricted period of neuronal development to impact sleep in adulthood.
(A) Conditional rescue of inc1 mutants using the ligand-inducible Q-system. Quinic acid relieves QS suppression of the pan-neuronally expressed Gal4QF transcriptional activator, inducing UAS-inc-HA in neurons. (B) Total sleep duration of controls (gray) and inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ animals exposed to quinic acid (+) or vehicle (−) at indicated life stages; embryos (E), larval stages (1–3), pupae (P), and adults (A). Bars represent mean ± standard error of the mean (SEM). n = 11–86. One-way analysis of variance (ANOVA) (F(7,397) = 86.73, p < 0.0001) and Tukey post hoc tests, *p < 0.01 for comparisons to inc1; UAS-inc-HA/+. (C) Average sleep profiles of flies in (B), with induction regimens indicated below. Shading indicates ± SEM. (D) Anti-HA staining of inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ brains from indicated induction regimens. Scale bars, 100 μm.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Additional sleep parameters for conditional rescue of inc1 mutants using the Q-system.
Sleep parameters for control (gray) and inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ animals exposed to quinic acid (+) or vehicle (−) at indicated life stages; embryos (E), larval stages (1–3), pupae (P), and adults (A). Bars represent mean ± SEM. n = 11–86 as in Figure 2B. (A) Nighttime sleep. One-way analysis of variance (ANOVA) (F(7,397 ) = 61.39, p < 0.0001) and Tukey post hoc tests, *p < 0.01 for comparisons to inc1; UAS-inc-HA/+. (B) Daytime sleep. One-way ANOVA (F(7, 397) = 66.67, p < 0.0001) and Tukey post hoc tests, *p < 0.01 for comparisons to inc1; UAS-inc-HA/+. (C) Sleep bout length. Kruskal–Wallis (p < 0.0001) and Dunn’s tests, *p < 0.01 compared to inc1; UAS-inc-HA/+ and to vehicle-treated inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ animals. (D) Sleep bout number. One-way ANOVA (F(7, 397) = 42.86, p < 0.0001) and Tukey post hoc tests, *p < 0.01. Note that effects of Q-system induction on sleep bout number are compared to vehicle-treated inc1; UAS-inc-HA/tub-QS; nsyb-GAL4QF/+ animals, which have significantly more sleep bouts than inc1; UAS-inc-HA/+ animals (p < 0.01).
Figure 3.
Figure 3.. Conditional rescue of inc2 mutants and conditional inc RNAi in larval and pupal neurons.
(A) Conditional neuronal rescue of inc2 mutants using the ligand-inducible Q-system. inc2 mutants contain a transposon insertion in the inc 5′UTR immediately upstream of the endogenous start codon. A UAS/TATA element within the transposon terminus permits Gal4-dependent restoration of inc expression (Stavropoulos and Young, 2011). (B) Total sleep duration in inc2; tub-QS/+; nysb-Gal4QF/+ animals exposed to vehicle or quinic acid at the late third instar larval and pupal stages. n = 20–83. One-way analysis of variance (ANOVA) (F(3, 170) = 70.66, p > 0.0001) and Tukey post hoc tests, *p < 0.01 for comparisons to inc2. (C) Average sleep profiles of indicated genotypes from (B). (D) Total sleep duration in tub-QS/UAS-inc-RNAi; nsyb-Gal4QF/UAS-dcr2 animals exposed to vehicle or quinic acid at the late third instar larval and pupal stages. n = 16–24. Student’s t-test, *p < 0.01 for comparison to vehicle-treated control. (E) Average sleep profiles of animals from (D). For (B) and (D), bars represent mean ± SEM. For (C) and (E), shading represents ± SEM.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Additional sleep parameters for conditional inc2 rescue in third instar larval and pupal neurons.
Sleep parameters for control, inc2, and inc2; tub-QS/+; nsyb-Gal4QF/+ animals exposed to vehicle or quinic acid at third instar larval and pupal stages. Bars represent mean ± SEM. Data are shown for the same animals in Figure 3B, C. n = 20–83. (A) Nighttime sleep. One-way analysis of variance (ANOVA) (F(3, 170) = 95.85, p < 0.0001) and Tukey post hoc tests, *p < 0.01. (B) Daytime sleep, one-way ANOVA (F(3, 170) = 23.10, p < 0.0001) and Tukey post hoc tests, *p < 0.01. (C) Sleep bout length. Kruskal–Wallis (p < 0.0001) and Dunn’s post hoc tests, *p < 0.01. (D) Sleep bout number (F(3, 170) = 46.14, p < 0.0001).
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. Additional sleep parameters for conditional inc RNAi in larval and pupal neurons.
Sleep parameters for tub-QS/UAS-inc-RNAi; nsyb-Gal4QF/UAS-dcr2 animals exposed to vehicle or quinic acid in late third instar larval and pupal neurons. (A) Nighttime sleep. (B) Daytime sleep. (C) Sleep bout length. (D) Sleep bout number. Bars represent mean ± SEM. n = 16–24 as in Figure 3D, E. *p < 0.01 for comparison to vehicle-treated controls using Student’s t-tests (A, B), and (D) or Mann–Whitney test (C).
Figure 4.
Figure 4.. The mushroom body is a critical brain region through which inc impacts sleep.
(A) Mean sleep is plotted for each line in a Gal4 rescue screen of inc2 animals. n ≥ 5 per genotype. (B) c253-Gal4 and c309-Gal4 rescue sleep in inc2 mutants. n = 14–78. One-way analysis of variance (ANOVA) (F(4, 172) = 20.36, p < 0.0001) and Tukey post hoc test, *p < 0.01 for comparisons to inc2. (C) Average sleep profiles of flies in (B). (D) Anti-GFP immunostaining of indicated genotypes. Scale bars, 100 μm. (E) MB-Gal80 suppresses sleep rescue in inc1 inc-Gal4; UAS-inc-HA/+ animals. n = 30–69. One-way ANOVA (F(3, 161) = 121.4, p < 0.0001) and Tukey post hoc tests, *p < 0.01. (F) Average sleep profiles of indicated genotypes from (E). For (B) and (E), bars represent mean ± SEM. For (C) and (F), shading represents ± SEM.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Rescue of inc sleep phenotypes by c253-Gal4 and c309-Gal4.
For all panels, bars represent mean ± SEM. n = 14–78 as in Figure 4B, C. **p < 0.01 and *p < 0.05 for post hoc comparisons to inc2. (A) Nighttime sleep. One-way analysis of variance (ANOVA) (F(4, 172) = 24.57, p < 0.0001) and Tukey post hoc tests. (B) Daytime sleep. One-way ANOVA (F(4, 172) = 9.664, p < 0.0001) and Tukey post hoc tests. (C) Sleep bout length. Kruskal–Wallis (p < 0.0001) and Dunn’s post hoc tests. (D) Sleep bout number. One-way ANOVA (F(4, 172) = 27.26, p < 0.0001) and Tukey post hoc tests.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Additional parameters for suppression of sleep rescue in inc1 inc-Gal4; UAS-inc-HA animals by MB-Gal80.
(A) Nighttime sleep. One-way analysis of variance (ANOVA) (F(3, 161) = 99.03, p < 0.0001) and Tukey post hoc tests, *p < 0.01. (B) Daytime sleep. One-way ANOVA (F(3, 161) = 104.8, p < 0.0001) and Tukey post hoc tests, *p < 0.01. (C) Sleep bout length. Kruskal–Wallis (p < 0.0001) and Dunn’s post hoc tests, *p < 0.01. (D) Sleep bout number. One-way ANOVA (F(3, 161) = 36.26, p < 0.0001) and Tukey post hoc tests, *p < 0.01. For all panels, n = 30–69 as in Figure 4E; bars represent mean ± SEM. ns, p > 0.5.
Figure 5.
Figure 5.. Sleep-promoting functions of the mushroom body are impaired in inc mutants.
(A) Thermogenetic activation of neuronal populations expressing TrpA1 in control and inc1 animals. Percent change in sleep (mean ± SEM) elicited by activation is shown. n = 31–144. Control and inc1 animals expressing dTrpA1 are compared to no-Gal4 controls (UAS-dTrpA1/+ and inc1; UAS-dTrpA1/+, respectively). *p < 0.01 for Dunnet’s post hoc comparisons after one-way analysis of variance (ANOVA) for control animals (F(8, 528) = 92.12, p < 0.0001) or inc1 mutants (F(8, 452) = 50.01, p < 0.0001). Green and pink bars indicate drivers that significantly promote or inhibit sleep, respectively; gray bars indicate no significant change with respect to controls. (B) Average sleep profiles of animals from (A) on the baseline day and during thermogenetic activation. Shading represents ± SEM.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Sleep profiles during baseline and neuronal activation.
Average sleep profiles of animals from Figure 5A on the baseline day and during thermogenetic activation. Shading represents ± SEM. Note that control animals (No Gal4) are identical to those in Figure 5B.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. A split-Gal4 driver expressed specifically in mushroom body (MB) neurons.
Anti-GFP (green) and anti-Brp (magenta) staining is shown for maximal projections of adult brain and ventral nerve cord expressing 20×UAS-IVS-CD8-GFP under the control of a mushroom body-specific split-Gal4 driver (MB004B). Inset shows maximal projections of a partial z-stack generated from posterior sections of a different brain, indicating broad expression in MB neuron somata. Scale bars, 100 and 50 μm (inset).
Figure 6.
Figure 6.. Sleep phenotypes for mushroom body ablation and inc mutants.
Sleep parameters for inc2 mutants and animals exposed to vehicle or hydroxyurea (HU). For all panels, n = 37–49; *p < 0.01 for post hoc tests. (A) Total sleep. One-way analysis of variance (ANOVA) (F(2, 122) = 132.9, p < 0.0001) and Tukey post hoc tests. (B) Average daily sleep profiles. Shading represents ± SEM. (C) Nighttime sleep. One-way ANOVA (F(2, 122) = 126.6, p < 0.0001) and Tukey post hoc tests. (D) Daytime sleep. One-way ANOVA (F(2, 122) = 74.32, p < 0.0001) and Tukey post hoc tests. (E) Sleep bout length. Kruskal–Wallis (p < 0.0001) and Dunn’s post hoc tests. (F) Sleep bout number. One-way ANOVA (F(2, 122) = 24.89, p < 0.0001) and Tukey post hoc tests. For (A) and (C–F), bars represent mean ± SEM.
Figure 7.
Figure 7.. inc regulates neurogenesis and anatomy of late-born mushroom body (MB) neurons.
(A) Adult control and inc1 brains expressing UAS-MyrGFP-2A-RedStinger in indicated MB neuron subtypes, stained with anti-GFP (cyan) and anti-dsRed (yellow). (B) MB neuron number per hemisphere. γd, n = 10–11; α ´/β ´, n = 7–10; α/βc, n = 16–18. *p < 0.01, Welch’s t-test. (C) Absolute difference in MB neuron number between left and right brain hemispheres; γd, n = 5–6; α ´/β ´, n = 3–5; α/βc, n = 8–9. *p < 0.01, Welch’s t-test. (D) Number of α/βc neuron clusters per hemisphere. n = 16–18. *p < 0.01, Welch’s t-test. (E) Numbers of dorsal fan-shaped body (dFB) and DH44+ neurons. dFB, n = 26; DH44+, n = 6–8. ns, p > 0.01, Welch’s t-test. (F) Adult control and inc1 brains expressing UAS-DenMark-smGdP-V5 in indicated MB neuron subtypes, stained with anti-GFP. (G) Dendrite volume per hemisphere. γd, n = 16–17; α ´/β ´, n = 19; α/βc, n = 14–16. *p < 0.01, Welch’s t-test. (H) Quantification of axonal projection defects for MB neuron subtypes. Colored bars represent the number of MB lobes in each brain entirely lacking axonal myr-GFP signal. See also panel (A). n = 10–25. (I) Adult control and inc1 brains expressing UAS-MyrGFP-2A-RedStinger in dFB neurons. All scale bars represent 100 μm. For (B–E) and (G), bars represent mean ± SEM.
Figure 7—figure supplement 1.
Figure 7—figure supplement 1.. Analysis of α/βc neuron clusters and projections.
High-resolution maximal-projection images of control (top) and inc1 (bottom) brains expressing UAS-MyrGFP-2A-RedStinger in mushroom body α/βc neurons. Anti-dsRed (yellow) and anti-GFP (cyan) signal is shown for left and right hemispheres of single brains; the same brains are shown at lower resolution in Figure 7A. White dashed lines mark discrete neuron clusters enumerated in single-blind analysis (see Methods); magenta dots indicate distinct projection fascicles from analysis of image stacks. Scale bars, 30 μm.
Figure 7—figure supplement 2.
Figure 7—figure supplement 2.. Analysis of dendrites in additional sleep-regulatory circuits.
(A) Adult brains from control animals and inc1 mutants expressing UAS-DenMark-smGdP-V5 in PDF and CRZ neurons, stained with anti-GFP (cyan). Scale bars, 100 μm. (B) Quantification of dendritic volume. Bars represent mean ± SEM. n = 9–10 (PDF); n = 14–17 (CRZ). ns, p > 0.05, Welch’s t-test.
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