Research ArticleGENETICS

Evolutionarily conserved regulation of sleep by epidermal growth factor receptor signaling

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Science Advances  13 Nov 2019:
Vol. 5, no. 11, eaax4249
DOI: 10.1126/sciadv.aax4249
  • Fig. 1 TGFa overexpression increases sleep.

    (A to D) ISH of egfra and tgfa in the 5-dpf zebrafish brain (schematic) (A). A, anterior; L, lateral; V, ventral; Ce, cerebellum; Hy, hypothalamus; TeO, tectum. (A′ and A″) Sagittal (A′) and dorsal (A″) views of egfra expression in juxtaventricular cells (white arrowheads). (B and B′) egfra coexpression with sox2 in these cells. (C and C′) Sagittal (C) and dorsal (C′) views of tgfa expression in cells just dorsal to juxtaventricular cells in the diencephalon (white arrowheads). (D and D′) tgfa coexpression with sox2 in these cells (white arrowheads). Dashed lines in (A′) and (C) indicate the horizontal planes shown in (A″) and (C′). Boxed regions in (B) and (D) are magnified in (B′) and (D′). Dashed line in (D) shows outline of brain. Scale bars, 30 μm (B, B′, D, and D′) and 50 μm (A″ and C′). (E to H) Following a 1-hour HS (yellow bars), Tg(hs:tgfa) animals were less active (E and F) and slept more (G and H) than their WT siblings. Pre-HS and post-HS are calculated for the day or night before, and the day or night after, HS, respectively. White and black bars indicate day (14 hours) and night (10 hours). Data are obtained from two pooled experiments. Bar graphs show mean ± SEM. n = number of animals. m/h, minutes/hour; s/h, second/hour. ***P < 0.0001 by two-way ANOVA with Holm-Sidak test.

  • Fig. 2 Loss of EGFR signaling decreases sleep.

    (A to D) egf−/−; tgfa−/− animals were more active during the day and slept less during the day and night than egf+/+; tgfa−/− siblings. Larvae were generated by mating egf+/−; tgfa+/− to egf+/−; tgfa−/− animals. For clarity, data for other genotypes are not shown. (E to H) egfra−/− animals were more active during the day and slept less during the day and night than egfra+/+ siblings. (I to L) Gefitinib-treated WT animals were more active and slept less during the day and night than their dimethyl sulfoxide (DMSO)–treated siblings. Pooled data from nine (A to D), eight (E to H), and six (I to L) experiments are shown. Bar graphs show mean ± SEM. n = number of animals. *P ≤ 0.05, **P < 0.01, and ***P < 0.005 for indicated comparisons by one-way ANOVA and Holm-Sidak test (A to H) or Student’s t test (I to L).

  • Fig. 3 EGFR signaling regulates arousal threshold and sleep homeostasis.

    Representative stimulus response curves for Tg(hs:tgfa) animals and their WT siblings following HS (A), gefitinib and DMSO vehicle-treated WT siblings (B) and erlotinib and DMSO vehicle-treated WT siblings (C). Data points indicate mean ± SEM fraction of animals that responded to the stimulus. Dashed lines mark ETP50 values. (A) Tg(hs:tgfa) animals had an ETP50 value of 22.2 versus 6.3 for WT siblings (252% increase; F1,834 = 20.95, P < 0.0001 by extra sum-of-squares F test). (B) Gefitinib-treated animals had an ETP50 of 2.7 versus 4.3 for DMSO-treated siblings (37% decrease; F1,834 = 18.16, P < 0.0001 by extra sum-of-squares F test). (C) Erlotinib-treated animals had an ETP50 of 3.5 versus 5.0 for DMSO-treated siblings (30% decrease; F1,834 = 4.9, P < 0.05 by extra sum-of-squares F test). a.u., arbitrary units. n = number of animals. (D) SD paradigm. (E and F) Sleep behavioral traces for animals treated with DMSO (E) or gefitinib (F) starting at 5 dpf, subjected to SD during the first 6 hours of the night at 7 dpf (P; orange), and monitored thereafter in the dark. The 4-hour periods of RS (purple) are indicated with dashed boxes. (G) Normalized sleep rebound in DMSO- or gefitinib-treated siblings following SD. Normalized sleep rebound = amount of sleep for each perturbed animal during the first 4 hours of RS divided by the average amount of sleep of all nonperturbed controls during this time period. (H to J) After a 1-hour HS in the middle of the day (yellow bar), Tg(hs:tgfa) animals showed increased daytime and nighttime sleep for ~24 hours (H and I), followed by decreased sleep (H and J). Sleep in (I) and (J) is quantified for the boxed regions in (H), which includes the entire night and the last 3 hours of each day. Pooled data from five (D to G) and two (H to J) experiments are shown. Black, white, and hatched bars under behavioral traces indicate night (10 hours), day (14 hours), and subjective day (14 hours), respectively. n = number of animals. **P < 0.01 and ***P < 0.001 by Mann-Whitney test.

  • Fig. 4 TGFa overexpression–induced sleep requires EGFRa.

    (A) Total sleep induced following TGFa overexpression quantified during the fourth hour after HS. (B and C) c-fos expression in a Tg(hs:tgfa) brain at 4 hours after HS shows activation of juxtaventricular cells in the telencephalon, hypothalamus, and hindbrain (arrowhead) (C), but not in the brain of an identically treated WT sibling (B). (D and D′) Double FISH of a Tg(hs:tgfa) brain fixed at 4 hours after HS shows coexpression of egfra and c-fos in juxtaventricular cells. Boxed region in (D) is magnified in (D′). Images are 2.9-μm-thick (D) and 0.7-μm-thick (D′) confocal sections. (E to G) TGFa overexpression increased sleep in transgenic animals compared to nontransgenic siblings in egfra+/+, but not in egfra−/− animals (red comparisons) (G). TGFa overexpression–induced sleep was significantly decreased in egfra−/− animals compared to egfra+/− and egfra+/+ (black comparisons) (G). Yellow bars indicate HS. Pre- and post-HS data are calculated for the day of HS. Data are obtained from six pooled experiments. Bar graphs show mean ± SEM. n.s., not significant. (H to L) Juxtaventricular c-fos expression that is induced by TGFa overexpression (white arrowheads) is suppressed in egfra−/− animals (J and K) compared to egfra+/+ animals (H and I) at 4 hours after HS. Hindbrain (Hb)–boxed regions in (H) and (J) are magnified in (I) and (K). (L) Quantification of c-fos expression in hindbrain and hypothalamus egfra-expressing cells [boxed regions in (H) and (J)]. Data points represent single brains. n = number of animals. *P < 0.05 and ***P < 0.005 by two-way ANOVA with Holm-Sidak test. Te, telencephalon; Hb, hindbrain. Scale bars, 30 μm.

  • Fig. 5 EGFR signaling promotes sleep via NPVF.

    (A to D) Larval zebrafish schematic. Red box indicates hypothalamic region shown in (A), (C), and (G). Immunostaining with an NPVF-specific antibody reveals higher NPVF protein levels at 2 hours after HS for Tg(hs:tgfa) animals compared to WT siblings (A and B) and lower NPVF protein levels in animals fixed at 3 hours after treatment with gefitinib compared to DMSO-treated controls (C and D). Representative images are shown in (A) and (C), and average pixel intensity of NPVF labeling is quantified in (B) and (D); each dot represents one animal. (E) TGFa-induced sleep was partially suppressed in npvf−/− animals compared to npvf+/− siblings. Animals were heat-shocked at 3 p.m. Pre- and post-HS quantify sleep for the day of HS. (F) egfra−/− animals slept less than their egfra+/+ siblings (black comparison), and RF9 suppressed sleep in egfra+/+ animals compared to DMSO-treated egfra+/+ siblings (blue comparison), but RF9 treatment did not further decrease sleep in egfra−/− animals compared to DMSO-treated egfra−/− siblings (red comparison). RF9 was added on the afternoon of 5 dpf, and sleep was quantified during the day at 6 and 7 dpf. (G) Representative images from a Tg(npvf:GCaMP6s-p2A-tdTomato) animal. An individual NPVF neuron is indicated with a white circle. (H) No difference in GCaMP6s/tdTomato values was observed between non–heat-shocked Tg(hs:tgfa) and WT siblings. (I) GCaMP6s/tdTomato values were significantly increased in Tg(hs:tgfa) animals compared to WT siblings at 2 hours after HS. (J) Normalized GCaMP6s/tdTomato values were significantly decreased in WT gefitinib-treated animals compared to DMSO-treated siblings. Normalized GCaMP6s/tdTomato = GCaMP6s/tdTomato value in each neuron at 2 hours after the addition of either gefitinib or DMSO divided by the value in the same neuron before treatment is shown. Bar graphs (E and F) and lines (B and D) indicate mean ± SEM. Numbers in parentheses indicate number of animals (E and F) or neurons (H to J). *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA with Holm-Sidak test (E and F) or by two-tailed Wilcoxon matched-pairs signed-rank test (H to J). Scale bars, 20 μm.

  • Fig. 6 Genetic variants at KSR2 and ERBB4 associate with human sleep quality and quantity.

    (A) Forest plot of associations between genetic variant rs7607363 in ERBB4 and rs1846644 in KSR2 with sleep quality and quantity measures. Black boxes indicate effect estimate, and gray lines represent 95% confidence interval. Data are based on 453,964 human subjects of European ancestry in the U.K. Biobank. Full results for each single-nucleotide polymorphism (SNP) are shown in table S1. (B) Regional association plot for genome-wide significant association between ERBB4 rs7607363 and EDS. (C to E). Regional association plots for genome-wide significant association between KSR2 rs1846644 and sleep duration (C), EDS (D), and daytime napping (E). Genes within each region are shown in the lower panels. Blue lines indicate recombination rate. Filled circles show the −log10 P value for each SNP, with the named SNP shown in purple. Additional SNPs in the locus are colored according to correlation (r2) with the lead SNP, estimated by LocusZoom based on the CEU HapMap haplotypes. Dashed lines represent the genome-wide significance threshold (P < 5 × 10−8).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax4249/DC1

    Fig. S1. Effects of gain and loss of EGFR signaling on sleep architecture.

    Fig. S2. Amino acid alignment of human and zebrafish TGFa, EGF, and EGFR.

    Fig. S3. Gefitinib does not enhance egfra−/− phenotype and effects of EGFR inhibitors on sleep architecture.

    Fig. S4. EGFR signaling is not required for behavioral circadian rhythms.

    Fig. S5. Validation of an SD assay, and EGFR signaling is required for normal homeostatic regulation of sleep.

    Fig. S6. Inhibition of MAPK/ERK signaling suppresses TGFa overexpression–induced sleep.

    Fig. S7. EGFR signaling regulates npvf expression, and TGFa overexpression–induced sleep is suppressed in npvf mutant animals.

    Fig. S8. Association of ERBB4 sleepiness allele with increased ERBB4 expression in humans and pharmacological inhibition of KSR2 or ERBB4 decrease sleep in zebrafish.

    Table S1. Variants at ERBB4 and KSR2 associate with self-reported measures of sleep quality and quantity in U.K. Biobank subjects.

    Table S2. Descriptive characteristics of U.K. Biobank subjects of European ancestry used for sleep trait analysis.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Effects of gain and loss of EGFR signaling on sleep architecture.
    • Fig. S2. Amino acid alignment of human and zebrafish TGFa, EGF, and EGFR.
    • Fig. S3. Gefitinib does not enhance egfra−/− phenotype and effects of EGFR inhibitors on sleep architecture.
    • Fig. S4. EGFR signaling is not required for behavioral circadian rhythms.
    • Fig. S5. Validation of an SD assay, and EGFR signaling is required for normal homeostatic regulation of sleep.
    • Fig. S6. Inhibition of MAPK/ERK signaling suppresses TGFa overexpression–induced sleep.
    • Fig. S7. EGFR signaling regulates npvf expression, and TGFa overexpression–induced sleep is suppressed in npvf mutant animals.
    • Fig. S8. Association of ERBB4 sleepiness allele with increased ERBB4 expression in humans and pharmacological inhibition of KSR2 or ERBB4 decrease sleep in zebrafish.
    • Table S1. Variants at ERBB4 and KSR2 associate with self-reported measures of sleep quality and quantity in U.K. Biobank subjects.
    • Table S2. Descriptive characteristics of U.K. Biobank subjects of European ancestry used for sleep trait analysis.

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