Forward-genetics analysis of sleep in randomly mutagenized mice

Journal name:
Nature
Volume:
539,
Pages:
378–383
Date published:
DOI:
doi:10.1038/nature20142
Received
Accepted
Published online

Abstract

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

At a glance

Figures

  1. Identification of Sik3 splicing mutation responsible for reduced total wake time.
    Figure 1: Identification of Sik3 splicing mutation responsible for reduced total wake time.

    a, Wake-time distribution of B023 N2 littermates (bars) and all mice screened (curve). Blue and purple bars indicates retrospectively genotyped Sik3+/+ and Sik3Slp/+ mice, respectively. b, Quantitative trait locus (QTL) analysis of B023 pedigree (n = 93) for total wake time. Inset shows LOD score peak between rs13480122 and rs29644859. c, Haplotype analysis of Sleepy mutant pedigrees, B021–B025, in terms of the presence of Sleepy phenotypes. d, Exome sequencing results from Sleepy mutant mice of B023 and B024 pedigrees together with wild-type mice within the region of the LOD score peak. e, Direct sequencing of the Sik3 gene. f, RT–PCR of Sik3 mRNA produced smaller bands specific to Sik3Slp mice. Ctx, cerebral cortex; Hypo, hypothalamus. g, Structures of wild-type and mutant SIK3 proteins. h, Immunoblotting of brain homogenates showing wild-type (+/+) and mutant SIK3 (Slp/+ and Slp/Slp) protein variants. i, RT–PCR of brain mRNA from ZFN-based Sik3Slp/+ mice showing smaller bands lacking exon 13, in addition to larger bands containing the exon 13. j, Sik3Slp(ZFN)/+ mice (n = 15) showed a shorter total wake time than wild-type littermates (n = 14). ***P < 0.001, two-way analysis of variance (ANOVA) followed by Tukey’s test. Data are mean ± s.e.m.

  2. Increased sleep need and normal wake-promoting response of Sik3 mutant mice.
    Figure 2: Increased sleep need and normal wake-promoting response of Sik3 mutant mice.

    a, b, Circadian variation in wakefulness (a) and NREMS (b) in Sik3+/+ (n = 22), Sik3Slp/+ (n = 32) and Sik3Slp/Slp (n = 31) mice. *P < 0.05 (black); *P < 0.001 (red), one-way repeated measures ANOVA followed by Tukey’s test. c, Time spent in wakefulness from zeitgeber time (ZT)4 to ZT5 after cage change at ZT5 of Sik3+/+ (n = 6), Sik3Slp/+ (n = 9) and Sik3Slp/Slp (n = 6) mice. ***P < 0.001, one-way repeated measures ANOVA followed by Tukey’s test. d, Wake time for 6 h after caffeine injection at ZT0 in Sik3+/+ (n = 6), Sik3Slp/+ (n = 6) and Sik3Slp/Slp (n = 6) mice. *P < 0.05 (black); ##P < 0.01 versus 10 mg kg−1 body weight (BW); *P < 0.01 (red) versus 15 mg kg−1 BW, two-way ANOVA followed by Tukey’s test. e, NREMS delta density of Sik3 mutant mice (Sik3+/+, n = 22; Sik3Slp/+, n = 32; Sik3Slp/Slp, n = 31) across the light–dark cycle. *P < 0.05; ***P < 0.001, one-way repeated ANOVA followed by Tukey’s test. f, Increase in NREMS delta power of Sik3+/+ (n = 7), Sik3Slp/+ (n = 7) and Sik3Slp/Slp (n = 10) mice after 6-h sleep deprivation. *P < 0.05, one-way ANOVA followed by Tukey’s test. g, Increase in NREMS delta power after 2, 4 and 6 h of sleep deprivation of Sik3+/+ (n = 11) and Sik3Slp/+ (n = 11) mice relative to NREMS delta power of the same ZT during basal sleep. *P < 0.05; **P < 0.01; ***P < 0.001, two-way ANOVA followed by Tukey’s test. h, Phosphorylation of Flag–SIK3 of Flag-Sik3+/+ brains with or without 4-h sleep deprivation. *P < 0.05, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  3. Role of Sik3 orthologues in invertebrate sleep-like behaviours.
    Figure 3: Role of Sik3 orthologues in invertebrate sleep-like behaviours.

    a, The phylogenetic conservation of the exon 13-encoded region of Sik3. b, c, Sleep time before and after induction of SIK3(Ser563Ala) by RU486 under constant darkness (32 per group). d, Sleep time of control and Sik3 hypomorphic mutant (Sik3(hypo)) in 12-h light:12-h dark (L–D) conditions (top, 16 per group, P < 0.001) and in constant darkness (D–D) (bottom, 16 per group, P < 0.001); one-way repeated measures ANOVA. CT, circadian time. e, Sik3-null mutant worms, kin-29(oy38) (n = 15), exhibited reduced quiescence during lethargus compared with wild-type worms (n = 10). kin-29(oy38);PH20::kin-29 worms (n = 9), in which wild-type kin-29 was expressed in neuronal cells, restored normal quiescence during lethargus. The fractions of quiescence out of lethargus were similar (P = 0.98). *P < 0.05; **P < 0.01; ***P < 0.001, one-way repeated measures ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  4. Missense mutation in Nalcn gene reduces REMS time and episode duration.
    Figure 4: Missense mutation in Nalcn gene reduces REMS time and episode duration.

    a, Histogram of REMS episode duration of G1 mice screened (mean ± s.d. = 1.41 ± 0.19 min). Arrow indicates the founder of Dreamless mutant pedigree. b, QTL analysis of Dreamless mutant pedigree (n = 56) for REMS episode duration. Inset shows LOD score peak near rs31233932. c, Direct sequencing of the Nalcn gene. d, e, Total REMS time (d) and REMS episode duration (e) of NalcnDrl/+ mice (n = 29) and Nalcn+/+ mice (n = 25) of the Dreamless mutant pedigree. f, g, Total REMS time (f) and REMS episode duration (g) of NalcnDrl/+ mice (n = 11) and Nalcn+/+ mice (n = 17) produced by CRISPR/Cas9 technology. ***P < 0.001, two-tailed Student’s t-test. Data are mean ± s.e.m.

  5. Dreamless mutation in Nalcn gene increases excitability of neurons in the ‘REM-off’ area.
    Figure 5: Dreamless mutation in Nalcn gene increases excitability of neurons in the ‘REM-off’ area.

    a, Schematic structure of NALCN protein. b, Phylogenetic conservation of Asn315 residue in NALCN. c, Representative traces of membrane currents in response to 300-ms step pulses ranging from −80 mV to +80 mV in 10-mV increments (holding potential Vh = 0 mV, bottom) recorded from HEK293T cells transfected with wild-type NALCN (top) or mutant NALCN(DRL) (middle). d, Mean current–voltage (I–V) curves in wild-type NALCN (n = 5, black circles and bottom right) or NALCN(DRL) (n = 7, purple circles). e, The conductance of NALCN(DRL)-transfected cells was larger than that of wild-type NALCN-transfected cells (NALCN, 0.09 ± 0.02 nS pF−1, n = 5; NALCN(DRL), 1.81 ± 0.62 nS pF−1, n = 7). **P < 0.01, Mann–Whitney U test. f, Representative trace of membrane potentials of DpMe neurons in Nalcn+/+ (top) and NalcnDrl/+ (bottom) mice. Dashed lines indicate 0 mV level. g, h, Mean membrane potentials (g) and spontaneous firing rates (h) of DpMe neurons (Nalcn+/+, n = 33; NalcnDrl/+, n = 31). *P < 0.05, Mann–Whitney U test. Data are mean ± s.e.m.

  6. Sleep/wakefulness screening of randomly mutagenized mice.
    Extended Data Fig. 1: Sleep/wakefulness screening of randomly mutagenized mice.

    a, ENU-treated G0 mice were mated with B6N females to obtain the offspring. The F1 mice were used for sleep/wakefulness analysis. A mouse showing any sleep abnormalities was crossed with B6N female mice. The N2 progeny was examined for heritability of sleep abnormality and for chromosomal mapping. b, B6J (n = 20) and B6N (n = 21) showed similar total wake time (left, P = 0.67, two-tailed Student’s t-test), NREMS time (centre, P = 0.66) and REMS time (right, P = 0.84). Data are mean ± s.e.m. c, The histogram shows total daily wake time of all mice screened. Total wake time of screened mice was 735 ± 66.9 min (mean ± s.d.). Arrows indicate the founders of Sleepy mutant pedigrees.

  7. QTL analysis of Sleepy mutant pedigrees and characterization of Sik3 transcript.
    Extended Data Fig. 2: QTL analysis of Sleepy mutant pedigrees and characterization of Sik3 transcript.

    a, QTL analysis of B021 (n = 119), B022 (n = 95), B024 (n = 59) and B025 (n = 112) pedigrees for total wake time produced a single LOD score peak on chromosome 9. b, Direct sequencing of the exon 12/13 boundary and exon 13/14 boundary of Sik3 mRNA of Sik13+/+ mouse. Direct sequencing of the short RT–PCR product specific to Sik3 mutant mice shows the direct transition from exon 12 to exon 14. c, d, Sik3 mRNA is expressed broadly in forebrain neurons (c). Sik3 mRNA is expressed throughout the cerebral cortex in the primary motor area (d). DG, dentate gyrus; LV, lateral ventricle; MHb, medial habenula. Scale bars, 1 mm (c) and 250 μm (d). e, RT–PCR of Sik3 mRNA from cerebral cortex and liver of Sik3+/+, Sik3Slp/+ and Sik3Slp/Slp mice. Normal Sik3 variant lacking exon 15 expressed in the cerebral cortex.

  8. Sleep/wakefulness of Sik3Slp knock-in mice.
    Extended Data Fig. 3: Sleep/wakefulness of Sik3Slp knock-in mice.

    a, The structure of the Sik3 genome and targeting vector for Sik3Slp. Neomycin resistance gene under the mouse phosphoglycerol kinase promoter (neo) was sandwiched with the flippase recognition target (FRT) sequences. The guanine at the fifth nucleotide from the beginning of the intron 13 was substituted with adenine. The neo cassette was deleted by crossing with ActbCAG-FLPknock-in mice. b, RT–PCR of Sik3 mRNA of Sik3Slp/+ knock-in mice. c, Total wake time of Sik3Slp/+ knock-in mice (n = 10) and Sik3+/+ littermates (n = 6). ***P < 0.001, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  9. Sleep/wakefulness behaviours of Sik3 mutant mice.
    Extended Data Fig. 4: Sleep/wakefulness behaviours of Sik3 mutant mice.

    a, Representative 8-s EEG and EMG for wake, NREMS and REMS of Sik3 mutant mice. b, Representative hypnogram of Sik3 mutant mice. Wake (blue), NREMS (green) and REMS (red) are indicated from ZT0 to ZT24. cg, Total wake time (c), NREMS time (d), REMS time (e), NREMS/total sleep ratio (f), REMS/total sleep ratio (g) and circadian variation of REMS (h) of Sik3+/+ (n = 22), Sik3Slp/+ (n = 32) and Sik3Slp/Slp (n = 31) mice. *P < 0.05; **P < 0.01; ***P < 0.001, two-way ANOVA followed by Tukey’s test (cg). *P < 0.05 (red); *P < 0.001 (black), one-way repeated measures ANOVA followed by Tukey’s test (h). i, Total wake time of female Sik3+/+ (n = 10), Sik3Slp/+ (n = 11) and Sik3Slp/Slp (n = 9) mice. ***P < 0.001, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  10. Characterization of sleep/wakefulness behaviours of Sik3 mutant mice.
    Extended Data Fig. 5: Characterization of sleep/wakefulness behaviours of Sik3 mutant mice.

    a, Wake time after cage change at ZT15 in Sik3+/+ (n = 5), Sik3Slp/+ (n = 10) and Sik3Slp/Slp (n = 5) mice. The graph shows time spent in wakefulness from ZT15 to ZT16 under a basal condition and after cage change from the home cage to a new cage at ZT15. *P < 0.05; ***P < 0.001 versus Sik3+/+; #P < 0.05; ###P < 0.001, one-way repeated measures ANOVA followed by Tukey’s test. b, Wake time increases for 3 h after modafinil injection at ZT0 in Sik3+/+ (n = 6), Sik3Slp/+ (n = 6) and Sik3Slp/Slp (n = 6) mice. *P < 0.05; versus modafinil 10 mg kg−1 in the same genotype, #P < 0.05, ##P < 0.01, two-way ANOVA followed by Tukey’s test. c, The circadian period under constant darkness in Sik3+/+ (n = 8), Sik3Slp/+ (n = 8) and Sik3Slp/Slp (n = 6) mice. P = 0.97, one-way ANOVA. d, Total wake time of Sik3+/+ (n = 9) and Sik3Slp/+ (n = 12) mice under constant darkness. ***P < 0.001, two-tailed Student’s t-test. e, EEG power spectra of Sik3+/+ (n = 22), Sik3Slp/+ (n = 32) and Sik3Slp/Slp (n = 31) mice. *P < 0.05; ***P < 0.001, one-way ANOVA followed by Tukey’s test. f, Increase in NREMS delta power after 2 h, 4 h and 6 h of sleep deprivation of Sik3+/+ (n = 11) and Sik3Slp/+ (n = 11) mice relative to mean NREMS delta power during basal sleep. **P < 0.01, two-way ANOVA followed by Tukey’s test. g, Phosphorylation of Flag–SIK3 of Flag-Sik3+/+ brains and of Flag–SIK3(SLP) of Flag-Sik3Slp/+ brains with or without 4-h sleep deprivation. *P < 0.05; ***P < 0.001, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  11. Characterization of Flag-Sik3 mice made by CRISPR/Cas9 technology.
    Extended Data Fig. 6: Characterization of Flag-Sik3 mice made by CRISPR/Cas9 technology.

    a, Exon 1 of the Sik3 gene contains the first and second methionine residues. The single-guide RNA was designed to target the second methionine-coding region. The donor oligonucleotide has a Flag-haemagglutinin (HA)-coding sequence immediately after the second methionine and 70-nucleotide long arms at both 5′ and 3′ ends. The Flag-HA-coding region is followed by an XbaI site. b, Immunoblotting of brain homogenates of Sik3+/+, Sik3Flag/Flag Sik3Flag,Slp/+ mice showed that anti-Flag antibody detected Flag–SIK3 protein of Sik3Flag/Flag brains and Flag–SIK3 (SLP) protein of Sik3Flag,Slp/+ brains, whereas anti-SIK3 antibody detected SIK3 proteins of all genotypes. c, RT–PCR of brain Sik3 mRNA of Sik3+/+, Sik3Flag/Flag, Sik3Flag,Slp/+ mice. d, Tryptic peptides of immunoprecipitated and gel-purified Flag–SIK3 protein were analysed by LC–MS and mapped on the reference SIK3 protein. The peptide fragments were mapped on almost entire SIK3 protein with high confidence.

  12. Phylogenetic conservation of the SIK3 protein.
    Extended Data Fig. 7: Phylogenetic conservation of the SIK3 protein.
  13. Identification of Nalcn mutation of the Dreamless mutant pedigree.
    Extended Data Fig. 8: Identification of Nalcn mutation of the Dreamless mutant pedigree.

    a, Histogram of REMS episode duration in N2 littermates of Dreamless mutant pedigree (bars) and all F1 mice examined (curve). b, Haplotype analysis of chromosome 14 of Dreamless mutant pedigree with or without short REMS episode duration. c, Whole-exome sequencing of Dreamless mutant N2 mice. All mice with short REMS episode duration had the single nucleotide substitution in exon 9 of the Nalcn gene.

  14. Sleep/wakefulness behaviour of Nalcn mutant mice.
    Extended Data Fig. 9: Sleep/wakefulness behaviour of Nalcn mutant mice.

    a, Representative 8-s EEG and EMG for wake, NREMS and REMS of Nalcn mutant mice b, Representative hypnogram of Nalcn+/+ mice (top) and NalcnDrl/+ mice (bottom). Wake (blue), NREMS (green) and REMS (red) are indicated from ZT0 to ZT12. c, Enlarged hypnogram of around ZT7 showed the frequent transitions between NREMS and REMS of NalcnDrl/+ mice. d, Total wake time and NREMS time of NalcnDrl/+ mice (n = 29) and Nalcn+/+ mice (n = 25). Wake, P = 0.58; NREMS, P = 0.17, one-way ANOVA. e, f, Circadian period length (e) and amplitude of circadian behaviour (f) in constant darkness of NalcnDrl/+ mice (n = 6) and Nalcn+/+ mice (n = 7). P = 0.76 (e); ***P < 0.001 (f), two-tailed Student’s t-test. g, Total REMS time of NalcnDrl/+ mice (n = 9) and Nalcn+/+ mice in constant darkness (n = 8). ***P < 0.001, two-tailed Student’s t-test. h, EEG power spectra of NalcnDrl/+ mice (n = 29) and Nalcn+/+ mice (n = 25). ***P < 0.001, one-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  15. Increased conductance of NALCN(DRL).
    Extended Data Fig. 10: Increased conductance of NALCN(DRL).

    ac, Nalcn mRNA is expressed in the ventrolateral periaqueductal grey mater (vlPAG) and deep mesencephalic nucleus (DpMe) of the upper pons (a), the lateral dorsal tegmental nucleus (LDT) and sublateral dorsal nucleus (SLD) of the lower pons (b), and the lateral paragigantocellular nucleus (LPGi) of the medulla (c). AQ, aqueduct; dscp, decussation of superior cerebellar peduncle; IO, inferior olive; scp, superior cerebellar peduncle. Scale bars, 500 μm. d, Representative traces of membrane currents in response to ramp pulses (Vh = 0 mV, from −100 mV to +100 mV in 1 s; lower) recorded from HEK293T cells cotransfected with UNC80, SRC(Tyr529Phe), and NALCN–GFP (top) or NALCN(DRL)–GFP (middle). The traces are averaged from three trials. The transient capacitance currents are also recorded. e, Mean current density in response to ramp pulses (NALCN, n = 5, black line; NALCN(DRL), n = 7, purple line). The data from NALCN are also shown on an expanded scale (bottom right). f, The charge transfer of NALCN(DRL)-transfected cells was larger than that of NALCN(WT)-transfected cells. **P < 0.01, Mann–Whitney U test. The recording data are same as in e. Data are mean ± s.e.m.

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Author information

  1. These authors contributed equally to this work.

    • Chika Miyoshi,
    • Tomoyuki Fujiyama,
    • Takeshi Kanda &
    • Makito Sato

Affiliations

  1. International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

    • Hiromasa Funato,
    • Chika Miyoshi,
    • Tomoyuki Fujiyama,
    • Takeshi Kanda,
    • Makito Sato,
    • Zhiqiang Wang,
    • Jing Ma,
    • Aya Ikkyu,
    • Miyo Kakizaki,
    • Noriko Hotta-Hirashima,
    • Satomi Kanno,
    • Haruna Komiya,
    • Fuyuki Asano,
    • Takato Honda,
    • Staci J. Kim,
    • Kanako Harano,
    • Hiroki Muramoto,
    • Toshiya Yonezawa,
    • Shinichi Miyazaki,
    • Linzi Connor,
    • Yu Hayashi,
    • Qinghua Liu,
    • Joseph S. Takahashi &
    • Masashi Yanagisawa
  2. Department of Anatomy, Faculty of Medicine, Toho University, Ota-ku, Tokyo 143-8540, Japan

    • Hiromasa Funato
  3. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Makito Sato &
    • Masashi Yanagisawa
  4. Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan

    • Shin Nakane,
    • Jun Tomita &
    • Kazuhiko Kume
  5. Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

    • Seiya Mizuno,
    • Fumihiro Sugiyama &
    • Satoru Takahashi
  6. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Vivek Kumar &
    • Joseph S. Takahashi
  7. The Jackson Laboratory, Bar Harbor, Maine 04609, USA

    • Vivek Kumar
  8. Technology and Development Team for Mouse Phenotype Analysis, RIKEN Bioresource Center, Tsukuba, Ibaraki 305-0074, Japan

    • Ikuo Miura,
    • Tomohiro Suzuki &
    • Shigeharu Wakana
  9. Laboratory of Research Advancement, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan

    • Atsushi Watanabe
  10. Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan

    • Manabu Abe &
    • Kenji Sakimura
  11. PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

    • Yu Hayashi
  12. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Qinghua Liu
  13. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Joseph S. Takahashi &
    • Masashi Yanagisawa
  14. Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

    • Masashi Yanagisawa

Contributions

H.F. and M.Y. were responsible for the overall experimental design, based on strategies conceived by M.Y. and J.S.T. M.S. constructed EEG analysis and database systems. C.M., S.K., N.H.-H., A.I., H.K., F.A., T.H., S.J.K. and K.H. conducted EEG recording and analysis. M.K. performed in situ hybridization. T.F., Se.M., F.S. and S.T. produced CRISPR-based gene-modified mice. M.A. and K.S. produced gene-modified mice. Sh.M., L.C. and Y.H. conducted roundworm experiments. T.K., H.M. and T.Y. conducted electrophysiological experiments. Z.W., J.M., A.W. and Q.L. conducted proteomics experiments. S.N., J.T. and K.K. conducted fruitfly experiments. ENU mice production and linkage analysis were conducted by I.M., T.S. and S.W. V.K. and J.S.T. designed B6 substrain-based screening. H.F. and M.Y. wrote the paper, which was reviewed by all authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Reviewer Information

Nature thanks D.-J. Dijk, A. Sehgal and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Sleep/wakefulness screening of randomly mutagenized mice. (203 KB)

    a, ENU-treated G0 mice were mated with B6N females to obtain the offspring. The F1 mice were used for sleep/wakefulness analysis. A mouse showing any sleep abnormalities was crossed with B6N female mice. The N2 progeny was examined for heritability of sleep abnormality and for chromosomal mapping. b, B6J (n = 20) and B6N (n = 21) showed similar total wake time (left, P = 0.67, two-tailed Student’s t-test), NREMS time (centre, P = 0.66) and REMS time (right, P = 0.84). Data are mean ± s.e.m. c, The histogram shows total daily wake time of all mice screened. Total wake time of screened mice was 735 ± 66.9 min (mean ± s.d.). Arrows indicate the founders of Sleepy mutant pedigrees.

  2. Extended Data Figure 2: QTL analysis of Sleepy mutant pedigrees and characterization of Sik3 transcript. (964 KB)

    a, QTL analysis of B021 (n = 119), B022 (n = 95), B024 (n = 59) and B025 (n = 112) pedigrees for total wake time produced a single LOD score peak on chromosome 9. b, Direct sequencing of the exon 12/13 boundary and exon 13/14 boundary of Sik3 mRNA of Sik13+/+ mouse. Direct sequencing of the short RT–PCR product specific to Sik3 mutant mice shows the direct transition from exon 12 to exon 14. c, d, Sik3 mRNA is expressed broadly in forebrain neurons (c). Sik3 mRNA is expressed throughout the cerebral cortex in the primary motor area (d). DG, dentate gyrus; LV, lateral ventricle; MHb, medial habenula. Scale bars, 1 mm (c) and 250 μm (d). e, RT–PCR of Sik3 mRNA from cerebral cortex and liver of Sik3+/+, Sik3Slp/+ and Sik3Slp/Slp mice. Normal Sik3 variant lacking exon 15 expressed in the cerebral cortex.

  3. Extended Data Figure 3: Sleep/wakefulness of Sik3Slp knock-in mice. (190 KB)

    a, The structure of the Sik3 genome and targeting vector for Sik3Slp. Neomycin resistance gene under the mouse phosphoglycerol kinase promoter (neo) was sandwiched with the flippase recognition target (FRT) sequences. The guanine at the fifth nucleotide from the beginning of the intron 13 was substituted with adenine. The neo cassette was deleted by crossing with ActbCAG-FLPknock-in mice. b, RT–PCR of Sik3 mRNA of Sik3Slp/+ knock-in mice. c, Total wake time of Sik3Slp/+ knock-in mice (n = 10) and Sik3+/+ littermates (n = 6). ***P < 0.001, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  4. Extended Data Figure 4: Sleep/wakefulness behaviours of Sik3 mutant mice. (425 KB)

    a, Representative 8-s EEG and EMG for wake, NREMS and REMS of Sik3 mutant mice. b, Representative hypnogram of Sik3 mutant mice. Wake (blue), NREMS (green) and REMS (red) are indicated from ZT0 to ZT24. cg, Total wake time (c), NREMS time (d), REMS time (e), NREMS/total sleep ratio (f), REMS/total sleep ratio (g) and circadian variation of REMS (h) of Sik3+/+ (n = 22), Sik3Slp/+ (n = 32) and Sik3Slp/Slp (n = 31) mice. *P < 0.05; **P < 0.01; ***P < 0.001, two-way ANOVA followed by Tukey’s test (cg). *P < 0.05 (red); *P < 0.001 (black), one-way repeated measures ANOVA followed by Tukey’s test (h). i, Total wake time of female Sik3+/+ (n = 10), Sik3Slp/+ (n = 11) and Sik3Slp/Slp (n = 9) mice. ***P < 0.001, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  5. Extended Data Figure 5: Characterization of sleep/wakefulness behaviours of Sik3 mutant mice. (311 KB)

    a, Wake time after cage change at ZT15 in Sik3+/+ (n = 5), Sik3Slp/+ (n = 10) and Sik3Slp/Slp (n = 5) mice. The graph shows time spent in wakefulness from ZT15 to ZT16 under a basal condition and after cage change from the home cage to a new cage at ZT15. *P < 0.05; ***P < 0.001 versus Sik3+/+; #P < 0.05; ###P < 0.001, one-way repeated measures ANOVA followed by Tukey’s test. b, Wake time increases for 3 h after modafinil injection at ZT0 in Sik3+/+ (n = 6), Sik3Slp/+ (n = 6) and Sik3Slp/Slp (n = 6) mice. *P < 0.05; versus modafinil 10 mg kg−1 in the same genotype, #P < 0.05, ##P < 0.01, two-way ANOVA followed by Tukey’s test. c, The circadian period under constant darkness in Sik3+/+ (n = 8), Sik3Slp/+ (n = 8) and Sik3Slp/Slp (n = 6) mice. P = 0.97, one-way ANOVA. d, Total wake time of Sik3+/+ (n = 9) and Sik3Slp/+ (n = 12) mice under constant darkness. ***P < 0.001, two-tailed Student’s t-test. e, EEG power spectra of Sik3+/+ (n = 22), Sik3Slp/+ (n = 32) and Sik3Slp/Slp (n = 31) mice. *P < 0.05; ***P < 0.001, one-way ANOVA followed by Tukey’s test. f, Increase in NREMS delta power after 2 h, 4 h and 6 h of sleep deprivation of Sik3+/+ (n = 11) and Sik3Slp/+ (n = 11) mice relative to mean NREMS delta power during basal sleep. **P < 0.01, two-way ANOVA followed by Tukey’s test. g, Phosphorylation of Flag–SIK3 of Flag-Sik3+/+ brains and of Flag–SIK3(SLP) of Flag-Sik3Slp/+ brains with or without 4-h sleep deprivation. *P < 0.05; ***P < 0.001, two-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  6. Extended Data Figure 6: Characterization of Flag-Sik3 mice made by CRISPR/Cas9 technology. (330 KB)

    a, Exon 1 of the Sik3 gene contains the first and second methionine residues. The single-guide RNA was designed to target the second methionine-coding region. The donor oligonucleotide has a Flag-haemagglutinin (HA)-coding sequence immediately after the second methionine and 70-nucleotide long arms at both 5′ and 3′ ends. The Flag-HA-coding region is followed by an XbaI site. b, Immunoblotting of brain homogenates of Sik3+/+, Sik3Flag/Flag Sik3Flag,Slp/+ mice showed that anti-Flag antibody detected Flag–SIK3 protein of Sik3Flag/Flag brains and Flag–SIK3 (SLP) protein of Sik3Flag,Slp/+ brains, whereas anti-SIK3 antibody detected SIK3 proteins of all genotypes. c, RT–PCR of brain Sik3 mRNA of Sik3+/+, Sik3Flag/Flag, Sik3Flag,Slp/+ mice. d, Tryptic peptides of immunoprecipitated and gel-purified Flag–SIK3 protein were analysed by LC–MS and mapped on the reference SIK3 protein. The peptide fragments were mapped on almost entire SIK3 protein with high confidence.

  7. Extended Data Figure 7: Phylogenetic conservation of the SIK3 protein. (123 KB)
  8. Extended Data Figure 8: Identification of Nalcn mutation of the Dreamless mutant pedigree. (352 KB)

    a, Histogram of REMS episode duration in N2 littermates of Dreamless mutant pedigree (bars) and all F1 mice examined (curve). b, Haplotype analysis of chromosome 14 of Dreamless mutant pedigree with or without short REMS episode duration. c, Whole-exome sequencing of Dreamless mutant N2 mice. All mice with short REMS episode duration had the single nucleotide substitution in exon 9 of the Nalcn gene.

  9. Extended Data Figure 9: Sleep/wakefulness behaviour of Nalcn mutant mice. (315 KB)

    a, Representative 8-s EEG and EMG for wake, NREMS and REMS of Nalcn mutant mice b, Representative hypnogram of Nalcn+/+ mice (top) and NalcnDrl/+ mice (bottom). Wake (blue), NREMS (green) and REMS (red) are indicated from ZT0 to ZT12. c, Enlarged hypnogram of around ZT7 showed the frequent transitions between NREMS and REMS of NalcnDrl/+ mice. d, Total wake time and NREMS time of NalcnDrl/+ mice (n = 29) and Nalcn+/+ mice (n = 25). Wake, P = 0.58; NREMS, P = 0.17, one-way ANOVA. e, f, Circadian period length (e) and amplitude of circadian behaviour (f) in constant darkness of NalcnDrl/+ mice (n = 6) and Nalcn+/+ mice (n = 7). P = 0.76 (e); ***P < 0.001 (f), two-tailed Student’s t-test. g, Total REMS time of NalcnDrl/+ mice (n = 9) and Nalcn+/+ mice in constant darkness (n = 8). ***P < 0.001, two-tailed Student’s t-test. h, EEG power spectra of NalcnDrl/+ mice (n = 29) and Nalcn+/+ mice (n = 25). ***P < 0.001, one-way ANOVA followed by Tukey’s test. Data are mean ± s.e.m.

  10. Extended Data Figure 10: Increased conductance of NALCN(DRL). (487 KB)

    ac, Nalcn mRNA is expressed in the ventrolateral periaqueductal grey mater (vlPAG) and deep mesencephalic nucleus (DpMe) of the upper pons (a), the lateral dorsal tegmental nucleus (LDT) and sublateral dorsal nucleus (SLD) of the lower pons (b), and the lateral paragigantocellular nucleus (LPGi) of the medulla (c). AQ, aqueduct; dscp, decussation of superior cerebellar peduncle; IO, inferior olive; scp, superior cerebellar peduncle. Scale bars, 500 μm. d, Representative traces of membrane currents in response to ramp pulses (Vh = 0 mV, from −100 mV to +100 mV in 1 s; lower) recorded from HEK293T cells cotransfected with UNC80, SRC(Tyr529Phe), and NALCN–GFP (top) or NALCN(DRL)–GFP (middle). The traces are averaged from three trials. The transient capacitance currents are also recorded. e, Mean current density in response to ramp pulses (NALCN, n = 5, black line; NALCN(DRL), n = 7, purple line). The data from NALCN are also shown on an expanded scale (bottom right). f, The charge transfer of NALCN(DRL)-transfected cells was larger than that of NALCN(WT)-transfected cells. **P < 0.01, Mann–Whitney U test. The recording data are same as in e. Data are mean ± s.e.m.

Supplementary information

PDF files

  1. Supplementary Information (1.6 MB)

    The file contains the raw data for Figure 1,f,h,i, and Extended Data Figures 2e, 3b, 6b,c.

Additional data