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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody
Cell ignaling Tech
Cat#9101
Gem monoclonal antibody (M01), clone 4B12
Abnova
Cat#H00002669-M01
c-Fos antibody
Abcam
Cat#ab7963
Chemicals, peptides, and recombinant proteins
Toluidine blue
Sigma-Aldrich
Cat#T3260
TRIzol reagent
Thermo
Cat#15596026
RNeasy micro kit
Qiagen
Cat#74004
Tetrodotoxin
Wako
Cat# 206-11071
One-Cycle Target Labelling and Control
Reagents Kit
Affymetrix
Discontinued
3,3’-Diaminobenzidine (DAB)
Wako
Cat#R-074N
Goat Biotinylated anti-rabbit IgG
Vector lab
Cat# BP-9100-50
Lipofectamine 2000
Thermo
Cat#11668027
Bicculine
Wako
Cat#0130
proteinase K
Wako
Cat#161-28701
RNAse A
Wako
Cat#318-06391
Avidin/biotinylated horseradish peroxidase
Vector lab
Cat#PK-4005
Diaminobenzidine
Wako
Cat#349-00903
Entellan
Merck
Cat#107960
SuperScript III First-Strand Synthesis SuperMix
Thermo
Cat#18080400
Platinum SYBR Green qPCR SuperMix-UDG
Thermo
Cat#11733038
PrimeScript RT reagent kits
Takara
Cat#RR037A
Thunderbird SYBR qPCR mix
Toyobo
Cat#QPS-201
2-mercaptoethanol
Nacalai
Cat#21417-52
RNeasy Mini Kit
Qiagen
Cat#74104
SuperScriptTM VILOTM cDNA Synthesis
Thermo
Cat#11754050
ROCHE Digoxigenin-11-UTP
MilliporeSigma
Cat#12352200
Nifedipine
Wako
Cat# 141-05783
Promega
E1910
Mouse: Gem/
In this paper
N/A
Mouse: Per1-promoter-luc
Curr Biol. 10(14):873-6,2000.
N/A
pGL3-Promoter
Promega
E1751
pRL-CMV Renilla reporter
Promega
E2261
GraphPad
https://www.graphpad.com/
Critical commercial assays
Dual-Luciferase Reporter Assay System
Experimental models: Organisms/strains
Recombinant DNA
Software and algorithms
Prism 5
RESOURCE AVAILABILITY
Lead contact
Further information and requests for reagents should be directed to and will be fulfilled by Hitoshi Okamura (e-mail: okamura.hitoshi.
4u@kyoto-u.ac.jp).
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Materials availability
All unique/stable reagents generated in this study are available from the lead contact without restriction.
Data and code availability
d All data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
We developed and mated male and female C57BL6 backcrossed Gem knockout mice (Gem/) and Gem+/+ mice in housing conditions under 12-h light: 12-h dark (LD) cycles at 22 ± 2 C, with food and water provided ad libitum. We used male mice to avoid the
effect of sexual cycle on circadian rhythms. At 8–12 weeks of age, mice were transferred to individually housed conditions under 12-h
light: 12-h dark (LD) cycles and kept there for at least one week before the behavioral analysis. For electrophysiological studies, adult
Gem/ and Gem+/+ mice (6- to 10-week-old) were housed in the same condition for at least 2 weeks prior to experiments. For organotypic slice culture study, we sampled SCN from both sexes of neonatal (4- to 7- day old) Gem/ and Gem+/+ mice harboring
Per1-luc transgene, and luciferase experiments were performed after 2 to 4 weeks of initiation of culture.
All animal experimental procedures were pre-approved by the Animal Experimentation Committee at Kyoto University and the
Institutional Animal Care and Use Committee of RIKEN Kobe Branch. Experimental protocols for mice were in accordance with
the guidelines for animal experiments approved by the Animal Ethics Committee at Kyoto University and RIKEN Regulations for
the Animal Experiments.
METHOD DETAILS
Screening strategy of SCN gene project
We first screened the involvement of RGK protein expressions in the SCN by in situ hybridization at various circadian time point by a
screening strategy called SCN-Gene Project (Okamura, 2007), in which we already found key signaling molecules for the circadian
generation and synchronization of cellular clocks in the SCN (Doi et al., 2011; Yamaguchi et al., 2013,; Matsuo et al., 2021). In the
project, we (i) used histochemistry to identify genes whose expression is enriched in the mouse SCN, (ii) generated mutant mice lacking candidate genes of interest, and (iii) measured the locomotor activity at various environmental conditions.
Generation of gem/ mice
The Gem knockout mice (Accession No. CDB0673K: http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) were generated. The
targeting construct, containing a part of the first exon and full range of the second exon of Gem, was prepared using genomic DNA fragments from a BAC clone. Briefly, 7.0 kb adjacent upstream of Gem and 3.4 kb downstream of Gem was amplified by PCR and sub-cloned
into PGK-Neo-pA/DT-ApA vector and verified by full sequencing. The targeting constructs were linearized with NotI and electroporated
into the TT2 embryonic stem (ES) cells (Yagi et al., 1993). The construct was introduced into C57BL/6/CBA background ES cells, and two
independent chimeric mouse lines were established. These lines were independently crossed with C57BL/6 mice to produce F1N0 heterozygous mice, and then crossed further for at least three times before use in experiments. Gem/ mice did not have gross abnormalities. They were fertile and were born at the expected Mendelian ratio. Disruption of Gem in the genome was confirmed by Southern blotting, conventional PCR, and in situ hybridization. We further confirmed targeting of Gem by western blotting with micro-punched SCN
extracts.
Genotypes were determined by Southern blot and PCR. NheI-digested DNA was Southern blotted and hybridized with a 32Plabeled external probe. Genotyping primers we used for detecting WT and KO alleles were 50 -CCGTGCATTGGCTTTATC-TT-30
(Fw primer for WT), 50 -TCGCCTTCTTGACGAGTTCT-30 (Fw primer for KO), and 50 -AGCTCAGGCCTCCTAAGTCC-30 (common
Rev primer for WT and KO).
Gem activates Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction (Krey et al.,
2013; Ward et al., 2002). Gem-knockout mice (Gem/) develop normally at young ages. We also assessed behaviors at 8–10weeks-of-age and found no evidence of significant neurological impairment such as cerebellar ataxia, vestibular ataxia, or chorea.
Examination of animal behavior
To determine the impact of Gem deficiency on the circadian clock system, we monitored the circadian activity of mice and their response
to a brief light pulse at 8-12-weeks-of-age. Gem/ mice and their Gem+/+ littermates were individually housed in circadian activity-monitoring chambers. Food and water were given ad libitum and daily activity of each mouse was recorded using infrared sensors. Locomotor
activity was detected with passive (pyroelectric) infrared sensors (FA-05 F5B; Omron), and the data obtained were analyzed with Clocklab software (Actimetrics) developed on MatLab (Mathworks). Mice were initially maintained on 12 h light/12 h dark (LD) cycles for at least
10 days to establish entrainment before being transferred to constant darkness (DD). A free-running period was determined with a linear
regression line fit to the activity onset of 14 consecutive days in DD.
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For the phase-dependent phase-shift experiments, mice in DD were exposed to a 30 min light pulse (200 lux) at eight different
designated time points: CT2, 5, 8, 11, 14, 17, 20, and 23. The effect of light intensity on phase-shift was examined by changing light
intensities (2, 30, 200, and 1000 lux) at CT14. Phase-shifts were quantified as the time difference between regression lines of activity
onset before and after light application. Regression lines were made based on the activity onset 7 days before light stimulation, and
7 days activity onset starting 3 days after the light stimulation.
All animal procedures described in this study were approved by the Animal Research Committee of Kyoto University (2010-43) and
the Committee for Animal Research of Kobe University (P060601).
Electrophysiological recordings
For electrophysiological studies, adult mice (6- to 10-week-old) were entrained for at least 2 weeks on a 12 h light/dark schedule prior
to experiments. SCN slices (250 mm-thick) were prepared with a Microslicer (DTK-300K, Dohsaka EM, Japan) in ice-cold reduced Ca2+
artificial cerebrospinal fluid (aCSF) containing (in mM): 120.0 NaCl, 2.5 KCl, 1.2 NaH2PO4, 10.0 glucose, 26.0 NaHCO3, 5.0 MgCl2, and
0.5 CaCl2 (Chuhma et al., 2001). Whole-cell patch-clamp recordings in voltage-clamp mode from SCN neurons were carried out. SCN
neurons were randomly selected. Patch pipettes were prepared from thick-walled borosilicate glass to a tip resistance of 5–8 MU. The
intracellular recording medium contained (in mM): 155.0 Cs-methanesulfonate, 5.0 NaCl, 10.0 HEPES, 0.2 ethylenediaminetetraacetic
acid (EGTA), and 3.0 MgCl2. Corrections were made for the liquid junction potential (18 mV). To reduce large Na+ currents and Cl
currents in SCN neurons, tetrodotoxin (TTX, 1 mM) and bicuculline (40 mM) were added to the recording medium. The membrane potential was held at 80 mV, and voltage steps were applied between 90 and +70 mV per 10 mV step. Active currents were generated
from approximately 20 mV. Current is normalized to cell membrane capacitance to produce current density for each cell (pA/pF).
Luciferase activity
Organotypic SCN slice cultures from Per1-luc neonatal transgenic mice (4- to 7-day-old) were obtained as described previously (Yamaguchi et al., 2003, 2000). Gem+/ mice were crossed to Per1-luc transgenic mice to generate Per1-luc transgenic mice harboring either a
Gem/ or Gem+/+ genotype. Our bioluminescent system is able to determine the phase of the SCN with a high time-resolution (5 min). To
align the phase condition between slices, tissues were entrained with a 36 h-bath application of 10 mg/mL cycloheximide (CHX) (Yamaguchi et al., 2003), and the phase of the circadian clock in each SCN was determined by the peak time of Per1-luciferase activity. SCN
slice cultures were maintained in a sealed 24-well cell culture plate; during bioluminescence recording, each well contained 240 mL culture medium with 1 mM luciferin at 35 C. For high K+ (50 mM) stimulation, SCN slice cultures were transferred 6 h after the first peak to
control medium (50% minimum essential medium, 50% Hank’s balanced salt solution, 36 mM glucose, and penicillin/streptomycin),
with or without K+ (50 mM) and/or nifedipine (20 mM), for 30 min at 35 C. The tissues were then washed three times with control medium
for 30 min at 35 C, and returned to the original culture medium. Sample sizes were vehicle-control (n = 10), high K+ (n = 11), high K+ plus
nifedipine (n = 11), and nifedipine (n = 10), respectively. A one-way ANOVA was used to analyze these data.
Radioisotopic in situ hybridization
In situ hybridization was performed as previously described (Shigeyoshi et al., 1997). Briefly, paraformaldehyde-fixed brains were
frozen and cryoprotected. Serial coronal sections (40 mm-thick) were prepared from the rostral end to the caudal end of the SCN
with a cryostat. Free-floating tissue sections were then transferred through 4 3 saline sodium citrate (SSC) buffer, proteinase K
(1 mg/mL) in 0.1 M Tris buffer [pH 8.0], 50 mM EDTA for 15 min at 37 C, 0.25% acetic anhydride in 0.1 M triethanolamine for
10 min, and 4 3 SSC for 10 min. The sections were then incubated in hybridization buffer [55% formamide, 10% dextran sulfate,
10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 0.6 M NaCl, 0.2% N-laurylsarcosine, 500 mg/mL tRNA, 1 3 Denhardt’s, 0.25%
SDS, and 10 mM dithiothreitol (DTT)] containing radiolabeled riboprobes for 16 h at 60 C.
Radiolabeled molecular-specific riboprobes were prepared based on the following sequences: for Gem, nucleotides 254–988 of
Gem (NM_010276.4); for Rad, nucleotides 1–543 of (NM_019662.1); for Rem1, nucleotides 100–629 of (NM_009047.4); for Rem2, nucleotides 103–700 of (NM_080726.2); for Cacna1s, nucleotides 2291–2851 of (XM_358335.4); for Cacna1c, nucleotides 5470–5914 of
(NM_009781.3); for Cacna1d, nucleotides 5504–5979 of (NM_028981.2); for Cacna1f, nucleotides 4837–5261 of (NM_019582.2); for
Cacna1a, nucleotides 3339–3736 of (NM_007578.3); for Cacna1b, nucleotides 5910–6406 of (NM_007579.1); for Cacna1e, nucleotides 1353–1832 of (AK171983.1); for Cacna1g, nucleotides 7155–7608 of (NM_009783.1); for Cacna1h, nucleotides 6968–7397 of
(NM_021415.3); for Cacna1i, nucleotides 6874–7255 of (NM_001044308.2); for Cacna2d1, nucleotides 2141–2683 of
(NM_001110843.1); for Cacna2d2, nucleotides 1172–1643 of (NM_001174047.1); for Cacna2d3, nucleotides 2443–2986
of (NM_009785.1); for Cacnb1, nucleotides 193–748 of (NM_031173.3); for Cacnb2, nucleotides 1434–2003 of (NM_023116.4); for
Cacnb3, nucleotides 1699–2179 of (NM_007581.2); for Cacnb4, nucleotides 1278–1799 of (NM_001037099.1). The corresponding
cDNA fragment was cloned and used as a template for the generation of riboprobes. The riboprobes were radiolabeled with [33P]
UTP (PerkinElmer, Waltham, MA), using a standard protocol for the cRNA synthesis. Following a high-stringency post-hybridization
wash, the sections were treated with RNase A. Air-dried sections were exposed to X-ray film (Kodak Biomax).
Autoradiographic films (Kodak, Biomax) were quantified with MCID imaging software (Imaging Research Inc., Canada) after conversion into the relative optical densities using 14C-autoradiographic microscales (Amersham, UK); ten SCN sections were then
summed.
Cell Reports 39, 110844, May 24, 2022 e3
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In situ hybridization of gem with digoxigenin-labeled riboprobes
Digoxigenin-labeled probes allow for a better resolution than isotope probes for analyzing the cellular distribution of mRNA. We,
therefore, prepared digoxigenin-labeled antisense cRNA probes using digoxigenin-UTP (Roche Diagnostics) following a standard
protocol for cRNA synthesis. Tissue preparation, pre-hybridization, hybridization, and post-hybridization washing were identical
as in isotope probe hybridization, with the exception that we used 20 mm-thick sections of the SCN. Brain sections hybridized
with the digoxigenin-labeled probe were processed for immunochemistry with a nucleic acid detection kit (Roche Diagnostics). Signals were visualized in a solution containing nitroblue tetrazolium salt (0.34 mg/mL) and 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt (0.18 mg/mL) (Roche Diagnostics).
Immunohistochemistry for pERK and cFos
For immunohistochemical labeling of pERK and cFos, animals were anesthetized and systemically perfused with 25 mL of cold fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (Doi et al., 2011). Isolated brains were transferred to the same
fixative for 12 h at 4 C, and then immersed in 20% sucrose in 0.1 M PB for cryoprotection. Coronal brain cryosections (20 mm-thick)
were processed for free-floating immunohistochemistry. We used a rabbit polyclonal antibody specific for the phosphorylated forms
of ERK 1 and 2 (Cell Signaling; catalog code 9101) and a rabbit antibody for cFos protein identification (Abcam; catalog code 7963).
Free-floating sections were pretreated with hydrogen peroxide (1.5% in 0.1 M PB, for 20 min at 4 C) and blocked with 5% horse
serum (in 0.1 M PB) for 1 h at room temperature. The sections were then incubated with pERK antibody [1:500 dilution, in 0.1 M
PB containing 0.3% Triton X-100 (PBX)], or with cFos antibody [1:8000] for 12 h at room temperature. After washing with PBX,
the sections were incubated with a biotinylated anti-rabbit IgG secondary antibody (1:500 dilution in PBX) (Vector Laboratories)
for 1 h at room temperature. The tissues were then subjected to standard avidin-biotin-immunoperoxidase staining (Vectorstain Elite
ABC kit, Vector Laboratories). Immunoreactivity was visualized with 3,30 -diaminobenzidine (DAB). Immunostained sections were
then washed with 50 mM Tris-HCl buffer (pH 7.5), dehydrated in ethanol, and coverslipped with Entellan mounting medi ...
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