(a) A photograph and diagram showing the aVSOP method. Each stain was traced, scanned and quantified by Image J 1.42 software. (b–d) Female Cx43^+/− mice had larger functional bladder capacity than sex-matched Cx43^+/+ littermates. (b) A photograph of urine spots on paper made by Cx43^+/+ (top) and Cx43^+/− (bottom) mice. The scale bar indicates 10 cm, corresponding to 1 hour. (c) Representative charts of UVVM of Cx43^+/+ (top) and Cx43^+/− (bottom) mice under light/dark conditions for 4 days. UVVM, urine volume voided per micturition. (d) UVVM per 6 hours in Cx43^+/+ and Cx43^+/− mice had diurnal variation (F(3[degrees of freedom (DF) for the time factor],9[error DF])=12.3 and 10.9, respectively; *P < 0.005 by one-way repeated measures ANOVA; #P < 0.05 in the late light [sleep] phase vs. late dark [active] phase, followed by Bonferroni’s post hoc test). Maximal correlations from a cosine curve (MaxCorr) of Cx43^+/+ and Cx43^+/− mice were 0.949 and 0.989, respectively. ZT, zeitgeber time: light-on at ZT0 and off at ZT12. UVVM was significantly different between Cx43^+/+ and Cx43^+/− mice (F(1[DF for the strain factor],6[error DF])=11.2, P < 0.05 by two-way repeated measures ANOVA; †P < 0.05 vs. Cx43^+/+ by Bonferroni’s post hoc test; n=4 for each group, with a total of 296 micturitions). Error bars represent s.e.m. (e) Relative Cx43 mRNA levels of the urinary bladder in Cx43^+/− and Cx43^+/+ mice used in the micturition analysis by real-time RT-PCR. Error bars represent s.d., n=4 for each mice. The value of Cx43^+/+ was set as 1. *P < 0.05 by Student’s t-test. (f) Cx43 protein expression of the urinary bladder in Cx43^+/+ and Cx43^+/− mice.

(a) A representative chart of UVVM of WT C57BL/6 mice under light/dark (LD) conditions followed by constant dark (DD) conditions. (b) Temporal UVVM every 4 hours in WT mice (n=5), for 8 days under LD conditions (940 micturitions) and 5 days under DD conditions (556 micturitions). Diurnal variation of UVVM in LD conditions (F(5[degrees of freedom (DF) for the time factor],20[error DF])=17.28, **P < 0.005 by one-way repeated measures ANOVA) was also observed in DD conditions (F(5,20)=8.23, *P < 0.05), with no significant difference among times in LD vs. DD by two-way repeated measures ANOVA. (c, d) Loss of circadian rhythm of UVVM in Cry-null mice under DD conditions. Age-matched female WT, 1493 micturitions; Cry-null, 1009 micturitions, n=5 each. (c) A representative chart of UVVM of Cry-null mice. (d) Temporal UVVM every 4 hours in Cry-null (red-diamond) and WT (black-diamond) mice. Diurnal variation detected in WT mice (F(5,20)=8.21, P < 0.05 by one-way repeated measures ANOVA) was not observed in Cry-null mice. (e) Temporal Per2, Bmal1 and (f) Cx43 mRNA accumulation in the bladder in WT and Cry-null mice (n=3 for each time point). MaxCorrs of Per2, Bmal1 and Cx43 were (0.96, 0.93 and 0.85) in WT and (0.19, 0.42 and 0.38) in Cry-null mice, respectively. There was no significant difference in temporal Cx43 mRNA levels in Cry-null mice by one-way ANOVA. (g) Immunoblots showing temporal changes in protein levels of Cx43 in WT-mouse bladder (three independent samples for each time point). (h) Immunostaining of the muscle layer in mouse urinary bladder showing a difference in immunoreactivity with a decrease in Cx43 at CT4 compared to CT16. Representative photographs of three replicated experiments with similar results are shown. Bar, 50 μm. *P < 0.05 and **P < 0.01 by one-way ANOVA with Tukey’s post hoc test in f and g. Error bars represent s.e.m. in b and d, and s.d. in e–g. For the relative levels, the maximal values of WT were set as 1 in e and f. F(x,y), x=DF for the time factor; y=error DF in b and c. CT, circadian time.

(a) Patterns of UVVM in female Sprague-Dawley rats under LD conditions for 2 days (n=15, 1001 micturitions; F(2.7[degrees of freedom [DF] for the time factor],38.3[error DF])=11.9; *P < 0.005 by one-way repeated measures ANOVA with a Greenhouse-Geisser correction). (b) Temporal mRNA accumulation of Per2 Bmal1 and Cx43 in the rat bladder under LD and DD conditions (n=5 and n=2 for each time point, respectively). MaxCorrs were 0.87, 0.90 and 0.84 in LD conditions, respectively, and 0.98, 0.95 and 0.93 in DD conditions, respectively. (c) Temporal Cx43 protein accumulation in the rat bladder under DD conditions as shown by immunoblotting. (d) Immunostaining of Cx43 in the rat bladder under DD conditions (red, Cx43; blue, DAPI). The scale bar indicates 100 μm. Error bars represent s.e.m. in a and s.d. in b. For the relative expression, maximal values were set as 1 in b.

(a) Oscillation of luminescence in bladder ex vivo slice culture obtained from mPer2^Luciferase knock-in (Per2::luc) mice. The period of oscillation was 24.92 ± 0.56 (mean ± s.d.) (n=10). The muscle layer of the bladder is shown by alpha smooth muscle actin (αSMA) immunostaining. m, muscle. The scale bar indicates 100 μm. The oscillation of luminescence is also shown by a movie in Supplementary Movie 1. (b) Temporal variation of Per2, Bmal1 and Cx43 mRNA levels in serum-shocked rat bladder smooth muscle cells (BSMC). *P < 0.01 against the nadir of each genes’ mRNA levels (time 12 for Per2, time 48 for Bmal1 and time 64 for Cx43) by one-way ANOVA with Dunnett’s post hoc test (n=3–6). SS, serum shock. For relative levels, the values before serum shock were set as 1. (c) Immunoblots showing temporal changes in Cx43 protein levels with αSMA as a loading control in serum-shocked rat BSMC. (d) Immunostaining of Cx43 at times 12, 24, 36 and 48 hours in serum-shocked rat BSMC (red, Cx43; blue, DAPI). Arrow heads indicate typical plaques of gap junctions. Representative images of two replicate experiments with similar results are shown in c and d. (e) Oscillation of gap junction function evaluated by Lucifer yellow (LY) microinjection in serum-shocked rat BSMC. One representative photograph of each time point (green, LY; blue, Hoechst 33342) and overall quantification of the degree of dye-coupling (n=6–9, a total of 71 injections) is shown. *P < 0.05 and **P < 0.01 vs. time 24 hours by one-way ANOVA with Tukey-Kramer’s post hoc test. Similar significant differences were obtained in two independent experiments. Error bars represent s.e.m. Scale bars in d and e indicate 100 μm.

(a) Dose-dependent activation of Cx43 transcription by Rev-erbα in HEK293T cells (n=3 for each dose). (b) Impaired activation of Cx43 transcription by a mutant of Rev-erbα without 127–206 amino acids from the N-terminal (Rev-Mut) in HEK293T cells (n=3 for each dose). (c) Activation of Cx43 transcription by Rev-erbα in rat BSMC (n=6 for each dose). *P < 0.01 vs. Rev-erbα (−) by one-way ANOVA with Dunnett’s post hoc test in a–c. Similar data obtained in three independent experiments for a and b, and in two independent experiments for c. (d) Suppression of Cx43 expression by knock-down of endogenous Rev-erbα in BSMC. Three types of Rev-erbα siRNAs, containing high (si-1), middle (si-2) and low (si-3) GC ratios or their controls containing corresponding GC ratios were transfected. (n=4). Messenger RNA and protein expression (data of si-3) was normalized by 18s ribosomal RNA and GAPDH, respectively. Interference of Rev-erbα mRNA significantly decreased mRNA expression of Cx43 and increased Bmal1 compared with their corresponding controls (F(1[DF for the treatment factor],18[error DF])=324 for Rev-erbα, 9.7 for Cx43 and 11.7 for Bmal1. *P < 0.01 by two-way ANOVA). Temporal bladder Rev-erbα mRNA accumulation in WT and Cry-null mice (n=3). (e) and in rats under LD (n=5) and DD (n=2) conditions (f). *P < 0.05 vs. CT8 and **P < 0.01 vs. CT0, 16 and 20 in WT by one-way ANOVA with Tukey’s post hoc test. No significant difference in Cry-null mice. MaxCorrs were WT, 0.98; Cry-null, 0.31; rats in LD, 0.84; in DD, 0.93. The maximal value of WT was set as 1. Error bars represent s.d. in a–f. For relative levels, Rev-erbα (−) was set as 1 in a–c. CT, circadian time; ZT, zeitgeber time.

(a) Sequences including Sp1 sites are indispensable for Cx43 promoter activation by Rev-erbα. *P < 0.001 vs. the control of each construct and †P < 0.001 vs. −54 (without Sp1 sequences) construct by two-way ANOVA with Bonferroni’s post hoc test (n=3 for each). (b–e) Rev-erbα and Sp1 activate Cx43 expression using Sp1 sites. (b) Diagram of Cx43 promoter sequences including three Sp1 sites, labelled as Sp1A, B and C. The asterisk indicates corresponding nucleotide sequences among humans, rats and mice. (c) Dose dependent activation of Cx43 transcription by Sp1 and Rev-erbα with Sp1. *P < 0.001 vs. the value -without Sp1 and Rev-erbα, and †P < 0.001 by one-way ANOVA with Tukey’s post hoc test (n=3 for each). (d) Immunoblot analysis of the effect of Sp1 and Rev-erbα on expression of Cx43 and Bmal1 (control of negative regulatory effect by Rev-erbα). (e) Impaired activation of pCx43 with the Sp1 sites mutation by Sp1 and Rev-erbα. *P < 0.001 vs. the controls of each construct, and †P < 0.001 vs. the MutC construct by two-way ANOVA with Bonferroni’s post hoc test (n=3 for each group). Error bars represent s.d. in a, c and e. Cells used were HEK293T in all transfection experiments. The control without Rev-erbα and Sp1 was set as 1 in a, c and e. One representative of two experiments with similar results is shown in a, c, d and e.

(a) Co-immunoprecipitation showing a complex formation between HA-tagged Rev-erbα and DDDDK-tagged Sp1 transfected in HEK293T cells, using antibodies for HA and DDDDK. One representative of three experiments with similar results is shown. (b) Chromatin immunoprecipitation (ChIP) assay using antibodies for HA and DDDDK in HEK 293T cells transfected with HA-Rev-erbα and DDDDK-Sp1. Analyses by real-time RT-PCR are shown, targeted against endogenous Sp1 sites of the human Cx43 promoter and its negative control sites, which are approximately 7 kbp up-(5’) and 10 kbp down- (3’) stream from the transcription start site. A ChIP assay using an antibody for RNA polymerase II and primers for human GAPDH promoter was used as a positive control. One representative of two experiments with similar results is shown. (c) Temporal Sp1 mRNA accumulation in the mouse bladder (n=3 for each time point). There were no significant differences among time points by one-way ANOVA. (d) Oscillations of Rev-erbα mRNA (left) and protein expression (right) in serum-shocked rat BSMC (top row). *P < 0.01 vs. the nadir value (time 8) by one-way ANOVA with Dunnett’s post hoc test (n=3–6). SS, serum shock. The ChIP assay, using an antibody for endogenous Rev-erbα, was analysed by RT-PCR targeted against endogenous Sp1 sites of the rat Cx43 promoter and negative control sites, which are approximately 8 kbp up- (5’ negative) and down- (3’ negative) stream (bottom row). β-actin is a positive control. Results of real-time RT-PCR are added; it was targeted against Sp1 sites of the Cx43 promoter, which was immunoprecipitated using an antibody for Rev-erbα (corresponding to the framed bands, bottom). One representative of two experiments with similar results is shown. (e) A mechanistic scheme of Cx43 oscillation, controlled by the Rev-erbα and Sp1 complex binding to Sp1 sites of the Cx43 promoter. Error bars represent s.d. in c and s.e.m. in d. For relative levels, the maximal value was set as 1 in c and the values before serum shock (time 0) was set as 1 in d.