Casein kinase 1δ activity: a key element in the zebrafish circadian timing system

Abstract

Zebrafish have become a popular model for studies of the circadian timing mechanism. Taking advantage of its rapid development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines, much knowledge has been gained about the circadian clock system in this species. However, the post-translational modifications of clock proteins, and in particular the phosphorylation of PER proteins by Casein kinase I delta and epsilon (CK1δ and CK1ε), have so far not been examined in the zebrafish. Using pharmacological inhibitors for CK1δ and CK1ε, a pan-CK1δ/ε inhibitor PF-670462, and a CK1ε -selective inhibitor PF-4800567, we show that CK1δ activity is crucial for the functioning of the circadian timing mechanism of zebrafish, while CK1ε plays a minor role. The CK1δ/ε inhibitor disrupted circadian rhythms of promoter activity in the circadian clock-containing zebrafish cell line, PAC-2, while the CK1ε inhibitor had no effect. Zebrafish larvae that were exposed to the CK1δ/ε inhibitor showed no rhythms of locomotor activity while the CK1ε inhibitor had only a minor effect on locomotor activity. Moreover, the addition of the CK1δ/ε inhibitor disrupted rhythms of aanat2 mRNA expression in the pineal gland. The pineal gland is considered to act as a central clock organ in fish, delivering a rhythmic hormonal signal, melatonin, which is regulated by AANAT2 enzymatic activity. Therefore, CK1δ plays a key role in the circadian timing system of the zebrafish. Furthermore, the effect of CK1δ inhibition on rhythmic locomotor activity may reflect its effect on the function of the central clock in the pineal gland as well as its regulation of peripheral clocks.

Figure 1. CK1δ inhibition disrupts the PAC-2 cell circadian clock.

Dose-response of PF-670462, a pan-CK1δ/ε inhibitor and PF-4800567, a selective inhibitor of CK1ε, in the zebrafish PAC-2 cell line. Bioluminescence assay of cells transfected with per1b:Luc (A and C) or Ebox:Luc (B and D), and treated with PF-670462 (A and B) or with PF-4800567 (C and D) at the indicated time (arrow). Cells were maintained under LD cycles, and then the inhibitor was added to the cell culture 1.5 h before lights on (black arrows) at different concentrations (PF-670462: 0.5 μM- light blue line, 1 μM- blue line, 2 μM-green line, 5 μM- red line. PF-4800567: 5 μM- green line, 7.5 μM- light blue line and 10 μM- blue line). Controls were treated with DMSO (black line) or with culture medium (gray line). After three LD cycles cell were transferred to DD. Bioluminescence is plotted on the y-axis and time (hours) on the x-axis. White/black bars show the light and dark periods, respectively. Clock-controlled rhythmic promoter was disrupted by PF-67046 but not by PF-4800567, thus CK1δ activity appears to be important for peripheral circadian clock function.

Figure 2. The effect of CK1δ -inhibition on peripheral circadian clocks is reversible.

Bioluminescence assay of cells transfected with per1b:Luc (A) and Ebox:Luc (B). Cells were maintained under LD cycles, and then the inhibitor, PF-670462 (5 μM), was added to the cell culture 1.5 h before lights on (black arrows). Cells were maintained in LD conditions for 2 days after which the inhibitor was washed away 3.5 h before lights on (red arrows). After one LD cycle for re-entrainment, the cells were transferred to constant darkness for 24 h. Control cells were treated with DMSO. Bioluminescence is plotted on the y-axis and time (hours) on the x-axis. White/black bars show the light and dark periods, respectively. The clock-controlled rhythmic promoter activity reappeared immediately following removal of the inhibitor. Thus the effects of CK1δ inhibition are reversible.

Figure 3. CK1δ inhibition disrupts clock-controlled rhythmic locomotor activity in zebrafish larvae.

Locomotor activity of zebrafish larvae was detected by the DanioVision observation chamber. The PF-670462 inhibitor was added to the water at 5 dpf at different concentrations, A-0.5 μM, B-1 μM and C-5 μM (red lines). Control larvae were treated with DMSO at the same concentrations (black lines). The larvae were kept under constant conditions (dim light) and the distance moved (cm, y-axis) was recorded over time (hours, x-axis). The horizontal bars represent the light conditions before and during the experiment. White boxes represent light, black boxes represent dark and grey boxes represent dim light. The rhythm of locomotor activity was completely abolished by the CK1δ inhibitor.

Figure 4. CK1ε inhibition does not affect clock-controlled rhythmic locomotor activity in zebrafish larvae.

Locomotor activity of zebrafish larvae was detected by the DanioVision observation chamber. Distance moved (cm) is plotted on the y-axis and time (hours) on the x-axis. The horizontal bars represent the lighting conditions before and during the experiment. White boxes represent light, black boxes represent dark and grey boxes represent dim light. The PF-480567 inhibitor was added to the water at two different concentrations, A-5 μM and B-10 μM (blue lines). Control larvae were treated with DMSO at the same concentrations (black lines). CK1ε inhibition did not affect the locomotor activity rhythm, but did have a slight phase shifting effect.

Figure 5. CK1δ inhibition abolishes rhythmic pineal aanat2 mRNA expression.

A. Top panel: Schematic representation of the experimental design. The horizontal bars represent the light conditions before and during sampling; white boxes represent light, grey boxes represent subjective day and black boxes represent dark. Middle panel: Whole-mount ISH signals for aanat2 mRNA (dorsal views of the heads) of representative specimens treated with DMSO (control, a) or with PF-670462 (5 μM) (a). Zeitgeber times (ZT) are indicated for each sample. ZT0 corresponds to “lights-on,” ZT12 to “lights-off”. ZT2b refers to the second day of the experiment. ISH signals in the pineal gland are indicated by arrows. Bottom chart: Quantification of signal intensities in the pineal glands of control (DMSO) larvae (black line) and PF-670462-treated larvae (grey line). Values represent the mean ± SE optical densities of the pineal signals. Aanat2 mRNA expression is significantly affected by treatment and sampling times (P<0.01 by two way ANOVA). Differences in sampling time within each treatment were determined by one-way ANOVA followed by Tukey's post-hoc test for each treatment. B. Whole-mount ISH signals for otx5 mRNA at ZT2. Photographs of pineal signals in (a) DMSO (control) and (b) CK1δ-treated larvae are presented in the bottom panel.

Figure 6. The effect of CK1δ inhibition on rhythmic pineal aanat2 mRNA expression is reversible.

Reversibility was determined after two (A) or three (B) LD cycles for re-entrainment. Top panels: Experimental design. The horizontal bars represent the light conditions before and during sampling; white boxes represent light, grey boxes represent subjective day and black boxes represent dark. Middle panels: Whole-mount ISH signals (dorsal views of the heads) of representative specimens treated with DMSO (control, a) or with PF-670462 (b). ZT 2-10b refers to the second 24 h cycle of the sampling. Bottom panels: Signal intensities in the pineal gland. Values represent the mean ± SE optical density of the pineal signals. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test for each treatment. Following removal of the inhibitor and re-entrainment, for two (A) or three (B) LD cycles, the rhythm of aanat2 mRNA re-appeared but seemed to be shifted.