Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior

Interactions between the circadian clock and TGF-β signaling pathway in zebrafish

<div><p>Background</p><p>TGF-β signaling is a cellular pathway that functions in most cells and has been shown to play a role in multiple processes, such as the immune response, cell differentiation and proliferation. Recent evidence suggests a possible interaction between TGF-β signaling and the molecular circadian oscillator. The current study aims to characterize this interaction in the zebrafish at the molecular and behavioral levels, taking advantage of the early development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines.</p><p>Results</p><p><i>Smad3a</i>, a TGF-β signaling-related gene, exhibited a circadian expression pattern throughout the brain of zebrafish larvae. Both pharmacological inhibition and indirect activation of TGF-β signaling in zebrafish Pac-2 cells caused a concentration dependent disruption of rhythmic promoter activity of the core clock gene <i>Per1b</i>. Inhibition of TGF-β signaling in intact zebrafish larvae caused a phase delay in the rhythmic expression of <i>Per1b</i> mRNA. TGF-β inhibition also reversibly disrupted, phase delayed and increased the period of circadian rhythms of locomotor activity in zebrafish larvae.</p><p>Conclusions</p><p>The current research provides evidence for an interaction between the TGF-β signaling pathway and the circadian clock system at the molecular and behavioral levels, and points to the importance of TGF-β signaling for normal circadian clock function. Future examination of this interaction should contribute to a better understanding of its underlying mechanisms and its influence on a variety of cellular processes including the cell cycle, with possible implications for cancer development and progression.</p></div

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

<p>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.</p

The effect of CK1δ inhibition on rhythmic pineal <i>aanat2</i> mRNA expression is reversible.

<p>Reversibility was determined after two (A) or three (B) LD cycles for re-entrainment. <b>Top panels</b>: 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.</p

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

<p>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.</p

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

<p>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.</p

TGF-β inhibition disrupts circadian locomotor activity rhythms under light:Dim light cycles.

<p>Larval rhythmic locomotor activity under LDim was significantly disrupted (<i>p<0</i>.<i>05</i>, <i>t-</i>test), but not completely abolished, after treatment with the TGF-β inhibitor LY-374947 (20µM) in comparison with the DMSO control group. Embryos were raised under LD for 3 days, raised under LDim in the DanioVision chamber for 2 days, the inhibitor was added and locomotor activity (distance moved every 10 min) was monitored under LDim cycles. The data is presented as a moving average (10 sliding points) for each group (n = 24/group). 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 (upper panel). TGF-β inhibitor treated larvae exhibited significantly lower g-factor values in comparison with control larvae (<i>p<0</i>.<i>001</i>, Kolmogorov-Smirnov test), indicating that their locomotor activity is significantly less circadian LDim cycles. The median is represented for each group (red line). This experiment was repeated twice, resulting in similar outcomes. The represented results are of one experiment.</p

TGF-β inhibition abolishes clock-controlled rhythmic locomotor activity in zebrafish larvae.

<p>Clock-controlled rhythmic locomotor activity of zebrafish larvae under constant dim light was abolished after treatment with the TGF-β inhibitor LY-374947 (20µM) in comparison to a control group (DMSO). Embryos were raised under LD for 3 days, raised under LDim in the DanioVision chamber for 2 days, the inhibitor was then applied and locomotor activity (distance moved every 10 min) was monitored under constant Dim. The data is presented as a moving average (10 sliding points) for each group (n = 24/group). Larvae exhibited a significant reduction in the amplitude of rhythmic locomotor activity (<i>p<0</i>.<i>001</i>, <i>t-test</i>, bottom right panel). 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 (upper panel). TGF-β inhibitor-treated larvae exhibited significantly lower g-factor values (fitness to a circadian rhythm) in comparison to control larvae (<i>p<0</i>.<i>001</i>, Kolmogorov-Smirnov test), indicating that their locomotor activity is less circadian (bottom left panel). The median is represented for each group (red line).</p

Locomotor activity levels in response to dark flashes is not effected by TGF-β inhibition.

<p>Larvae were kept under LD cycles. On day 5 the inhibitor, or DMSO as control, was added on day 6. Larvae were subjected to 3 dark flashes of 10 seconds each, which are known to induce startle response, with 15 minutes intervals of light between flashes, and their activity was recorded (upper panel). No statistical difference was observed between the activity of control (DMSO) and the TGF-β inhibitor (LY-374947, 20μM) treated groups during the dark flashes (<i>p = 0</i>.<i>28</i>, <i>t</i>-test), indicating that TGF-β inhibition does not impair larval mobility (lower panel). Each line represents the average of three succeeding trials, which measured the average movement per second of each group of larvae, recorded from 10 second before the flash, during the flash, and 10 second after the flash. Black and white horizontal boxes represent the light phase and dark flashes, respectively. This experiment was repeated twice, resulting in similar outcomes. The represented results are of one experiment.</p

<i>Per1b</i> mRNA circadian expression pattern in zebrafish larvae is phase-shifted by TGF-β inhibition.

<p>Zebrafish larvae were treated with TGF-β inhibitor LY-364947 (20 μM), and the expression pattern of <i>Per1b</i> was evaluated by whole mount ISH. <i>Per1b</i> expression was detected throughout the head region and its circadian expression pattern was altered in the presence of the TGF-β inhibitor, exhibiting a phase delay of circadian expression in comparison to a control group (DMSO). <i>Per1b</i> mRNA expression was significantly affected by sampling time (<i>p<0</i>.<i>001</i>, two-way ANOVA), and by an interaction between treatment and sampling time (<i>p<0</i>.<i>001</i>, two-way ANOVA) (n = 15/group). (A) Schematic representation of the experimental design. The horizontal bars represent the light conditions before and during sampling; white boxes represent light and black boxes represent dark periods. <i>Bottom panel</i>: whole mount ISH signals for <i>Per1b</i> mRNA (dorsal views of the heads) of representative specimens. Grey bars represent subjective day and black bars represent subjective night. Circadian times are indicated for each sample. CT0 corresponds to “subjective lights on”, CT12 to “subjective lights-off”. (B) <i>Left</i>: Quantification of signal intensities in the heads of treated and control larvae. Values represent the mean ± SE optical densities of the head signals. <i>Right</i>: Different letters represent statistically different values within each treatment (<i>p<0</i>.<i>05</i>, one-way ANOVA, Tukey’s test). This experiment was repeated twice, resulting in similar outcomes. The represented results are of one experiment.</p