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

Clock-controlled rhythmic gene expression is disrupted in ΔCLK-expressing pineal glands.

<p>(A) Experimental procedure for transcriptome analysis. Adult fish were kept under DD and pineal glands were sampled at 12 time points (indicated by arrows) throughout two daily cycles. Black and gray bars correspond to subjective night and day, respectively. (B) The mRNA-seq analysis resulted in the identification of 29 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish compared with 290 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP) control fish.</p

Generation of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish.

<p>(A) Schematic representation of the transgenic construct used for the generation of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish. The <i>pT2-aanat2</i>:<i>EGFP-2A-Δclocka-5×MYC</i> plasmid consists of two arms from the Tol2 transposable element (black), <i>aanat2</i> regulatory regions (gray), EGFP coding sequence (CDS; green), 2A peptide sequence (orange), ΔCLK CDS (blue), 5×Myc tags (purple) and SV40 poly(A) signal (yellow). (B and B') EGFP expression is restricted to the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae. Dorsal views of the head region of 7-day post-fertilization (dpf) larvae, anterior to the top; confocal z-stack projection (B) and a single confocal plane (B'). (C) Immunostaining with anti-Myc antibody confirms that ΔCLK is specifically expressed in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae. Dorsal view of the head region of a 5-dpf larva, anterior to the top; confocal z-stack projection.</p

Diverse effects of ΔCLK on expression profiles of clock-controlled genes in the pineal gland.

<p>Representative examples of expression profiles of CCGs in the pineal gland of control Tg(<i>aanat2</i>:EGFP) fish (control; blue trendline) compared with the expression profiles of these genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish (ΔCLK; red trendline). Black and gray bars denote subjective night and day, respectively. CT, circadian time. While the majority of CCGs became arrhythmic (A–I), a few maintained their circadian profile in the Tg(<i>aanat2</i>:EGFP-ΔCLK) pineal gland (J–L). For some of the CCGs that became arrhythmic in the Tg(<i>aanat2</i>:EGFP-ΔCLK) pineal gland the overall basal expression levels remained relatively intermediate or high (A–D), whereas for others the expression was down-regulated (E–H) or abolished (I).</p

Circadian rhythms of <i>per1b</i> promoter activity in whole larvae are not affected by the pineal ΔCLK mutation.

<p>(A) Mean bioluminescence of Tg[<i>aanat2</i>:EGFP-ΔCLK;(−3.1)<i>per1b</i>::luc] larvae (ΔCLK; green trace; <i>n</i> = 23) and Tg(−3.1)<i>per1b</i>::luc larvae (control; black trace; <i>n</i> = 55), starting from 5 dpf for two daily cycles under DD. Circadian time (CT) is plotted on the x-axis. Gray and black bars represent subjective day and subjective night, respectively. Error bars represent SD. (B) Distribution of the G-factors (a representation of the extent to which the frequency of the luciferase activity pattern corresponds to a 24-hr period; see 'Fourier analysis' in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006445#pgen.1006445.s013" target="_blank">S1 Text</a>) of ΔCLK and control larvae. The median G-factor value for each group is indicated (red lines).</p

Clock-controlled rhythms of melatonin production are disturbed in ΔCLK-expressing pineal glands.

<p>Circadian rhythms of melatonin release from cultured pineal glands of Tg(<i>aanat2</i>:EGFP-ΔCLK) adult fish are disturbed under DD (<i>p</i><0.05, Kolmogorov-Smirnov test). Yellow bar represents the light period, black bars represent dark, and gray bars represent dark during subjective day. Values indicate the normalized amount of melatonin produced by the glands. Error bars represent SE (<i>n</i> = 3). CT, circadian time.</p

Circadian rhythms of locomotor activity under DD and DimDim, but not under LL or LD cycles, are affected by blocking the pineal clock.

<p>Analysis of locomotor activity of 6–8 dpf Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae (ΔCLK) and control larvae under various lighting conditions. A–D, left chart: The average distance moved (cm/10 min) is plotted on the y-axis and circadian time (CT) is plotted on the x-axis; error bars stand for SE (<i>n</i> = 24); black, white and diagonally lined bars represent dark, light and dim light, respectively. A–D, right chart: Distribution of the G-factors (see 'Fourier analysis' in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006445#pgen.1006445.s013" target="_blank">S1 Text</a>) of Tg(<i>aanat2</i>:EGFP-ΔCLK) and control larvae; the median G-factor value for each group is indicated (red lines). (A) Circadian rhythms of locomotor activity under DD, after entrainment by 5 LD cycles, are affected by blocking the pineal clock; a significant difference in the distribution of G-factors was found between Tg(<i>aanat2</i>:EGFP-ΔCLK) and control larvae (<i>p</i><0.0001, Kolmogorov-Smirnov test). (B) Circadian rhythms of locomotor activity under DimDim, after entrainment by 3 LD cycles and 2 light-dim light (LDim) cycles, are affected by blocking the pineal clock; a significant difference in the distribution of the G-factors was found between Tg(<i>aanat2</i>:EGFP-ΔCLK) and control larvae (<i>p</i><0.0001, Kolmogorov-Smirnov test). (C) Circadian rhythms of locomotor activity under LL, after entrainment by 5 LD cycles, are NOT affected by blocking the pineal clock. (D) Circadian rhythms of locomotor activity under LD cycles are NOT affected by blocking the pineal clock.</p

Clock-controlled <i>aanat2</i> mRNA rhythm is abolished in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae.

<p>(A) Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae (ΔCLK) exhibit arrhythmic pineal <i>aanat2</i> mRNA levels under DD compared with a robust rhythm in their WT siblings (<i>p</i><0.0001, two-way ANOVA), indicating that the pineal gland molecular clock is disrupted. Each value represents the mean optical density ± SE of the pineal <i>aanat2</i> whole-mount ISH signal (<i>n</i> = 10−15 larvae). Different letters represent statistically different values (<i>p</i><0.05, Tukey's test). (B) Representative whole-mount ISH <i>aanat2</i> signals (8 dpf, dorsal view) in the pineal glands of WT sibling larvae (upper panel) and Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae (ΔCLK; bottom panel). Black and gray bars represent subjective night and day, respectively. CT, circadian time.</p