Codeset information

CodeSet information (gene names, accession numbers, target sequences) for the NanoString probe set used in this study

Gene expression data

Results obtained from the NanoString analysis of gene expression during zebrafish development (raw data, normalised data, and analysis)

Expression of light sensitive genes, <i>cry1a</i> and <i>per2</i>, in the adult zebrafish brain.

<p>A & C) Wild type adult zebrafish were kept in the dark for 3 days and then either exposed to light for 3 hours or remained in the dark. The zebrafish were killed at CT22 and their brain and hearts dissected, RNA extracted and qPCR performed. The majority of samples collected from different brain regions showed the light responsive genes, <i>cry1a</i> and <i>per2</i>, were increased in light pulsed tissues compared to dark control. Numerous brain parts showed an increase in <i>cry1a</i> and <i>per2</i> in the light pulsed samples (p<0.0001 and p = 0.0015 respectively, Two way ANOVA, n = 3). The light pulsed zebrafish had significantly higher c<i>ry1a</i> and <i>per2</i> levels in both the heart and brain (p<0.002, Two way ANOVA, n = 3). B & D) Wild type adult zebrafish brain parts, whole brains and hearts were cultured in L15-media for four days. Samples were exposed to a 3 hour light pulse or kept in the dark and collected at CT22, RNA extracted, and qPCR performed. <i>Cry1a</i> and <i>per2</i> is induced by light in both the brain and heart (p<0.0001, Two way ANOVA, n = 3–5). The light pulsed brain part cultures had significantly higher <i>cry1a</i> and <i>per2</i> levels than the dark controls (p<0.0001 and p = 0.0013 respectively, Two way ANOVA, n = 3). E) Wild type adult zebrafish were kept in the dark for 3 days and then either exposed to light for 3 hours or remained in the dark. The zebrafish were killed at CT22 and their brain dissected, fixed, frozen and sectioned. Chromogenic <i>in situ</i> hybridisation was performed to determine the location of <i>per2</i> mRNA expression. There was minimal or undetectable expression in the dark samples. In the light pulsed samples expression of <i>per2</i> was increased in the i) PPp and SCN, ii) PGZ, TL, Val_gra and Vam_gra, iii) Hc and Hd, iv) EG, CCe_gra, and LCa_gra.</p

Expression of <i>c-fos</i> in the adult zebrafish brain.

<p>A) Zebrafish were kept on 2 days of 14:10 LD followed by 2 days of DD, and brains dissected at the times indicated. RNA was extracted and qPCR was performed to evaluate the relative expression of <i>c-fos</i> mRNA. In LD there was a peak at ZT1 and ZT15 on day 1 and 2, (p<0.0001, One way ANOVA, n = 4–9). The statistical significance is shown from the post-hoc Dunnett's multiple comparison test, which used the calibrator Day 1 ZT9. In DD there was a peak at CT21 on day 3 and trough at CT9 on day 3 and day 4 (p<0.001, One way ANOVA, n = 4–9). The statistical significance is shown from the post-hoc Dunnett's multiple comparison test, which used the calibrator CT9 on day 4. The above white and black bars indicate the lighting schedule, and the shades of green reflect the light, dark and subjective dark phases. B) Adult zebrafish on a 14:10LD were given a 30 minute light pulse or kept in the dark at ZT21. Brains were dissected, RNA extracted, and qPCR performed to determine levels of <i>c-fos</i> mRNA as an indicator of neuronal activity. <i>C-fos</i> expression was five-fold higher in the brains of the light pulsed zebrafish (p<0.0001, unpaired two-tailed t-test, n = 7–8). C) <i>c-fos</i> is induced in specific brain regions in response to a light pulse in the night. Adult zebrafish maintained on a 14L:10D LD cycle were exposed to a 30-min light pulse at ZT21 or kept in the dark. <i>In situ</i> hybridisation was performed on brain sections to determine the levels of <i>c-fos</i> mRNA. Regions that show increased <i>c-fos</i> expression in response to light include i) PPp and SCN, ii) TeO, TL, Val_gra and Vam_gra and iii) Hc and Hd. iv) There is no change in expression in the rhombencephalon. Abbreviations: PPp (dorsal pretectum), SCN (suprachiasmatic nuclei), TeO (optic tectum), TL (torus longitudinalis), Val_gra (lateral valvula cerebelli), Vam_gra (medial valvula cerebelli), Hc (caudal hypothalamus) and Hd (dorsal hypothalamus).</p

Regional <i>per3</i> expression in the adult zebrafish brain.

<p>Adult zebrafish were kept on a 14:10LD cycle and brains were collected at ZT3 and ZT15. <i>In situ</i> hybridization was performed to show expression of <i>per3</i> mRNA. A) Schematics of the brain containing the diencephalon, mesencephalon and rhombencephalon are shown. At ZT3 there is expression of <i>per3</i> in the i) PPp and SCN, ii) PGZ, TL, Val_gra and Vam_gra, iii) CM, Hc and Hd, iv) EG, CCe_gra, and LCa_gra. At ZT15 there is either low or undetectable levels of <i>per3</i> in these same regions. B) The antisense (AS) probe shows the <i>per3</i> expression and the sense (S) control shows the background signal.</p

Circadian Rhythmicity and Light Sensitivity of the Zebrafish Brain

<div><p>Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the <i>period3 (per3)-luciferase</i> transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, <i>in situ</i> hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including <i>per3</i>, <i>cryptochrome1a</i> (<i>cry1a</i>) and <i>per2</i>. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of <i>c-fos,</i> a marker for neuronal activity, was examined. <i>c-fos</i> mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, <i>c-fos</i> shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker complexity that was not previously appreciated.</p></div

<i>Per3</i> is a highly rhythmic circadian clock gene in whole brains <i>in vivo</i> and <i>in vitro.</i>

<p>A) Zebrafish were kept on 2 days of 14:10 LD followed by 2 days of DD, and brains dissected at the times indicated. RNA was extracted and qPCR was performed to evaluate the relative expression of <i>per3</i> mRNA. In LD there was a peak at ZT3 and trough at ZT15 (p<0.0001, One way ANOVA, n = 7–10). In DD there was also a peak at CT3 and trough at CT15 (p<0.0001, One way ANOVA, n = 3–5). The statistical significance is shown from the post-hoc Dunnett's multiple comparison test, which used the calibrator day 1 ZT15 for LD conditions, and day 4 CT15 for DD conditions. B) Whole brains were dissected, cultured and kept on a 14:10 LD cycle for 3 days. Samples were collected at the times indicated, RNA extracted and qPCR performed to determine the expression of <i>per3</i> mRNA. There was a peak at ZT3, and a trough between ZT15 and ZT 21 (p<0.001, One-way ANOVA, n = 3–4). The statistical significance is shown from the Dunnett's multiple comparison post-hoc test are, using the trough, ZT21, on day 5 as the calibrator. C) Whole brains were dissected as above, cultured, but this time maintained on a 14:10 LD cycle for one day before being placed into constant darkness for two additional days. Samples were collected at the times indicated, mRNA extracted, and qPCR performed to measure the levels of <i>per3</i>. The rhythm persisted in <i>per3</i> for one cycle under free-running conditions <i>in vitro</i> before damping on the second cycle in the dark. The above white black bars represent the lighting conditions, with the different plotted histogram shades representing light, dark or subjective dark phases.</p

<i>Per3</i> rhythms in all isolated regional brain cultures from <i>per3-luc</i> zebrafish show entrainment to LD cycles and free-running in DD.

<p>Brains were dissected from adult <i>per3-luc</i> zebrafish and monitored for bioluminescence in either (A, C, E, G, J) 6 days of 12:12LD, 4 days of DD and 4 days back into LD, or (B, D, F, H, K) 7 days of 12:12LD followed by five cycles of 12:12DL. The mean bioluminescence in counts per second (CPS) is plotted (n = 3–4). All brain regions entrain to a 24-hour period in the LD cycle with a peak around ZT4–6 and free-run with a longer period in DD (p<0.0001, two-tailed paired t-test, n = 3–4 per tissue). All regions rapidly entrain to a new LD cycle or DL cycle with a peak at ZT3-5. Black and white boxes indicate the lighting regime and arrows indicate when this changes.</p

Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

<div><p>The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as <i>cdc2</i>, <i>wee1</i>, <i>p21, PCNA</i> and <i>cdk2</i>, but only weakly influences <i>cyclin B1, cyclin B2</i> and <i>cyclin E1</i> expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally.</p> </div

Intestinal circadian clock and cell cycle genes are food-entrainable in zebrafish.

<p>(A) In DD, clock genes <i>per1</i>, <i>cry1a</i> and <i>per2</i> are rhythmically expressed during restricted feeding, when food is provided at noon or midnight. The rhythms in clock gene expression retain a stable phase relationship between the two opposite feeding schedules. (B–C) Key cell cycle regulators show corresponding entrainment to the two opposite feeding regimes. <i>cdc2</i> peaks are observed at midnight for noon fed animals and at midday for the midnight fed animals. <i>wee1</i> expression shows peak values 6 hours after the feeding time. (D) The table illustrates the time difference between the feeding regime, noon or midnight, and peak expression for all the genes studied, as well as the phase difference between the two experiments. (E) A schematic of the food pulse experimental design. (F) There is no acute effect of feeding for <i>per1</i> and <i>cry1a</i> expression. In contrast, <i>per2</i> expression after 3h is increased compared to the unfed control. Red arrow represents timing of the food pulse. Grey backgrounds represent constant dark conditions. Data represents the mean ± SEM from 3 or 4 fish per time point. For panels A, B and C, samples collected at noon are compared to those collected at midnight using a Student’s t-test. For panel D, at each time point after the food pulse, a Student’s t-test is employed to compare the two conditions (* represents a significant statistical difference of p<0.05).</p