7 research outputs found
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. (BâC) 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 Studentâs t-test. For panel D, at each time point after the food pulse, a Studentâs t-test is employed to compare the two conditions (* represents a significant statistical difference of p<0.05).</p
Random feeding in LD and DD alters cell cycle gene oscillations.
<p>(A) Entrained to a LD cycle, the rhythm and amplitude of <i>per1</i> expression is not altered by the feeding regime, either normal (NF) or random (RF). In DD and NF, <i>per1</i> is rhythmic, but under RF and DD conditions, <i>per1</i> ceases to show a robust, precise daily rhythm. (B) <i>Cyclin</i> gene expression (<i>cyB1</i>, <i>cyB2</i> and <i>cyE1</i>) shows a large reduction in the level of expression and very shallow oscillations in RF compared to NF. Under RF, expression of <i>cdc2</i>, <i>wee1</i>, <i>PCNA</i> and <i>cdk2</i> displays a similar rhythmicity to NF, but with significantly reduced amplitude. <i>p21</i> expression is not altered by the feeding regime. (C) When fish free-run in DD and are fed at random times, all the genes studied (<i>cyB1</i>, <i>cyB2</i>, <i>cdc2</i>, <i>wee1</i>, <i>PCNA</i>, <i>cdk2</i> and <i>cyE1</i>), except <i>p21</i>, display a disrupted profile compared to NF. No clear rhythmicity is observed. White and grey backgrounds represent light and dark phases, respectively. Uninterrupted grey backgrounds represent constant dark condition. Data represents the mean ± SEM from 3 or 4 fish per time point. For panels A, B and C, NF data are compared to RF at each time point using a Studentâs t-test (* represents a significant statistical difference of p<0.05).</p
M phase is affected by starvation.
<p>(A) Cell division is rhythmic under a normal feeding schedule (NF) (peak and trough shown), but this rhythm is lost when fish are starved (SF). DAPI is used here as a nuclear counterstain. (B) Cell division is largely abolished when no food is given. (C) The <i>per1</i> rhythm is unaltered in NF and SF fish. However, all the M-phase genes studied (<i>cyB1</i>, <i>cyB2</i>, <i>cdc2</i> and <i>wee1</i>) and most G1/S-phase genes (<i>PCNA</i>, <i>cdk2</i> and <i>cyE1</i>) show reduced levels of expression, and a general loss of rhythmicity during starvation. <i>p21</i> expression is the one exception, showing a relatively small response to starvation. White and grey backgrounds represent light and dark phases, respectively. Cell cycle gene data represents the mean ± SEM from 8 to 12 fish per time point. For panels B and C, NF data are compared to SF using a Studentâs t-test (* p<0.05, ** p<0.01 and *** p<0.001).</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
Zebrafish intestine possesses a directly light-responsive circadian pacemaker.
<p>(A) After entrainment to a LD cycle (14L:10D), the expression of the core clock component <i>per1</i> is rhythmic in the intestine with a peak at ZT3. The oscillation is maintained when animals free-run in DD. Data represents the mean ± SEM from 8 fish per indicated zeitgeber or circadian time (ZT or CT), where ZT0 is lights on. (BâBâ) A three-hour light pulse induces expression of <i>cry1a</i> and <i>per2</i> compared to a dark control. Data represent the mean ± SEM from 5 fish. (C) The adult intestine of <i>per3-luciferase</i> zebrafish entrained to 4 days of LD, 4 days of DD and returned to 4 days in LD was monitored. Gut <i>per3</i> expression is rhythmic in LD with a peak at ZT5 and free-runs in DD with a damped amplitude. The mean bioluminescence in counts per seconds (CPS) is plotted (n=3-4). (D) Intestine of adult <i>per3-luciferase</i> zebrafish were entrained to 5 days of LD then transferred in DL for 6 days. Gut <i>per3</i> is able to re-entrain to a new, reversed light regime. The mean bioluminescence in CPS is plotted (n=3-4). White and grey backgrounds represent light and dark phases, respectively.</p
Pituitary Hormones mRNA Abundance in the Mediterranean Sea Bass Dicentrarchus labrax: Seasonal Rhythms, Effects of Melatonin and Water Salinity
In fish, most hormonal productions of the pituitary gland display daily and/or seasonal rhythmic patterns under control by upstream regulators, including internal biological clocks. The pineal hormone melatonin, one main output of the clocks, acts at different levels of the neuroendocrine axis. Melatonin rhythmic production is synchronized mainly by photoperiod and temperature. Here we aimed at better understanding the role melatonin plays in regulating the pituitary hormonal productions in a species of scientific and economical interest, the euryhaline European sea bass Dicentrarchus labrax. We investigated the seasonal variations in mRNA abundance of pituitary hormones in two groups of fish raised one in sea water (SW fish), and one in brackish water (BW fish). The mRNA abundance of three melatonin receptors was also studied in the SW fish. Finally, we investigated the in vitro effects of melatonin or analogs on the mRNA abundance of pituitary hormones at two times of the year and after adaptation to different salinities. We found that (1) the reproductive hormones displayed similar mRNA seasonal profiles regardless of the fish origin, while (2) the other hormones exhibited different patterns in the SW vs. the BW fish. (3) The melatonin receptors mRNA abundance displayed seasonal variations in the SW fish. (4) Melatonin affected mRNA abundance of most of the pituitary hormones in vitro; (5) the responses to melatonin depended on its concentration, the month investigated and the salinity at which the fish were previously adapted. Our results suggest that the productions of the pituitary are a response to multiple factors from internal and external origin including melatonin. The variety of the responses described might reflect a high plasticity of the pituitary in a fish that faces multiple external conditions along its life characterized by marked daily and seasonal changes in photoperiod, temperature and salinity