Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish

The authors show that a circadian clock that regulates locomotor activity in larval zebrafish develops gradually over the first 4 days of life and that exposure to entraining signals late in embryonic development is necessary for initiation of robust behavioral rhythmicity. When zebrafish larvae were transferred from a light-dark (LD) cycle to constant darkness (DD) on the third or fourth day postfertilization, the locomotor activity of almost all fish was rhythmic on days 5 to 9 postfertilization, with peak activity occurring during the subjective day. Rhythm amplitude was higher after four LD cycles than after three LD cycles. When embryos were transferred from LD to DD on the second day postfertilization, only about half of the animals later displayed statistically significant activity rhythms. These rhythms were noisier and of lower amplitude, but phased normally. When zebrafish were raised in DD beginning at 14 h postfertilization, only 22% of them expressed significant circadian rhythmicity as larvae. These rhythms were of low amplitude and phase-locked to the time of handling on the third day rather than to the maternal LD cycle. These results show that behavioral rhythmicity in zebrafish is regulated by a pacemaking system that is sensitive to light by the second day of embryogenesis but continues to develop into the fourth day. This pacemaking system requires environmental signals to initiate or synchronize circadian rhythmicity.     

Differential effects of transient constant light-dark conditions on daily rhythms of Period and Clock transcripts during Senegalese sole metamorphosis

Studies on the developmental onset of the teleost circadian clock have been carried out in zebrafish and, recently, in rainbow trout and Senegalese sole, where rhythms of clock gene expression entrained by light-dark (LD) cycles have been reported from the first days post fertilization. However, investigations of molecular clock rhythms during crucial developmental phases such as metamorphosis are absent in vertebrates. In this study, we documented the daily expression profile of Per1, Per2, Per3, and Clock during Senegalese sole pre-, early-, middle-, and post-metamorphic stages under LD 14:10 cycles (LD group), as well as under transient exposure to constant light (LL-LD group) or constant dark (DD-LD group) conditions. Our results revealed that robust rhythms of clock genes were maintained along the metamorphic process, although with declining amplitudes and expression levels. All daily profiles were affected by transient constant conditions, in particular Per1, Per3, and Clock amplitudes and Per2 acrophase. Rhythm parameters were progressively restored upon reversion to LD cycles but even after 9?d under cycling conditions, a prolonged effect on clock function was observed, especially in the LL-LD group. These results reflect the differential sensitivity of clock machinery of sole to transitory light cues, being Per1 and Per3 predominantly clock regulated and supporting the role of Per2 as part of the light input pathway. Interestingly, there is no reversal in the phase of clock gene rhythms between pre- and post-metamorphic animals that would be coincident with the switch from diurnal to nocturnal locomotor activity, which occurs in this species just before the beginning of this process. Whether specialized central pacemakers dictate the phase of locomotor activity or this control is exerted outside of the core clock mechanism remains to be elucidated. Our results emphasize the importance of maintaining cycling light-dark conditions in aquaculture practices during ontogeny of Senegalese sole. (Author correspondence: munoz.cueto@uca.es or carlos.pendon@uca.es)     

Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes)

One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.     

Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

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 cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 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.     

Daily rhythms of clock gene expression and feeding behavior during the larval development in gilthead seabream,Sparus aurata

Light is the main environmental time cue which synchronizes daily rhythms and the molecular clock of vertebrates. Indeed, alterations in photoperiod have profound physiological effects in fish (e.g. reproduction and early development). In order to identify the changes in clock genes expression in gilthead seabream larvae during ontogeny, three different photoperiods were tested: a regular 12L:12D cycle (LD), a continuous light 24L:0D (LL) and a two-phases photoperiod (LL?+?LD) in which the photoperiod changed from LL to LD on day 15 after hatching (dph). Larvae were sampled on 10, 18, 30 and 60 days post-hatch (dph) during a 24?h cycle. In addition to the expression of clock genes (clock, bmal1, cry1 and per3), food intake was measured. Under LD photoperiod, larvae feed intake and clock genes expression showed a rhythmic pattern with a strong light synchronization, with the acrophases occurring at the same hour in all tested ages. Under LL photoperiod, the larvae also showed a rhythmic pattern but the acrophases occurred at different times depending on the age, although at the end of the experiment (60 dph) clock genes expression and feed intake rhythms were similar to those larvae exposed to LD photoperiod. Moreover, the expression levels of bmal1 and cry1 were much lower than in LD photoperiod. Under the LL?+?LD photoperiod, the 10 dph larvae showed the same patterns as LL treatment while 18 and 30 dph larvae showed the same patterns as LD treatment. These results revealed the presence of internal factors driving rhythmic physiological responses during larvae development under constant environmental conditions. The LL?+?LD treatment demonstrates the plasticity of the clock genes expression and the strong effect of light as synchronizer in developing fish larvae.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Distinct signaling pathways from the circadian clock participate in regulation of rhythmic conidiospore development in Neurospora crassa

Several different environmental signals can induce asexual spore development (conidiation) and expression of developmentally regulated genes in Neurospora crassa. However, under constant conditions, where no environmental cues for conidiation are present, the endogenous circadian clock in N. crassa promotes daily rhythms in expression of known developmental genes and of conidiation. We anticipated that the same pathway of gene regulation would be followed during clock-controlled conidiation and environmental induction of conidiation and that the circadian clock would need only to control the initial developmental switch. Previous experiments showed that high-level developmental induction of the clock-controlled genes eas (ccg-2) and ccg-1 requires the developmental regulatory proteins FL and ACON-2, respectively, and normal developmental induction of fl mRNA expression requires ACON-2. We demonstrate that the circadian clock regulates rhythmic fl gene expression and that fl rhythmicity requires ACON-2. However, we find that clock regulation of eas (ccg-2) is normal in an fl mutant strain and ccg-1 expression is rhythmic in an acon-2 mutant strain. Together, these data point to the endogenous clock and the environment following separate pathways to regulate conidiation-specific gene expression.     

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)     

Light regulates the cell cycle in zebrafish

The timing of cell proliferation is a key factor contributing to the regulation of normal growth. Daily rhythms of cell cycle progression have been documented in a wide range of organisms. However, little is known about how environmental, humoral, and cell-autonomous factors contribute to these rhythms. Here, we demonstrate that light plays a key role in cell cycle regulation in the zebrafish. Exposure of larvae to light-dark (LD) cycles causes a range of different cell types to enter S phase predominantly at the end of the day. When larvae are raised in constant darkness (DD), a low level of arrhythmic S phase is observed. In addition, light-entrained cell cycle rhythms persist for several days after transfer to DD, both observations pointing to the involvement of the circadian clock. We show that the number of LD cycles experienced is essential for establishing this rhythm during larval development. Furthermore, we reveal that the same phenomenon exists in a zebrafish cell line. This represents the first example of a vertebrate cell culture system where circadian rhythms of the cell cycle are observed. Thus, we implicate the cell-autonomous circadian clock in the regulation of the vertebrate cell cycle by light.     

Rhythmic patterns in phagocytosis and the production of reactive oxygen species by zebrafish leukocytes

Multiple components of vertebrate immune systems have been shown to exhibit circadian fluctuations. While the zebrafish is currently generating a wealth of information on the molecular pacemakers that may control circadian rhythms, there have been no reports of rhythmic activity in zebrafish leukocytes. In this study, we found that phagocytosis and the production of reactive oxygen species by zebrafish leukocytes varied significantly throughout twenty-four hour periods. A distinct peak in cellular ROS levels occurred before dawn, while the kinetics of respiratory burst responses were least rapid at this time of day. Phagocytosis of E. coli peaked late in the day, whereas there was no daily variation in phagocytosis of S. aureus. As seen in other species, the number of bacteria ingested per cell peaked during the night. These data provide direct evidence of rhythmic immune system activity, and demonstrate that zebrafish can be a valuable model in which to study the relationships between circadian gene expression, systemic pacemakers, and the activity of vertebrate immune system cells.     

Circadian oscillations outside the optic lobe in the cricket Gryllus bimaculatus

Although circadian rhythms are found in many peripheral tissues in insects, the control mechanism is still to be elucidated. To investigate the central and peripheral relationships in the circadian organization, circadian rhythms outside the optic lobes were examined in the cricket Gryllus bimaculatus by measuring mRNA levels of period (per) and timeless (tim) genes in the brain, terminal abdominal ganglion (TAG), anterior stomach, mid-gut, testis, and Malpighian tubules. Except for Malpighian tubules and testis, the tissues showed a daily rhythmic expression in either both per and tim or tim alone in LD. Under constant darkness, however, the tested tissues exhibited rhythmic expression of per and tim mRNAs, suggesting that they include a circadian oscillator. The amplitude and the levels of the mRNA rhythms varied among those rhythmic tissues. Removal of the optic lobe, the central clock tissue, differentially affected the rhythms: the anterior stomach lost the rhythm of both per and tim; in the mid-gut and TAG, tim expression became arrhythmic but per maintained rhythmic expression; a persistent rhythm with a shifted phase was observed for both per and tim mRNA rhythms in the brain. These data suggest that rhythms outside the optic lobe receive control from the optic lobe to different degrees, and that the oscillatory mechanism may be different from that of Drosophila.     

Diurnal rhythm in expression and release of yolk protein in the testis of Spodoptera littoralis (Lepidoptera: Noctuidae)

Circadian clocks (oscillators) regulate multiple life functions in insects. The circadian system located in the male reproductive tract of Lepidoptera is one of the best characterized peripheral oscillators in insects. Our previous research on the cotton leafworm, Spodoptera littoralis, demonstrated that this oscillator controls the rhythm of sperm release from the testis and coordinates sperm maturation in the upper vas deferens (UVD). We demonstrated previously that a protein that functions as yolk protein in females is also produced in cyst cells surrounding sperm bundles in the testis, and is released into the UVD. Here, we investigated the temporal expression of the yolk protein 2 (yp2) gene at the mRNA and protein level in the testis of S. littoralis, and inquired whether their expression is regulated by PER-based molecular oscillator. We describe a circadian rhythm of YP2 accumulation in the UVD seminal fluid, where this protein interacts with sperm in a circadian fashion. However, we also demonstrate that yp2 mRNA and YP2 protein levels within cyst cells show only a diurnal rhythm in light/dark (LD) cycles. These rhythms do not persist in constant darkness (DD), suggesting that they are non-circadian. Interestingly, the per gene mRNA and protein levels in cyst cells are rhythmic in LD but not in DD. Nevertheless, per appears to be involved in the diurnal timing of YP2 protein accumulation in cyst cells.     

Circadian Rhythms of Embryonic Development and Hatching in Fish: A Comparative Study of Zebrafish (Diurnal), Senegalese Sole (Nocturnal), and Somalian Cavefish (Blind)

During early development, most organisms display rhythmic physiological processes that are shaped by daily changes in their surrounding environment (i.e., light and temperature cycles). In fish, the effects of daily photocycles and their interaction with temperature during early developmental stages remain largely unexplored. We investigated the existence of circadian rhythms in embryonic development and hatching of three teleost species with different daily patterns of behavior: diurnal (zebrafish), nocturnal (Senegalese sole), and blind, not entrained by light (Somalian cavefish). To this end, fertilized eggs were exposed to three light regimes: 12 h of light: 12 h of darkness cycle (LD), continuous light (LL), or continuous darkness (DD); and three species-appropriate temperature treatments: 24°C, 28°C, or 32°C for zebrafish and cavefish and 18°C, 21°C, or 24°C for sole. The results pointed to the existence of daily rhythms of embryonic development and hatching synchronized to the LD cycle, with different acrophases, depending on the species: zebrafish embryos advanced their developmental stage during the light phase, whereas sole did so during the dark phase. In cavefish, embryogenesis occurred within 24 h post fertilization (hpf) at the same pace during day or night. The hatching rhythms appeared to be controlled by a clock mechanism that restricted or “gated” hatching to a particular time of day/night (window), so that embryos that reached a certain developmental state by that time hatch, whereas those that have not wait until the next available window. Under LL and DD conditions, hatching rhythms and the gating phenomenon persisted in cavefish, in zebrafish they split into ultradian bouts of hatching occurring at 12–18-h intervals, whereas in sole DD and LL produced a 24-h delay and advance, respectively. Hatching rates were best under the LD cycle and the reported optimal temperature for each species (95.2?±?2.7% of the zebrafish and 83.3?±?0.1% of the cavefish embryos hatched at 28°C, and 93.1?±?2.9% of the sole embryos hatched at 21°C). In summary, these results revealed that hatching rhythms in fish are endogenously driven by a time-keeping mechanism, so that the day and time of hatching are determined by the interplay between the developmental state (temperature-sensitive) and the circadian clock (temperature-compensated), with the particular phasing being determined by the diurnal/nocturnal behavior of the species. (Author correspondence: javisan@um.es)     

Clock controls angiogenesis

Circadian rhythms control multiple physiological and pathological processes, including embryonic development in mammals and development of various human diseases. We have recently, in a developing zebrafish embryonic model, discovered that the circadian oscillation controls developmental angiogenesis. Disruption of crucial circadian regulatory genes, including Bmal1 and Period2, results in marked impairment or enhancement of vascular development in zebrafish. At the molecular level, we show that the circadian regulator Bmal1 directly targets the promoter region of the vegf gene in zebrafish, leading to an elevated expression of VEGF. These findings can reasonably be extended to developmental angiogenesis in mammals and even pathological angiogenesis in humans. Thus, our findings, for the first time, shed new light on mechanisms that underlie circadian clock-regulated angiogenesis.     

Light Activates Output from Evening Neurons and Inhibits Output from Morning Neurons in the Drosophila Circadian Clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

FEEDING ENTRAINMENT OF DAILY RHYTHMS OF LOCOMOTOR ACTIVITY AND CLOCK GENE EXPRESSION IN ZEBRAFISH BRAIN

Light and feeding cycles strongly synchronize daily rhythms in animals, which may, as a consequence, develop food anticipatory activity (FAA). However, the light/food entraining mechanisms of the central circadian oscillator remain unknown. In this study, we investigate the existence of FAA in seven groups of zebrafish subjected to a light/dark (LD) cycle or constant light (LL) and different feeding regimes (random, fasting, and feeding in the middle of the light phase or dark phase). The aim was to ascertain whether the daily rhythm of behavior and clock gene (per1 and cry1) expression in the zebrafish brain was entrained by the light and feeding regime. The results revealed that FAA developed in zebrafish fed daily at a fixed time, under LD and under LL. Zebrafish displayed locomotor activity mostly during the daytime, although the percentage of activity during the light phase varied depending on feeding time (ranging from 93.2% to 63.1% in the mid-light and mid-dark fed groups, respectively). However, the different feeding regimes failed to modify the daily rhythm of per1 and cry1 expression in the zebrafish brain under LD (approximate acrophases [peak times] at ZT22 and ZT4, respectively; lights-on =?ZT0). Under LL, per1 and cry1 expression did not show significant daily rhythmicity, regardless of the feeding regime. These findings indicate that, although schedule-fed zebrafish developed FAA as regards locomotor activity, feeding had little effect on clock gene expression in whole brain homogenates, suggesting the feeding-entrainable oscillator may be located elsewhere or at specific brain sites. (Author correspondence: jasanchez@um.es)     

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。