Evolution during millions of years in perpetual darkness leads to mutations in non-visual opsin genes (Melanopsin and TMT opsin) and an aberrant, blind circadian clock in cavefish.

Objective-Diurnal variations in levels of factor VII (FVII), FVIII, proteins C and S, antithrombin, plasminogen activator inhibitor-1, prothrombin fragment F 1ϩ2 , and D-dimers in healthy humans point to the existence of circadian rhythms of coagulation factors. We sought for temporal fluctuations of tissue factor pathway inhibitor (TFPI) activity in human and mouse plasma. Methods and Results-TFPI activity showed significant daily variations with highest levels in the morning in healthy men (ϩ11%) and in mice at the light-to-dark transition (ϩ63%), the beginning of the physically active period. Variations in FVII activity paralleled those in TFPI. In mice, the feeding schedule had a strong impact on these rhythms. Although restricted feeding and fasting shifted the peak of TFPI, the FVII peak disappeared. Investigation of temporal fluctuations in constant darkness indicated the existence of daily rhythms for TFPI and of true circadian rhythms for FVII. Conclusions-For the first time, we report, both in humans and mice, temporal variations in TFPI activity. The coherent variations in FVII and TFPI activity could interplay to maintain the coagulation equilibrium. The chronobiological patterns should be considered to analyze activity levels of these factors. Moreover, the mouse model could be exploited to investigate modifiers of coagulation rhythms potentially associated to morning peaks of cardiovascular events. Key Words: factor VII Ⅲ TFPI Ⅲ circadian Ⅲ feeding schedule Ⅲ mouse model F requencies of thromboembolic events in humans exhibit marked diurnal variations, 1-3 with peaks from morning to noon. Temporal variations in the occurrence of hemorrhagic events have also been reported. 4 Fluctuations in coagulation factor levels able to influence the hemostatic balance might contribute to these adverse outcomes. Diurnal rhythms in levels of factor VII (FVII), 5 FVIII, 6 proteins C and S 7 , antithrombin, 7 and plasminogen activator inhibitor (PAI)-1 8 have been described in healthy humans. Temporal oscillations in prothrombin fragment F 1ϩ2 5-6 and D-dimer, 6 markers of thrombin generation and fibrinolysis, have been also described. These variations could reflect the existence of circadian rhythms of blood coagulation factors. Circadian rhythms are the overt expression of an internal timing mechanism measuring daily time, with the fundamental adaptive function of providing optimal temporal organization of physiological processes in relation to the environment. 9 Because formal assessment of circadian rhythms in coagulation factor levels is hardly feasible in humans, a circadian control has been so far demonstrated in a mouse model for PAI-1 10 -11 and fibrinogen 12 mRNA expression.Among factors interacting with circadian rhythms, daily availability of food represents a major component. Several studies suggested that postprandial and fasting lipoproteins are associated with plasma levels or activation state of coagulation factors, and particularly of FVII 13-17 that plays a key role in the initiation of the clott...

The circadian clock is a highly conserved cell-autonomous mechanism that directs daily rhythms in most aspects of biology. Daily entrainment by environmental signals, notably light, is essential for its function. However, our understanding of the mechanisms and the evolution of photic entrainment remains incomplete. Fish represent attractive models for exploring how light regulates the circadian clock due to the direct light sensitivity of their peripheral clocks. Central to this property is the light induced expression of clock genes that is mediated by D-box enhancer elements. Here, using zebrafish cells, we reveal that the light responsive D-box enhancer serves as a nuclear target for reactive oxygen species (ROS). We demonstrate that exposure to short wavelengths of visible light triggers increases in ROS levels via NADPH oxidase activity. Elevated ROS activates the JNK and p38 MAP kinases and in turn, induces clock gene expression via the D-box. In blind cavefish and mammals, where peripheral clocks are no longer entrained by direct illumination, ROS levels are still increased upon light exposure. However, in these species ROS no longer induces D-box driven clock gene transcription. Thus, during evolution, alterations in ROS-responsive signal transduction pathways underlie fundamental changes in peripheral clock photoentrainment.

Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD + RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD + RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture

Intensive and trait‐selective mortality of fish and wildlife can cause evolutionary changes in a range of life‐history and behavioural traits. These changes might in turn alter the circadian system due to co‐evolutionary mechanisms or correlated selection responses both at behavioural and molecular levels, with knock‐on effects on daily physiological processes and behavioural outputs. We examined the evolutionary impact of size‐selective mortality on group risk‐taking behaviour and the circadian system in a model fish species. We exposed zebrafish Danio rerio to either large or small size‐selective harvesting relative to a control over five generations, followed by eight generations during which harvesting was halted to remove maternal effects. Size‐selective mortality affected fine‐scale timing of behaviours. In particular, small size‐selective mortality, typical of specialized fisheries and gape‐limited predators targeting smaller size classes, increased group risk‐taking behaviuor during feeding and after simulated predator attacks. Moreover, small size‐selective mortality increased early peaks of daily activity as well as extended self‐feeding daily activity to the photophase compared to controls. By contrast large size‐selective mortality, typical of most wild capture fisheries, only showed an almost significant effect of decreasing group risk‐taking behaviour during the habituation phase and no clear changes in fine‐scale timing of daily behavioural rhythms compared to controls. We also found changes in the molecular circadian core clockwork in response to both size‐selective mortality treatments. These changes disappeared in the clock output pathway because both size‐selected lines showed similar transcription profiles. This switch downstream to the molecular circadian core clockwork also resulted in similar overall behavioural rhythms (diurnal swimming and self‐feeding in the last hours of darkness) independent of the underlying molecular clock. To conclude, our experimental harvest left an asymmetrical evolutionary legacy in group risk‐taking behaviour and in fine‐scale daily behavioural rhythms. Yet, the overall timing of activity showed evolutionary resistance probably maintained by a molecular switch. Our experimental findings suggest that size‐selective mortality can have consequences for behaviour and physiological processes.