Mutations in blind cavefish target the light-regulated circadian clock gene, period 2

Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.Universidad de Ferrara | Ref. FAR2014-201

Evolution shapes the responsiveness of the D-box enhancer element to light and reactive oxygen species in vertebrates

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.Universidad de Ferrara | Ref. FAR2014–201

Evolution shapes the responsiveness of the D-box enhancer element to light and reactive oxygen species in vertebrates

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

A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception

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

Circadian Clock and OxInflammation: Functional Crosstalk in Cutaneous Homeostasis

Circadian rhythms are biological oscillations that occur with an approximately 24 h period and optimize cellular homeostasis and responses to environmental stimuli. A growing collection of data suggests that chronic circadian disruption caused by novel lifestyle risk factors such as shift work, travel across time zones, or irregular sleep-wake cycles has long-term consequences for human health. Among the multiplicity of physiological systems hypothesized to have a role in the onset of pathologies in case of circadian disruption, there are redox-sensitive defensive pathways and inflammatory machinery. Due to its location and barrier physiological role, the skin is a prototypical tissue to study the influence of environmental insults induced OxInflammation disturbance and circadian system alteration. To better investigate the link among outdoor stressors, OxInflammation, and circadian system, we tested the differential responses of keratinocytes clock synchronized or desynchronized, in an in vitro inflammatory model exposed to O3. Being both NRF2 and NF-κB two key redox-sensitive transcription factors involved in cellular redox homeostasis and inflammation, we analyzed their activation and expression in challenged keratinocytes by O3. Our results suggest that a synchronized circadian clock not only facilitates the protective role of NRF2 in terms of a faster and more efficient defensive response against environmental insults but also moderates the cellular damage resulting from a condition of chronic inflammation. Our results bring new insights on the role of circadian clock in regulating the redox-inflammatory crosstalk influenced by O3 and possibly can be extrapolated to other pollutants able to affect the oxinflammatory cellular processes

Circadian clock as possible protective mechanism to pollution induced keratinocytes damage

Ozone is among the most toxic environmental stressors to which we are continuously exposed. Due to its critical location, skin is one of the most susceptible tissues to oxidative stress damaging effect of ozone. An increasing collection of data suggests a significant role of circadian system in regulation of cellular response to oxidative stress. However, the molecular mechanism linking circadian clock and antioxidant pathway it is not completely understood. Here we investigated a possible protective role of entrained circadian clock to ozone induced damage in keratinocytes, the main cellular component of human epidermis. Our results showed that, clock-synchronized keratinocytes compared to arrhythmic ones exhibited a more efficient antioxidant response, attested by a faster activation of the master antioxidant regulatory factor NRF2. Moreover, analysis of clock gene expression profiles reveals a more rapid induction of the cardinal clock gene Bmal1 in entrained cells. Based on these findings, we suppose that an adequate coordination of circadian system and antioxidant pathway might be essential to maintain homeostasis in the skin. Alteration of metabolic pathways occurred in neurological diseases or in irregular schedule of life activity could negatively influence tissue gene expression programs and associated organ physiology via its effect on the circadian system

Daily rhythms of the expression of key genes involved in steroidogenesis and gonadal function in zebrafish

Fish present daily and seasonal rhythms in spawning and plasmatic levels of steroids that control reproduction. However, the existence of the rhythms of expression of the genes that underlie the endocrine mechanisms responsible for processes such as steroidogenesis and reproduction in fish have still been poorly explored to date. Here we investigated the daily pattern of the expression of key genes involved in sex steroid production that ultimately set the sex ratio in fish. Adult zebrafish were maintained under a 12:12 h light-dark cycle at a constant temperature of 27°C and were sampled every 4 h during a 24-hour cycle. The expression of key genes in the gonads and brains of female and male individuals were analyzed. In gonads, the expression of aromatase (cyp19a1a, ovarian aromatase) and the antimüllerian hormone (amh, testis) was rhythmic, with almost opposite acrophases: ZT 5:13 h (in the light phase) and ZT 15:39 h (at night), respectively. The expression of foxl2 (forkhead box L2) was also rhythmic in the ovary (acrophase located at ZT 5:02 h) and the expression of dmrt1 (doublesex and mab-3-related transcription factor 1) was rhythmic in testes (acrophase at ZT 18:36 h). In the brain, cyp19a1b (brain aromatase) and cyp11b (11beta-hydroxylase) presented daily differences, especially in males, where the expression peaked at night. These results provide the first evidence for marked time-of-the-day-dependent differences in the expression of the genes involved in sex ratio control, which should be considered when investigating processes such as reproduction, sex differentiation and steroidogenesis in fish

Size-selective mortality induces evolutionary changes in group risk-taking behavior and the circadian system in a fish

Marine Evolution 1st Italian Congress (EvolMar 2020), 23-25 November 2020Aim: Size-selective mortality is a major evolutionary driver of life-history and behavioral traits in human-dominated marine ecosystems. These changes might in turn alter the circadian system and therefore affect daily physiological processes and behavioral outputs. Methods: The proximate mechanisms governing evolution are difficult to disentangle in the wild. We overcome this limitation by studying the evolutionary impact of size-selective harvesting on group risk-taking behavior 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 halted. Results: Size-selective mortality affected fine-scale timing of behaviours. In particular, small size-selective mortality, typical of gape-limited predators targeting smaller size classes, increased group risk-taking behavior. 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 behavior and no clear changes in fine-scale timing of daily behavioral 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 suggesting the presence of a molecular switch. Main conclusion: To conclude, our experimental harvest left an asymmetrical evolutionary legacy in group risk-taking behavior and in fine-scale daily behavioral rhythms. Yet, the overall timing of activity showed evolutionary resistance probably maintained by a molecular switch. Our experimental findings suggest that human-induced changes in size selective mortality can have consequences for behavior and physiological processes in marine ecosystemsPeer reviewe