Role of melanocortin system in the locomotor activity rhythms and melatonin secretion as revealed by agouti-signalling protein (asip1) overexpression in zebrafish.

Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish.

Regulation of ddb2 expression in blind cavefish and zebrafish reveals plasticity in the control of sunlight-induced DNA damage repair.

We have gained considerable insight into the mechanisms which recognize and repair DNA damage, but how they adapt to extreme environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the evolution of DNA repair in the complete absence of UV-induced DNA damage and light. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. Here, we explore whether other systems repairing UV-induced DNA damage have been similarly affected in this cavefish model. By performing a comparative study using P. andruzzii and the surface-dwelling zebrafish, we provide evidence for a conservation of sunlight-regulated Nucleotide Excision Repair (NER). Specifically, the expression of the ddb2 gene which encodes a key NER recognition factor is robustly induced following exposure to light, UV and oxidative stress in both species. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Interestingly, despite the loss of D-box-regulated photolyase gene expression in P. andruzzii, the D-box is required for ddb2 induction by visible light and oxidative stress in cavefish. However, in the cavefish ddb2 gene this D-box-mediated induction requires cooperation with an adjacent, highly conserved E2F element. Furthermore, while in zebrafish UV-induced ddb2 expression results from transcriptional activation accompanied by stabilization of the ddb2 mRNA, in P. andruzzii UV induces ddb2 expression exclusively via an increase in mRNA stability. Thus, we reveal plasticity in the transcriptional and post transcriptional mechanisms regulating the repair of sunlight-induced DNA damage under long-term environmental challenges.

Photoreceptor Diversification Accompanies the Evolution of Anthozoa.

Anthozoan corals are an ecologically important group of cnidarians, which power the productivity of reef ecosystems. They are sessile, inhabit shallow, tropical oceans and are highly dependent on sun- and moonlight to regulate sexual reproduction, phototaxis, and photosymbiosis. However, their exposure to high levels of sunlight also imposes an increased risk of UV-induced DNA damage. How have these challenging photic environments influenced photoreceptor evolution and function in these animals? To address this question, we initially screened the cnidarian photoreceptor repertoire for Anthozoa-specific signatures by a broad-scale evolutionary analysis. We compared transcriptomic data of more than 36 cnidarian species and revealed a more diverse photoreceptor repertoire in the anthozoan subphylum than in the subphylum Medusozoa. We classified the three principle opsin classes into distinct subtypes and showed that Anthozoa retained all three classes, which diversified into at least six subtypes. In contrast, in Medusozoa, only one class with a single subtype persists. Similarly, in Anthozoa, we documented three photolyase classes and two cryptochrome (CRY) classes, whereas CRYs are entirely absent in Medusozoa. Interestingly, we also identified one anthozoan CRY class, which exhibited unique tandem duplications of the core functional domains. We next explored the functionality of anthozoan photoreceptors in the model species Exaiptasia diaphana (Aiptasia), which recapitulates key photo-behaviors of corals. We show that the diverse opsin genes are differentially expressed in important life stages common to reef-building corals and Aiptasia and that CRY expression is light regulated. We thereby provide important clues linking coral evolution with photoreceptor diversification.

Medaka as a model for seasonal plasticity: Photoperiod-mediated changes in behaviour, cognition, and hormones.

Teleosts display the highest level of brain plasticity of all vertebrates. Yet we still know little about how seasonality affects fish behaviour and the underlying cognitive mechanisms since the common neurobehavioral fish models are native to tropical environments where seasonal variation is absent or reduced. The medaka, Oryzias latipes, which inhabits temperate zone habitats, represents a promising model in this context given its large phenotypic changes associated with seasonality and the possibility to induce seasonal plasticity by only manipulating photoperiod. Here, we report the first extended investigation of seasonal plasticity in medaka behaviour and cognition, as well as the potential underlying molecular mechanisms. We compared medaka exposed to summer photoperiod (16 h light:8 h dark) with medaka exposed to winter photoperiod (8 h light:16 h dark), and detected substantial differences. Medaka were more active and less social in summer photoperiod conditions, two effects that emerged in the second half of an open-field and a sociability test, respectively, and might be at least in part related to habituation to the testing apparatus. Moreover, the cognitive phenotype was significantly affected: in the early response to a social stimulus, brain functional lateralisation shifted between the two hemispheres under the two photoperiod conditions, and inhibitory and discrimination learning performance were reduced in summer conditions. Finally, the expression of genes encoding key pituitary hormones, tshß and gh, and of the tshß regulatory transcription factor tef in the brain was increased in summer photoperiod conditions. This work reveals remarkable behavioural and cognitive phenotypic plasticity in response to photoperiod in medaka, and suggests a potential regulatory role for the same hormones involved in seasonal plasticity of other vertebrates.

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day-night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator's period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep-wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Evolution Shapes the Gene Expression Response to Oxidative Stress.

Reactive oxygen species (ROS) play a key role in cell physiology and function. ROS represents a potential source of damage for many macromolecules including DNA. It is thought that daily changes in oxidative stress levels were an important early factor driving evolution of the circadian clock which enables organisms to predict changes in ROS levels before they actually occur and thereby optimally coordinate survival strategies. It is clear that ROS, at relatively low levels, can serve as an important signaling molecule and also serves as a key regulator of gene expression. Therefore, the mechanisms that have evolved to survive or harness these effects of ROS are ancient evolutionary adaptations that are tightly interconnected with most aspects of cellular physiology. Our understanding of these mechanisms has been mainly based on studies using a relatively small group of genetic models. However, we know comparatively little about how these mechanisms are conserved or have adapted during evolution under different environmental conditions. In this review, we describe recent work that has revealed significant species-specific differences in the gene expression response to ROS by exploring diverse organisms. This evidence supports the notion that during evolution, rather than being highly conserved, there is inherent plasticity in the molecular mechanisms responding to oxidative stress.

Cavefish eye loss in response to an early block in retinal differentiation progression.

The troglomorphic phenotype shared by diverse cave-dwelling animals is regarded as a classical example of convergent evolution. One unresolved question is whether the characteristic eye loss in diverse cave species is based on interference with the same genetic program. Phreatichthys andruzzii, a Somalian cavefish, has evolved under constant conditions in complete darkness and shows severe troglomorphic characteristics, such as complete loss of eyes, pigments and scales. During early embryonic development, a complete eye is formed but is subsequently lost. In Astyanax mexicanus, another blind cavefish, eye loss has been attributed to interference during eye field patterning. To address whether similar pathways have been targeted by evolution independently, we investigated the retinal development of P. andruzzii, studying the expression of marker genes involved in eye patterning, morphogenesis, differentiation and maintenance. In contrast to Astyanax, patterning of the eye field and evagination of the optic vesicles proceeds without obvious deviation. However, the subsequent differentiation of retinal cell types is arrested during generation of the first-born cell type, retinal ganglion cells, which also fail to project correctly to the optic tectum. Eye degeneration in both species is driven by progressive apoptosis. However, it is retinal apoptosis in Phreatichthys that progresses in a wave-like manner and eliminates progenitor cells that fail to differentiate, in contrast to Astyanax, where lens apoptosis appears to serve as a driving force. Thus, evolution has targeted late retinal differentiation events, indicating that there are several ways to discontinue the development and maintenance of an eye.

The light-induced transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian clockwork by light.

Light constitutes a primary signal whereby endogenous circadian clocks are synchronized ('entrained') with the day/night cycle. The molecular mechanisms underlying this vital process are known to require gene activation, yet are incompletely understood. Here, the light-induced transcriptome in the zebrafish central clock organ, the pineal gland, was characterized by messenger RNA (mRNA) sequencing (mRNA-seq) and microarray analyses, resulting in the identification of multiple light-induced mRNAs. Interestingly, a considerable portion of the molecular clock (14 genes) is light-induced in the pineal gland. Four of these genes, encoding the transcription factors dec1, reverbb1, e4bp4-5 and e4bp4-6, differentially affected clock- and light-regulated promoter activation, suggesting that light-input is conveyed to the core clock machinery via diverse mechanisms. Moreover, we show that dec1, as well as the core clock gene per2, is essential for light-entrainment of rhythmic locomotor activity in zebrafish larvae. Additionally, we used microRNA (miRNA) sequencing (miR-seq) and identified pineal-enhanced and light-induced miRNAs. One such miRNA, miR-183, is shown to downregulate e4bp4-6 mRNA through a 3'UTR target site, and importantly, to regulate the rhythmic mRNA levels of aanat2, the key enzyme in melatonin synthesis. Together, this genome-wide approach and functional characterization of light-induced factors indicate a multi-level regulation of the circadian clockwork by light.

Systematic identification of rhythmic genes reveals camk1gb as a new element in the circadian clockwork.

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA-seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.

A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception.

The circadian clock is synchronized with the day-night cycle primarily by light. Fish represent fascinating models for deciphering the light input pathway to the vertebrate clock since fish cell clocks are regulated by direct light exposure. Here we have performed a comparative, functional analysis of the circadian clock involving the zebrafish that is normally exposed to the day-night cycle and a cavefish species that has evolved in perpetual darkness. Our results reveal that the cavefish retains a food-entrainable clock that oscillates with an infradian period. Importantly, however, this clock is not regulated by light. This comparative study pinpoints the two extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin as essential upstream elements of the peripheral clock light input pathway.

Reactive Oxygen Species Signaling and Oxidative Stress: Transcriptional Regulation and Evolution.

Since the evolution of the aerobic metabolism, reactive oxygen species (ROS) have represented significant challenges to diverse life forms. In recent decades, increasing knowledge has revealed a dual role for ROS in cell physiology, showing they serve as a major source of cellular damage while also functioning as important signaling molecules in various biological processes. Our understanding of ROS homeostasis and ROS-mediated cellular signaling pathways has presumed that they are ancient and highly conserved mechanisms shared by most organisms. However, emerging evidence highlights the complexity and plasticity of ROS signaling, particularly in animals that have evolved in extreme environments. In this review, we focus on ROS generation, antioxidative systems and the main signaling pathways that are influenced by ROS. In addition, we discuss ROS's responsive transcription regulation and how it may have been shaped over the course of evolution.

Sporadic feeding regulates robust food entrainable circadian clocks in blind cavefish.

The circadian clock represents a key timing system entrained by various periodic signals that ensure synchronization with the environment. Many investigations have pointed to the existence of two distinct circadian oscillators: one regulated by the light-dark cycle and the other set by feeding time. Blind cavefish have evolved under extreme conditions where they completely lack light exposure and experience food deprivation. Here, we have investigated feeding regulated clocks in two cavefish species, the Somalian cavefish Phreatichthys andruzzii and the Mexican cavefish Astyanax mexicanus, in comparison with the surface-dwelling zebrafish Danio rerio. Our results reveal that feeding represents an extremely strong synchronizer for circadian locomotor rhythmicity in subterranean cavefish. Indeed, we showed that consuming just one meal every 4 days is sufficient to entrain circadian rhythmicity in both cavefish species, but not in zebrafish. These profound adaptations to an extreme environment provide insight into the connections between feeding and circadian clocks.

Oseltamivir phosphate (Tamiflu) alters neurobehavior of zebrafish larvae by inducing mitochondrial dysfunction.

Antiviral drugs are widely used, yet their potential risks during early development, particularly within the central nervous system, remain contentious. Oseltamivir phosphate (OSE), a commonly prescribed antiviral, is increasingly detected in various environments. However, its toxicity to organisms and the underlying mechanisms are not well understood. In this study, we employed the zebrafish model to evaluate the developmental neurotoxic effects of OSE at environmentally and therapeutically relevant doses, through high-throughput behavioral analysis, in vivo two-photon imaging, transcriptomic sequencing, pharmacological intervention, and biochemical and molecular assays. Our results indicated that OSE exposure increased heart rate and induced pericardial edema in zebrafish larvae. Additionally, OSE-exposed larvae exhibited hyperactive behavior, impaired social interactions, and reduced habitual learning capacity. Although OSE at our selected levels did not significantly affect neuron count in the brain, it activated neuroinflammatory responses, altered blood vessel morphology, modulated neurotransmitter levels and the expression of neurodevelopment-related genes. Transcriptomic analysis revealed upregulation of mitochondria-related genes associated with oxidative phosphorylation. Further assessments of mitochondrial function demonstrated altered activities of respiratory chain complexes, reduced mitochondrial membrane potential (MMP), and decreased ATP content. Notably, co-treatment with mitochondrial protectants acetyl-l-carnitine-hydrochloride (ALC) or nicotinamide riboside (NR) effectively mitigated OSE-induced neurobehavioral disorders. These findings suggest that overuse of OSE can pose neurodevelopmental risks for both humans and animals, potentially attributable to mitochondrial dysfunction.

Photobiology: Fish eggs go sunny side up.

The UV radiation in sunlight can damage organisms. A new study reveals that female zebrafish deposit a chemical sunscreen into their eggs to protect their developing embryos, a feat that has been lost in fish species whose embryos never experience sunlight.

Negative phototaxis in the photosymbiotic sea anemone Aiptasia as a potential strategy to protect symbionts from photodamage.

Photosymbiotic cnidarians generally seek bright environments so that their symbionts can be photosynthetically active. However, excess light may result in a breakdown of symbiosis due to the accumulation of photodamage in symbionts causing symbiont loss (bleaching). It is currently unknown if photosymbiotic cnidarians sense light only to regulate spawning time and to facilitate predation, or whether they also use their light-sensing capacities to protect their symbionts from photodamage. In this study, we examined how the sea anemone Aiptasia changes its behaviour when exposed to excess light. We reveal that Aiptasia polyps, when carrying symbionts, contract their bodies when exposed to high light intensities and subsequently migrate away in a direction perpendicular to the light source. Interestingly, this negative phototaxis was only evident under blue light and absent upon UV, green and red light exposure. Non-symbiotic Aiptasia did not exhibit this light response. Our study demonstrates that photosymbiotic Aiptasia polyps display negative phototactic behaviour in response to blue light, and that they also can perceive its direction, despite lacking specialized eye structures. We postulate that Aiptasia uses blue light, which penetrates seawater efficiently, as a general proxy for sunlight exposure to protect its symbionts from photodamage.

Establishment of cell lines from individual zebrafish embryos.

With the increasing use of fish as model species for research, cell cultures derived from caudal fin explants as well as pre-hatching stage embryos have provided powerful in vitro tools that can complement or serve as an ethically more acceptable alternative to live animal experiments. The widely-used protocols to establish these lines require, as a starting point, homogeneous pools of embryos or viable adult fish which are large enough for collecting sufficient fin tissue. This excludes the use of fish lines with adverse phenotypes or lines that exhibit mortality at early developmental stages and so can only be propagated as heterozygotes. Specifically, when no visually overt mutant phenotype is detectable for identifying homozygous mutants at early embryonic stages, it is then impossible to sort pools of embryos with the same genotypes to generate cell lines from the progeny of a heterozygote in-cross. Here, we describe a simple protocol to generate cell lines on a large scale starting from individual early embryos that can subsequently be genotyped by polymerase chain reaction. This protocol should help to establish fish cell culture models as a routine approach for the functional characterization of genetic changes in fish models such as the zebrafish. Furthermore, it should contribute to a reduction of experiments which are ethically discouraged to avoid pain and distress.

Ferroptosis contributes to nickel-induced developmental neurotoxicity in zebrafish.

Nickel (Ni) is a widely utilized heavy metal that can cause environmental pollution and health hazards. Its safety has attracted the attention of both the environmental ecology and public health fields. While the central nervous system (CNS) is one of the main targets of Ni, its neurotoxicity and the underlying mechanisms remain unclear. Here, by taking advantage of the zebrafish model for live imaging, genetic analysis and neurobehavioral studies, we reveal that the neurotoxic effects induced by exposure to environmentally relevant levels of Ni are closely related to ferroptosis, a newly-described form of iron-mediated cell death. In vivo two-photon imaging, neurobehavioral analysis and transcriptome sequencing consistently demonstrate that early neurodevelopment, neuroimmune function and vasculogenesis in zebrafish larvae are significantly affected by environmental Ni exposure. Importantly, exposure to various concentrations of Ni activates the ferroptosis pathway, as demonstrated by physiological/biochemical tests, as well as the expression of ferroptosis markers. Furthermore, pharmacological intervention of ferroptosis via deferoxamine (DFO), a classical iron chelating agent, strongly implicates iron dyshomeostasis and ferroptosis in these Ni-induced neurotoxic effects. Thus, this study elucidates the cellular and molecular mechanisms underlying Ni neurotoxicity, with implications for our understanding of the physiologically damaging effects of other environmental heavy metal pollutants.

Light directs zebrafish period2 expression via conserved D and E boxes.

For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.

Encephalic photoreception and phototactic response in the troglobiont Somalian blind cavefish Phreatichthys andruzzii.

Many physiological and behavioural responses to changes in environmental lighting conditions are mediated by extraocular photoreceptors. Here we investigate encephalic photoreception in Phreatichthys andruzzii, a typical cave-dwelling fish showing an extreme phenotype with complete anophthalmy and a reduction in size of associated brain structures. We firstly identified two P. andruzzii photopigments, orthologues of rod opsin and exo-rod opsin. In vitro, both opsins serve as light-absorbing photopigments with λ(max) around 500 nm when reconstituted with an A(1) chromophore. When corrected for the summed absorption from the skin and skull, the spectral sensitivity profiles shifted to longer wavelengths (rod opsin: 521 nm; exo-rod opsin: 520 nm). We next explored the involvement of both opsins in the negative phototaxis reported for this species. A comparison of the spectral sensitivity of the photophobic response with the putative A(2) absorbance spectra corrected for skin/skull absorbance indicates that the A(2) versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.