CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.

Elevated DNA Damage without signs of aging in the short-sleeping Mexican Cavefish.

Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish, Astyanax mexicanus provides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and cellular stress, we compared the DNA Damage Response (DDR) and oxidative stress levels between A. mexicanus populations. We measured markers of chronic sleep loss and discovered elevated levels of the DNA damage marker γH2AX in the brain, and increased oxidative stress in the gut of cavefish, consistent with chronic sleep deprivation. Notably, we found that acute UV-induced DNA damage elicited an increase in sleep in surface fish but not in cavefish. On a transcriptional level, only the surface fish activated the photoreactivation repair pathway following UV damage. These findings suggest a reduction of the DDR in cavefish compared to surface fish that coincides with elevated DNA damage in cavefish. To examine DDR pathways at a cellular level, we created an embryonic fibroblast cell line from the two populations of A. mexicanus. We observed that both the DDR and DNA repair were diminished in the cavefish cells, corroborating the in vivo findings and suggesting that the acute response to DNA damage is lost in cavefish. To investigate the long-term impact of these changes, we compared the transcriptome in the brain and gut of aged surface fish and cavefish. Strikingly, many genes that are differentially expressed between young and old surface fish do not transcriptionally vary by age in cavefish. Taken together, these findings suggest that have developed resilience to sleep loss, despite possessing cellular hallmarks of chronic sleep deprivation.

Venom trade-off shapes interspecific interactions, physiology, and reproduction.

The ability of an animal to effectively capture prey and defend against predators is pivotal for survival. Venom is often a mixture of many components including toxin proteins that shape predator-prey interactions. Here, we used the sea anemone Nematostella vectensis to test the impact of toxin genotypes on predator-prey interactions. We developed a genetic manipulation technique to demonstrate that both transgenically deficient and a native Nematostella strain lacking a major neurotoxin (Nv1) have a reduced ability to defend themselves against grass shrimp, a native predator. In addition, secreted Nv1 can act indirectly in defense by attracting mummichog fish, which prey on grass shrimp. Here, we provide evidence at the molecular level of an animal-specific tritrophic interaction between a prey, its antagonist, and a predator. Last, this study reveals an evolutionary trade-off, as the reduction of Nv1 levels allows for faster growth and increased reproductive rates.

Radial astrocyte synchronization modulates the visual system during behavioral-state transitions.

Glial cells support the function of neurons. Recent evidence shows that astrocytes are also involved in brain computations. To explore whether and how their excitable nature affects brain computations and motor behaviors, we used two-photon Ca2+ imaging of zebrafish larvae expressing GCaMP in both neurons and radial astrocytes (RAs). We found that in the optic tectum, RAs synchronize their Ca2+ transients immediately after the end of an escape behavior. Using optogenetics, ablations, and a genetically encoded norepinephrine sensor, we observed that RA synchronous Ca2+ events are mediated by the locus coeruleus (LC)-norepinephrine circuit. RA synchronization did not induce direct excitation or inhibition of tectal neurons. Nevertheless, it modulated the direction selectivity and the long-distance functional correlations among neurons. This mechanism supports freezing behavior following a switch to an alerted state. These results show that LC-mediated neuro-glial interactions modulate the visual system during transitions between behavioral states.

Tsh Induces Agrp1 Neuron Proliferation in Oatp1c1-Deficient Zebrafish.

Thyroid hormones (THs), thyroxine (T4), and triiodothyronine (T3), regulate growth, metabolism, and neurodevelopment. THs secretion is controlled by the pituitary thyroid-stimulating hormone (TSH) and the hypothalamic-pituitary-thyroid (HPT) axis. The organic anion-transporting polypeptide 1C1 (OATP1C1/SLCO1C1) and the monocarboxylate transporter 8 (MCT8/SLC16A2) actively transport THs, which bind to their nuclear receptors and induce gene expression. A mutation in OATP1C1 is associated with brain hypometabolism, gradual neurodegeneration, and impaired cognitive and motor functioning in adolescent patients. To understand the role of Oatp1c1 and the mechanisms of the disease, we profiled the transcriptome of oatp1c1 mutant (oatp1c1 -/-) and mct8 -/- xoatp1c1 -/- adult male and female zebrafish brains. Among dozens of differentially expressed genes, agouti-related neuropeptide 1 (agrp1) expression increased in oatp1c1 -/- adult brains. Imaging in the hypothalamus revealed enhanced proliferation of Agrp1 neurons in oatp1c1 -/- larvae and adults, and increased food consumption in oatp1c1 -/- larvae. Similarly, feeding and the number of Agrp1 neurons increased in thyroid gland-ablated zebrafish. Pharmacological treatments showed that the T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid), but not T4, normalized the number of Agrp1 neurons in oatp1c1 -/- zebrafish. Since the HPT axis is hyperactive in the oatp1c1 -/- brain, we used the CRISPR-Cas9 system to knockdown tsh in oatp1c1 -/- larvae, and inducibly enhanced the HPT axis in wild-type larvae. These manipulations showed that Tsh promotes proliferation of Agrp1 neurons and increases food consumption in zebrafish. The results revealed upregulation of both the HPT axis-Agrp1 circuitry and feeding in a zebrafish model for OATP1C1 deficiency.SIGNIFICANCE STATEMENT Mutation in the thyroid hormone (TH) transporter OATP1C1 is associated with cognitive and motor functioning disturbances in humans. Here, we used an oatp1c1 -/- zebrafish to understand the role of organic anion-transporting polypeptide 1C1 (Oatp1c1), and the characteristics of OATP1C1 deficiency. Transcriptome profiling identified upregulation of agrp1 expression in the oatp1c1 -/- brain. The oatp1c1 -/- larvae showed increased thyroid-stimulating hormone (tsh) levels, proliferation of Agrp1 neurons and food consumption. Genetic manipulations of the hypothalamic-pituitary-thyroid (HPT) axis showed that Tsh increases the number of Agrp1 neurons and food consumption. The T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid) normalizes the number of Agrp1 neurons and may have potential for the treatment of Oatp1c1 deficiency. The findings demonstrate a functional interaction between the thyroid and feeding systems in the brain of zebrafish and suggest a neuroendocrinological mechanism for OATP1C1 deficiency.

Genetic and Neurological Deficiencies in the Visual System of mct8 Mutant Zebrafish.

Thyroid hormones (THs; T3 and T4) enter cells using specific transporters and regulate development and metabolism. Mutation in the TH transporter monocarboxylate transporter 8 (MCT8, SLC16A2) is associated with brain hypothyroidism and neurological impairment. We established mct8 mutant (mct8-/-) zebrafish as a model for MCT8 deficiency, which causes endocrinological, neurological, and behavioral alterations. Here, we profiled the transcriptome of mct8-/- larvae. Among hundreds of differentially expressed genes, the expression of a cluster of vision-related genes was distinct. Specifically, the expression of the opsin 1 medium wave sensitive 2 (opn1mw2) decreased in two mct8 mutants: mct8-/- and mct8-25bp-/- larvae, and under pharmacological inhibition of TH production. Optokinetic reflex (OKR) assays showed a reduction in the number of conjugated eye movements, and live imaging of genetically encoded Ca2+ indicator revealed altered neuronal activity in the pretectum area of mct8-25bp-/- larvae. These results imply that MCT8 and THs regulate the development of the visual system and suggest a mechanism to the deficiencies observed in the visual system of MCT8-deficiency patients.

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.

Parp1 promotes sleep, which enhances DNA repair in neurons.

The characteristics of the sleep drivers and the mechanisms through which sleep relieves the cellular homeostatic pressure are unclear. In flies, zebrafish, mice, and humans, DNA damage levels increase during wakefulness and decrease during sleep. Here, we show that 6 h of consolidated sleep is sufficient to reduce DNA damage in the zebrafish dorsal pallium. Induction of DNA damage by neuronal activity and mutagens triggered sleep and DNA repair. The activity of the DNA damage response (DDR) proteins Rad52 and Ku80 increased during sleep, and chromosome dynamics enhanced Rad52 activity. The activity of the DDR initiator poly(ADP-ribose) polymerase 1 (Parp1) increased following sleep deprivation. In both larva zebrafish and adult mice, Parp1 promoted sleep. Inhibition of Parp1 activity reduced sleep-dependent chromosome dynamics and repair. These results demonstrate that DNA damage is a homeostatic driver for sleep, and Parp1 pathways can sense this cellular pressure and facilitate sleep and repair activity.

Flatfoot in Africa, the cirripede Chthamalus in the west Indian Ocean.

Barnacles of the genus Chthamalus are commonly encountered rocky intertidal shores. The phylogeography of the different species in the Western Indian Ocean is unclear. Using morphological characteristics as well as the molecular markers mitochondrial cytochrome oxygenase subunit I (COI) and the nuclear sodium-potassium ATPase (NaKA), we identified four clades representing four species in the Western Indian Ocean and its adjacent seas. Among these species, a newly identified species, Chthamalus barilani, which was found in Madagascar, Zanzibar and Tanzania. Chthamalus from the coasts of Tanzania and Zanzibar is identified morphologically as C. malayensis, and clusters with C. malayensis from the Western Pacific and the Indo Malayan regions. C. malayensis is regarded as a group of four genetically differentiated clades representing four cryptic species. The newly identified African clade is genetically different from these clades and the pairwise distances between them justify the conclusion that it is an additional cryptic species of C. malayensis. This type of genetic analyses offers an advantage over morphological characterization and allowed us to reveal that another species, C. barnesi, which is known from the Red Sea, is also distributed in the Arabian Sea and the Persian Gulf. We could also confirm the presence of the South African species C. dentatus in the Mozambique channel. This represents the Northeastern limit of C. dentatus, which is usually distributed along the coast of southern Africa up to the Islands of Cape Verde in West Africa. Altogether, based on a combination of morphology and genetics, we distinct between four clusters of Chthamalus, and designate their distribution in the West Indian Ocean. These distinctions do not agree with the traditional four groups reported previously based merely on morphological data. Furthermore, these findings underline the importance of a combining morphological and genetics tools for constructing barnacle taxonomy.

Heterogeneity of Hypocretin/Orexin Neurons.

The multifunctional, hypothalamic hypocretin/orexin (HCRT)-producing neurons regulate an array of physiological and behavioral states including arousal, sleep, feeding, emotions, stress, and reward. How a presumably uniform HCRT neuron population regulates such a diverse set of functions is not clear. The role of the HCRT neuropeptides may vary depending on the timing and localization of secretion and neuronal activity. Moreover, HCRT neuropeptides may not mediate all functions ascribed to HCRT neurons. Some could be orchestrated by additional neurotransmitters and neuropeptides that are expressed in HCRT neurons. We hypothesize that HCRT neurons are segregated into genetically, anatomically and functionally distinct subpopulations. We discuss accumulating data that suggest the existence of such HCRT neuron subpopulations that may effectuate the diverse functions of these neurons in mammals and fish.

Systematic identification of A-to-I RNA editing in zebrafish development and adult organs.

A-to-I RNA editing is a common post transcriptional mechanism, mediated by the Adenosine deaminase that acts on RNA (ADAR) enzymes, that increases transcript and protein diversity. The study of RNA editing is limited by the absence of editing maps for most model organisms, hindering the understanding of its impact on various physiological conditions. Here, we mapped the vertebrate developmental landscape of A-to-I RNA editing, and generated the first comprehensive atlas of editing sites in zebrafish. Tens of thousands unique editing events and 149 coding sites were identified with high-accuracy. Some of these edited sites are conserved between zebrafish and humans. Sequence analysis of RNA over seven developmental stages revealed high levels of editing activity in early stages of embryogenesis, when embryos rely on maternal mRNAs and proteins. In contrast to the other organisms studied so far, the highest levels of editing were detected in the zebrafish ovary and testes. This resource can serve as the basis for understanding of the role of editing during zebrafish development and maturity.

Thyroid Hormones Regulate Goblet Cell Differentiation and Fgf19-Fgfr4 Signaling.

Hypothyroidism is a common pathological condition characterized by insufficient activity of the thyroid hormones (THs), thyroxine (T4), and 3,5,3'-triiodothyronine (T3), in the whole body or in specific tissues. Hypothyroidism is associated with inadequate development of the intestine as well as gastrointestinal diseases. We used a zebrafish model of hypothyroidism to identify and characterize TH-modulated genes and cellular pathways controlling intestine development. In the intestine of hypothyroid juveniles and adults, the number of mucus-secreting goblet cells was reduced, and this phenotype could be rescued by T3 treatment. Transcriptome profiling revealed dozens of differentially expressed genes in the intestine of hypothyroid adults compared to controls. Notably, the expression of genes encoding to Fgf19 and its receptor Fgfr4 was markedly increased in the intestine of hypothyroid adults, and treatment with T3 normalized it. Blocking fibroblast growth factor (FGF) signaling, using an inducible dominant-negative Fgfr transgenic line, rescued the number of goblet cells in hypothyroid adults. These results show that THs inhibit the Fgf19-Fgfr4 signaling pathway, which is associated with inhibition of goblet cell differentiation in hypothyroidism. Both the TH and Fgf19-Fgfr4 signaling pathways can be pharmaceutical targets for the treatment of TH-related gastrointestinal diseases.

Cavefish brain atlases reveal functional and anatomical convergence across independently evolved populations.

Environmental perturbation can drive behavioral evolution and associated changes in brain structure and function. The Mexican fish species, Astyanax mexicanus, includes eyed river-dwelling surface populations and multiple independently evolved populations of blind cavefish. We used whole-brain imaging and neuronal mapping of 684 larval fish to generate neuroanatomical atlases of surface fish and three different cave populations. Analyses of brain region volume and neural circuits associated with cavefish behavior identified evolutionary convergence in hindbrain and hypothalamic expansion, and changes in neurotransmitter systems, including increased numbers of catecholamine and hypocretin/orexin neurons. To define evolutionary changes in brain function, we performed whole-brain activity mapping associated with behavior. Hunting behavior evoked activity in sensory processing centers, while sleep-associated activity differed in the rostral zone of the hypothalamus and tegmentum. These atlases represent a comparative brain-wide study of intraspecies variation in vertebrates and provide a resource for studying the neural basis of behavioral evolution.

Publisher Correction: Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hunger Potentiates the Habenular Winner Pathway for Social Conflict by Orexin-Promoted Biased Alternative Splicing of the AMPA Receptor Gene.

Many animals fight for dominance between conspecifics. Because winners could obtain more resources than losers, fighting outcomes are important for the animal's survival, especially in a situation with insufficient resources, such as hunger. However, it remains unclear whether and how hunger affects fighting outcomes. Herein, we investigate the effects of food deprivation on brain activity and fighting behaviors in zebrafish. We report that starvation induces winning in social conflicts. Before the fights, starved fish show potentiation of the lateral subregion of the dorsal habenula (dHbL)-dorsal/intermediate interpeduncular nucleus (d/iIPN) pathway, which is known to be essential for and potentiated after winning fights. Circuit potentiation is mediated by hypothalamic orexin/hypocretin neuropeptides, which prolong AMPA-type glutamate receptor (AMPAR) activity by increasing the expression of a flip type of alternative splicing variant of the AMPAR subunit. This mechanism may underlie how hungry vertebrates win fights and may be commonly shared across animal phylogeny.

Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior.

The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of stress response in general. Alternative splicing of PAC1 results in multiple gene products, which differ in their mode of signalling and tissue distribution. However, the roles of distinct splice variants in the regulation of stress behavior is poorly understood. Alternative splicing of a short exon, which is known as the "hop cassette", occurs during brain development and in response to stressful challenges. To examine the function of this variant, we generated a splice-specific zebrafish mutant lacking the hop cassette, which we designated 'hopless'. We show that hopless mutant larvae display increased anxiety-like behavior, including reduced dark exploration and impaired habituation to dark exposure. Conversely, adult hopless mutants displayed superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-like responses to an ensuing novelty stress. We propose that the developmental loss of a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the long term, predisposes the organism's stress response towards a resilient phenotype. Our study presents a unique genetic model demonstrating how early-life state of anxiety paradoxically correlates with reduced stress susceptibility in adulthood.

Neurotensin Enhances Locomotor Activity and Arousal and Inhibits Melanin-Concentrating Hormone Signaling.

BACKGROUND: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. MATERIALS AND METHODS: In order to study the role of Nts, nts mutant (nts-/-), and overexpressing zebrafish were generated. RESULTS: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts-/- larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts-/- larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts-/- larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts-/- larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts-/- larvae. CONCLUSION: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.

Neural Alterations and Hyperactivity of the Hypothalamic-Pituitary-Thyroid Axis in Oatp1c1 Deficiency.

Background: The thyroid hormones (THs) triiodothyronine (T3) and thyroxine (T4) are crucial regulators of brain development and function. Cell-specific transporter proteins facilitate TH uptake and efflux across the cell membrane, and insufficient TH transport causes hypothyroidism and mental retardation. Mutations in the TH transporters monocarboxylate transporter 8 (MCT8, SLC16A2) and the organic anion-transporting polypeptide 1C1 (OATP1C1, SLCO1C1) are associated with the psychomotor retardation Allan-Herndon-Dudley syndrome and juvenile neurodegeneration, respectively. Methods: To understand the mechanisms and test potential treatments for the recently discovered OATP1C1 deficiency, we established an oatp1c1 mutant (oatp1c1-/-) zebrafish. Results:oatp1c1 is expressed in endothelial cells, neurons, and astrocytes in zebrafish. The activity of the hypothalamic-pituitary-thyroid axis and behavioral locomotor activity increased in oatp1c1-/- larvae. Neuropathological analysis revealed structural alteration in radial glial cells and shorter neuronal axons in oatp1c1-/- larvae and adults. Notably, oatp1c1-/- and oatp1c1-/-Xmct8-/- adults exhibit an enlarged thyroid gland (goiter). Pharmacological assays showed that TH analogs, but not THs, can reduce the size and improve the color of the thyroid gland in adult mutant zebrafish. Conclusion: These results establish a vertebrate model for OATP1C1 deficiency that demonstrates endocrinological, neurological, and behavioral alterations mimicking findings observed in an OATP1C1-deficient patient. Further, the curative effect of TH analogs in the oatp1c1-/- zebrafish model may provide a lead toward a treatment modality in human patients.

Neuronal noise as an origin of sleep arousals and its role in sudden infant death syndrome.

In addition to regular sleep/wake cycles, humans and animals exhibit brief arousals from sleep. Although much is known about consolidated sleep and wakefulness, the mechanism that triggers arousals remains enigmatic. Here, we argue that arousals are caused by the intrinsic neuronal noise of wake-promoting neurons. We propose a model that simulates the superposition of the noise from a group of neurons, and show that, occasionally, the superposed noise exceeds the excitability threshold and provokes an arousal. Because neuronal noise decreases with increasing temperature, our model predicts arousal frequency to decrease as well. To test this prediction, we perform experiments on the sleep/wake behavior of zebrafish larvae and find that increasing water temperatures lead to fewer and shorter arousals, as predicted by our analytic derivations and model simulations. Our findings indicate a previously unrecognized neurophysiological mechanism that links sleep arousals with temperature regulation, and may explain the origin of the clinically observed higher risk for sudden infant death syndrome with increased ambient temperature.

Neuronal Mechanisms for Sleep/Wake Regulation and Modulatory Drive.

Humans have been fascinated by sleep for millennia. After almost a century of scientific interrogation, significant progress has been made in understanding the neuronal regulation and functions of sleep. The application of new methods in neuroscience that enable the analysis of genetically defined neuronal circuits with unprecedented specificity and precision has been paramount in this endeavor. In this review, we first discuss electrophysiological and behavioral features of sleep/wake states and the principal neuronal populations involved in their regulation. Next, we describe the main modulatory drives of sleep and wakefulness, including homeostatic, circadian, and motivational processes. Finally, we describe a revised integrative model for sleep/wake regulation.