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Salient sensory stimuli are perceived by the brain, which guides both the timing and outcome of behaviors in a context-dependent manner. Light is such a stimulus, which is used in treating mood disorders often associated with a dysregulated hypothalamic-pituitary-adrenal stress axis. Relationships between the emotional valence of light and the hypothalamus, and how they interact to exert brain-wide impacts remain unclear. Employing larval zebrafish with analogous hypothalamic systems to mammals, we show in free-swimming animals that hypothalamic corticotropin releasing factor (CRFHy) neurons promote dark avoidance, and such role is not shared by other hypothalamic peptidergic neurons. Single-neuron projection analyses uncover processes extended by individual CRFHy neurons to multiple targets including sensorimotor and decision-making areas. In vivo calcium imaging uncovers a complex and heterogeneous response of individual CRFHy neurons to the light or dark stimulus, with a reduced overall sum of CRF neuronal activity in the presence of light. Brain-wide calcium imaging under alternating light/dark stimuli further identifies distinct and distributed photic response neuronal types. CRFHy neuronal ablation increases an overall representation of light in the brain and broadly enhances the functional connectivity associated with an exploratory brain state. These findings delineate brain-wide photic perception, uncover a previously unknown role of CRFHy neurons in regulating the perception and emotional valence of light, and suggest that light therapy may alleviate mood disorders through reducing an overall sum of CRF neuronal activity.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

Background and Aims: The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC‐to‐hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. Approach and Results: Here, using zebrafish models of LPC‐mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC‐to‐hepatocyte differentiation through the mitogen‐activated ERK kinase (MEK)‐extracellular signal‐regulated kinase (ERK)–sex‐determining region Y‐box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC‐to‐hepatocyte differentiation as well as genetic suppression of the EGFR‐ERK‐SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short‐term treatment with EGFR inhibitors resulted in better liver recovery over the long term. Conclusions: The EGFR‐ERK‐SOX9 axis suppresses LPC‐to‐hepatocyte differentiation during LPC‐mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

Significance Membrane and cytoskeletal dynamics are critical to cell motility. Extensively studied in cell culture, their roles in cell movement in vivo are less understood, especially in higher vertebrates. We use dynamic imaging to visualize membrane and cytoskeletal behavior in migrating neural crest cells in living tissue. We found that forward movement of individual neural crest cells is accompanied by circular membrane flow, from anterior-to-posterior apically and posterior-to-anterior basally, coupled with internalization of lipid vesicles via macropinocytosis in the soma. Macropinosomes become wrapped with actin, then undergo anterograde translocation via microtubules toward the lamellipodium, resulting in its expansion. We elucidate how actin dynamics and membrane flow are interacted to drive forward locomotion of individual cells., Individual cell migration requires front-to-back polarity manifested by lamellipodial extension. At present, it remains debated whether and how membrane motility mediates this cell morphological change. To gain insights into these processes, we perform live imaging and molecular perturbation of migrating chick neural crest cells in vivo. Our results reveal an endocytic loop formed by circular membrane flow and anterograde movement of lipid vesicles, resulting in cell polarization and locomotion. Rather than clathrin-mediated endocytosis, macropinosomes encapsulate F-actin in the cell body, forming vesicles that translocate via microtubules to deliver actin to the anterior. In addition to previously proposed local conversion of actin monomers to polymers, we demonstrate a surprising role for shuttling of F-actin across cells for lamellipodial expansion. Thus, the membrane and cytoskeleton act in concert in distinct subcellular compartments to drive forward cell migration.

Prokineticin receptors (PROKR1 and PROKR2) are G protein-coupled receptors which control human central and peripheral reproductive processes. Importantly, allelic variants of PROKR2 in humans are associated with altered migration of GnRH neurons, resulting in congenital hypogonadotropic hypogonadism (CHH), a heterogeneous disease characterized by delayed/absent puberty and/or infertility. Although this association is established in humans, murine models failed to fully recapitulate the reproductive and olfactory phenotypes observed in patients harboring PROKR2 mutations. Here, taking advantage of zebrafish model we investigated the role of prokr1b (ortholog of human PROKR2) during early stages of GnRH neuronal migration. Real-Time PCR and whole mount in situ hybridization assays indicate that prokr1b spatial-temporal expression is consistent with gnrh3. Moreover, knockdown and knockout of prokr1b altered the correct development of GnRH3 fibers, a phenotype that is rescued by injection of prokr1b mRNA. These results suggest that prokr1b regulates the development of the GnRH3 system in zebrafish. Analysis of gonads development and mating experiments indicate that prokr1b is not required for fertility in zebrafish, although its loss determine changes also at the testis level. Altogether, our results support the thesis of a divergent evolution in the control of vertebrate reproduction and provide a useful in vivo model for deciphering the mechanisms underlying the effect of PROKR2 allelic variants on CHH.

Experiments with biological samples require precise control of environmental conditions. In our work we use zebrafish (Danio rerio) to understand the neurobiology of sleep, which requires precise control of temperature and lighting. Like many labs, lighting and temperature in the animal facility are centrally controlled in the building. During behavioral experiments and microscopy sessions, we use custom-built heating systems and perform occasional manual checks of conditions. However, without a system to precisely record conditions, gradual changes in temperature can go unnoticed for a long time, and temporary failures may be missed entirely. Here we present the design and characterization of affordable open-source tools to record temperature and light conditions during animal experiments using an Arduino microcontroller or a Raspberry Pi compact computer. The waterproof temperature sensor has high stability over 50 days of recording and is precise to 0.1°C. The Arduino device can be used through a common serial port interface for which we present code in Python and MATLAB. The Raspberry Pi version can be accessed through a web interface, for which we provide an installation guide. We use the device to record and review temperature and lighting conditions in two zebrafish animal facilities. We use our platform to add a water heating system to maintain temperature at 28°C during in vivo light-sheet imaging of larval zebrafish. We show that a change in temperature from 28°C to 32°C affects resting heart rate of the animal, highlighting the importance of maintaining and recording conditions. The protocols presented here do not require advanced engineering, fabrication, or software skills, and provide an approach to accurately record and report experimental conditions.

Although several sleep-regulating neuronal populations have been identified, little is known about how they interact with each other to control sleep/wake states. We previously identified neuropeptide VF (NPVF) and the hypothalamic neurons that produce it as a sleep-promoting system (Lee et al., 2017). Here we show using zebrafish that npvf-expressing neurons control sleep via the serotonergic raphe nuclei (RN), a hindbrain structure that is critical for sleep in both diurnal zebrafish and nocturnal mice. Using genetic labeling and calcium imaging, we show that npvf-expressing neurons innervate and can activate serotonergic RN neurons. We also demonstrate that chemogenetic or optogenetic stimulation of npvf-expressing neurons induces sleep in a manner that requires NPVF and serotonin in the RN. Finally, we provide genetic evidence that NPVF acts upstream of serotonin in the RN to maintain normal sleep levels. These findings reveal a novel hypothalamic-hindbrain neuronal circuit for sleep/wake control.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene,dreammist(dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expresseddmistgene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of eitherfxyd1oratp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Sinceatp1a3aanddmistmutants have elevated intracellular Na+levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+pump function modulates neuronal excitability to maintain normal sleep behaviour.

Prokineticin receptors (PROKR1 and PROKR2) are G protein-coupled receptors which control human central and peripheral reproductive processes. Importantly, allelic variants of PROKR2 in humans are associated with altered migration of GnRH neurons, resulting in congenital hypogonadotropic hypogonadism (CHH), a heterogeneous disease characterized by delayed/absent puberty and/or infertility. Although this association is established in humans, murine models failed to fully recapitulate the reproductive and olfactory phenotypes observed in patients harboring PROKR2 mutations. Here, taking advantage of zebrafish model we investigated the role of prokr1b (ortholog of human PROKR2) during early stages of GnRH neuronal migration. Real-Time PCR and whole mount in situ hybridization assays indicate that prokr1b spatial-temporal expression is consistent with gnrh3. Moreover, knockdown and knockout of prokr1b altered the correct development of GnRH3 fibers, a phenotype that is rescued by injection of prokr1b mRNA. These results suggest that prokr1b regulates the development of the GnRH3 system in zebrafish. Analysis of gonads development and mating experiments indicate that prokr1b is not required for fertility in zebrafish, although its loss determine changes also at the testis level. Altogether, our results support the thesis of a divergent evolution in the control of vertebrate reproduction and provide a useful in vivo model for deciphering the mechanisms underlying the effect of PROKR2 allelic variants on CHH.

Although several sleep-regulating neurons have been identified, little is known about how they interact with each other for sleep/wake control. We previously identified neuropeptide VF (NPVF) and the hypothalamic neurons that produce it as a sleep-promoting system (Lee et al., 2017). Here we use zebrafish to describe a neural circuit in which neuropeptide VF (npvf)-expressing neurons control sleep via the serotonergic raphe nuclei (RN), a hindbrain structure that promotes sleep in both diurnal zebrafish and nocturnal mice. Using genetic labeling and calcium imaging, we show that npvf-expressing neurons innervate and activate serotonergic RN neurons. We additionally demonstrate that optogenetic stimulation of npvf-expressing neurons induces sleep in a manner that requires NPVF and is abolished when the RN are ablated or lack serotonin. Finally, genetic epistasis demonstrates that NPVF acts upstream of serotonin in the RN to maintain normal sleep levels. These findings reveal a novel hypothalamic-hindbrain circuit for sleep/wake control.

Epidermal growth factor receptor signaling regulates sleep homeostasis in zebrafish and is implicated in regulating human sleep., The genetic bases for most human sleep disorders and for variation in human sleep quantity and quality are largely unknown. Using the zebrafish, a diurnal vertebrate, to investigate the genetic regulation of sleep, we found that epidermal growth factor receptor (EGFR) signaling is necessary and sufficient for normal sleep levels and is required for the normal homeostatic response to sleep deprivation. We observed that EGFR signaling promotes sleep via mitogen-activated protein kinase/extracellular signal–regulated kinase and RFamide neuropeptide signaling and that it regulates RFamide neuropeptide expression and neuronal activity. Consistent with these findings, analysis of a large cohort of human genetic data from participants of European ancestry revealed that common variants in genes within the EGFR signaling pathway are associated with variation in human sleep quantity and quality. These results indicate that EGFR signaling and its downstream pathways play a central and ancient role in regulating sleep and provide new therapeutic targets for sleep disorders.

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

Summary Sleep is a cross-species phenomenon whose evolutionary and biological function remain poorly understood. Clinical and animal studies suggest that sleep disturbance is significantly associated with disruptions in protein homeostasis—or proteostasis—in the brain, but the mechanism of this link has not been explored. In the cell, the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway modulates proteostasis by transiently inhibiting protein synthesis in response to proteostatic stress. In this study, we examined the role of the PERK pathway in sleep regulation and provide the first evidence that PERK signaling is required to regulate normal sleep in both vertebrates and invertebrates. We show that pharmacological inhibition of PERK reduces sleep in both Drosophila and zebrafish, indicating an evolutionarily conserved requirement for PERK in sleep. Genetic knockdown of PERK activity also reduces sleep in Drosophila, whereas PERK overexpression induces sleep. Finally, we demonstrate that changes in PERK signaling directly impact wake-promoting neuropeptide expression, revealing a mechanism through which proteostatic pathways can affect sleep and wake behavior. Taken together, these results demonstrate that protein synthesis pathways like PERK could represent a general mechanism of sleep and wake regulation and provide greater insight into the relationship between sleep and proteostasis.

GnRH neurons integrate internal and external cues to control sexual maturation and fertility. Homeostasis of energy balance and food intake correlates strongly with the status of reproduction. Neuropeptides secreted by the hypothalamus involved in modulating energy balance and feeding may play additional roles in the regulation of reproduction. Hypocretin (Hcrt) (also known as orexin) is one such peptide, primarily controlling sleep/wakefulness, food intake, and reward processing. There is a growing body of evidence indicating that Hcrt/orexin (Hcrt) modulates reproduction through interacting with the hypothalamo-pituitary-gonadal axis in mammals. To explore potential morphological and functional interactions between the GnRH and Hcrt neuronal systems, we employed a variety of experimental approaches including confocal imaging, immunohistochemistry, and electrophysiology in transgenic zebrafish, in which fluorescent proteins are genetically expressed in GnRH3 and Hcrt neurons. Our imaging data revealed close apposition and direct connection between GnRH3 and Hcrt neuronal systems in the hypothalamus during larval development through adulthood. Furthermore, the Hcrt receptor (HcrtR) is expressed in GnRH3 neurons. Electrophysiological data revealed a reversible inhibitory effect of Hcrt on GnRH3 neuron electrical activity, which was blocked by the HcrtR antagonist almorexant. In addition, Hcrt had no effect on the electrical activity of GnRH3 neurons in the HcrtR null mutant zebrafish (HcrtR-/-). Our findings demonstrate a close anatomical and functional relationship between Hcrt and GnRH neuronal systems in zebrafish. It is the first demonstration of a link between neuronal circuits controlling sleeping/arousal/feeding and reproduction in zebrafish, an important animal model for investigating the molecular genetics of development.

Sleep remains a major mystery of biology. In particular, little is known about the mechanisms that account for the drive to sleep. In an unbiased screen of more than 12,000 Drosophila lines, we identified a single gene, nemuri, that induces sleep. The NEMURI protein is an antimicrobial peptide that can be secreted ectopically to drive prolonged sleep (with resistance to arousal) and to promote survival after infection. Loss of nemuri increased arousability during daily sleep and attenuated the acute increase in sleep induced by sleep deprivation or bacterial infection. Conditions that increase sleep drive induced expression of nemuri in a small number of fly brain neurons and targeted it to the sleep-promoting, dorsal fan-shaped body. We propose that NEMURI is a bona fide sleep homeostasis factor that is particularly important under conditions of high sleep need; because these conditions include sickness, our findings provide a link between sleep and immune function.

The hypothalamus plays an important role in regulating sleep, but few hypothalamic sleep-promoting signaling pathways have been identified. Here we demonstrate a role for the neuropeptide QRFP (also known as P518 and 26RFa) and its receptors in regulating sleep in zebrafish, a diurnal vertebrate. We show that QRFP is expressed in ∼10 hypothalamic neurons in zebrafish larvae, which project to the hypothalamus, hindbrain, and spinal cord, including regions that express the two zebrafish QRFP receptor paralogs. We find that the overexpression of QRFP inhibits locomotor activity during the day, whereas mutation ofqrfpor its receptors results in increased locomotor activity and decreased sleep during the day. Despite the restriction of these phenotypes to the day, the circadian clock does not regulateqrfpexpression, and entrained circadian rhythms are not required for QRFP-induced rest. Instead, we find that QRFP overexpression decreases locomotor activity largely in a light-specific manner. Our results suggest that QRFP signaling plays an important role in promoting sleep and may underlie some aspects of hypothalamic sleep control.SIGNIFICANCE STATEMENTThe hypothalamus is thought to play a key role in regulating sleep in vertebrate animals, but few sleep-promoting signaling pathways that function in the hypothalamus have been identified. Here we use the zebrafish, a diurnal vertebrate, to functionally and anatomically characterize the neuropeptide QRFP. We show that QRFP is exclusively expressed in a small number of neurons in the larval zebrafish hypothalamus that project widely in the brain. We also show that QRFP overexpression reduces locomotor activity, whereas animals that lack QRFP signaling are more active and sleep less. These results suggest that QRFP signaling participates in the hypothalamic regulation of sleep.

The zebrafish (Danio rerio) is a useful vertebrate model system in which to study neural circuits and behavior, but tools to modulate neurons in freely behaving animals are limited. As poikilotherms that live in water, zebrafish are amenable to thermal and pharmacological perturbations. We exploit these properties by using transient receptor potential (TRP) channels to activate or ablate specific neuronal populations using the chemical and thermal agonists of heterologously expressed TRPV1, TRPM8 and TRPA1.

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

Prior to the 21st century, genetic mechanisms that regulate sleep were largely unknown. In 1998, de Lecea et al. [1] (Proc. Natl. Acad. Sci. U. S. A. 1998; 95:322–327) and Sakurai et al. [2] (Cell 1998; 92: 573–585) reported the discovery of a gene they named hypocretin and orexin, respectively, which led to a revolution in our understanding of genetic and neuronal mechanisms that regulate sleep.

The duration of sleep varies dramatically between species, yet little is known about genetic bases or evolutionary factors driving this variation in behavior. The Mexican cavefish,Astyanax mexicanus, exists as surface populations that inhabit rivers, and multiple independently derived cave populations with convergent evolution on sleep loss. The number of Hypocretin/Orexin (HCRT)-positive hypothalamic neurons is increased significantly in cavefish, and HCRT is upregulated at both the transcript and protein levels. Pharmacological or genetic inhibition of HCRT signaling increases sleep duration in cavefish without affecting sleep in surface fish, suggesting enhanced HCRT signaling underlies sleep loss in cavefish. Ablation of the lateral line or starvation, manipulations that selectively promote sleep in cavefish, inhibithcrtexpression in cavefish while having little effect in surface fish. These findings provide the first evidence of genetic and neuronal changes that contribute to the evolution of sleep loss, and support a conserved role for HCRT in sleep regulation.

Sleep is an essential and phylogenetically conserved behavioral state, but it remains unclear to what extent genes identified in invertebrates also regulate vertebrate sleep. RFamide-related neuropeptides have been shown to promote invertebrate sleep, and here we report that the vertebrate hypothalamic RFamide neuropeptide VF (NPVF) regulates sleep in the zebrafish, a diurnal vertebrate. We found that NPVF signaling and npvf-expressing neurons are both necessary and sufficient to promote sleep, that mature peptides derived from the NPVF preproprotein promote sleep in a synergistic manner, and that stimulation of npvf-expressing neurons induces neuronal activity levels consistent with normal sleep. These results identify NPVF signaling and npvf-expressing neurons as a novel vertebrate sleep-promoting system and suggest that RFamide neuropeptides participate in an ancient and central aspect of sleep control.

Light affects sleep and wake behaviors by providing an indirect cue that entrains circadian rhythms and also by inducing a direct and rapid regulation of behavior. While circadian entrainment by light is well characterized at the molecular level, mechanisms that underlie the direct effect of light on behavior are largely unknown. In zebrafish, a diurnal vertebrate, we found that both overexpression and mutation of the neuropeptide prokineticin 2 (Prok2) affect sleep and wake behaviors in a light-dependent but circadian-independent manner. In light, Prok2 overexpression increases sleep and induces expression of galanin (galn), a hypothalamic sleep-inducing peptide. We also found that light-dependent, Prok2-induced sedation requires prokineticin receptor 2 (prokr2) and is strongly suppressed in galn mutants. These results suggest that Prok2 antagonizes the direct wake-promoting effect of light in zebrafish, in part through the induction of galn expression in the hypothalamus.

The role of serotonin (5-HT) in sleep is controversial: early studies suggested a sleep-promoting role, but eventually the paradigm shifted toward a wake-promoting function for the serotonergic raphe. Here, we provide evidence from zebrafish and mice that the raphe are critical for the initiation and maintenance of sleep. In zebrafish, genetic ablation of 5-HT production by the raphe reduces sleep, sleep depth, and the homeostatic response to sleep deprivation. Pharmacological inhibition or ablation of the raphe reduces sleep, while optogenetic stimulation increases sleep. Similarly, in mice, ablation of the raphe increases wakefulness and impairs the homeostatic response to sleep deprivation, whereas tonic optogenetic stimulation at a rate similar to baseline activity induces sleep. Interestingly, burst optogenetic stimulation induces wakefulness in accordance with previously described burst activity of the raphe during arousing stimuli. These results indicate that the serotonergic system promotes sleep in both diurnal zebrafish and nocturnal rodents. VIDEO ABSTRACT.

Pharmacological studies in mammals and zebrafish suggest that histamine plays an important role in promoting arousal. However, genetic studies using rodents with disrupted histamine synthesis or signaling have revealed only subtle or no sleep/wake phenotypes. Studies of histamine function in mammalian arousal are complicated by its production in cells of the immune system and its roles in humoral and cellular immunity, which can have profound effects on sleep/wake states. To avoid this potential confound, we used genetics to explore the role of histamine in regulating sleep in zebrafish, a diurnal vertebrate in which histamine production is restricted to neurons in the brain. Similar to rodent genetic studies, we found that zebrafish that lack histamine due to mutation of histidine decarboxylase (hdc) exhibit largely normal sleep/wake behaviors. Zebrafish containing predicted null mutations in several histamine receptors also lack robust sleep/wake phenotypes, although we are unable to verify that these mutants are completely nonfunctional. Consistent with some rodent studies, we found that arousal induced by overexpression of the neuropeptide hypocretin (Hcrt) or by stimulation ofhcrt-expressing neurons is not blocked inhdcorhrh1mutants. We also found that the number ofhcrt-expressing or histaminergic neurons is unaffected in animals that lack histamine or Hcrt signaling, respectively. Thus, while acute pharmacological manipulation of histamine signaling has been shown to have profound effects on zebrafish and mammalian sleep, our results suggest that chronic loss of histamine signaling due to genetic mutations has only subtle effects on sleep in zebrafish, similar to rodents.

Zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have been shown to induce targeted mutations, but they have not been extensively tested in any animal model. Here, we describe a large-scale comparison of ZFN and TALEN mutagenicity in zebrafish. Using deep sequencing, we found that TALENs are significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs. We observed a strong correlation between somatic and germ-line mutagenicity, and identified germ line mutations using ZFNs whose somatic mutations rates are well below the commonly used threshold of 1%. Guidelines that have previously been proposed to predict optimal ZFN and TALEN target sites did not predict mutagenicity in vivo. However, we observed a significant negative correlation between TALEN mutagenicity and the number of CpG repeats in TALEN target sites, suggesting that target site methylation may explain the poor mutagenicity of some TALENs in vivo. The higher mutation rates and ability to target essentially any sequence make TALENs the superior technology for targeted mutagenesis in zebrafish, and likely other animal models.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of approximately 20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.

Neuromodulation of arousal states ensures that an animal appropriately responds to its environment and engages in behaviors necessary for survival. However, the molecular and circuit properties underlying neuromodulation of arousal states such as sleep and wakefulness remain unclear. To tackle this challenge in a systematic and unbiased manner, we performed a genetic overexpression screen to identify genes that affect larval zebrafish arousal. We found that the neuropeptide neuromedin U (Nmu) promotes hyperactivity and inhibits sleep in zebrafish larvae, whereas nmu mutant animals are hypoactive. We show that Nmu-induced arousal requires Nmu receptor 2 and signaling via corticotropin releasing hormone (Crh) receptor 1. In contrast to previously proposed models, we find that Nmu does not promote arousal via the hypothalamic-pituitary-adrenal axis, but rather probably acts via brainstem crh-expressing neurons. These results reveal an unexpected functional and anatomical interface between the Nmu system and brainstem arousal systems that represents a novel wake-promoting pathway.

Egfr/Ras signaling promotes vein cell fate specification in the developing Drosophila wing. While the importance of Ras signaling in vein determination has been extensively documented, the mechanisms linking Ras activity to vein differentiation remain unclear. We found that Ras signaling regulates both the levels and subcellular localization of the cell adhesion molecule DE-cadherin/Shotgun (Shg) in the differentiating wing epithelium. High Ras activity in presumptive vein cells directs the apical localization of Shg containing adherens junctions, whereas low Ras activity in intervein cells allows Shg to relocalize basally. These alterations in Shg-mediated adhesion control cell shape changes that are essential for vein morphogenesis. While Decapentaplegic (Dpp) acts downstream of Ras to maintain vein cell identity in the pupal wing, our results indicate that Ras controls Shg localization via a Dpp-independent mechanism. Ras, therefore, regulates both the transcriptional responses necessary for vein cell identity, and the cell adhesive changes that determine vein and intervein cell morphology.

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine β-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep, dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons. DOI: http://dx.doi.org/10.7554/eLife.07000.001, eLife digest Although the neural circuits that regulate sleep and wakefulness have yet to be fully identified, the importance of at least two brain regions is well established. These are the hypothalamus, a structure deep within the brain that controls a number of basic activities including hunger, thirst and sleep; and the brainstem, which connects the brain with the spinal cord. Specific neurons within the hypothalamus and brainstem regulate the sleep–wake cycle by signaling to one another using chemicals called neurotransmitters and neuropeptides. Throughout the day, some hypothalamic neurons release a neuropeptide called hypocretin, which helps maintain wakefulness. Hypocretin acts on neurons within the brainstem and causes them to release other neurotransmitters that promote wakefulness. However, the identity of these molecules is unclear. One candidate is norepinephrine. Drugs that enhance the effects of norepinephrine increase wakefulness, whereas those that block norepinephrine signaling promote sleep. Despite this, mice that have been genetically modified to lack the enzyme that produces norepinephrine exhibit relatively normal sleep. This may be because in mammals, norepinephrine also has important roles outside the brain, thus complicating the effects of this genetic modification on behavior. Alternatively, while zebrafish that lack norepinephrine are healthy, mice containing this modification die early in development. Treating these mice with a specific drug allows them to survive, but might affect their behavior. To clarify the role of norepinephrine and its interaction with hypocretin, Singh, Oikonomou and Prober created a new animal model by genetically modifying zebrafish. In contrast to mice, zebrafish that were unable to make norepinephrine slept more than normal fish, although they were also lighter sleepers and were more prone to being startled. A genetic modification that increases hypocretin signaling induces insomnia; Singh, Oikonomou and Prober found that this occurs only in animals with normal levels of norepinephrine. Thus, these experiments indicate that hypocretin does indeed promote wakefulness though norepinephrine. The work of Singh, Oikonomou and Prober has clarified the role of norepinephrine in regulating the sleep–wake cycle. These findings could help in the development of drugs that target the neurons that make hypocretin, which may improve treatments for sleep disorders. DOI: http://dx.doi.org/10.7554/eLife.07000.002

Loss of neurons that express the neuropeptide hypocretin (Hcrt) has been implicated in narcolepsy, a debilitating disorder characterized by excessive daytime sleepiness and cataplexy. Cell replacement therapy, using Hcrt-expressing neurons generated in vitro, is a potentially useful therapeutic approach, but factors sufficient to specify Hcrt neurons are unknown. Using zebrafish as a high-throughput system to screen for factors that can specify Hcrt neurons in vivo, we identified the LIM homeobox transcription factor Lhx9 as necessary and sufficient to specify Hcrt neurons. We found that Lhx9 can directly induce hcrt expression and we identified two potential Lhx9 binding sites in the zebrafish hcrt promoter. Akin to its function in zebrafish, we found that Lhx9 is sufficient to specify Hcrt-expressing neurons in the developing mouse hypothalamus. Our results elucidate an evolutionarily conserved role for Lhx9 in Hcrt neuron specification that improves our understanding of Hcrt neuron development.

Sleep is an essential and evolutionarily conserved behavioral state whose regulation remains poorly understood. To identify genes that regulate vertebrate sleep, we recently performed a genetic screen in zebrafish, and here we report the identification of neuropeptide Y (NPY) as both necessary for normal daytime sleep duration and sufficient to promote sleep. We show that overexpression of NPY increases sleep, whereas mutation of npy or ablation of npy-expressing neurons decreases sleep. By analyzing sleep architecture, we show that NPY regulates sleep primarily by modulating the length of wake bouts. To determine how NPY regulates sleep, we tested for interactions with several systems known to regulate sleep, and provide anatomical, molecular, genetic, and pharmacological evidence that NPY promotes sleep by inhibiting noradrenergic signaling. These data establish NPY as an important vertebrate sleep/wake regulator and link NPY signaling to an established arousal-promoting system.

Trabajo presentado en la IV International Meeting on Marine Research (IMMR 2014), celebrada en Peniche (Portugal) el 10 y 11 de julio de 2014.

A great deal of work has focused on how oncogenes regulate the cell cycle during normal development and in cancer, yet their roles in regulating cell growth have been largely unexplored. Recent work in several model organisms has demonstrated that homologs of several oncogenes regulate cell growth and has suggested that some of the effects of oncogenes on the cell cycle may be a result of growth promotion. These studies have also suggested how growth and cell-cycle progression may be coupled.

The Ras GTPase links extracellular mitogens to intracellular mechanisms that control cell proliferation. To understand how Ras regulates proliferation in vivo, we activated or inactivated Ras in cell clones in the developing Drosophila wing. Cells lacking Ras were smaller, had reduced growth rates, accumulated in G1, and underwent apoptosis due to cell competition. Conversely, activation of Ras increased cell size and growth rates and promoted G1/S transitions. Ras upregulated the growth driver dMyc, and both Ras and dMyc increased levels of cyclin E posttranscriptionally. We propose that Ras primarily promotes growth and that growth is coupled to G1/S progression via cyclin E. Interestingly, upregulation of growth by Ras did not deregulate G2/M progression or a developmentally regulated cell cycle exit.

Transcription factors of the Myc proto-oncogene family promote cell division, but how they do this is poorly understood. Here we address the functions of Drosophila Myc (dMyc) during development. Using mosaic analysis in the fly wing, we show that loss of dMyc retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rates and cell size. dMyc-induced growth promotes G1/S progression but fails to accelerate cell division because G2/M progression is independently controlled by Cdc25/String. We also show that the secreted signal Wingless patterns growth in the wing primordium by modulating dMyc expression. Our results indicate that dMyc links patterning signals to cell division by regulating primary targets involved in cellular growth and metabolism.

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal’s well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish has emerged as a promising model system for sleep and arousal research. Here we review experimental evidence that the zebrafish, a diurnal vertebrate, exhibits fundamental behavioral and neurochemical characteristics of mammalian sleep and arousal. We also propose how specific advantages of the zebrafish can be harnessed to advance the field. These include tractable genetics to identify and manipulate molecular and cellular regulators of behavioral states, optical transparency to facilitate in vivo observation of neural structure and function, and amenability to high-throughput drug screens to discover novel therapies for neurological disorders.