Large-scale Analysis of Sleep in Zebrafish.

Over the past decade, zebrafish have emerged as a powerful model for the study of vertebrate sleep and wake behaviors. Experimental evidence has demonstrated behavioral, anatomical, genetic, and pharmacological conservation of sleep between zebrafish and mammals, suggesting that discoveries in zebrafish can inform our understanding of mammalian sleep. Here, we describe a protocol for performing sleep behavioral experiments in larval zebrafish, using a high-throughput video tracking system. We explain how to set up a sleep behavioral experiment and provide guidelines on how to analyze the data. Using this protocol, a typical experiment can be completed in less than five days, and this method provides a scalable platform to perform genetic and pharmacological screens in a simple and cost-effective vertebrate model. By combining high-throughput behavioral assays with several advantageous features of zebrafish, this model system provides new opportunities to make discoveries that clarify the genetic and neurological mechanisms that regulate sleep.

Attenuating the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase-Sex-Determining Region Y-Box 9 Axis Promotes Liver Progenitor Cell-Mediated Liver Regeneration in Zebrafish.

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.

Neuropeptide VF neurons promote sleep via the serotonergic raphe.

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.

Evolutionarily Conserved Regulation of Sleep by the Protein Translational Regulator PERK.

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.

Evolutionarily conserved regulation of sleep by epidermal growth factor receptor signaling.

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.

Genetic and neuronal regulation of sleep by neuropeptide VF.

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.

A Zebrafish Genetic Screen Identifies Neuromedin U as a Regulator of Sleep/Wake States.

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.

The LIM homeodomain factor Lhx2 is required for hypothalamic tanycyte specification and differentiation.

Hypothalamic tanycytes, a radial glial-like ependymal cell population that expresses numerous genes selectively enriched in embryonic hypothalamic progenitors and adult neural stem cells, have recently been observed to serve as a source of adult-born neurons in the mammalian brain. The genetic mechanisms that regulate the specification and maintenance of tanycyte identity are unknown, but are critical for understanding how these cells can act as adult neural progenitor cells. We observe that LIM (Lin-11, Isl-1, Mec-3)-homeodomain gene Lhx2 is selectively expressed in hypothalamic progenitor cells and tanycytes. To test the function of Lhx2 in tanycyte development, we used an intersectional genetic strategy to conditionally delete Lhx2 in posteroventral hypothalamic neuroepithelium, both embryonically and postnatally. We observed that tanycyte development was severely disrupted when Lhx2 function was ablated during embryonic development. Lhx2-deficient tanycytes lost expression of tanycyte-specific genes, such as Rax, while also displaying ectopic expression of genes specific to cuboid ependymal cells, such as Rarres2. Ultrastructural analysis revealed that mutant tanycytes exhibited a hybrid identity, retaining radial morphology while becoming multiciliated. In contrast, postnatal loss of function of Lhx2 resulted only in loss of expression of tanycyte-specific genes. Using chromatin immunoprecipitation, we further showed that Lhx2 directly regulated expression of Rax, an essential homeodomain factor for tanycyte development. This study identifies Lhx2 as a key intrinsic regulator of tanycyte differentiation, sustaining Rax-dependent activation of tanycyte-specific genes while also inhibiting expression of ependymal cell-specific genes. These findings provide key insights into the transcriptional regulatory network specifying this still poorly characterized cell type.

Dietary and sex-specific factors regulate hypothalamic neurogenesis in young adult mice.

The hypothalamus is the central regulator of a broad range of homeostatic and instinctive physiological processes, such as the sleep-wake cycle, food intake, and sexually dimorphic behaviors. These behaviors can be modified by various environmental and physiological cues, although the molecular and cellular mechanisms that mediate these effects remain poorly understood. Recently, it has become clear that both the juvenile and adult hypothalamus exhibit ongoing neurogenesis, which serve to modify homeostatic neural circuitry. In this report, we share new findings on the contributions of sex-specific and dietary factors to regulating neurogenesis in the hypothalamic mediobasal hypothalamus, a recently identified neurogenic niche. We report that high fat diet (HFD) selectively activates neurogenesis in the median eminence (ME) of young adult female but not male mice, and that focal irradiation of the ME in HFD-fed mice reduces weight gain in females but not males. These results suggest that some physiological effects of high fat diet are mediated by the stimulation of ME neurogenesis in a sexually dimorphic manner. We discuss these results in the context of recent advances in understanding the cellular and molecular mechanisms that regulate neurogenesis in postnatal and adult hypothalamus.

Feed your head: neurodevelopmental control of feeding and metabolism.

During critical periods of development early in life, excessive or scarce nutritional environments can disrupt the development of central feeding and metabolic neural circuitry, leading to obesity and metabolic disorders in adulthood. A better understanding of the genetic networks that control the development of feeding and metabolic neural circuits, along with knowledge of how and where dietary signals disrupt this process, can serve as the basis for future therapies aimed at reversing the public health crisis that is now building as a result of the global obesity epidemic. This review of animal and human studies highlights recent insights into the molecular mechanisms that regulate the development of central feeding circuitries, the mechanisms by which gestational and early postnatal nutritional status affects this process, and approaches aimed at counteracting the deleterious effects of early over- and underfeeding.

Functional interrogation of adult hypothalamic neurogenesis with focal radiological inhibition.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

Efficacy and safety of ingredients found in preworkout supplements.

PURPOSE: Published evidence on common ingredients of "energy drinks" and other dietary supplements widely used by consumers in hopes of enhancing athletic performance is reviewed. SUMMARY: Preworkout products- unregulated dietary supplements- typically contain "proprietary blends" of multiple ingredients, including caffeine, dimethylamylamine, creatine, arginine, ß-alanine, taurine, and phosphates. While some dietary supplement labels instruct consumers to seek the advice of a health care professional before using the products, the labels usually do not disclose all ingredients or their precise amounts, and evidence to support the purported performance-enhancing benefits is generally lacking. There is limited evidence to support the use of some preworkout supplement ingredients. For example, in one small placebo-controlled study (n = 12), the use of the energy drink Red Bull (containing caffeine and taurine) 40 minutes before a simulated cycling time trial appeared to provide a meaningful ergogenic benefit; in another small study (n = 12), the use of a similar caffeine-containing product (Redline) by strength-trained athletes was found to improve reaction time, energy, and mental focus relative to placebo use. However, published evidence on the use of the other ingredients listed above is scant, inconclusive, or conflicting. Adverse effects reported in association with preworkout supplements include gastrointestinal symptoms, cardiac arrhythmia, blood pressure increases, and potential effects on lipids and blood glucose. CONCLUSION: Although evidence exists to support the performance-enhancement efficacy of some preworkout ingredients as standalone agents, published data on combination products are scant, inconclusive, or conflicting. The safety of these products may be compromised if users consume larger-than-recommended amounts or use more than one product.

A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly.

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.

Functional implications of hypothalamic neurogenesis in the adult mammalian brain.

Adult neurogenesis represents a striking example of structural plasticity in the mature brain. Research on adult mammalian neurogenesis today focuses almost exclusively on two areas: the subgranular zone (SGZ) in the dentate gyrus of the hippocampus, and the subventricular zone (SVZ) of the lateral ventricles. Numerous studies, however, have also reported adult neurogenesis in the hypothalamus, a brain structure that serves as a central homeostatic regulator of numerous physiological and behavioral functions, such as feeding, metabolism, body temperature, thirst, fatigue, aggression, sleep, circadian rhythms, and sexual behavior. Recent studies on hypothalamic neurogenesis have identified a progenitor population within a dedicated hypothalamic neurogenic zone. Furthermore, adult born hypothalamic neurons appear to play a role in the regulation of metabolism, weight, and energy balance. It remains to be seen what other functional roles adult hypothalamic neurogenesis may play. This review summarizes studies on the identification and characterization of neural stem/progenitor cells in the mammalian hypothalamus, in what contexts these stem/progenitor cells engage in neurogenesis, and potential functions of postnatally generated hypothalamic neurons.

WITHDRAWN: Functional implications of hypothalamic neurogenesis in the adult mammalian brain.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi 10.1016/j.ijdevneu.2012.07.003. The duplicate article has therefore been withdrawn.

Tanycytes of the hypothalamic median eminence form a diet-responsive neurogenic niche.

Adult hypothalamic neurogenesis has recently been reported, but the cell of origin and the function of these newborn neurons are unknown. Using genetic fate mapping, we found that median eminence tanycytes generate newborn neurons. Blocking this neurogenesis altered the weight and metabolic activity of adult mice. These findings reveal a previously unreported neurogenic niche in the mammalian hypothalamus with important implications for metabolism.

The orphan nuclear hormone receptor ERRbeta controls rod photoreceptor survival.

Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor estrogen-related receptor beta (ERRbeta) as selectively expressed in rod photoreceptors. Overexpression of ERRbeta induces expression of rod-specific genes in retinas of wild-type as well as Nrl(-/-) mice, which lack rod photoreceptors. Mutation of ERRbeta results in dysfunction and degeneration of rods, whereas inverse agonists of ERRbeta trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRbeta. ERRbeta coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRbeta activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRbeta is a critical regulator of rod photoreceptor function and survival, and suggest that ERRbeta agonists may be useful in the treatment of certain retinal dystrophies.

A genomic atlas of mouse hypothalamic development.

The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.