Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen.

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.

QRFP and Its Receptors Regulate Locomotor Activity and Sleep in Zebrafish.

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 of qrfp or 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 regulate qrfp expression, 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 STATEMENT: The 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.

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.

TRP channel mediated neuronal activation and ablation in freely behaving zebrafish.

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.

Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9.

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.

Regulation of zebrafish sleep and arousal states: current and prospective approaches.

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.

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.

Exchange of genetic sequences of long terminal repeat and the env gene by a promiscuous primate type D retrovirus.

Squirrel monkey retrovirus (SMRV) is a New World primate type D retrovirus. It was shown that SMRV-related sequences could be detected in another New World species, the skunk. It was further suggested that SMRV and an Old World primate type C retrovirus, baboon endogenous virus (BaEV), may have exchanged their env gene sequences. In this study, we sought to understand which sequences were exchanged between the genomic DNAs of SMRV and skunk. We also sought to determine the sequences exchanged between SMRV and BaEV. Here, we demonstrate that the long terminal repeat of SMRV is present in the skunk genome. We also show, by nucleotide sequence analysis, that the env gene that encodes the p15E glycoprotein of BaEV was most likely transduced from the corresponding gene of a primate type D retrovirus. Our results demonstrate that SMRV is a promiscuous virus with its pol gene homologous to the pol genes of type A, type B and avian type C viruses and a portion of its env gene homologous to the env genes of primate type C retroviruses. However, the primer binding sequence is unique to type D retroviruses. These kinds of recombination are likely to occur more than once in the evolution of retroviruses. The promiscuous nature of retroviruses and the recent incidence of unintended retroviral integration into a gene therapy patient underscore the importance of understanding how retroviral sequences are recombined among themselves and how they are integrated into the mammalian genome.