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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.

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains has recently been found to contain cryptic open reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here we show that two putative lincRNAs (linc-mipep and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes – evolutionarily newer cell types that develop postnatally in humans. These findings highlight the power of screening for unexplored micropeptide functions by revealing a key missing link in the evolution of vertebrate brain cell development and illustrating a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

Sleep-wake disturbances are among the earliest symptoms of Alzheimer’s disease (AD) and contribute to disease severity. Since a major driver of AD—the accumulation of amyloid-beta (Aβ) in the brain—is modulated by sleep, a “vicious” feedforward cycle has been proposed in which Aβ buildup disrupts sleep, leading to more Aβ secretion and further worsening of sleep and AD. Consistent with this idea, mouse models of AD develop early sleep phenotypes, and sleep is acutely modulated by exogenous Aβ. However, these overexpression paradigms leave unclear whether endogenous Aβ signaling contributes to sleep regulation. To tackle this question, we generated loss of function mutations in the zebrafish orthologs of Amyloid Precursor Protein (APP) and monitored larval sleep behavior. Larvae with mutations in appa had reduced waking activity levels but normal sleep patterns, while larvae that lacked appb had reduced sleep due to an inability to maintain sleep bout durations. In addition, larvae exposed to the γ-secretase inhibitor DAPT, which inhibits Aβ production from APP, also have shorter sleep bouts at night. Although γ- secretase inhibition impacts the proteolytic cleavage of many proteins, appb mutants were insensitive to the sleep bout shortening effects of DAPT, suggesting that loss of γ-secretase dependent proteolytic cleavage products of Appb are responsible for the reduced sleep maintenance phenotypes. These results are consistent with a model in which endogenous Aβ directly modulates sleep in zebrafish and supports the idea that sleep disturbances may be a useful early-onset biomarker for AD.

Please cite 10.7554/eLife.59683 if you use this protocol. Note; our eLife publication used Version 2 of this protocol. Later version are subsequent improvements/simplifications. Get in touch for questions/suggestions twitter –@francois_kroll email –francois@kroll.be

Research over the last 20 years has firmly established the existence of sleep states across the animal kingdom. Work in non-mammalian animal models such as nematodes, fruit flies, and zebrafish has now uncovered many evolutionarily conserved aspects of sleep physiology and regulation, including shared circuit architecture, homeostatic and circadian control elements, and principles linking sleep physiology to function. Non-mammalian sleep research is now shedding light on fundamental aspects of the genetic and neuronal circuit regulation of sleep, with direct implications for the understanding of how sleep is regulated in mammals.

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockoutcrystalfish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

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.

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. SCAR20 is understood to involve subcellular disruption to autophagy and lipid metabolism. Previously reported studies on the phenotypic consequences of SNX14 mutations have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. In addition, studies have investigated the biochemical roles of SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) which have demonstrated an important role during lipid biogenesis. This study investigates the impact of constitutive Snx14 mutations in laboratory species: mice and zebrafish. Loss of SNX14 in mice was found to be embryonic lethal around mid-gestation. This is due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. Zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation contrasts with other vertebrates, being both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the overall consequences of loss of function varies considerably between species. New mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

Data and code for preprint Kroll, F. et al.2020. A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes. https://www.biorxiv.org/content/10.1101/2020.06.04.133462v3 You can also navigate the files here:https://github.com/francoiskroll/f0knockout (some of the heavier files are only included in the Zenodo repository) If you use some of the data or code, please cite the preprint! ___ Contact: Twitter: @francois_kroll Email: francois@kroll.be

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockoutcrystalfish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Animal behavior is dynamic, evolving over multiple timescales from milliseconds to days and even across a lifetime. To understand the mechanisms governing these dynamics, it is necessary to capture multi-timescale structure from behavioral data. Here, we develop computational tools and study the behavior of hundreds of larval zebrafish tracked continuously across multiple 24-h day/night cycles. We extracted millions of movements and pauses, termed bouts, and used unsupervised learning to reduce each larva’s behavior to an alternating sequence of active and inactive bout types, termed modules. Through hierarchical compression, we identified recurrent behavioral patterns, termed motifs. Module and motif usage varied across the day/night cycle, revealing structure at sub-second to day-long timescales. We further demonstrate that module and motif analysis can uncover novel pharmacological and genetic mutant phenotypes. Overall, our work reveals the organization of larval zebrafish behavior at multiple timescales and provides tools to identify structure from large-scale behavioral datasets.

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14−/− mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14−/− zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.

The explosion of genome-wide association studies and whole genome sequencing of tens of thousands of individual humans have led to the rapid discovery of many genetic alterations implicated in neurodevelopmental and neuropsychiatric disorders, including schizophrenia, intellectual disability, and autism spectrum disorder (ASD). This wealth of genetic information brings new challenges to uncover how these genes work, alone and in concert, to sculpt the development and function of neuronal circuits that impact behavior. The zebrafish is an excellent system in which to tackle this challenge, as the zebrafish model combines the facility to quickly and in parallel generate mutations in dozens or hundreds of genes with a reasonable fidelity to human biology, including a highly conserved genome and a vertebrate brain plan. Moreover, their rapid development and complex behavioral repertoire at young ages grant experimental access to both early and late stages of nervous system form and function. Although no animal model can completely capture all aspects of human neurodevelopmental disorders, which can impact unique human traits such as language, the zebrafish does have many clinically important behaviors, including sleep, learning and memory, and complex social interactions, and these behaviors are governed by conserved genetic and neuronal processes. This chapter will examine the use of zebrafish to investigate the neurodevelopmental disorder, ASD. In particular, we will discuss the genetics of ASD, the zebrafish models of ASD that have been generated to date, and the scope for examining critical aspects of ASD biology, especially social interactions, in the zebrafish model.

The zebrafish represents an excellent compromise between system complexity and practical simplicity, features that make it useful for modeling and mechanistic analysis of complex brain disorders. Also promising are screens for psychoactive drugs with effects on larval and adult zebrafish behavior. This review, based upon a recent symposium held at the 2016 IBNS Congress, provides different perspectives on how the zebrafish may be utilized to advance research into human central nervous system disorders. It starts with a discussion on an important bottleneck in zebrafish research, measuring the behavior of this species (specifically shoaling), and continues with examples on research on autism spectrum disorder in larval zebrafish, on screening natural products for compounds with psychoactive properties in adult zebrafish, and on the development of a zebrafish model of fetal alcohol spectrum disorders. By providing information on a broad spectrum of brain disorders, experimental methods, and scientific approaches using both larval and adult zebrafish, the review is intended to showcase this underutilized laboratory species for behavioral neuroscience and psychopharmacology research.

Animal behaviour is dynamic, evolving over multiple timescales from milliseconds to days and even across a lifetime. To understand the mechanisms governing these dynamics, it is necessary to capture multi-timescale structure from behavioural data. Here, we develop computational tools and study the behaviour of hundreds of larval zebrafish tracked continuously across multiple 24-hour day/night cycles. We extracted millions of movements and pauses, termed bouts, and used unsupervised learning to reduce each larva’s behaviour to an alternating sequence of active and inactive bout types, termed modules. Through hierarchical compression, we identified recurrent behavioural patterns, termed motifs. Module and motif usage varied across the day/night cycle, revealing structure at sub-second to day-long timescales. We further demonstrate that module and motif analysis can uncover novel pharmacological and genetic mutant phenotypes. Overall, our work reveals the organisation of larval zebrafish behaviour at multiple timescales and provides tools to identify structure from large-scale behavioural datasets.

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-β, the toxic driver of Alzheimer’s disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges. Electronic supplementary material The online version of this article (10.1007/s00401-019-02091-z) contains supplementary material, which is available to authorized users.

SUMMARYDisrupted sleep is a major feature of Alzheimer’s Disease (AD), often arising years before symptoms of cognitive decline. Prolonged wakefulness exacerbates the production of amyloid-beta (Aβ) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which Aβ affects sleep are unknown. We demonstrate in zebrafish that Aβ acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short Aβ oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer Aβ forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signalling cascade. Our data indicate that Aβ can trigger a bi-directional sleep/wake switch. Alterations to the brain’s Aβ oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signalling events.HIGHLIGHTSAmyloid beta oligomers can drive either sleep or wakefulness, depending on their sizeWakefulness driven by short amyloid beta oligomers requires binding partners Adrenergic Beta Receptor 2 and Pgrmc1Long amyloid beta oligomers drive sleep through interaction with Prion ProteinThe in vivo sleep effects of amyloid beta can be pharmacologically blocked by targeting several steps of the Amyloid beta-Prion Protein signalling cascade.

Sleep pressure homeostatically increases during wake and dissipates during sleep, but the molecular signals and neuronal substrates that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates acute, short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole brain mapping with the neuronal activity marker phosphorylated extracellular signal–regulated kinase (pERK) identified preoptic Galanin-expressing neurons as selectively active during rebound sleep, and the relative induction of galanin transcripts was predictive of total rebound sleep time. Galanin is required for sleep homeostasis, as galanin mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galanin plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat. (word count: 158).

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

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 lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain. DOI: http://dx.doi.org/10.7554/eLife.25932.001

All animals have a fundamental and unavoidable requirement for rest, yet we still do not fully understand the processes that initiate, maintain, and regulate sleep. The larval zebrafish is an optically translucent, genetically tractable model organism that exhibits sleep states regulated by conserved sleep circuits, thereby offering a unique system for investigating the genetic and neural control of sleep. Recent studies using high throughput monitoring of larval sleep/wake behaviour have unearthed novel modulators involved in regulating arousal and have provided new mechanistic insights into the role of established sleep/wake modulators. In addition, the application of computational tools to large behavioural datasets has allowed for the identification of neuroactive compounds that alleviate sleep symptoms associated with genetic neurological disorders.

Detailed descriptions of brain-scale sensorimotor circuits underlying vertebrate behavior remain elusive. Recent advances in zebrafish neuroscience offer new opportunities to dissect such circuits via whole-brain imaging, behavioral analysis, functional perturbations, and network modeling. Here, we harness these tools to generate a brain-scale circuit model of the optomotor response, an orienting behavior evoked by visual motion. We show that such motion is processed by diverse neural response types distributed across multiple brain regions. To transform sensory input into action, these regions sequentially integrate eye- and direction-specific sensory streams, refine representations via interhemispheric inhibition, and demix locomotor instructions to independently drive turning and forward swimming. While experiments revealed many neural response types throughout the brain, modeling identified the dimensions of functional connectivity most critical for the behavior. We thus reveal how distributed neurons collaborate to generate behavior and illustrate a paradigm for distilling functional circuit models from whole-brain data.

Understanding how metals contribute to brain function is a major health priority. A new study combining pharmacology and genetics implicates the accumulation of copper in a brain arousal center as a regulator of zebrafish activity.

Many studies in diverse organisms, including humans, have demonstrated a fundamental role for sleep in the formation of memories. A new study by Berry et al. indicates that, in fruit flies, sleep accomplishes this in part by preventing an active process of forgetting.

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.

Darkness serves as a stimulus for vertebrate photoreceptors; they are actively depolarized in the dark and hyperpolarize in the light. Here, we show that larval zebrafish essentially turn off their visual system at night when they are not active. Electroretinograms recorded from larval zebrafish show large differences between day and night; the responses are normal in amplitude throughout the day but are almost absent after several hours of darkness at night. Behavioral testing also shows that larval zebrafish become unresponsive to visual stimuli at night. This phenomenon is largely circadian driven as fish show similar dramatic changes in visual responsiveness when maintained in continuous darkness, although light exposure at night partially restores the responses. Visual responsiveness is decreased at night by at least two mechanisms: photoreceptor outer segment activity decreases and synaptic ribbons in cone pedicles disassemble.

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.

Light affects animal physiology and behavior more than simply through classical visual, image-forming pathways. Nonvisual photoreception regulates numerous biological systems, including circadian entrainment, DNA repair, metabolism, and behavior. However, for the majority of these processes, the photoreceptive molecules involved are unknown. Given the diversity of photophysiological responses, the question arises whether a single photopigment or a greater diversity of proteins within the opsin superfamily detect photic stimuli. Here, a functional genomics approach identified the full complement of photopigments in a highly light-sensitive model vertebrate, the zebrafish (Danio rerio), and characterized their tissue distribution, expression levels, and biochemical properties. The results presented here reveal the presence of 42 distinct genes encoding 10 classical visual photopigments and 32 nonvisual opsins, including 10 novel opsin genes comprising four new pigment classes. Consistent with the presence of light-entrainable circadian oscillators in zebrafish, all adult tissues examined expressed two or more opsins, including several novel opsins. Spectral and electrophysiological analyses of the new opsins demonstrate that they form functional photopigments, each with unique chromophore-binding and wavelength specificities. This study has revealed a remarkable number and diversity of photopigments in zebrafish, the largest number so far discovered for any vertebrate. Found in amphibians, reptiles, birds, and all three mammalian clades, most of these genes are not restricted to teleosts. Therefore, nonvisual light detection is far more complex than initially appreciated, which has significant biological implications in understanding photoreception in vertebrates.

The extraordinary cellular heterogeneity of the mammalian nervous system has largely hindered the molecular analysis of neuronal identity and diversity. In order to uncover mechanisms involved in neuronal differentiation and diversification, we have monitored the expression profiles of individual neurons and progenitor cells collected from dissociated tissue or captured from intact slices. We demonstrate that this technique provides a sensitive and reproducible representation of the single-cell transcriptome. In the olfactory system, hundreds of transcriptional differences were identified between olfactory progenitors and mature sensory neurons, enabling us to define the large variety of signaling pathways expressed by individual progenitors at a precise developmental stage. Finally, we show that regional differences in gene expression can be predicted from transcriptional analysis of single neuronal precursors isolated by laser capture from defined areas of the developing brain.