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Supplementary Table S1, Gene sets that are enriched in Hmga2/GFP-positive and -negative PDAC cells; Supplementary Table S2, A stringent list of Blimp1-dependent genes that are either induced or repressed under hypoxia; Supplementary Table S3, GO terms enriched in hypoxia-induced, Blimp1-dependent genes by FuncAssociate 3.0.

Supplementary Figure S1. Isolation of the Hmga2-GFPpos PDAC sub-population from the KPCcolors mice and GFPpos PDAC cells are a highly metastatic state;Supplementary Figure S2. Highly metastatic PDAC cells have a gene signature that is not enriched for CSC markers and distal PDAC metastases reveal minor gene expression changes related to glucose metabolism;Supplementary Figure S3. Identification of top candidate pro-metastatic genes and interrogation of Blimp1 function in PDAC metastasis;Supplementary Figure S4. Hmga2positive PDAC areas overlap with hypoxic areas and Hmga2 protein is slightly induced by hypoxia but not critical to the expression of hypoxia-induced target genes;Supplementary Figure S5. Hypoxia-induced Blimp1 expression is linked to functional HRE motifs 240 kb upstream of its transcription start site;Supplementary Figure S6. Blimp1 may contribute to migratory and clonal growth ability and is critical for a subset of hypoxia-induced gene expression changes that are independent of changes in chromatin accessibility;Supplementary Figure S7. Blimp1 regulates a subset of hypoxia-induced genes; Supplementary Figure S8. Blimp1 is required for hypoxia-induced cell cycle arrest and the expression of metastasis modulators.

All animals carefully studied sleep, suggesting that sleep as a behavioral state exists in all animal life. Such evolutionary maintenance of an otherwise vulnerable period of environmental detachment suggests that sleep must be integral in fundamental biological needs. Despite over a century of research, the knowledge of what sleep does at the tissue, cellular or molecular levels remain cursory. Currently, sleep is defined based on behavioral criteria and physiological measures rather than at the cellular or molecular level. Physiologically, sleep has been described as two main states, non-rapid eye moment (NREM) and REM/paradoxical sleep (PS), which are defined in the neocortex by synchronous oscillations and paradoxical wake-like activity, respectively. For decades, these two sleep states were believed to be defining characteristics of only mammalian and avian sleep. Recent work has revealed slow oscillation, silencing, and paradoxical/REM-like activities in reptiles, fish, flies, worms, and cephalopods suggesting that these sleep dynamics and associated physiological states may have emerged early in animal evolution. Here, we discuss these recent developments supporting the conservation of neural dynamics (silencing, oscillation, paradoxical activity) of sleep states across phylogeny.

INTRODUCTION: Sleep destabilization is strongly associated with aging and cognitive function decline. Despite sleep fragmentation being central to the most prevalent complaints of sleep problems in elderly populations, the mechanistic underpinnings of sleep instability remain elusive. Fragmented sleep during aging has been observed across species, indicating conserved underlying mechanisms across the phylogenetic tree. Therefore, understanding why the aging brain fails to consolidate sleep may shed light on translational applications for improving the sleep quality of aged individuals. RATIONALE: We hypothesized that the decline in sleep quality could be due to malfunction of the neural circuits associated with sleep/wake control. It has been established that hypocretin/orexin (Hcrt/OX) neuronal activity is tightly associated with wakefulness and initiates and maintains the wake state. In this study, we investigated whether the intrinsic excitability of Hcrt neurons is altered, leading to a destabilized control of sleep/wake states during aging. RESULTS: Aged mice exhibited sleep fragmentation and a significant loss of Hcrt neurons. Hcrt neurons manifested a more frequent firing pattern, driving wake bouts and disrupting sleep continuity in aged mice. Aged Hcrt neurons were capable of eliciting more prolonged wake bouts upon optogenetic stimulations. These results suggested that hyperexcitability of Hcrt neurons emerges with age. Patch clamp recording in genetically identified Hcrt neurons revealed distinct intrinsic properties between the young and aged groups. Aged Hcrt neurons were hyperexcitable with depolarized membrane potentials (RMPs) and a smaller difference between RMP and the firing threshold. Aged Hcrt neurons expressing ChR2-eYFP were more sensitive to optogenetic stimulations, with a smaller-amplitude attenuation upon repetitive light pulse stimulations. More spikelets were generated in aged Hcrt neurons upon current injections. Recording from non-Hcrt neurons postsynaptic to Hcrt neurons revealed that optogenetic stimulation of Hcrt neurons expressing ChR2-eYFP reliably evoked time-locked postsynaptic currents (PSCs) after optogenetic stimulation more often in the aged group. Aged Hcrt neurons were characterized with a functional impairment of repolarizing M-current mediated by KCNQ2/3 channels and an anatomical loss of KCNQ2, revealed with array tomography at ultrastructural resolution. Single-nucleus RNA-sequencing (snRNA-seq) revealed molecular adaptions, including up-regulated prepro-Hcrt mRNA expression and a smaller fraction of Kcnq family subtypes Kcnq1/2/3/5 in aged Hcrt neurons. CRISPR/SaCas9–mediated disruption of Kcnq2/3 genes selectively in Hcrt neurons was sufficient to recapitulate the aging-associated sleep fragmentation trait in young mice. Pharmacological augmentation of M-current repolarized the RMP, suppressed spontaneous firing activity in aged Hcrt neurons, and consolidated sleep stability in aged mice. Sleep fragmentation in a narcolepsy mouse model with genetic ablation of Hcrt neurons at young ages manifested a mechanism other than hyperexcitable arousal-promoting Hcrt neurons that drives sleep fragmentation during healthy aging. CONCLUSION: Our data indicate that emerging hyperexcitability of arousal-promoting Hcrt neurons is strongly associated with fragmented sleep in aged mice, which display a lowered sleep-to-wake transition threshold defined for Hcrt neuronal activity. We have demonstrated that the down-regulation of KCNQ2/3 channels compromising repolarization drives Hcrt neuronal hyperexcitability, which leads to sleep instability during aging. Pharmacological remedy of sleep continuity through targeting KCNQ2/3 channels in aged mice confers a potential translational therapy strategy for improving sleep quality in aged individuals.

Neurological disorders encompass an extremely broad range of conditions, including those that present early in development and those that progress slowly or manifest with advanced age. Although these disorders have distinct underlying etiologies, the activation of shared pathways, e.g., integrated stress response (ISR) and the development of shared phenotypes (sleep deficits) may offer clues toward understanding some of the mechanistic underpinnings of neurologic dysfunction. While it is incontrovertibly complex, the relationship between sleep and persistent stress in the brain has broad implications in understanding neurological disorders from development to degeneration. The convergent nature of the ISR could be a common thread linking genetically distinct neurological disorders through the dysregulation of a core cellular homeostasis pathway.

William C. Dement, one of the founders of sleep science and medicine, died on 17 June. He was 91. Bill devoted his life to helping the public appreciate the importance of sleep health. His pioneering work, which included the discovery of rapid eye movement (REM) sleep, touched countless lives. Born in Wenatchee, Washington, in 1928, Bill earned a bachelor's degree from the University of Washington in Seattle in basic medical science in 1951. He then started medical school at the University of Chicago, where he conducted groundbreaking work even before receiving his M.D. in 1955 and his Ph.D. in neurophysiology in 1957. He earned his medical license at Mount Sinai Hospital in New York City and then stayed on to continue his sleep research. In 1963, Bill joined the psychiatry department at Stanford University in Stanford, California, where he remained for the rest of his career. Bill first encountered sleep research at the University of Chicago, where he joined the lab of physiologist and sleep researcher Nathaniel Kleitman. A fellow graduate student, Eugene Aserinsky, had begun to study eye movements in sleep in 1953. Bill helped discover and describe REM sleep, a term he later coined. From 1954 through 1957, he described the relationship between REM sleep and dreaming, established the all-night sleep patterns in human beings, discovered REM sleep in animals and newborn babies, and demonstrated that the patterns of specific rapid eye movements are related to the visual dream experience. Upon completion of his internship at Mount Sinai Hospital, Bill obtained a research grant to establish a sleep laboratory, which he housed in his apartment. His ingenuity and persuasive powers were evident in the grant stipulations: Because the sleep research took place at night and he needed to be near his young family, the funding covered half of his apartment rent. Among the research subjects coming to his sleep research lab were members of the Radio City Rockettes. In 1960, he published a paper in Science based on that work, in which he described the effect of dream deprivation. In 1963, Bill developed a sleep research program at Stanford that drew international attention. He collaborated with many of the scientists who would move the new field forward, including neurophysiologists Michel Jouvet and Christian Guilleminault. In 1970, he opened the Stanford Sleep Disorders Clinic. Bill invented the term polysomnography to convince insurance companies to reimburse patients for the costs of clinical sleep studies. This coding change, from experimental treatment to sanctioned medical procedure, marked the beginning of the modern field of clinical sleep medicine. Bill helped create the multiple sleep latency test, now the international standard for evaluating all forms of hypersomnia, and he established the use of clinical sleep studies in pharmacology trials. Sleep-onset REM periods, another of Bill's discoveries, became a pathophysiological marker of narcolepsy. His narcoleptic dog colony at Stanford eventually helped establish the cause of narcolepsy in humans. REM sleep phenomena have now been uncovered in dragon lizards, fishes, and even cephalopods, broadening the influence of Bill's work to more than half a billion years of animal evolution. As the field's founder, Bill worked to provide sleep research with the necessary resources. He helped found the Sleep Research Society in 1961 and the Society for Neuroscience in 1969. He published Sleep , the first journal devoted to the field. The founding president of the American Sleep Disorders Association (now the American Academy of Sleep Medicine), he served in that role for 12 years. He created Stanford's incredibly popular university course Sleep and Dreams. Out of necessity, he wrote the first college textbook on the subject. Bill was such a pioneer that he had to sign his own certificate when he became board certified in sleep medicine. An intellectually curious and bold man who took advantage of every opportunity, Bill anticipated the great potential for the sleep field to improve public health and worked tirelessly to promote it. He chaired the National Commission on Sleep Disorders Research, advocating awareness of sleep conditions, and spurred Congress to establish the National Center on Sleep Disorders Research at the National Institutes of Health. His work raising awareness about the dangers of drowsy driving and drowsiness in the workplace has undoubtedly saved innumerable lives. I (R.P.) met Bill in 1993 when I applied for a fellowship at Stanford. When the famous Dr. Dement burst into the room and extended his hand, he immediately put me at ease. From that day on, Bill always made me smile. Later, we co-taught his Sleep and Dreams course, traveling together to class in his golf cart. Bill was just as welcoming to me (P.M.). When I met him in 2005, I was shocked that he asked me, a junior faculty member, to share my opinions about how to improve the definition of sleep. Kind and generous to colleagues and students alike, Bill had a positive energy and good humor that motivated everyone who worked with him. While stationed in Japan as a journalist for the army after World War II, Bill performed professionally as a jazz musician. Surrounded by talented young musicians such as Ray Charles and Quincy Jones, Bill later would say that he realized he could “make a better living as a mediocre physician than as a mediocre musician.” However, he never lost his love of performing. When asked, he would sing a cappella to the students in class. His musical performances were often the highlight of our professional conferences. Bill served as a role model by always keeping an open mind and embracing scientific inquiry with the goal of helping others. A living legend, beloved by his friends and family, he enjoyed his celebrity status. He died secure in the knowledge that his colleagues and students would honor and carry on his work. Sleep researchers have lost a giant, but Bill's legacy will guide the field as it continues to grow.

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte–stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers., Author summary Melanocytes produce melanin, a natural skin pigment, for body coloration which helps to protect and camouflage an organism and to attract mates. Melanocytes are ubiquitous pigment cells in vertebrates and the genes underlying their development are well conserved, making fishes that possess the ability to modify their pigmentation, biologically relevant and successful models for human skin disorders. Many human skin diseases including albinism, vitiligo, and melanoma are derived from mutations in conserved pigmentation genes. However, much of the conserved molecular mechanisms behind these diseases and human pigmentation remain unknown. For instance, melanin concentrating hormone (MCH) was originally identified as a peptide that when injected, could make fish paler by promoting melanin aggregation but no mutants demonstrating an endogenous function for MCH in pigmentation have been reported. Here, we use zebrafish mutants of MCH and the MCH receptor to determine their specific genetic function in pigmentation. Additionally, we demonstrate that MCH has an antagonistic pigmentation function to the melanocortin system, where MCH expression promotes lighter pigmentation and melanocortin activity promotes darkening. Thus, we find that the balance between the MCH and melanocortin system activities are likely required for skin pigmentation and dysregulation of these pathways could underlie adverse human skin conditions.

Most of an animal’s neuronal cells are present at birth and continue to function until the animal’s death. Thus, in contrast to a skin, blood or liver cell, whose life cycle ranges from days to months, a neuron may need to maintain its integrity across decades, all the while maintaining its capacity to connect to other neurons in an ever-changing environment. This unique cellular aging constraint requires permanent homeostatic and plastic capabilities to maintain circuitry over years and create new connections and controlling ad hoc neurochemical changes within the neuronal cells, as well as in their extracellular environment. While it is unclear how neural tissues achieve such a feat, a recurring period of our lives may be critical for the survival, maintenance, and plasticity of our brain: sleep. Although often described to as a behavior, we hypothesize that sleep and its stages are a recurring biological state critical for the cellular maintenance of plasticity of the central nervous system and associated cognitive abilities. Over the average 75-year life span, an individual may sleep over 10,000 full days. However, those 10,000 days of sleep are not distributed evenly across a lifetime; sleep duration often declines with age. There is strong evidence in young to middle-aged adults that reduced sleep quality correlates with cognitive impairment, which suggests that life-span changes in sleep contribute to the widespread changes in cognitive functioning commonly observed in older adults. If so, then improving sleep might delay or reverse cognitive aging. However, there is a massive gap between describing comorbidity of sleep disturbances and cognitive decline in aging to understanding the mechanistic interlinks between sleep, aging, and cognition. The goal of this perspective is to provide new insights on the role(s) of sleep on the nuclear, membrane/synapse, and extracellular components of the brain tissue during aging.

Thousands of human disease-associated single nucleotide polymorphisms (SNPs) lie in the non-coding genome, but only a handful have been demonstrated to affect gene expression and human biology. We computationally identified risk-associated SNPs in deeply conserved non-exonic elements (CNEs) potentially contributing to 45 human diseases. We further demonstrated that human CNE1/rs17421627 associated with retinal vasculature defects showed transcriptional activity in the zebrafish retina, while introducing the risk-associated allele completely abolished CNE1 enhancer activity. Furthermore, deletion of CNE1 led to retinal vasculature defects and to a specific downregulation of microRNA-9, rather than MEF2C as predicted by the original genome-wide association studies. Consistent with these results, miR-9 depletion affects retinal vasculature formation, demonstrating MIR-9-2 as a critical gene underpinning the associated trait. Importantly, we validated that other CNEs act as transcriptional enhancers that can be disrupted by conserved non-coding SNPs. This study uncovers disease-associated non-coding mutations that are deeply conserved, providing a path for in vivo testing to reveal their cis-regulated genes and biological roles.

The duration of sleep varies dramatically between species, yet little is known about the genetic basis or evolutionary factors driving this variation in behavior. The Mexican cavefish, Astyanax mexicanus, exists as surface populations that inhabit rivers, and multiple 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 in cavefish, suggesting enhanced HCRT signaling underlies the evolution of sleep loss. Ablation of the lateral line or starvation, manipulations that selectively promote sleep in cavefish, inhibit hcrt expression in cavefish while having little effect on 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 essential to all animals with a nervous system. Nevertheless, the core cellular function of sleep is unknown, and there is no conserved molecular marker to define sleep across phylogeny. Time-lapse imaging of chromosomal markers in single cells of live zebrafish revealed that sleep increases chromosome dynamics in individual neurons but not in two other cell types. Manipulation of sleep, chromosome dynamics, neuronal activity, and DNA double-strand breaks (DSBs) showed that chromosome dynamics are low and the number of DSBs accumulates during wakefulness. In turn, sleep increases chromosome dynamics, which are necessary to reduce the amount of DSBs. These results establish chromosome dynamics as a potential marker to define single sleeping cells, and propose that the restorative function of sleep is nuclear maintenance., Do single neurons require sleep and what is the conserved cellular function of sleep? In this paper, the authors use real-time imaging of chromosomes in individual cells within live zebrafish to show that sleep increases chromosome dynamics, which are necessary to reduce DNA damage that is accumulated during wakefulness.

Summary Prolonged behavioral challenges can cause animals to switch from active to passive coping strategies to manage effort-expenditure during stress; such normally adaptive behavioral state transitions can become maladaptive in psychiatric disorders such as depression. The underlying neuronal dynamics and brainwide interactions important for passive coping have remained unclear. Here, we develop a paradigm to study these behavioral state transitions at cellular-resolution across the entire vertebrate brain. Using brainwide imaging in zebrafish, we observed that the transition to passive coping is manifested by progressive activation of neurons in the ventral (lateral) habenula. Activation of these ventral-habenula neurons suppressed downstream neurons in the serotonergic raphe nucleus and caused behavioral passivity, whereas inhibition of these neurons prevented passivity. Data-driven recurrent neural network modeling pointed to altered intra-habenula interactions as a contributory mechanism. These results demonstrate ongoing encoding of experience features in the habenula, which guides recruitment of downstream networks and imposes a passive coping behavioral strategy.

In most species, females time reproduction to coincide with fertility. Thus, identifying factors that signal fertility to the brain can provide access to neural circuits that control sexual behaviors. In vertebrates, levels of key signaling molecules rise at the time of fertility to prime the brain for reproductive behavior [1-11], but how and where they regulate neural circuits is not known [12, 13]. Specifically, 17α,20β-dihydroxyprogesterone (DHP) and prostaglandin F2α (PGF2α) levels rise in teleost fish around the time of ovulation [10, 14, 15]. In an African cichlid fish, Astatotilapia burtoni, fertile females select a mate and perform a stereotyped spawning routine, offering quantifiable behavioral outputs of neural circuits. We show that, within minutes, PGF2α injection activates a naturalistic pattern of sexual behavior in female A. burtoni. We also identify cells in the brain that transduce the prostaglandin signal to mate and show that the gonadal steroid DHP modulates mRNA levels of the putative receptor for PGF2α (Ptgfr). We use CRISPR/Cas9 to generate the first targeted gene mutation in A. burtoni and show that Ptgfr is necessary for the initiation of sexual behavior, uncoupling sexual behavior from reproductive status. Our findings are consistent with a model in which PGF2α communicates fertility status via Ptgfr to circuits in the brain that drive female sexual behavior. Our targeted genome modification in a cichlid fish shows that dissection of gene function can reveal basic control mechanisms for behaviors in this large family of species with diverse and fascinating social systems [16, 17].

Due to anatomical and physiological similarities to humans, the common marmoset (Callithrix jacchus) is an ideal organism for the study human diseases. Researchers are currently leveraging genome-editing technologies such as CRISPR/Cas9 to genetically engineer marmosets for the in vivo biomedical modeling of human neuropsychiatric and neurodegenerative diseases. The genome characterization of these cell lines greatly reinforces these transgenic efforts. It also provides the genomic contexts required for the accurate interpretation of functional genomics data. We performed haplotype-resolved whole-genome characterization for marmoset ESC line cj367 from the Wisconsin National Primate Research Center. This is the first haplotype-resolved analysis of a marmoset genome and the first whole-genome characterization of any marmoset ESC line. We identified and phased single-nucleotide variants (SNVs) and Indels across the genome. By leveraging this haplotype information, we then compiled a list of cj367 ESC allele-specific CRISPR targeting sites. Furthermore, we demonstrated successful Cas9 Endonuclease Dead (dCas9) expression and targeted localization in cj367 as well as sustained pluripotency after dCas9 transfection by teratoma assay. Lastly, we show that these ESCs can be directly induced into functional neurons in a rapid, single-step process. Our study provides a valuable set of genomic resources for primate transgenics in this post-genome era.

Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show—using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate—that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago. Fluorescence-based polysomnography in zebrafish reveals two major sleep signatures that share features with those of amniotes, which suggests that common neural sleep signatures emerged in the vertebrate brain over 450 million years ago.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character. Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184–99. ©2017 AACR. See related commentary by Vakoc and Tuveson, p. 1067. This article is highlighted in the In This Issue feature, p. 1047

In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.

RFamide neuropeptide VF (NPVF) is expressed by neurons in the hypothalamus and has been implicated in nociception, but the circuit mechanisms remain unexplored. Here, we studied the structural and functional connections from NPVF neurons to downstream targets in the context of nociception, using novel transgenic lines, optogenetics, and calcium imaging in behaving larval zebrafish. We found a specific projection from NPVF neurons to serotonergic neurons in the ventral raphe nucleus (vRN). We showed NPVF neurons and vRN are suppressed and excited by noxious stimuli, respectively. We combined optogenetics with calcium imaging and pharmacology to demonstrate that stimulation of NPVF cells suppresses neuronal activity in vRN. During noxious stimuli, serotonergic neurons activation was due to a suppression of an inhibitory NPVF-ventral raphe peptidergic projection. This study reveals a novel NPVF-vRN functional circuit modulated by noxious stimuli in vertebrates.

Video processing is increasingly becoming a standard procedure in zebrafish behavior investigations as it enables higher research throughput and new or better measures. This trend, fostered by the ever increasing performance-to-price ratio of the required recording and processing equipment, should be expected to continue in the foreseeable future, with video-processing based methods permeating more and more experiments and, as a result, expanding the very role of behavioral studies in zebrafish research. To assess whether the routine video tracking of zebrafish larvae directly in the Petri dish is a capability that can be expected in the near future, the key processing concepts are discussed and illustrated on published zebrafish studies when available or other animals when not.

Sleep is tightly regulated by the circadian clock and homeostatic mechanisms. Although the sleep/wake cycle is known to be associated with structural and physiological synaptic changes that benefit the brain, the function of sleep is still debated. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate various functions including feeding, reward, sleep, and wake. Continuous imaging of single neuronal circuits in live animals is vital to understanding the role of sleep in regulating synaptic dynamics, and the transparency of the zebrafish model enables time-lapse imaging of single synapses during both day and night. Here, we use the gephyrin (Gphnb) protein, a central inhibitory synapse organizer, as a fluorescent post-synaptic marker of inhibitory synapses. Double labeling showed that Gphnb-tagRFP and collybistin-EGFP clusters co-localized in dendritic inhibitory synapses. Using a transgenic hcrt:Gphnb-EGFP zebrafish, we showed that the number of inhibitory synapses in the dendrites of Hcrt neurons was increased during development. To determine the effect of sleep on the inhibitory synapses, we performed two-photon live imaging of Gphnb-EGFP in Hcrt neurons during day and night, under light/dark and constant light and dark conditions, and following sleep deprivation (SD). We found that synapse number increased during the night under light/dark conditions but that these changes were eliminated under constant light or dark conditions. SD reduced synapse number during the night, and the number increased during post-deprivation daytime sleep rebound. These results suggest that rhythmic structural plasticity of inhibitory synapses in Hcrt dendrites is independent of the circadian clock and is modulated by consolidated wake and sleep.

Sleep is a fundamental and evolutionarily conserved aspect of animal life. Recent studies have shed light on the role of sleep in synaptic plasticity. Demonstrations of memory replay and synapse homeostasis suggest that one essential role of sleep is in the consolidation and optimization of synaptic circuits to retain salient memory traces despite the noise of daily experience. Here, we review this recent evidence, and suggest that sleep creates a heightened state of plasticity, which may be essential for this optimization. Furthermore, we discuss how sleep deficits seen in diseases such as Alzheimer’s disease and autism spectrum disorders might not just reflect underlying circuit malfunction, but could also play a direct role in the progression of those disorders.

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant ( hcrtr −/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr −/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr −/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr −/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

Melanin-concentrating hormone (MCH) regulates feeding and complex behaviors in mammals and pigmentation in fish. The relationship between fish and mammalian MCH systems is not well understood. Here, we identify and characterize two MCH genes in zebrafish, Pmch1 and Pmch2. Whereas Pmch1 and its corresponding MCH1 peptide resemble MCH found in other fish, the zebrafish Pmch2 gene and MCH2 peptide share genomic structure, synteny, and high peptide sequence homology with mammalian MCH. Zebrafish Pmch genes are expressed in closely associated but non-overlapping neurons within the hypothalamus, and MCH2 neurons send numerous projections to multiple MCH receptor-rich targets with presumed roles in sensory perception, learning and memory, arousal, and homeostatic regulation. Preliminary functional analysis showed that whereas changes in zebrafish Pmch1 expression correlate with pigmentation changes, the number of MCH2-expressing neurons increases in response to chronic food deprivation. These findings demonstrate that zebrafish MCH2 is the putative structural and functional ortholog of mammalian MCH and help elucidate the nature of MCH evolution among vertebrates.

The hypocretin/orexin (HCRT/ORX) excitatory neuropeptides are expressed in a small population of lateral hypothalamic cells in mammals and fish. In humans, loss of these cells causes the sleep disorder narcolepsy. Identification of genes expressed in HCRT-producing cells may be revealing as to the regulation of sleep and the pathophysiology of narcolepsy. In this study, in situ hybridization analyses were performed to characterize the expression pattern of receptors and enzyme, which regulate ATP-mediated transmission in hypocretin cells of zebrafish larvae. The zebrafish cDNA encoding the ecto-nucleoside triphosphate diphosphohydrolase 3 (ENTPD3/NTPDase3) was isolated. This transcript was found to be expressed in zebrafish HCRT cells as previously reported in mammals. It was also expressed in the cranial nerves (gV, gVII, gIV and gX) and in primary sensory neurons (i.e., Rohon–Beard neurons) in the spinal cord. The expression of known zebrafish p2rx purinergic receptor family members was next studied and found to overlap with the entpd3 expression pattern. Specifically, p2rx2, p2rx3.1, p2rx3.2 and p2rx8 were expressed in the trigeminal ganglia and subsets of Rohon–Beard neurons. In contrast to mammals, p2rx2 was not expressed in HCRT cells; rather, p2rx8 was expressed with entpd3 in this hypothalamic region. The conservation of expression of these genes in HCRT cells and sensory neurons across vertebrates suggests an important role for ATP mediated transmission in the regulation of sleep and the processing of sensory inputs.

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.

Two independent high-throughput zebrafish behavioral screens of tens of thousands of compounds identify the 'finazines', a novel group of antipsychotics, and their endogenous genetic target, the σ1 receptor.

Transgene-induced post-transcriptional gene silencing (PTGS) results from specific degradation of RNAs that are homologous with the transgene transcribed sequence. This phenomenon, also known as cosuppression in plants and quelling in fungi, resembles RNA interference (RNAi) in animals. Indeed, cosuppression/quelling/RNAi require related PAZ/PIWI proteins (AGO1/QDE-2/RDE-1), indicating that these mechanisms are related. Unlike Neurospora crassa qde-2 and Caenorhabditis elegans rde-1 mutants, which are morphologically normal, the 24 known Arabidopsis ago1 mutants display severe developmental abnormalities and are sterile. Here, we report the isolation of hypomorphic ago1 mutants, including fertile ones. We show that these hypomorphic ago1 mutants are defective for PTGS, like null sgs2, sgs3, and ago1 mutants, suggesting that PTGS is more sensitive than development to perturbations in AGO1. Conversely, a mutation in ZWILLE/PINHEAD, another member of the Arabidopsis AGO1 gene family, affects development but not PTGS. Similarly, mutations in ALG-1 and ALG-2, two members of the C. elegans RDE-1 gene family, affect development but not RNAi, indicating that the control of PTGS/RNAi and development by PAZ/PIWI proteins can be uncoupled. Finally, we show that hypomorphic ago1 mutants are hypersensitive to virus infection, confirming the hypothesis that in plants PTGS is a mechanism of defense against viruses.

Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals. © 2001 Wiley-Liss, Inc.

In plants, transgenes can be silenced at both the transcriptional [1] and post-transcriptional levels [2]. Methylation of the transgene promoter correlates with transcriptional gene silencing (TGS) [3] whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS) [4]. In animals, TGS requires methylation and changes in chromatin conformation [5]. The involvement of methylation during PTGS in plants is unclear and organisms with non-methylated genomes such as Caenorhabditis elegans or Drosophila can display RNAinterference (RNAi), a silencing process mechanistically related to PTGS [6]. Here, we crossed Arabidopsis mutants impaired in a SWI2/SNF2 chromatin component (ddm1[7]) or in the major DNA methyltransferase (met1[8] and E. Richards, personal communication) with transgenic lines in which a reporter consisting of the cauliflower mosaic virus 35S promoter fused to the β-glucuronidase (GUS) gene (35S–GUS) was silenced by TGS or PTGS. We observed an efficient release of 35S–GUS TGS by both the ddm1 and met1 mutations and stochastic release of 35S–GUS PTGS by these two mutations during development. These results show that DNA methylation and chromatin structure are common regulators of TGS and PTGS.

The recent development of gene transfer methods for almost all eukaryotes has revealed that transgenes can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene, thus limiting the potential application of genetic transformation. Despite this limitation, transgene-induced gene silencing events were considered originally as anecdotal phenomena. However, as more and more similarities were found between transgene-induced gene silencing and natural epigenetic phenomena, considerable interest has been devoted to this subject (for recent reviews see Depicker and Van Montagu, 1997; Stam et al., 1997b). Epigenetics is commonly defined as ‘the study of mitotically and/or meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence’ (Russo et al., 1996). For a long time, DNA was considered as the only target for epigenetic modifications. Epigenetic changes corresponding to changes in chromatin structure and affecting transcription have been reported in almost all eukaryotes: yeast, fungi, Drosophila, plants and mammals (Dorer, 1997; Foss and Selker, 1991; Rossignol and Faugeron, 1994; Ye and Signer, 1996). However, recent studies have suggested that, besides DNA, other molecules can be modified in a manner that resembles epigenetic DNA changes. First, it was shown that proteins can be converted into molecules of aberrant conformation called prions in yeast and mammals (Lacroute, 1971; Prusiner, 1982). More recently, the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes (Cogoni et al., 1996; Fire et al., 1998; Napoli et al., 1990). This review will focus on transgene-induced silencing phenomena in plants. The number of copies of a transgene that integrate into the genome of a transformed plant and

Contact, a new zebrafish transforming growth factor-beta (TGF-beta) member is most closely related to mouse GDF5 and to human CDMP-1 responsible, when mutated, for limb brachypodism phenotype and Hunter-Thompson syndrome, respectively. Contact exhibits a dynamic spatial expression pattern in the pharyngeal arches and the pectoral fin buds that much prefigures cartilage formation. Within the fin buds, contact expression is detected in the proximal mesenchyme from which the endoskeleton will develop. Exogeneously applied retinoic acid (RA) induces duplication of the pectoral fin rudiment in zebrafish embryos as well as contact expression along the proximal margin of the fin mesenchyme showing that both endoskeleton and exoskeleton can be duplicated.

Nitrate assimilation is a fundamental function in any plant including those that can fix atmospheric nitrogen in symbiosis with soil bacteria. In recent years, attempts have been made to understand the biological significance of the complex regulation of this pathway using genetic engineering techniques. Transgenic plants that either over- or under-express genes of the nitrate assimilation pathway were created in order to determine whether such directed changes affect the regulation of the metabolism. Apart from interesting physiological results, unexpected gene silencing phenomena have been observed resulting from the introduction of five different transgenes derived from either the tobacco Nia or Nii genes encoding nitrate reductase and nitrite reductase, respectively. In this review, each of these five silencing phenomena is described, with the emphasis on the advantages provided by the use of both Nia and Nii genes to analyze the molecular and genetic basis of gene silencing in transgenic plants.

Recent advances in imaging tools are inspiring zebrafish researchers to tackle ever more ambitious questions in the neurosciences. Behaviorally fundamental conserved neural networks can now be potentially studied using zebrafish from a brain-wide scale to molecular resolution. In this perspective, we offer a roadmap by which a zebrafish researcher can navigate the course from collecting neural activities across the brain associated with a behavior, to unraveling molecular identities and testing the functional relevance of active neurons. In doing so, important insights will be gained as to how neural networks generate behaviors and assimilate changes in synaptic connectivity.

Rapid technological advances over the past decade have moved us closer to a high throughput molecular approach to neurobiology, where we see the merging of neurogenetics, genomics, physiology, imaging and pharmacology. This is the case more in zebrafish than in any other model organism commonly used. Recent improvements in the generation of transgenic zebrafish now allow genetic manipulation and live imaging of neuronal development and function in early embryonic, larval, and adult animals. The sequenced zebrafish genome and comparative genomics give unprecedented insights into genome evolution and its relation to genome structure and function. There is now information on embryonic and larval expression of over 12,000 genes and just under 1000 mutant phenotypes. We review the remarkable similarity of the zebrafish genetic blueprint for the nervous system to that of mammals and assess recent technological advances that make the zebrafish a model of choice for elucidating the development and function of neuronal circuitry, transgene-based neuroanatomy, and small molecule neuropharmacology.

SummaryDuring gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1–3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3–6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-β/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.