IGDB-2, an Ig/FNIII protein, binds the ion channel LGC-34 and controls sensory compartment morphogenesis in C. elegans.

Sensory organ glia surround neuronal receptive endings (NREs), forming a specialized compartment important for neuronal activity, and reminiscent of glia-ensheathed synapses in the central nervous system. We previously showed that DAF-6, a Patched-related protein, is required in glia of the C. elegans amphid sensory organ to restrict sensory compartment size. LIT-1, a Nemo-like kinase, and SNX-1, a retromer component, antagonize DAF-6 and promote compartment expansion. To further explore the machinery underlying compartment size control, we sought genes whose inactivation restores normal compartment size to daf-6 mutants. We found that mutations in igdb-2, encoding a single-pass transmembrane protein containing Ig-like and fibronectin type III domains, suppress daf-6 mutant defects. IGDB-2 acts in glia, where it localizes to glial membranes surrounding NREs, and, together with LIT-1 and SNX-1, regulates compartment morphogenesis. Immunoprecipitation followed by mass spectrometry demonstrates that IGDB-2 binds to LGC-34, a predicted ligand-gated ion channel, and lgc-34 mutations inhibit igdb-2 suppression of daf-6. Our findings reveal a novel membrane protein complex and suggest possible mechanisms for how sensory compartment size is controlled.

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.

Opposing activities of LIT-1/NLK and DAF-6/patched-related direct sensory compartment morphogenesis in C. elegans.

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

Some, but not all, retromer components promote morphogenesis of C. elegans sensory compartments.

The endings of sensory receptor cells often lie within specialized compartments formed by glial cells. The main sensory organ of Caenorhabditis elegans, the amphid, provides a powerful setting for studying glial compartment morphogenesis. Our previous studies showed that amphid compartment size is controlled by opposing activities of the Nemo-like kinase LIT-1, which promotes compartment expansion, and the Patched-related protein DAF-6, which restricts compartment growth. From a genetic screen for mutations able to suppress the bloated sensory compartments of daf-6 mutants, we identified an allele of the sorting nexin gene snx-1. SNX-1 protein is a component of the retromer, a protein complex that facilitates recycling of transmembrane proteins from the endosome to the Golgi network. We find that snx-1 functions cell autonomously within glia to promote sensory compartment growth, and that SNX-1 protein is enriched near the surface of the sensory compartment. snx-1 interacts genetically with lit-1 and another regulator of compartment size, the Dispatched-related gene che-14. Mutations in snx-3 and vps-29, also retromer genes, can suppress daf-6 defects. Surprisingly, however, remaining retromer components seem not to be involved. Our results suggest that a novel assembly of retromer components is important for determining sensory compartment dimensions.

Large-scale Analysis of Sleep in Zebrafish.

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

The glia of Caenorhabditis elegans.

Glia have been, in many ways, the proverbial elephant in the room. Although glia are as numerous as neurons in vertebrate nervous systems, technical and other concerns had left research on these cells languishing, whereas research on neurons marched on. Importantly, model systems to study glia had lagged considerably behind. A concerted effort in recent years to develop the canonical invertebrate model animals, Drosophila melanogaster and Caenorhabditis elegans, as settings to understand glial roles in nervous system development and function has begun to bear fruit. In this review, we summarize our current understanding of glia and their roles in the nervous system of the nematode C. elegans. The recent studies we describe highlight the similarities and differences between C. elegans and vertebrate glia, and focus on novel insights that are likely to have general relevance to all nervous systems.

Neuropeptide VF neurons promote sleep via the serotonergic raphe.

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

The Serotonergic Raphe Promote Sleep in Zebrafish and Mice.

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

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

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

Attacking sleep from a new angle: contributions from zebrafish.

Sleep consumes a third of our lifespan, but we are far from understanding how it is initiated, maintained and terminated, or what purposes it serves. To address these questions, alternative model systems have recently been recruited. The diurnal zebrafish holds the promise of bridging the gap between simple invertebrate systems, which show little neuroanatomical conservation with mammals, and well-established, but complex and nocturnal, murine systems. Zebrafish larvae can be monitored in a high-throughput fashion, pharmacologically tested by adding compounds into the water, genetically screened using transient transgenesis, and optogenetically manipulated in a non-invasive manner. Here we discuss work that has established the zebrafish as a powerful system for the study of sleep, as well as novel insights gained by exploiting its particular advantages.

Genetic Analysis of Histamine Signaling in Larval Zebrafish Sleep.

Pharmacological studies in mammals and zebrafish suggest that histamine plays an important role in promoting arousal. However, genetic studies using rodents with disrupted histamine synthesis or signaling have revealed only subtle or no sleep/wake phenotypes. Studies of histamine function in mammalian arousal are complicated by its production in cells of the immune system and its roles in humoral and cellular immunity, which can have profound effects on sleep/wake states. To avoid this potential confound, we used genetics to explore the role of histamine in regulating sleep in zebrafish, a diurnal vertebrate in which histamine production is restricted to neurons in the brain. Similar to rodent genetic studies, we found that zebrafish that lack histamine due to mutation of histidine decarboxylase (hdc) exhibit largely normal sleep/wake behaviors. Zebrafish containing predicted null mutations in several histamine receptors also lack robust sleep/wake phenotypes, although we are unable to verify that these mutants are completely nonfunctional. Consistent with some rodent studies, we found that arousal induced by overexpression of the neuropeptide hypocretin (Hcrt) or by stimulation of hcrt-expressing neurons is not blocked in hdc or hrh1 mutants. We also found that the number of hcrt-expressing or histaminergic neurons is unaffected in animals that lack histamine or Hcrt signaling, respectively. Thus, while acute pharmacological manipulation of histamine signaling has been shown to have profound effects on zebrafish and mammalian sleep, our results suggest that chronic loss of histamine signaling due to genetic mutations has only subtle effects on sleep in zebrafish, similar to rodents.

Light-Dependent Regulation of Sleep and Wake States by Prokineticin 2 in Zebrafish.

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.

Genetic and neuronal regulation of sleep by neuropeptide VF.

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

Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish.

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine ß-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep, dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons.

Melatonin is required for the circadian regulation of sleep.

Sleep is an evolutionarily conserved behavioral state whose regulation is poorly understood. A classical model posits that sleep is regulated by homeostatic and circadian mechanisms. Several factors have been implicated in mediating the homeostatic regulation of sleep, but molecules underlying the circadian mechanism are unknown. Here we use animals lacking melatonin due to mutation of arylalkylamine N-acetyltransferase 2 (aanat2) to show that melatonin is required for circadian regulation of sleep in zebrafish. Sleep is dramatically reduced at night in aanat2 mutants maintained in light/dark conditions, and the circadian regulation of sleep is abolished in free-running conditions. We find that melatonin promotes sleep downstream of the circadian clock as it is not required to initiate or maintain circadian rhythms. Additionally, we provide evidence that melatonin may induce sleep in part by promoting adenosine signaling, thus potentially linking circadian and homeostatic control of sleep.

On the morphogenesis of glial compartments in the sensory organs of Caenorhabditis elegans.

Glial cells surround neuronal endings and isolate them within specialized compartments. This architecture is found at synapses in the central nervous system, as well as at receptive endings of sensory neurons. Recent studies are beginning to uncover the contributions of glial compartments to the functions of the ensheathed neurons. However, the cellular and molecular processes that guide compartment morphogenesis remain unknown. The main sensory organ of Caenorhabditis elegans, the amphid, provides an experimentally tractable setting in which to address the mechanisms underlying glial compartment formation. Amphid development is stereotyped and amphid structure is easily assayed. We recently uncovered a molecular tug of war that regulates the size of the amphid sensory compartment. The Nemo-like kinase LIT-1 interacts with the glial cytoskeleton to promote compartment growth, a process that also involves components of the retromer complex, while the Patched-related transmembrane protein DAF-6 keeps this expansion in check. Here we discuss how regulation of secretion by the cytoskeleton could guide the sculpting of glial compartments.

Transient complete atrioventricular block associated with herb intake.

We report a case of transient complete atrioventricular block in a 38-year-old man, after intake of a mixture of herbs, intended to aid cigarette smoking cessation. Since all other causes of conduction disturbances were excluded, a side-effect of the herbal remedy was identified as the most likely diagnosis. Given that most patients are unaware of the potential risks of the intake of various herbs, we would urge that their usage be regulated.