Sensory Organ Morphogenesis in Caenorhabditis Elegans

Sensory organs are the gates through which information flows into the nervous system. In most animals, such organs consist of sensory neurons, which can transform stimuli into changes in their membrane potential, and glial cells, which establish a niche important for the morphogenesis and function of the neurons. Although similar glial compartments are seen throughout the nervous system, their morphogenesis is poorly understood. In the work presented here, I use the main sensory organ of Caenorhabditis elegans, the amphid, as a model system for understanding how glia form these compartments. First, by the interpretation of electron microscopy reconstructions of the developing amphid, I was able to uncover a role for daf-6/Patched, an established regulator of amphid morphogenesis, in restricting the size of the sensory compartment. Second, I sought to identify genes acting in the opposite direction, in expanding the sensory compartment, by cloning and characterizing suppressors of daf-6. Through this approach I discovered that lit-1/Nlk acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, my 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. I show that actin also lines the sensory compartment, and that WASP is important for compartment expansion, potentially by functioning in the same pathway as LIT-1. These results suggest that the daf-6 and lit-1 glial pathways constitute a rheostat used to control sensory compartment size. Finally, I also identify a role for the retromer complex, a module involved in the recycling of transmembrane proteins and membrane material from the endosomes to the Golgi apparatus, in amphid morphogenesis. Similar to lit-1, mutations of retromer components suppress daf-6, suggesting that the retromer could also act in promoting sensory compartment expansion

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

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 β-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

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

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

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

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