Hermaphrodite-specific Neurons Research Articles

Neuropeptide and serotonin co-transmission sets the activity pattern in the C. elegans egg-laying circuit

Neurons typically release both a neurotransmitter and one or more neuropeptides, but how these signals are integrated within neural circuits to generate and tune behaviors remains poorly understood. We studied how the two hermaphrodite-specific neurons (HSNs) activate the egg-laying circuit of Caenorhabditis elegans by releasing both the neurotransmitter serotonin and NLP-3 neuropeptides. Egg laying occurs in a temporal pattern with approximately 2-min active phases, during which eggs are laid, separated by approximately 20-min inactive phases, during which no eggs are laid. To understand how serotonin and NLP-3 neuropeptides together help produce this behavior pattern, we identified the G-protein-coupled receptor neuropeptide receptor 36 (NPR-36) as an NLP-3 neuropeptide receptor using genetic and molecular experiments. We found that NPR-36 is expressed in, and promotes egg laying within, the egg-laying muscle cells, the same cells where two serotonin receptors also promote egg laying. During the active phase, when HSN activity is high, we found that serotonin and NLP-3 neuropeptides each have a different effect on the timing of egg laying. During the inactive phase, HSN activity is low, which may result in release of only serotonin, yet mutants lacking either serotonin or nlp-3 signaling have longer inactive phases. This suggests that NLP-3 peptide signaling may persist through the inactive phase to help serotonin signaling terminate the inactive phase. We propose a model for neural circuit function in which multiple signals with short- and long-lasting effects compete to generate and terminate persistent internal states, thus patterning a behavior over tens of minutes.

A full-body transcription factor expression atlas with completely resolved cell identities in C. elegans

Invariant cell lineage in C. elegans enables spatiotemporal resolution of transcriptional regulatory mechanisms controlling the fate of each cell. Here, we develop RAPCAT (Robust-point-matching- And Piecewise-affine-based Cell Annotation Tool) to automate cell identity assignment in three-dimensional image stacks of L1 larvae and profile reporter expression of 620 transcription factors in every cell. Transcription factor profile-based clustering analysis defines 80 cell types distinct from conventional phenotypic cell types and identifies three general phenotypic modalities related to these classifications. First, transcription factors are broadly downregulated in quiescent stage Hermaphrodite Specific Neurons, suggesting stage- and cell type-specific variation in transcriptome size. Second, transcription factor expression is more closely associated with morphology than other phenotypic modalities in different pre- and post-differentiation developmental stages. Finally, embryonic cell lineages can be associated with specific transcription factor expression patterns and functions that persist throughout postembryonic life. This study presents a comprehensive transcription factor atlas for investigation of intra-cell type heterogeneity.

The Immunoglobulin Superfamily Members syg-2 and syg-1 Regulate Neurite Development in C. elegans.

Neurons form elaborate networks by guiding axons and dendrites to appropriate destinations. Neurites require information about the relative body axes during the initial projection from the cell body, and failure to receive or interpret those cues correctly can result in outgrowth errors. We identified a mutation in the Ig superfamily member syg-2 in a screen for animals with anterior/posterior (A/P) axon guidance defects. We found that syg-2 and its cognate Ig family member syg-1 appear to function in a linear genetic pathway to control the outgrowth of GABAergic axons. We determined that this pathway works in parallel to Wnt signaling. Specifically, mutations in syg-2 or syg-1 selectively affected the embryonically derived Dorsal D-type (DD) GABAergic neurons. We found no evidence that these mutations affected the Ventral D-type neurons (VD) that form later, during the first larval stage. In addition, mutations in syg-1 or syg-2 could result in the DD neurons forming multiple processes, becoming bipolar, rather than the expected pseudounipolar morphology. Given SYG-2′s essential function in synaptogenesis of the hermaphrodite-specific neurons (HSNs), we also examined DD neuron synapses in syg-2 mutants. We found syg-2 mutants had a decreased number of synapses formed, but synaptic morphology was largely normal. These results provide further evidence that the GABAergic motorneurons use multiple guidance pathways during development.

Repulsive guidance molecule acts in axon branching in Caenorhabditis elegans

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) extend dorsal branches at the region abutting the vulval muscles. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of the axons. Although DRAG-1 is normally expressed in the ventral hypodermis excepting the vulval region, its ectopic expression in vulval precursor cells was sufficient to induce the branching. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function, suggesting that DRAG-1 should be anchored to the cell surface. Genetic analyses suggested that the membrane receptor UNC-40 acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the ventral hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.

Networks of Causal Linkage Between Eigenmodes Characterize Behavioral Dynamics of Caenorhabditis elegans.

Behavioral phenotyping of model organisms has played an important role in unravelling the complexities of animal behavior. Techniques for classifying behavior often rely on easily identified changes in posture and motion. However, such approaches are likely to miss complex behaviors that cannot be readily distinguished by eye (e.g., behaviors produced by high dimensional dynamics). To explore this issue, we focus on the model organism Caenorhabditis elegans, where behaviors have been extensively recorded and classified. Using a dynamical systems lens, we identify high dimensional, nonlinear causal relationships between four basic shapes that describe worm motion (eigenmodes, also called “eigenworms”). We find relationships between all pairs of eigenmodes, but the timescales of the interactions vary between pairs and across individuals. Using these varying timescales, we create “interaction profiles” to represent an individual’s behavioral dynamics. As desired, these profiles are able to distinguish well-known behavioral states: i.e., the profiles for foraging individuals are distinct from those of individuals exhibiting an escape response. More importantly, we find that interaction profiles can distinguish high dimensional behaviors among divergent mutant strains that were previously classified as phenotypically similar. Specifically, we find it is able to detect phenotypic behavioral differences not previously identified in strains related to dysfunction of hermaphrodite-specific neurons.

Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain.

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

Development and improvement of ‘functional neural cellomics’ to elucidate the structure‐function relationships of neural networks of Caenorhabditis elegans

BackgroundA nervous system generates complex behaviors. However, relationships between neural computation and behaviors have hardly been elucidated. Optogenetics is a powerful technique for investigating mechanisms of neural computation. However, conventional optogenetics has three problems: (1) a hypothesis is required before an experiment; (2) the throughput is low because we should generate appropriate transgenic animals to test each hypothesis; (3) single‐cell analysis is difficult because few single‐cell‐specific promoters are known. To solve these problems, we have developed a new methodology ‘functional neural cellomics’ which enables comprehensive elucidation of relationships between behaviors and neural networks of Caenorhabditis elegans.MethodThe core technology of functional neural cellomics is the stochastic labeling of opsin in the neural network of C. elegans. The stochastic labeling of opsin was implemented by applying the Cre‐lox system. We designed a genetics circuit in which two sets of lox variants, lox2272 and loxP sequences, are inserted alternately downstream of pan‐neuronal F25B3.3 promoter. In addition, mCherry and a transcription factor, QF2w, are interposed between these lox sequences. After the induction of Cre recombinase by heat shock, a Cre‐lox recombination event occurs exclusively either between lox2272 sequences or between loxP sequences. If Cre is allowed to act on lox2272 sequences, QF2w is expressed. Then, QF2w induces expression of ChR2::GFP. In this way, we can easily acquire a C. elegans library in which ChR2 is labeled in a stochastic manner in each animal.Results & DiscussionBy inducing Cre recombinase, we confirmed that ChR2::GFP was labeled in a stochastic manner in each animal. To verify the feasibility of functional neural cellomics, we tried to demonstrate that neurons involved in the egg‐laying behavior of C. elegans could be identified in a high‐throughput manner. The C. elegans library was irradiated with blue light to activate ChR2, and we found some animals laid eggs in a blue‐light‐dependent manner. We identified neurons producing ChR2 using a confocal microscope, and confirmed that ChR2::GFP was expressed in HSNs (hermaphrodite‐specific neurons) in the egg‐laying individuals, but was not in the non‐egg laying individuals [1].In the above experiment, we found that the labeling rate of opsin was about 30%. If opsin is labeled in too many neurons, it becomes difficult to distinguish the function of individual neurons. To enable precise control of the labeling rate, we generated a library of randomized lox sequences by PCR. We evaluated excision efficiencies of the lox variants by using NGS, and successfully identified lox variant sequences which showed excision efficiencies ranging from 0.01% to 100%. Using these lox variants, we will be able to improve functional neural cellomics for comprehensive intervention in neural networks and high‐throughput identification of neural functions.Support or Funding InformationThis work was supported by PRESTO, JST (grant No. JPMJPR16F1), JSPS KAKENHI (grant No. JP17K19452), and Kyoto University Live Imaging Center.

Analysis of neural networks of Caenorhabditis elegans by functional cellomics

BackgroundNeural networks generate complex behaviors, but relationships between neural networks and complex behaviors have not been understood well. Although optogenetics has been a powerful tool which can provide knowledges on mechanisms of neural computation, conventional optogenetics has mainly three difficulties: 1. A hypothesis is necessary to conduct experiments in advance; 2. Optogenetics is low‐throughput because different transformants are required to test different hypothesis; 3. Single cell analysis is difficult due to lack of single‐cell‐specific promoters.Therefore, we have developed a method called ‘functional cellomics’ in which we can annotate functions of neural networks of Caenorhabditis elegans in hypothesis‐free, highthroughput, and single‐cell‐resolution manner.MethodOne of the core technologies of ‘functional cellomics’ is a stochastic Cre/lox system. By applying Cre/lox system, we have designed a methodology to enable stochastic labeling of any effectors to C. elegans neurons. To develop ‘functional cellomics’, we constructed four plasmids. In the initial state, pan‐neuronal F25B3.3 promoter drives production of mCherry in all neurons. After induction of Cre recombinase by heat shock, Cre catalyzes recombination of DNA exclusively either between loxP sequences or between lox2272 sequences. If a recombination occurs between lox2272 sequences, a transcription factor QF2w is produced. Then, QF2w drives production of ChR2::GFP under QUAS promoter. In this way, we can obtain a C. elegans library in which ChR2 is labeled in a stochastic manner.Results & DiscussionBy applying heat shock against the C. elegans library, we confirmed that ChR2::GFP was labeled in a stochastic manner. To demonstrate feasibility of ‘functional cellomics’, we chose the egg‐laying behavior of C. elegans as a model. The C. elegans library was illuminated with the blue light. We identified that ChR2::GFP was produced in HSNs (hermaphrodite‐specific neurons) in all of egg‐laying individuals, but it was not in the non‐egg‐laying individuals.Our results that HSNs can be identified by ‘functional cellomics’ in a high‐throughput manner proves the feasibility of proposed ‘functional cellomics’ [1].Support or Funding InformationThis work was supported by PRESTO, JST (grant No. JPMJPR16F1), JSPS KAKENHI (grant No. JP17K19452), and Kyoto University Live Imaging Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serotonin and neuropeptides are both released by the HSN command neuron to initiate Caenorhabditis elegans egg laying.

Neurons typically release both a small-molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how this co-released neuropeptide might modulate serotonin signaling. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit which have been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, similar to those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, and in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs. Specifically, the vm2 muscles cells, which are direct postsynaptic targets of the HSN, failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant co-transmitters that together stimulate and coordinate activity of the target cells onto which they are released.

Cadmium-induced serotonergic neuron and reproduction damages conferred lethality in the nematode Caenorhabditis elegans

Cadmium is a ubiquitous environmental toxicant. The use of Caenorhabditis elegans as a model for monitoring cadmium exposure has revealed several conserved signaling pathways. However, little is known about the killing process during lethality assay. In the present study, we investigated the effects serotonergic neuronal and reproductive damages on cadmium exposure in C. elegans. We found that sterile hermaphrodites, males and worms that passed reproduction span presented high cadmium resistance compared to those of young adults. The results demonstrated that reproduction process other than reproduction capacity conferred cadmium sensitivity. Cadmium exposure resulted in high ratio bagging phenotype, which was a severe reproductive deficit with embryos hatched internally that could cause worms to die early. The mechanism of bagging formation was ascribed to cadmium-induced egg laying deficiency that led embryos to retain and hatch in uterus. The addition of serotonin and imipramine promoted egg laying and thereby increased cadmium resistance. The results demonstrated that vulval muscles responsible for egg laying were still functional, while the serotonergic hermaphrodite specific neurons might be dysfunctional under cadmium exposure. Cadmium exposure resulted in shrinkage of serotonergic neuronal body and reduced expressions of tryptophan hydroxylase, the key enzyme for serotonin synthesis. The protection of serotonergic neuron through transient thermal preconditioning improved survival rate. In conclusion, our study demonstrated that damages of serotonergic neurons and reproduction conferred to cadmium-induced lethality.

Cellomics approach for high-throughput functional annotation of Caenorhabditis elegans neural network

In Caenorhabditis elegans, which has only 302 neurons, relationships between behaviors and neural networks are not easily elucidated. In this study, we proposed a novel cellomics approach enabling high-throughput and comprehensive exploration of the functions of a single neuron or a subset of neurons in a complex neural network on a particular behavior. To realize this, we combined optogenetics and Brainbow technologies. Using these technologies, we established a C. elegans library where opsin is labeled in a randomized pattern. Behavioral analysis on this library under light illumination enabled high-throughput annotation of neurons affecting target behaviors. We applied this approach to the egg-laying behavior of C. elegans and succeeded in high-throughput confirmation that hermaphrodite-specific neurons play an important role in the egg-laying behavior. This cellomics approach will lead to the accumulation of neurophysiological and behavioral data of the C. elegans neural network, which is necessary for constructing neuroanatomically grounded models of behavior.

Homeostatic Feedback Modulates the Development of Two-State Patterned Activity in a Model Serotonin Motor Circuit in Caenorhabditis elegans.

Neuron activity accompanies synapse formation and maintenance, but how early circuit activity contributes to behavior development is not well understood. Here, we use the Caenorhabditis elegans egg-laying motor circuit as a model to understand how coordinated cell and circuit activity develops and drives a robust two-state behavior in adults. Using calcium imaging in behaving animals, we find the serotonergic hermaphrodite-specific neurons (HSNs) and vulval muscles show rhythmic calcium transients in L4 larvae before eggs are produced. HSN activity in L4 is tonic and lacks the alternating burst-firing/quiescent pattern seen in egg-laying adults. Vulval muscle activity in L4 is initially uncoordinated but becomes synchronous as the anterior and posterior muscle arms meet at HSN synaptic release sites. However, coordinated muscle activity does not require presynaptic HSN input. Using reversible silencing experiments, we show that neuronal and vulval muscle activity in L4 is not required for the onset of adult behavior. Instead, the accumulation of eggs in the adult uterus renders the muscles sensitive to HSN input. Sterilization or acute electrical silencing of the vulval muscles inhibits presynaptic HSN activity and reversal of muscle silencing triggers a homeostatic increase in HSN activity and egg release that maintains ∼12-15 eggs in the uterus. Feedback of egg accumulation depends upon the vulval muscle postsynaptic terminus, suggesting that a retrograde signal sustains HSN synaptic activity and egg release. Our results show that egg-laying behavior in C. elegans is driven by a homeostat that scales serotonin motor neuron activity in response to postsynaptic muscle feedback.SIGNIFICANCE STATEMENT The functional importance of early, spontaneous neuron activity in synapse and circuit development is not well understood. Here, we show in the nematode Caenorhabditis elegans that the serotonergic hermaphrodite-specific neurons (HSNs) and postsynaptic vulval muscles show activity during circuit development, well before the onset of adult behavior. Surprisingly, early activity is not required for circuit development or the onset of adult behavior and the circuit remains unable to drive egg laying until fertilized embryos are deposited into the uterus. Egg accumulation potentiates vulval muscle excitability, but ultimately acts to promote burst firing in the presynaptic HSNs which results in egg laying. Our results suggest that mechanosensory feedback acts at three distinct steps to initiate, sustain, and terminate C. elegans egg-laying circuit activity and behavior.

Regulation of chromatin states and gene expression during HSN neuronal maturation is mediated by EOR-1/PLZF, MAU-2/cohesin loader, and SWI/SNF complex

Newborn neurons mature by distinct and sequential steps through the timely induction of specific gene expression programs in concert with epigenetic changes. However, it has been difficult to investigate the relationship between gene expression and epigenetic changes at a single-cell resolution during neuronal maturation. In this study, we investigated the maturation of hermaphrodite-specific neurons (HSNs) in C. elegans, which provided the link between chromatin dynamics, gene expression, and the degree of neuronal maturation at a single-cell resolution. Our results demonstrated that chromatin composition in the promoter region of several genes acting for neuronal terminal maturation was modulated at an early developmental stage, and is dependent on the function of the transcription factor EOR-1/PLZF and the cohesin loader MAU-2/MAU2. Components of the SWI/SNF chromatin remodeling complex were also required for the proper expression of terminal maturation genes. Epistasis analyses suggested that eor-1 functions with mau-2 and swsn-1 in the same genetic pathway to regulate the maturation of HSNs. Collectively, our study provides a novel approach to analyze neuronal maturation and proposes that predefined epigenetic modifications, mediated by EOR-1, MAU-2, and the SWI/SNF complex, are important for the preparation of future gene expression programs in neuronal terminal maturation.

Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified. Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Comparative genetic, proteomic and phosphoproteomic analysis of C. elegans embryos with a focus on ham-1/STOX and pig-1/MELK in dopaminergic neuron development

Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) – an open access, searchable online database.

G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway.

Environmental CO2 inhibits Caenorhabditis elegans egg-laying by modulating olfactory neurons and evokes widespread changes in neural activity

Carbon dioxide (CO2) gradients are ubiquitous and provide animals with information about their environment, such as the potential presence of prey or predators. The nematode Caenorhabditis elegans avoids elevated CO2, and previous work identified three neuron pairs called "BAG," "AFD," and "ASE" that respond to CO2 stimuli. Using in vivo Ca(2+) imaging and behavioral analysis, we show that C. elegans can detect CO2 independently of these sensory pathways. Many of the C. elegans sensory neurons we examined, including the AWC olfactory neurons, the ASJ and ASK gustatory neurons, and the ASH and ADL nociceptors, respond to a rise in CO2 with a rise in Ca(2+). In contrast, glial sheath cells harboring the sensory endings of C. elegans' major chemosensory neurons exhibit strong and sustained decreases in Ca(2+) in response to high CO2. Some of these CO2 responses appear to be cell intrinsic. Worms therefore may couple detection of CO2 to that of other cues at the earliest stages of sensory processing. We show that C. elegans persistently suppresses oviposition at high CO2. Hermaphrodite-specific neurons (HSNs), the executive neurons driving egg-laying, are tonically inhibited when CO2 is elevated. CO2 modulates the egg-laying system partly through the AWC olfactory neurons: High CO2 tonically activates AWC by a cGMP-dependent mechanism, and AWC output inhibits the HSNs. Our work shows that CO2 is a more complex sensory cue for C. elegans than previously thought, both in terms of behavior and neural circuitry.

Combinatorial roles of heparan sulfate proteoglycans and heparan sulfates in Caenorhabditis elegans neural development.

Heparan sulfate proteoglycans (HSPGs) play critical roles in the development and adult physiology of all metazoan organisms. Most of the known molecular interactions of HSPGs are attributed to the structurally highly complex heparan sulfate (HS) glycans. However, whether a specific HSPG (such as syndecan) contains HS modifications that differ from another HSPG (such as glypican) has remained largely unresolved. Here, a neural model in C. elegans is used to demonstrate for the first time the relationship between specific HSPGs and HS modifications in a defined biological process in vivo. HSPGs are critical for the migration of hermaphrodite specific neurons (HSNs) as genetic elimination of multiple HSPGs leads to 80% defect of HSN migration. The effects of genetic elimination of HSPGs are additive, suggesting that multiple HSPGs, present in the migrating neuron and in the matrix, act in parallel to support neuron migration. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a linear signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function together in a pathway that is parallel to sdn-1 and hst-6. These results suggest core protein specific HS modifications that are critical for HSN migration. In C. elegans, the core protein specificity of distinct HS modifications may be in part regulated at the level of tissue specific expression of genes encoding for HSPGs and HS modifying enzymes. Genetic analysis reveals that there is a delicate balance of HS modifications and eliminating one HS modifying enzyme in a compromised genetic background leads to significant changes in the overall phenotype. These findings are of importance with the view of HS as a critical regulator of cell signaling in normal development and disease.

Neuronal migration is regulated by endogenous RNAi and chromatin-binding factor ZFP-1/AF10 in Caenorhabditis elegans.

Endogenous short RNAs and the conserved plant homeodomain (PHD) zinc-finger protein ZFP-1/AF10 regulate overlapping sets of genes in Caenorhabditis elegans, which suggests that they control common biological pathways. We have shown recently that the RNAi factor RDE-4 and ZFP-1 negatively modulate transcription of the insulin/PI3 signaling-dependent kinase PDK-1 to promote C. elegans fitness. Moreover, we have demonstrated that the insulin/IGF-1-PI3K-signaling pathway regulates the activity of the DAF-16/FOXO transcription factor in the hypodermis to nonautonomously promote the anterior migrations of the hermaphrodite-specific neurons (HSNs) during embryogenesis of C. elegans. In this study, we implicate the PHD-containing isoform of ZFP-1 and endogenous RNAi in the regulation of HSN migration. ZFP-1 affects HSN migration in part through its negative effect on pdk-1 transcription and modulation of downstream DAF-16 activity. We also identify a novel role for ZFP-1 and RNAi pathway components, including RDE-4, in the regulation of HSN migration in parallel with DAF-16. Therefore, the coordinated activities of DAF-16, ZFP-1, and endogenous RNAi contribute to gene regulation during development to ensure proper neuronal positioning.