Takaaki Hirotsu

Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans

Too Close for Comfort Pheromones are often used for sexual communications in animals, but they can also serve as a measure of population density. Now, Yamada et al. (p. 1647) have found that population density in the nematode worm Caenorhabditis elegans regulates plasticity of olfactory behavior, in which attraction to an odorant decreases after prolonged exposure. Using two rounds of genetic screens, a peptide named SNET-1 and a homolog of a mammalian transmembrane peptidase neprilysin were found to mediate pheromonal regulation. This regulation of olfactory behavior may serve to coordinate the behavior of individual animals in relation to the status of the whole population. A nematode odor response is regulated by population density through dauer pheromone, a neuropeptide, and neprilysin peptidase. Population density–dependent dispersal is a well-characterized strategy of animal behavior in which dispersal rate increases when population density is higher. Caenorhabditis elegans shows positive chemotaxis to a set of odorants, but the chemotaxis switches from attraction to dispersal after prolonged exposure to the odorants. We show here that this plasticity of olfactory behavior is dependent on population density and that this regulation is mediated by pheromonal signaling. We show that a peptide, suppressor of NEP-2 (SNET-1), negatively regulates olfactory plasticity and that its expression is down-regulated by the pheromone. NEP-2, a homolog of the extracellular peptidase neprilysin, antagonizes SNET-1, and this function is essential for olfactory plasticity. These results suggest that population density information is transmitted through the external pheromone and endogenous peptide signaling to modulate chemotactic behavior.

Behavioral choice between conflicting alternatives is regulated by a receptor guanylyl cyclase, GCY-28, and a receptor tyrosine kinase, SCD-2, in AIA interneurons of Caenorhabditis elegans.

Animals facing conflicting sensory cues make a behavioral choice between competing alternatives through integration of the sensory cues. Here, we performed a genetic screen to identify genes important for the sensory integration of two conflicting cues, the attractive odorant diacetyl and the aversive stimulus Cu2+, and found that the membrane-bound guanylyl cyclase GCY-28 and the receptor tyrosine kinase SCD-2 regulate the behavioral choice between these alternatives in Caenorhabditis elegans. The gcy-28 mutants and scd-2 mutants show an abnormal bias in the behavioral choice between the cues, although their responses to each individual cue are similar to those in wild-type animals. Mutants in a gene encoding a cyclic nucleotide gated ion channel, cng-1, also exhibit the defect in sensory integration. Molecular genetic analyses suggested that GCY-28 and SCD-2 regulate sensory integration in AIA interneurons, where the conflicting sensory cues may converge. Genetic ablation or hyperpolarization of AIA interneurons showed nearly the same phenotype as gcy-28 or scd-2 mutants in the sensory integration, although this did not affect the sensory response to each individual cue. In gcy-28 or scd-2 mutants, activation of AIA interneurons is sufficient to restore normal sensory integration. These results suggest that the activity of AIA interneurons regulates the behavioral choice between the alternatives. We propose that GCY-28 and SCD-2 regulate sensory integration by modulating the activity of AIA interneurons.

A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans

The molecular mechanism by which neurites are selected for elimination or incorporation into the mature circuit during developmental pruning remains unknown. The trophic theory postulates that local cues provided by target or surrounding cells act to inhibit neurite elimination. However, no widely conserved factor mediating this trophic function has been identified. We found that the developmental survival of specific neurites in Caenorhabditis elegans largely depends on detection of the morphogen Wnt by the Ror kinase CAM-1, which is a transmembrane tyrosine kinase with a Frizzled domain. Mutations in Wnt genes or in cam-1 enhanced neurite elimination, whereas overexpression of cam-1 inhibited neurite elimination in a Wnt-dependent manner. Moreover, mutations in these genes counteracted the effect of a mutation in mbr-1, which encodes a transcription factor that promotes neurite elimination. These results reveal the trophic role of an atypical Wnt pathway and reinforce the classical model of developmental pruning.

Odour concentration-dependent olfactory preference change in C. elegans

The same odorant can induce attractive or repulsive responses depending on its concentration in various animals including humans. However, little is understood about the neuronal basis of this behavioural phenomenon. Here we show that Caenorhabditis elegans avoids high concentrations of odorants that are attractive at low concentrations. Behavioural analyses and computer simulation reveal that the odour concentration-dependent behaviour is primarily generated by klinokinesis, a behavioural strategy in C. elegans. Genetic analyses and lesion experiments show that distinct combinations of sensory neurons function at different concentrations of the odorant; AWC and ASH sensory neurons have critical roles for attraction to or avoidance of the odorant, respectively. Moreover, we found that AWC neurons respond to only lower concentrations of the odorant, whereas ASH neurons respond to only higher concentrations of odorant. Hence, our study suggests that odour concentration coding in C. elegans mostly conforms to the labelled-line principle where distinct neurons respond to distinct stimuli.

Screening of Odor-Receptor Pairs in Caenorhabditis elegans Reveals Different Receptors for High and Low Odor Concentrations

Distinct receptors and neuronal circuits enable worms to move toward low and away from high concentrations of the same chemical. Detecting Too Much of a Good Thing Worms, such as the nematode Caenorhabditis elegans, rely on chemosensory perception to move toward nutrient sources and away from noxious chemicals. However, some chemicals are beneficial at low concentrations but hazardous at high concentrations. Taniguchi et al. used an RNA interference screen to match several odorant molecules to genes encoding 194 G protein–coupled receptors, creating a useful launching point for studying chemosensory perception signaling. C. elegans used different receptors in different neurons to respond to high or low concentrations of diacetyl, a chemical that attracts worms at low concentrations and repulses them at high concentrations. Olfactory systems sense and respond to various odorants. Olfactory receptors, which in most organisms are G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors, directly bind volatile or soluble odorants. Compared to the genomes of mammals, the genome of the nematode Caenorhabditis elegans contains more putative olfactory receptor genes, suggesting that in nematodes there may be combinatorial complexity to the receptor-odor relationship. We used RNA interference (RNAi) screening to identify nematode olfactory receptors necessary for the response to specific odorants. This screening identified 194 candidate olfactory receptor genes linked to 11 odorants. Additionally, we identified SRI-14 as being involved in sensing high concentrations of diacetyl. Rescue and neuron-specific RNAi experiments demonstrated that SRI-14 functioned in ASH neurons, specific chemosensory neurons, resulting in avoidance responses. Calcium imaging revealed that ASH neurons responded to high diacetyl concentrations only, whereas another class of chemosensory neurons, AWA neurons, reacted to both low and high concentrations. Loss of SRI-14 function hampered ASH responses to high diacetyl concentrations, whereas loss of ODR-10 function reduced AWA responses to low odorant concentrations. Chemosensory neurons ectopically expressing SRI-14 responded to a high concentration of diacetyl. Thus, nematodes have concentration-dependent odor-sensing mechanisms that are segregated at the olfactory receptor and sensory neuron levels.

GPC-1, a G Protein γ-Subunit, Regulates Olfactory Adaptation in Caenorhabditis elegans

Caenorhabditis elegans genome carries two Gγ genes, gpc-1 and gpc-2, and two Gβ genes, gpb-1 and gpb-2. Of these, gpc-2 and gpb-1 are expressed ubiquitously and are essential for viability. Through a genetic screen, we identified gpc-1 as essential for olfactory adaptation. While wild-type animals show decreased chemotaxis to the odorant benzaldehyde after a short preexposure to the odorant, gpc-1 mutants are still attracted to the odorant after the same preexposure. Cell-specific rescue experiments show that gpc-1 acts in the AWC olfactory neurons. Coexpression of GPC-1 and GPB-1, but not GPB-2, caused enhanced adaptation, indicating that GPC-1 may act with GPB-1. On the other hand, knock down of gpc-2 by cell-targeted RNAi caused reduced chemotaxis to the odorant in unadapted animals, indicating that GPC-2 mainly act for olfactory sensation and the two Gγs have differential functions. Nonetheless, overexpression of gpc-2 in AWC neurons rescued the adaptation defects of gpc-1 mutants, suggesting partially overlapping functions of the two Gγs. We further tested genetic interaction of gpc-1 with several other genes involved in olfactory adaptation. Our analyses place goa-1 Goα and let-60 Ras in parallel to gpc-1. In contrast, a gain-of-function mutation in egl-30 Gqα was epistatic to gpc-1, suggesting the possibility that gpc-1 Gγ may act upstream of egl-30 Gqα.

A highly accurate inclusive cancer screening test using Caenorhabditis elegans scent detection

Early detection and treatment are of vital importance to the successful eradication of various cancers, and development of economical and non-invasive novel cancer screening systems is critical. Previous reports using canine scent detection demonstrated the existence of cancer-specific odours. However, it is difficult to introduce canine scent recognition into clinical practice because of the need to maintain accuracy. In this study, we developed a Nematode Scent Detection Test (NSDT) using Caenorhabditis elegans to provide a novel highly accurate cancer detection system that is economical, painless, rapid and convenient. We demonstrated wild-type C. elegans displayed attractive chemotaxis towards human cancer cell secretions, cancer tissues and urine from cancer patients but avoided control urine; in parallel, the response of the olfactory neurons of C. elegans to the urine from cancer patients was significantly stronger than to control urine. In contrast, G protein α mutants and olfactory neurons-ablated animals were not attracted to cancer patient urine, suggesting that C. elegans senses odours in urine. We tested 242 samples to measure the performance of the NSDT, and found the sensitivity was 95.8%; this is markedly higher than that of other existing tumour markers. Furthermore, the specificity was 95.0%. Importantly, this test was able to diagnose various cancer types tested at the early stage (stage 0 or 1). To conclude, C. elegans scent-based analyses might provide a new strategy to detect and study disease-associated scents.

Temporally-regulated quick activation and inactivation of Ras is important for olfactory behaviour

Responses to environmental stimuli are mediated by the activation and inactivation of various signalling proteins. However, the temporal dynamics of these events in living animals are not well understood. Here we show real-time imaging of the activity of the key regulator of the MAP kinase pathway, Ras, in living Caenorhabditis elegans and that Ras is transiently activated within a few seconds in olfactory neurons in response to increase in the concentration of odorants. This fast activation of Ras is dependent on the olfactory signalling pathway and Ras guanyl nucleotide-releasing protein (RasGRP). A negative feedback loop then quickly leads to Ras inactivation despite the continued presence of the odorant. Phenotypes of Ras mutants suggest this rapid activation and inactivation of Ras is important for regulation of interneuron activities and olfactory behaviours. Our results reveal novel kinetics and biological implication of transient activation of Ras in olfactory systems.

A role for Ras in inhibiting circular foraging behavior as revealed by a new method for time and cell-specific RNAi.

BackgroundThe nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. In particular, RNAi is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible RNAi or cell-specific RNAi. However, conventional RNAi is insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions.ResultsHere, we investigated the Ras gene for its role in exploratory behavior in C. elegans. We found that, under poor environmental conditions, mutations in the Ras-MAPK signaling pathway lead to circular locomotion instead of normal exploratory foraging. Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion. We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons. To investigate the temporal- and cell-specific profiles of the functions of Ras, we developed a new RNAi method that enables simultaneous time- and cell-specific knockdown. In this method, one RNA strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded RNA in the target cell when heat shock is induced. This technique revealed that control of GLR-1 localization in RMD neurons requires Ras at the adult stage. Further, we demonstrated the application of this method to other genes.ConclusionsWe have established a new RNAi method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the Ras-MAPK pathway in the control of exploratory behavior under poor environmental conditions.

Corrigendum: The Ras–MAPK pathway is important for olfaction in Caenorhabditis elegans

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