RESUMEN
Much is known about molecular mechanisms by which animals detect pathogenic microbes, but how animals sense beneficial microbes remains poorly understood. The roundworm Caenorhabditis elegans is a microbivore that must distinguish nutritive microbes from pathogens. We characterized a neural circuit used by C. elegans to rapidly discriminate between nutritive bacteria and pathogens. Distinct sensory neuron populations responded to chemical cues from nutritive Escherichia coli and pathogenic Enterococcus faecalis, and these neural signals are decoded by downstream AIB interneurons. The polyamine metabolites cadaverine, putrescine, and spermidine produced by E. coli activate this neural circuit and elicit positive chemotaxis. Our study shows how polyamine odorants can be sensed by animals as proxies for microbe identity and suggests that, hence, polyamines might have widespread roles brokering host-microbe interactions.
Asunto(s)
Caenorhabditis elegans , Poliaminas , Animales , Poliaminas/metabolismo , Caenorhabditis elegans/metabolismo , Escherichia coli/metabolismo , Espermidina , PutrescinaRESUMEN
Balance and movement are impaired in many neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics but without the throughput and scalability necessary to screen candidate genes/potential therapeutics. Here, we present a scalable apparatus to measure posture and locomotion (SAMPL). SAMPL includes extensible hardware and open-source software with real-time processing and can acquire data from D. melanogaster, C. elegans, and D. rerio as they move vertically. Using SAMPL, we define how zebrafish balance as they navigate vertically and discover small but systematic variations among kinematic parameters between genetic backgrounds. We demonstrate SAMPL's ability to resolve differences in posture and navigation as a function of effect size and data gathered, providing key data for screens. SAMPL is therefore both a tool to model balance and locomotor disorders and an exemplar of how to scale apparatus to support screens.
Asunto(s)
Caenorhabditis elegans , Drosophila melanogaster , Animales , Pez Cebra , Conducta Animal , Locomoción , PosturaRESUMEN
The nematode C. elegans uses mechanosensitive neurons to detect bacteria, which are food for worms. These neurons release dopamine to suppress foraging and promote dwelling. Through a screen of genes highly expressed in dopaminergic food-sensing neurons, we identify a K2P-family potassium channel-TWK-2-that damps their activity. Strikingly, loss of TWK-2 restores mechanosensation to neurons lacking the NOMPC-like channel transient receptor potential 4 (TRP-4), which was thought to be the primary mechanoreceptor for tactile food sensing. The alternate mechanoreceptor mechanism uncovered by TWK-2 mutation requires three Deg/ENaC channel subunits: ASIC-1, DEL-3, and UNC-8. Analysis of cell-physiological responses to mechanostimuli indicates that TRP and Deg/ENaC channels work together to set the range of analog encoding of stimulus intensity and to improve signal-to-noise characteristics and temporal fidelity of food-sensing neurons. We conclude that a specialized mechanosensory modality-tactile food sensing-emerges from coordination of distinct force-sensing mechanisms housed in one type of sensory neuron.
Asunto(s)
Proteínas de Caenorhabditis elegans , Percepción del Tacto , Animales , Caenorhabditis elegans/metabolismo , Canales Iónicos/genética , Tacto/fisiología , Células Receptoras Sensoriales/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canales Iónicos Sensibles al Ácido , Canales Epiteliales de SodioRESUMEN
Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL). SAMPL includes extensible imaging hardware and low-cost open-source acquisition software with real-time processing. We first demonstrate that SAMPL's hardware and acquisition software can acquire data from from D. melanogaster, C. elegans, and D. rerio as they move vertically. Next, we leverage SAMPL's throughput to rapidly (two weeks) gather a new zebrafish dataset. We use SAMPL's analysis and visualization tools to replicate and extend our current understanding of how zebrafish balance as they navigate through a vertical environment. Next, we discover (1) that key kinematic parameters vary systematically with genetic background, and (2) that such background variation is small relative to the changes that accompany early development. Finally, we simulate SAMPL's ability to resolve differences in posture or vertical navigation as a function of affect size and data gathered -- key data for screens. Taken together, our apparatus, data, and analysis provide a powerful solution for labs using small animals to investigate balance and locomotor disorders at scale. More broadly, SAMPL is both an adaptable resource for labs looking process videographic measures of behavior in real-time, and an exemplar of how to scale hardware to enable the throughput necessary for screening.
RESUMEN
At chemical synapses, neurotransmitters are packaged into synaptic vesicles that release their contents in response to depolarization. Despite its central role in synaptic function, regulation of the machinery that loads vesicles with neurotransmitters remains poorly understood. We find that synaptic glutamate signaling in a C. elegans chemosensory circuit is regulated by antagonistic interactions between the canonical vesicular glutamate transporter EAT-4/VGLUT and another vesicular transporter, VST-1. Loss of VST-1 strongly potentiates glutamate release from chemosensory BAG neurons and disrupts chemotaxis behavior. Analysis of the circuitry downstream of BAG neurons shows that excess glutamate release disrupts behavior by inappropriately recruiting RIA interneurons to the BAG-associated chemotaxis circuit. Our data indicate that in vivo the strength of glutamatergic synapses is controlled by regulation of neurotransmitter packaging into synaptic vesicles via functional coupling of VGLUT and VST-1.
Asunto(s)
Transporte Biológico , Caenorhabditis elegans/metabolismo , Ácido Glutámico/metabolismo , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Interneuronas/metabolismo , Locomoción , Neuronas , Alineación de Secuencia , Sinapsis/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
During development, the nervous system generates neurons that serve highly specialized roles and, accordingly, possess unique functional attributes. The chemosensory BAG neurons of C. elegans are striking exemplars of this. BAGs sense the respiratory gas carbon dioxide (CO2) and, in a context-dependent manner, switch from mediating avoidance of CO2 to supporting CO2 attraction. To determine mechanisms that support the physiology and plasticity of BAG neurons, we used tandem ChIP-seq and cell targeted RNA-seq to identify gene targets of the transcription factor ETS-5, which is required for BAG development. A functional screen of ETS-5 targets revealed that NHR-6, the sole C. elegans NR4A-type nuclear receptor, is required for BAG-mediated avoidance of CO2 and regulates expression of a subset of BAG-specific genes. Unlike ets-5 mutants, which are defective for both attraction to and avoidance of CO2, nhr-6 mutants are fully competent for attraction. These data indicate that the remarkable ability of BAGs to adaptively assign positive or negative valence to a chemosensory stimulus requires a gene-regulatory program supported by an evolutionarily conserved type of nuclear receptor. We suggest that NHR-6 might be an example of a developmental mechanism for modular encoding of functional plasticity in the nervous system.
Asunto(s)
Caenorhabditis elegans/citología , Caenorhabditis elegans/fisiología , Plasticidad Neuronal/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica/genéticaRESUMEN
Natural products are a major source of small-molecule therapeutics, including those that target the nervous system. We have used a simple serotonin-dependent behavior of the roundworm Caenorhabditis elegans, egg laying, to perform a behavior-based screen for natural products that affect serotonin signaling. Our screen yielded agonists of G protein-coupled serotonin receptors, protein kinase C agonists, and a microbial metabolite not previously known to interact with serotonin signaling pathways: the disulfide-bridged 2,5-diketopiperazine gliotoxin. Effects of gliotoxin on egg-laying behavior required the G protein-coupled serotonin receptors SER-1 and SER-7, and the Gq ortholog EGL-30. Furthermore, mutants lacking serotonergic neurons and mutants that cannot synthesize serotonin were profoundly resistant to gliotoxin. Exogenous serotonin restored their sensitivity to gliotoxin, indicating that this compound synergizes with endogenous serotonin to elicit behavior. These data show that a microbial metabolite with no structural similarity to known serotonergic agonists potentiates an endogenous serotonin signal to affect behavior. Based on this study, we suggest that microbial metabolites are a rich source of functionally novel neuroactive molecules.
Asunto(s)
Conducta Animal , Caenorhabditis elegans/fisiología , Microbiota , Serotonina/metabolismo , Conducta Sexual Animal , Simbiosis , Animales , Biomarcadores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Gliotoxina/farmacología , Neuronas/metabolismoRESUMEN
Nervous system development is instructed by genetic programs and refined by distinct mechanisms that couple neural activity to gene expression. How these processes are integrated remains poorly understood. Here, we report that the regulated release of insulin-like peptides (ILPs) during development of the Caenorhabditis elegans nervous system accomplishes such an integration. We find that the p38 MAP kinase PMK-3, which is required for the differentiation of chemosensory BAG neurons, limits an ILP signal that represses expression of a BAG neuron fate. ILPs are released from BAGs themselves in an activity-dependent manner during development, indicating that ILPs constitute an autocrine signal that regulates the differentiation of BAG neurons. Expression of a specialized neuronal fate is, therefore, coordinately regulated by a genetic program that sets levels of ILP expression during development, and by neural activity, which regulates ILP release. Autocrine signals of this kind might have general and conserved functions as integrators of deterministic genetic programs with activity-dependent mechanisms during neurodevelopment.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Regulación del Desarrollo de la Expresión Génica , Insulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sistema Nervioso/embriología , Células Receptoras Sensoriales/metabolismo , Alelos , Animales , Comunicación Autocrina , Calcio/metabolismo , Linaje de la Célula , Genotipo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/metabolismo , Mutación , Péptidos/química , RNA-Seq , Transducción de Señal , TransgenesRESUMEN
Developmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cromatina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Neuronas/citología , Animales , Diferenciación Celular/fisiología , Células Madre Embrionarias , Humanos , Neuronas/metabolismoRESUMEN
The sensory nervous system of C. elegans comprises cells with varied molecular and functional characteristics, and is, therefore, a powerful model for understanding mechanisms that generate neuronal diversity. We report here that VAB-3, a C. elegans homolog of the homeodomain-containing protein Pax6, has opposing functions in regulating expression of a specific chemosensory fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes gene expression and cell function. In other cells, a long isoform of VAB-3, comprising a Paired homology domain and a homeodomain, represses expression of ETS-5, a transcription factor required for expression of BAG fate. Repression of ets-5 requires the Eyes Absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. We determined sequences that mediate high-affinity binding of ETS-5, VAB-3 and CEH-32. The ets-5 locus is enriched for ETS-5-binding sites but lacks sequences that bind VAB-3 and CEH-32, suggesting that these factors do not directly repress ets-5 expression. We propose that a promoter-selection system together with lineage-specific expression of accessory factors allows VAB-3/Pax6 to either promote or repress expression of specific cell fates in a context-dependent manner. This article has an associated 'The people behind the papers' interview.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Bleomicina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Clorambucilo/metabolismo , Cisplatino/metabolismo , Ciclofosfamida/metabolismo , Dactinomicina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción/genética , Vinblastina/metabolismoRESUMEN
Many behaviors promote reproduction or food finding. These critical functions of behavior can conflict; successful reproductive strategies can grow populations to the point where food is depleted. In this issue of Neuron, Wu et al. (2019) show how the nematode C. elegans detects crowding to change feeding behavior by coupling pheromone sensing to signaling via insulin-like peptides.
Asunto(s)
Proteínas de Caenorhabditis elegans , Insulina , Animales , Caenorhabditis elegans , Péptidos , Feromonas , Cambio SocialRESUMEN
Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.
Asunto(s)
Amidina-Liasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos/metabolismo , Neuropéptidos/metabolismo , Amidina-Liasas/química , Amidina-Liasas/genética , Secuencia de Aminoácidos , Animales , Vías Biosintéticas , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cobre/metabolismo , Eliminación de Gen , Humanos , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutación , Neuropéptidos/genética , Alineación de Secuencia , Espectrometría de Masas en TándemRESUMEN
Sensory neurons often possess cilia with elaborate membrane structures that are adapted to the sensory modality of the host cell. Mechanisms that target sensory transduction proteins to these specialized membrane domains remain poorly understood. Here, we show that a homolog of the human retinal dystrophy gene Retinal Degeneration 3 (RD3) is a Golgi-associated protein required for efficient trafficking of a sensory receptor, the receptor-type guanylate cyclase GCY-9, to cilia in chemosensory neurons of the nematode Caenorhabditis elegans The trafficking defect caused by mutation of the nematode RD3 homolog is suppressed in vivo by mutation of key components of the retromer complex, which mediates recycling of cargo from endosomes to the Golgi. Our data show that there exists a critical balance in sensory neurons between the rates of anterograde and retrograde trafficking of cargo destined for the sensory cilium and this balance requires molecular specialization at an early stage of the secretory pathway.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Cilios/fisiología , Proteínas del Ojo/metabolismo , Transporte de Proteínas/fisiología , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas del Ojo/clasificación , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Células Receptoras Sensoriales/fisiologíaRESUMEN
A landmark study has revealed that an interleukin-17-like signaling system modulates a neural circuit that controls the aggregation behavior of nematodes.
Asunto(s)
Caenorhabditis elegans , Interleucina-17 , Animales , NeurotransmisoresRESUMEN
Serotonin is an evolutionarily ancient molecule that functions in generating and modulating many behavioral states. Although much is known about how serotonin acts on its cellular targets, how serotonin release is regulated in vivo remains poorly understood. In the nematode C. elegans, serotonin neurons that drive female reproductive behavior are directly modulated by inhibitory neuropeptides. Here, we report the isolation of mutants in which inhibitory neuropeptides fail to properly modulate serotonin neurons and the behavior they mediate. The corresponding mutations affect the T-type calcium channel CCA-1 and symmetrically re-tune its voltage-dependencies of activation and inactivation towards more hyperpolarized potentials. This shift in voltage dependency strongly and specifically bypasses the behavioral and cell physiological effects of peptidergic inhibition on serotonin neurons. Our results indicate that T-type calcium channels are critical regulators of a C. elegans serotonergic circuit and demonstrate a mechanism in which T-type channels functionally gate inhibitory modulation in vivo.
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Caenorhabditis elegans/fisiología , Canales de Calcio Tipo T/metabolismo , Neuropéptidos/metabolismo , Neuronas Serotoninérgicas/efectos de los fármacos , Neuronas Serotoninérgicas/fisiología , AnimalesRESUMEN
Animals must decide when to consume precious fat stores in order to sustain life. In this issue of Cell Reports, Witham et al. report how oxygen-sensing neurons ensure this decision is made under environmental conditions that favor metabolic efficiency.
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Metabolismo de los Lípidos , Oxígeno/metabolismo , Animales , QuemadurasRESUMEN
Toll-like receptors (TLRs) play critical roles in innate immunity in many animal species. The sole TLR of C. elegans--TOL-1--is required for a pathogen-avoidance behavior, yet how it promotes this behavior is unknown. We show that for pathogen avoidance TOL-1 signaling is required in the chemosensory BAG neurons, where it regulates gene expression and is necessary for their chemosensory function. Genetic studies revealed that TOL-1 acts together with many conserved components of TLR signaling. BAG neurons are activated by carbon dioxide (CO2), and we found that this modality is required for pathogen avoidance. TLR signaling can therefore mediate host responses to microbes through an unexpected mechanism: by promoting the development and function of chemosensory neurons that surveil the metabolic activity of environmental microbes.
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Caenorhabditis elegans/genética , Expresión Génica , Serratia marcescens/fisiología , Transducción de Señal , Animales , Reacción de Prevención , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dióxido de Carbono/metabolismo , Señales (Psicología) , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
Many proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown. Here we identify a mechanism that favours cilium disassembly and maintains the disassembled state. We show that co-localization of the S/G2 phase kinase, Nek2 and Kif24 triggers Kif24 phosphorylation, inhibiting cilia formation. We show that Kif24, a microtubule depolymerizing kinesin, is phosphorylated by Nek2, which stimulates its activity and prevents the outgrowth of cilia in proliferating cells, independent of Aurora A and HDAC6. Our data also suggest that cilium assembly and disassembly are in dynamic equilibrium, but Nek2 and Kif24 can shift the balance toward disassembly. Further, Nek2 and Kif24 are overexpressed in breast cancer cells, and ablation of these proteins restores ciliation in these cells, thereby reducing proliferation. Thus, Kif24 is a physiological substrate of Nek2, which regulates cilia disassembly through a concerted mechanism involving Kif24-mediated microtubule depolymerization.
Asunto(s)
Cilios/fisiología , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Carcinogénesis , Ciclo Celular , Línea Celular Tumoral , Regulación de la Expresión Génica/fisiología , Humanos , Cinesinas/genética , Quinasas Relacionadas con NIMA , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Animals from diverse phyla possess neurons that are activated by the product of aerobic respiration, CO2. It has long been thought that such neurons primarily detect the CO2 metabolites protons and bicarbonate. We have determined the chemical tuning of isolated CO2 chemosensory BAG neurons of the nematode Caenorhabditis elegans. We show that BAG neurons are principally tuned to detect molecular CO2, although they can be activated by acid stimuli. One component of the BAG transduction pathway, the receptor-type guanylate cyclase GCY-9, suffices to confer cellular sensitivity to both molecular CO2 and acid, indicating that it is a bifunctional chemoreceptor. We speculate that in other animals, receptors similarly capable of detecting molecular CO2 might mediate effects of CO2 on neural circuits and behavior.