ASJ Neurons Research Articles

Neuroendocrine gene expression coupling of interoceptive bacterial food cues to foraging behavior of C. elegans

Animal internal state is modulated by nutrient intake, resulting in behavioral responses to changing food conditions. The neural mechanisms by which internal states are generated and maintained are not well understood. Here, we show that in the nematode Caenorhabditis elegans, distinct cues from bacterial food – interoceptive signals from the ingestion of bacteria and gustatory molecules sensed from nearby bacteria – act antagonistically on the expression of the neuroendocrine TGF-beta ligand DAF-7 from the ASJ pair of sensory neurons to modulate foraging behavior. A positive-feedback loop dependent on the expression of daf-7 from the ASJ neurons acts to promote transitions between roaming and dwelling foraging states and influence the persistence of roaming states. SCD-2, the C. elegans ortholog of mammalian anaplastic lymphoma kinase (ALK), which has been implicated in the central control of metabolism of mammals, functions in the AIA interneurons to regulate foraging behavior and cell-non-autonomously control the expression of DAF-7 from the ASJ neurons. Our data establish how a dynamic neuroendocrine daf-7 expression feedback loop regulated by SCD-2 functions to couple sensing and ingestion of bacterial food to foraging behavior. We further suggest that this neuroendocrine feedback loop underlies previously characterized exploratory behaviors in C. elegans. Our data suggest that the expression of daf-7 from the ASJ neurons contributes to and is correlated with an internal state of ‘unmet need’ that regulates exploratory foraging behavior in response to bacterial cues in diverse physiological contexts.

Neuroendocrine gene expression coupling of interoceptive bacterial food cues to foraging behavior of C. elegans.

Animal internal state is modulated by nutrient intake, resulting in behavioral responses to changing food conditions. The neural mechanisms by which internal states are generated and maintained are not well understood. Here, we show that in the nematode Caenorhabditis elegans, distinct cues from bacterial food - interoceptive signals from the ingestion of bacteria and gustatory molecules sensed from nearby bacteria - act antagonistically on the expression of the neuroendocrine TGF-beta ligand DAF-7 from the ASJ pair of sensory neurons to modulate foraging behavior. A positive-feedback loop dependent on the expression of daf-7 from the ASJ neurons acts to promote transitions between roaming and dwelling foraging states and influence the persistence of roaming states. SCD-2, the C. elegans ortholog of mammalian anaplastic lymphoma kinase (ALK), which has been implicated in the central control of metabolism of mammals, functions in the AIA interneurons to regulate foraging behavior and cell-non-autonomously control the expression of DAF-7 from the ASJ neurons. Our data establish how a dynamic neuroendocrine daf-7 expression feedback loop regulated by SCD-2 functions to couple sensing and ingestion of bacterial food to foraging behavior. We further suggest that this neuroendocrine feedback loop underlies previously characterized exploratory behaviors in C. elegans. Our data suggest that the expression of daf-7 from the ASJ neurons contributes to and is correlated with an internal state of 'unmet need' that regulates exploratory foraging behavior in response to bacterial cues in diverse physiological contexts.

Left-right asymmetry in oxidative stress sensing neurons in C. elegans.

Perception of oxidative stress in nematodes involves specific neurons expressing antioxidant enzymes. Here, we carefully characterized GFP knock-in lines for C. elegans peroxiredoxin PRDX-2 and thioredoxin TRX-1, and uncovered that left and right I2, PHA and ASJ neurons reproducibly express an asymmetric level of each enzyme. We observed that high-expressing neurons are in most cases associated with a particular side, indicating a directional rather than stochastic type of asymmetry. We propose that the biological relevance of this left-right asymmetry is to fine-tune H 2 O 2 or light sensing, which remains to be investigated.

A serine/threonine-specific protein kinase of Haemonchus contortus with a role in the development.

In the free-living nematode Caenorhabditis elegans, the serine/threonine-specific protein kinase, AKT, is known to play a key role in dauer formation, life-span, and stress-resistance through the insulin-like signaling pathway. Although the structure and function of AKT-coding genes of C. elegans are understood, this is not the case for homologous genes in parasitic nematodes. In the present study, we explored a C. elegans akt-1 gene homolog in the parasitic nematode Haemonchus contortus, investigated its transcript isoforms (Hc-akt-1a and Hc-akt-1b), and studied expression and function using both homologous and heterologous functional genomic tools. In C. elegans, we showed that the predicted promoter of Hc-akt-1 drives substantial expression in ASJ neurons of the N2 (wild-type) strain. In H. contortus (Haecon-5 stain), RNAi (soaking) led to a significantly decreased transcript abundance for both Hc-akt-1a and Hc-akt-1b, and reduced larval development in larval stages in vitro. Chemical inhibition was also shown to block larval development. Taken together, the evidence from this study points to a key functional role for Hc-akt-1 in H. contortus.

Intestinal infection regulates behavior and learning via neuroendocrine signaling.

The recognition of pathogens and subsequent activation of defense responses are critical for the survival of organisms. The nematode Caenorhabditis elegans recognizes pathogenic bacteria and elicits defense responses by activating immune pathways and pathogen avoidance. Here we show that chemosensation of phenazines produced by pathogenic Pseudomonas aeruginosa, which leads to rapid activation of DAF-7/TGF-β in ASJ neurons, is insufficient for the elicitation of pathogen avoidance behavior. Instead, intestinal infection and bloating of the lumen, which depend on the virulence of P. aeruginosa, regulates both pathogen avoidance and aversive learning by modulating not only the DAF-7/TGF-β pathway but also the G-protein coupled receptor NPR-1 pathway, which also controls aerotaxis behavior. Modulation of these neuroendocrine pathways by intestinal infection serves as a systemic feedback that enables animals to avoid virulent bacteria. These results reveal how feedback from the intestine during infection can modulate the behavior, learning, and microbial perception of the host.

PDF-1 neuropeptide signaling regulates sexually dimorphic gene expression in shared sensory neurons of C. elegans.

Sexually dimorphic behaviors are a feature common to species across the animal kingdom, however how such behaviors are generated from mostly sex-shared nervous systems is not well understood. Building on our previous work which described the sexually dimorphic expression of a neuroendocrine ligand, DAF-7, and its role in behavioral decision-making in C. elegans (Hilbert and Kim, 2017), we show here that sex-specific expression of daf-7 is regulated by another neuroendocrine ligand, Pigment Dispersing Factor (PDF-1), which has previously been implicated in regulating male-specific behavior (Barrios et al., 2012). Our analysis revealed that PDF-1 signaling acts sex- and cell-specifically in the ASJ neurons to regulate the expression of daf-7, and we show that differences in PDFR-1 receptor activity account for the sex-specific effects of this pathway. Our data suggest that modulation of the sex-shared nervous system by a cascade of neuroendocrine signals can shape sexually dimorphic behaviors.

Counteracting Ascarosides Act through Distinct Neurons to Determine the Sexual Identity of C. elegans Pheromones

Sex pheromones facilitate reproduction by attracting potential mates and altering their behavior and physiology. In C.elegans, males and hermaphrodites secrete similar blends of pheromone molecules, two of which are present in different relative concentrations: ascr#3, which is more abundant in hermaphrodites, and ascr#10, which is more abundant in males. It is not currently understood how this compositional difference results in sex-specific effects, for example, the slower aging of the hermaphrodite germline in the presence of physiologically relevant concentrations of male pheromones. Here we report three key elements of the mechanism responsible forthis phenomenon. First, ascr#3 counters the activity of ascr#10. This antagonism decreases the magnitude and the sensitivity of the hermaphrodite response to the male pheromone, restricting it to situations in which the presence of a male could be inferred with high confidence. Second, hermaphrodites recognize pheromone as male if the concentration of ascr#10 is higher than that of ascr#3. Third, the response to ascr#10 requires TRPV channel function in the ADL neurons and the daf-7 signaling from the ASI neurons, whereas the response to ascr#3 relies on cyclic guanosine monophosphate (cGMP)-gated channels and activity of the ASJ, AWB, and AWC neurons. These results argue that the counteracting activities of distinct neuronal circuits determine the sexual identity of the pheromone. The parallels between this mechanism and other signaling systems suggest that diverse organisms may perform particular neuronal computations using similar general principles.

Regulatory changes in two chemoreceptor genes contribute to a Caenorhabditis elegans QTL for foraging behavior.

Natural isolates of C. elegans differ in their sensitivity to pheromones that inhibit exploratory behavior. Previous studies identified a QTL for pheromone sensitivity that includes alternative alleles of srx-43, a chemoreceptor that inhibits exploration through its activity in ASI sensory neurons. Here we show that the QTL is multigenic and includes alternative alleles of srx-44, a second chemoreceptor gene that modifies pheromone sensitivity. srx-44 either promotes or inhibits exploration depending on its expression in the ASJ or ADL sensory neurons, respectively. Naturally occurring pheromone insensitivity results in part from previously described changes in srx-43 expression levels, and in part from increased srx-44 expression in ASJ, which antagonizes ASI and ADL. Antagonism between the sensory neurons results in cellular epistasis that is reflected in their transcription of insulin genes that regulate exploration. These results and genome-wide evidence suggest that chemoreceptor genes may be preferred sites of adaptive variation in C. elegans.

Inhibition of Lithium-Sensitive Phosphatase BPNT-1 Causes Selective Neuronal Dysfunction in C. elegans

Lithium has been a mainstay for the treatment of bipolar disorder, yet the molecular mechanisms underlying its action remain enigmatic. Bisphosphate 3'-nucleotidase (BPNT-1) is a lithium-sensitive phosphatase that catalyzes the breakdown of cytosolic 3'-phosphoadenosine 5'-phosphate (PAP), a byproduct of sulfation reactions utilizing the universal sulfate group donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) [1-3]. Loss of BPNT-1 leads to the toxic accumulation of PAP in yeast and non-neuronal cell types in mice [4, 5]. Intriguingly, BPNT-1 is expressed throughout the mammalian brain [4], and it has been hypothesized that inhibition of BPNT-1 could contribute to the effects of lithium on behavior [5]. Here, we show that loss of BPNT-1 in Caenorhabditis elegans results in the selective dysfunction of two neurons, the bilaterally symmetric pair of ASJ chemosensory neurons. As a result, BPNT-1 mutants are defective in behaviors dependent on the ASJ neurons, such as dauer exit and pathogen avoidance. Acute treatment with lithium also causes dysfunction of the ASJ neurons, and we show that this effect is reversible and mediated specifically through inhibition of BPNT-1. Finally, we show that the selective effect of lithium on the nervous system is due in part to the limited expression of the cytosolic sulfotransferase SSU-1 in the ASJ neuron pair. Our data suggest that lithium, through inhibition of BPNT-1 in the nervous system, can cause selective toxicity to specific neurons, resulting in corresponding effects on behavior of C.elegans.

Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans.

Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance.

TRX-1 Regulates SKN-1 Nuclear Localization Cell Non-autonomously in Caenorhabditis elegans.

The Caenorhabditis elegans oxidative stress response transcription factor, SKN-1, is essential for the maintenance of redox homeostasis and is a functional ortholog of the Nrf family of transcription factors. The numerous levels of regulation that govern these transcription factors underscore their importance. Here, we add a thioredoxin, encoded by trx-1, to the expansive list of SKN-1 regulators. We report that loss of trx-1 promotes nuclear localization of intestinal SKN-1 in a redox-independent, cell non-autonomous fashion from the ASJ neurons. Furthermore, this regulation is not general to the thioredoxin family, as two other C. elegans thioredoxins, TRX-2 and TRX-3, do not play a role in this process. Moreover, TRX-1-dependent regulation requires signaling from the p38 MAPK-signaling pathway. However, while TRX-1 regulates SKN-1 nuclear localization, classical SKN-1 transcriptional activity associated with stress response remains largely unaffected. Interestingly, RNA-Seq analysis revealed that loss of trx-1 elicits a general, organism-wide down-regulation of several classes of genes; those encoding for collagens and lipid transport being most prevalent. Together, these results uncover a novel role for a thioredoxin in regulating intestinal SKN-1 nuclear localization in a cell non-autonomous manner, thereby contributing to the understanding of the processes involved in maintaining redox homeostasis throughout an organism.

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.

Cis- and trans-regulatory mechanisms of gene expression in the ASJ sensory neuron of Caenorhabditis elegans.

The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons.

Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans.

Temperature is a critical environmental stimulus that has a strong impact on an organism’s biochemistry. Animals can respond to changes in ambient temperature through behaviour or altered physiology. However, how animals habituate to temperature is poorly understood. The nematode C. elegans stores temperature experiences and can induce temperature habituation-linked cold tolerance. Here we show that light and pheromone-sensing neurons (ASJ) regulate cold habituation through insulin signalling. Calcium imaging reveals that ASJ neurons respond to temperature. Cold habituation is abnormal in a mutant with impaired cGMP signalling in ASJ neurons. Insulin released from ASJ neurons is received by the intestine and neurons regulating gene expression for cold habituation. Thus, temperature sensation in a light and pheromone-sensing neuron produces a robust effect on insulin signalling that controls experience-dependent temperature habituation.

Optogenetic Long-Term Manipulation of Behavior and Animal Development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.

The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons ( Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene ( Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm.

The thioredoxin TRX-1 modulates the function of the insulin-like neuropeptide DAF-28 during dauer formation in Caenorhabditis elegans.

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.

TRP‐ing off the p53 apoptotic switch

TRP‐ing off the p53 apoptotic switch

Protection from Afar

A factor secreted from anterior neurons in Caenorhabditis elegans protects germ cells from apoptosis.

Sulfated Signal from ASJ Sensory Neurons Modulates Stomatin-dependent Coordination in Caenorhabditis elegans

The neuronal stomatin-like proteins UNC-1 and UNC-24 play important roles in the nervous system of Caenorhabditis elegans. These neuronal stomatin-like proteins are putative chaperone proteins that can modify volatile anesthetic sensitivity and disrupt coordinated locomotion. A suppressor of unc-1 and unc-24, named ssu-1(fc73) (for suppressor of stomatin uncoordination), suppresses three phenotypes of neuronal stomatin-like protein deficiency as follows: volatile anesthetic sensitivity, uncoordinated locomotion, and a constitutive alternative developmental phenotype known as dauer. Here we provide the first phenotypic characterization of ssu-1, predicted to be the only C. elegans cytosolic alcohol sulfotransferase, a family of enzymes that catalyze a sulfate linkage with the alcohol group of small molecules for the purposes of detoxification or modification of signaling. In vitro enzyme analysis of bacterially expressed SSU-1 demonstrates sulfotransferase activity and thus confirms the function predicted by protein sequence similarities. Whereas unc-1 is expressed in the majority of neurons of C. elegans, expression of SSU-1 protein in only the two ASJ amphid interneurons is sufficient to restore the wild type phenotype. This work demonstrates that SSU-1 is a functional sulfotransferase that likely modifies endocrine signaling in C. elegans. The expression of SSU-1 in the ASJ neurons refines the understanding of the function of these cells and supports their classification as endocrine tissue. The relationship of unc-1, unc-24, and ssu-1 is the first association of neuronal stomatin-like proteins sharing regulatory roles with a sulfotransferase enzyme.