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Curr Biol. Author manuscript; available in PMC 2016 August 31. Published in final edited form as: Curr Biol. 2015 August 31; 25(17): 2228–2237. doi:10.1016/j.cub.2015.07.037.
Toll-like receptor signaling promotes the development and function of sensory neurons required for a C. elegans pathogenavoidance behavior Julia P. Brandt1 and Niels Ringstad1 1The
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Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, Molecular Neurobiology Program, and Department of Cell Biology, NYU Langone Medical Center, New York, NY 10016
Summary
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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 TOL-1 signaling is required in the chemosensory BAG neurons for pathogen avoidance, where it regulates gene expression and is necessary for BAG 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.
Introduction
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Toll-like receptors (TLRs) are integral membrane proteins found in many metazoans that have remained linked to host-pathogen interactions throughout evolution [1]. Drosophila Toll, the first characterized TLR, was discovered as a factor required for dorsoventral patterning of the early embryo [2]. Subsequently, it was found that Toll mutants are highly susceptible to infection and have defects in pathogen-induced expression of antifungal peptides [3]. Mammalian TLRs were found to play similar roles in immune signaling [4, 5], where they promote the inflammatory response and expression of pathogen-defense molecules such as interferons [6]. In phylogenetically distant animals, therefore, TLRs constitute a first line of defense against pathogens during exposure and infection. In both mammals and insects, TLRs play instructive roles in immune signaling. Drosophila Toll is activated by pathogens when extracellular receptors for pathogen-derived cues
Corresponding author is Niels Ringstad, [email protected]. Author contributions JPB and NR designed experiments, executed experiments, interpreted data and wrote this manuscript. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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initiate a proteolytic cascade that activates latent Spätzle, a BMP-like ligand for Toll [7]. Vertebrate TLRs directly bind pathogen-derived cues such as lipopolysaccharide and viral nucleic acids [8]. Although the mechanisms by which TLRs are activated in response to infection in mammals and insects differ, once liganded, TLRs initiate conserved signaling pathways that drive inflammatory and immune responses [9]. Canonical TLR signaling activates kinase signaling cascades and negatively regulates the transcriptional inhibitor IκB/Cactus. Downstream of IkB is a master regulator of the inflammatory response, NFκB/ Relish, which drives expression of pathogen-defense molecules such as antimicrobial peptides in insects and interferons and cytokines in mammals [10].
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In contrast to TLR signaling in mammals and flies, little is known about this pathway in C. elegans. C. elegans uses conserved signaling pathways to mediate immunity to a broad spectrum of pathogens [11, 12, 13]. However, the sole TLR of C. elegans, TOL-1, has not been linked to these pathways. Instead, TOL-1 has been shown to be required for a behavioral response to the pathogenic microbe Serratia marcescens [14], although how TOL-1 functions in pathogen-avoidance is unknown. That TOL-1 is required for an avoidance behavior to pathogenic microbes suggests that in nematodes, TLR signaling might function in the nervous system.
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Here we show that TOL-1 and associated signaling molecules are required in a pair of chemosensory neurons – the BAG neurons - for avoidance of Serratia marcescens. TLR signaling regulates gene expression in BAG neurons and is required for their sensory function. BAG neurons are gas-sensing neurons that are activated by hypoxia and CO2 [15, 16, 17]. We find that their CO2-sensing modality is required for avoidance of Serratia marcescens. Together, our data indicate that CO2 produced by microbial respiration is a molecular cue used by C. elegans to avoid metabolically active pathogens. In C. elegans, therefore, a TLR functions in microbe-sensing through a distinct and, to our knowledge, previously unknown mechanism: by promoting the development of neurons that are required for sensory detection of microbes.
Results The p38 MAP kinase PMK-3 is required for proper gene expression by chemosensory BAG neurons
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C. elegans BAG neurons are ciliated chemosensory neurons that sense increases in ambient CO2 [15, 16]. Studies of BAG neurons have identified several factors required for CO2sensing: the GCY-9 receptor-type guanylate cyclase, which is specifically expressed in BAG cells and likely functions as a CO2 receptor, and cyclic nucleotidegated (CNG) ion channels comprising TAX-2 and TAX-4 subunits [18, 19, 20]. Transcription factors required for the specification and differentiation of BAG neurons have been identified [21, 22, 23] but our knowledge of gas-sensing gene regulation remains incomplete. To better understand mechanisms that function in the development of BAG cells and promote CO2chemosensation, we performed a reporter-based screen for mutants with abnormal expression of a marker for differentiated BAG neurons (Supplemental Fig. 1). One set of non-complementing mutations isolated by the screen caused reduced expression of a BAG cell marker, although the BAG cell itself was present and easily identified on the basis of its Curr Biol. Author manuscript; available in PMC 2016 August 31.
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characteristic position and nuclear morphology (Fig. 1A). This complementation group consisted of four alleles of the p38 Mitogen Activated Protein (MAP) kinase gene pmk-3 (Fig. 1B).
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We tested a panel of genes expressed by BAG neurons for regulation by pmk-3. In addition to several neuropeptide genes [24], BAG neurons express genes that are required for their chemosensory functions [17, 18]. We found that a subset of these genes were dysregulated in pmk-3 mutants (Fig. 1C). Expression of the neuropeptide gene flp-17 (Supplemental Fig. 1) and the guanylate cyclase gene gcy-33 (Fig. 1A) was greatly reduced in BAG neurons and expression of the cGMP-gated channel gene tax-2 was partly reduced (Fig. 1C). By contrast, expression of the guanylate cyclase gene gcy-31 was increased in pmk-3 mutant BAG neurons. gcy-9, which encodes a guanylate cyclase required for CO2-sensing, the neuropeptide genes flp-10 and flp-19, and the CNG channel gene tax-4 were unaffected by pmk-3 mutation (Fig. 1C). BAG cell-specific expression of pmk-3 restored expression of the Pflp-17::GFP reporter in pmk-3(wz31) mutants (Fig. 1D), indicating that pmk-3 acts cell autonomously to promote the expression of BAG-specific genes. pmk-3 therefore, is required for normal expression of some but not all markers of a BAG neuron fate. In addition to abnormal gene expression, pmk-3 mutants exhibited a low penetrance morphological defect. 30% of BAG cell axons in pmk-3 mutants failed to form proper commissures in the nerve ring (Supplemental Fig. 2B) suggesting that in addition to regulating gene expression, pmk-3 plays a role in axon morphogenesis in BAG cells.
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To determine when pmk-3 acts to promote expression of BAG neuron fate markers, we carried out heat-shock experiments to transiently induce pmk-3 expression in embryos, larvae and adults. Inducing pmk-3 expression in embryos and larvae was sufficient to restore expression of a Pflp-17::GFP reporter in BAG cells (Fig. 1E). However, induction of pmk-3 expression in the adult failed to restore transgene expression, indicating that PMK-3 is required during embryonic and larval stages to promote the expression of a BAG cell fate. While these data show that pmk-3 is required during development, we note that it is possible that pmk-3 is also required post-developmentally in the BAG neurons.
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We next tested whether pmk-3 mutation affects BAG neuron function by measuring the CO2-avoidance behavior of pmk-3 mutants. BAG neurons are directly activated by CO2 and required for chemotaxis down a CO2 gradient (Fig. 2A). pmk-3 mutants were severely defective in CO2-avoidance and failed to navigate down a steep (0–10%) CO2 gradient (Fig. 2B and Supplemental Fig. 3). Mutation of pmk-3 caused defects in CO2 avoidance assays as severe as loss of the CO2-receptor gene gcy-9 (Fig. 2B). pmk-3 is in an operon with two other p38 MAPK genes, pmk-1 and pmk-2 [25]. pmk-1 and pmk-2 function in neuronal development and stress responses [11, 26]. To determine whether pmk-3 mutation affected BAG cells by altering expression of the entire operon, we tested pmk-1 and pmk-2 mutants for CO2 avoidance and found them to be wild-type in this assay (Fig. 2B). Furthermore, expression of only pmk-3 in BAG cells restored CO2 avoidance behavior to pmk-3 mutants. Therefore, the abnormally differentiated BAG neurons of pmk-3 mutants cannot mediate CO2-sensing.
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We used heat-shock-mediated induction of pmk-3 expression to determine whether pmk-3 gene function was required for BAG cell function at the same time that it is required for gene expression by BAG neurons. Inducing pmk-3 expression in embryos and larvae restored CO2 avoidance behavior to pmk-3 mutant adults (Fig. 2C). By contrast, induction of pmk-3 expression in adults failed to restore their CO2 avoidance behavior. These data indicate that PMK-3 functions during the same window of developmental time to promote both the proper gene expression and function of BAG cells. Furthermore, these data show that pmk-3 plays a permissive role in CO2-sensing by acting during BAG neuron development as opposed to an instructive role during sensory signaling. PMK-3 functions with the Toll-like receptor TOL-1 and TLR signaling factors to promote BAG neuron function
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Because p38 MAPKs are canonically regulated by a kinase cascade driven by MAP kinase kinase kinases (MAP3Ks), we sought to identify a MAP3K that acts upstream of PMK-3 in BAG cells [27]. The C. elegans genome encodes three such kinases: DLK-1, NSY-1 and MOM-4. DLK-1 is known to activate PMK-3 in motor neurons to promote synaptic development and axonal regeneration [28, 29]. However, dlk-1 mutants were wild-type for CO2-sensing (Fig. 3A), indicating that DLK-1 does not regulate PMK-3 to promote BAG neuron function. Although we could not measure CO2 avoidance by nsy-1 mutants, which are uncoordinated, we found that mutants for its downstream partner SEK-1 [30] were wildtype for CO2 avoidance (Fig. 3A). mom-4 mutants, however, were strongly defective in CO2 avoidance. BAG cell-specific expression of mom-4 rescued this defect, suggesting that MOM-4 functions with PMK-3 in BAG cells (Fig. 3A).
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MOM-4 is a homolog of TGF-beta-activated kinase (TAK), which is regulated by a small number of cell-surface receptors, including TLRs [31]. We tested the sole C. elegans TLR, TOL-1, for a role in BAG neurons. A partial loss-of-function allele (nr2033) and a null allele (nr2013) of tol-1 each caused CO2-avoidance defects (Fig. 3B). BAG-specific RNAi knockdown of tol-1 caused a defect as severe as that of a tol-1 null mutant, demonstrating that TOL-1 is required in BAG neurons for their function (Fig. 3B). Also, BAG cell-specific expression of tol-1 in tol-1(nr2013) mutants restored CO2 avoidance behavior to tol-1 mutants, demonstrating that tol-1 expression in BAG neurons can account for a significant amount of its function in CO2-avoidance behavior. tol-1 expression was previously reported in sensory neurons but its expression in BAG cells had not been documented [14, 32, 33]. We made a Ptol-1::GFP reporter carrying 5 kilobases of upstream sequences from the tol-1 translational start site. As previously reported, we observed robust expression by the URY sensory neurons (Supplemental Fig. 4). However, we also observed expression by BAG cells (Fig. 3C), consistent with our observation that tol-1 is required in these cells for their function. We used fosmid-based reporter transgenes to determine the expression of pmk-3 and mom-4 and found that these genes were also expressed by BAG cells (Supplemental Fig. 5A and B). Like pmk-3 mutant cells, we found that 30% of tol-1 mutant BAG cells exhibited abnormal axonal commissures (Supplemental Fig. 2C). To further determine the similarities between tol-1 mutants and pmk-3 mutants, we tested whether tol-1 promotes gene expression in BAG
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neurons as does pmk-3. We found that like pmk-3 mutation, loss of tol-1 function reduced expression of gcy-33 and increased expression of gcy-31, suggesting that pmk-3 and tol-1 function together to regulate these BAG cell genes (Fig. 3D). We could not detect a role for tol-1 in regulating expression of flp-17 or tax-2 reporters, suggesting that pmk-3 has gene regulatory functions that are independent of tol-1. Furthermore, we found that tol-1 is required for proper expression of flp-19, a neuropeptide gene that is not regulated by pmk-3 (Fig. 1C), indicating that tol-1 can regulate gene expression independently of pmk-3 (Fig. 3D). Taken together these data suggest that in addition to having distinct functions, pmk-3 and tol-1 coordinately regulate expression of a subset of genes in BAG neurons.
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To determine whether TOL-1, MOM-4 and PMK-3 function together in a signaling pathway we performed a series of genetic interaction tests using constitutively active MKK-4, a kinase that can directly activate PMK-3 and bypass any requirement for endogenous PMK-3 regulators [28]. MKK-4(gf) expression suppressed the CO2-avoidance defects of mom-4 and tol-1 mutants but had no effect on pmk-3 mutants (Fig. 4A), consistent with the hypothesis that TOL-1 and MOM-4 act upstream of PMK-3 in BAG neurons. In other contexts, TLR signaling promotes gene expression by inhibiting IκB, an inhibitor of gene expression [34]. We found that deletion of ikb-1, which encodes an IκB-like protein [14], suppressed the CO2 avoidance defect caused by loss of either tol-1 or pmk-3 function (Fig. 4B). These data suggest that TLR signaling in BAG neurons involves a canonical and evolutionarily conserved regulator of transcription, IκB. The C. elegans genome also encodes two other homologs of TLR signaling genes: pik-1, which encodes the Interleukin Receptor Activated Kinase (IRAK) homolog, and trf-1, which encodes a homolog of the TNF-receptor associated factor (TRAF) [14]. We tested whether pik-1 and trf-1 mutants were defective for CO2 avoidance behavior and found that each of these mutants exhibited severe behavioral defects (Fig. 4C). Furthermore, the defects of trf-1 and pik-1 were partially bypassed by activated MAPK signaling specifically in the BAG neurons (Fig. 4C). Together, our data support a role for TLR signaling in promoting the development and function of the chemosensory BAG neurons. Figure 4D depicts a pathway model for TOL-1 signaling that integrates our observations with established models of mammalian TLR signaling [9]. Loss of TLR signaling disrupts the physiology of chemosensory BAG neurons
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We next sought to determine whether behavioral defects of TLR signaling mutants might be caused by defective sensory transduction. We used in vivo calcium imaging to determine whether TLR signaling was required for activation of BAG neurons by CO2 stimuli [22]. Wild-type BAG neurons showed robust calcium responses to CO2 (Fig. 5A). By contrast, tol-1 mutants had blunted CO2 responses that were, on average, 33% of the wild-type response (Fig. 5B and C). These data indicate that the BAG neurons of tol-1 mutants have a primary defect in sensory detection of CO2, suggesting that some genes regulated by TLR signaling function in sensory transduction. Chemosensory BAG neurons are the site of TOL-1 action in microbe-sensing Previously, TOL-1 was shown to be required for a pathogen-avoidance behavior [14] but its site and mechanism of action were unknown. Does a role for TOL-1 in BAG cell development and function relate to its role in pathogen avoidance? To measure pathogen
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avoidance we used a simple behavioral assay in which the distribution of animals in an arena is measured after exposure either to pathogenic or to non-pathogenic microbes (Fig. 6A). As previously reported [14], C. elegans exposed to non-pathogenic bacteria rarely foraged away from the bacterial lawn while C. elegans exposed to the pathogen Serratia marcescens dispersed (Fig. 6B). By contrast, tol-1 mutants did not disperse when exposed to S. marcescens (Fig. 6B). We found that the TLR signaling pathway that promotes BAG cell development and function is also required for this pathogen avoidance behavior. Like tol-1 mutants, pmk-3 mutants failed to avoid S. marcescens (Fig. 6B). Importantly, loss of tol-1 function specifically in BAG cells recapitulated the pathogen avoidance defect of tol-1 mutants, and BAG cell-specific rescue of pmk-3 and tol-1 restored pathogen avoidance behavior to the corresponding mutants (Fig. 6B). Therefore, BAG cells are a site of tol-1 action in both CO2-avoidance and pathogen avoidance, suggesting a link between CO2sensing and microbe-sensing.
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To determine whether CO2-sensing per se is required for pathogen avoidance, we tested CO2-blind gcy-9 mutants [18]. gcy-9 mutants were as defective in avoidance of S. marcescens as tol-1 and pmk-3 mutants (Fig. 6C). In addition to CO2, BAG neurons sense hypoxia through a distinct mechanism that requires the heteromeric GCY-31/GCY-33 guanylate cyclases [17]. We tested gcy-31 mutants and found that they robustly avoided pathogen (Fig. 6C), indicating that hypoxia-sensing by BAG neurons is not required for S. marcescens avoidance. Defects in the CO2-sensing modality of BAG cells, therefore, account for the pathogen-avoidance defects of TLR signaling mutants. We further found that C. elegans did not avoid heat-killed S. marcescens or the attenuated S. marcescens strain Db1140 (Fig. 6D). These data suggest that CO2 produced by the respiration of living microbes is an important factor in avoidance behavior.
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BAG neurons detect CO2 as a contextual cue for pathogen-avoidance
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We observed that pathogenic and non-pathogenic microbes used in our studies evolved comparable amounts of CO2 under conditions used for behavioral studies (Supplemental Fig. 6), making it unlikely that the lawn leaving behavior evoked by S. marcescens is simply another manifestation of CO2 avoidance behavior. Is detection of microbial CO2, then, part of a mechanism that triggers pathogen avoidance? To test this possibility we exposed animals dwelling on non-pathogenic E. coli to uniform concentrations of either S. marcescens odorant or to artificial atmospheres containing various concentrations of CO2 and measured lawn-leaving behavior (Fig. 7A). S. marcescens odorant that contained 1% CO2, caused lawn leaving by the wild type but not by CO2-blind gcy-9 mutants, BAGablated animals, or mutants of the TLR pathway (Fig. 7B and Supplemental Fig. 7). Furthermore, BAG cell-specific expression of pmk-3 in pmk-3 mutants restored lawn leaving in response to S. marcescens odorant (Supplemental Fig. 7). Artificial atmospheres containing the same amount of CO2 as S. marcescens odorant did not induce lawn leaving, but a higher concentration of CO2 (10%) did, indicating that intense BAG cell activation is sufficient to drive lawn leaving to some extent. Because there is no CO2 gradient under these experimental conditions, these data indicate that CO2 is a necessary part of a microbederived signal that triggers dispersal rather than a cue used for navigation away from the bacterial lawn.
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Strikingly, S. marcescens odorant was more effective than CO2 concentration-matched artificial atmospheres in triggering lawn leaving (Fig. 7B). To determine whether this was because S. marcescens odorant contains a component that activates BAG neurons independently of CO2 we compared the ability of S. marcescens odorant and CO2-matched artificial atmospheres to activate BAG cells in vivo. S. marcescens odorant and CO2matched artificial atmospheres evoked indistinguishable responses in BAG cells (Fig. 7C), indicating that BAG cells principally detect the CO2 component of S. marcescens odorant. Importantly, gcy-9 mutant BAG cells failed to respond to S. marcescens odorant, providing further evidence that BAG neurons detect microbially produced CO2 (Fig. 7C).
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Our observation that detection of microbially produced CO2 is necessary for pathogen avoidance but that CO2 itself is not able to effectively trigger the behavior support a model in which CO2 acts as a contextual cue that functions together with pathogen-specific factors to evoke a behavioral response. Therefore, detection of microbial respiration is required for C. elegans to respond to the detection of pathogen-specific cues (Fig. 7D). Our data further suggest that pathogen-specific cues are sensed not by BAG neurons but by other chemosensory neurons that collaborate with BAG neurons to trigger avoidance behavior (Fig. 7D).
Discussion
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The ability to detect and respond to pathogens is critical for all animals. C. elegans, which are bacteriovores, must differentiate pathogens from food. Pathogenic microbes express many factors that are inherently attractive to C. elegans. For example, our data demonstrate that heat-killed S. marcescens is attractive to C. elegans as are non-pathogenic E. coli. Others have demonstrated that näive C. elegans are more attracted to pathogenic bacteria than they are to E. coli [35]. Pathogen-avoidance, therefore, requires that the valence of a complex suite of sensory stimuli produced by pathogenic microbes switch from attractive to aversive. Our data indicate that BAG neurons play an important role in this process by sensing microbially-produced CO2. In the case of S. marcescens avoidance, our data demonstrate that the absence or presence of a product of microbial respiration – CO2 - tips the balance from attraction to aversion. It will be interesting to determine whether the same mechanism functions in behavioral responses to other pathogenic microbes known to infect C. elegans [36].
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We found evidence that S. marscesens odorant contains factors that synergize with CO2 to trigger avoidance behavior. We imagine that a sensory circuit involving BAG neurons and other olfactory sensory neurons decodes the complex olfactory stimulus that is bacterial odorant. Such a circuit would likely collaborate with other sensory systems to determine how C. elegans responds to microbes. For example, C. elegans has been shown to sense an aversive gustant - the surfactant serrawettin - produced by S. marcescens [32]. Also, pathogen-derived metabolites such as phenazine-1-carboxamide and pyochelin exert effects on C. elegans behavior over long periods of time by inducing expression of a TGF-beta-like factor, which in turn alters the function of oxygen-sensing circuits [37]. It is therefore likely that C. elegans uses a combination of olfactory, gustatory, gas-sensing and neuromodulatory
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modalities over multiple time scales to sculpt behavioral responses to environmental microbes. Which raises a question: why use CO2-sensing neurons at all in a pathogen-detection mechanism? Both pathogenic and non-pathogenic microbes respire aerobically and produce CO2, so the presence of this factor does not indicate whether the microbes that produced it are harmful. We propose that by monitoring microbial CO2 production, C. elegans is able to determine whether environmental microbes are metabolically active. By integrating a measure of microbial metabolism into mechanisms that detect pathogen-specific cues, C. elegans might be able to reserve the option to feed on attenuated or dead microbes that would otherwise be pathogenic, thereby maximizing its opportunity to capture food resources in its environment.
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Strikingly, the BAG sensory neurons, which are required for an innate behavioral response of C. elegans to the pathogenic microbe S. marcescens, require TLR signaling for proper gene expression and function. We found that TLR signaling is required during development to regulate gene expression, and our data strongly suggest that TLR target genes are required for the function of chemosensory BAG neurons. TLR signaling in C. elegans, therefore, functions in microbe-sensing in an unexpected manner: by promoting the differentiation of chemosensory neurons that function in a sensory system that evaluates the qualities of environmental microbes to instruct foraging behavior. Unlike TLRs in insect and vertebrate innate immunity, where TLRs are activated acutely by the presence of pathogen-derived factors, C. elegans TOL-1 signaling plays a permissive role in pathogen-detection. In fact, our data show that it is insufficient to restore function to this pathway post-developmentally and suggest that there is a critical period of development during which TOL-1 signaling in BAG neurons establishes the program of gene expression required for cell function.
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C. elegans lacks several components of the TLR signaling pathways that mediate innate immunity in insects and mammals, including the Myd88 adapter, the transcription factor NF-κB and its regulatory IκB kinases (IKKs) [14, 38]. We propose that TOL-1 signaling uses different molecules to couple TLR activation to kinase signaling cascades and IκB, and that novel transcriptional regulators will act downstream of TOL-1 to promote expression of neuronal genes. Interestingly, our data indicate that the nr2033 mutant receptor, which lacks much of its cytoplasmic domain including the Toll-Interleukin Receptor homology (TIR) domain, still retains some activity. This suggests that TOL-1 can couple to intracellular signaling pathways in BAG neurons through an unconventional i.e. TIR domainindependent mechanisms. We speculate that counterparts of these mechanisms exist in mammals, where they might define new branches of TLR signaling in innate immunity or link TLR signaling to biological processes such as differentiation of specific cell types during development.
Experimental Procedures Strains All strains and their genotypes used in this study are listed in Supplemental Table 1. C. elegans strains were grown under standard conditions [39] at 20°C except tol-1(nr2013)
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mutants, which were grown at 15°C to promote viability and shifted to 20°C one day prior to behavioral assays. Plasmids Plasmids were constructed by standard methods. Transcriptional reporters were made by cloning 5′ regulatory sequences of genes into the plasmid pPD95.75. cDNA expression constructs were made using the plasmid pPD49.26 [40]. pmk-3 and mom-4 reporter transgenes were translational fusions with GFP made by fosmid recombineering [41]. Plasmids used in this study are: pJB203 Pflp-17::pmk-3; pJB254 Ptax-2::dsRed pJB227 Pflp-17::mom-4; pJB252 Pflp-17::tol-1; pJB237 Pflp-17::tol-1sense; pJB234 Pflp-17::tol-1antisense; pJB24: Pflp-17::mkk-4(gf); pJB252 Ptol-1::GFP; pJB134 Pflp-17::dsRed ; pRM60 Pflp-17::cameleon; pJB240 mom-4::GFP; pNR519 pmk-3::GFP; pJB221 Phsp-16.41::pmk-3; pJB258 Pflp-19::GFP
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Genetic screen for genes regulating BAG cell development The BAG-neuron-specific reporter transgene ynIs64[Pflp-17::GFP] was used to isolate mutants with defects in BAG neuron development. Synchronized animals carrying ynIs64[Pflp-17::GFP], were grown to mid-L4 stage and treated with 47 mM ethyl methane sulfonate (EMS) as described [39]. Animals were distributed onto 10 cm NGM plates seeded with E. coli OP50 and screened in the F2 generation for abnormal GFP expression. Screening was carried out on a Leica M165 FC fluorescence dissecting microscope. Mapping and cloning of pmk-3 alleles
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An initial round of screening identified the non-complementing alleles wz27 and wz31. High resolution SNP-mapping of wz31 was performed by crossing wz31 mutants to the polymorphic strain CB4856 and identifying crossovers using restriction fragment polymorphisms (snip-SNPs) [42]. This experiment placed wz31 in a 10 Mb interval on LG IV. Whole-genome sequencing of wz31 and wz27 mutants revealed that both mutants carry coding mutations in pmk-3: wz31 contains a G->A mutation predicted to change Glu320 to Lys, and wz27 contains a C->T mutations predicted to change Gln339 to an Ochre stop. Further screening and complementation tests identified two additional alleles of pmk-3: wz34 and wz36. Sanger sequencing revealed that wz34 is a C->T mutation predicted to change Arg251 to Cys, and wz36 is a G->A mutation predicted to change Glu297 to Lys. Transgene expression analysis
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Animals were mounted on 2% agarose pads made in M9 medium and immobilized with 30 mM sodium azide. Reporter transgene expression was quantified on a Zeiss AxioImager M2 upright microscope equipped with an EM-CCD camera (Andor) using a 20× 0.8 NA air objective. For each genotype 20–30 animals were imaged and mean pixel values in a 50 pixel-radius circular region of interest centered in the BAG soma were measured using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2011). Pixel values were then normalized to the mean of measurements of wild-type cells and plotted.
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Heat-shock experiments
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Mutant pmk-3(wz31) animals carrying a Phsp16.41::pmk-3 transgene were placed in a 30°C water bath for three hours. After heat-shock, animals were placed at 20°C to recover. Heatshocked embryos and larvae were assayed when they reached adulthood, while heat-shocked adults were assayed 24 hours later. Separate heat-shock experiments were carried out for gene expression and behavior experiments. For gene expression assays, the percentage of animals expressing Pflp-17::GFP after heat-shock was counted on a Leica M165 FC fluorescence dissecting microscope. For CO2 behavior experiments, animals were assayed as described below. CO2 avoidance assays
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20–25 adult hermaphrodites were selected from a staged culture and placed on the surface of an unseeded 10 cm NGM plate. In experiments measuring behavior of transgenic animals, non-transgenic siblings were used as controls. Otherwise, animals were selected blindly from a strain for inclusion in behavioral assays. A custom-made chamber fitted with inlets for air and 10% CO2 was placed on the plate confining the animals to an arena. A thin layer of glycerol had been applied to the bottom of the chamber to seal the chamber to the plate surface and confine animals. Gas mixes were pushed into the chamber at 1.5 mL/minute using a syringe pump. After 30 minutes, the number of animals on the air and CO2 sides of the chamber were counted. Avoidance indices (A.I.) were computed according to the following equation:
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Cell-specific RNAi of tol-1 in BAG cells Two plasmids were generated that used the flp-17 promoter to drive transcription of a 404 bp fragment of the tol-1 cDNA in either the sense and antisense directions. These constructs were co-injected at a concentration of 100 ng μl−1 each to generate an array that expresses dsRNA targeting tol-1 specifically in BAG cells. Confocal Microscopy Young adults were mounted on a 2% agarose pad made in M9 medium and anaesthetized with 30 mM sodium azide. Z stacks were obtained with a Zeiss LSM510 confocal microscope and maximum projection images were created using ImageJ. Imaging BAG cell-calcium responses in restrained animals
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In vivo calcium imaging was performed as previously described [20]. Briefly, animals expressing the ratiometric calcium indicator YC3.60 were glued to the surface of an agarose pad and placed in a custom chamber for imaging and stimulus delivery. The YC3.60 indicator was excited with 435 nm light from a monochromator (Till Photonics). YC3.60 blue and yellow emissions were separated using a Photometrics DV2 image splitter and simultaneously imaged by projecting two images onto the detector of an EM-CCD camera (Andor). Stimulus delivery and image acquisition was controlled by the Live Acquisition
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software package (Till Photonics) and calcium signals were analyzed and plotted using custom scripts written for Matlab (Mathworks). S. marcescens avoidance assays 100 μL of S. marcescens Db10 culture was spotted onto 6 cm NGM plates and grown overnight at room temperature. The following day, 20–25 adult hermaphrodites were placed on the center of the lawn. After 16–24 hours at 20° C, animals were scored as either off the lawn or on the lawn/bordering. A dwelling index was then calculated as shown in Fig. 4A. The same method was used to score dwelling indices of animals on E. coli OP50 and S. marcescens Db1140 lawns. Assaying responses to S. marcescens odorant
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To obtain S. marcescens odorant we aspirated the headspace above stationary phase cultures of S. marcescens using a 60 mL syringe. CO2 in the headspace was measured using an Alphasense IRC-A1 NDIR CO2 transmitter, and samples were diluted with room air to 1% CO2. Bacterial odorant or artificial atmospheres were flowed into a covered 6 cm NGM plate seeded with OP50 at 1 mL/minute for 1 hour using a syringe pump. Following this treatment, the number of animals on/bordering the lawn and those off the lawn were counted to compute the dwelling index. Statistical analysis
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Standard errors and P values were calculated using GraphPad Prism. P values of avoidance and dwelling indices were calculated using a Mann-Whitney test. P values for comparisons of transgene expression were calculated using Fisher’s exact test. All P values were corrected for multiple comparisons. Supplemental Table 2 contains P values for comparisons denoted in the figures.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments This work was supported by NIH grants R01GM098320 and R01GM113182, The Edward Mallinckrodt Jr. Foundation and The Pew Scholars Program. We thank Dennis Kim, Roger Pocock, Christine Li and Cori Bargmann for reagents, Jeremy Nance, Jessica Treisman and the Ringstad lab for helpful comments and discussions, Luis Martinez-Velazquez for assistance with genome sequence analysis, Rouzbeh Mashayekhi for plasmid construction and Tugba Colak-Champollion for isolation of two pmk-3 alleles. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
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Figure 1. The p38 MAP kinase PMK-3 is required for proper gene expression by chemosensory BAG neurons. A
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Overlays of differential interference contrast (DIC) and fluorescence micrographs of wildtype and pmk-3(wz31) mutants carrying a marker of differentiated BAG cells, Pgcy-33::GFP. White arrows indicate the BAG neuron nucleus. B. Structure of the pmk-3 locus showing four alleles isolated by our screen - wz27, wz31, wz34 and wz36 – and a deletion allele ok169. C. Expression of BAG neuron markers in the wild type and pmk-3 mutants, normalized to wild-type levels. Promoters from the indicated genes were used to drive expression of either GFP or mCherry (see methods). D. Percent animals expressing Pflp-17::GFP in the wild type, pmk-3(wz31) and pmk-3(wz31) mutants expressing pmk-3 specifically in BAG. Data from three independent transgenic lines are shown. E. Percent animals expressing Pflp-17::GFP in the wild type and pmk-3(wz31) mutants carrying a transgene for heat-shock-inducible expression of pmk-3 (Phsp-16.41::pmk-3) that had been heat-shocked at the indicated developmental stages. Bar graph data are plotted as means ± SEM. N ≥ 20 for all measurements. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons. P values for these and other comparisons in subsequent figures are presented in Supplementary Table 1.
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Author Manuscript Figure 2. PMK-3 is required for function of the CO2-sensing BAG neurons
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A. A schematic of a behavior assay for BAG neuron function. B. CO2-avoidance indices of the wild type, gcy-9(tm2816), pmk-3(ok169), pmk-3(wz31), pmk-1(km25), pmk-2(qd284) and pmk-3(wz31) mutants carrying a BAG cell-specific rescuing transgene. C. CO2-avoidance indices of the wild type, and pmk-3(wz31) mutants carrying a transgene for heat-shockinducible expression of pmk-3 (Phsp-16.41::pmk-3) that had been heat-shocked at the indicated developmental stages. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Author Manuscript Author Manuscript Figure 3. The TAK homolog MOM-4 and the TLR TOL-1 are required for the function of CO2sensing BAG neurons
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A. CO2-avoidance indices of the wild type, dlk-1(ju475), sek-1(km4), mom-4(gk563) and mom-4(gk563) mutants carrying a transgene for BAG-specific expression of mom-4. B. CO2-avoidance assays of the wild type, tol-1(nr2033) and tol-1(nr2013) mutants, animals carrying a transgene for BAG cell-specific RNAi of tol-1 and tol-1(nr2013) mutants carrying a transgene for BAG cell-specific expression of tol-1. C. Fluorescence micrograph showing a single optical section of an animal carrying a Ptol-1::GFP transcriptional reporter and the BAG cell-specific reporter Pflp-17::dsRed. This section did not capture strong GFP expression in URY neurons (see Supplemental Figure 3). D. Expression of transgene markers of differentiated BAG neurons in the wild type and animals carrying a transgene for BAG cell-specific RNAi of tol-1, normalized to wild-type levels, which are re-plotted from Figure 1C. Promoters from the indicated genes were used to drive expression of either GFP or mCherry. For these bar graphs data are plotted as means ± SEM. N ≥ 20 for all measurements. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Figure 4. PMK-3 functions in a Toll-like receptor signaling pathway
A. CO2-avoidance indices of the wild type, tol-1(nr2013), mom-4(gk563) and pmk-3(wz31) mutants with and without a transgene driving mkk-4(gf) specifically in BAG cells. B. Avoidance indices of pmk-3(wz31) mutants and tol-1(RNAi knockdown specifically in BAG cells) animals with and without the ikb-1 deletion allele nr2027. C. CO2-avoidance indices of the wild type and two other TLR signaling mutants: trf-1(nr2014) and pik-1(gk345127) with and without a transgene driving mkk-4(gf) specifically in the BAG neurons. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons. D. A model of TLR signaling that acts in BAG cells to promote CO2-sensing.
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Author Manuscript Figure 5. Toll-like receptor signaling is required for chemotransduction in CO2-sensing BAG neurons
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A. Calcium responses of wild-type BAG neurons to CO2 stimuli. B. Calcium responses of tol-1(nr2013) mutant BAG cells to CO2 stimuli. Responses to 10% CO2 were measured using the ratiometric calcium indicator YC3.60. Individual responses are plotted in light gray, while the black line shows average responses. In each plot stimulus presentation is indicated as a black bar. N = 11 for wild-type measurements and N = 15 for tol-1 mutant measurements. C. Peak ratio changes of wild-type and tol-1 mutant BAG cells. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Author Manuscript Author Manuscript Figure 6. C. elegans avoidance of the pathogen S. marcescens requires the CO2-sensing modality of BAG neurons
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A. A schematic of pathogen-avoidance behavior and the calculation of a dwelling index. B. Dwelling indices for the wild type, tol-1(nr2033), pmk-3(wz31), pmk-3(wz31) mutants carrying a BAG-specific rescuing construct, tol-1 RNAi in BAG cells and tol-1(nr2033) mutants carrying a BAG cell-specific rescuing construct on non-pathogenic E. coli lawns and S. marcescens lawns. C. Dwelling indices of animals lacking BAG cells, gcy-9(tm2816) mutants, which fail to detect CO2 (14), and gcy-31(ok296) mutants, which fail to detect hypoxia (17), on E. coli and S. marcescens lawns. D. Dwelling indices of the wild type on lawns of living or heat-killed S. marcescens (Db10), or the attenuated strain Db1140. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Figure 7. Microbial CO2 acts together with other factors to trigger avoidance behavior
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A. Schematic showing delivery of S. marcescens odorant to animals dwelling on nonpathogenic E. coli. B. Dwelling indices of the wild type and gcy-9(tm2816) mutants on E. coli after one hour of exposure to air, artificial atmospheres containing CO2, and S. marcescens odorant containing 1% CO2. C. Calcium responses of wild-type BAG neurons to 1% CO2 (N = 10) or S. marcescens odorant containing 1% CO2 (N = 10), and responses of gcy-9(tm2816) mutant BAG cells to S. marcescens odorant containing 1% CO2, (N = 8). Individual responses are plotted in light gray, while the black line shows average responses. In all plots stimulus presentation is indicated as a black bar. D. A model for the role of BAG neurons in pathogen-evoked avoidance behavior. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Curr Biol. Author manuscript; available in PMC 2016 August 31. Published in final edited form as: Curr Biol. 2015 August 31; 25(17): 2228–2237. doi:10.1016/j.cub.2015.07.037.
Toll-like receptor signaling promotes the development and function of sensory neurons required for a C. elegans pathogenavoidance behavior Julia P. Brandt1 and Niels Ringstad1 1The
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Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, Molecular Neurobiology Program, and Department of Cell Biology, NYU Langone Medical Center, New York, NY 10016
Summary
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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 TOL-1 signaling is required in the chemosensory BAG neurons for pathogen avoidance, where it regulates gene expression and is necessary for BAG 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.
Introduction
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Toll-like receptors (TLRs) are integral membrane proteins found in many metazoans that have remained linked to host-pathogen interactions throughout evolution [1]. Drosophila Toll, the first characterized TLR, was discovered as a factor required for dorsoventral patterning of the early embryo [2]. Subsequently, it was found that Toll mutants are highly susceptible to infection and have defects in pathogen-induced expression of antifungal peptides [3]. Mammalian TLRs were found to play similar roles in immune signaling [4, 5], where they promote the inflammatory response and expression of pathogen-defense molecules such as interferons [6]. In phylogenetically distant animals, therefore, TLRs constitute a first line of defense against pathogens during exposure and infection. In both mammals and insects, TLRs play instructive roles in immune signaling. Drosophila Toll is activated by pathogens when extracellular receptors for pathogen-derived cues
Corresponding author is Niels Ringstad, [email protected]. Author contributions JPB and NR designed experiments, executed experiments, interpreted data and wrote this manuscript. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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initiate a proteolytic cascade that activates latent Spätzle, a BMP-like ligand for Toll [7]. Vertebrate TLRs directly bind pathogen-derived cues such as lipopolysaccharide and viral nucleic acids [8]. Although the mechanisms by which TLRs are activated in response to infection in mammals and insects differ, once liganded, TLRs initiate conserved signaling pathways that drive inflammatory and immune responses [9]. Canonical TLR signaling activates kinase signaling cascades and negatively regulates the transcriptional inhibitor IκB/Cactus. Downstream of IkB is a master regulator of the inflammatory response, NFκB/ Relish, which drives expression of pathogen-defense molecules such as antimicrobial peptides in insects and interferons and cytokines in mammals [10].
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In contrast to TLR signaling in mammals and flies, little is known about this pathway in C. elegans. C. elegans uses conserved signaling pathways to mediate immunity to a broad spectrum of pathogens [11, 12, 13]. However, the sole TLR of C. elegans, TOL-1, has not been linked to these pathways. Instead, TOL-1 has been shown to be required for a behavioral response to the pathogenic microbe Serratia marcescens [14], although how TOL-1 functions in pathogen-avoidance is unknown. That TOL-1 is required for an avoidance behavior to pathogenic microbes suggests that in nematodes, TLR signaling might function in the nervous system.
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Here we show that TOL-1 and associated signaling molecules are required in a pair of chemosensory neurons – the BAG neurons - for avoidance of Serratia marcescens. TLR signaling regulates gene expression in BAG neurons and is required for their sensory function. BAG neurons are gas-sensing neurons that are activated by hypoxia and CO2 [15, 16, 17]. We find that their CO2-sensing modality is required for avoidance of Serratia marcescens. Together, our data indicate that CO2 produced by microbial respiration is a molecular cue used by C. elegans to avoid metabolically active pathogens. In C. elegans, therefore, a TLR functions in microbe-sensing through a distinct and, to our knowledge, previously unknown mechanism: by promoting the development of neurons that are required for sensory detection of microbes.
Results The p38 MAP kinase PMK-3 is required for proper gene expression by chemosensory BAG neurons
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C. elegans BAG neurons are ciliated chemosensory neurons that sense increases in ambient CO2 [15, 16]. Studies of BAG neurons have identified several factors required for CO2sensing: the GCY-9 receptor-type guanylate cyclase, which is specifically expressed in BAG cells and likely functions as a CO2 receptor, and cyclic nucleotidegated (CNG) ion channels comprising TAX-2 and TAX-4 subunits [18, 19, 20]. Transcription factors required for the specification and differentiation of BAG neurons have been identified [21, 22, 23] but our knowledge of gas-sensing gene regulation remains incomplete. To better understand mechanisms that function in the development of BAG cells and promote CO2chemosensation, we performed a reporter-based screen for mutants with abnormal expression of a marker for differentiated BAG neurons (Supplemental Fig. 1). One set of non-complementing mutations isolated by the screen caused reduced expression of a BAG cell marker, although the BAG cell itself was present and easily identified on the basis of its Curr Biol. Author manuscript; available in PMC 2016 August 31.
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characteristic position and nuclear morphology (Fig. 1A). This complementation group consisted of four alleles of the p38 Mitogen Activated Protein (MAP) kinase gene pmk-3 (Fig. 1B).
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We tested a panel of genes expressed by BAG neurons for regulation by pmk-3. In addition to several neuropeptide genes [24], BAG neurons express genes that are required for their chemosensory functions [17, 18]. We found that a subset of these genes were dysregulated in pmk-3 mutants (Fig. 1C). Expression of the neuropeptide gene flp-17 (Supplemental Fig. 1) and the guanylate cyclase gene gcy-33 (Fig. 1A) was greatly reduced in BAG neurons and expression of the cGMP-gated channel gene tax-2 was partly reduced (Fig. 1C). By contrast, expression of the guanylate cyclase gene gcy-31 was increased in pmk-3 mutant BAG neurons. gcy-9, which encodes a guanylate cyclase required for CO2-sensing, the neuropeptide genes flp-10 and flp-19, and the CNG channel gene tax-4 were unaffected by pmk-3 mutation (Fig. 1C). BAG cell-specific expression of pmk-3 restored expression of the Pflp-17::GFP reporter in pmk-3(wz31) mutants (Fig. 1D), indicating that pmk-3 acts cell autonomously to promote the expression of BAG-specific genes. pmk-3 therefore, is required for normal expression of some but not all markers of a BAG neuron fate. In addition to abnormal gene expression, pmk-3 mutants exhibited a low penetrance morphological defect. 30% of BAG cell axons in pmk-3 mutants failed to form proper commissures in the nerve ring (Supplemental Fig. 2B) suggesting that in addition to regulating gene expression, pmk-3 plays a role in axon morphogenesis in BAG cells.
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To determine when pmk-3 acts to promote expression of BAG neuron fate markers, we carried out heat-shock experiments to transiently induce pmk-3 expression in embryos, larvae and adults. Inducing pmk-3 expression in embryos and larvae was sufficient to restore expression of a Pflp-17::GFP reporter in BAG cells (Fig. 1E). However, induction of pmk-3 expression in the adult failed to restore transgene expression, indicating that PMK-3 is required during embryonic and larval stages to promote the expression of a BAG cell fate. While these data show that pmk-3 is required during development, we note that it is possible that pmk-3 is also required post-developmentally in the BAG neurons.
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We next tested whether pmk-3 mutation affects BAG neuron function by measuring the CO2-avoidance behavior of pmk-3 mutants. BAG neurons are directly activated by CO2 and required for chemotaxis down a CO2 gradient (Fig. 2A). pmk-3 mutants were severely defective in CO2-avoidance and failed to navigate down a steep (0–10%) CO2 gradient (Fig. 2B and Supplemental Fig. 3). Mutation of pmk-3 caused defects in CO2 avoidance assays as severe as loss of the CO2-receptor gene gcy-9 (Fig. 2B). pmk-3 is in an operon with two other p38 MAPK genes, pmk-1 and pmk-2 [25]. pmk-1 and pmk-2 function in neuronal development and stress responses [11, 26]. To determine whether pmk-3 mutation affected BAG cells by altering expression of the entire operon, we tested pmk-1 and pmk-2 mutants for CO2 avoidance and found them to be wild-type in this assay (Fig. 2B). Furthermore, expression of only pmk-3 in BAG cells restored CO2 avoidance behavior to pmk-3 mutants. Therefore, the abnormally differentiated BAG neurons of pmk-3 mutants cannot mediate CO2-sensing.
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We used heat-shock-mediated induction of pmk-3 expression to determine whether pmk-3 gene function was required for BAG cell function at the same time that it is required for gene expression by BAG neurons. Inducing pmk-3 expression in embryos and larvae restored CO2 avoidance behavior to pmk-3 mutant adults (Fig. 2C). By contrast, induction of pmk-3 expression in adults failed to restore their CO2 avoidance behavior. These data indicate that PMK-3 functions during the same window of developmental time to promote both the proper gene expression and function of BAG cells. Furthermore, these data show that pmk-3 plays a permissive role in CO2-sensing by acting during BAG neuron development as opposed to an instructive role during sensory signaling. PMK-3 functions with the Toll-like receptor TOL-1 and TLR signaling factors to promote BAG neuron function
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Because p38 MAPKs are canonically regulated by a kinase cascade driven by MAP kinase kinase kinases (MAP3Ks), we sought to identify a MAP3K that acts upstream of PMK-3 in BAG cells [27]. The C. elegans genome encodes three such kinases: DLK-1, NSY-1 and MOM-4. DLK-1 is known to activate PMK-3 in motor neurons to promote synaptic development and axonal regeneration [28, 29]. However, dlk-1 mutants were wild-type for CO2-sensing (Fig. 3A), indicating that DLK-1 does not regulate PMK-3 to promote BAG neuron function. Although we could not measure CO2 avoidance by nsy-1 mutants, which are uncoordinated, we found that mutants for its downstream partner SEK-1 [30] were wildtype for CO2 avoidance (Fig. 3A). mom-4 mutants, however, were strongly defective in CO2 avoidance. BAG cell-specific expression of mom-4 rescued this defect, suggesting that MOM-4 functions with PMK-3 in BAG cells (Fig. 3A).
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MOM-4 is a homolog of TGF-beta-activated kinase (TAK), which is regulated by a small number of cell-surface receptors, including TLRs [31]. We tested the sole C. elegans TLR, TOL-1, for a role in BAG neurons. A partial loss-of-function allele (nr2033) and a null allele (nr2013) of tol-1 each caused CO2-avoidance defects (Fig. 3B). BAG-specific RNAi knockdown of tol-1 caused a defect as severe as that of a tol-1 null mutant, demonstrating that TOL-1 is required in BAG neurons for their function (Fig. 3B). Also, BAG cell-specific expression of tol-1 in tol-1(nr2013) mutants restored CO2 avoidance behavior to tol-1 mutants, demonstrating that tol-1 expression in BAG neurons can account for a significant amount of its function in CO2-avoidance behavior. tol-1 expression was previously reported in sensory neurons but its expression in BAG cells had not been documented [14, 32, 33]. We made a Ptol-1::GFP reporter carrying 5 kilobases of upstream sequences from the tol-1 translational start site. As previously reported, we observed robust expression by the URY sensory neurons (Supplemental Fig. 4). However, we also observed expression by BAG cells (Fig. 3C), consistent with our observation that tol-1 is required in these cells for their function. We used fosmid-based reporter transgenes to determine the expression of pmk-3 and mom-4 and found that these genes were also expressed by BAG cells (Supplemental Fig. 5A and B). Like pmk-3 mutant cells, we found that 30% of tol-1 mutant BAG cells exhibited abnormal axonal commissures (Supplemental Fig. 2C). To further determine the similarities between tol-1 mutants and pmk-3 mutants, we tested whether tol-1 promotes gene expression in BAG
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neurons as does pmk-3. We found that like pmk-3 mutation, loss of tol-1 function reduced expression of gcy-33 and increased expression of gcy-31, suggesting that pmk-3 and tol-1 function together to regulate these BAG cell genes (Fig. 3D). We could not detect a role for tol-1 in regulating expression of flp-17 or tax-2 reporters, suggesting that pmk-3 has gene regulatory functions that are independent of tol-1. Furthermore, we found that tol-1 is required for proper expression of flp-19, a neuropeptide gene that is not regulated by pmk-3 (Fig. 1C), indicating that tol-1 can regulate gene expression independently of pmk-3 (Fig. 3D). Taken together these data suggest that in addition to having distinct functions, pmk-3 and tol-1 coordinately regulate expression of a subset of genes in BAG neurons.
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To determine whether TOL-1, MOM-4 and PMK-3 function together in a signaling pathway we performed a series of genetic interaction tests using constitutively active MKK-4, a kinase that can directly activate PMK-3 and bypass any requirement for endogenous PMK-3 regulators [28]. MKK-4(gf) expression suppressed the CO2-avoidance defects of mom-4 and tol-1 mutants but had no effect on pmk-3 mutants (Fig. 4A), consistent with the hypothesis that TOL-1 and MOM-4 act upstream of PMK-3 in BAG neurons. In other contexts, TLR signaling promotes gene expression by inhibiting IκB, an inhibitor of gene expression [34]. We found that deletion of ikb-1, which encodes an IκB-like protein [14], suppressed the CO2 avoidance defect caused by loss of either tol-1 or pmk-3 function (Fig. 4B). These data suggest that TLR signaling in BAG neurons involves a canonical and evolutionarily conserved regulator of transcription, IκB. The C. elegans genome also encodes two other homologs of TLR signaling genes: pik-1, which encodes the Interleukin Receptor Activated Kinase (IRAK) homolog, and trf-1, which encodes a homolog of the TNF-receptor associated factor (TRAF) [14]. We tested whether pik-1 and trf-1 mutants were defective for CO2 avoidance behavior and found that each of these mutants exhibited severe behavioral defects (Fig. 4C). Furthermore, the defects of trf-1 and pik-1 were partially bypassed by activated MAPK signaling specifically in the BAG neurons (Fig. 4C). Together, our data support a role for TLR signaling in promoting the development and function of the chemosensory BAG neurons. Figure 4D depicts a pathway model for TOL-1 signaling that integrates our observations with established models of mammalian TLR signaling [9]. Loss of TLR signaling disrupts the physiology of chemosensory BAG neurons
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We next sought to determine whether behavioral defects of TLR signaling mutants might be caused by defective sensory transduction. We used in vivo calcium imaging to determine whether TLR signaling was required for activation of BAG neurons by CO2 stimuli [22]. Wild-type BAG neurons showed robust calcium responses to CO2 (Fig. 5A). By contrast, tol-1 mutants had blunted CO2 responses that were, on average, 33% of the wild-type response (Fig. 5B and C). These data indicate that the BAG neurons of tol-1 mutants have a primary defect in sensory detection of CO2, suggesting that some genes regulated by TLR signaling function in sensory transduction. Chemosensory BAG neurons are the site of TOL-1 action in microbe-sensing Previously, TOL-1 was shown to be required for a pathogen-avoidance behavior [14] but its site and mechanism of action were unknown. Does a role for TOL-1 in BAG cell development and function relate to its role in pathogen avoidance? To measure pathogen
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avoidance we used a simple behavioral assay in which the distribution of animals in an arena is measured after exposure either to pathogenic or to non-pathogenic microbes (Fig. 6A). As previously reported [14], C. elegans exposed to non-pathogenic bacteria rarely foraged away from the bacterial lawn while C. elegans exposed to the pathogen Serratia marcescens dispersed (Fig. 6B). By contrast, tol-1 mutants did not disperse when exposed to S. marcescens (Fig. 6B). We found that the TLR signaling pathway that promotes BAG cell development and function is also required for this pathogen avoidance behavior. Like tol-1 mutants, pmk-3 mutants failed to avoid S. marcescens (Fig. 6B). Importantly, loss of tol-1 function specifically in BAG cells recapitulated the pathogen avoidance defect of tol-1 mutants, and BAG cell-specific rescue of pmk-3 and tol-1 restored pathogen avoidance behavior to the corresponding mutants (Fig. 6B). Therefore, BAG cells are a site of tol-1 action in both CO2-avoidance and pathogen avoidance, suggesting a link between CO2sensing and microbe-sensing.
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To determine whether CO2-sensing per se is required for pathogen avoidance, we tested CO2-blind gcy-9 mutants [18]. gcy-9 mutants were as defective in avoidance of S. marcescens as tol-1 and pmk-3 mutants (Fig. 6C). In addition to CO2, BAG neurons sense hypoxia through a distinct mechanism that requires the heteromeric GCY-31/GCY-33 guanylate cyclases [17]. We tested gcy-31 mutants and found that they robustly avoided pathogen (Fig. 6C), indicating that hypoxia-sensing by BAG neurons is not required for S. marcescens avoidance. Defects in the CO2-sensing modality of BAG cells, therefore, account for the pathogen-avoidance defects of TLR signaling mutants. We further found that C. elegans did not avoid heat-killed S. marcescens or the attenuated S. marcescens strain Db1140 (Fig. 6D). These data suggest that CO2 produced by the respiration of living microbes is an important factor in avoidance behavior.
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BAG neurons detect CO2 as a contextual cue for pathogen-avoidance
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We observed that pathogenic and non-pathogenic microbes used in our studies evolved comparable amounts of CO2 under conditions used for behavioral studies (Supplemental Fig. 6), making it unlikely that the lawn leaving behavior evoked by S. marcescens is simply another manifestation of CO2 avoidance behavior. Is detection of microbial CO2, then, part of a mechanism that triggers pathogen avoidance? To test this possibility we exposed animals dwelling on non-pathogenic E. coli to uniform concentrations of either S. marcescens odorant or to artificial atmospheres containing various concentrations of CO2 and measured lawn-leaving behavior (Fig. 7A). S. marcescens odorant that contained 1% CO2, caused lawn leaving by the wild type but not by CO2-blind gcy-9 mutants, BAGablated animals, or mutants of the TLR pathway (Fig. 7B and Supplemental Fig. 7). Furthermore, BAG cell-specific expression of pmk-3 in pmk-3 mutants restored lawn leaving in response to S. marcescens odorant (Supplemental Fig. 7). Artificial atmospheres containing the same amount of CO2 as S. marcescens odorant did not induce lawn leaving, but a higher concentration of CO2 (10%) did, indicating that intense BAG cell activation is sufficient to drive lawn leaving to some extent. Because there is no CO2 gradient under these experimental conditions, these data indicate that CO2 is a necessary part of a microbederived signal that triggers dispersal rather than a cue used for navigation away from the bacterial lawn.
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Strikingly, S. marcescens odorant was more effective than CO2 concentration-matched artificial atmospheres in triggering lawn leaving (Fig. 7B). To determine whether this was because S. marcescens odorant contains a component that activates BAG neurons independently of CO2 we compared the ability of S. marcescens odorant and CO2-matched artificial atmospheres to activate BAG cells in vivo. S. marcescens odorant and CO2matched artificial atmospheres evoked indistinguishable responses in BAG cells (Fig. 7C), indicating that BAG cells principally detect the CO2 component of S. marcescens odorant. Importantly, gcy-9 mutant BAG cells failed to respond to S. marcescens odorant, providing further evidence that BAG neurons detect microbially produced CO2 (Fig. 7C).
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Our observation that detection of microbially produced CO2 is necessary for pathogen avoidance but that CO2 itself is not able to effectively trigger the behavior support a model in which CO2 acts as a contextual cue that functions together with pathogen-specific factors to evoke a behavioral response. Therefore, detection of microbial respiration is required for C. elegans to respond to the detection of pathogen-specific cues (Fig. 7D). Our data further suggest that pathogen-specific cues are sensed not by BAG neurons but by other chemosensory neurons that collaborate with BAG neurons to trigger avoidance behavior (Fig. 7D).
Discussion
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The ability to detect and respond to pathogens is critical for all animals. C. elegans, which are bacteriovores, must differentiate pathogens from food. Pathogenic microbes express many factors that are inherently attractive to C. elegans. For example, our data demonstrate that heat-killed S. marcescens is attractive to C. elegans as are non-pathogenic E. coli. Others have demonstrated that näive C. elegans are more attracted to pathogenic bacteria than they are to E. coli [35]. Pathogen-avoidance, therefore, requires that the valence of a complex suite of sensory stimuli produced by pathogenic microbes switch from attractive to aversive. Our data indicate that BAG neurons play an important role in this process by sensing microbially-produced CO2. In the case of S. marcescens avoidance, our data demonstrate that the absence or presence of a product of microbial respiration – CO2 - tips the balance from attraction to aversion. It will be interesting to determine whether the same mechanism functions in behavioral responses to other pathogenic microbes known to infect C. elegans [36].
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We found evidence that S. marscesens odorant contains factors that synergize with CO2 to trigger avoidance behavior. We imagine that a sensory circuit involving BAG neurons and other olfactory sensory neurons decodes the complex olfactory stimulus that is bacterial odorant. Such a circuit would likely collaborate with other sensory systems to determine how C. elegans responds to microbes. For example, C. elegans has been shown to sense an aversive gustant - the surfactant serrawettin - produced by S. marcescens [32]. Also, pathogen-derived metabolites such as phenazine-1-carboxamide and pyochelin exert effects on C. elegans behavior over long periods of time by inducing expression of a TGF-beta-like factor, which in turn alters the function of oxygen-sensing circuits [37]. It is therefore likely that C. elegans uses a combination of olfactory, gustatory, gas-sensing and neuromodulatory
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modalities over multiple time scales to sculpt behavioral responses to environmental microbes. Which raises a question: why use CO2-sensing neurons at all in a pathogen-detection mechanism? Both pathogenic and non-pathogenic microbes respire aerobically and produce CO2, so the presence of this factor does not indicate whether the microbes that produced it are harmful. We propose that by monitoring microbial CO2 production, C. elegans is able to determine whether environmental microbes are metabolically active. By integrating a measure of microbial metabolism into mechanisms that detect pathogen-specific cues, C. elegans might be able to reserve the option to feed on attenuated or dead microbes that would otherwise be pathogenic, thereby maximizing its opportunity to capture food resources in its environment.
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Strikingly, the BAG sensory neurons, which are required for an innate behavioral response of C. elegans to the pathogenic microbe S. marcescens, require TLR signaling for proper gene expression and function. We found that TLR signaling is required during development to regulate gene expression, and our data strongly suggest that TLR target genes are required for the function of chemosensory BAG neurons. TLR signaling in C. elegans, therefore, functions in microbe-sensing in an unexpected manner: by promoting the differentiation of chemosensory neurons that function in a sensory system that evaluates the qualities of environmental microbes to instruct foraging behavior. Unlike TLRs in insect and vertebrate innate immunity, where TLRs are activated acutely by the presence of pathogen-derived factors, C. elegans TOL-1 signaling plays a permissive role in pathogen-detection. In fact, our data show that it is insufficient to restore function to this pathway post-developmentally and suggest that there is a critical period of development during which TOL-1 signaling in BAG neurons establishes the program of gene expression required for cell function.
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C. elegans lacks several components of the TLR signaling pathways that mediate innate immunity in insects and mammals, including the Myd88 adapter, the transcription factor NF-κB and its regulatory IκB kinases (IKKs) [14, 38]. We propose that TOL-1 signaling uses different molecules to couple TLR activation to kinase signaling cascades and IκB, and that novel transcriptional regulators will act downstream of TOL-1 to promote expression of neuronal genes. Interestingly, our data indicate that the nr2033 mutant receptor, which lacks much of its cytoplasmic domain including the Toll-Interleukin Receptor homology (TIR) domain, still retains some activity. This suggests that TOL-1 can couple to intracellular signaling pathways in BAG neurons through an unconventional i.e. TIR domainindependent mechanisms. We speculate that counterparts of these mechanisms exist in mammals, where they might define new branches of TLR signaling in innate immunity or link TLR signaling to biological processes such as differentiation of specific cell types during development.
Experimental Procedures Strains All strains and their genotypes used in this study are listed in Supplemental Table 1. C. elegans strains were grown under standard conditions [39] at 20°C except tol-1(nr2013)
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mutants, which were grown at 15°C to promote viability and shifted to 20°C one day prior to behavioral assays. Plasmids Plasmids were constructed by standard methods. Transcriptional reporters were made by cloning 5′ regulatory sequences of genes into the plasmid pPD95.75. cDNA expression constructs were made using the plasmid pPD49.26 [40]. pmk-3 and mom-4 reporter transgenes were translational fusions with GFP made by fosmid recombineering [41]. Plasmids used in this study are: pJB203 Pflp-17::pmk-3; pJB254 Ptax-2::dsRed pJB227 Pflp-17::mom-4; pJB252 Pflp-17::tol-1; pJB237 Pflp-17::tol-1sense; pJB234 Pflp-17::tol-1antisense; pJB24: Pflp-17::mkk-4(gf); pJB252 Ptol-1::GFP; pJB134 Pflp-17::dsRed ; pRM60 Pflp-17::cameleon; pJB240 mom-4::GFP; pNR519 pmk-3::GFP; pJB221 Phsp-16.41::pmk-3; pJB258 Pflp-19::GFP
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Genetic screen for genes regulating BAG cell development The BAG-neuron-specific reporter transgene ynIs64[Pflp-17::GFP] was used to isolate mutants with defects in BAG neuron development. Synchronized animals carrying ynIs64[Pflp-17::GFP], were grown to mid-L4 stage and treated with 47 mM ethyl methane sulfonate (EMS) as described [39]. Animals were distributed onto 10 cm NGM plates seeded with E. coli OP50 and screened in the F2 generation for abnormal GFP expression. Screening was carried out on a Leica M165 FC fluorescence dissecting microscope. Mapping and cloning of pmk-3 alleles
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An initial round of screening identified the non-complementing alleles wz27 and wz31. High resolution SNP-mapping of wz31 was performed by crossing wz31 mutants to the polymorphic strain CB4856 and identifying crossovers using restriction fragment polymorphisms (snip-SNPs) [42]. This experiment placed wz31 in a 10 Mb interval on LG IV. Whole-genome sequencing of wz31 and wz27 mutants revealed that both mutants carry coding mutations in pmk-3: wz31 contains a G->A mutation predicted to change Glu320 to Lys, and wz27 contains a C->T mutations predicted to change Gln339 to an Ochre stop. Further screening and complementation tests identified two additional alleles of pmk-3: wz34 and wz36. Sanger sequencing revealed that wz34 is a C->T mutation predicted to change Arg251 to Cys, and wz36 is a G->A mutation predicted to change Glu297 to Lys. Transgene expression analysis
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Animals were mounted on 2% agarose pads made in M9 medium and immobilized with 30 mM sodium azide. Reporter transgene expression was quantified on a Zeiss AxioImager M2 upright microscope equipped with an EM-CCD camera (Andor) using a 20× 0.8 NA air objective. For each genotype 20–30 animals were imaged and mean pixel values in a 50 pixel-radius circular region of interest centered in the BAG soma were measured using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2011). Pixel values were then normalized to the mean of measurements of wild-type cells and plotted.
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Heat-shock experiments
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Mutant pmk-3(wz31) animals carrying a Phsp16.41::pmk-3 transgene were placed in a 30°C water bath for three hours. After heat-shock, animals were placed at 20°C to recover. Heatshocked embryos and larvae were assayed when they reached adulthood, while heat-shocked adults were assayed 24 hours later. Separate heat-shock experiments were carried out for gene expression and behavior experiments. For gene expression assays, the percentage of animals expressing Pflp-17::GFP after heat-shock was counted on a Leica M165 FC fluorescence dissecting microscope. For CO2 behavior experiments, animals were assayed as described below. CO2 avoidance assays
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20–25 adult hermaphrodites were selected from a staged culture and placed on the surface of an unseeded 10 cm NGM plate. In experiments measuring behavior of transgenic animals, non-transgenic siblings were used as controls. Otherwise, animals were selected blindly from a strain for inclusion in behavioral assays. A custom-made chamber fitted with inlets for air and 10% CO2 was placed on the plate confining the animals to an arena. A thin layer of glycerol had been applied to the bottom of the chamber to seal the chamber to the plate surface and confine animals. Gas mixes were pushed into the chamber at 1.5 mL/minute using a syringe pump. After 30 minutes, the number of animals on the air and CO2 sides of the chamber were counted. Avoidance indices (A.I.) were computed according to the following equation:
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Cell-specific RNAi of tol-1 in BAG cells Two plasmids were generated that used the flp-17 promoter to drive transcription of a 404 bp fragment of the tol-1 cDNA in either the sense and antisense directions. These constructs were co-injected at a concentration of 100 ng μl−1 each to generate an array that expresses dsRNA targeting tol-1 specifically in BAG cells. Confocal Microscopy Young adults were mounted on a 2% agarose pad made in M9 medium and anaesthetized with 30 mM sodium azide. Z stacks were obtained with a Zeiss LSM510 confocal microscope and maximum projection images were created using ImageJ. Imaging BAG cell-calcium responses in restrained animals
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In vivo calcium imaging was performed as previously described [20]. Briefly, animals expressing the ratiometric calcium indicator YC3.60 were glued to the surface of an agarose pad and placed in a custom chamber for imaging and stimulus delivery. The YC3.60 indicator was excited with 435 nm light from a monochromator (Till Photonics). YC3.60 blue and yellow emissions were separated using a Photometrics DV2 image splitter and simultaneously imaged by projecting two images onto the detector of an EM-CCD camera (Andor). Stimulus delivery and image acquisition was controlled by the Live Acquisition
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software package (Till Photonics) and calcium signals were analyzed and plotted using custom scripts written for Matlab (Mathworks). S. marcescens avoidance assays 100 μL of S. marcescens Db10 culture was spotted onto 6 cm NGM plates and grown overnight at room temperature. The following day, 20–25 adult hermaphrodites were placed on the center of the lawn. After 16–24 hours at 20° C, animals were scored as either off the lawn or on the lawn/bordering. A dwelling index was then calculated as shown in Fig. 4A. The same method was used to score dwelling indices of animals on E. coli OP50 and S. marcescens Db1140 lawns. Assaying responses to S. marcescens odorant
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To obtain S. marcescens odorant we aspirated the headspace above stationary phase cultures of S. marcescens using a 60 mL syringe. CO2 in the headspace was measured using an Alphasense IRC-A1 NDIR CO2 transmitter, and samples were diluted with room air to 1% CO2. Bacterial odorant or artificial atmospheres were flowed into a covered 6 cm NGM plate seeded with OP50 at 1 mL/minute for 1 hour using a syringe pump. Following this treatment, the number of animals on/bordering the lawn and those off the lawn were counted to compute the dwelling index. Statistical analysis
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Standard errors and P values were calculated using GraphPad Prism. P values of avoidance and dwelling indices were calculated using a Mann-Whitney test. P values for comparisons of transgene expression were calculated using Fisher’s exact test. All P values were corrected for multiple comparisons. Supplemental Table 2 contains P values for comparisons denoted in the figures.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments This work was supported by NIH grants R01GM098320 and R01GM113182, The Edward Mallinckrodt Jr. Foundation and The Pew Scholars Program. We thank Dennis Kim, Roger Pocock, Christine Li and Cori Bargmann for reagents, Jeremy Nance, Jessica Treisman and the Ringstad lab for helpful comments and discussions, Luis Martinez-Velazquez for assistance with genome sequence analysis, Rouzbeh Mashayekhi for plasmid construction and Tugba Colak-Champollion for isolation of two pmk-3 alleles. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
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Figure 1. The p38 MAP kinase PMK-3 is required for proper gene expression by chemosensory BAG neurons. A
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Overlays of differential interference contrast (DIC) and fluorescence micrographs of wildtype and pmk-3(wz31) mutants carrying a marker of differentiated BAG cells, Pgcy-33::GFP. White arrows indicate the BAG neuron nucleus. B. Structure of the pmk-3 locus showing four alleles isolated by our screen - wz27, wz31, wz34 and wz36 – and a deletion allele ok169. C. Expression of BAG neuron markers in the wild type and pmk-3 mutants, normalized to wild-type levels. Promoters from the indicated genes were used to drive expression of either GFP or mCherry (see methods). D. Percent animals expressing Pflp-17::GFP in the wild type, pmk-3(wz31) and pmk-3(wz31) mutants expressing pmk-3 specifically in BAG. Data from three independent transgenic lines are shown. E. Percent animals expressing Pflp-17::GFP in the wild type and pmk-3(wz31) mutants carrying a transgene for heat-shock-inducible expression of pmk-3 (Phsp-16.41::pmk-3) that had been heat-shocked at the indicated developmental stages. Bar graph data are plotted as means ± SEM. N ≥ 20 for all measurements. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons. P values for these and other comparisons in subsequent figures are presented in Supplementary Table 1.
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Author Manuscript Figure 2. PMK-3 is required for function of the CO2-sensing BAG neurons
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A. A schematic of a behavior assay for BAG neuron function. B. CO2-avoidance indices of the wild type, gcy-9(tm2816), pmk-3(ok169), pmk-3(wz31), pmk-1(km25), pmk-2(qd284) and pmk-3(wz31) mutants carrying a BAG cell-specific rescuing transgene. C. CO2-avoidance indices of the wild type, and pmk-3(wz31) mutants carrying a transgene for heat-shockinducible expression of pmk-3 (Phsp-16.41::pmk-3) that had been heat-shocked at the indicated developmental stages. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Author Manuscript Author Manuscript Figure 3. The TAK homolog MOM-4 and the TLR TOL-1 are required for the function of CO2sensing BAG neurons
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A. CO2-avoidance indices of the wild type, dlk-1(ju475), sek-1(km4), mom-4(gk563) and mom-4(gk563) mutants carrying a transgene for BAG-specific expression of mom-4. B. CO2-avoidance assays of the wild type, tol-1(nr2033) and tol-1(nr2013) mutants, animals carrying a transgene for BAG cell-specific RNAi of tol-1 and tol-1(nr2013) mutants carrying a transgene for BAG cell-specific expression of tol-1. C. Fluorescence micrograph showing a single optical section of an animal carrying a Ptol-1::GFP transcriptional reporter and the BAG cell-specific reporter Pflp-17::dsRed. This section did not capture strong GFP expression in URY neurons (see Supplemental Figure 3). D. Expression of transgene markers of differentiated BAG neurons in the wild type and animals carrying a transgene for BAG cell-specific RNAi of tol-1, normalized to wild-type levels, which are re-plotted from Figure 1C. Promoters from the indicated genes were used to drive expression of either GFP or mCherry. For these bar graphs data are plotted as means ± SEM. N ≥ 20 for all measurements. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Figure 4. PMK-3 functions in a Toll-like receptor signaling pathway
A. CO2-avoidance indices of the wild type, tol-1(nr2013), mom-4(gk563) and pmk-3(wz31) mutants with and without a transgene driving mkk-4(gf) specifically in BAG cells. B. Avoidance indices of pmk-3(wz31) mutants and tol-1(RNAi knockdown specifically in BAG cells) animals with and without the ikb-1 deletion allele nr2027. C. CO2-avoidance indices of the wild type and two other TLR signaling mutants: trf-1(nr2014) and pik-1(gk345127) with and without a transgene driving mkk-4(gf) specifically in the BAG neurons. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons. D. A model of TLR signaling that acts in BAG cells to promote CO2-sensing.
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Author Manuscript Figure 5. Toll-like receptor signaling is required for chemotransduction in CO2-sensing BAG neurons
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A. Calcium responses of wild-type BAG neurons to CO2 stimuli. B. Calcium responses of tol-1(nr2013) mutant BAG cells to CO2 stimuli. Responses to 10% CO2 were measured using the ratiometric calcium indicator YC3.60. Individual responses are plotted in light gray, while the black line shows average responses. In each plot stimulus presentation is indicated as a black bar. N = 11 for wild-type measurements and N = 15 for tol-1 mutant measurements. C. Peak ratio changes of wild-type and tol-1 mutant BAG cells. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Author Manuscript Author Manuscript Figure 6. C. elegans avoidance of the pathogen S. marcescens requires the CO2-sensing modality of BAG neurons
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A. A schematic of pathogen-avoidance behavior and the calculation of a dwelling index. B. Dwelling indices for the wild type, tol-1(nr2033), pmk-3(wz31), pmk-3(wz31) mutants carrying a BAG-specific rescuing construct, tol-1 RNAi in BAG cells and tol-1(nr2033) mutants carrying a BAG cell-specific rescuing construct on non-pathogenic E. coli lawns and S. marcescens lawns. C. Dwelling indices of animals lacking BAG cells, gcy-9(tm2816) mutants, which fail to detect CO2 (14), and gcy-31(ok296) mutants, which fail to detect hypoxia (17), on E. coli and S. marcescens lawns. D. Dwelling indices of the wild type on lawns of living or heat-killed S. marcescens (Db10), or the attenuated strain Db1140. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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Figure 7. Microbial CO2 acts together with other factors to trigger avoidance behavior
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A. Schematic showing delivery of S. marcescens odorant to animals dwelling on nonpathogenic E. coli. B. Dwelling indices of the wild type and gcy-9(tm2816) mutants on E. coli after one hour of exposure to air, artificial atmospheres containing CO2, and S. marcescens odorant containing 1% CO2. C. Calcium responses of wild-type BAG neurons to 1% CO2 (N = 10) or S. marcescens odorant containing 1% CO2 (N = 10), and responses of gcy-9(tm2816) mutant BAG cells to S. marcescens odorant containing 1% CO2, (N = 8). Individual responses are plotted in light gray, while the black line shows average responses. In all plots stimulus presentation is indicated as a black bar. D. A model for the role of BAG neurons in pathogen-evoked avoidance behavior. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.
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