A conserved neuronal DAF-16/FoxO plays an important role in conveying pheromone signals to elicit repulsion behavior in Caenorhabditis elegans AbstractAnimals use pheromones as a conspecific chemical language to respond appropriately to environmental changes. The soil nematode Caenorhabditis elegans secretes ascaroside pheromones throughout the lifecycle, which influences entry into dauer phase in early larvae, in addition to sexual attraction and aggregation. In adult hermaphrodites, pheromone sensory signals perceived by worms usually elicit repulsion as an initial behavioral signature. However, the molecular mechanisms underlying neuronal pheromone sensory process from perception to repulsion in adult hermaphrodites remain poorly understood. Here, we show that pheromone signals perceived by GPA-3 is conveyed through glutamatergic neurotransmission in which neuronal DAF-16/FoxO plays an important modulatory role by controlling glutaminase gene expression. We further provide evidence that this modulatory role for DAF-16/FoxO seems to be conserved evolutionarily by electro-physiological study in mouse primary hippocampal neurons that are responsible for glutamatergic neurotransmission. These findings provide the basis for understanding the nematode pheromone signaling, which seems crucial for adaptation of adult hermaphrodites to changes in environmental condition for survival. IntroductionPheromones serve as a chemical language through which organisms of the same species communicate in response to environmental changes, including the presence of stress, different sexes and food scarcity. The soil nematode Caenorhabditis elegans, one of the most genetically well-understood metazoans secretes pheromones termed daumones or ascaroside pheromones throughout the lifecycle. For instance, the nematode ascaroside pheromones have been known to signal worms to enter dauer phase, a non-aging state, under unfavorable growth conditions 1鈥?. In addition, these ascaroside pheromones (pheromones) are involved in diverse biological processes (e.g., sexual attraction, aggregation, and fungal traps) depending on their developmental stage (early larvae vs. adults) and sex (hermaphrodites vs. male) 6鈥?. Especially, it is well known that these pheromones act as a signal to the nematode that the surrounding environment is an unfavorable condition. Thus, when pheromone signals are recognized, young larvae enter the dauer phase, a non-aging state, for a long-term survival1,2,3,4. However, when adult hermaphrodites sense the pheromones, they elicit repulsion response as an initial behavioral output10. Although this repulsion serves as a signature of pheromone sensory process, molecular mechanism underlying pheromone sensory process after an initial perception remains less characterized.In search of pheromone signaling perception, there have been a few reports on the putative pheromone receptors that are demonstrated to mediate neuronal responses to ascr#1鈥? in ASK neurons11 (锘縎RBC64/66) or receptors in ASI neurons that respond to ascr#212 (DAF-37/38). Additionally, Bargmann group reported the identification of srg-36/37 genes encode G-protein-coupled receptors for ascr#5 (C3)13. Despite decades-long research on pheromone function, its perception, the molecular pathway in adult hermaphrodites that starts from an initial pheromone perception to elicit behavioral outputs as a repulsive response is not fully understood. Here, we show that pheromone sensory signals are likely conveyed through glutamatergic neurotransmission in which neuronal DAF-16/FoxO plays an important modulatory role.Results and DiscussionPerception of pheromone sensory signaling by GPA-3 via insulin/IGF-1 pathwayTo identify some molecular components involved in perception of pheromones, we screened G-protein subunit genes by assessing the chemotaxis index of C. elegans that had been exposed to three major ascaroside pheromones (daumones 1鈥? or ascr#1鈥?) as a measure of perception of pheromones8 (Fig.聽S1a). These three pheromones have been known as most abundant and highly active on pheromone functions among those identified so far3, 5, 14, 15. In our experiment, we used both plate-based assays and drop assays in our study. For instance, the plate-based chemotaxis assay10 was used to determine the stationary response to the aversive chemicals at the period of certain times (duration), whereas the drop assay was used especially when the rescuing transgenic animals were not stable lines16. Transmission rate of the extrachromosomal arrays in transgenic worms are variable between lines. Some siblings from transgenic worms tend to lose transgene. Thus transgenic animals, which only showed myo-3P::dsRed marker (proof of carrying transgene) were individually picked and tested for the drop assay. However, these two assays produced essentially the same results against all three pheromones.When we determined net movement rather than individual real-time movements of worms across 1鈥塰 (Fig.聽S1a), N2 wild type worms showed a dose- and time-dependent repulsion response to all three individual pheromones (Fig.聽S1b,c), to which late larvae (L4) and young adult worms responding more strongly than early larvae (L1) (Fig.聽S1d). Notably, all three pheromones elicited a similar pattern and intensity of repulsion responses. Therefore, we used a single pheromone rather than blend of their combination for the plate-based chemotaxis assay in most cases. It appears that they share the common repulsion behavior in response to any of three pheromones. Because the daumone 1 (ascr#1) induced the repulsion of hermaphrodites as well as induction of dauers and fungal traps3, 9, 14, we used mostly daumone 1 in chemotaxis screening of G-protein subunit mutant strains and related experiments.As the initial pheromone sensory process relies on G-proteins (e.g., GPA-2 and 3)17, we further sought to define the specific G伪 subunit that primarily transfers the initial detection signals of the three major pheromones. Of the 17 G伪 subunits examined, only gpa-3 mutant worms showed defective repulsion responses to all three pheromones (Figs聽S1e and聽S2a), suggesting that gpa-3 is the major G-protein subunit gene that is involved in the perception of these pheromones. Previously, we reported that gpa-3 negatively controls both insulin/IGF-1 signaling (IIS) and TGF-beta signaling18. Thus, to identify the downstream cell signaling pathway involved in GPA-3 signaling, we assessed repulsion responses in worms with mutations in downstream effectors of IIS or TGF-尾 pathways. Interestingly, daf-16/FoxO mutant worms showed reduced repulsion responses to all three pheromones (Fig.聽S2b), whereas daf-3/Smad and daf-5/SnoSki mutant worms showed similar repulsion responses as wild-type worms (Fig.聽S2c). These results suggest that at least part of the IIS pathway, but not the TGF-尾 pathway, participates in pheromone sensory signaling in hermaphrodites (Fig.聽S2b,c). It also indicated that DAF-16/FoxO may play an important role in pheromone sensory signaling process.Role of neuronal DAF-16/FoxO in glutamatergic neurotransmission of pheromone sensory signalsSince DAF-2 normally suppresses nuclear localization of DAF-16/FoxO19, we examined whether daf-16/FoxO and/or its isoforms participate in the pheromone sensory process. To this end, daf-2(e1370);daf-16/FoxO (mgDf50) double mutants were subjected to rescue experiment by microinjection of those constructs containing each daf-16/FoxO isoform20. All isoforms examined rescued the repulsion responses of daf-16/FoxO mutants, suggesting their common roles in conveying pheromone sensory signals (Fig.聽1a). Notably, the rescue effect by the daf-16b/FoxO isoform20 suggests that neuronal DAF-16/FoxO might play an important role (activation or suppression) in conveying pheromone signals. To test whether the tissue specificity of DAF-16/FoxO is important in conveying pheromone signals, we performed rescue experiments using ges-1 promoter-driven intestine-specific daf-16/FoxO and unc-119 promoter-driven pan-neuronal daf-16/FoxO. Whereas intestine-specific DAF-16/FoxO did not rescue repulsion responses, neuron-specific DAF-16/FoxO recovered the repulsion responses of daf-16/FoxO mutants (Fig.聽1b), indicating a tissue-specific (neuronal) expression of DAF-16/FoxO seems critical in conveying pheromone sensory signals in head neurons.Figure 1Glutamate signaling mediates pheromone sensory signals to produce repulsion response through the insulin/IGF-1 signaling. (a) Rescuing the daf-2(e1370);daf-16/FoxO (mgDf50) phenotype with different daf-16/FoxO isoforms (wild type, n鈥?鈥?45; daf-2;daf-16/FoxO, n鈥?鈥?96; daf-2;daf-16/FoxO;DAF-16/FoxOa, n鈥?鈥?46; daf-2;daf-16/FoxO;DAF-16/FoxOdf, n鈥?鈥?18; daf-2;daf-16;DAF-16/FoxOb, n鈥?鈥?33). (b) Tissue-specific rescue of daf-2;daf-16/FoxO phenotype (wild type, n鈥?鈥?49; daf-2;daf-16/FoxO, n鈥?鈥?25; daf-2;daf-16/FoxO;ges-1P::DAF-16/FoxO, n鈥?鈥?50; daf-2;daf-16/FoxO;unc-119P::DAF-16/FoxO, n鈥?鈥?42). DAF-16/FoxO cDNA was expressed under control of intestine- (ges-1 P) or pan-neuronal-specific (unc-119 P) promoters. (c) eat-4(ky5) mutants deficient in glutamate transporter showed defective repulsion responses. (d) Genetic epistasis between daf-2 and eat-4 mutants (wild type, n鈥?鈥?8; eat-4, n鈥?鈥?7; daf-2, n鈥?鈥?8; daf-2 eat-4, n鈥?鈥?2). (e) DAF-16/FoxO in glutamatergic neurons rescued daf-2;daf-16/FoxO repulsion responses (wild type, n鈥?鈥?9; daf-2;daf-16/FoxO, n鈥?鈥?4; daf-2;daf-16/FoxO; eat-4P::daf-16/FoxO, n鈥?鈥?0). DAF-16/FoxO cDNA was expressed under control of the eat-4 promoter. *P 0.05, ns: not significant compared to wild type. **P 0.05 compared to daf-2;daf-16/FoxO. Significance was determined using two-tailed, unpaired t-tests. In these experiments, daumone 1 (1 渭锘匡豢M) was singly used in isolation.Full size image To identify neurotransmitters required for the transmission of pheromone sensory signals and to define the role of neuronal daf-16/FoxO, we tested several strains with mutations of genes involved in neurotransmitter signaling for their elicitation of repulsive behaviors. They are; eat-4 (glutamate transporter), tph-1 (serotonin biosynthesis), cat-2 (dopamine biosynthesis), egl-3 (pro-neuropeptide processing), cha-1 (acetylcholine biosynthesis), and unc-49 锘縂ABA 锘縭eceptor. Of these, only eat-4(ky5) mutants showed defective repulsion responses (Fig.聽1c), suggesting that neuronal pheromone signals may share (or converted to) glutamate signals to elicit repulsion responses. When we repeated this experiment with another allele of eat-4 (ad819) mutant, the results remained essentially the same (data not shown) (data in Fig.聽1c).We next assessed the genetic epistatic relationship between glutamate and daf-2 IIS by comparing single daf-2 or eat-4 mutants with daf-2(e1370) eat-4(ky5) double mutants. Whereas daf-2 mutants, in which DAF-16/FoxO is highly activated, showed strong repulsion responses similar to those of wild-type worms, daf-2 eat-4 double mutants showed defective repulsion responses similar to those of eat-4 single mutants (Fig.聽1d), suggesting that eat-4- mediated glutamatergic signaling may occur downstream of daf-2 activity. However, we cannot exclude the possibility that daf-2 signaling could be in parallel with glutamatergic signaling by modulating other related metabolic pathways. Next, to test whether daf-16/FoxO functions in glutamatergic cell autonomously or not, we examined if daf-16/FoxO expression in eat-4-expressing neurons is enough for the recovery of repulsion behavior in daf-16 mutants by generating transgenic worms in which daf-16 is expressed under control of the eat-4 promoter in daf-2(e1370);daf-16(mgDf50) mutants. Interestingly, eat-4 promoter-driven daf-16/FoxO rescued the daf-16/FoxO mutant phenotype (Fig.聽1e), providing evidence of a cell-autonomous function of DAF-16/FoxO in controlling the glutamatergic neurotransmission central to pheromone sensory signaling. In addition, when we examined whether DAF-16 influences eat-4 expression, we found that the transcript levels of eat-4 remained unchanged in daf-16 mutants (Fig.聽S2d). We also found that the reduced daf-16/FoxO response in daf-16(mgDf50) single mutant was comparable to that of daf-2; daf-16/FoxO double mutants (Fig.聽S3). Taken together, our data suggests that daf-2 signaling maybe genetically upstream of glutamatergic signaling not by regulating expression of eat-4 expression level but presumably by altering another components in glutamate signaling pathway.Neuronal DAF-16/FoxO controls glutaminase gene expressionWe next addressed a question as to what would be the potential role of neuronal DAF-16/FoxO in conveying the pheromone sensory signals through the glutamate neurotransmission to elicit repulsion behavior. The levels of neuronal glutamate are tightly regulated by glutaminase activity in conjunction with energy metabolism in astrocytes of mammalian brain21. In fact, mammalian brain is a high-energy demand organ and glucose is the primary source of energy. The C. elegans genome contains three glutaminase genes: glna-1, glna-2, and glna-3. To test whether DAF-16/FoxO modulates glutaminase gene expression thereby conveying pheromone sensory signals, we examined the relative expression of these genes and found that only glna-3 expression was reduced in daf-16/FoxO mutants (Fig.聽2a), which was also supported by RNAi knockdown results (Fig.聽2b). We also found that the expression of DAF-16/FoxO in glna-3-expressing neurons rescued the repulsion responses of daf-2 (e1370); daf-16/FoxO (mgDf50) mutants (Fig.聽2c). And the reduced response of daf-16 mutant was fully rescued by overexpression of glna-3(glna-3P::glna-3), which suggests that glna-3 acts downstream of daf-16 to regulate the pheromone response (Fig.聽2d, Fig.聽S7a). In this rescue experiment, independent transgenic lines were also tested and they all showed essentially the same results (Fig.聽S7a). Taken together, these results strengthen the notion that glutamatergic neuronal activity responsible for conveying pheromone sensory signals to elicit repulsion behavior appears to be regulated at the level of glna-3 expression by neuronal DAF-16/FoxO. To corroborate the involvement of glutamate receptors in conveying pheromone sensory signals, we next tested worms with mutation of mgl-1, a homolog of the human type II metabotropic receptor GRM3, which is predicted to locate at the presynaptic glutamate neuron that inhibits glutamate release22. As expected, mgl-1(tm1811) mutants elicited a stronger repulsion response than wild-type worms (Fig.聽S4), perhaps due to enhanced presynaptic glutamate release, indicating the potential role of MGL-1 as a gate for pheromone-elicited glutamatergic repulsion responses. However, it remains to be determined whether additional downstream components of glutamate signaling (e.g., glr-1, mgl-2, and nmr-2) in post-synaptic neurons contribute to repulsion responses. Because the mgl-1(tm1811) strain exhibited a hypersensitive phenotype at a lower concentration of pheromone (i.e., 1.0鈥塶M), we normalized the repulsion responses of mgl-1 mutants to those of wild-type worms.Figure 2DAF-16/FoxO regulates glna-3 expression. (a) Glutaminase gene transcript levels in daf-16/FoxO mutants. Bars represent the mean of three independent biological replicates. *P 0.05, ns: not significant compared to wild type. (b) RNAi against glna-3 in a neuronal RNAi-sensitive strain (unc-119P::sid-1). F1 animals were hatched and grown on control or glna-3 RNAi plates. F1 young adults were transferred to new RNAi plates and allowed to lay eggs, and F2 young adults were tested (Ctrl RNAi, n鈥?鈥?48; glna-3 RNAi, n鈥?鈥?63). *P 0.05 compared to Ctrl RNAi (c) DAF-16/FoxO in glna-3-expressing neurons rescued the daf-2;daf-16/FoxO phenotype (wild type, n鈥?鈥?8; daf-2;daf-16/FoxO, n鈥?鈥?01; daf-2;daf-16/FoxO; glna-3P::DAF-16/FoxO, n鈥?鈥?2). DAF-16/FoxO cDNA was expressed under control of the glna-3 promoter. *P 0.05 compared to wild type, **P 0.05 compared to daf-2;daf-16/FoxO. (d) glna-3P::glna-3 rescued daf-16/FoxO mutant phenotype (wild type, n鈥?鈥?50; daf-16/FoxO, n鈥?鈥?49; daf-16/FoxO;glna-3P::glna-3, n鈥?鈥?50). *:daf-16(-) vs daf-16(-); glna-3P::daf-16. Bars represent the mean of three independent biological replicates. Significance was determined using two-tailed, unpaired t-tests.Full size image Cellular and transcriptional expression of glna-3 With respect to glna-3-expressing neurons, we observed that glna-3P::gfp expression, driven by a 1422-bp segment in the 5鈥?upstream region of the glna-3 gene, was localized in head neurons (Fig.聽3a,b). Specifically, glna-3P::gfp expression and a dye filling assay, which stains chemosensory amphid neurons, showed that glna-3 is expressed in AWB neurons (Fig.聽3a), consistent with previous findings that eat-4/vGlut1 is expressed in AWB neurons23. By contrast, ASI, ADL, ASK, ASH, and ASJ neurons did not express glna-3P::gfp (Fig.聽3b).Figure 3Expression pattern of glna-3P:gfp and ChIP analysis of DAF-16/FoxO::GFP bound to the upstream region of glna-3 gene (a) and (b) Expression pattern of glna-3P::gfp. Worms were also stained with DiI dye to visualize chemosensory amphid neurons. (c) str-1P::glna-3 rescued daf-16/FoxO mutant phenotype (wild type, n=160; daf-16/FoxO, n=155; str-1P::glna-3; daf-16/FoxO, n=150.) *P 0.05 compared to wild type, **P 0.05 compared to daf-16/FoxO.聽(d) Putative DAF-16/FoxO binding sites in the 5鈥?upstream region and first intron of the glna-3 gene. (e) ChIP of DAF-16/FoxO::GFP with anti-GFP antibody in daf-2;DAF-16/FoxO and daf-2;DAF-16/FoxO;DAF-16/FoxO::gfp mutants. Bars represent the mean of three independent biological replicates. *P 0.05 compared to daf-2;DAF-16/FoxO. Significance was determined using two-tailed, unpaired t-tests.Full size image However, the glna-3P::gfp reporter used in our study24 was expressed in a limited number of head neurons compared to that of previously reported23. This is presumably because our promoter construct did not include the first intron sequence. At least three pairs of amphid neurons (ASH, ADL, and AWB) are required for detecting either attractants or repellents25, with the AWB neuron being required for repulsion responses to 2-nonanone26. To address whether AWB neurons are involved in transmitting dauer pheromone-mediated hermaphrodite repulsion behavior, glna-3 was specifically expressed in daf-16 mutant under the control of AWB specific str-1 promoter26 (Figs聽3c, S7b锘?/a>). The str-1P::glna-3 partially rescued the reduced repulsion phenotype of daf-16 mutant, suggesting that AWB neuron, at least in part, plays a role in pheromone-induced repulsion response. We further tested lim-4(ky403) and ceh-37 (ok272) mutants. In lim-4 and che-37 mutants, neuronal cell fate of AWB neurons is altered, as a result, AWB neurons adopt AWC neuronal characteristics27, 28. Our study showed that the lim-4 and ceh-37 mutants conferred reduced repulsion behavior upon exogenous dauer pheromone (Fig.聽S5). Thus, it is likely that pheromone sensing signal that is initially perceived by GPA-3 is transmitted through AWB glutamatergic neuron where neuronal聽DAF-16/FoxO modulates glutaminase gene expression, resulting in elicitation of repulsion behavior. Of course, we cannot exclude the possibility that other neurons may also be involved in this process.These results also raised additional questions: (1) What are the molecular mechanisms by which DAF-16/FoxO transcriptionally regulates glna-3 expression? (2) Similar to nematode DAF-16 /FoxO, can the corresponding mammalian mFoxO3 regulate glutamate transmission in the hippocampus, a specific expression site of mFoxO3, a close homolog of daf-16 29. To answer the first question as to the transcriptional regulation mechanism by which DAF-16/FoxO controls glna-3 expression, we examined seven predicted putative DAF-16/FoxO binding domains (BDs) located within the 5鈥?upstream and first intron region of the glna-3 gene (Fig.聽3d) for their binding to DAF-16/FoxO. To determine whether DAF-16/FoxO could bind to these sites, we performed chromatin immunoprecipitation (ChIP) in daf-2(e1370); daf-16(mgDf50); daf-16/FoxO::gfp animals using anti-GFP antibody. ChIP assay showed that DAF-16/FoxO binding is more enriched in BD1 (upstream) and BD6/7 (intron region) of the glna-3 gene regulatory region, suggesting that neuronal DAF-16/FoxO may regulate glna-3 transcription by binding to at least these two upstream regions of the glna-3 gene in neurons (Fig.聽3e). This result is also supported by a recent report that glna-3 levels were up-regulated in daf-2 mutants compared to daf-2;daf-16/FoxO double mutants30. Together, it is suggested that the DAF-16/FoxO transcription factor may modulate neuronal glutamate homeostasis by regulating glutaminase expression, which subsequently produces the repulsion behavior in response to the exogenous pheromones. However, it remains to further delineate the interactions between DAF-16/FoxO and the specific DNA sequences within the BD1 and BD6/7 of the glna-3 gene.A conserved modulatory role of DAF-16/FoxO in glutamatergic neurotransmissionTo answer the second question as to conservation of FoxO function between nematodes and mammals, we performed electrophysiological experiments in mice. Whereas C. elegans has only one FoxO transcription factor (DAF-16/FoxO), humans and mice have four FoxO transcription factors (FoxO1, 3, 4, and 6). DAF-16/FoxO shares the highest sequence homology with mammalian FoxO329, whereas the expression pattern of FoxO6 is enriched in brain tissues31. To examine similarities between mammalian FoxO (mFoxO) and nematode DAF-16/FoxO in glutamatergic transmission regulatory function that is crucial for pheromone sensory transmission, we knocked down both mFoxO3 and mFoxO6 expression in cultures of mouse primary hippocampal neurons, which are known to express both mFoxO3 and mFoxO632, by shRNA-mediated viral infection. Our experiment was also based on the earlier report that neurons in hippocampus mainly release glutamate and GABA33. After shRNA constructs were initially tested in NIH/3T3 cell lines before viral packaging, we chose two shRNA constructs for each gene (Fig.聽S6). Whole-cell patch recordings were obtained from primary hippocampal neurons 5 days after infection with scrambled adeno-associated virus (AAV-Scr), AAV-shFoxO3, or AAV-shFoxO6 at 10 days in vitro 33. The efficiency of viral infection was confirmed by mCherry expression as a marker of AAV vector (Fig.聽4a). Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in the presence of picrotoxin (50鈥壩糓) to exclude inhibitory postsynaptic currents (Fig.聽4b). Both sEPSCs and large-amplitude burst oscillations were observed in primary hippocampal neurons, as previously reported32. The frequency of sEPSCs was reduced in AAV-shFoxO3-infected neurons compared with control, AAV-Scr-, or AAV-shFoxO6-infected neurons, whereas the frequency of sEPSCs was unchanged in AAV-shFoxO6 infected neurons (Fig.聽4c). There were no differences between groups in sEPSC amplitude (Fig.聽4d). AAV-shFoxO3-infected neurons also showed reduced burst oscillation frequency (Fig.聽4e) and amplitude (Fig.聽4f ). These results indicate that the specific knockdown of mFoxO3 suppresses glutamatergic transmission in mammalian neurons, which is consistent with our results in C. elegans. Our findings demonstrate that FoxO plays a conserved pivotal role in maintaining glutamate homeostasis in the mouse hippocampus and the head of C. elegans.Figure 4Knockdown of FoxO3 reduced sEPSCs, burst oscillations in mouse primary hippocampal neurons. (a) Primary hippocampal neurons without (control) or with infection of AAV-Scr, AAV-shFoxO3, or AAV-shFoxO6 in bright-field (top) and mCherry fluorescence (bottom) images. AAV-infected groups had increased infection rates (38.1%, AAV-Scr; 42.5%, AAV-shFoxO3; 56.7% AAV-shFoxO6). (b) Representative traces of sEPSCs and burst oscillations from primary hippocampal neurons held at 鈭?0 mV in voltage clamping mode in the presence of picrotoxin. The part of each trace in the left panel marked with an upper line is enlarged in the right panel. (c) AAV-shFoxO3 neurons (n鈥?鈥?) exhibited fewer sEPSCs than control (n鈥?鈥?), AAV-Scr (n鈥?鈥?), or AAV-shFoxO6 (n鈥?鈥?) neurons. (d) There were no differences in sEPSC amplitude. (e) and (f) Burst oscillation frequency and amplitude were reduced in AAV-shFoxO3 neurons. *P 0.05, **p 0.01, ***p 0.001.Full size image Conclusions and perspectivesIn this work, we demonstrate how information contained in pheromones is processed internally by neural circuit to yield behavioral response. Furthermore, we provide a previously unexplored basic framework for neuronal components that are likely involved in neurotransmission of pheromone signals and a potential modulatory role of neuronal DAF-16/FoxO in this process. The potential components that participate in pheromone sensory processing leading to repulsion behavior include, but are not limited to, GPA-3, EAT-4, DAF-16/FoxO, GLNA-3, and MGL-1 (Fig.聽5). Because gpa-3 is not expressed in AWB neurons17, we may draw the conclusion that once pheromones are sensed in gpa-3 expressing neurons such as ASI, ADL, or ASK, their signals are conveyed to glna-3 expressing AWB neurons to elicit repulsion behaviors (Fig.聽5). Moreover, this sensory process appears to be modulated by evolutionally conserved neuronal DAF-16/mFoxO3. Given that eliciting repulsion behaviors may be important for various pheromone activities1, 2, 4, our work on the identification of pheromone sensory signaling pathway may mark a major breakthrough in this field. 锘縏his work could also stimulate investigations on general pheromone signaling in animals including mammals as well as its potential application to related neuronal disorders. As many important regulatory functions of FoxO across species are being explored, it may also be possible to conduct integrated studies that link neuronal FoxO-mediated pheromone sensation to neurological diseases caused by disturbances in glutamatergic neurotransmission in humans such as Alzheimer鈥檚 disease.Figure 5A proposed model of neuronal DAF-16/FoxO-mediated pheromone sensory signal transduction pathway. The pathway from pheromone perception to repulsion behavior includes at least five components: GPA-3, DAF-16/FoxO, GLNA-3, EAT4, and MGL-1. By binding to putative pheromone receptors (not shown), pheromones may stimulate GPA-3 and subsequently activate glutamatergic neurotransmission in AWB neurons, which is transcriptionally modulated by neuronal DAF-16/FoxO via GLNA-3 activation. The production of glutamate signals is likely gated by MGL-1/mGRM3.鈥搖nconfirmed relation; 鈹€ confirmed relation.Full size image Methods C. elegans strains and culture C. elegans were cultured using standard techniques34. The strains used in this work were N2 Bristol (wild-type), DAF-16/FoxO(mu86), DAF-16/FoxO(m26), DAF-16/FoxO(mgDf50); daf-2(e1370), DAF-16/FoxO(mgDf50); daf-2(e1370) unc-119(ed3); lpIs12[DAF-16/FoxOa::RFP鈥?鈥?i>unc-119(+)], DAF-16/FoxO(mgDf50); daf-2(e1370) unc-119(ed3); lpIs13[DAF-16/FoxOb::CFP鈥?鈥?i>unc-119(+)], DAF-16/FoxO(mgDf50); daf-2(e1370) unc-119(ed3); lpIs14[DAF-16/FoxOf::GFP鈥?鈥?i>unc-119(+)], gpa-1(pk15), gpa-2(pk16), gpa-3(pk35), gpa-4(pk381), gpa-5(pk376), gpa-6(pk480), gpa-8(pk345), gpa-9(pk438), gpa-10(pk362), gpa-11(pk349), gpa-12(pk322), gpa-13(pk1270), gpa-14(pk342), gpa-15(pk477), goa-1(n1134), odr-3(n2105), eat-4(ad819), mgl-1(tm1811), tbh-1(n3247), cmk-1(ok287), osm-6(p811), cat-2(e1112), egl-3(gk238), cha-1(n2411), che-37(ok272), lim-4(ky403) and unc-49(e382), DAF-16/FoxO(mu86); ykpEx025[鈥? glna-3P::glna-3鈥?鈥?i>myo-3P::rfp], and DAF-16/FoxO(mu86); ykpEx026[鈥? str-1P::glna-3鈥?鈥?i>
myo-3P::RFP]. Worms were grown on nematode growth media seeded with E. coli OP50 as a food source.Transgenic wormsRescue constructs of DAF-16/FoxO were generated by PCR fusion of the regulatory regions of unc-119 (1200鈥塨p), eat-4 (2196鈥塨p), or glna-3 (1422鈥塨p) upstream of the start codon of DAF-16/FoxO::gfp amplified from the TJ356 strain. Transgenes were microinjected in the germline of daf-2; daf-16/FoxO mutants with myo-3P::dsRed as a transgene marker. NC1478, a strain harboring wdEx584[glna-3P::gfp, unc-119(+)]; unc-119(ed3), was gift from Dr. David Miller III. Rescue construct of glna-3 and AWB neuron-specific glna-3 rescue construct were also generated by PCR fusion of the 1422鈥塨p upstream regions of glna-3 or 4000鈥塨p upstream region of str-1 gene26 to glna-3 cDNA including 3鈥?UTR. Each transgenes were microinjected in the germline of daf-16(mu86) mutants with Pmyo-3::RFP as a transgene marker, to generate glna-3 rescue worms and AWB neuron-specific glna-3 rescue worms.Ascaroside PheromonesAll ascaroside pheromones (daumones 1鈥? or ascr#1鈥?) were chemically synthesized and characterized at our laboratory as previously described2, 5, 15. Pheromones were dissolved in absolute ethanol and prepared in a stock solution (10鈥塵M with ethanol). A serial dilution of pheromones were diluted into in M13 buffer in Eppendorf tubes to the final concentration of pheromone for the plate-based chemotaxis assay or drop assay (see below).Chemotaxis assayFor the plate-based chemotaxis assay, L1-synchronized worms were collected and grown to the young adult stage, washed three times with S-basal buffer to remove E. coli, and transferred to the center of a plate using aspirator tube assemblies for calibrated microcapillary pipettes (Sigma, St. Louis, MO). Chemotaxis index values were determined by counting worms that moved to different zones of the plate according to the following formula (see Fig. S1a): (A鈭払)/(A鈥?鈥塀), where A is the number of worms that moved to zone A (containing pheromone [1 渭M]) and B is the number of worms that moved to zone B (containing EtOH only). Unless otherwise indicated, chemotaxis index values were calculated 1鈥塰 after placement on the plate. In this assay, any anesthetizing drugs were not used. Worms that crawled up the side of the plate were not counted. To avoid errors in measurement due to reduced movement, we used strains with no motility deficits. Each data point represents 100鈥?50 worms. Statistics were performed using GraphPad Prism 5. The drop assay for ascaroside pheromone-induced repulsion behavior was previously described16. For drop assay, worms were stage-synchronized with egg-preparation assay in prior to the assay. Twenty young adult animals (total 140鈥?50 worms in each assay) were moved onto unseeded NGM plate (55 mm diameter) at 20鈥壜癈 with the platinum wire. A serial dilution of pheromones (stock of 10鈥塵M with ethanol) diluted into in M13 buffer in Eppendorf tubes to the final concentration of 1 渭M of pheromone. Glass capillary was utilized to deliver pheromone to the head of a forward moving worm, then, scored the positive and negative responses. The repulsion behavior was monitored by putting a small drop of the ascaroside pheromone ahead of the forward moving worm and observed the two to three turns of backward movements as 鈥榬epulsive鈥?and the fraction of worms 鈥榬epulsive鈥?was calculated by comparing the with buffer controls. The synthetic daumone 1 used for the mgl-1 mutant behavior was a different batch of other experiments.DiI staining and MicroscopyDiI staining was performed to visualize ciliated chemosensory neurons as described previously in Michael Koelle鈥檚 protocol (www.wormatlas.org/EMmethods/DiDiO.htm), with minor modifications. Briefly, DiI (1.1鈥?dilinoleyl-3,3,3鈥?3鈥?tetramethylindocarbocyanine perchlorate, Molecular Probes) stock solution was prepared in 2鈥塵g/ml concentration in dimethyl formamide, stored at 鈭?0鈥壜癈. The DiI stock solution was diluted 1:200 in M9 and 150鈥壩糽 of solution was put in a glass tube, where L2 worms were transferred and DiI-stained for 2鈥塰ours at 20鈥壜癈. After staining, worms were washed with M9 and transferred to NGM plate to crawl on a bacterial lawn for 1鈥塰our to destain. Worms were visualized by using confocal microscope LSM 700 (Carl Zeiss). Images were analyzed with Carl Zeiss Zen 2.1 (Ver. 11.0) software.shRNA design and vectorpLKO.1-puro constructs containing scrambled (SHC002, Sigma), shFoxO3 (TRCN0000071616, Sigma), or shFoxO6 (TRCN000008777, Sigma) sequences were transfected into NIH/3T3 cells to confirm knock-down efficiency. The mouse shFoxO3 nucleotide targeted the FoxO3 sequence from 1441 to 1461鈥塨p (5鈥?CGGCACCATGAATCTGAATGA-3鈥? NM_019740.2), and the mouse shFoxO6 nucleotide targeted the FoxO6 sequence from 830 to 850鈥塨p (5鈥?CCTCGCCACTCATGTACCCAA-3鈥? NM_194060.1).For AAV packaging, scrambled, shFoxO3, or shFoxO6 sequences were cloned into pAAV-U6-shRNA-CMV-mCherry vector by the site-directed mutagenesis method (Enzynomics). Each construct was synthesized using complementary primers; scrambled: 5鈥?AGAGATTGGTGCTCTTCATCTTGTTGTTTTTTCTCGAGTACTAGGA-3鈥?(sense), 5鈥?TGAATTGGTGCTCTTCATCTTGTTGAAACAAGGCTTTTCTCCAAG-3鈥?(antisense); shFoxO3: 5鈥?AGAGATCATTCAGATTCATGGTGCCGTTTTTTCTCGAGTACTAGGA-3鈥?(sense), 5鈥?TGAATCATTCAGATTCATGGTGCCGAAACAAGGCTTTTCTCCAAG-3鈥?(antisense); shFoxO6: 5鈥?AGAGATTGGGTACATGAGTGGCGAGGTTTTTTCTCGAGTACTAGGA-3鈥?(sense), 5鈥?TGAATTGGGTACATGAGTGGCGAGGAAACAAGGCTTTTCTCCAAG-3鈥?(antisense).Primary hippocampal neuron culturesFor primary hippocampal neuron cultures, hippocampi were isolated from mice on postnatal day 0鈥? and maintained in ice-cold Ca2+- and Mg2+-free Hank鈥檚 balanced salt solution (HBSS). They were then incubated with HBSS containing trypsin (0.15鈥塵g/ml) and L-cystein (0.5鈥塵g/ml) for 20鈥塵in at 37鈥壜癈 and triturated into single cells. After centrifugation, cells were suspended in Neurobasal A medium with B-27 supplement and 2鈥塵M glutamine and then plated on coverslips coated with poly-D-lysine (1鈥塵g/ml) at a concentration of 5鈥壝椻€?05 cells/ml. Half of the medium was replaced every 4 days. Neuronal cultures were infected with AAV-Scr, AAV-shFoxO3, or AAV-shFoxO6 at 10 days in vitro and used for experiments at 15 days in vitro.qRT-PCR analysisTotal RNA was isolated from age-synchronized young adult worms using Trizol reagent (Invitrogen) followed by clean-up with RNeasy spin columns (Qiagen, Valencia, CA). cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and used for qRT-PCR. All the relative expression data of worms by qRT-PCR was normalized by act-2 gene expression.ElectrophysiologyThis experiment was performed as previously described35. Primary hippocampal neurons isolated from mice and cultured on coverslips were placed in a recording chamber (Warner Instrument, Hamden, CT) mounted to an upright microscope (EX51WI, Olympus, Japan) and camera (ORCA-R2, Hamamatsu, Japan). The recording chamber was perfused continually with artificial cerebrospinal fluid containing (in mM) 124 NaCl, 3 KCl, 1.3 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 2.4 CaCl2鈭?H2O, and 10 glucose aerated with 95% O2/5% CO2 at room temperature. Borosilicate glass capillaries (GC150F-10, Warner Instrument Corp., Hamden, CT) for fabricating patch electrodes (4 to 6 M惟) were made using a pipet puller (P-97, Sutter Instrument, Novato, CA). Synaptic currents were measured in whole-cell configuration and amplified using Multiclamp 700B (Molecular Devices, Sunnyvale, CA). Data acquisition was performed using a Digitizer 1440鈥堿 (Molecular Devices) and Clampex 10.3 (Molecular Devices). Analysis of data was conducted using Clampfit 10.3 (Molecular Devices) and the MiniAnalysis program (Synaptosoft, Fort Lee, NJ). The intracellular pipette solution for voltage-clamp recordings contained (in mM) 130 CsCl, 10 MgCl2, 10 HEPES, 5 Mg-ATP, 5 QX-314, 0.5 Na-GTP, and 0.1 EGTA, at pH 7.3 and 282鈥塵Osm. For measurement of bursting, membrane potential was held at 鈭?0 mV, and 50鈥壩糓 picrotoxin (Sigma) was added to the bath for 5鈥塵in. References1.Golden, J. W. Riddle, D. L. A Pheromone influences larval development in the nematode Caenorhabditis elegans. Science 218, 578鈥?80 (1982).ADS聽 CAS聽 Article聽 PubMed聽Google Scholar聽 2.Jeong, P. Y. et al. Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature 433, 541鈥?45 (2005).ADS聽 CAS聽 Article聽 PubMed聽Google Scholar聽 3.Butcher, R. A., Fujita, M., Schroeder, F. C. Clardy, J. Small-molecule pheromones that control dauer development in Caenorhabditis elegans. Nat. Chem Biol. 3, 420鈥?22 (2007).CAS聽 Article聽 PubMed聽Google Scholar聽 4.Fielnbach, N. Antebi, A. C. elegans dauer formation and the molecular basis of plasticity. Genes and Dev. 22, 2149鈥?165 (2008).Article聽Google Scholar聽 5.Kim, K. Y. et al. Development of a Method to Quantitate Nematode Pheromone for Study of Small-Molecule Metabolism in Caenorhabditis elegans. Anal. Chem. 85, 2681鈥?688 (2013).CAS聽 Article聽 PubMed聽Google Scholar聽 6.Srinivasan, J. et al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115鈥?118 (2008).ADS聽 CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 7.von Reuss, S. H. et al. Comparative Metabolomics Reveals Biogenesis of Ascarosides, a Modular Library of Small-Molecule Signals in C. elegans. J. Am. Chem. Soc. 134, 1817鈥?824 (2012).Article聽Google Scholar聽 8.Srinivasan, J. et al. A Modular Library of Small Molecule Signals Regulates Social Behaviors in Caenorhabditis elegans. Plos Biology 10, e1001237 (2012).CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 9.Hsueh, Y. P., Mahanti, P., Schroeder, F. C. Sternberg, P. W. Nematode-trapping fungi eavesdrop on nematode pheromones. Curr Biol. 23, 83鈥?6 (2013).CAS聽 Article聽 PubMed聽Google Scholar聽 10.Macosko, E. Z. et al. A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 458, 1171鈥?175 (2009).ADS聽 CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 11.Kim, K. et al. Two Chemoreceptors Mediate Developmental Effects of Dauer Pheromone in C. elegans. Science 326, 994鈥?98 (2009).ADS聽 CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 12.Park, D. et al. Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 109, 9917鈥?922 (2012).ADS聽 CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 13.McGrath, P. T. et al. Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes. Nature 477, 321鈥?25 (2011).ADS聽 CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 14.Joo, H.-J. et al. Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis. Biochem. J. 422, 61鈥?1 (2009).CAS聽 Article聽 PubMed聽Google Scholar聽 15.Joo, H.-J. et al. Contribution of the peroxisomal acox gene to the dynamic balance of daumone production in Caenorhabditis elegans. J. Biol. Chem. 285, 29319鈥?9325 (2010).CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 16.Jang, H. Bargmann, C. I. Acute behavioral responses to pheromones in C. elegans (adult behaviors: attraction, repulsion). Methods in Molecular Biology (Clifton, NJ) 1068, 285鈥?92 (2013).CAS聽 Article聽Google Scholar聽 17.Zwaal, R. R., Mendel, J. E., Sternberg, P. W. Plasterk, R. H. Two neuronal G proteins are involved in chemosensation of the Caenorhabditis elegans Dauer-inducing pheromone. Genetics 145, 715鈥?27 (1997).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 18.Hahm, J.-H., Kim, S. Paik, Y.-K. Endogenous cGMP regulates adult longevity via the insulin signaling pathway in Caenorhabditis elegans. Aging Cell 8, 473鈥?83 (2009).CAS聽 Article聽 PubMed聽Google Scholar聽 19.Henderson, S. T. Johnson, T. E. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr. Biol. 11, 1975鈥?980 (2001).CAS聽 Article聽 PubMed聽Google Scholar聽 20.Kwon, E.-S., Narasimhan, D., Yen, K. Tissenbaum, H. A. A new DAF-16/FOXO isoform regulates longevity. Nature 466, 498鈥?02 (2010).ADS聽 CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 21.Belanger, M., Allaman, I. Magistretti, P. J. Brain Energy Metabolism: Focus on Astrocyte-Neuron Metabolic Cooperation. Cell Met. 14, 724鈥?38 (2011).CAS聽 Article聽Google Scholar聽 22.Dillon, J., Hopper, N. A., Holden-Dye, L. O鈥機onnor, V. Molecular characterization of the metabotropic glutamate receptor family in Caenorhabditis elegans. Biochem. Soc. Trans. 34, 942鈥?48 (2006).CAS聽 Article聽 PubMed聽Google Scholar聽 23.Serrano-Saiz, E. et al. Modular Control of Glutamatergic Neuronal Identity in C. elegans by Distinct Homeodomain Proteins. Cell 155, 659鈥?73 (2013).CAS聽 Article聽 PubMed聽Google Scholar聽 24.Watson, J. D. et al. Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system. BMC genomics 9, 84 (2008).Article聽 PubMed聽 PubMed Central聽Google Scholar聽 25.Bargmann, C. I. Chemosensation in C. elegans. WormBook 1鈥?9, doi:10.1895/wormbook.1.123.1 (2006).26.Troemel, E. R., Kimmel, B. E. Bargmann, C. I. Reprogramming chemotaxis responses: Sensory neurons define olfactory preferences in C. elegans. Cell 91, 161鈥? (1997).CAS聽 Article聽 PubMed聽Google Scholar聽 27.Sagasti, A., Hobert, O., Troemel, E. R., Ruvkun, G. Bargmann, C. I. Alternative olfactory neuron fates are specified by the LIM homeobox gene lim-4. Genes Dev. 13, 1794鈥?806 (1999).CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 28.Lanjuin, A., VanHoven, M. K., Bargmann, C. I., Thompson, J. K. Sengupta, P. Otx/otd homeobox genes specify distinct sensory neuron identities in C. elegans. Dev. Cell 5, 621鈥?33 (2003).CAS聽 Article聽 PubMed聽Google Scholar聽 29.Ogg, S. et al. The Fork head transcription factor DAF-16/FOXO transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994鈥?99 (1997).ADS聽 CAS聽 Article聽 PubMed聽Google Scholar聽 30.Kaletsky, R. et al. The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators. Nature 529, 92鈥?6 (2016).ADS聽 CAS聽 Article聽 PubMed聽Google Scholar聽 31.Hoekman, M. F. M., Jacobs, F. M. J., Smidt, M. P. Burbach, J. P. H. Spatial and temporal expression of FoxO transcription factors in the developing and adult murine brain. Gene Expr. Patterns 6, 134鈥?40 (2006).CAS聽 Article聽 PubMed聽Google Scholar聽 32.Bacci, A., Verderio, C., Pravettoni, E. Matteoli, M. Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons in culture. Eur. J. Neurosci. 11, 389鈥?97 (1999).CAS聽 Article聽 PubMed聽Google Scholar聽 33.Kullmann D. The Hippocampus Book, Oxford University Press, 203鈥?41 (2007).34.Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71鈥?4 (1974).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 35.Halder, D. et al. Combining Suppression of Stemness with Lineage-Specific Induction Leads to Conversion of Pluripotent Cells into Functional Neurons. Chem Biol. 22, 1512鈥?520 (2015).CAS聽 Article聽 PubMed聽Google Scholar聽 Download referencesAcknowledgementsWe thank the Caenorhabditis Genetics Center (funded by the NIH Office of Research Infrastructure Programs (P40 OD10440)) for providing mutant worms. We thank Dr. David Miller III for the gift of glna-3P::gfp transgenic animals. We thank Dr. E.-S. Kwon for his gifts of DAF-16/FoxO isoforms and related double mutants for this work. This work was supported by the National Research Foundation of Korea to YKP (2011-0028112) and DP (2013R1A1A2009033).Author informationAuthor notesJeong-Hoon Hahm聽 聽Sunhee KimPresent address: Center for Plant Aging Research, Institute for Basic Science (IBS), Daegu, 42988, Republic of KoreaDonha Park and Jeong-Hoon Hahm contributed equally to this work.AffiliationsDepartment of Biochemistry, Yonsei University, Seoul, KoreaDonha Park,聽Jeong-Hoon Hahm,聽Haelim Jeong,聽Sunhee Kim聽 聽Young-Ki PaikYonsei Proteome Research Center, Yonsei University, Seoul, KoreaDonha Park,聽Jeong-Hoon Hahm,聽Haelim Jeong,聽Heekyeong Kim,聽Sunhee Kim聽 聽Young-Ki PaikDepartment of Integrated Omics for Biomedical Science, Yonsei University, Seoul, KoreaSaeram Park聽 聽Young-Ki PaikDepartment of Biotechnology, and College of Life Science and Biotechnology, Yonsei University, Seoul, KoreaGo Ha,聽Gyeong-Eon Chang聽 聽Eunji CheongAuthorsDonha ParkView author publicationsYou can also search for this author in PubMed聽Google ScholarJeong-Hoon HahmView author publicationsYou can also search for this author in PubMed聽Google ScholarSaeram ParkView author publicationsYou can also search for this author in PubMed聽Google ScholarGo HaView author publicationsYou can also search for this author in PubMed聽Google ScholarGyeong-Eon ChangView author publicationsYou can also search for this author in PubMed聽Google ScholarHaelim JeongView author publicationsYou can also search for this author in PubMed聽Google ScholarHeekyeong KimView author publicationsYou can also search for this author in PubMed聽Google ScholarSunhee KimView author publicationsYou can also search for this author in PubMed聽Google ScholarEunji CheongView author publicationsYou can also search for this author in PubMed聽Google ScholarYoung-Ki PaikView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsY.K.P. conceived of the project; D.P., J.H.H., G.E.H., G.E.C., H.J., S.P., and S.H.K. performed experiments and analyzed the data; H.K. provided reagents; Y.K.P., J.H.H., D.P., H.J., and E.C. wrote the manuscript.Corresponding authorCorrespondence to Young-Ki Paik.Ethics declarations Competing Interests The authors declare that they have no competing interests. Additional information Publisher\'s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic supplementary material Supplementary InformationRights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article鈥檚 Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article鈥檚 Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articlePark, D., Hahm, JH., Park, S. et al. A conserved neuronal DAF-16/FoxO plays an important role in conveying pheromone signals to elicit repulsion behavior in Caenorhabditis elegans Sci Rep 7, 7260 (2017). https://doi.org/10.1038/s41598-017-07313-6Download citationReceived: 07 March 2017Accepted: 27 June 2017Published: 03 August 2017DOI: https://doi.org/10.1038/s41598-017-07313-6 Jun Young Park, Mi Cheong Cheong, Jin-Young Cho, Hyeon-Sook Koo Young-Ki Paik Scientific Reports (2020) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.
