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Intestine-to-neuronal signaling alters risk-taking behaviors in food-deprived Caenorhabditis elegans [1]

['Molly A. Matty', 'Molecular Neurobiology Laboratory', 'The Salk Institute For Biological Studies', 'La Jolla', 'California', 'United States Of America', 'Hiu E. Lau', 'Division Of Biological Sciences', 'University Of California', 'San Diego']

Date: 2022-07

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

We have all experienced behavioral changes when we are hungry—the pang in our stomach that can cause us to behave erratically. In particular, animals, including humans, are known to pursue more risky behaviors when they are hungry as compared to when they are well-fed. Here we explore the molecular details of this behavior in the invertebrate animal model C. elegans. We show that C. elegans displays reduced copper sensitivity when hungry. As copper is toxic and repellant to C. elegans, this reduced avoidance suggests that these animals employ riskier food search strategies when food-deprived. Moreover, we find that this hunger-induced behavioral change is reversible upon re-feeding and is not caused by an increased attraction to food or depletion of fat stores, but rather insulin signaling between the intestine and neurons. We use genetic tools, microscopy, and behavioral tests to determine that this risky behavior involves a sensation of “lack of food” in the intestine, release of signaling molecules, and engagement with neurons. Our work highlights new and potentially evolutionarily conserved ways in which intestinal cells and neurons communicate and produce behavioral changes, highlighting the importance of the gut-brain-axis.

Funding: This work was supported by grants from The Rita Allen Foundation ( http://ritaallen.org ), The W.M. Keck Foundation ( http://wmkeck.org ), NIH R01 MH096881 (S.H.C.); NSF Postdoctoral Research Fellowship in Biology Grant No. 2011023 (M.A.M.); Glenn Foundation Fellowship (http:// https://www.salk.edu/science/research-centers/glenn-center-for-research-on-aging/ ) (A.S.); NSF Socrates Award 742551 (H.E.L.); and NSF Graduate Research Fellowships (H.E.L. and J.A.H.). S.H.C. received salary support from grants from The Rita Allen Foundation, The W.M. Keck Foundation and NIH R01MH096881; M.A.M. received support from the NSF Postdoctoral fellowship; A.S. received support from the Glenn Foundation Fellowship; while H.E.L. and J.A.H. received support from the NSF fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we used C. elegans to dissect the machinery required to integrate internal food signals and modify behaviors. We combined food deprivation over multiple hours with a behavioral assay that quantifies the animal’s ability to respond to both toxic and food-related signals, mimicking a simplified ecologically relevant scenario. In this sensory integration assay, animals cross a toxic copper barrier (repellent) and chemotax towards a point source of a volatile food-associated odor, diacetyl (attractant) [ 30 ]. We show that animals that have been food-deprived for multiple hours have reduced sensitivity to the repellent and cross the copper barrier more readily than well-fed animals. Next, we show that two transcription factors change their localization patterns in the intestinal nuclei during multiple hours of food deprivation. We confirm a role for these transcription factors and identify the downstream peptide released by the intestine to relay the “lack of food” signal to other tissues. Finally, we show that neurons likely receive these intestine-released peptides. This allows food-deprived animals to use a higher risk strategy and search for food by reducing their avoidance to repellents.

The nematode Caenorhabditis elegans, with just 302 neurons [ 11 ], and 20 intestinal cells [ 12 ], provides a unique opportunity for a high-resolution analysis of how the nervous system integrates internal signals. Previous studies have shown that, similar to mammals, C. elegans exhibits a number of behavioral, physiological, and metabolic changes in response to altered nutritional status. Hermaphroditic C. elegans retain eggs [ 13 ], are unlikely to mate with males [ 14 ], initiate altered foraging behaviors [ 15 – 17 ], and change their responses to environmental CO 2 [ 18 ], salt [ 19 ], and pheromones [ 20 ] upon food deprivation. Moreover, many molecules that signal hunger are conserved between C. elegans and vertebrates. For example, neuropeptide Y (NPY) signaling influences feeding behaviors in both nematodes and mammals [ 21 – 23 ]. Similar effects are also seen with insulin and dopamine signaling, which modify chemosensory neuronal activity in nematodes [ 24 , 25 ] and mammals [ 26 – 28 ] leading to changes in feeding behavior. While neuronal pathways responding to food-deprivation on the multiple-minute timescales have been mapped [ 17 , 29 ], those integrating these signals on the multiple hour timescales are poorly understood.

Animals evaluate their environment, integrating prior experiences and internal state information in order to optimize their behavior for maximum reward and threat avoidance [ 1 ]. Thus, changes in internal states play a critical role in adjusting the animal’s responses to external stimuli [ 2 , 3 ]. One critical internal state is hunger, which has a profound effect on animal survival and elicits dramatic changes in food-seeking behaviors [ 2 , 4 ]. Multiple species, including humans, have been shown to alter their chemosensory behavior during periods of starvation [ 5 – 10 ]. Despite this, little is known about how the nervous system receives and interprets information about hunger status.

Results

Acute food deprivation specifically alters repellent-driven behaviors Animals simultaneously receive and interpret both attractant and repellent signals from their environment and use that information to generate appropriate behavioral responses. To mimic these ecological interactions, we employed a sensory integration assay in which animals must cross a repellent copper barrier (CuSO 4 ) towards a gradient of a volatile attractant, diacetyl [30] (described in Methods). The proportion of animals that cross the toxic copper barrier are counted and expressed as a chemotactic index (Fig 1A). We analyzed the behavior of well-fed, wildtype animals and found that only ~20% cross the copper barrier and locomote towards the spot of diacetyl (black bars, Fig 1B and 1C and S1 Movie). In contrast, animals food-deprived for at least 2 hrs were more likely to cross the copper barrier (blue bars, Fig 1B and S2 Movie) with a maximal effect at 3 hrs (Fig 1B and 1C). Next, we tested whether the food-deprivation effect was reversible. We food-deprived animals for 3 hrs and then returned them to food for increasing durations and analyzed animal behavior after the food experience. We found that 3-hour food-deprived animals that had been returned to food for at least 3 hrs reverted to the “well-fed” state (Fig 1C). Taken together, these results indicate that food deprivation reversibly modifies sensory integration behavior. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Starvation reduces copper avoidance. A) Schematic of the sensory integration assay. ~100–200 day 1 adult animals (n) are placed in the black rectangle. Blue barrier represents copper barrier (or other repellant) and star represents diacetyl or other attractant. Chemotactic Index is the number of animals that have crossed the barrier (odor side) divided by the total number of animals on the plate (odor + origin sides). Experiments with well-fed (WF) animals will appear with black dots and those with food-deprived (FD) animals will be indicated with blue dots. Unless otherwise noted, FD is 3 hrs with no food. Each dot represents a single plate (N) of animals (n). B) Animals are deprived of food for increasing periods of time (15 mins, 30 mins, 2 hrs, 3 hrs). Animals are exposed to 50 mM CuSO 4 repellant and 1:500 (0.2%) diacetyl attractant. N = 8. C) Sensory integration behaviors of animals that have been starved for 3 hrs and 6 hrs. Animals that have been starved for 3 hours are allowed to recover for 1, 3, or 5 hrs on OP50. Well-fed matched partners are kept on OP50 plates for the entire length of the experiment. Animals are exposed to 50 mM CuSO 4 repellant and 1:500 (0.2%) diacetyl attractant. N≥6. D) Animals are exposed to increasing concentrations of other repellants (Fructose, NaCl, Quinine) with the attractant 0.05% diacetyl (1:2000) in each condition N≥7. E) Animals are exposed to decreasing concentrations of diacetyl (DiA) (0.2%, 0.1% and 0.05%, or 1:200, 1:1000, and 1:2000, respectively) and other volatile attractants 0.1% Benzaldehyde (BZ) and 0.05% Isoamyl Alcohol (IAA). 50 mM CuSO 4 is the repellant in each condition N≥6. F) Animals are exposed to CuSO 4 in increasing concentrations (5 mM, 25 mM, 50 mM, 100 mM) without any attractant N≥8. G) Animals are exposed to diacetyl alone in decreasing concentrations (0.2%, 0.1%, 0.05%). Full assay (0.2% diacetyl and 50 mM CuSO 4 ) is included as a control N≥7. H) Animals are exposed to 1:500 diacetyl and increasing concentrations of CuSO 4 (5 mM, 25 mM, 50 mM, 100 mM) N≥6. All graphs are analyzed using a two-way ANOVA, determined to have significant differences across well-fed and food-deprived conditions. WF/FD comparisons were then performed as pairwise comparisons within each genotype or treatment as t-tests with Bonferroni corrections for multiple comparisons. * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001, ns p>0.05. Error bars are S.D. https://doi.org/10.1371/journal.pgen.1010178.g001 We then tested whether this food deprivation-evoked change in sensory integration behavior was specific to the copper repellent and diacetyl attractant used in the assay. We observed that in this sensory integration assay layout and with the diacetyl concentration used (1:2000), food-deprived animals did not cross the repellent barrier when diacetyl was paired with other repellents like fructose (with the exception of one intermediate concentration), sodium chloride, or quinine (Fig 1D). In contrast, when copper was paired with other attractants like benzaldehyde and isoamyl alcohol, food-deprived animals continued to cross the copper barrier more readily than well-fed animals (Fig 1E). Collectively, these data suggest that multiple food-associated volatile attractants can promote repellent barrier crossing in food-deprived animals. Consistently, a previous study showed that food-deprived animals are more likely to cross the repellent barrier when paired with a bacterial lawn [31]. To test whether food-deprivation differentially affected copper or diacetyl responses, we analyzed responses of these animals to varying concentrations of copper or diacetyl alone. We found that food-deprived animals crossed the copper barrier more readily than well-fed animals, suggesting that their responsiveness to copper is reduced even in the absence of an attractant (Fig 1F). In contrast, food-deprived animals did not discernably alter their attraction to diacetyl in the absence of the copper repellant, except at one intermediate concentration (Fig 1G). Given the small number of well-fed animals that cross the copper barrier alone (Fig 1F), we continued to pair copper with the diacetyl attractant for further analysis. We found that food-deprived animals were significantly more likely than well-fed animals to cross the repellent barrier above a threshold of 5 mM copper concentration (Fig 1H). To gain further confirmation of this copper-specific change, we tested food-deprived animals in a single animal copper drop assay (S2A Fig). In this assay, the response of a single animal to a drop of 1.5 mM CuSO 4 solution placed in its path was monitored. Most repellents can be tested in this assay with animals generating a robust avoidance response [32]. We found that food-deprived animals showed a significant deficit in their copper-avoidance response (S2B Fig). Collectively, these data show that food-deprived animals display reduced avoidance of copper repellent, which we dissected further using genetic methods and tracking software.

Lack of food and not changes in fat drives the food-deprivation induced behavioral change Given that the change in sensory integration behavior requires multiple hours of food deprivation, we hypothesized that metabolic signals like changes in fat content might play a crucial role. Previous studies have shown that prolonged starvation can deplete fat stores in C. elegans, which in turn can affect behavior [35,36]. We tested whether 3 hrs of food deprivation alters the fat content of animals. Oil-Red O (ORO), a fat-soluble dye that stains triglycerides and lipoproteins, and has been used to label and quantify fat stores in C. elegans (Fig 3A and 3B) [37]. We used this dye and found that 3 hrs of food-deprivation altered neither the ORO signal nor the area of the animal labeled by this stain (Fig 3C and 3D). In contrast, we observed a significant change in the both the intensity of the signal and area of animal stained in 6-hr food-deprived animals, consistent with previous studies [38]. These data suggest that changes in sensory integration behavior, which occur after 3 hours of food deprivation, are likely to be independent of fat metabolism as measured by Oil-Red O staining. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Lack of food, not fat or physical interactions, drive behavioral changes. (A) Schematic of Oil Red O experiments. Animals are raised together to day 1 of adulthood and separated into three groups: well-fed (on food), 3 hr food-deprived, and 6 hr food-deprived. Animals are stained using Oil Red O and then imaged using a color camera. (B) Representative images of well-fed (WF, black), 3 hour food-deprived (3hr FD, blue), and 6 hr food-deprived (6 hr FD, green). Inset images are shown, highlighting the regions where there is the most difference in staining. Black arrows highlight regions of no Oil Red O stain in 6 hr FD. (C) Graph showing the percent change in Oil Red O staining when compared to the average of the area of Oil Red O signal above a threshold value in the well-fed group within each independent experiment. N = 3, n>20 within each experimental treatment group. (D) Graph showing the percent of the animals’ area that contains Oil Red O signal above threshold N = 3, n>20 within each experimental treatment group. Same data as in C, shown as non-normalized values. (E) A schematic representing the experiment in F, in which populations of animals are either well-fed or food-deprived in the presence or absence of Sephadex beads before performing the sensory integration assay. (F) Prior to the sensory integration assay, animals are exposed to either standard OP50 (“no beads WF”) or empty plates (“no beads FD”), or Sephadex gel beads as chemosensory input. Alternatively, animals were exposed to beads and no food (“beads FD”) or OP50 with Sephadex beads on top (“beads WF”) for 3 hours. Animals were then exposed to standard Sensory Integration Assay set-up with 50 mM CuSO 4 and 1 μl of 0.2% diacetyl. N≥18. (G) A schematic representing the experiment in H, in which populations of animals are either well-fed or food-deprived in the presence of OP50-containing agar plugs on the lid of the plate or agar alone plugs on the lid of the plate before performing the sensory integration assay. (H) Prior to the sensory integration assay, animals are exposed to either standard OP50 empty plates, covered with lids containing either agar plugs (agar) or agar plugs with OP50 lawns (OP50 odor) as a chemosensory input for 3 hours. Animals were then exposed to standard Sensory Integration Assay set-up with 50 mM CuSO 4 and 1 μl of 0.2% diacetyl. N = 12 per condition. C and D were analyzed using Welch’s ANOVA test with Dunnett’s multiple comparisons test. * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001, ns p>0.05. F and G were analyzed using a full model two-way ANOVA, determined to have significant differences across well-fed and food-deprived conditions but no difference between “bead”/“no bead” groups or “odor”/”agar” groups. Those comparisons are shown to indicate no difference between “beads” and “no beads”. Pairwise comparisons within each treatment were performed as t-tests with Tukey’s multiple comparisons test. Error bars are S.D. https://doi.org/10.1371/journal.pgen.1010178.g003 Next, we sought to identify the relevant aspects of the bacterial experience contributing to the food deprivation-triggered behavioral change. C. elegans has been shown to evaluate multiple aspects of the food experience, including changes in food distribution, oxygen and carbon dioxide concentrations, small molecule metabolites, and others [39–41]. To uncouple the tactile and chemosensory input of the bacteria from the nutritional value of ingesting bacteria, we analyzed the effect of using Sephadex gel beads on animal behavior. Animals exposed to gel beads experienced the tactile input, but not the nutritional value of food (Fig 3E) [15]. Notably, we found that animals exposed to Sephadex beads in the absence of E. coli (OP50) for 3 hrs behaved similarly to food-deprived animals in the sensory integration assay (Fig 3F) suggesting that mechanosensory input is not involved in this food-deprivation invoked behavior. Next, we tested whether chemosensory information from bacteria can affect animal behavior in the sensory integration assay. We exposed animals to the volatile odors of OP50 using agar plugs on the lid of the dish prior to performing the assay (Fig 3G). Animals exposed to the volatile odors of OP50 in the absence of food for 3 hrs behaved similarly to food-deprived animals exposed to agar plugs with no OP50 volatile odors (Fig 3H). Together, these results suggest that the absence of volatile chemosensory and mechanosensory cues do not reduce the animal’s copper avoidance behavior. Rather, the lack of food in the C. elegans intestine may be causing the observed food-deprivation behavior.

Transcription factors mediate food deprivation-induced behavioral change Our study suggests that the lack of food inside the animal is responsible for the reversible reduction in copper avoidance. To gain insights into the underlying molecular machinery, we investigated the role of nutritional-responsive transcription factors in the sensory integration assay. In mammalian cells, glucose is rapidly converted to glucose-6-phosphate, whose levels are sensed by two basic-helix-loop-helix-leucine zipper transcription factors, MondoA and ChREBP (Carbohydrate Response Element Binding Protein). In well-fed conditions, MondoA binds excess glucose-6-phosphate and Mlx (Max-like protein X) and translocates to the nucleus where it activates transcription of glucose-responsive genes. In the absence of glucose, MondoA remains in the cytoplasm [42,43] (Fig 4A). C. elegans orthologs of MondoA and Mlx have been identified as MML-1 and MXL-2, respectively [44]. MML-1/MondoA has previously been shown to translocate into the intestinal nuclei under well-fed conditions (Fig 4A) [45]. We predicted that mml-1 mutants would be unable to sense the lack of food and thereby unable to reduce copper sensitivity after food deprivation. Consistently, we found that mml-1, but not mxl-2 mutants were defective in their integration responses after food deprivation (Fig 4B). We then tested whether food deprivation alters the sub-cellular localization of the MML-1 protein. We monitored the GFP fluorescence under well-fed and food-deprived conditions in a mml-1 knockout transgenic animal expressing GFP fused to the full-length coding sequence of MML-1/MondoA under well-fed and food-deprived conditions [45]. We found that 3 hrs of food-deprivation resulted in an increased translocation of MML-1/MondoA from the nucleus to the cytoplasm of the intestinal cells (Fig 4C and 4D). We suggest that this cytosolic MML-1/MondoA reduces copper avoidance by modifying signaling between tissues. PPT PowerPoint slide

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TIFF original image Download: Fig 4. mml-1 and hlh-30 are required for sensory integration change upon food deprivation, correlated with shifts in their intestinal localization. (A) Schematic showing the 20 intestinal cells in a day 1 adult C. elegans. Our findings for mml-1::gfp and hlh-30::gfp transgenic animals are shown in the dotted box, while previously published paradigms are within the solid line box. Addition of glucose has been shown to induce nuclear localization of MondoA. Autophagy has been shown to increase nuclear localization of HLH-30. (B) Standard sensory integration assay with mml-1(ok849) and mxl-2(tm1516) and wildtype controls. N = 20. (C) Representative images of MML-1::GFP localization in day 1 adult animals (data quantified in D). All images were collected with the same exposure time and laser power. (D) Intestinal MML-1::GFP expression in animals during static timepoints food deprivation. Only intestinal expression was characterized as “nuclear”, “nuclear/cytoplasmic”, or “cytoplasmic”. Each dot represents the proportion of animals within an experiment with the phenotype. N = 6, n = 296.(E) Standard sensory integration assay with hlh-30(tm1978) mutant animals and wildtype controls. N = 9. (F) Representative images of HLH-30::GFP localization in day 1 adult animals (data quantified in G). All images were collected with the same exposure time and laser power. (G) Intestinal HLH-30::GFP expression in animals during static timepoints of food deprivation. Only intestinal expression was characterized as “nuclear”, “nuclear/cytoplasmic”, or “cytoplasmic”. Each dot represents the proportion of animals within an experiment with the phenotype. N = 3, n = 149. (H) Intestinal HLH-30::GFP expression in animals during time lapse imaging. The proportion of animals (n = 3, n = 5, n = 6) with nuclear localization are plotted over time, with images taken every 10 minutes. The areas shaded in yellow correspond to the timepoints that match those in the separate experiments in Fig 4G, with the average of the timepoints within that period of time in parentheses. The shaded region labeled “prep” denotes time that the animals are off food but cannot be imaged due to preparation constraints. B and E were analyzed using two-way ANOVA, determined to have significant differences across well-fed and food-deprived conditions. WF/FD comparisons were then performed as pairwise comparisons within each genotype or treatment as t-tests with Bonferroni’s multiple comparisons test. D and G were analyzed using Two-Way ANOVA, determined to have significant differences across localization and an interaction between time of food deprivation and localization. Within each localization group, pairwise comparisons were performed across each time point and tested for significance using Tukey’s multiple comparisons test. * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001, ns p>0.05. Error bars are S.D. https://doi.org/10.1371/journal.pgen.1010178.g004 Previous studies have shown that MML-1 regulates the activity and nuclear localization of a second bHLH transcription factor HLH-30 (C. elegans TFEB, Fig 4A) [46]. In multiple animal models, HLH-30/TFEB functions as a key regulator of longevity pathways by promoting autophagy and lysosome biogenesis [47–50]. We tested whether HLH-30/TFEB was also required for food deprivation-evoked change in sensory integration. We found that, unlike wild-type animals, hlh-30 null mutants did not show a change in their behavior after food-deprivation in the sensory integration assay (Fig 4E), but were mobile in the absence of a copper barrier (S1 Table). We then tested whether the subcellular localization of HLH-30/TFEB was also affected by food deprivation. We observed an initial decrease in cytosolic GFP fluorescence at 1 hour of food-deprivation, with a concomitant increase in nuclear localization in HLH-30::GFP transgenic animals [47] (Fig 4F and 4G). Subsequently, at 3 hrs of food-deprivation, we found a robust increase in cytosolic HLH-30::GFP fluorescence with a decrease in nuclear localization at 2 and 3 hrs of food deprivation. Further analysis of HLH-30::GFP animals throughout an in vivo time course of food deprivation suggested that HLH-30 nuclear intestinal localization was dynamic (Fig 4H and S3 Movie), implying a complex role for this transcription factor. Moreover, the change in localization of HLH-30 during food deprivation corresponded to the timing of behavioral changes (after 1 hour, Fig 1B), suggesting that HLH-30 might transcribe a “hunger” signal. Collectively, these data show that both MML-1 and HLH-30 change their localization in response to food-deprivation and are required for behavioral change in sensory integration.

Intestine-to-neuron signaling involves insulin signaling Previous studies have shown that the C. elegans intestine is a major site for the transcriptional regulation of insulin-like peptide genes in response to starvation [51]. In addition, HLH-30/TFEB has been shown to act upstream of the insulin-signaling pathway in regulating the expression of neuronal chemoreceptor genes [52]. The C. elegans genome encodes about 40 insulin-like peptides [53] and all of these ligands are thought to bind and signal via a single tyrosine kinase DAF-2 receptor [54,55]. We hypothesized that insulin-like peptides might also act downstream of HLH-30/TFEB in relaying food status signals from the intestine to other tissues. Consistent with our hypothesis, multiple insulin-like peptides including INS-3, INS-4, INS-6, INS-10, INS-11, INS-17, INS-18, INS-23, and INS-31 contain HLH-30/TFEB binding sites in their promoters [52]. In addition, INS-7, INS-8, and INS-37 have been shown to affect the subcellular localization of HLH-30/TFEB in the C. elegans intestine after mating [56] (summarized in Fig 5A). We tested mutants in these insulin-like peptide genes for their ability to alter sensory integration behavior after food deprivation. Some alleles of ins-3 (ok2488 and tm3603), ins-4 (ok3534), and ins-18 (tm339) show altered chemotactic behaviors, where well-fed and food-deprived indices are similar (Fig 5B). However, upon testing additional alleles we find that animals carrying ins-3(ok2487), ins-4(tm3620), or ins-18(ok1672) alleles as well as ins-6(tm2416), ins-10(tm3498), ins-11(tn1053), ins-17(tm790), and ins-23(tm1875) displayed significantly different chemotactic indices when food deprived, making their responses similar to wild-type animals (Fig 5B). In contrast, we found that null mutants in the insulin-like peptide ins-31(tm3543) were unable to respond to food deprivation. Specifically, these mutant animals did not display an increased ability to cross the repellent copper barrier when food deprived (Fig 5B), implying that INS-31 might be a candidate signal relaying food status signals. Also, mutants in other insulin-like peptides that affect the localization of HLH-30/TFEB, ins-7(tm2001), ins-8(tm4144), and ins-37(tm6061), were similar to wild-type animals in their ability to cross the copper barrier in both well-fed and food-deprived conditions (Fig 5C). To directly assess whether INS-31 is produced or secreted from the intestinal cells, we generated a rescue construct to drive expression of ins-31 cDNA in either the intestine (from the gly-19 promoter) or in neurons (from the H20 promoter) in the background of ins-31(tm3543) mutant animals. We observe that intestinal, but not neuronal-selective expression of ins-31 cDNA was sufficient to restore wild-type behavior to the ins-31(tm3543) null mutants. Also, neuron-selective expression of ins-31 cDNA reduces chemotactic behaviors in both well-fed and food-deprived animals (Fig 5D), indicating that aberrant neuronal expression of this transgene might affect chemotaxis behaviors. Taken together, these data suggest that the C. elegans intestine likely releases INS-31 to relay hunger information to other tissues. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Sensory integration changes require HLH-30-regulated insulin while daf-2 is required in neurons. (A) HLH-30 interacts with C. elegans insulin peptides. Of the 40 insulin-like peptides encoded in the C. elegans genome, 22% have an HLH-30 binding motif (CANNTG E-box, blue) in the 5’ UTR (< 300bp upstream of start site) [52]. 7% of insulins have been shown to regulate the localization of HLH-30 but do not contain an E-box (orange, “HLH-30 modifiers”). An illustration of a representative insulin peptide with two yellow exons and an upstream E-box with HLH-30 initiating transcription. (B) All insulins known to contain an HLH-30 binding motif in the 5’ UTR were tested using the standard sensory integration assay. When available, more than one allele was tested (N≥8) for each insulin, with wildtype (N2) animals tested with each mutant. (C) Insulins previously shown to regulate HLH-30 localization (ins-7, ins-8, ins-37) were tested using the standard sensory integration assay alongside wildtype (N2) control. N≥7. (D) ins-31 mutants and tissue-specific rescues are tested in the standard sensory integration assay. N ≥8 for each strain tested alongside wildtype N2. ins-31 is rescued in neurons and intestines using tissue-specific promoters. (E) daf-2 mutants and tissue-specific rescues are tested in the standard sensory integration assay N ≥ 9 for each strain tested alongside wildtype N2. daf-2 is rescued in neurons, intestines, and pharynx using tissue-specific promoters. (F) Schematic showing requirement of ins-31 in the intestine and daf-2 in neurons. CI phenotype means Chemotactic Index phenotype, where wildtype animals display a chemotactic index of WF < FD. All graphs were analyzed using a two-way ANOVA, determined to have significant differences across well-fed and food-deprived conditions. WF/FD comparisons were then performed as pairwise comparisons within each genotype or treatment as t-tests with Bonferroni’s multiple comparisons test. * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001, ns p>0.05. https://doi.org/10.1371/journal.pgen.1010178.g005 Next, we probed the role of the insulin receptor, DAF-2, in affecting 3 hour-food deprivation evoked changes in sensory integration. Consistent with our analysis of mutants in various insulin-like peptide genes, we found that two different alleles (m596 and e1370) in the insulin receptor, DAF-2, were also defective in their response to food deprivation (Fig 5E). To localize the site of DAF-2 action, we analyzed the effect of rescuing this receptor in different tissues. We found that re-expressing daf-2 under neuronal (rgef-1), but not intestinal (ges-1) or pharyngeal muscle (myo-2) promoters [57] restored normal behavior to daf-2(m596) mutants (Fig 5E). Taken together, these results suggest that neuronally expressed DAF-2 receptors might detect INS-31 peptides released from the intestine (Fig 5F).

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