Co-option of neurotransmitter signaling for inter-organismal communication in C. elegans

Abstract

Biogenic amine neurotransmitters play a central role in metazoan biology, and both their chemical structures and cognate receptors are evolutionarily conserved. Their primary roles are in cell-to-cell signaling, as biogenic amines are not normally recruited for communication between separate individuals. Here, we show that in the nematode C. elegans, a neurotransmitter-sensing G protein-coupled receptor, TYRA-2, is required for avoidance responses to osas#9, an ascaroside pheromone that incorporates the neurotransmitter, octopamine. Neuronal ablation, cell-specific genetic rescue, and calcium imaging show that tyra-2 expression in the nociceptive neuron, ASH, is necessary and sufficient to induce osas#9 avoidance. Ectopic expression in the AWA neuron, which is generally associated with attractive responses, reverses the response to osas#9, resulting in attraction instead of avoidance behavior, confirming that TYRA-2 partakes in the sensing of osas#9. The TYRA-2/osas#9 signaling system represents an inter-organismal communication channel that evolved via co-option of a neurotransmitter and its cognate receptor.

Fig. 1

osas#9 is repulsive to starved animals. a Structural and functional diversity of ascarosides. osas#9 is involved in avoidance, icas#3 attracts hermaphrodites, and ascr#8 attracts males at low concentrations and induces dauer formation at high concentrations. b Avoidance to osas#9 is dependent on the physiological state of C. elegans. Avoidance index of young adult (YA), wild type (N2) animals in response to solvent control (black dotted line), and 1 µM osas#9 (black solid line) after different time points after removal from food. After 40 minutes of starvation, animals begin to avoid osas#9, with the response reaching a plateau at 60 min. n ≥ 3 trials. Note for all other assays, unless otherwise stated, animals are starved for at least 60 min. c All life stages of hermaphrodites, and adult males, avoid osas#9 when starved. n ≥ 4 trials. d Avoidance index for starved young adult (YA) wild-type worms in response 1 µM osas#9. n = 8 trials. 1 µM osas#9 concentration was used in all other assays unless stated otherwise. Data presented as mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001, one factor ANOVA with Sidak’s multiple comparison posttest, except for Fig. 1d, where a Student’s t-test was used. Individual data points for each bar graph are represented as gray circles

Fig. 2

tyra-2 is required for osas#9 mediated aversive responses independent of tyramine. a Screen for receptors required to mediate osas#9 avoidance. tyra-2 lof animals are defective in osas#9 avoidance response. n ≥ 4 trials. b Two alleles of tyra-2 lof animals (tm1846 and tm1815), are defective in osas#9 avoidance behavior. n ≥ 4 trials. c wild type, wt, and tyra-2 lof mutants showed no significant differences when subjected to known chemical deterrents. n ≥ 3 trials. d Wild type animals avoided osas#9 at 1 and 10 µM, while both tyra-2 lof mutants avoid at only 100 µM. n ≥ 8 trials. e Expression of tyra-2 receptor is dependent on the physiological state of the animal. RT-qPCR analysis starved animals indicates a nearly twofold upregulation of tyra-2. Data are displayed as the ratio of endogenous tyra-2 mRNA to ama-1 mRNA from three independent RT-qPCR experiments. n = 3 trials. f osas#9 avoidance response is not dependent on endogenous tyramine. Two different alleles of tdc-1 lof animals (n3419 and n3420) which are deficient in tyramine biosynthesis, exhibit normal response to osas#9. n ≥ 7 trials. Data presented as mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001, one factor ANOVA with Sidak’s multiple comparison posttest except for 2D and 2E, where Student’s t-test was used

Fig. 3

TYRA-2 is required in ASH for sensation of osas#9. a Cellular localization of TYRA-2 in sensory neurons. Expression is seen in ASE, ASG, ASH, ASH, and NSM neurons (×40 magnification, scale bar denotes 10 µm). b Chemosensory neurons required for osas#9 response. Neurons expressing tyra-2 reporter were ablated using laser microbeam. ASH ablations resulted in abolished response to osas#9 that was indistinguishable from solvent control. ASE and ASI ablated animals showed reduced but not complete loss of avoidance. n ≥ 3 trials (≥10 ablated animals precondition). c, d Calcium dynamics of ASH upon osas#9 exposure. c ASH::GCaMP3 animals (black) exhibit calcium transients when exposed to 1 µM osas#9. tyra-2 lof animals (red) did not display a change in fluorescence upon stimulation. Shaded region gray depicts time animals were subjected to osas#9, n = 10 animals. d Maximum fluorescence intensity before (solvent control, gray) and during exposure to 1 µM osas#9 of ASH::GCaMP3 animals (black) and tyra-2 lof animals (red). e osas#9 elicits calcium transients in ASH neurons at a broad range of concentrations: 1 pM (black), 1 nM (red), 100 nM (blue), 1 µM (orange), 10 µM (magenta), 100 µM (cyan), n ≥ 10 animals per condition. f Maximum fluorescence intensity before and during exposure to varying osas#9 concentrations. g Tyramine elicits avoidance only at high concentrations in wild-type animals, n ≥ 5 trials. h Calcium dynamics in ASH upon exposure to different concentrations of tyramine, for ASH::GCaMP3 1 µM (magenta) and 1 mM (cyan) and tyra-2 lof 1 mM tyramine (red). Tyramine exposure resulted in a significant increase in calcium transients in ASH at concentrations of 1 mM, but not 1 µM, n ≥ 10 animals. i Maximum fluorescence intensity before (solvent control) and during exposure to varying tyramine concentrations for ASH::GCaMP3 animals and tyra-2 lof animals. Data presented as mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001. Figure 3b, one factor ANOVA with Sidak’s multiple comparison posttest. Figure 3d, f, i Student’s t-test was used to compare the solvent control to stimulus max peak fluorescence

Fig. 4

tyra-2 expression in sensory neurons is required for sensitivity to osas#9. a A transcriptional rescue construct, pnhr-79::tyra-2::RFP, exhibits expression of tyra-2 in both ASH and ADL neurons (×40 magnification, scale bar denotes 10 µm). b Rescue of tyra-2 in ASH/ADL neurons fully reconstituted behavioral response to 1 µM osas#9. n ≥ 4 trials. c Rescue of tyra-2 in ASH neurons in a tyra-2 lof (blue) background show no difference compared to calcium transients of wild type animals expressing GCaMP3 in ASH neurons (black) when exposed to 1 µM osas#9 (gray-shaded region), while tyra-2 lof animals show a clear loss of calcium transcients in ASH (red), n ≥ 10 animals. d Maximum fluorescence intensity in transgenic worms before (solvent control) and during exposure to 1 µM osas#9. e Sub-cellular localization of tyra-2. A translational reporter of the entire tyra-2 genomic locus (ptyra-2::tyra-2::GFP) was injected into tyra-2 lof animals at 1 ng/µL, revealing expression of the receptor in both soma and sensory cilia. (60x magnification, scale bar denotes 20 µm). f Expression of the translational reporter restores wild type behavior in a tyra-2 lof background, n ≥ 5 trials. Data presented as mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001. One factor ANOVA with Sidak’s multiple comparison posttest

Fig. 5

Ectopic expression of tyra-2 confers the ability to respond to osas#9. a Misexpression of tyra-2 in ADL neurons confers avoidance behavior in response to osas#9. nhr-79 promoter driving tyra-2 expression in ASH and ADL sensory neurons rescues osas#9 avoidance. Ablation of either ADL or ASH neurons does not affect osas#9 avoidance in the rescue lines, suggesting that misexpression of tyra-2 in ADL neurons is sufficient for osas#9 response. Ablation of both ASH and ADL completely abolished avoidance. n ≥ 5 trials. b Schematic illustration of cellular ablations in the transgenic rescue lines expressing tyra-2 under the nhr-79 promoter. c Animals with reprogrammed AWA sensory neurons in tyra-2 lof background do not avoid 1 µM osas#9. n ≥ 4 trials. d AWA neurons (black) do not exhibit calcium transients in response to 1 µM osas#9, while reprogrammed AWA::tyra-2 neurons (red) are hyperpolarized upon exposure (gray-shaded region), n = 10 animals. e Maximum fluorescence intensity in transgenic worms before (solvent control) and during exposure to 1 µM osas#9. f Schematic illustration of the leaving assay to measure osas#9 attraction. g Wild type (black), tyra-2 lof (red), and AWA::tyra-2 lines (1 [magenta], 2 [cyan]) were subjected to 10 pM osas#9 in the leaving assay. Wild-type animals left the osas#9 solution spot quicker than the tyra-2 lof animals, whereas the misexpression lines remained closer to osas#9, n ≥ 3 trials. h Reprogrammed AWA::tyra-2 animals have an increased reversal rate in comparison to both wild type and tyra-2 lof animals in 10 pM osas#9, n ≥ 3. Data presented as mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001. One factor ANOVA with Sidak’s multiple comparison posttest

Fig. 6

GPA-6 functions in ASH sensory neurons to mediate osas#9 response. a Screen of mutations in Gα subunits resulted in identification of the Gα subunit, gpa-6, which was defective in avoidance response to osas#9, n ≥ 3 trials. b Expression of gpa-6 in ASH neurons using nhr-79 promoter reconstituted avoidance response similar to wild-type animals, n ≥ 3 trials. c gpa-6 localizes to the soma and cilia in ASH neurons. Translational fusion of the entire gpa-6 genomic region displayed localization of the subunit to the soma of AWA, AWB, and ASH neurons. In addition, we also observed ciliary localization in ASH neurons (×40 magnification, scale bar denotes 10 µm). Data presented as mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001. One factor ANOVA with Sidak’s multiple comparison posttest

Fig. 7

osas#9 serves as a dispersal cue for C. elegans. An animal navigating its environment encounters a food source, where offspring grow and reproduce rapidly, eventually depleting their food source. Eggs hatch on the depleted food patch and halt development as L1 arrest animals. L1 arrest animals secrete the aversive compound, osas#9, signaling for conspecifics to disperse away from the unfavorable condition