The head mesodermal cell couples FMRFamide neuropeptide signaling with rhythmic muscle contraction in C. elegans

Abstract

FMRFamides are evolutionarily conserved neuropeptides that play critical roles in behavior, energy balance, and reproduction. Here, we show that FMRFamide signaling from the nervous system is critical for the rhythmic activation of a single cell of previously unknown function, the head mesodermal cell (hmc) in C. elegans. Behavioral, calcium imaging, and genetic studies reveal that release of the FLP-22 neuropeptide from the AVL neuron in response to pacemaker signaling activates hmc every 50 s through an frpr-17 G protein-coupled receptor (GPCR) and a protein kinase A signaling cascade in hmc. hmc activation results in muscle contraction through coupling by gap junctions composed of UNC-9/Innexin. hmc activation is inhibited by the neuronal release of a second FMRFamide-like neuropeptide, FLP-9, which functions through its GPCR, frpr-21, in hmc. This study reveals a function for two opposing FMRFamide signaling pathways in controlling the rhythmic activation of a target cell through volume transmission.

Fig. 1. AVL controls aBoc through peptidergic signaling to hmc.

a Schematic showing the circuit controlling aBoc identified in this study. b Quantification of the number of aBocs per defecation cycle in adult animals of the indicated genotypes. “intestinal nlp-40” denotes nlp-40 cDNA expressed under the intestine-specific ges-1 promoter. “AVL ICE”, “AVL aex-2”, and “AVL TeTx” denote ICE, aex-2 cDNA or TeTx expressed in AVL using the nmur-3(1 kb) promoter. n = 6, 3, 3, 3, 6, 5, 3 independent animals. c Quantification of the number of aBocs per defecation cycle in adult animals of the indicated genotypes. flp-22(vj229) and frpr-17(vj265) null mutants were analyzed. “GABAergic flp-22” and “cholinergic flp-22” denote flp-22 cDNA expressed under the GABAergic-specific unc-47 and cholinergic-specific unc-129 promoter, respectively. “AVL flp-22” denotes flp-22 cDNA expressed in AVL using the nmur-3(1 kb) promoter. “neuronal frpr-17” denotes frpr-17 cDNA expressed under the pan-neuronal rab-3 promoter. “muscle frpr-17” denotes frpr-17 cDNA expressed under the muscle-specific myo-3 promoter. “hmc frpr-17” denotes frpr-17 cDNA expressed in hmc using the nmur-3(Δ) promoter. n = 6, 6, 4, 3, 8, 5, 4, 6, 3, 5 independent animals. d Left: schematic of the location of AVL and hmc in the neck region and representative images showing GFP transcriptional reporters in wild-type and hlh-8 mutants, scale bar, 40 μm. “AVL + hmc GFP” denotes expressing GFP under the numr-3(3 kb) promoter. GFP fluorescence is not detectable in hlh-8 mutants (0 out of 20 animals exhibited GFP fluorescence in hmc). Right: quantification of the number of aBocs per defecation cycle in adult animals of the indicated genotypes. “hmc ICE” and “hmc miniSOG” denote transgenes expressing ICE or miniSOG in hmc under control of the nmur-3(Δ) promoter. n = 6, 3, 5, 8, 5, 3, 11 independent animals. Data are presented as mean values ± SEM. ***P < 0.001, **P < 0.0,1 and *P < 0.05 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant.

Fig. 2. hmc is rhythmically activated by FLP-22 and FRPR-17 signaling.

a Representative images from videos showing GCaMP fluorescence in the intestine, AVL, and hmc before cycle start, at cycle start indicated by the intestinal calcium oscillation, and at the onset of AVL and hmc activation in animals expressing GCaMP3 in the intestine (under the nlp-40 promoter) and GCaMP6 in AVL and hmc (under the nmur-3(3 kb) promoter). Scale bar, 40 μm. b Representative trace showing the calcium dynamics in the AVL soma and hmc cell body during three sequential cycles of the defecation motor program in adult animals. The calcium oscillation in the intestine marks the start of each cycle (dotted line). c Left: normalized GCaMP fluorescence intensities in AVL and hmc and aBoc contractions averaged from time lapse images from 5 cycles for AVL calcium spikes, 10 cycles for hmc calcium spikes, and 10 cycles for aBoc. The x axis denotes seconds relative to the initiation of the calcium spike in AVL. The times that the average aBoc contraction begins (start), reaches maximum contraction (max), and begins relaxation (relaxation) are indicated. Right: quantification of the indicated parameters relative to the onset of the calcium spike in AVL. Data are presented as mean values ± SEM. d–f Quantification of the number of calcium spikes per cycle observed in AVL and hmc in adult animals of the indicated genotypes. “AVL aex-2” denotes aex-2 cDNA expressed in AVL under the nmur-3(1 kb) promoter. “AVL flp-22” denotes flp-22 cDNA expressed in AVL under the nmur-3(1 kb) promoter. “hmc frpr-17” denotes frpr-17 cDNA expressed in hmc under the nmur-3(Δ) promoter. Wild-type: 33 cycles in 8 animals, aex-2: 36 cycles in 7 animals, aex-2; AVL aex-2: 20 cycles in 4 animals, AVL ICE: 43 cycles in 9 animals, flp-22: 49 cycles in 9 animals, frpr-17: 56 cycles in 13 animals, flp-22; frpr-17: 43 cycles in 9 animals, frpr-17; hmc frpr-17: 20 cycles in 7 animals, flp-22; frpr-17; hmc frpr-17: 42 cycles in 9 animals. ***P < 0.001 and *P < 0.05 in two-sided chi-square test with Bonferroni’s correction for multiple comparisons; n.s., not significant. g Left: average traces of calcium dynamics in the indicated genotypes in hmc aligned to the calcium spike initiation time. The solid lines indicate average fold change in GCaMP intensity and the shades indicate SEM. Right: quantification of the average peak amplitude, rise time, and half-decay time. Data are presented as mean values ± SEM. n = 10, 6, 6, 5 cycles from different animals. **P < 0.01 and *P < 0.05 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant.

Fig. 3. A PKA signaling cascade functions in hmc to facilitate hmc activation.

a Quantification of the number of aBocs per cycle in adult animals of the indicated genotypes. “hmc PKA[DN]” denotes a transgene in which a PKA dominant negative variant is expressed in hmc using the nmur-3(Δ) promoter. RNAi denotes RNA interference-mediated knockdown of the indicated gene by feeding. Data are presented as mean values ± SEM. n = 6, 3, 9, 3, 4, 2 independent animals. ***P < 0.001 in one-way ANOVA with Dunnett’s correction for multiple comparisons; n.s. not significant. b Quantification of the number of hmc calcium spikes observed per cycle in adult animals of the indicated genotype. “hmc PKA[DN]” and “hmc PKA[CA]” denote transgenes expressing PKA dominant negative or PKA constitutively active variants in hmc under the control of the nmur-3(Δ) promoter. For (b) and (e), wild-type: 33 cycles in 8 animals, hmc PKA[DN]: 63 cycles in 21 animals, frpr-17: 56 cycles in 13 animals, frpr-17; hmc PKA[CA]: 27 cycles in 6 animals. ***P < 0.001 in two-sided chi-square test with Bonferroni’s correction for multiple comparisons; n.s. not significant. c Violin plots of calcium spike initiation time in hmc after the end of the intestinal calcium oscillation. Dashed lines refer to median and dotted lines refer to quartiles. ***P < 0.001 and *P < 0.05 in Kruskal–Wallis test with Dunn’s correction for multiple comparisons. d Left: average traces of calcium dynamics in hmc aligned to the calcium spike initiation time in the indicated mutants. “hmc PKA[DN]” and “hmc PKA[CA]” denote transgenes expressing PKA dominant negative or PKA constitutively active variants in hmc under the control of the nmur-3(Δ) promoter. The solid lines indicate average fold change in GCaMP intensity and the shades indicate SEM. Dotted line represents a possible critical threshold for aBoc. Right: quantification of the average peak amplitude and half-decay time. Data are presented as mean values ± SEM n = 10, 7, 5 cycles from different animals. ***P < 0.001 and **P < 0.01 in one-way ANOVA with Bonferroni’s correction for multiple comparisons and in two-tailed Student’s t-test. e Quantification of the number aBocs observed per cycle in the indicated mutants in the time lapse calcium images. “hmc PKA[DN]” and “hmc PKA[CA]” denote transgenes expressing PKA dominant negative or PKA constitutively active variants in hmc under the control of the nmur-3(Δ) promoter. ***P < 0.001 in two-sided chi-square test with Bonferroni’s correction for multiple comparisons; n.s. not significant.

Fig. 4. Gap junctions composed of UNC-9/innexin are required for hmc signaling to neck muscles.

a Transmission electron micrograph images of cross sections on the ventral side of the neck showing the AVL and hmc processes, adapted from. AVL and hmc are separated by the pseudocoelom (PS). A large gap junction appears as an electron dense area where the plasma membranes of the neck muscle arm and hmc contact each other. b Quantification of the number of aBocs per cycle in adult animals of the indicated genotypes. “hmc unc-9” and “muscle unc-9” denote expressing unc-9::mTur2 fusion proteins or unc-9 cDNA under the nmur-3(Δ) and myo-3 promoter, respectively. Data are presented as mean values ± SEM. n = 6, 9, 4, 7 independent animals. ***P < 0.001 and **P < 0.01 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant. c Representative images of the neck region of an animal co-expressing NLS::mCherry (arrow), FRPR-17::Venus (plasma membrane of hmc), and UNC-9::mTur2 (arrowheads) in hmc under the nmur-3(Δ) promoter. The dotted line in the top panel shows the position of hmc. The asterisk denotes autofluorescence in the intestine. Similar expression patterns were observed in all 16 animals examined. Scale bar, 40 μm. d Quantification of the number of calcium spikes observed in AVL and hmc during DMP in adult unc-9 mutants. For d and f, unc-9: 27 cycles in 9 animals. Two-sided Fisher’s exact test; n.s., not significant. e Average traces of calcium dynamics in hmc aligned to the calcium spike initiation time in the indicated mutants. The solid lines indicate average fold change in GCaMP intensity and the shades indicate SEM. Quantification of the average peak amplitude, rise time, and half-decay time. unc-9 (no aBoc) refers to cycles with calcium spike followed by no aBoc and unc-9 (aBoc) refers to cycles with calcium spike followed by aBoc. Data are presented as mean values ± SEM. n = 10, 6, 6 cycles from different animals. **P < 0.01 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant. f Quantification of the number aBocs observed per cycle in the indicated mutants in the time lapse calcium images. ***P < 0.001 in two-sided chi-square test.

Fig. 5. hmc activity is negatively regulated by the flp-9 FMRFamide-like neuropeptide and the frpr-21 GPCR.

a Quantification of the number of aBocs per cycle in adult animals of the indicated genotypes. Data are presented as mean values ± SEM. n = 6, 6, 5, 5 independent animals. ***P < 0.001 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant. b Quantification of the number of calcium spikes observed per cycle in hmc in adult animals of the indicated genotypes. “hmc frpr-21” denotes expressing frpr-21 cDNA under the nmur-3(Δ) promoter. “Pflp-9 flp-9” denotes expressing flp-9 cDNA under a 4.8 kb flp-9 promoter fragment. “GABAergic flp-9” denotes expressing flp-9 cDNA under the unc-47 promoter. Wild-type: 33 cycles in 8 animals, frpr-21: 23 cycles in 9 animals, frpr-17: 56 cycles in 13 animals, frpr-21; frpr-17: 32 cycles in 10 animals, frpr-21; frpr-17; hmc frpr-21: 38 cycles in 7 animals, flp-9; frpr-17: 47 cycles in 8 animals, frpr-21; flp-9; frpr-17: 44 cycles in 7 animals. ***P < 0.001 and *P < 0.05 in two-sided chi-square test with Bonferroni’s correction for multiple comparisons; n.s. not significant. c Left: average traces of calcium dynamics in hmc aligned to the calcium spike initiation time in the indicated mutants. The solid lines indicate average fold change in GCaMP intensity and the shades indicate SEM. Right: quantification of the average peak amplitude, rise time, and half-decay time from the traces on the left. Data are presented as mean values ± SEM. n = 10, 9, 5, 6, 6, 5 cycles from different animals. **P < 0.01 and *P < 0.05 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant. d Representative image of hmc cell body (arrow) and processes from adults expressing FRPR-21::GFP in hmc under the nmur-3(Δ) promoter. Similar expression patterns were observed in all 15 animals examined. Scale bar, 40 μm. e Quantification of the number of aBocs per cycle in adult animals of the indicated genotypes. “flp-9 (OE)” denotes expression of flp-9 cDNA under the GABAergic-specific unc-47 promoter. Data are presented as mean values ± SEM. n = 6, 3, 3, 4 independent animals. **P < 0.01 in one-way ANOVA with Bonferroni’s correction for multiple comparisons; n.s. not significant. f Quantification of the number of calcium spikes observed per cycle in hmc in adult animals of the indicated genotypes. “frpr-21 (OE)” denotes expressing frpr-21 cDNA in hmc using the nmur-3(Δ) promoter. Wild-type: 33 cycles in 8 animals, flp-9: 27 cycles in 8 animals, flp-9 (OE): 27 cycles in 5 animals, frpr-21; flp-9 (OE): 23 cycles in 5 animals, frpr-21 (OE): 46 cycles in 7 animals, frpr-21; frpr-21 (OE): 29 cycles in 8 animals. ***P < 0.001 in two-sided chi-square test with Bonferroni’s correction for multiple comparisons; n.s. not significant.

Fig. 6. Working model for the aBoc step.

During the defecation motor program, a calcium oscillation occurs in the intestine every 50 s, which leads to NLP-40 release from the intestine. NLP-40 activates its receptor, AEX-2, on AVL which results in a calcium spike. The calcium spike triggers secretion of the neuropeptide FLP-22 from AVL, which in turn activates its receptor, FRPR-17, on hmc. Activation of FRPR-17 leads to a calcium spike in hmc possibly by activating a signaling cascade composed of, GSA-1/Gαs and KIN-1/PKA. The calcium spike in hmc is transmitted to muscles through the gap junction protein UNC-9, resulting in a contraction of anterior body-wall muscles which is aBoc. Additional GABAergic neuron(s) may also secrete FLP-22 to activate hmc. FLP-9 activates its receptor, FRPR-21, on hmc which inhibits hmc activation. Additional peptidergic input to hmc, possibly from AVL, contributes to hmc activation in some cycles.