cGMP signaling in ASJ neurons contributes to H2S avoidance

(A–H) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT (N2) and daf-19(m86) (A); WT and dyf-3(m185) (B); WT and dyf-7(m539) (C); WT, daf-11(m47) and tax-4(p678) (D); WT and tax-2(p691) (E); WT, daf-2(e1370), daf-16(mgDf47) and daf-2(e1370); daf-16(mgDf47) double mutants (F); WT, daf-7(e1372), daf-3(mgDf90) and daf-7(e1372); daf-3(mgDf90) double mutants (G); WT and gcy-9(tm7632) (H). In this and subsequent figures for H2S assay, red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively in Figure 1D, F, G, J and K) used for statistical analysis unless otherwise indicated. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test. (I) Comparison between the acute locomotory response of WT animals to 150 ppm H2S balanced with 7% O2 and the acute locomotory response to 5% CO2 balanced with 7% O2. Red bars on the x-axis indicate two intervals (3-4 minutes and 11-12 minutes) used for statistical analysis. * = p < 0.05, ns = not significant, Mann–Whitney U test. (J) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, daf-11(m47) and transgenic daf-11(m47) expressing daf-11 genomic DNA in ASJ neurons (two independent lines). *** = p < 0.001, ** = p < 0.01, * = p < 0.05, Mann–Whitney U test. (K) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, tax-4(p678) and transgenic tax-4(p678) expressing tax-4 cDNA in ASJ neurons (two independent lines). *** = p < 0.001, ** = p < 0.01, * = p < 0.05, Mann–Whitney U test.

Activation of O2 sensing circuit antagonizes H2S avoidance

(A) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals, wild isolates CB4858 and CB4855. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analyses. ** = p < 0.01, * = p < 0.05, Mann–Whitney U test. (B, C) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and npr-1(ad609) (B); WT, npr-1(ad609) and transgenic npr-1(ad609) expressing npr-1 in RMG neurons using Cre-LoxP system (Macosko et al., 2009) (C). Red bars on the x-axis indicate two intervals (4-5 minutes and 11-12 minutes) used for statistical analysis. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, Mann–Whitney U test. (D–G) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, npr-1(ad609), gcy-35(ok769) and npr-1(ad609); gcy-35(ok769) double mutants (D); WT, npr-1(ad609), tax-4(p678) and npr-1(ad609); tax-4(p678) double mutants (E); WT, npr-1(ad609), qaIs2241(genetic ablation of AQR, PQR and URX neurons) and npr-1(ad609); qaIs 2241 (F); WT, npr-1(ad609), npr-1(ad609); gcy-35(ok769) double mutants and transgenic npr-1(ad609); gcy-35(ok769) double mutants expressing gcy-35 cDNA under gcy-37 promoter, which drives expression in O2 sensing neurons (G). Red bars on the x-axis indicate two intervals (4-5 minutes and 11-12 minutes) used for statistical analysis. *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test.

Prolonged H2S exposure reprograms gene expression in C. elegans

(A) Venn diagram displaying the number of differentially expressed genes after 1-hour exposure to 50 ppm or 150 ppm H2S balanced with 7% O2 in WT animals. p<1e-10. (B) Venn diagram displaying the number of differentially expressed genes after 1-hour, 2-hour and 12-hour exposure to 150 ppm H2S balanced with 7% O2 in WT animals. p<1e-10. (C, D) Significantly enriched GO categories for differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm (C) or 150 ppm (D) H2S balanced with 7% O2 for 1 hour. (E, F) Volcano plots showing the differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm (E) or 150 ppm (F) H2S balanced with 7% O2 for 1 hour. A set of genes involved in H2S detoxification, cysteine metabolism and stress response were highlighted in red.

Acute response to H2S is modulated by HIF-1 signaling

(A–D) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and hif-1(ia4) (A); WT, egl-9(sa307) and vhl-1(ok161) (B); WT, egl-9(sa307), hif-1(ia4) and egl-9(sa307); hif-1(ia4) double mutants (C); WT, vhl-1(ok161), hif-1(ia4) and vhl-1(ok161); hif-1(ia4) double mutants (D). Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analyses. *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test. (E) Locomotory responses of WT animals to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 after 0, 12, 24 or 36 hours in 1% O2. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analyses. *** = p < 0.001, ** = p < 0.01, ns = not significant, Mann–Whitney U test. (F) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals carrying the non-degradable form of HIF-1. Non-degradable form of HIF-1 (P621A and P621G) are expressed from a pan-neuronal promoter unc-14 or form hif-1 endogenous promoter, respectively. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analyses. ** = p < 0.01, ns = not significant, Mann–Whitney U test. (G–J) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and sqrd-1(tm3378) (G); WT and ethe-1(yum2895) (H); WT, cysl-1(ok762), cysl-2(ok3516) and cysl-3(yum4) (I); WT and semo-1(yum2889) (J). Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analyses. *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test.

Labile iron pool is critical to sustain the locomotory activity in H2S

(A, B) Volcano plots showing the relative expression of the genes involved in the regulation of iron homeostasis in WT animals after 2-hour exposure with 50 ppm (A) or 150 ppm (B) H2S balanced with 7% O2. (C) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals, WT animals pretreated with 100 μM 2,2′-Bipyridyl (BP) and WT animals pretreated with 5 mg/ml ferric ammonium citrate (FAC) in the presence of food for 16 hours. Red bars on the x-axis represent two intervals (4–5 minutes and 14–15 minutes, labeled as a and b respectively), used for statistical analyses. ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test. (D) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, smf-3(ok1305) and ftn-1(ok3625). Red bars on the x-axis represent two intervals (4–5 minutes and 14–15 minutes, labeled as a and b respectively), used for statistical analysis. *** = p < 0.001, * = p < 0.05, ns = not significant, Mann–Whitney U test. (E) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals and animals overexpressing ftn-1 genomic DNA under its own promoter (#1 and #2 indicate two independent lines). Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analysis. **** = p < 0.0001, ** = p < 0.01, ns = not significant, Mann–Whitney U test. (F, G) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, hif-1(ia4) and hif-1(ia4) mutants pretreated with 5 mg/ml ferric ammonium citrate (FAC) for 16 hours (F); WT, hif-1(ia4), ftn-1(ok3625) and hif-1(ia4); ftn-1(ok3625) double mutants (G). Red bars on the x-axis represent two intervals (4–5 minutes and 8–9 minutes) used for statistical analysis. * = p < 0.05, ns = not significant, Mann– Whitney U test.

Mitochondrial function is required for acute response to H2S

(A–D) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and gas-1(fc21) (A); WT and clk-1(qm30) (B); WT and mev-1(kn1) (C); WT and isp-1(qm150) (D). Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes) used for statistical analysis. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05, Mann–Whitney U test. (E) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals pretreated with 10 μM rotenone for 0 minute, 10 minutes, 30 minutes, 60 minutes or 120 minutes. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analysis. *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test.

Acute locomotory responses of C. elegans to hydrogen sulfide.

(A) Changes in the locomotory activity of WT animals evoked by a switch from 7% O2 to 150 ppm H2S balanced with 7% O2. (B) Reorientation movements (omega turns) of WT animals evoked by a switch from 7% O2 to 150 ppm H2S balanced with 7% O2. (C) Changes in reversal frequency in WT animals experiencing a switch from 7% O2 to 150 ppm H2S balanced with 7% O2. (D) Locomotory activity of WT was recorded for 2 minutes at 7% O2 and 148 minutes at 150 ppm H2S balanced with 7% O2 followed by 2 additional minutes at 7% O2. (E) Locomotory activity of WT animals evoked by acute hypoxia after preincubation for 148 minutes at 150 ppm H2S balanced with 7% O2. Locomotory activity was recorded for 2 minutes at each gas interval. (F) Locomotory responses of WT animals to different concentrations of H2S balanced with 7% O2. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively) used for statistical analysis. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test.

Acute response to H2S is regulated by multiple signaling pathways

(A–E) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and tax-6(ok2065) (A); WT and cnb-1(ok276) (B); WT and nhr-49(nr2041) (C); WT animals and WT animals starved for 24 hours (D); WT and daf-2(e1370) (E). To assay daf-2 animals, WT and daf-2 mutants were kept in 15°C. L4 animals were picked, maintained at 25°C until day-one adults and assayed at room temperature. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes), used for statistical analysis. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, ns = not significant, Mann–Whitney U test. (F–I) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, daf-11(m47) and transgenic daf-11 mutants expressing daf-11 genomic DNA in different subsets of neurons (F–H); The detailed expression pattern of each promoter is listed in (I). Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled as a and b respectively), used for statistical analysis. * = p < 0.05, ns = not significant, Mann–Whitney U test.

NPR-1 signaling modulates locomotory response to H2S

(A) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals and wild isolate CB4856. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes), used for statistical analysis. ** = p < 0.01, ns = not significant, Mann–Whitney U test. (B) Optogenetic stimulation of RMG neurons using channelrhodopsin-2 (ChR2). Expressing ChR2 in RMG neurons were achieved using Cre-LoxP system (Macosko et al., 2009). L4 animals were picked, grown for 16 hours on the seeded plates containing ATR, and assayed as day-one adults. Blue light was delivered throughout the assay. Red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes), used for statistical analysis. ** = p < 0.01, ns = not significant, Mann–Whitney U test. (C) Activation of O2 sensing neurons by expressing a gain-of-function allele of pkc-1 under gcy-37 promoter. Red bars on the x-axis represent two intervals (4–5 minutes and 11– 12 minutes), used for statistical analysis. * = p < 0.05, ns = not significant, Mann–Whitney U test.

Transcriptome reprogramming induced by H2S exposure

(A) Venn diagram displaying the number of differentially expressed genes after exposure in 50 ppm H2S balanced with 7% O2 in WT animals for 1 hour, 2 hours or 12 hours. Adjusted p value <1e-10. (B, C) Significantly enriched GO categories for differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm (B) or 150 ppm (C) H2S balanced with 7% O2 for 2 hours. (D, E) Volcano plot showing the differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm (D) or 150 ppm (E) H2S balanced with 7% O2 for 2 hours. A set of genes involved in H2S detoxification, cysteine metabolism and stress response were highlighted in red. (F, G) The number of HIF-1(F) and SKN-1(G) target genes induced by 50 ppm or 150 ppm H2S exposure for 1hour, 2 hours or 12 hours in WT animals, with Adjusted p value < 1e-30. (H–J) Venn diagrams displaying the number of differentially expressed genes (adjusted p value <1e-10) in WT animals of the indicated conditions: animals were exposed to 50 ppm or 150 ppm H2S for 1hour (H); for 2hours (I); and for 12 hours (J). (K, L) Volcano plots showing the differentially expressed genes (adjusted p value <1e-10) in WT animals that were exposed to either 50 ppm (K) or 150 ppm (L) H2S balanced with 7% O2 for 12 hours. A set of genes involved in H2S detoxification, cysteine metabolism and stress response were highlighted in red.

ftn-1 expression is consistently repressed in H2S

(A–D) Volcano plots showing the relative expression of the genes involved in the regulation of iron homeostasis (ftn-1, ftn-2 and smf-3) in WT animals of the indicated conditions: 1-hour exposure in 50 ppm H2S balanced with 7% O2 (A); 1-hour exposure in 150 ppm H2S balanced with 7% O2 (B); 12-hour exposure in 50 ppm H2S balanced with 7% O2 (C); and 12-hour exposure in 150 ppm H2S balanced with 7% O2 (D). (E, F) Locomotory responses to a switch to 7% to 1% O2 for animals of the indicated treatments or genotypes: WT animals, WT animals treated with 100 μM 2,2′-Bipyridyl (BP), and WT animals treated with 5 mg/ml ferric ammonium citrate (FAC) for 16 hours (E); WT and WT animals overexpressing ftn-1 genomic DNA under its own promoter (#1 and #2 indicate two independent lines) (F). Red bar on the x-axis represents time interval (3–4 minutes) used for statistical analysis. * = p < 0.05, ns = not significant, Mann–Whitney U test.