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ROS and cGMP signaling modulate persistent escape from hypoxia in Caenorhabditis elegans [1]

['Lina Zhao', 'Umeå Centre For Molecular Medicine', 'Umeå University', 'Umeå', 'Wallenberg Centre For Molecular Medicine', 'Umeå Centre For Microbial Research', 'Lorenz A. Fenk', 'Max Planck Institute For Brain Research', 'Frankfurt Am Main', 'Lars Nilsson']

Date: 2022-07

The ability to detect and respond to acute oxygen (O 2 ) shortages is indispensable to aerobic life. The molecular mechanisms and circuits underlying this capacity are poorly understood. Here, we characterize the behavioral responses of feeding Caenorhabditis elegans to approximately 1% O 2 . Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. While the behavioral responses to 1% O 2 closely resemble those evoked by 21% O 2 , they have distinct molecular and circuit underpinnings. Disrupting phosphodiesterases (PDEs), specific G proteins, or BBSome function inhibits escape from 1% O 2 due to increased cGMP signaling. A primary source of cGMP is GCY-28, the ortholog of the atrial natriuretic peptide (ANP) receptor. cGMP activates the protein kinase G EGL-4 and enhances neuroendocrine secretion to inhibit acute responses to 1% O 2 . Triggering a rise in cGMP optogenetically in multiple neurons, including AIA interneurons, rapidly and reversibly inhibits escape from 1% O 2 . Ca 2+ imaging reveals that a 7% to 1% O 2 stimulus evokes a Ca 2+ decrease in several neurons. Defects in mitochondrial complex I (MCI) and mitochondrial complex I (MCIII), which lead to persistently high reactive oxygen species (ROS), abrogate acute hypoxia responses. In particular, repressing the expression of isp-1, which encodes the iron sulfur protein of MCIII, inhibits escape from 1% O 2 without affecting responses to 21% O 2 . Both genetic and pharmacological up-regulation of mitochondrial ROS increase cGMP levels, which contribute to the reduced hypoxia responses. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute responses to hypoxia by C. elegans.

The remarkable ability of C. elegans to tolerate extreme O 2 concentrations, from near anoxia to 100% O 2 [ 20 ], makes it a good model to study O 2 responses in an intact, behaving, animal. By contrast to our detailed understanding of avoidance and escape from hyperoxia, little is known about how C. elegans responds to acute hypoxia [ 20 , 21 ]. Here, we characterize changes in C. elegans’ locomotory behavior when O 2 levels drop abruptly to hypoxic levels and delineate the importance of intracellular cGMP and mitochondrial ROS in the regulation of these responses.

Several studies implicate cGMP signaling in acute hypoxia sensing, with cGMP inhibiting the O 2 sensitivity of chemosensory organs [ 9 – 12 ]. For example, atrial natriuretic peptide (ANP), which enhances cGMP production, dampens acute vasoconstriction of the pulmonary artery in response to hypoxia [ 9 , 12 ]. Similarly, nitric oxide (NO) stimulated cGMP production represses the sensory activity of the carotid body [ 11 ]. How a rise in cGMP blocks the sensitivity of O 2 sensors is unclear. By contrast, it is well established in the nematode Caenorhabditis elegans that cGMP is the primary second messenger mediating responses to acute hyperoxia (21% O 2 ). C. elegans avoids both atmospheric (21%) and hypoxic (1%) O 2 concentrations [ 13 ]. When O 2 levels rise from 7% to 21%, atypical soluble guanylate cyclases in the O 2 sensing neurons URX, AQR, and PQR that directly bind O 2 become progressively activated, elevating cGMP levels, which opens cyclic nucleotide gated (CNG) channels and causes neuron depolarization [ 13 – 19 ].

Animals have evolved sophisticated mechanisms to maintain cellular homeostasis when encountering changes in oxygen (O 2 ) availability. O 2 -sensing mechanisms, including in the carotid body, vascular smooth muscle cells, and chromaffin cells of fetal adrenal medulla, react acutely to hypoxia to ensure sufficient oxygenation of critical organs [ 1 – 5 ]. O 2 -sensitive cells respond to hypoxia by closure of K + channels and subsequent opening of voltage-gated Ca 2+ channels. A variety of models have been proposed for how a drop in O 2 levels is detected and transmitted to the downstream K + channels. Accumulating evidence suggests that the rapid decrease of O 2 is detected by the mitochondria, which rapidly generate reactive oxygen species (ROS) to inhibit K + channels on the plasma membrane [ 1 , 6 – 8 ]. Perturbation of mitochondrial complex I (MCI) or mitochondrial complex III (MCIII) leads to continuous ROS production, which subsequently prevents acute response to hypoxia [ 1 , 6 – 8 ]. However, it remains unclear whether mitochondrial ROS directly modifies K + channels on the plasma membrane.

Results

Hypoxia evokes immediate and sustained changes in C. elegans behavior We set out to examine the effect of acute hypoxia on C. elegans locomotory behavior. A sudden decrease from 7% to 1% O 2 elicited a bout of turning maneuvers (omega turns and reversals, S1A and S1B Fig) followed by a switch to rapid forward movement (S1C Fig). Strikingly, while the increased turning behavior was transient, the locomotory arousal elicited by hypoxia was sustained for as long as O 2 levels remained low and showed no sign of adapting to repeated stimulation (S1D and S1E Fig). Acute hypoxia thus evokes both transient and sustained changes in C. elegans locomotory behavior, implying the existence of fast and slowly adapting neuronal mechanisms. To explore behavioral responses to more severe hypoxia, we shifted animals from 7% O 2 to pure nitrogen. The microfluidic chamber arena we used for our behavioral assays was not completely gastight, so pumping pure nitrogen into the chamber resulted in measured O 2 levels of approximately 0.2% (henceforth “near-anoxia”), whereas we measured approximately 1.2% O 2 (hypoxia) when we pumped 1% O 2 into the chamber. A shift to near-anoxia elicited a sharp increase in speed of locomotion that peaked soon after stimulation and then decreased slowly (S1F Fig). The speeds reached by animals in near-anoxia were lower than those of animals in hypoxia (S1F Fig). When switched back from near-anoxia to 7% O 2 , animals did not reduce speed immediately and often transiently increased their locomotory activity before slowing down (S1F Fig). These observations, together with earlier work [22,23], suggest acute behavioral responses to hypoxia and anoxia differ both quantitatively and qualitatively [20]. The behavioral responses to rapid up- and downshifts in O 2 , from 7% to 21% and from 7% to 1% O 2 , were remarkably similar [14,16], prompting us to ask if they share sensory pathways. Avoidance and escape from 21% O 2 are driven principally by AQR, PQR, and URX neurons and requires the heteromeric O 2 -binding soluble guanylate cyclase GCY-35/GCY-36 that acts upstream of the TAX-4/TAX-2 cGMP-gated ion channels [13–18]. Since the standard C. elegans lab strain, N2, responds weakly to 21% O 2 , due to a gain-of-function mutation in the npr-1 neuropeptide gene [13,24], we used an npr-1 null background to examine acute responses to 21% and 1% O 2 in the same animals. npr-1 mutants responded normally to 1% O 2 (S1G Fig). While gcy-35; npr-1 and tax-4; npr-1 null mutants showed defective responses to 21% O 2 , they reacted robustly to 1% O 2 (Fig 1A and 1B). Thus, distinct mechanisms underlie acute detection of hyperoxia and hypoxia in C. elegans. Responses to chronic hypoxia require the transcription factor HIF-1 and its negative regulator EGL-9 [25]. Both hif-1 and egl-9 mutants dramatically increased their locomotory speed in response to 1% O 2 , suggesting that responses to acute hypoxia do not require the core machinery mediating chronic hypoxia responses (Fig 1C). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Defects in IFT disrupt escape from hypoxia. (A and B) Locomotory responses to switches from 7% to 21% O 2 and 7% to 1% O 2 of animals of indicated genotype: npr-1(ad609) and npr-1(ad609); gcy-35(ok769) in (A) and npr-1(ad609) and npr-1(ad609); tax-4(p678) in (B). In this and subsequent figures, speed was recorded for 2 minutes at each O 2 interval. Red bars on the x-axis indicate time intervals used for statistical analysis. Data for each genotype are from 3 to 4 assays, >80 animals. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05, NS = not significant, Mann–Whitney U test. (C) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT (N2), hif-1(ia4), and egl-9(sa307) animals. NS = not significant. ANOVA, Tukey multiple comparison. (D) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT, dyf-3(m185), and dyf-3(m185) expressing dyf-3 cDNA from its endogenous promoter. Statistical comparison of the locomotory speed at 7% and 1% O 2 of each genotype: WT (****), dyf-3(m185) (NS), and dyf-3(m185) expressing dyf-3 cDNA from its endogenous promoter (****). **** = p < 0.0001, NS = not significant. ANOVA, Tukey multiple comparison. Statistical analysis of the locomotory speed at 1% O 2 of different genotypes were displayed in the figure. **** = p < 0.0001, NS = not significant. ANOVA, Tukey multiple comparison. (E) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT, bbs-9(gk471), and bbs-9(gk471) expressing bbs-9 cDNA in ciliated neurons from the osm-6 promoter. Statistical comparison of the locomotory speed at 7% and 1% O 2 of each genotype: WT (****), bbs-9(gk471) (NS), and bbs-9(gk471) expressing bbs-9 cDNA in ciliated neurons from the osm-6 promoter (****). **** = p < 0.0001, NS = not significant. ANOVA, Tukey multiple comparison. Statistical analysis of the locomotory speed at 1% O 2 of different genotypes were displayed in the figure. **** = p < 0.0001, NS = not significant. ANOVA, Tukey multiple comparison. (F) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT, dyf-3(m185), and dyf-3(m185) expressing dyf-3 cDNA in ciliated neurons from the osm-6 promoter, bbs-9(gk471), and bbs-9(gk471) expressing bbs-9 cDNA in ciliated neurons from the osm-6 promoter. Statistical comparison of the locomotory speed at 7% and 1% O 2 of each genotype: WT (****), dyf-3(m185) (NS), and dyf-3(m185) expressing dyf-3 cDNA in ciliated neurons from the osm-6 promoter (****), bbs-9(gk471) (NS), and bbs-9(gk471) expressing bbs-9 cDNA in ciliated neurons from the osm-6 promoter (****). **** = p < 0.0001, NS = not significant. ANOVA, Tukey multiple comparison. Statistical analysis of the locomotory speed at 1% O 2 of different genotypes were displayed in the figure. **** = p < 0.0001. ANOVA, Tukey multiple comparison. (G) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT, dyf-3(m185), and dyf-3(m185) mutants expressing dyf-3 cDNA from the sra-6 (ASH), srh-220 (ADL), and gpa-11 (ASH and ADL) promoters. Statistical comparison of the locomotory speed at 7% and 1% O 2 of each genotype: WT (****), dyf-3(m185) (*), and dyf-3(m185) mutants expressing dyf-3 cDNA from the sra-6 promoter (***), srh-220 promoter (****), and gpa-11 promoter (****). **** = p < 0.0001, *** = p < 0.001, * = p < 0.05. ANOVA, Tukey multiple comparison. Statistical analysis of the locomotory speed at 1% O 2 of different genotypes were displayed in the figure. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05. ANOVA, Tukey multiple comparison. (H) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT, bbs-9(gk471), and bbs-9(gk471) mutants expressing bbs-9 cDNA from the sra-6 (ASH), srh-220 (ADL), and gpa-11 (ASH and ADL) promoters. Statistical comparison of the locomotory speed at 7% and 1% O 2 of each genotype: WT (****), bbs-9(gk471) (NS), and bbs-9(gk471) mutants expressing bbs-9 cDNA from the sra-6 promoter (***), srh-220 promoter (****), and gpa-11 promoter (****). **** = p < 0.0001, *** = p < 0.001, NS = not significant. ANOVA, Tukey multiple comparison. Statistical analysis of the locomotory speed at 1% O 2 of different genotypes were displayed in the figure. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05. ANOVA, Tukey multiple comparison. The source code underlying behavioral data can be found at https://github.com/wormtracker/zentracker. IFT, intraflagellar transport; O 2 , oxygen; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001684.g001

BBSome acts in ciliated neurons to indirectly modulate cGMP production in AIA interneurons The fact that cGMP levels are elevated in bbs-9 mutants, and that GCY-28 is a major contributor to cGMP production, prompted us to speculate that GCY-28 expression or localization might be altered in bbs-9 mutants. However, we observed reduced instead of increased expression of GFP-tagged GCY-28.C and GCY-28.D in bbs-9 mutants, and no obvious change in the subcellular localization of GCY-28-GFP when expressed either from a single copy MosSCI insertion (GCY-28.C) or from extrachromosomal arrays (GCY-28.D) (S8A and S8B Fig). It is possible that GCY-28 activity, not expression, is affected by loss of the BBSome. However, since GCY-28 is not localized to the cilia [47] and its main action site does not overlap with BBSome expressing neurons, its activity is unlikely to be directly targeted by the BBSome. Moreover, bbs-9 mutants showed increased cGMP production even in the absence of GCY-28 (Fig 3D, columns 3 and 4), suggesting that other guanylate cyclases may be activated in a bbs-9 mutant background. Supporting this, mutations in several other guanylate cyclase genes besides gcy-28 partially suppressed the hypoxia response defects of bbs-9 mutants (S5B Fig). Taken together, we suspect that GCY-28 may not be directly involved in abnormal cGMP generation in bbs-9 mutants. However, because GCY-28 is a major guanylate cyclase in cGMP production (Fig 3D), loss of GCY-28 could effectively neutralize any increases in cGMP generated by other guanylate cyclases in bbs-9 mutants, thereby reversing their inhibition of acute responses to hypoxia. How does the BBSome, which acts in ciliated neurons, modulate cGMP production in downstream circuits such as the AIA neurons? Disrupting daf-19, which encodes an RFX transcription factor essential for ciliogenesis [49], attenuated but did not abolish hypoxia responses (S1 Table). This makes it unlikely that loss of acute responses to hypoxia in bbs-9 mutants reflects loss of cilia function. An alternative hypothesis, supported by previous studies, is that bbs mutants have acquired ectopic signaling capability, perhaps because they accumulate signaling components in their cilia due to defective retrograde traffic [50–54]. To test this, we asked if blocking accumulation of signaling molecules in cilia rescues the hypoxia response defects of bbs mutants. Indeed, eliminating the core IFT component OSM-6 suppressed the hypoxia response defects of bbs-9 to the levels found in osm-6 single mutants (Fig 5A). PPT PowerPoint slide

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TIFF original image Download: Fig 5. G protein signaling regulates acute responses to hypoxia. (A) Locomotory responses to 7% to 1% O 2 stimuli of animals with indicated genotypes: WT, bbs-9(gk471), osm-6(p811), and bbs-9(gk471); osm-6(p811) double mutants. *** = p < 0.001, NS = not significant. ANOVA, Tukey multiple comparison. (B) Locomotory responses to 7% to 1% O 2 stimuli of animals treated with 10mM histamine: WT, bbs-9(gk471), bbs-9(gk471) expressing HisCl1 from sra-6 (ASH), srh-220 (ADL) and gpa-11 (ASH and ADL) promoters. *** = p < 0.001, ** = p < 0.01, and NS = not significant. ANOVA, Tukey multiple comparison. (C–E) Locomotory responses to 7% to 1% O 2 stimuli of animals with indicated genotypes: WT, gpa-3(pk35), odr-3(n1605), and gpa-3(pk35) odr-3(n1605) (C); WT, egl-4(n478), gpa-3(pk35) odr-3(n1605), and gpa-3(pk35) odr-3(n1605); egl-4(n478) triple mutants (D); and WT, gcy-28(yum32), gpa-3(pk35) odr-3(n1605), and gpa-3(pk35) odr-3(n1605); gcy-28(yum32) triple mutants (E). **** = p < 0.0001, ** = p < 0.01, and NS = not significant. ANOVA, Tukey multiple comparison. (F) Total cGMP in worm lysates determined by cGMP enzyme immunoassay of indicated genotypes: WT and gpa-3(pk35) odr-3(n1605) double mutants. *** = p < 0.001. t test. (G and H) Locomotory responses to 7% to 1% O 2 stimuli of animals with indicated genotypes: WT, bbs-9(gk471), transgenic bbs-9(gk471) expressing gpa-3 and odr-3 transgenes from the gpa-3 promoter and their non-transgenic bbs-9(gk471) siblings (G); WT, gpa-3(pk35) odr-3(n1605), gpa-3(pk35) odr-3(n1605) expressing gpa-3 cDNA in ADL and ASH neurons (gpa-11p), in ASH (sra-6p) or ADL (srh-220p) alone (H). **** = p < 0.0001, *** = p < 0.001, and * = p < 0.05. ANOVA, Tukey multiple comparison. (I and J) Representative images (upper panel) and quantification (lower panel) of GFP-tagged markers in the cilia and neuronal cell bodies of WT and bbs-9(gk471) animals. Arrowheads point to cilia; arrows indicate neuronal cell bodies. The plots quantify GFP in cilia (left) and cell bodies (right). The average values in WT were arbitrarily set to 1, and the GFP signal in bbs-9(gk471) mutants normalized to WT. (I), ODR-3-GFP expressed from the gpa-11 promoter, ** = p < 0.01, * = p < 0.05, t test. (J), GPA-3-GFP expressed from the gpa-11 promoter, ** = p < 0.01, * = p < 0.05, t test. (K) ODR-10-GFP expression in the cilia of AWA neurons. Arrowheads indicate the AWA cilia. The GFP intensity of WT was arbitrarily set to 1, and the GFP signal of bbs-9(gk471) was normalized to WT. **** = p < 0.0001, t test. The underlying data can be found in S1 Data, and the source code can be found at https://github.com/wormtracker/zentracker. O 2 , oxygen; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001684.g005 Since bbs-9 hypoxia response defects were rescued by expressing bbs-9 cDNA in ADL and ASH neurons, we surmised that signals from these 2 neurons could be heightened in bbs-9 mutants. If this was the case, silencing ADL and ASH neurons should suppress the defects of bbs-9 mutants. Indeed, simultaneously but not individually inhibiting ADL and ASH neurons using the histamine-gated chloride channel HisCl1 partially restored locomotory responses to bbs-9 mutants (Fig 5B). By contrast, inhibiting or ablating these 2 neurons in the wild-type background did not affect responses to acute hypoxia (S8C and S8D Fig). These data suggest that abnormal signaling from ADL and ASH in bbs-9 mutants is at least partially responsible for loss of acute hypoxia responses in these animals.

BBSome modulates cGMP production through GPCR signaling We next probed the nature of the ectopic signaling in bbs mutants in ADL and ASH neurons. Mutants lacking the Gi/Go-like protein GPA-3, similar to bbs mutants, have high cGMP levels [55]. Disrupting gpa-3 weakly reduced the locomotory response to hypoxia (Fig 5C). By assaying mutants in other Gα subunits, we found that simultaneously disrupting gpa-3 and odr-3 strongly attenuated responses to acute hypoxia (Fig 5C). Mutations in gcy-28 or egl-4 suppressed this defect (Fig 5D and 5E) and, consistent with this, gpa-3 odr-3 double mutants showed significantly elevated cGMP (Fig 5F), suggesting that GPA-3 and ODR-3 redundantly inhibit cGMP production. We examined whether the BBSome and G proteins act in parallel or in the same pathway to modulate GCY-28 signaling. We found that simultaneously overexpressing gpa-3 and odr-3 restored hypoxia-evoked behavioral responses to bbs-9 mutants, suggesting that in genetic terms the BBSome acts upstream of G protein signaling to promote acute hypoxia responses (Fig 5G). We next examined if GPA-3 and ODR-3 act in the same neurons as BBSome. The behavioral defects of gpa-3 odr-3 double mutants were partially rescued by expressing gpa-3 under the gpa-11 promoter (Fig 5H). These observations suggest that G proteins, similar to the BBSome, act in ADL and ASH neurons to modulate locomotory responses to hypoxia. Since both GPA-3 and ODR-3 are localized to sensory cilia, we tested whether this localization was affected in bbs-9 mutants. The expression of both G proteins was significantly decreased in bbs-9 mutants compared to wild type, both in neuronal cell bodies and in cilia of ADL and ASH neurons (Fig 5I and 5J). ODR-3 expression in the cilia of AWA neurons showed the same tendency (S8E Fig). By contrast, and consistent with previous observations, levels of the ODR-10 olfactory GPCR in cilia increased >2-fold in bbs-9 mutants (Fig 5K) [53]. Together, these data suggest that disrupting the BBSome alters G protein localization and signaling in ciliated neurons to modulate cGMP production in downstream circuits.

Abnormal neuroendocrine secretion disrupts acute hypoxia responses BBSome mutants have been shown to enhance neuropeptide secretion from ADL neurons [51]. This increased neurosecretion can be abrogated by disrupting aex-6, the C. elegans ortholog of human RAB27, or by mutations in the RAB27 regulator RBF-1. These findings prompted us to ask if increased cGMP levels inhibit acute responses to hypoxia by promoting neurotransmission. Consistent with this, deleting aex-6 or rbf-1 restored acute hypoxia responses to bbs-9 mutants (Fig 6A and 6B). Moreover, enhanced secretion of the insulin DAF-28 and the FMRF-like peptide FLP-21 in bbs-9 mutants was suppressed by a mutation in gcy-28, although loss of gcy-28 did not alter release of these peptides in WT (Fig 6C–6F). In addition, the defective acute hypoxia responses in animals overexpressing gcy-28.c under its endogenous promoter were suppressed by mutations in rbf-1 (Fig 6G). These observations suggest that increased cGMP signaling enhances neuroendocrine secretion, which, in turn, inhibits behavioral responses to 1% O 2 . PPT PowerPoint slide

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TIFF original image Download: Fig 6. Increased neuropeptide secretion inhibits acute hypoxia responses. (A and B) Locomotory responses to 7% to 1% O 2 stimuli for indicated genotypes: WT, bbs-9(gk471), aex-6(sa24), and bbs-9(gk471); aex-6(sa24) double mutants in (A); and WT, bbs-9(gk471), rbf-1(js232), and bbs-9(gk471); rbf-1(js232) double mutants in (B). **** = p < 0.0001, ** = p < 0.01, ANOVA, Tukey multiple comparison. (C) Coelomocyte accumulation of DAF-28-mCherry secreted from ADL neurons in WT, bbs-9(gk471), gcy-28(yum32), and bbs-9(gk471) gcy-28(yum32) double mutants. Arrows indicate posterior coelomocytes. (D) Quantification of DAF-28-mCherry fluorescent intensity in one of the posterior coelomocytes. The mCherry intensity in WT (n = 40) was arbitrarily set to 1, and the mCherry signal in bbs-9(gk471) (n = 36), gcy-28(yum32) (n = 40), and bbs-9(gk471) gcy-28(yum32) (n = 40) mutants was normalized to WT. **** = p < 0.0001, NS = not significant. ANOVA, Tukey multiple comparison. (E) Coelomocyte accumulation of FLP-21-mCherry in WT, bbs-9(gk471), gcy-28(yum32), and bbs-9(gk471) gcy-28(yum32) double mutants. Arrows indicate posterior coelomocytes. (F) Quantification of FLP-21-mCherry fluorescent intensity in one of the coelomocytes. The mCherry intensity in WT (n = 30) was arbitrarily set to 1, and the mCherry signal in bbs-9(gk471) (n = 30), gcy-28(yum32) (n = 27), and bbs-9(gk471) gcy-28(yum32) (n = 28) mutants was normalized to WT. **** = p < 0.0001, ** = p < 0.01, and NS = not significant. ANOVA, Tukey multiple comparison. (G) Locomotory responses to 7% to 1% O 2 stimuli for animals of the indicated genotype: WT, rbf-1(js232) and gcy-28.c overexpression from the gcy-28.c promoter in WT and rbf-1(js232) background. *** = p < 0.001, NS = not significant, ANOVA, Tukey multiple comparison. The underlying data can be found in S1 Data, and the source code can be found at https://github.com/wormtracker/zentracker. O 2 , oxygen; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001684.g006

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