GLOBIN-5-Dependent O₂ Responses Are Regulated by PDL-1/PrBP That Targets Prenylated Soluble Guanylate Cyclases to Dendritic Endings

The neuroglobin GLB-5 regulates C. elegans responses to hypoxic exposure

Transcription of many globin genes, including some C. elegans globins, is induced by hypoxia (Sun et al., 2001; Hoogewijs et al., 2007). This regulated expression is proposed to protect against hypoxia/re-oxygenation damage (Burmester and Hankeln, 2009). To investigate if GLB-5 altered C. elegans responses to hypoxia/re-oxygenation stress, we exposed animals bearing the functional glb-5(Haw) allele found in natural isolates or the loss-of-function glb-5(Bri) allele found in N2 Bristol lab strain, to 1% O₂, returned them to 21% O₂, and looked for differences in behavior. In our assays we used animals that also carried the npr-1 215F allele or the npr-1(ad609) functional null allele, since the npr-1 215V gain-of-function allele found in N2 suppresses the activity of some O₂-sensing circuits (Gray et al., 2004; Cheung et al., 2005; Rockman and Kruglyak, 2009; Weber et al., 2010). We incubated the glb-5(Bri); npr-1(ad609) and glb-5(Haw); npr-1(ad609) animals (hereafter referred to as npr-1 and glb-5(Haw); npr-1 animals, respectively), and animals from the Hawaiian wild strain CB4856 (bearing the natural glb-5(Haw) and npr-1(215F) alleles) in 1% O₂ for 3, 6, 9, and 18 h, transferred them to 21% O₂, and examined their behavior after 30 min. When grown in 21% O₂ animals from all three strains gather where the bacteria grow thickest, at the border of the lawn (Fig. 1A; de Bono and Bargmann, 1998). Upon exposure to 1% O₂, animals from each strain left the food and accumulated outside the bacteria. When returned to normoxia, glb-5(Haw); npr-1 and CB4856 animals immediately accumulated at the lawn border, regardless of time in hypoxia. In contrast, npr-1 animals incubated in hypoxia for 18 h showed little preference for the border when returned to 21% O₂ (Fig. 1B). The hypoxia-induced inhibition of bordering was reversed within 4 h of return to normoxia (Fig. 1C). Bordering reflects an O₂ preference, with [O₂] ∼13% in the thicker bacteria at the border, and ∼17% O₂ at the lawn center (Gray et al., 2004). These results suggest that GLB-5(Haw) signaling enables C. elegans exposed to hypoxia and returned to normoxia to remain sensitive to these O₂ differences and to border.

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Figure 1.

GLB-5(Haw) modifies C. elegans responses to hypoxia/re-oxygenation. A, Bordering measures accumulation of animals where the bacterial food lawn grows thickest, at the border. B, Bordering behavior after incubation at 1% O₂ for various lengths of time. Bordering was measured 30 min after animals were returned to 21% O₂. glb-5(Haw) is the natural wild allele (yellow and red bars); glb-5(Bri) is a loss-of-function allele of glb-5 (blue bar) selected during domestication that expresses a truncated GLB-5 protein. Asterisks indicate significance compared with glb-5(Haw); npr-1 animals. C, Time course of recovery of bordering by animals previously exposed to hypoxia. Asterisks indicate significance for comparisons with glb-5(Haw); npr-1(ad609) animals. The schematic drawings on the bottom-right corner represent the full-length active GLB-5 (Hawaii) and the truncated nonactive GLB-5(Bri). D, A schematic diagram of pdl-1, highlighting the splice acceptor site mutation in the db805 allele; n = 4 or more assays performed over at least 3 d. ***p < 0.001, Kruskal–Wallis test with Dunn's post-test. Error bars represent SEM.

C. elegans PDE6δ/PrBP prenyl binding protein is required for GLB-5 function

How does GLB-5(Haw) alter O₂ sensing? To investigate this question we turned to forward genetics, and sought genes required for glb-5 signaling. Originally, we identified the glb-5(Haw) allele because it enables natural isolates of C. elegans to switch from slow to rapid movement on a bacterial lawn when O₂ rises from 17 to 21% (Persson et al., 2009). Although robust, this assay is too labor intensive for forward genetic screens. In contrast, identifying mutations that prevent glb-5(Haw); npr-1 animals from bordering after hypoxia exposure is amenable to this approach. We mutagenized glb-5(Haw); npr-1 animals and sought mutants that, after experiencing 1% O₂, failed to border immediately after return to 21% O₂. To exclude mutations that indiscriminately disrupted bordering, we selected lines that resumed bordering after recovering at 21% O₂ for 4 h. We isolated four mutants that satisfied these criteria. By combining Snip SNP mapping with Illumina whole genome sequencing (Wicks et al., 2001; Sarin et al., 2008) we mapped one allele, db805, to a 3.8 Mb genomic interval on chromosome II, and identified 10 mutations in this interval. Only one of these mutations altered a predicted open reading frame, disrupting a splice acceptor site in pdl-1 (Fig. 1D). pdl-1 encodes a 17 kDa protein orthologous to mammalian PDE6δ (phosphodiesterase 6, δ-subunit). PDL-1 and human PDE6δ share 69% amino acid identity at the protein level.

PDE6δ was originally identified as a subunit of PDE6 from bovine retina (Gillespie et al., 1989). Biochemical studies showed that it is a prenyl binding protein (PrBP) that can extract prenylated proteins such as small lipidated GTPases (e.g., Ras) from membranes, sequestering them in the cytoplasm, and facilitating their traffic to different membrane compartments (Chandra et al., 2012). Mice lacking PDE6δ are small but viable; they exhibit progressive cone–rod dystrophy and defective localization of prenylated rhodopsin kinase (GRK1) and PDE6 catalytic subunits to rod outer segments (Zhang et al., 2007).

To confirm the pdl-1 phenotype, we obtained a deletion allele, gk157, which removed most of the pdl-1 coding region and ∼0.5 kb of the upstream promoter region. pdl-1(gk157) delayed the recovery of bordering in glb-5(Haw); npr-1 animals exposed to hypoxia more strongly than the pdl-1(db805) mutation (Fig. 2A). Since pdl-1(gk157) is probably a null allele, we used it in subsequent experiments, unless otherwise indicated.

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Figure 2.

PDL-1 acts in the AQR, PQR, URX, and BAG neurons to promote GLB-5(Haw)-dependent O₂ responses. A, Disrupting pdl-1 inhibits the rapid recovery of bordering in glb-5(Haw); npr-1 animals returned to 21% O₂ after exposure to hypoxia. Bordering was measured 30 min after return to 21% O₂. Asterisks indicate significance for comparisons with glb-5(Haw); npr-1 animals. Kruskal–Wallis test with Dunn's post-test. B, pdl-1 is strongly expressed in the AQR, PQR, URX, and BAG O₂-sensing neurons, which are highlighted by a pglb-5::glb-5(Haw)::RFP fiduciary marker. Scale bars: 10 μm. C, Fast recovery of bordering behavior after prolonged hypoxia requires PDL-1 activity in the AQR, PQR, URX, and BAG neurons. Promoters used for transgenic rescue drive expression in the following: pglb-5 (AQR, PQR, URX, and BAG); pdl-1 promoter (multiple neurons); pgcy-33 (BAG); and pgcy-37 (AQR, PQR, and URX). Asterisks indicate significance for comparisons to glb-5(Haw); npr-1 animals after hypoxia treatment. Kruskal–Wallis test with Dunn's post-test. D, PDL-1 is required for GLB-5(Haw) to retune C. elegans O₂ sensitivity, and to enable glb(Haw);npr-1 animals to switch from rapid to slow movement when O₂ drops from 21 to 17%. Asterisks indicate significance for comparisons with glb-5(Haw); npr-1 at 17% O₂. Kruskal–Wallis test with Dunn's post-test. E, Disrupting pdl-1 slightly but significantly reduces C. elegans' speed of movement at 21% O₂ when glb-5 is defective. Asterisks indicate significance for comparisons with npr-1. One-way ANOVA with Bonferroni's multiple-comparison test. F, PDL-1 functions in O₂-sensing neurons to promote O₂-evoked changes in locomotor behavior; transgenes used are the same as in C. Kruskal–Wallis test with Dunn's post-test. Asterisks indicate significance for comparisons with glb-5(Haw); npr-1 animals at 17% O₂. G, RNAi knockdown of pdl-1 in AQR, PQR, and URX suppresses the retuning of O₂ responses mediated by glb-5(Haw). Asterisks indicate significance for comparisons with pdl-1; glb-5(Haw); npr-1; Ex ppdl-1::pdl-1::gfp at 17% O₂. Kruskal–Wallis test with Dunn's post-test; n ≥ 4, *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent SEM.

PDL-1 functions in the same neurons as GLB-5

Where does PDL-1 function to modify GLB-5-dependent behaviors? glb-5 is expressed in the AQR, PQR, URX, and BAG O₂-sensing neurons. Previous work showed that selectively expressing a glb-5(Haw) transgene in AQR, PQR, and URX neurons confers on npr-1 animals the sharply tuned O₂ responses of glb-5(Haw); npr-1 animals (McGrath et al., 2009; Persson et al., 2009). To identify where PDL-1 is expressed, we generated a transgene in which genomic DNA for pdl-1 was fused in-frame with a sequence-encoding GFP (Fig. 2B). Using behavioral assays, we showed that this transgene was fully functional (Fig. 2C,F). We then crossed it with a strain expressing a functional pglb-5::glb-5(Haw)::mCherry fusion construct, and looked for colocalization of green and red fluorescence in animals bearing both transgenes. pdl-1 and glb-5(Haw) were coexpressed in the AQR, PQR, URX, and BAG neurons (Fig. 2B). pdl-1 was also expressed in other neurons in the head and tail.

Is PDL-1 generally required for GLB-5(Haw) signaling to modify O₂ sensing, or are its effects specific to the hypoxia paradigm? To address this, we asked whether pdl-1 mutations affected the ability of normoxia-cultivated glb-5(Haw); npr-1 animals to switch from rapid movement at 21% O₂ to slow movement at 17% O₂ (McGrath et al., 2009; Persson et al., 2009). Consistent with a general role, pdl-1; glb-5(Haw); npr-1 animals moved much faster than glb-5(Haw); npr-1 animals at 17% O₂ (Fig. 2D). These data suggest that GLB-5(Haw) effects on O₂ responses depend on correct trafficking of prenylated proteins. Counterintuitively, disrupting pdl-1 also slightly but significantly reduced the rate of movement of npr-1 animals at 21% O₂ (Fig. 2E), suggesting PDL-1 can influence O₂ sensing independently of GLB-5, perhaps by affecting soluble guanylate cyclases. Does PDL-1 act in the same neurons as GLB-5 to regulate O₂ responses? Replacing the pdl-1 promoter with the glb-5 promoter enabled pdl-1; glb-5(Haw); npr-1 animals exposed to hypoxia to resume bordering rapidly after re-oxygenation, consistent with PDL-1 and GLB-5 functioning in the same cells (Fig. 2C). However, expressing pdl-1 specifically in the AQR, PQR, and URX neurons, by replacing the pdl-1 promoter with the gcy-37 promoter, or in BAG neurons, using the gcy-33 promoter, did not rescue the hypoxia-evoked decrease in bordering (Fig. 2C), suggesting pdl-1 is required in AQR, PQR, URX, and BAG. Consistent with this, coexpressing the pgcy-33::pdl-1 and pgcy-37::pdl-1 transgenes rescued bordering behavior after hypoxia (Fig. 2C).

PDL-1 also acts in the AQR, PQR, URX, and BAG neurons to enable normoxia-cultivated glb-5(Haw); npr-1 animals to switch from rapid movement at 21% O₂ to slow movement at 17% O₂. This phenotype could be rescued by expressing pdl-1 from its own promoter, or from the glb-5 promoter (Fig. 2F). As for bordering behavior, expressing pdl-1 specifically in the AQR, PQR, and URX neurons, using the gcy-37 promoter, or only in BAG neurons, using the gcy-33 promoter, failed to rescue glb-5(Haw)-dependent responses to a switch from 21 to 17% O₂. In contrast, coexpressing the two transgenes rescued the behaviors (Fig. 2F). To confirm our results, we made RNAi constructs under the control of the gcy-37 promoter that targeted pdl-1 or GFP sequences. We injected these into pdl-1; glb-5(Haw); npr-1 mutant animals that expressed a pdl-1(cDNA)-GFP fusion construct from the pdl-1 promoter. Whereas animals not expressing the RNAi constructs strongly reduced movement when switched from 21 to 17% O₂, animals expressing the RNAi transgenes did not (Fig. 2G). These data suggest that PDL-1 acts in AQR, PQR, URX, and BAG neurons to promote glb-5(Haw)-dependent O₂ responses in behavioral paradigms that operate at very different timescales.

PDL-1/PrBP mediates localization of soluble guanylate cyclases to dendritic endings

Mammalian PrBP/PDE6δ interacts with prenylated proteins and nonprenylated small GTPases and regulates their traffic (Hanzal-Bayer et al., 2002). GLB-5 is in neither of these categories, but three of the atypical soluble guanylate cyclases required for O₂ responses—GCY-35 and GCY-36 in AQR, PQR and URX neurons, and GCY-33 in BAG neurons—are predicted to have C-terminal -CAAX sequences, suggesting they are prenylated (Cheung et al., 2004, 2005; Gray et al., 2004; Persson et al., 2009; Zimmer et al., 2009; Busch et al., 2012). These heme proteins are thought to directly bind O₂, andO₂ binding is thought to modulate their cGMP production and to change neural activity by gating cGMP ion channels (Couto et al., 2013). The functional importance of the CAAX motif has been directly tested for the GCY-36 soluble guanylate cyclase. Mutating sequences encoding the CAAX motif to encode SAAX disrupted GCY-36 localization to dendritic endings and resulted in a nonfunctional gcy-36 gene (Cheung et al., 2004).

To examine if PDL-1 regulates trafficking of GCY-35 and GCY-33, we made transgenes that expressed functional fluorescently tagged versions of these proteins. Tagging GCY-35 N terminally or C terminally interfered with its biological activity. We therefore inserted gfp cDNA into sequences encoding a predicted flexible loop in the GCY-35 C-terminal tail. Like GLB-5(Haw), GCY-35-GFP was enriched at the dendritic tips of URX neurons (Fig. 3A). Moreover, this transgene largely (although not completely) restored O₂ control of locomotor activity to gcy-35; npr-1 mutants (Fig. 3B). We compared the accumulation of GCY-35::GFP in the dendrite and cell body of URX neurons in gcy-35; npr-1 and gcy-35; pdl-1; npr-1 animals (Fig. 3C,D). While fluorescence in the cell body was very similar in the two genotypes, loss of pdl-1 essentially abolished GCY-35-GFP accumulation at the tip of URX dendrites. Thus, pdl-1 is required for correct localization of GCY-35 at dendritic endings. However, localization of GCY-35 to dendrites is not essential for O₂-evoked responses.

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Figure 3.

PDL-1 regulates GCY-35 and GCY-33 localization. A, GCY-35::GFP accumulates at the dendritic tips of URX neurons. Scale bars: 10 μm. B, Expressing a gcy-35::GFP fusion protein in the AQR, PQR, and URX neurons rescues gcy-35; npr-1 mutant phenotypes. Asterisks indicate significant differences from gcy-35; npr-1 animals at 21% O₂. Kruskal–Wallis test with Dunn's post-test. C, PDL-1 is required for GCY-35::GFP to accumulate at the dendritic tips of URX dendrites, but not in the cell body. Scale bars: 10 μm. D, Quantitation of GCY-35::GFP accumulation in dendrites and cell bodies of gcy-35; npr-1 and gcy-35; pdl-1; npr-1 animals; n ≥ 10, Mann–Whitney test. E, Hypoxia does not reduce GCY-35::GFP accumulation in the URX dendrites in gcy-35; npr-1 or gcy-35; glb-5(Haw); npr-1 animals; n ≥ 16, Mann–Whitney test. F, Yeast two-hybrid assay. Diploid growth on selectable plates containing SD/-Leu/-Trp, X-α-Gal, and Aureobasidin A. Top, PDL-1 interacts strongly with GCY-35 but not ARL-3 or ARL-13; the interactions of SV40-T with p53 and lamin provide positive and negative controls, respectively. Mutating the CAAX prenylation motif to SAAX inhibits GCY-35–PDL-1 interaction. Middle and lower left, Colonies of diploids coexpressing PDL-1 with RAC-2 or GCY-33 were much smaller compared withPDL-1::GCY-35 colonies, indicating that the interaction between these proteins and PDL-1 is weak. Supporting this, PDL-1::GCY-33 diploids did not grow on a more restrictive media (-Trp/-Leu/-Ade/-His/Aureobasidin A/X-α-Gal; lower right). G, Disrupting PDL-1 reduces accumulation of mcherry-tagged GLB-5(Haw) in both dendrites and cell bodies. Scale bar, 10 μm. H, Quantification of GLB-5(Haw)::mCherry fluorescence in the dendritic ending and cell body of glb-5-expressing head neurons in npr-1 and pdl-1; npr-1 animals. Data represent an average of at least 10 animals, Mann–Whitney test. I, PDL-1 regulates GCY-33::GFP localization in BAG neurons. Disrupting pdl-1 reduces accumulation of GCY-33::GFP at the tips of BAG dendrites (marked by arrows). Scale bar, 10 μm. J, Quantification of GCY-33::GFP fluorescence at the dendritic ending and cell body of BAG neurons in gcy-33; npr-1 and pdl-1; gcy-33; npr-1 animals. Data represent an average of at least 10 animals, Mann–Whitney test. *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent SEM.

We also examined if prolonged exposure to hypoxia (18 h) altered levels and distribution of GCY-35 in the URX neurons. We incubated gcy-35; npr-1 and gcy-35; glb-5(Haw); npr-1 animals in 1% or 21% O₂ and quantified GCY-35-GFP fluorescence in dendrites (Fig. 3E). Exposure to hypoxia did not change GCY-35 levels in the dendrites of npr-1 animals or glb-5(Haw); npr-1 animals. We observed reduced cell body expression of GCY-35::GFP in glb-5(Haw); npr-1 animals following hypoxia, but this may reflect regulation of transcription from the gcy-37 promoter (Cheung et al., 2005).

To test if PDL-1 can directly interact with GCY-35, we performed yeast two-hybrid experiments (Y2H). We used PDL-1 as bait, mated it with a prey strain expressing GCY-35, and examined diploid growth on selective plates (Fig. 3F). The rapid growth of the pdl-1/gcy-35 diploids suggested that GCY-35 and PDL-1 physically interact. To determine whether this interaction required prenylation of GCY-35, we disrupted the CAAX prenylation motif by a cysteine-to-serine codon change. PDL-1 failed to interact with this mutant form of GCY-35. These results suggest that PDL-1, like its mammalian counterpart, is a prenyl binding protein involved in protein transport. More specifically, it is required to traffic GCY-35 to the nonciliated dendritic endings of URX by a prenylation-dependent physical interaction.

Mammalian PrBP/PDE6δ can also interact with the nonprenylated Arf-like (Arl) small GTPases, Arl2-GTP and Arl3-GTP. We therefore used the Y2H assay to examine if PDL-1 can also interact with arl-3 and arl-13. No significant growth was observed (Fig. 3F). Interestingly, we did observe a weak but reproducible interaction with the rac-2 GTPase (Fig. 3F).

Like GCY-35-GFP, GLB-5(Haw)-mcherry is enriched at the dendritic endings of URX neurons (McGrath et al., 2009; Persson et al., 2009). We therefore tested if GLB-5(Haw)-mcherry localization or expression was also regulated by pdl-1. GLB-5(Haw)-mcherry levels were significantly decreased in both URX dendrites and cell bodies (Fig. 3G,H). Thus, PDL-1 directly or indirectly regulates the expression and/or stability of GLB-5(Haw).

Our expression studies and cell-specific rescue experiments suggested that pdl-1 functions not only in AQR, PQR, and URX but also in BAG sensory neurons (Fig. 2C,F). BAG neurons express GCY-33, a soluble guanylate cyclase that also has a C-terminal CAAX prenylation motif. GCY-33::GFP accumulated strongly at the dendritic endings of BAG (Fig. 3I). This dendritic fluorescence was reduced in pdl-1; gcy-33; npr-1 mutants although not abolished (Fig. 3I,J), suggesting that PDL-1 contributes to but is not solely responsible for localizing GCY-33 to BAG dendritic endings. Levels of GCY-33::GFP in the cell body were not altered in pdl-1 mutants (Fig. 3J).

To test if PDL-1 and GCY-33 can interact directly, we set up Y2H experiments, as described previously for GCY-35. GCY-33/PDL-1-expressing diploids formed detectable but much smaller colonies than GCY-35/PDL-1 diploids, suggesting a weaker interaction. Consistent with this, when we tested the ability of PDL-1/GCY-33 diploids to grow on a more restrictive medium, containing six selectable markers (-Trp/-Leu/-Ade/-His/Aureobasidin A/X-α-Gal), the PDL-1/GCY-33 diploids did not form colonies whereas the GCY-35/PDL-1 diploids did (Fig. 3F, button, right). Our results suggest that although GCY-33 localization in BAG dendritic endings is regulated by PDL-1, the interaction between PDL-1 and GCY-33 may involve additional proteins. Moreover, PDL-1-independent pathways exist to traffic GCY-33, since a small but significant amount of GCY-33 is found in BAG dendritic endings even in the absence of PDL-1 (Fig. 3I,J).

O₂-evoked Ca²⁺ responses in URX in pdl-1 mutants

To investigate how loss of pdl-1 and mislocalization of soluble guanylate cyclases altered the physiology of O₂-sensing neurons, we monitored O₂-evoked responses in these neurons using the Ca²⁺ sensor YC2.60 (Nagai et al., 2004). YC2.60 has higher Ca²⁺ affinity than the YC3.60 sensor we used previously, providing a better readout of intermediate Ca²⁺ levels (Persson et al., 2009; Busch et al., 2012). The ratiometric nature of YC Ca²⁺ sensors permits a steady-state level of Ca²⁺ to be compared across genotypes, which is informative since O₂-sensing neurons signal tonically (Persson et al., 2009; Busch et al., 2012). We began by examining animals grown at normoxia.

We compared Ca²⁺ responses evoked in URX by two O₂ profiles: 21%–17%–21% O₂ and 21%–7%–21% O₂. We included the 21%–7%–21% profile as a control because the neuronal activity of the O₂-sensing neurons and the speed of npr-1 animals at 7% O₂ is the same regardless of the glb-5 genotype (Persson et al., 2009). The 21%–17%–21% O₂ stimulus train probes the ability of GLB-5(Haw) to alter the dynamic range of the O₂ response (Fig. 4A,B; Persson et al., 2009). As expected, the glb-5(Haw) allele sharpened the tuning of URX: at 17% O₂ Ca²⁺ was significantly lower in glb-5(Haw); npr-1 animals compared with npr-1 animals, and was indistinguishable from Ca²⁺ at 7% O₂ (Fig. 4A,B). These results mirror the behavioral data: glb-5(Haw); npr-1 animals switch to slow movement at much higher O₂ levels than npr-1 animals (Persson et al., 2009). Despite the GCY-35 localization defects, and consistent with our behavioral observations, O₂ stimuli evoked robust Ca²⁺ responses in URX in both pdl-1; npr-1 and pdl-1; glb-5(Haw); npr-1 animals (Fig. 4C,D). However, URX Ca²⁺ at 17% O₂ was significantly higher in pdl-1; glb-5(Haw); npr-1 compared with glb-5(Haw); npr-1 (Fig. 4E). This correlated with the greater locomotor activity of pdl-1; glb-5(Haw); npr-1 animals kept at 17% O₂ compared with glb-5(Haw); npr-1 animals (Fig. 2D,F). Our results suggest that localizing the GCY-35 O₂ sensor to the dendritic endings of URX is not essential for transducing O₂ stimuli, but that PDL-1 activity modifies O₂-evoked Ca²⁺ signaling in URX.

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Figure 4.

Disrupting PDL-1 alters O₂-evoked Ca²⁺ responses in the URX neurons. A–D, Ca²⁺ responses evoked in URX by 21%–7%–21% and 21%–17%–21% O₂ stimulus trains measured using the YC2.60 sensor in npr-1 (A), glb-5(Haw); npr-1 (B), pdl-1; npr-1 (C), and pdl-1; glb-5(Haw); npr-1 (D) animals. Asterisks indicate significant differences in Ca²⁺ levels at 17 and 7% O₂ in the 30 s before the transition to 21% O₂, Mann–Whitney test. Gray shading represents SEM. E, Bar graph comparing tonic URX Ca²⁺ levels at 17% O₂ in various genotypes (data taken from A–D). Asterisks indicate significant differences compared with glb-5(Haw); npr-1. Kruskal–Wallis test with Dunn's post-test. Error bars represent SEM, *p < 0.05, ***p < 0.001.

GLB-5 and PDL-1 modify O₂-evoked Ca²⁺ responses in BAG neurons

We next investigated how the glb-5(Haw) allele and pdl-1 contributed to O₂-evoked Ca²⁺ responses in BAG neurons in normoxia-grown animals. Previous work showed that a fall in O₂ concentration evokes a rise in Ca²⁺ in BAG neurons (Zimmer et al., 2009). Our behavioral experiments suggested BAG neurons were part of the circuit that enables feeding glb-5(Haw); npr-1 animals to reduce their locomotor activity sharply when O₂ decreases from 21 to 17% (Fig. 2F). To monitor BAG Ca²⁺ we expressed YC2.60 using the flp-17 promoter. We measured Ca²⁺ changes evoked by a series of O₂ downsteps in well fed animals in the absence of food. We could detect small but robust rises in Ca²⁺ when O₂ fell from 21 to 17%; as expected Ca²⁺ responses became bigger as we reduced O₂ levels further (Fig. 5A). Thus, in glb-5(Haw); npr-1 animals even small decreases in O₂ evoked an increase In BAG Ca²⁺.

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Figure 5.

O₂-evoked Ca²⁺ responses in the BAG neurons. A, Dynamic range of O₂-evoked responses in BAG measured using YC2.60. Asterisks indicate significant differences between Ca²⁺ levels at 21% O₂ and at the lower O₂ tension during indicated time intervals, Mann–Whitney test. B, 21%–7%–21% O₂ stimulus trains evoked strong Ca²⁺ responses in BAG in npr-1, glb-5(Haw); npr-1, and pdl-1; glb-5(Haw); npr-1 animals, but not in gcy-33, npr-1 animals. Asterisks indicate significant differences for each genotype between Ca²⁺ levels at 21% O₂ and at 7% O₂ during indicated time intervals (Mann–Whitney test) or significant differences between npr-1 and other genotypes at 7% O₂ during indicated time intervals. Kruskal–Wallis test with Dunn's post-test. C, Deleting pdl-1 attenuates O₂-evoked Ca²⁺ responses in BAG in npr-1 animals. Asterisks indicate significant differences between npr-1 and pdl-1; npr-1 animals exposed to 7% O₂ at times indicated, as well as significant differences in Ca²⁺ at 21 and 7% O₂ within each strain. Mann–Whitney test. The data presented in B were collected on a different imaging setup from that used in A and C. *p < 0.05, **p < 0.01, and ***p < 0.001. Gray shading represents SEM. D, GLB-5(Haw) restores localization of GCY-33-GFP to the cilia of BAG neurons in pdl-1; gcy-33; npr-1 mutants. ****p < 0.0001, Mann–Whitney test. Error bars represent SEM.

The small size of the Ca²⁺ response evoked in BAG by a switch from 21 to 17% O₂ (Fig. 5A) precluded us from genetically dissecting this response. Instead, we studied responses evoked by a switch from 21 to 7% O₂. As expected from previous work (Zimmer et al., 2009), O₂-evoked Ca²⁺ responses in BAG were eliminated in mutants defective in gcy-33 (Fig. 5B).

To investigate how pdl-1 modifies BAG signaling, we first compared O₂-evoked responses in npr-1 and pdl-1; npr-1 mutants. Disrupting pdl-1 attenuated the rise in Ca²⁺ evoked by a switch from 21 to 7% O₂ (Fig. 5C). Unexpectedly, the presence of GLB-5(Haw) compensated for the loss of pdl-1; Ca²⁺ responses evoked by a 21–7% O₂ stimulus in pdl-1; glb-5(Haw); npr-1 and glb-5(Haw); npr-1 animals were similar (Fig. 5B). Why? If the BAG response defect in pdl-1; npr-1 animals reflected loss of GCY-33 localization to cilia, then GLB-5(Haw) should rescue not only the Ca²⁺ response of pdl-1; npr-1 animals, but also the GCY-33 localization defect. This is in fact the case: GLB-5(Haw) restored GCY-33 accumulation at the dendritic endings of BAG to pdl-1; npr-1 animals, without affecting GCY-33 levels in the cell body (Fig. 5D). Together, our results suggest that GCY-33 localization at the dendritic-endings of BAG is important for BAG O₂ sensing. Moreover, since GLB-5(Haw) is involved in GCY-33 trafficking to or retention in cilia, our data suggest that GLB-5 and GCY-33 are part of a complex in vivo.

Hypoxia modifies O₂-sensor physiology

Having explored how GLB-5(Haw) and PDL-1 activity shaped the O₂ responses of BAG and URX neurons in animals grown in normoxia, we explored how these genes influenced the physiology of O₂-sensing neurons after hypoxia experience. We kept animals in 1% O₂ for 18 h and then imaged O₂-evoked Ca²⁺ responses. For these experiments, individual worms were transferred from the hypoxia chamber and rapidly glued to agarose pads in a 21% O₂ environment. All experiments were done in the presence of food as described previously, and begun within 5 min from the time of transfer. For URX, we chose a 21%–17%–21% O₂ stimulus train to evoke neuronal activity, since it mimics the O₂ concentration difference between the bacterial lawn (∼17% O₂) and the surrounding agar (∼21% O₂) in the bordering assay plates. Ca²⁺ responses were undetectable in npr-1 animals following hypoxic cultivation (Fig. 6A). In contrast, glb-5(Haw); npr-1 animals retained strong O₂-evoked Ca²⁺ responses in URX after exposure to hypoxia (Fig. 6B). These data suggest that hypoxia/re-oxygenation reconfigures the activity of URX in npr-1 animals, and that the glb-5(Haw) allele either suppresses this reconfiguration or enables rapid recovery. In hypoxia-conditioned pdl-1; glb-5(Haw); npr-1 animals, URX neurons responded less robustly than glb-5(Haw); npr-1 controls to O₂ stimuli (Fig. 6C,D), suggesting that the protective effect of GLB-5(Haw) is partly dependent on PDL-1. These imaging results are consistent with our behavioral observations that hypoxia experience inhibits bordering in npr-1 and pdl-1; glb-5(Haw); npr-1 but not glb-5(Haw); npr-1 animals (Fig. 2A,C).

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Figure 6.

O₂-evoked Ca²⁺ responses in URX neurons after prolonged hypoxia. Ca²⁺ responses evoked by 21%–17%–21% O₂ stimulus trains in URX after 18 h incubation at 1% O₂ measured using YC2.60. A, B, Prolonged hypoxia abolishes URX responsiveness to changes in O₂ in npr-1 animals (A), but has much weaker effects on glb-5(Haw); npr-1 animals (B). C, Deleting pdl-1 reduced URX responsiveness of glb-5(Haw); npr-1 animals in both normoxia and hypoxia cultivated animals, and led to higher Ca²⁺ levels at 17% O₂. D, Bar graph comparing tonic URX Ca²⁺ levels at 21% O₂, just after the switch from 17% O₂ to 21% O₂, in various genotypes; data taken from A–C (21%–17%–21% O₂ responses after 18 h in hypoxia); baseline normalized ratios. Asterisks indicate significant differences compared with glb-5(Haw); npr-1. Mann–Whitney test, *p < 0.05. Error bars represent SEM. E, 21%–7%–21% O₂ stimulus trains evoked strong Ca²⁺ responses in BAG in npr-1, glb-5(Haw); npr-1, and pdl-1; glb-5(haw); npr-1 animals. F, The 21%–7%–21% O₂-evoked Ca²⁺ responses in BAG were smaller after hypoxia (for comparison see E). Asterisks indicate significant differences between Ca²⁺ level just after the shift from 21 to 7% O₂ at times indicated by the bars, Mann–Whitney test. Gray shading represents SEM. G, Disrupting hif-1 did not prevent the fast recovery of bordering behavior in glb-5(Haw); npr-1 animals following exposure to hypoxia. Asterisks indicate significance for comparisons with glb-5(Haw); npr-1 animals. Kruskal–Wallis test with Dunn's post-test, n ≥ 4, *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent SEM.

To explore if the physiology of BAG neurons was also reconfigured by hypoxia, we compared O₂-evoked Ca²⁺ responses in animals kept for 18 h at 21% or 1% O₂ (Fig. 6E,F). The 21%–7%–21% O₂ stimulus trains elicited smaller Ca²⁺ responses in npr-1 and glb-5(Haw); npr-1 animals after exposure to hypoxia (Fig. 6E,F). These results suggest that like in URX, hypoxia experience reconfigures BAG O₂ responses; however, unlike in URX, the presence of GLB-5(Haw) or absence of PDL-1 did not appear to have strong effects. One caveat is that expression of the YC2.60 sensor in BAG neurons was decreased in hypoxia-treated animals, complicating comparisons across O₂ conditions.

GLB-5(Haw) suppresses behavioral reconfiguration by hypoxia in an HIF-1-independent mechanism

The reconfiguration of bordering behavior by hypoxia occurred over hours (Fig. 1B), suggesting that transcriptional regulation is involved. Since HIF-1 is a key factor in adaptation to hypoxia (Semenza and Wang, 1992; Jiang et al., 2001), we wondered if HIF-1 was involved. To test this, we examined the bordering behavior of hif-1(ia4); npr-1; pglb-5:glb-5(Haw) animals after hypoxia (Fig. 6G). hif-1(ia4) animals survived well in 24 h hypoxia, and upon exposure to 1% O₂, left food and accumulated outside the bacteria. When returned to 21% O₂, hif-1(ia4); npr-1; pglb-5:glb-5(Haw) animals rapidly resumed bordering behavior, like glb-5(Haw); npr-1 animals. These results suggest that HIF-1 is not required for GLB-5(Haw) to promote rapid recovery from hypoxia exposure, although we have not examined if it required for npr-1 animals to inhibit bordering following hypoxia experience.

GLB-5(Haw) enables URX neurons adapted to hypoxia to rapidly retune their O₂ response set point upon return to normoxia

GLB-5(Haw) counteracts the effects of prolonged hypoxia on behavior and URX physiology after animals are returned to normoxia. We speculated that GLB-5 could do this in one of two ways: by preventing hypoxia from reconfiguring the physiology of URX or by enabling rapid recovery of URX response properties when animals exposed to prolonged hypoxia are returned to 21% O₂. In the second model URX recovery would need to be fast, since glb-5(Haw); npr-1 animals showed substantial O₂-evoked Ca²⁺ responses within 5 min of transfer from hypoxia to 21% O₂ (Fig. 6B).

To differentiate between these possibilities we built an airtight microfluidic-imaging chamber that enabled us to measure URX response properties in hypoxia-grown animals without prior exposure to 21% O₂. Since gluing animals in the hypoxia chamber was difficult, we immobilized animals using the cholinergic agonist levamisole (see Materials and Methods). We grew npr-1 and glb-5(Haw); npr-1 animals for 18 h in either 1% or 21% O₂, transferred them to the imaging scope, and shifted them from 1 to 7% O₂. To compare the effects of acute and prolonged hypoxia, we also exposed npr-1 and glb-5(Haw); npr-1 animals cultivated at 21% O₂ to a 21%–1%–7–21% O₂ stimulus train. Animals adapted to 1% O₂ for 18 h exhibited large persistent increases in URX Ca²⁺ when shifted to 7% O₂ (Fig. 7A,B), whereas animals exposed to 1% O₂ for only 5 min exhibited a smaller, transient increase in Ca²⁺ to the same 7% O₂ stimulus (Fig. 7C–E). The effects of prolonged hypoxia on URX activity after O₂ levels were raised for the first time from 1 to 7% were independent of GLB-5(Haw) activity (Fig. 7A,B). This contrasts with the marked difference in O₂-evoked Ca²⁺ responses in URX when hypoxia-treated npr-1 and glb-5(Haw); npr-1 animals were exposed to 21% O₂ (Fig. 6A,B). These data suggest that hypoxia can reprogram URX activity in both npr-1 and glb-5(Haw); npr-1 animals, but that recovery after further exposure to 21% O₂ is much faster in glb-5(Haw); npr-1 than npr-1 animals.

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Figure 7.

Prolonged hypoxia reconfigures URX O₂ responses in npr-1 and glb-5(Haw); npr-1 animals. A, B. Ca²⁺ responses evoked in URX by 7% O₂ after 18 h incubation in 1% O₂. The Ca²⁺ sensor is YC2.60. Animals that were incubated in 1% O₂ did not experience 21% O₂ before the 7% O₂ stimulus. The transition from hypoxia to 7% O₂ elicited an increase in Ca²⁺ in npr-1 and glb-5(Haw); npr-1 animals. Asterisks indicate significant differences at times indicated by bars between the Ca²⁺ levels before the transition to 7% O₂, Mann–Whitney test. C, Brief exposure to 1% O₂ followed by a 7% O₂ stimulus did not elicit a strong and enduring increase in Ca²⁺ concentration in npr-1 and glb-5(Haw); npr-1 animals. Asterisks indicate significant differences compared with the Ca²⁺ level in 1% O₂, at times indicated by bars just before the transition to 7% O₂, Mann–Whitney test. D, E, Prolonged exposure to hypoxia reconfigures URX O₂ responses. A comparison of responses evoked by a switch from 1 to 7% O₂ in npr-1 and glb-5(Haw); npr-1 animals (from A and C; B and C, baseline normalized ratios). Asterisks indicate significant differences compared with the Ca²⁺ level in 1% O₂ after 3 min in 7% O₂. Mann–Whitney test, *p < 0.05, **p < 0.01, and ***p < 0.001. Gray shading represents SEM. F, Model for PDL-1 activity in the URX and BAG neurons. Prenylated GCY-33 and GCY-35 interact with PDL-1, which facilitates their traffic to dendritic endings. There, they act together with GLB-5(Haw) to tune the dynamic range of O₂ responses, and to rapidly retune O₂-sensing properties after return to normoxia following prolonged hypoxia.