Caenorhabditis elegans HIF-1 Is Broadly Required for Survival in Hydrogen Sulfide

Abstract

Hydrogen sulfide is common in the environment, and is also endogenously produced by animal cells. Although hydrogen sulfide is often toxic, exposure to low levels of hydrogen sulfide improves outcomes in a variety of mammalian models of ischemia-reperfusion injury. In Caenorhabditis elegans, the initial transcriptional response to hydrogen sulfide depends on the hif-1 transcription factor, and hif-1 mutant animals die when exposed to hydrogen sulfide. In this study, we use rescue experiments to identify tissues in which hif-1 is required to survive exposure to hydrogen sulfide. We find that expression of hif-1 from the unc-14 promoter is sufficient to survive hydrogen sulfide. Although unc-14 is generally considered to be a pan-neuronal promoter, we show that it is active in many nonneuronal cells as well. Using other promoters, we show that pan-neuronal expression of hif-1 is not sufficient to survive exposure to hydrogen sulfide. Our data suggest that hif-1 is required in many different tissues to direct the essential response to hydrogen sulfide.

Keywords: hydrogen sulfide; hypoxia; tissue-specific expression.

Figure 1

HIF-1 expression from the unc-14 promoter rescues the H₂S lethality of hif-1(ia4) mutant animals. (A) Survival of animals exposed to H₂S. All animals have the null hif-1(ia4) mutation. The otIs197 integrated array expresses a nondegradable HIF-1 variant. Other constructs were extrachromosomal arrays that express wild-type HIF-1. The unc-14 promoter is expressed pan-neuronally (Ogura et al. 1997), the rab-3 promoter is expressed in most, if not all, neurons (Nonet et al. 1997), unc-17 is expressed in cholinergic neurons (Rand et al. 2000), and unc-47 is expressed in GABAergic neurons (Eastman et al. 1999). Animals were exposed to 50 ppm H₂S starting at L4. (B) HIF-1 gene structure and predicted A and E isoforms (Wormbase 2017). The P621A mutation that prevents degradation of hif-1 included in otIs197 is marked with *. (C) Survival of animals expressing HIF-1 from unc-14 promoter exposed to H₂S. All animals have the null hif-1(ia4) mutation. Expression of HIF-1 was from extrachromosomal arrays. The yakEx137 array expresses nondegradable HIF-1(P621A) and the yakEx144 array expresses wild-type hif-1. For all panels, animals were exposed to 50 ppm H₂S starting at L4. Average of three independent experiments is shown, each with n = 20–40 animals. Error bars are SEM. In all panels, statistical comparisons were to hif-1(ia4) controls. Statistically significant differences are indicated with *** P < 0.001 (Fisher’s exact test).

Figure 2

The unc-14 promoter is active in many nonneuronal cells. (A) Visualization of GFP expressed from Prab-3::hif-1::operon::GFP::H2B (transgene yakEx125). Tail, head, and ventral cord neurons are shown from the ventral aspect of the same animal. VG, ventral ganglia; RVG, retrovesicular ganglia. In all images: bar, 10 μm. (B) Representative images of adult hif-1(ia4); Punc-14::hif-1::operon::GFP::H2B (transgene yakEx144) animals. GFP expression in hypodermal and intestinal cells is shown. Bar, 10 μm. (C and D) Representative images of (C) L1 and (D) adult transgenic animals expressing Punc-14::GFP (transgene yakEx142). Representative animals are shown with GFP expression in hypodermis, intestine, muscle, uterus, pharynx, and neurons. Bar, 5 μm in (C) and 10 μm in (D).

Figure 3

Survival in H₂S requires broad expression of hif-1. Survival of animals exposed to H₂S. All animals have the null hif-1(ia4) mutation. (A) Lethality of animals that express hif-1 only in hypodermis (Pdpy-7::hif-1; yakEx143), intestine (Pvha-6::hif-1; yakEx136), or muscle (Punc-120::hif-1(P621A); otEx3165). As a control, hif-1 was expressed from a ubiquitous promoter (eef-1A.1::hif-1; yakEx131). Expression was from extrachromosomal arrays. Wild-type hif-1 was used for all constructs except the Punc-120::hif-1(P621A), which expresses the nondegradable variant. (B) Survival of hif-1(ia4); yakEx146 animals exposed to H₂S that express hif-1 simultaneously in intestine (Pvha-6::hif-1), hypodermis (Pdpy-7::hif-1), and neurons (Prab-3::hif-1). Average of three independent experiments is shown, each with n = 20–35 animals. Error bars are SEM. In all panels, statistical comparisons were to hif-1(ia4) controls. Statistically significant differences are indicated with *** P < 0.001 (Fisher’s exact test).