Research ArticleStress responses

The receptor tyrosine kinase HIR-1 coordinates HIF-independent responses to hypoxia and extracellular matrix injury

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Science Signaling  02 Oct 2018:
Vol. 11, Issue 550, eaat0138
DOI: 10.1126/scisignal.aat0138

Help during hypoxia from HIR-1

Metazoans require a constant supply of oxygen, and inappropriate hypoxic adaptation is pathological—an example of which occurs in solid tumors. Using the model organism Caenorhabditis elegans, Vozdek et al. searched for pathways involved in coping with hypoxic environments and identified a receptor tyrosine kinase that they called HIR-1. Worms exposed to hypoxia had compromised cuticles, and this damage was recapitulated by loss of HIR-1. Moreover, worms lacking HIR-1 showed changes in the expression of genes involved in cuticle integrity and impaired recovery after severe hypoxia. If a human ortholog of HIR-1 is found, the authors speculate that it could be targeted to treat solid tumors.

Abstract

Inadequate tissue oxygen, or hypoxia, is a central concept in the pathophysiology of ischemic disorders and cancer. Hypoxia promotes extracellular matrix (ECM) remodeling, cellular metabolic adaptation, and cancer cell metastasis. To discover new pathways through which cells respond to hypoxia, we performed a large-scale forward genetic screen in Caenorhabditis elegans and identified a previously uncharacterized receptor tyrosine kinase named HIR-1. Loss of function in hir-1 phenocopied the impaired ECM integrity associated with hypoxia or deficiency in the oxygen-dependent dual oxidase, heme peroxidases, or cuticular collagens involved in ECM homeostasis. Genetic suppressor screens identified NHR-49 and MDT-15 as transcriptional regulators downstream of HIR-1. Furthermore, hir-1 mutants showed defects in adapting to and recovering from prolonged severe hypoxia. We propose that C. elegans HIR-1 coordinates hypoxia-inducible factor–independent responses to hypoxia and hypoxia-associated ECM remodeling through mechanisms that are likely conserved in other organisms.

INTRODUCTION

Oxygen is essential for aerobic metabolism of life. Varying oxygen levels occur in natural environments and in the tissues of living organisms, eliciting highly orchestrated organismic and cellular responses to maintain proper metabolic and physiological homeostasis. For example, mammals adjust pulmonary ventilation and blood circulation to improve oxygen delivery to target tissues under hypoxic conditions; the nematode Caenorhabditis elegans behaviorally navigates for preferred oxygen levels across a gradient of ambient hypoxia and elicits rapid locomotory response upon severe hypoxia and restoration of oxygen (13). Hypoxia is also a common pathophysiological condition in human disorders characterized by a low supply of oxygen, including myocardial ischemia, stroke, and solid tumors (4). Pathological hypoxic conditions can lead to tissue necrosis and fibrosis, degeneration, inflammation, and tumor metastasis—driving disease progression and leading to organismic mortality (5).

Animals respond to chronic hypoxia through evolutionarily conserved molecular pathways and cellular mechanisms that regulate gene expression and reprogram metabolism (4, 6, 7). The hypoxia-inducible factor (HIF) is a master transcriptional regulator of hypoxic responses. Genetic studies of C. elegans have led to the discovery of the evolutionarily conserved family of HIF hydroxylases [egg laying defective 9 (EGL-9) in C. elegans and EGL nine homolog 2 (EGLN2) in humans] that link oxygen sensing to HIF-1 activation and transcriptional responses to hypoxia (8, 9). Hydroxylated HIF under normoxia is recognized by the Von Hippel-Lindau tumor suppressor and thereby targeted for proteasomal degradation, whereas hypoxia impairs HIF hydroxylation, leading to transcriptional activation of HIF target genes (10). In C. elegans, the HIF-1 pathway mediates various physiological and behavioral responses to hypoxia (2, 6, 11, 12). The transcriptional targets of mammalian HIF include LOX, LOXL2, and LOXL4, which encode copper-dependent lysyl oxidases that promote cross-linking of extracellular matrix (ECM) components for enhanced tissue stiffness, a proposed trigger of tumor metastasis (5). Hypoxia-induced ECM remodeling, in turn, activates intracellular signaling cascades to regulate cell fate, metastasis, and adaptation to hypoxia (13, 14). How cells sense and respond to hypoxia-induced ECM remodeling remains undefined.

Collagens are major integral components of ECM that are extensively modified by oxygen-dependent enzymes including prolyl hydroxylases, lysyl oxidases and hydroxylases, and dual oxidases. Beyond HIF-dependent mechanisms, it is unknown whether hypoxia can alter ECM integrity directly by impairing oxygen-dependent collagen modification and how ECM remodeling might elicit subsequent HIF-independent gene regulation. In mammalian cells, vascular endothelial growth factor (VEGF) can be induced by hypoxia through both HIF-dependent and HIF-independent mechanisms (15). Various transcription factors other than HIF respond to hypoxia in an HIF-independent manner to regulate gene expression (16, 17). Mitogen-activated protein kinases or integrin-linked protein kinases can transduce remodeled ECM signals to influence cell fate and resistance to hypoxia through transcriptional regulation (13, 14). C. elegans has also been extensively used as a model organism to investigate HIF-independent responses that mediate physiological and behavioral adaptation to severe hypoxia (1828). Nonetheless, the precise roles and cellular mechanisms of HIF-independent transcriptional response to hypoxia-induced ECM remodeling in physiology and diseases remain poorly understood.

We sought to identify new genes and pathways that transduce hypoxic signals to gene regulation independently of HIF-1. We generated C. elegans transgenic animals carrying green fluorescent protein (GFP) reporters that were robustly activated by hypoxia even in HIF-1–deficient animals. We performed large-scale forward genetic screens to isolate mutants with constitutively activated reporters under normoxia and carried out suppressor screens to identify requisite transcriptional regulators. We found that exposure to hypoxia was associated with the remodeling of ECM, which in turn triggers intracellular HIF-independent transcriptional responses that were mimicked by loss of function (LOF) in a gene encoding a cell transmembrane receptor tyrosine kinase (RTK) that we named HIR-1 (hypoxia-inhibited RTK).

RESULTS

The reporter comt-5p::GFP is induced by hypoxia independently of HIF-1

To identify genes and pathways that mediate HIF-1–independent transcriptional response by hypoxia, we used RNA sequencing (RNA-seq) analysis to compare transcriptomes of wild-type (WT) C. elegans under normoxia (21% oxygen) or severe hypoxia (nearly 0.5% oxygen) for 2 hours and egl-9 null mutants (in which HIF-1 is constitutively activated) under normoxia. We identified 72 genes that showed increased expression after hypoxia in WT animals but not in egl-9 null mutants (fig. S1A and data file S1). We generated transgenic animals with GFP driven by the promoters of these genes and focused on one of the reporters constructed, dmaIs1, that showed robust induction of GFP in the hypodermal and intestinal cells within 24 hours in 0.5% O2 (Fig. 1, A and B). dmaIs1 carries GFP driven by the promoter of comt-5, which encodes a predicted catechol-O-methyltransferase (Fig. 1C). We crossed animals carrying the dmaIs1 transgene with egl-9(sa307) or hif-1(ia04) LOF deletion mutants, and as expected, we did not observe activation of comt-5p::GFP in egl-9 mutants, whereas both WT and hif-1 mutants exhibited increased expression of comt-5p::GFP by hypoxia (Fig. 1, A and D).

Fig. 1 comt-5p::GFP is up-regulated by hypoxia in an hif-1–independent manner.

(A) Fluorescent images of WT and hif-1(ia04) mutants carrying the genome-integrated dmaIs1 [comt-5p::GFP] transgene, exposed to normoxia or hypoxia. Representative of >100 animals. Scale bars, 200 μm. (B) Enlarged gray scale images of C. elegans carrying comt-5p::GFP under normoxia and hypoxia. Arrows in normoxia image indicate head neurons with GFP signal. Arrowheads in hypoxia image indicate hypodermal cells with GFP signal. Representative of >100 animals. Scale bars, 40 μm. (C) Schematic diagram of the dmaIs1 transgene. Upstream sequence (2010 bp) of the comt-5 gene was cloned before the GFP-coding sequence, followed by 3′ untranslated region (3′UTR) of the unc-54. The dmaIs1 transgene also carries unc-54p::mCherry as an additional marker of expression. (D) Analysis of the GFP signal by Western blot analysis of 20 randomly picked transgenic animals carrying the dmaIs1 [comt-5p::GFP] transgene. Histone H3 was used as a loading control. Transgenic animals express a cryptic nonspecific protein recognized by the GFP antibody (33 kDa) that does not contribute to fluorescence in living animals. Representative of three independent experiments. (E) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of comt-5 expression in normoxia- or hypoxia-exposed WT and hif-1(ia04) animals. n ≥ 200 total animals of mixed stages for each group analyzed in three independent biological replicates. ***P < 0.001. (F) qRT-PCR analysis of comt-5 expression in animals exposed to hypoxia (1% O2), anoxia (0% O2), or hypoxia-mimicking conditions (CoCl2, NaSH, or KCN). n ≥ 200 total animals of mixed stages for each group analyzed in three independent biological replicates. ***P < 0.001.

To verify that endogenous comt-5 was induced by hypoxia independently of hif-1, we quantified its expression by qRT-PCR and found that comt-5 expression was increased by hypoxia in both WT and hif-1(ia04) mutants (Fig. 1E). Other stress conditions that mimic hypoxia in activating HIF-1, including exposure of animals to CoCl2, H2O2, CN, or H2S, did not induce the comt-5p::GFP reporter expression or increase endogenous comt-5 expression (Fig. 1F). These results identify comt-5 as a specific and robust reporter gene that is activated by hypoxia independently of EGL-9 and HIF-1.

HIR-1 is a cell-autonomous regulator of comt-5

To identify genes and pathways mediating HIF-independent regulation of comt-5p::GFP, we performed large-scale forward genetic screens using ethyl methanesulfonate (EMS)–induced mutagenesis and isolated many mutants with constitutively activated comt-5p::GFP reporters even under normoxia (Table 1). Genetic linkage analysis, whole-genome sequencing, and subsequent RNA interference (RNAi) of candidate genes identified dma51 as an allele of the previously uncharacterized gene C24G6.2 (which we called hir-1), which encodes a predicted transmembrane protein belonging to the RTK superfamily (according to InterPro scanning) (29). dma51 causes a LOF nonsense mutation W551Stop in HIR-1. We used clustered regularly interspaced short palindromic repeats (CRISPR) to generate a whole-gene deletion allele dma101 for hir-1, which also showed constitutive comt-5p::GFP induction. A similar phenotype was also observed in mutants with partial in-frame deletion (tm3911) or an out-of-frame deletion (tm4098) that covers the exons encoding the intracellular domain of HIR-1 (Fig. 2, A and B). A transcriptional reporter hir-1p::GFP revealed ubiquitous expression of hir-1 that was especially strong in the pharynx and hypodermal and seam cells in all developmental stages (Fig. 2C and fig. S2A). To determine whether hir-1 regulates comt-5p::GFP cell-autonomously in the hypoderm, where comt-5p::GFP is induced by hypoxia, we generated transgenic animals with hypodermal-specific expression of hir-1(+) driven by the dpy-7 promoter (Fig. 2C). We observed that transgenic dpy-7p::hir-1(+) extrachromosomal arrays rescued the hir-1(LOF)–induced hypodermal activation of comt-5p::GFP (Fig. 2, D and E). Transgenic animals with neuronal-specific expression of hir-1(+) driven by the pan-neuronal promoter ric-19 did not rescue hir-1(−)–induced activation of comt-5p::GFP (fig. S2B and Fig. 2E). These results identify hir-1 as a cell-autonomous negative regulator of comt-5p::GFP.

Table 1 Mutants isolated from EMS mutagenesis with constitutive comt-5p::GFP expression.
View this table:
Fig. 2 HIR-1 is a cell-autonomous inhibitor of comt-5.

(A) Fluorescent images of transgenic animals carrying the comt-5p::GFP transgene with various hir-1 alleles. Representative of >100 animals. (B) Schematic diagram of hir-1 with indicated LOF alleles isolated from EMS mutagenesis (dma51), CRISPR–Cas9 (CRISPR-associated protein 9) (dma101), and ultraviolet (UV) mutagenesis (tm3911 and tm4098). (C) Schematic map of various hir-1 transgenes. (D) Fluorescent images of transgenic animals expressing comt-5p::GFP (worm 1) with hir-1(dma101) (worm 2) and with hir-1(dma101); hir-1(+) array dmaEx113 (worm 3). Animals carrying extrachromosomal transgene dmaEx113 [dpy-7p::hir-1;myo-2::mCherry] have mCherry fluorescence in the pharynx. Representative of >100 animals. Scale bars, 300 μm. (E) Percentages of animals of indicated genotypes as in (D) with activated comt-5p::GFP. n ≥ 100 total animals of mixed stages for each group with three independent biological replicates. ***P < 0.001.

Inactivation of genes essential for ECM integrity mimics the transcriptional responses induced by hypoxia or hir-1(LOF)

Using genetic linkage mapping and whole-genome sequencing, we identified additional genes defined by other mutants isolated from EMS screens with constitutive comt-5p::GFP expression. dma52 is a Q4R mutation in dpy-2. dma22 is a G162R mutation in dpy-3. dma236 defines a previously uncharacterized gene C46A5.4, which we named to perl-1 because it encodes a heme peroxidase–like protein (Table 1 and Fig. 3, A and B). RNAi directed against dpy-2, dpy-3, or perl-1 or partial deletion alleles of these genes phenocopied EMS-derived mutations in activating constitutive expression of comt-5p::GFP under normoxia (table S1). Both dpy-2 and dpy-3 encode cuticle collagens (30), and perl-1 is paralogous to mlt-7, which encodes a heme peroxidase required for proper cross-linking of ECM collagens (Fig. 3C) (31). Both dpy-3 and dpy-2 are expressed in the hypoderm, and their LOF mutations, including EMS-derived alleles, resulted in abnormal animal morphology (specifically, a “dumpy” phenotype) and mimicked hypoxia-induced comt-5p::GFP activation (Fig. 3A). We performed eight independent genetic screens of more than 100,000 haploid genomes and observed several comt-5p::GFP–activating mutants that were unable to propagate. These mutants commonly exhibited cuticle morphological defects, such as blistering or dumpy phenotypes. We used RNAi to knock down additional genes directly involved in cuticle biosynthesis and observed that RNAi against cuticular collagens (bli-6), a collagenase inhibitor (bli-5), another heme peroxidase (mlt-7), or a subtilisin-like protease (bli-4) activated comt-5p::GFP expression in the hypoderm (table S1 and fig. S3A). Cuticle biosynthesis in the hypoderm is essential for molting, and several cuticular genes are regulated by molting-controlling transcription factors, such as NHR-23 (32). RNAi against these transcription factor genes resulted in molting defects accompanied by activation of comt-5p::GFP expression (table S1 and fig. S3A). These results show that diverse genetic manipulations that impair hypodermal ECM integrity are associated with activation of comt-5p::GFP expression.

Fig. 3 Deficiency of specific cuticular genes mimics hypoxia-induced comt-5p::GFP activation.

(A) Fluorescent images of comt-5p::GFP expression in the hypoderm of WT animals or mutants lacking dpy-2, dpy-3, or perl-1. Representative of >100 animals. (B) Schematic gene structures of dpy-2, dpy-3, and perl-1 showing the positions of EMS-derived LOF alleles. (C) Protein domain organization of dumpy 2 (DPY-2), DPY-3, and peroxidase-like 1 (PERL-1), with an asterisk indicating the positions of EMS-derived mutations. The G162R mutation in DPY-3 is in the conserved collagen domain; the Q4R mutation in DPY-2 is in the signal peptide. The missense mutations in the PERL-1 are in the N-terminal peroxidase-like domain. (D) Fluorescent images of animals carrying the col-19::GFP translational reporter exposed to normoxia, hypoxia (24 hours of ≤0.1% O2), or dpy-18(−) and RNAi directed against let-268, perl-1, and dpy-3. Representative of >30 animals. Scale bars, 25 μm. (E) Percentages of animals with COL-19::GFP disorganization with indicated genotypes and conditions. n = 10 1-day-old adults for each group analyzed in three independent biological replicates. ***P < 0.001. (F) Fluorescent images of comt-5p::GFP expression in animals treated with control or RNAi against the indicated genes essential for cuticle integrity. Representative of >100 animals. Scale bars, 200 μm.

Hypoxic stress is associated with changes in the ECM and cuticle disintegration

Because genetically induced impairment of hypodermal ECM integrity increased the expression of comt-5p::GFP, we wondered whether hypoxia, which also activates comt-5p::GFP, is associated with the remodeling and/or impairment of hypodermal ECM. The cuticular reporter COL-19::GFP normally localizes to circumferential annular rings and longitudinal alae of adult exoskeleton (33). We examined COL-19::GFP in 1-day-old WT animals exposed to severe hypoxia for 24 hours (nearly 0% O2) and observed disorganized COL-19::GFP distribution proximal to the alae (Fig. 3D). Cuticle biosynthesis begins in the endoplasmic reticulum (ER) lumen when collagens are hydroxylated by prolyl hydroxylases, followed by trimerization, secretion, extracellular propeptide cleavage, and cross-linking (34, 35). There are four characterized ER procollagen hydroxylases in C. elegans (dpy-18, phy-2, phy-3, and phy-4), which are partially genetically redundant (36). The hypomorphic allele of dpy-18 caused disorganization of COL-19::GFP (Fig. 3D), which was similar to the pattern observed in animals exposed to hypoxia or with inactivated collagen–cross-linking dual oxidase BLl (blistered cuticle)–3 (33). We also observed cuticle disorganization in animals with inactivated perl-1 and let-268, the sole C. elegans lysyl hydroxylase, whereas inactivated dpy-3 resulted in an altered GFP pattern characterized by a complete loss of the cuticular furrows (Fig. 3, D and E).

In addition, WT animals exposed to severe hypoxia for 24 hours (nearly 0% O2); perl-1, dpy-3, or dpy-18 mutants; or animals with RNAi-mediated knockdown of let-268 by RNAi exhibited exacerbated sensitivity to osmotic stress, which is characteristic of mutants with disrupted cuticle integrity (37) and is likely caused by increased cuticle permeability to water (fig. S3B). Furthermore, direct permeability assays with the cuticle impermeable dye Hoechst 22358 (38) revealed nuclear intercalation of the dye only in perl-1 and dpy-3 mutants, let-268 knockdowns, and hypoxia-treated WT animals but not WT animals under normoxia (fig. S3, C and D). These data provide evidence that cuticles in animals exposed to hypoxia or deficient in various oxygen-dependent collagen-modifying enzymes exhibit altered integrity characterized by increased permeability.

Inactivation of oxygen-dependent dual oxidase activates comt-5p::GFP

We next examined the effect of oxygen-dependent collagen-modifying enzymes on comt-5p::GFP expression. Whereas RNAi against genes encoding procollagen hydroxylases (dpy-18, phy-2, phy-3, and phy-4) had negligible effect on comt-5p::GFP expression, inactivation of the genes encoding a dual oxidase complex (bli-3 and tsp-15) led to a robust increase in comt-5p::GFP expression (Fig. 3F and fig. S3A). In addition, inactivation of phy-2, phy-3, or phy-4 in the dpy-18 mutant did not further increase comt-5p::GFP expression, although RNAi directed against phy-2 led to larval arrest with strong cuticle defects (fig. S3E). RNAi directed against let-268 led to comt-5p::GFP activation in the developmentally arrested larva but not in adults (Fig. 3F). Because LET (lethal)–268 activity appears to be limited to collagen type IV localized in the basement membrane but not cuticular collagens (39, 40), let-268 likely indirectly regulates comt-5p::GFP by affecting the maturation of ECM proteins in the ER (40). Consistently, RNAi against emb-9 encoding the collagen type IV did not increase expression of comt-5p::GFP (fig. S3E). Because BLI-3 mediates dityrosine cross-linking of collagen by oxygen-dependent generation of radicals to maintain cuticular integrity, its hypoxia sensitivity and key roles in regulating comt-5p::GFP expression suggest that it could be involved in an oxygen-sensing pathway that mediates transcriptional responses to hypoxia.

NHR-49 and MDT-15 mediate comt-5 transcriptional response to hypoxia

To identify specific transcription factors that drive comt-5p::GFP expression in response to hypoxia, we sought to identify second-site suppressor mutations of the most penetrant comt-5p::GFP–activating mutation dma11 isolated from EMS screens (Table 1). The gene defined by dma11 remains to be identified. We performed two independent genetic screens of more than 100,000 haploid genomes, isolated two independent suppressing alleles of dma11, dma54, and dma55, and used linkage analysis and RNAi phenocopying to identify them as mutations of mdt-15 and nhr-49, respectively. To verify that LOF of nhr-49 also suppressed hir-1– and dpy-3–induced comt-5p::GFP activation, we crossed the nhr-49 null allele nr2041 with hir-1 and dpy-3 mutants and found that comt-5p::GFP expression was suppressed in the hir-1; nhr-49 and the dpy-3; nhr-49 double mutants (Fig. 4A and fig. S4A). Moreover, hypoxia did not activate comt-5p::GFP expression in nhr-49 null mutants, whereas the gain-of-function (GOF) mutations nhr-49(et7) and mdt-15(et14) showed constitutively activated comt-5p::GFP expression under normoxia (Fig. 4A). Western blot confirmed the requirement for nuclear hormone receptor family 49 (NHR-49) and mediator 15 (MDT15) in the activation of comt-5p::GFP expression in hir-1 mutants (Fig. 4B). qRT-PCR analysis also confirmed that both nhr-49 and mdt-15 were required for the hypoxic induction of endogenous comt-5 (fig. S4B).

Fig. 4 HIR-1–mediated regulation of comt-5p::GFP requires NHR-49 and MDT-15.

(A) Fluorescent images of transgenic animals carrying comt-5p::GFP, and LOF and GOF of the indicated genes. Representative of >100 animals. Scale bars, 200 μm. (B) Western blot analysis of comt-5p::GFP expression in hir-1, hir-1; hif-1, and hir-1; nhr-49 mutants. (C) Schematic gene structures of nhr-49 and mdt-15 showing the position of EMS-derived mutations. (D) Protein sequence alignment of animal proteins orthologous to NHR-49 showing that Gly33 in NHR-49 is fully conserved in orthologs, including human hepatocyte nuclear factor 4 (HNF4). (E) Graph showing penetrance for increased comt-5p::GFP expression during developmental stages for each group. n ≥ 200 total animals of mixed stages for each group analyzed in three independent biological replicates. L1 to L4, larva 1 to larva 4; YA, young adult. (F) Penetrance analysis for increased comt-5p::GFP expression in the indicated strains. n ≥ 200 total adult animals for each group analyzed in three independent biological replicates. ***P < 0.001.

dma54 is a nonsense mutation leading to a premature stop codon in mdt-15, and dma55 is a missense mutation G33R in nhr-49 (Fig. 4C). Protein sequence analysis of orthologous nuclear hormone receptors revealed that Gly33 is in the conserved DNA binding domain (Fig. 4D). NHR-49 and MDT-15 are transcriptional regulators that physically interact to regulate lipid homeostasis and mediate responses to changes in lipid metabolic cues and temperature (11, 4144). Hypoxia or hir-1 inactivation did not appear to up-regulate the cold-sensitive NHR-49 target gene fat-7 (data file S2), indicating that HIR-1 signaling acts independently of temperature stress. We observed that comt-5p::GFP induction by hir-1(LOF) was limited to adulthood, whereas nhr-49 GOF or dpy-3 LOF activated comt-5p::GFP at all developmental stages (Fig. 4E). These results support a model in which NHR-49 not only mediates HIR-1–dependent transcriptional response in adults but also plays broader roles than HIR-1. Moreover, hir-1; nhr-49 or dpy-3; nhr-49 mutants exhibited comt-5p::GFP in 3 or 6% of animals, respectively, whereas RNAi of bli-3 in nhr-49 mutants led to comt-5p::GFP activation in nearly 100% of animals (Fig. 4F). We generated a transgenic strain expressing nhr-49::Venus under the ubiquitous rpl-28 promoter. The lack of changes in the subcellular localization of NHR-49::Venus after hypoxia (fig. S4C) suggests that NHR-49 is not regulated at the level of nuclear translocation. Thus, NHR-49 is essential for HIR-1 to regulate downstream gene expression, but the underlying mechanism remains to be elucidated.

Hypoxia and ECM rearrangements are associated with altered expression of HIR-1

Because hir-1 inactivation phenocopied exposure to hypoxia in terms of comt-5p::GFP activation, we wondered whether hypoxia directly inhibited HIR-1. The intracellular domain of HIR-1 is structurally similar to the proto-oncogene receptor RET (rearranged during transfection) and fibroblast growth factor (FGF) receptors (FGFRs), with conserved catalytic sites essential for autophosphorylation (fig. S5A). Thus, HIR-1 kinase activity could be inhibited by depletion of adenosine 5′- triphosphate (ATP) upon severe hypoxia. However, expression of comt-5p::GFP did not increase in animals exposed to the adenosine triphosphatase inhibitor oligomycin, the complex I inhibitor rotenone, or the uncoupling agent carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (Fig. 5A). These results indicate that ATP depletion through inhibited mitochondrial oxidative phosphorylation does not phenocopy inactivation of hir-1 and thus is unlikely to underlie effects of hypoxia in regulating HIR-1.

Fig. 5 Regulation of HIR-1 by hypoxia and ECM remodeling.

(A) Fluorescent images of merged comt-5p::GFP and unc-54p::mCherry expression in animals treated with control or indicated drugs or RNAi against let-756. Scale bars, 200 μm. (B) Schematic map of hir-1 transgenes expressing translational reporters. (C) Fluorescent images of transgenic animals carrying the hir-1 translational reporter [rpl-28p::hir-1sig::GFP::hir-1cod; myo-2p::mCherry]. Hypoxia indicates 0% O2 for 24 hours. Arrows indicate GFP::HIR-1 foci. The transgenic marker myo-2p::mCherry expressed in the pharyngeal muscles is also shown. n ≥ 10 total animals for each group analyzed in ≥3 independent biological replicates. Scale bars, 300 μm. (D) Percentages of animals with GFP::HIR-1 foci. n = 10 total adult animals for each group analyzed in three independent biological replicates. ***P < 0.001. (E) SDS–polyacrylamide gel electrophoresis (PAGE) in the presence of the reducing agent β-mercaptoethanol and Western blot analysis of crude extracts of transgenic animals expressing HIR-1::GFP. Hypoxia indicates 0% O2 for 24 hours. The asterisk indicates full-length species of GFP::HIR-1, and the X indicates processed GFP::HIR-1. Representative of three independent experiments. (F) Domain arrangement in HIR-1. (G) SDS-PAGE in the presence of the reducing agent β-mercaptoethanol and Western blot analysis of crude extracts of transgenic animals expressing HIR-1::FLAG. ^, insoluble GFP::HIR-1; *, expected full-length species of HIR-1::FLAG; X, partial fragment of GFP::HIR-1. Hypoxia indicates 0% O2 for 24 hours. Representative of three independent experiments. (H) Quantification of GFP intensity of expressed comt-5p::GFP in the indicated strains. Hypoxia indicates 0.5% O2 for 24 hours. n ≥ 3 total adult animals for each group with three independent biological replicates. ***P < 0.001. a.u., arbitrary units.

We generated a translational GFP reporter of HIR-1 (Fig. 5B) and observed that hypoxia induced formation of HIR-1::GFP foci without affecting the transgenic co-injection marker unc-54p::mCherry in the pharyngeal muscles (Fig. 5, C and D). Western blot analysis revealed that a range of partial-length HIR-1::GFP species increased in abundance in hypoxia-treated animals (Fig. 5E). Moreover, hypoxia increased the abundance of HIR-1::GFP in the protein pool that did not migrate in the SDS-PAGE, indicating increased insolubility of HIR-1 associated with hypoxic exposure (Fig. 5E).

The extracellular domain of HIR-1 resembles an immunoglobulin-like fold (amino acids 80 to 446), including fibronectin type III–like fold (amino acids 84 to 170), domains common in many ECM protein-interacting receptors (Fig. 5F) (45, 46). FGF4 is a ligand implicated in linking ECM sensing and regulation of trophoblast stem cell fate (13). Inactivation of the C. elegans FGF-encoding gene let-756 by RNAi led to constitutive activation of comt-5p::GFP under normoxia, phenocopying hir-1(LOF) mutants (Fig. 5A). Knockdown of either dpy-3 (which encodes cuticular collagen) and bli-3 (which encodes a cross-linking enzyme) resulted in formation of HIR-1::GFP foci, similar to those in WT animals exposed to hypoxia (Fig. 5, C and D), suggesting potential regulation of HIR-1 by ECM alterations. We generated FLAG-tagged hir-1 transgenic animals and found that full-length HIR-1::FLAG abundance markedly decreased in animals exposed to hypoxia and animals with let-756 RNAi (Fig. 5G). In addition, we generated transgenic animals that ubiquitously expressed human influenza hemagglutinin (HA)–tagged let-756 (fig. S5B) and observed hypoxia-induced abundance decrease in LET-756::HA (fig. S5C). These findings indicate that hypoxia and disrupted ECM integrity can affect the abundance of HIR-1 and LET-756.

On the basis of the genetic evidence connecting hypoxia, ECM integrity, and hir-1, we hypothesized that exposure to hypoxia induces ECM rearrangements that inhibit HIR-1 and subsequently activate comt-5p::GFP. To probe the genetic interactions between hir-1 and collagen-encoding dpy-3 in controlling the expression of comt-5, we generated hir-1; dpy-3 double mutants. qRT-PCR analysis showed that hypoxia induced endogenous comt-5 expression in mixed-stage WT, hir-1, dpy-3, and hir-1; dpy-3 mutants (fig. S5D). To determine the role of hir-1 specifically in the adult stage, we measured comt-5p::GFP intensity in the hypoderm. We found that, unlike dpy-3 single mutation or hir-1; dpy-3 double mutation, hir-1 mutation did not affect comt-5p::GFP intensity in the larval stage and that hypoxia increased GFP intensity in all three mutants (fig. S5E). These mutants also exhibited increased hypodermal comt-5p::GFP intensity in adult animals under normoxia, which was not further increased by hypoxia (Fig. 5H). These data are consistent with a model in which dpy-3 acts in the same genetic pathway with hir-1 for hypoxic induction of comt-5p::GFP in the adult hypoderm.

HIR-1 LOF affects the expression of genes involved in ECM homeostasis

We performed RNA-seq analysis of hir-1 mutants and compared transcriptomes of WT animals under normoxia or hypoxia with hir-1 mutants (figs. S1B and S6, A and B). Expression of genes that regulate comt-5p::GFP, including dpy-2, dpy-3, and perl-1, was increased by both hypoxia and hir-1 inactivation (fig. S6C). We used qRT-PCR to quantify the expression levels of genes encoding collagens, procollagen hydroxylases, peroxidases, and transcription factors mediating molting processes in hir-1, hir-1; nhr-49, and hir-1; hif-1 mutants. All examined genes showed increased expression in hir-1 and hir-1; hif-1 mutants and decreased expression in hir-1; nhr-49 mutants (fig. S6D). Furthermore, we found that genes involved in molting and cuticle integrity showed increased expression in hir-1 mutants, including members of collagen-encoding dpy genes whose inactivation can cause the dumpy phenotype (data file S2). Several collagen-encoding non-dumpy genes, including col-17 and col-41, were suppressed (fig. S6B). Collagens are the main structural components of the ECM and cuticle, whereas the epicuticle contains lipids that regulate its permeability (47). We found that hir-1 mutants showed increased expression of genes involved in lipid metabolism, including acs-2, a target gene of NHR-49 and key regulator of fatty acid homeostasis (fig. S6, B and C) (41).

We generated transgenic animals expressing a fluorescent Venus-tagged transgene of dpy-3, a downstream target regulated by hir-1. We observed that the DPY-3::Venus signal localized to the cuticle furrows (Fig. 6A). Exposure of DPY-3::Venus animals to hypoxia caused interrupted furrow lanes (Fig. 6, A and B). Compared with WT animals, adult hir-1 mutants also exhibited increased cuticle furrow interruptions (Fig. 6, A and B). Disrupted cuticle integrity in hir-1 adults was also observed on the basis of COL-19::GFP and cuticle permeability assays (Fig. 6, C and D, and fig. S7, A to C). These results indicate that HIR-1 LOF results in altered expression of cuticle-related genes and suggest that HIR-1 could be involved in maintaining ECM homeostasis in adults. The cuticle defects of hir-1 mutants were not rescued by nhr-49 LOF (Fig. 6, C and D), suggesting that defects in the ECM integrity of hir-1 mutants involve either nontranscriptional mechanisms or abnormal activation of additional unidentified transcription factors.

Fig. 6 hir-1(LOF) is associated with altered ECM homeostasis and defective behavioral recovery after hypoxia.

(A) Schematic map of dpy-3::Venus transgene (top) and fluorescent images of animals carrying the dpy-3::Venus translational reporter exposed to normoxia, hypoxia (24 hours of ≤0.1% O2), or RNAi against hir-1. Representative of >30 animals. Scale bars, 10 μm. (B) Percentages of animals with disrupted DPY-3::Venus pattern. n = 10 1-day-old adults for each group analyzed in three independent biological replicates. ***P < 0.001. (C) Fluorescent images of animals expressing col-19::GFP in the presence or absence of hir-1 RNAi. Representative of >30 animals. Scale bars, 25 μm. (D) Percentages of animals with COL-19::GFP disorganization. n = 10 1-day-old adults for each group analyzed in three independent biological replicates. ***P < 0.001. (E) WormLab plots showing representative locomotion tracks of WT and hir-1 animals in a 10-min time frame recorded after 24 hours of reoxygenation. Position is detected on the basis of the midpoint of the worm. (F) Displacement of WT and hir-1 mutants in a 10-min time frame recorded before exposure to hypoxia and after 0 to 10 min, 2 hours, and 24 hours of reoxygenation. n = 10 animals for each group analyzed in three independent biological replicates; ***P < 0.001. (G) Schematic model of the proposed HIR-1 pathway. Inactive components are shown in gray, and activated components are shown in black. In WT animals, hypoxia-induced changes in ECM transiently inhibit HIR-1 that permits comt-5 activation and ECM remodeling to maintain ECM homeostasis, whereas hir-1 mutants exhibit constitutive comt-5 activation, maladapted ECM, and decreased locomotor recovery after severe hypoxia.

HIR-1 deficiency results in defects in behavioral adaptation and recovery after severe hypoxia

We next tested whether inactivation of HIR-1 affects animal responses to severe hypoxia followed by reoxygenation. We exposed L1/L2 larvae and 1-day-old WT and hir-1(dma101) mutants to anoxia for 40 hours and subsequently compared their locomotor behavior after reoxygenation. Although neither WT nor hir-1 larvae exhibited locomotion defects, all adult animals were in suspended animation-like state unable to respond to external stimuli (namely, mechanical touch or UV light) during anoxia and immediately after reoxygenation. Suspended animation was followed by behavioral recovery in locomotion displacement and pharyngeal pumping. We found that the displacement of reoxygenated WT adults gradually recovered, whereas hir-1 mutants had markedly severe locomotion defects that did not improve even after 24 hours of reoxygenation (Fig. 6, E and F). The acute locomotion recovery after anoxia does not necessarily require restored cuticle integrity because WT animals with recovered locomotion still exhibited cuticle defects (fig. S7D). Together, our data support a model in which HIR-1 is transiently regulated by hypoxia-induced changes in the ECM and in turn affects the expression of genes involved in ECM integrity and homeostasis, behavioral adaptation, and locomotion recovery after severe hypoxia (Fig. 6G).

DISCUSSION

How cells in multicellular organisms sense hypoxia-remodeled ECM to promote ECM homeostasis and facilitate animal adaptation to severe hypoxia is unknown. From genetic screens, we identified HIR-1 as a component of the hif-1–independent transcriptional response to hypoxia. Altered expression pattern of GFP-tagged HIR-1 in animals exposed to hypoxia and in mutants with disrupted cuticle suggests that HIR-1 may be regulated by hypoxia-induced changes in ECM. The observed HIR-1::GFP foci are reminiscent of aggregation and proteolytic processing, a previously observed effect of hypoxia on other proteins in C. elegans (27, 28). We do not exclude the possibility that receptor clustering, internalization, or block in membrane trafficking could also affect HIR-1 or its interacting partners. Our genetic evidence suggests that LET-756, which is homologous to mammalian FGFs and whose binding to HIR-1 might be modulated by hypoxia-induced changes in ECM, could be a ligand for HIR-1. HIR-1 does not have the discoidin domain, which is responsible for interaction with collagens to sense ECM remodeling (48). Detailed mechanisms of how HIR-1 is regulated by ECM in coordination with its ligand and, in turn, transduces intracellular signaling for ECM homeostasis await further studies.

The integrity and composition of the ECM are sensitive to oxygen availability because of the oxygen-dependent activities of many enzymes, including prolyl hydroxylases in the ER and dual oxidases in the extracellular space (31, 49). We found that inactivation of the dual oxidase BLI-3 by RNAi phenocopied hypoxia-induced activation of comt-5p::GFP, suggesting that BLI-3 likely mediates hypoxic responses directly to affect change in cuticle integrity. In turn, HIR-1– and NHR-49–regulated genetic program might constitute a homeostatic response in attempt to eventually restore cuticle integrity. Depending on the tissue-specific range of oxygen levels to which enzymes are sensitive, extracellular oxidases such as BLI-3 could act as cellular oxygen sensors to mediate responses to varying degrees of hypoxia. We propose that such oxygen sensing in the extracellular space acts in parallel to cytosolic EGLN oxygen sensors to mediate HIF-independent transcriptional programs to induce ECM remodeling and inhibit HIR-1 to maintain ECM homeostasis and animal adaptation to hypoxia and reoxygenation.

Hypoxia-induced ECM remodeling occurs in cultured tumor cells (5), and the ECM surrounding bone marrow tumor cells exhibits increased stiffness that triggers metastasis (50). Increased stiffness of the ECM due to enhanced cross-linking of ECM proteins is mediated by increased expression of lysyl hydroxylases or oxidases and collagen deposition (5, 51, 52). Similarly, we also found that hypoxia increased the expression of genes encoding procollagen lysyl oxidase, ER hydroxylases, and collagens in C. elegans, indicating conserved transcriptional responses that promote homeostatic ECM remodeling, which may have been coopted by tumors to facilitate survival during hypoxia and metastasis.

We found that loss of HIR-1 in C. elegans resulted in defective adaptation to hypoxic stress and reoxygenation and changes in the expression of genes encoding not only ECM components but also a non-ECM component, comt-5. comt-5 is predicted to encode a catecholamine-degrading enzyme, and its up-regulation by hypoxia may help to decrease catecholamine levels to alleviate the toxicity of their oxidized forms under hypoxia. Dopamine targets peripheral tissues to maintain xenobiotic stress resistance in C. elegans (53). However, whether up-regulation of comt-5 is required for proper resistance to hypoxic and/or xenobiotic stress awaits further studies. Although we used primarily comt-5::GFP reporters for gene and pathway discovery, transcriptomic analysis revealed that loss of HIR-1 resulted in expression changes in genes responsible for remodeling of ECM, altering cuticle integrity and reprogramming lipid metabolism. ECM remodeling during aging through differential regulation of collagen-encoding genes contributes to extension of C. elegans longevity (54), in which HIR-1 could contribute by promoting homeostatic ECM remodeling in response to hypoxia. hir-1–regulated lipid metabolism genes may also contribute to hypoxic adaptation because lipid metabolic reprogramming also contributes to tumor development and hypoxic resistance in mammals (55).

Upstream of NHR-49, the regulatory axis from oxygen, the dual oxidase, and collagen to RTKs appears to share features common in both C. elegans and humans. Numerous human RTKs, including FGFRs, have been implicated in driving tumor survival and metastasis (56, 57). ECM remodeling is essential for the progression of cancer, tissue fibrosis, and many other diseases involving ECM dysregulation (58). Because HIR-1 may promote ECM remodeling to enhance resistance to hypoxia, the human counterpart of HIR-1, once verified, may be a promising therapeutic target for solid tumors that survive severe hypoxia and metastasize through ECM remodeling.

MATERIALS AND METHODS

C. elegans strains

Animals were maintained under standard procedure with nematode growth media (NGM) plates, unless otherwise stated. Bristol strain N2 was used as WT, and Hawaiian strain CB4856 was used for the linkage analysis of the mutants (59, 60). hir-1 null alleles were generated by CRISPR to induce double-stranded breaks, and subsequent nonhomologous end joining caused a deletion of hir-1. Feeding RNAi was performed as previously described (61). Transgenic strains were generated by germline transformation as described (62). Transgenic constructs were coinjected with dominant unc-54p::mCherry or myo-2::mCherry markers, and stable extrachromosomal lines of mCherry+ animals were established. Transgenic strains used were dmaIs1[comt-5p::GFP], dmaIs1; egl-9(sa307), dmaIs1; hif-1(ia4), dmaIs1; hir-1(dma51), dmaIs1; hir-1(tm4098), dmaIs1; hir-1(tm3911), dmaIs1; hir-1(dma101), dmaIs1; hir-1(dma101); dmaEx113[dpy-7p::hir-1(+); myo-2::mCherry], dmaIs1; hir-1(dma101); dmaEx113[ric-19p::hir-1(+); myo-2::mCherry], dmaIs1; dpy-3(dma22), dmaIs1; dpy-2(dma52), dmaIs1; perl-1(dma53), dmaIs1; perl-1(dma236), dmaIs1; dma11, dmaIs1; dma11; mdt-15(dma54), dmaIs1; dma11; nhr-49(dma55), dmaIs1; nhr-49(nr2041), dmaIs1; hir-1(dma101); nhr-49(nr2041), dmaIs1; hir-1(dma101); hif-1(ia4), dmaIs1; dpy-3(ok2263), dmaIs1; dpy-3(ok2263); nhr-49(nr2041), dmaIs1; hir-1(dma101); dpy-3(ok2263), dmaIs1; hir-1(dma101); dpy-3(ok2263); nhr-49(nr2041), dmaIs1; nhr-49(et7), dmaIs1; mdt-15(et14), dmaEx114[rpl-28p::nhr-49::Venus; myo-2::mCherry], dmaEx79[hir-1p::GFP; unc-54p::mCherry], dmaEx121[rpl-28p::GFP::hir-1; myo-2p::mCherry], dmaEx229[hir-1p::hir-1::Flag; unc-54p::mCherry], dmaEx229; dpy-3(ok2263), dmaEx206[snr-2p::let-756::HA; unc-54p::mCherry], dmaEx211[dpy-3p::dpy-3::FLAG; unc-54p::mCherry], dmaEx257[snr-2p::let-756::HA], DMS1101_dmaIs1; dpy-18(e364), dmaEx258[dpy-7p::dpy-3::Venus; unc-54p::mCherry], dmaEx258; hir-1(dma101), kaIs12[col-19::GFP], kaIs12; dpy-18(e364), kaIs12; hir-1(dma101), and kaIs12; hir-1(dma101); nhr-49(nr2041).

Genetic screens

A stereo-epifluorescence dissecting microscope (Nikon SMZ18) was used to isolate mutants with constitutive expression of comt-5p::GFP reporters or with suppressed expression of comt-5p::GFP reporters in the activated mutants after EMS-induced mutagenesis, as described previously (2, 11). Mutants were mapped genetically by single-nucleotide polymorphisms-based linkage analysis using the Hawaiian C. elegans strain CB4856 and then were sequenced by whole-genome sequencing to obtain lists of candidate genes. RNAi directed against genes with putative causal mutations was performed to confirm phenocopying, and the causality of mutation was subsequently confirmed by transformation rescue of mutants with WT alleles as transgenes.

Environmental stress assays

In the hypoxia stress assay, animals were placed on the NGM plate with the lid and put into a hypoxic chamber containing a plate with water to maintain normal humidity. A hypoxia chamber with ProOx 110 oxygen controller (BioSpherix) was used to induce hypoxic stress with 0.5 to 21% oxygen concentrations. A hypoxia incubator chamber (Applied StemCell) with constant nitrogen flow delivery was used to achieve severe hypoxia with nearly 0% oxygen (anoxia stress). In the hydrogen peroxide stress assay, animals were placed on the plate with 10 mM peroxide in the NGM and observed in the 1- to 48-hour intervals for GFP activation. To determine the effects of HIF-activating compounds on comt-5p::GFP, 5 mM CoCl2 and 5 mM KCN containing NGM plates were used. To measure hydrogen sulfide, 0.1 mg of NaHS powder was placed onto an NGM agar plate (10 ml of agar) with a lid sealed by parafilm to prevent leaking of the released H2S gas. To determine the effects of inhibitors of oxidative phosphorylation on comt-5p::GFP, 1 mM rotenone, 1 mM oligomycin, and 1 mM FCCP containing NGM plates were used. Animals were screened for GFP induction in the subsequent 1 to 48 hours and collected after 2 hours of exposure for qRT-PCR analysis.

Western blot analysis

Animals were lysed in the Laemmli sample buffer (Bio-Rad) with reducing agent β-mercaptoethanol, followed by boiling the samples for 10 min. The worm lysates were separated by SDS-PAGE and subsequently detected by GFP goat polyclonal antibody (AF424, Fisher Scientific) with histone H3 antibody (ab1791, Abcam) as a loading control. For FLAG-tagged and HA-tagged proteins, we used FLAG mouse monoclonal antibody (F1804, Sigma-Aldrich) and HA antibody (ab9110, Abcam), respectively.

Sample and library preparation for RNA-seq

All C. elegans strains [N2(WT), egl-9(sa307), egl-9(sa307) hif-1(ia04), and hir-1(tm4098)] were maintained at 20°C before RNA extraction. For anoxic stress, we placed N2 animals into a hypoxia incubator chamber (Applied StemCell) with constant nitrogen delivering for 2 hours before lysis. One microgram of total RNA from each sample was purified by the RNeasy Mini Kit from Qiagen and used for sequencing library construction. The NEBNext rRNA Depletion Kit, Agencourt RNAClean XP Beads (Beckman Coulter), NEBNext Ultra Directional RNA Library Prep Kit (Illumina), Agencourt AMPure XP (Beckman Coulter), and NEBNext Multiplex Oligos (Illumina) were used to prepare sequencing libraries, as per the manufacturers’ instruction. The Q5 Hot Start HiFi PCR Master Mix was used for PCR enrichment of the adaptor-ligated DNA. The libraries were submitted to 100 base pair (bp)–paired-end high-throughput sequencing using HiSeq-3000 by the Center for Advanced Technology of the University of California, San Francisco.

RNA-seq data analysis

The PRINSEQ-lite software (version 0.20.4) was used (63) to trim and filter raw reads. Reads longer than 30 bp, together with a minimum quality score of >15, were used for subsequent analyses. The Pairfq script was used for separation of paired and single reads. Clean reads were mapped to the C. elegans genome using HISAT2 (version 2.0.5) (64) with default parameters. The number of mapped reads was counted by featureCounts (version 1.5.0) (65). Differential gene expression analysis was performed using the DESeq2 package (66). An adjusted P value ≤ 0.05 was used as the threshold to identify the differentially expressed genes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for the differentially expressed genes were conducted using the Cytoscape plugins BiNGO (67) and ClueGO (68), respectively. Plots for the mapped reads were generated by igvtools (69). The accession numbers for the hir-1 samples are SRX3564686, SRX3564687, and SRX3564688; the accession numbers for the controls are SRX3563003, SRX3563021, and SRX3563026—all under the Sequence Read Archive (SRA) BioProject PRJNA430003.

Quantitative reverse transcription polymerase chain reaction

Total RNAs were isolated from animals of mixed stages, with 50-μl pellet animals (>200 animals) resuspended in 250 μl of lysis buffer of Quick-RNA MiniPrep kit (R1055, Zymo Research) and subsequently lysed by TissueRuptor (Motor unit “8” for 1 min). Total RNAs were extracted following the manufacturer’s instructions (R1055, Zymo Research). Two micrograms of RNA per sample was reverse-transcribed into complementary DNA (B24408, BioTools). Real-time PCR was performed by using Roche LightCycler 96 and SYBR Green (FERK1081, Thermo Fisher Scientific) as a double-stranded DNA–specific binding dye. qRT-PCR condition was set to 95°C for denaturation, followed by 45 cycles of 10 s at 95°C, 10 s at 60°C, and 20 s at 72°C. Melting curve analysis was performed after the final cycle to examine the specificity of primers in each reaction. Gene expression changes were calculated by the ΔΔCt method, with act-3 used as the reference gene. Primer sequences are listed in table S2.

Imaging

Animals were mounted onto a 2% agarose pad containing 10 mM sodium azide and imaged with an EVOS FL auto digital microscope for epifluorescence imaging or a confocal Leica SPE microscope for high-resolution COL-19::GFP and DPY-3::Venus confocal imaging. At least three biological replicates (≥10 animals for each replicate) were used for quantification of the designated phenotype.

Hoechst staining

Animals were placed into liquid drops containing the Hoechst 22358 dye (2 μg/ml) diluted in M9 buffer for 15 min. Animals were then picked into fresh M9 drops and subsequently placed onto 2% agarose pad containing 10 mM sodium azide for imaging with a confocal Leica SPE microscope. At least three biological replicates (10 animals for each group) were used for quantification of stained animals.

Osmotic shock assay

Five-day-old adult hermaphrodites were picked and placed into 200 μl of liquid drops of PCR-grade distilled water dropped on plastic lid of Petri plate. Time when the cuticle burst (release of insides) was monitored by stereoscope inspection during 15-s intervals. Three biological replicates (10 animals per assay) were used for statistical analysis.

Anoxia sensitivity behavioral assay

Animals were grown at 25°C for two continuous generations in nonstarving, nonstressed conditions. One-day-old adult hermaphrodites were placed on NGM plates into the anoxia chamber for 40 hours. Animals were subsequently screened for paralysis or movement in the indicated time intervals. WormLab System (MBF Bioscience) was used to quantify displacement, moving average speed, and tracking based on the midpoint position. At least three biological replicates (10 animals per assay) were used for statistical analysis.

Statistical analysis

Data are presented as means ± SD with P values calculated by unpaired Student’s t tests and one-way analysis of variance (ANOVA). Data with nonnormal distribution, including qRT-PCR and penetrance results, were assessed by nonparametric Mann-Whitney and Fisher’s exact test, respectively.

SUPPLEMENTARY MATERIALS

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Fig. S1. Genes are differentially regulated by egl-9, hif-1, hypoxia, and hir-1.

Fig. S2. Expression pattern of hir-1.

Fig. S3. Hypoxia- and ECM-related genes similarly impair cuticle integrity.

Fig. S4. Role of NHR-49 in hir-1 signaling.

Fig. S5. Hypoxia activates comt-5 through a regulatory cascade involving ECM components (LET-756 and DPY-3) and HIR-1.

Fig. S6. RNA-seq and qRT-PCR analysis of HIR-1–regulated genes.

Fig. S7. Impaired cuticle integrity in hir-1 mutants under various conditions.

Table S1. Genes that regulate comt-5p::GFP expression.

Table S2. Primer sequences.

Data file S1. RNA-seq analysis of egl-9, hif-1, and hypoxia-regulated genes.

Data file S2. Gene ontology and KEGG pathway analyses of genes differentially regulated in hir-1 mutants compared with WT animals.

REFERENCES AND NOTES

Acknowledgments: We thank the Caenorhabditis Genetics Center, National BioResource Project in Japan and the Million Mutation Project for C. elegans strains. We thank B. Wang and E. Chuang for technical assistance and M. Asahina and N. Singhal for discussion. Funding: The work was supported by NIH grants R01GM117461 and R00HL116654, American Diabetes Association grant 1-16-IBS-197, the Pew Scholar Award, the Alfred P. Sloan Foundation Fellowship, and the Packard Fellowship in Science and Engineering (to D.K.M.) and Larry L. Hillblom start-up grant (to D.K.M.) and fellowship (to R.V.). Author contributions: R.V. and D.K.M. designed, performed, and analyzed the experiments and wrote the manuscript. Y.L. performed RNA-seq and bioinformatic analysis. D.K.M. supervised the project. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The RNA-seq data have been deposited to the SRA BioProject PRJNA430003. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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