A genetic screen for aldicarb resistance of Caenorhabditiselegans dauer larvae uncovers 2 alleles of dach-1, a cytochrome P450 gene

Abstract

Animals exhibit phenotypic plasticity through the interaction of genes with the environment, and little is known about the genetic factors that change synaptic function at different developmental stages. Here, we investigated the genetic determinants of how animal's sensitivity to drugs that alter synaptic activity is regulated at a specific developmental stage using the free-living nematode Caenorhabditis elegans. C. elegans enters the stress-resistant dauer larval stage under harsh conditions. Although dauer is known to have reduced permeability and increased resistance to most known exogenous chemicals, we discovered that dauer is hypersensitive to a cholinesterase inhibitor, aldicarb. To investigate genes regulating dauer-specific acetylcholine transduction, we first screened for aldicarb-resistant mutations in dauer and then performed a secondary screen to rule out aldicarb-resistant mutations that also affect adults. We isolated 2 different mutations of a single gene called cyp-34A4 or dach-1 encoding a cytochrome P450. In the nondauer stages, dach-1 is mainly expressed in the intestine, but its expression is robustly increased in the epidermis of dauers. By tissue-specific rescue experiments, we found that dach-1 modulates aldicarb sensitivity in a cell nonautonomous manner. In addition, dach-1 plays pleiotropic functions in dauers by regulating quiescence and surviving heat shock and hyperosmolar stress. Our study reveals novel functions of the cytochrome P450 in synaptic and physiological changes during the developmental plasticity.

Keywords: cyp-34A1; aldicarb; cytochrome P450; dauer; dauer-specific acetylcholine defect-1 (dach-1); phenotypic plasticity.

Fig. 1.

Altered acetylcholine transmission in dauer stage of C. elegans. a) Dauer animals (n = 61) are sensitive to aldicarb compared to L3 stage animals (n = 60). b) Dauer animals (n = 21) are sensitive to aldicarb compared to L4 stage animals (n = 19). c) Dauer animals (n = 147) are resistant to 7% ethanol compared to L3 animals (n = 130). d) Dauer animals (n = 59) are sensitive to trichlorfon compared to L3 animals (n = 55). e) Dauer animals (n = 55) are not sensitive to levamisole compared to L3 (n = 61). f) Postdauer-L4 animals (n = 30) are not sensitive compared to dauer (n = 31). Error bar indicates SE. Statistical analysis is performed by log-rank (Mantel-Cox) test.

Fig. 2.

dach-1 is the cytochrome P450 regulating acetylcholine transmission in the dauer stage. a) Experimental scheme of forward genetics for dauer-specific aldicarb-resistance. b) Compared to daf-2(e1370), ys51 and ys52 mutants show aldicarb-resistant phenotype in the dauer stage. The percentage of animals not paralyzed after 80 min of aldicarb treatment was compared. n = 4 for each experiment. Error bar indicates SE. ****P < 0.0001 (1-way ANOVA, Dunnett’s posttest). c) ys51 and ys52 harbor distinct nonsense mutations in T09H2.1. d) Feeding RNAi of T09H2.1 in a RNAi-sensitive rrf-3;daf-2 phenocopied aldicarb-resistant phenotype in the dauer stage. The percentage of animals not paralyzed after 80 min of aldicarb treatment was compared. n = 6 for both experiments. Error bar indicates SE. ****P < 0.0001 (unpaired t-test).

Fig. 3.

dach-1 is increased in epidermis in the dauer stage and acts in cell nonautonomous manner. a) The 864-bp upstream from start codon of dach-1 was fused to a GFP minigene. Expression of dach-1 was shown in intestine at the L4 stage. Bar, 20 μm. b) dach-1 promoter showed increased expression at the dauer stage. Bar, 20 μm. c) dach-1 promoter was coexpressed with epidermis marker Pdpy-7::mCherry. Their expressions overlapped in epidermis. Bar, 20 μm. d) Tissue-specific expression of dach-1::SL2::GFP construct in dach-1 (n = 5), dpy-7 (n = 3), myo-3 (n = 6), and egl-3 (n = 6) promoter all rescued aldicarb-resistant phenotype of dach-1 mutants (n = 4) compared to wild-type N2 (n = 3). e) ace-3 fully suppresses the aldicarb resistance of dach-1. n = 3 for each experiment. d, e) The percentage of animals not paralyzed after 80 min of aldicarb treatment was compared. Error bar indicates SE. ****P < 0.0001 (1-way ANOVA, Dunnett’s posttest).

Fig. 4.

dach-1 plays a role in quiescence and stress resistance in dauer. a) dach-1 dauer shows increased spontaneous movement compared to N2. n = 3 for both strains. *P < 0.05 (2-tailed t-test). dach-1 dauer is sensitive to heat stress at 37°C for 4 h (b) and high osmotic stress at 1,500 mM NaCl for 20 h compared to N2 (c). The sensitivity of dach-1 to heat stress and osmotic stress is rescued by the expression of Pdach-1::dach-1::SL2::GFP. n = 3 for each experiment. Error bar indicates SE. ****P < 0.0001 (1-way ANOVA, Dunnett’s posttest).