A role for worm cutl-24 in background- and parent-of-origin-dependent ER stress resistance

Abstract

Background: Organisms in the wild can acquire disease- and stress-resistance traits that outstrip the programs endogenous to humans. Finding the molecular basis of such natural resistance characters is a key goal of evolutionary genetics. Standard statistical-genetic methods toward this end can perform poorly in organismal systems that lack high rates of meiotic recombination, like Caenorhabditis worms.

Results: Here we discovered unique ER stress resistance in a wild Kenyan C. elegans isolate, which in inter-strain crosses was passed by hermaphrodite mothers to hybrid offspring. We developed an unbiased version of the reciprocal hemizygosity test, RH-seq, to explore the genetics of this parent-of-origin-dependent phenotype. Among top-scoring gene candidates from a partial-coverage RH-seq screen, we focused on the neuronally-expressed, cuticlin-like gene cutl-24 for validation. In gene-disruption and controlled crossing experiments, we found that cutl-24 was required in Kenyan hermaphrodite mothers for ER stress tolerance in their inter-strain hybrid offspring; cutl-24 was also a contributor to the trait in purebred backgrounds.

Conclusions: These data establish the Kenyan strain allele of cutl-24 as a determinant of a natural stress-resistant state, and they set a precedent for the dissection of natural trait diversity in invertebrate animals without the need for a panel of meiotic recombinants.

Keywords: Caenorhabdis elegans; Statistical genetics; Stress resistance.

Fig. 1

Tunicamycin resistance phenotypes of wild C. elegans isolates. The y-axis reports the proportion of eggs from the indicated wild isolate that developed to adulthood in the presence of tunicamycin, normalized to the analogous quantity from wild-type ED3077. For a given column, each dot represents results from one replicate population, and the bar height reports the mean. ****, unpaired two-tailed t-test p < 0.0001. Raw data are reported in Supplementary Figure 1

Fig. 2

Tunicamycin resistance of inter-strain hybrids depends on the parent of origin. The y-axis reports the proportion of eggs from the indicated cross that developed to adulthood in the presence of tunicamycin, normalized to the analogous quantity from wild-type ED3077. For a given column, each dot represents results from one replicate population; the white cross reports the mean; box and whiskers report the interquartile range and the 10-90 percentile range, respectively, of the replicate measurement distribution. ED, ED3077. *, unpaired two-tailed t-test p < 0.05; ***, p < 0.001. Raw data are reported in Supplementary Figure 2

Fig. 3

Making hemizygote mutants for RH-seq. RH-seq requires hemizygote hybrids (purple) from crosses between mutants of one background (red) and wild-types of another (blue). Top: arrays (large ovals) harboring the Mos1 transposon (green) and heat-shock-inducible transposase enzyme gene (orange) in the red background come together into one strain. Center: after heat shock, a transposon copy integrates into the genome (straight black line) of an egg of the red background, which is fertilized by a wild-type male of the blue background. Bottom: the resulting F1 hybrids are hemizygous throughout the soma and are used as input into a sequencing-based tunicamycin resistance assay

Fig. 4

RH-seq reveals cutl-24 as a candidate gene at which inter-strain variation contributes to tunicamycin resistance. In a given panel, in each row the left-hand cartoon represents the region of cutl-24 in the hybrid genome (red line, ED3077 chromosome; blue line, N2 chromosome), and the triangle denotes the position of insertion of a Mos1 transposon as detected by transposon sequencing. The right-hand cell reports the log₂ of the abundance of the respective mutant, detected by sequencing, after development in tunicamycin, relative to the analogous quantity from development in untreated control conditions. The p-value reports the result of a two-tailed Mann-Whitney statistical test for a difference in the abundance after tunicamycin selection, relative to the abundance in an untreated control, of hemizygotes harboring transposon insertions in the two parents’ orthologs. Supplementary Table 4 reports Mos1 insertion positions and raw quantitation data, with the Benjamini-Hochberg method used to correct for multiple testing. Top, hemizygotes harboring transposon insertions in the N2 allele; bottom, hemizygotes harboring transposon insertions in the ED3077 allele

Fig. 5

cutl-24 in ED3077 mothers is required for tunicamycin resistance in their inter-strain hybrid progeny. a The y-axis reports tunicamycin resistance measurements in F1 hybrid animals from crosses between wild-type or cutl-24 mutant ED3077 and N2 as indicated, normalized per experiment with respect to the mean value from the wild-type ED3077 hermaphrodite x N2 male F1. b Data are as in (a) except that strains were purebred cutl-24 mutants or wild-type controls, and measurements were normalized to the mean value from wild-type ED3077. For a given column, each dot represents results from one replicate population; the white cross reports the mean; box and whiskers report the interquartile range and the 10-90 percentile range, respectively, of the replicate measurement distribution. ED, ED3077. *, unpaired two-tailed t-test p < 0.05; **, p < 0.01; ****, p < 0.0001. Raw data are reported in Supplementary Figure 5

Fig. 6

cutl-24 expression across C. elegans developmental stages. In a given panel, each bar reports average expression (FPKM, Fragments Per Kilobase of transcript per Million mapped reads) of cutl-24 in wild-type animals of the N2 background from [39, 40] in the indicated developmental stage (a) or sex of adult animals (b). Error bars report standard deviation (n = 2)

Fig. 7

cutl-24 is expressed in neurons. In a given panel, each bar reports cutl-24 expression (TPM, Transcripts Per Million kilobases) in the indicated cell type from single cell transcriptomes [41], in embryos at the indicated developmental time in minutes (a) or in L2 larvae (b). The highest expression was detected in inner labial socket (ILso), cephalic socket (CEPso), and phasmid socket (PHso) cells; for other abbreviations in labels, see Methods