Figure 3. Dithiothreitol (DTT) causes developmental toxicity via rips-1.

(A) Table summarizing the alleles of rips-1 identified by whole-genome sequencing in different DTT-resistant strains. The corresponding amino acid changes in the RIPS-1 protein are also shown. (B) Mapping of the rips-1 alleles identified in the forward genetic screen. (C) Representative images of N2 and rips-1(gk231506) animals after 84 hr of hatching at 20°C on E. coli OP50 diets containing either 0 or 10 mM DTT. Scale bar = 1 mm. (D) Fraction L4 or adult N2 and rips-1(gk231506) animals after 84 hr of hatching at 20°C on E. coli OP50 diets containing either 0 or 10 mM DTT. ***p < 0.001 via the t-test (n = 3 biological replicates; animals per condition per replicate >50). (E) Fraction L4 or adult rips-1(jsn11), rips-1(jsn11);jsnEx1, and rips-1(jsn11);jsnEx2 animals after 72 hr of hatching at 20°C on E. coli OP50 diet containing 10 mM DTT. jsnEx1 and jsnEx2 represent two independent extrachromosomal arrays containing rips-1p::rips-1::SL2::GFP and myo-2p::mCherry. ***p < 0.001 via the t-test (n = 3 biological replicates; animals per condition per replicate >50). (F) Representative fluorescence images of jsnIs1[rips-1p::GFP +myo-2p::mCherry] animals. The white arrows point at the intestinal regions showing GFP expression. Scale bar = 100 µm. (G) Gene expression analysis of N2 animals grown on E. coli OP50 diet containing 0 mM DTT until the young adult stage, followed by incubation on E. coli OP50 diet containing 0 or 10 mM DTT for 4 hr. ***p < 0.001 and **p < 0.01 via the t-test. n.s., nonsignificant (n = 3 biological replicates). (H) Representative fluorescence (top) and the corresponding brightfield (bottom) images of rips-1p::GFP animals grown on E. coli OP50 diet containing 0 mM DTT until the young adult stage, followed by incubation on E. coli OP50 diet containing 0 mM or 10 mM DTT for 8 hr. Scale bar = 200 µm. (I) Quantification of GFP levels of rips-1p::GFP animals grown on E. coli OP50 diet containing 0 mM DTT until the young adult stage, followed by incubation on E. coli OP50 diet containing 0 or 10 mM DTT for 8 hr. ***p < 0.001 via the t-test (n = 14 worms each). (J) Model depicting the effects of DTT and vitamin B12 on C. elegans development via the methionine–homocysteine cycle.

Figure 3—figure supplement 1. Forward genetic screen resulted in the isolation of 12 dithiothreitol (DTT)-resistant mutants.

(A) Representative images of N2 and DTT-resistant mutants (JSJ1 to JSJ12) on E. coli OP50 diet containing 10 mM DTT after 72 hr of hatching at 20°C. Scale bar = 1 mm. (B) Fraction L4 or adult N2 and DTT-resistant mutants on E. coli OP50 diet containing 10 mM DTT after 72 hr of hatching at 20°C.

Figure 3—figure supplement 2. Mapping of the mutations by whole-genome sequencing.

The frequencies of single-nucleotide polymorphisms (SNPs) were plotted against the positions of different chromosomes or linkage groups (LG). The high frequency of SNPs on a particular chromosome compared to other regions of the genome represents the region linked with the causative mutation(s). All mutants showed a high frequency of SNPs on chromosome V.

Figure 3—figure supplement 3. Dithiothreitol (DTT) causes developmental toxicity via rips-1.

(A) Representative images of N2 animals grown on RNA interference (RNAi) bacteria targeting the mentioned genes in the presence of 10 mM DTT after 72 hr of hatching at 20°C. EV, empty vector RNAi control. Scale bar = 1 mm. (B) Quantification of different developmental stages of N2 animals grown on RNAi bacteria targeting the mentioned genes in the presence of 10 mM DTT after 72 hr of hatching at 20°C (n = 3 biological replicates; animals per condition per replicate >100). (C) Quantification of different developmental stages of the nontransgenic and transgenic progenies of jsnEx1[rips-1p::rips-1::SL2::GFP + myo-2p::mCherry] animals after 72 hr of hatching at 20°C on E. coli OP50 diet containing 0, 2.5, and 5 mM DTT (n = 3 biological replicates; animals per condition per replicate >40). (D) Representative image of jsnEx1 animals after 4 days of hatching at 20°C on E. coli OP50 diet containing 5 mM DTT. Fluorescent (green) and nonfluorescent (nongreen) worms represent transgenic and nontransgenic progeny, respectively. Scale bar = 0.5 mm.

Figure 3—figure supplement 4. Dithiothreitol (DTT) upregulates the mitochondrial unfolded protein response (UPR).

(A) Representative fluorescence images (top) and the corresponding brightfield images (bottom) of hsp-6p::GFP animals grown on E. coli OP50 diet containing 0 mM DTT until the young adult stage, followed by incubation on E. coli OP50 diet containing 0, 10, and 10 mM DTT supplemented with 50 nM vitamin B12 for 24 hr. Scale bar = 200 µm. (B) Quantification of GFP levels of hsp-6p::GFP animals grown on E. coli OP50 diet containing 0 mM DTT until the young adult stage, followed by incubation on E. coli OP50 diet containing 0, 10, and 10 mM DTT supplemented with 50 nM vitamin B12 for 24 hr. p values are relative to the control. ***p < 0.001 via the t-test. n.s., nonsignificant (n = 15 for 0 mM DTT, and 16 each for 10 mM DTT and 10 mM DTT supplemented with 50 nM vitamin B12).