C22 disrupts embryogenesis and extends C. elegans lifespan

Abstract

Caenorhabditis elegans is an instrumental model in aging research due to its large brood size, short lifespan, and malleable genetics. However, maintaining a synchronous nematode population for longevity studies is challenging and time consuming due to their quick rate of development and reproduction. Multiple methods are employed in the field, ranging from worm strains with temperature dependent sterility to DNA replication inhibitors such as 5'-fluorodeoxyuridine (FUdR). In this study, we characterize a small molecule (C22) that impairs eggshell integrity and disrupts early embryogenesis to determine its applicability as a potential FUdR alternative. We find that C22 prevents egg hatching in a concentration dependent manner. However, it extends the lifespan of wild type worms and can induce FMO-2, a longevity regulating enzyme downstream of dietary restriction. Our results suggest that C22 is unlikely to be widely useful as an alternative to FUdR but its mechanism for lifespan extension may be worth further investigation.

Keywords: C. elegans; C22; FUdR; embryogenesis; lifespan.

FIGURE 1

C22 disrupts embryogenesis and extends wildtype C. elegans lifespan. (A) Images of F2 eggs hatching from F1 worms grown on NGM and DMSO condition plates from egg compared to F2 eggs not hatching from F1 worms grown on C22 test plates from egg at 20°C. Scale bar, 1.0 mm. (B) Images of day 1 adult N2 worms on DR displaying internal hatching on NGM and 3 µM C22 but not on FUdR/DMSO and 5 µM C22. Scale bar, 0.5 mm. (C) Quantification of percent worms with internal hatching under DR. N = 3 experiments. n > 30 worms per condition. (D,E) Percent alive of N2 worms fed live OP50 on C22 condition plates from (D) egg and (E) young adulthood. N = 2 experiments. n ∼ 100 worms per condition. Datasets are available in the source data file. One-way ANOVA with Tukey post hoc analysis was used to derive p-values for internal hatching comparisons. The log-rank test was used to derive p-values for lifespan comparisons. All error bars shown in figures represent the standard error of the mean (SEM) **** denotes p-value < 0.0001.

FIGURE 2

C22 induces fmo-2 under fed conditions and requires it for lifespan extension. (A) Pumping rate (pumps per 30 s) of day 2 adult fed N2 worms on C22 and control conditions. N = 2 experiments. n = 10 worms per condition. (B) Images of fmo-2 induction in day 2 adult worms after overnight exposure to C22 using the fmo-2p::mCherry reporter. N = 2 experiments. n > 30 worms per condition. Scale bar, 1 mm. (C) Quantification of mCherry fluorescence normalized to DMSO control. (D) Percent alive of fmo-2 worms fed live OP50 on C22 condition plates from egg. N = 2 experiments. n ∼ 100 worms per condition. Datasets are available in the source data file. A two-tailed t-test was used to derive p-values for pumping rate comparisons. One-way ANOVA with Tukey post hoc analysis was used to derive p-values for relative fluorescence comparisons. The log-rank test was used to derive p-values for lifespan comparisons. All error bars shown in figures represent the standard error of the mean (SEM) ns, no significant difference, * denotes p-value < 0.05, ** denotes p-value < 0.01, and **** denotes p-value < 0.0001.

FIGURE 3

C22 does not further extend DR mediated fmo-2 induction or lifespan extension. (A) Images of fmo-2 induction in day 2 adult worms after overnight exposure to C22 under fed and DR (overnight fast) using the fmo-2p::mCherry reporter. N = 2 experiments. n > 25 worms per condition. Scale bar, 1 mm. (B) Quantification of mCherry fluorescence normalized to fed controls. (C) Pumping rate (pumps per 30 s) of day 2 adult DR N2 worms on C22 and control conditions. N = 3 experiments. n = 10 worms per condition. (D) Percent alive of N2 worms on C22 condition plates from egg (separated to fed and sDR groups at Day 3 of adulthood). N = 2 experiments. n ∼ 100 worms per condition. Datasets are available in the source data file. One-way ANOVA with Tukey post hoc analysis was used to derive p-values for relative fluorescence and pumping assay. The log-rank test was used to derive p-values for lifespan comparisons. All error bars shown in figures represent the standard error of the mean (SEM) ns, no significant difference, *** denotes p-value < 0.001, and **** denotes p-value < 0.0001.

FIGURE 4

C22 directly impacts the lifespan and healthspan of the worms partially independent of bacteria metabolism. (A,B) Percent alive of (A) N2 and (B) fmo-2 worms fed dead OP50 on C22 condition plates from egg. N = 2 experiments. n ∼ 100 worms per condition. (C) Images of fmo-2 induction in day 2 adult worms after overnight exposure to C22 using the fmo-2p::mCherry reporter on live and dead OP50. N = 2 experiments. n > 20 worms per condition. Scale bar, 1 mm. (D) Quantification of mCherry fluorescence normalized to fed live controls. Datasets are available in the source data file. The log-rank test was used to derive p-values for lifespan comparisons. One-way ANOVA with Tukey post hoc analysis was used to derive p-values for relative fluorescence comparisons. All error bars shown in figures represent the standard error of the mean (SEM) ns, no significant difference, *** denotes p-value < 0.001, and **** denotes p-value < 0.0001.