An excreted small molecule promotes C. elegans reproductive development and aging

Abstract

Excreted small-molecule signals can bias developmental trajectories and physiology in diverse animal species. However, the chemical identity of these signals remains largely obscure. Here we report identification of an unusual N-acylated glutamine derivative, nacq#1, that accelerates reproductive development and shortens lifespan in Caenorhabditis elegans. Produced predominantly by C. elegans males, nacq#1 hastens onset of sexual maturity in hermaphrodites by promoting exit from the larval dauer diapause and by accelerating late larval development. Even at picomolar concentrations, nacq#1 shortens hermaphrodite lifespan, suggesting a trade-off between reproductive investment and longevity. Acceleration of development by nacq#1 requires chemosensation and is dependent on three homologs of vertebrate steroid hormone receptors. Unlike ascaroside pheromones, which are restricted to nematodes, fatty acylated amino acid derivatives similar to nacq#1 have been reported from humans and invertebrates, suggesting that related compounds may serve signaling functions throughout metazoa.

Figure 1 ∣. Excreted small molecules accelerate development in C. elegans.

a, Schematic of pre-conditioning experiment: worms on plates conditioned by single males or groups of hermaphrodites develop faster and have a shorter lifespan than worms raised on non-conditioned plates^,,. b, Developmental progression of N2 hermaphrodites grown on plates conditioned with exo-metabolomes of N2 hermaphrodites or males (n = 5 biologically independent samples, where each replicate was one plate with ~30 animals, also see Supplementary Dataset). c, Developmental acceleration, measuring onset of egg lay, in daf-22 hermaphrodites exposed to wildtype (N2, >99% hermaphrodites) and him-5 (~30% males) exo-metabolome (n = 10 worms for N2 and n = 9 worms for him-5)). For data, see Supplementary Table 2. Error bars represent mean ± s.e.m. and statistics were performed using the using the Χ² test (Fig. 1b) or two-tailed t-test (Fig. 1c); n.s., not significant.

Figure 2 ∣. Identification of nacq#1, a signaling molecule primarily produced by males.

a, Experimental set up for comparative metabolomics with male-enriched him-5 and wildtype N2 worm cultures. b, Ion chromatograms comparing HPLC fractions 12, 13 and 14 for m/z 293.1506 revealing presence of nacq#1 and nacq#2 in fraction 13 (this fractionation was performed once). c, HPLC-MS-based quantification of nacq#1 in the exo-metabolomes collected during different time intervals, corresponding to different developmental stages. The insert shows data for cumulative excretion of nacq#1, nacq#2, and ascr#3 up to specific time points during the L4-to-young adult transition. See Methods for details (n = 3 biologically independent samples per condition, except for the 61 h data point, n = 2). d, Relative amounts of nacq#1 excreted by hermaphrodites and males (n = 3 biologically independent samples). e, Chemical structures of compounds in f and enzymatic roles of fat-1 and fat-2. f, HPLC-MS-based quantification of nacq#1, linoleic acid, α-linolenic acid, and ascr#3 in the exo-metabolomes of wildtype (N2), fat-1(ok2323), fat-2(wa17), and daf-22(ok693) worms (n = 3 biologically independent samples). Error bars represent mean ± s.e.m.

Figure 3 ∣. Biological properties of nacq#1.

a, Synthesis of nacq#1 and related molecules. b, Developmental acceleration assays measuring the onset of egg laying in daf-22 worms exposed to nacq#1 (numbers of worms tested for each condition as indicated in Figure, also see Supplementary Table 5). c, Singled wild type hermaphrodites reach adulthood faster on plates conditioned with nacq#1 than animals on control plates (n = 25 (control) and n = 21 (nacq#1) worms per time point). Also see Supplementary Fig. 7c. d, Fractions of GFP-positive mlt-10::GFP animals developing on plates containing 100 pM nacq#1 compared to controls (n = 99 worms, 13-24 and 36-47 h (control), n = 98 worms, 25-35, 48-59 h (control), n = 101 worms, 13-24, 36-47 h (nacq#1 treated), n = 95 worms, 25-35, 48-59 h (nacq#1 treated). Boundaries between larval stages are designated at GFP expression minima. e, Progeny production during the first day of reproduction. Each dot represents offspring of one parent (n = 25 hermaphrodite parents). f, Survival of groups of hermaphrodites (25 worms/plate, blue) and singled hermaphrodites (1 worm/plate) in the presence (red) or absence of nacq#1 (black). Assays were repeated 3 times for grouped worms and 6 times for singled worms. See Supplementary Table 6 for total number of worms. g, Survival of singled hermaphrodites on plates containing nacq#1, ascr#10 or both compounds. Assays were repeated 5 times. Also see Supplementary Fig. 10 and Supplementary Tables 6-9. Error bars represent mean ± s.e.m. and statistics were performed using two-tailed t-tests.

Figure 4 ∣. nacq#1 and ascarosides are mutually antagonistic signals.

a, Ascarosides, e.g. ascr#2 and ascr#3, induce dauer diapause, a stress-resistant alternate larval stage. b, Ascarosides counteract developmental acceleration of daf-22 mutants treated with nacq#1 (numbers of worms tested for each condition are indicated in the Figure). For data, see Supplementary Table 11. c,d, nacq#1 promotes exit from the dauer stage in daf-7 (c) and daf-2 (d) mutants (n = 6 (daf-7) and n = 9 (daf-2) biologically independent samples). e, nacq#1 counteracts the effects of ascarosides in dauer exit (n = 6 biologically independent samples). For dauer data, see Supplementary Table 12. Error bars represent mean ± s.e.m. and statistics were performed using two-tailed t-tests.

Figure 5 ∣. nacq#1 signals via conserved signaling pathways.

a, Acceleration of development by the indicated concentrations of nacq#1 in wild type, daf-16 and daf-7 mutants (numbers of worms tested for each condition are indicated in the Figure). b, Acceleration of development by nacq#1 (100 pM) in wild type, nhr-8, nhr-48, daf-12, osm-6, and che-13 mutants (numbers of worms tested for each condition are indicated in the Figure). c, HPLC-MS-based quantification of nacq#1 in the exo- and endo-metabolomes of C. briggsae, collected after the worms reached the young adult stage (n = 2 biologically independent samples). d, Acceleration of development indicated concentrations of nacq#1 in wild type (N2) and genetic ablations of the ASI, ASK, or ASJ chemosensory neurons (numbers of worms tested for each condition are indicated in the Figure). e, Competing small molecule signals regulate development via NHR signaling. f, Chemical structures of N-acyl amino acids similar to nacq#1 identified from other phyla. See Supplementary Tables 13-15 for data in a, b, and d. Error bars represent mean ± s.e.m. and statistics were performed using two-tailed t-tests.