A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

Abstract

Small molecule metabolites play important roles in Caenorhabditis elegans biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate C. elegans development and behavior. Low concentrations of ascarosides attract males and thus appear to be part of the C. elegans sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage, an alternative, nonaging larval stage. The ascarosides act synergistically, which presented challenges for their identification via traditional activity-guided fractionation. As a result the chemical characterization of the dauer and male attracting pheromones remained incomplete. Here, we describe the identification of several additional pheromone components by using a recently developed NMR-spectroscopic approach, differential analysis by 2D NMR spectroscopy (DANS), which simplifies linking small molecule metabolites with their biological function. DANS-based comparison of wild-type C. elegans and a signaling-deficient mutant, daf-22, enabled identification of 3 known and 4 previously undescribed ascarosides, including a compound that features a p-aminobenzoic acid subunit. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure-activity relationships. Using a combination of the three most active ascarosides allowed full reconstitution of the male-attracting activity of wild-type pheromone extract. Our results highlight the efficacy of DANS as a method for identifying small-molecule metabolites and placing them within a specific genetic context. This study further supports the hypothesis that ascarosides represent a structurally diverse set of nematode signaling molecules regulating major life history traits.

Fig. 1.

Structures and male-attracting activity of ascarosides in C. elegans. (A) Previously identified ascarosides via activity-guided fractionation (blue). (B) Additional ascarosides identified in this study via DANS (red). (C) Wild-type (N2) metabolite extract has strong male-attracting activity, whereas daf-22 mutant metabolite extract is inactive. A mixture of previously identified ascr#2 and ascr#3 in amounts corresponding to those present in the wild-type metabolite extract (20 fmol each) added to the inactive daf-22 metabolite extract resulted in significant male attraction, but was much less attractive than wild-type metabolite extract containing similar amounts of ascr#2 and ascr#3.

Fig. 2.

DANS-based comparison of daf-22 and wild-type metabolomes. (A) Schematic representation of DANS. C. elegans wild-type metabolite extract has dauer-inducing and male-attracting activity, whereas daf-22 metabolite extract is inactive (8, 19). Comparison of wild-type and daf-22 metabolomes via DANS reveals candidate molecules for biological evaluation (red). (B) Aliphatic section of the dqfCOSY spectrum of C. elegans wild-type metabolite extract prior to differential analysis, revealing the complexity of this extract. (C) DANS overlay of the wild-type spectrum in Fig. 2B with the corresponding region of the daf-22 spectrum. Signals present in both wild-type and daf-22 spectra cancel or change color (blue), whereas signals representing compounds only present in wild type remain unaffected (brown), most prominently signals representing part of the side chains of ascr#2 and ascr#3.

Fig. 3.

Identification of daf-22-dependent metabolites ascr#6.1, ascr#7, and ascr#8. (A) DANS overlay of daf-22 and wild-type spectra, aromatic region, showing signals representing carbonyl-conjugated double bonds of ascr#3, ascr#7, and ascr#8 (red), the p-substituted aromatic ring in ascr#8 (blue), and several indole derivatives, of which the major component is indole-3-acetic acid (green). Signals corresponding to the methyl esters of ascr#3 and ascr#8, artifacts that arose from storage in methanol, have been removed for clarity. (B) Synthesis of ascr#6.1, ascr#7, and ascr#8. a, Mg/THF, 82%; b, TBDMSCl/DMF, 71%; c, O₃/(CH₃)₂S, 54%; d, (CH₃O)₂POCH₂CO₂CH₃/LiCl/DIEA/CH₃CN, 90%; e, 40% HF/H₂O, 88%; f, LiOH/dioxane 60 °C, 93%; g, (I) Oxalylchloride/DCM/DMF (II) p-aminobenzoic acid/DIEA/DCM, 31%; h, TMSOTF/hexanediol/DCM, 72%; i, KOH, 93%; j, TMSOTF and 4 in DCM– 36%; k, LiOH/THF, 100%; l: TMSOTF and 5 in DCM – 89%.

Fig. 4.

Biological evaluation of daf-22-dependent ascarosides identified via DANS. (A) Differential activity of the identified ascarosides in the male attraction assay. ascr#3 showed maximal activity followed by ascr#8 and ascr#2. All other ascarosides did not exhibit significant activity. All compounds were assayed in amounts of 1 pmol. (B) Concentration dependence of male attraction for ascr#2, ascr#3, and ascr#8. ascr#8 shows a broader range of activity than ascr#2 and ascr#3. Data for ascr#2 and ascr#3 have been published (8). (C) Synergistic interactions between the three most active ascarosides. Combinations of ascr#2 and ascr#3 as well as ascr#2 and ascr#8 displayed strong synergy, whereas ascr#3 and ascr#8 did not. ascr#2 and ascr#8 were tested at 100 fmol and ascr#3 at 10 fmol (8). (D) Dauer induction of the different ascarosides. All compounds were assayed at concentrations of 40 nM and 200 nM, and each compound was tested on at least 5 different days. ascr#2 was the most potent dauer inducer and showed significant dauer induction at both concentrations tested. ascr#3, ascr#6.1, and ascr#8 show significant dauer induction only at 200 nM, whereas ascr#7 does not show significant dauer formation at both of the concentrations tested. (E) Reconstitution of male attraction activity of wild-type metabolite extract by combining synthetic ascarosides. A mixture of 20 fmol of ascr#2 and 20 fmol of ascr#3 resulted in significant male attraction, but was less attractive than wild-type metabolite extract containing similar amounts of ascr#2 and ascr#3. However, a ternary mixture of 20 fmol of each ascr#2, ascr#3, and ascr#8 was as active as wild-type metabolite extract. Adding daf-22 metabolite extract does not further increase activity of this ternary mixture. For each data point in Fig. 4 A–C and E, n ≥ 30 animals were used. Error bars, SEM; *, P < 0.01; **, P < 0.001; ***, P < 0.0001, unpaired test with Welch's correction (A and E) and one-factor ANOVA with Tukey–Kramer post test (C).