Metabolite induction of Caenorhabditis elegans dauer larvae arises via transport in the pharynx

Abstract

Caenorhabditis elegans sense natural chemicals in their environment and use them as cues to regulate their development. This investigation probes the mechanism of sensory trafficking by evaluating the processing of fluorescent derivatives of natural products in C. elegans. Fluorescent analogs of daumone, an ascaroside, and apigenin were prepared by total synthesis and evaluated for their ability to induce entry into a nonaging dauer state. Fluorescent imaging detailed the uptake and localization of every labeled compound at each stage of the C. elegans life cycle. Comparative analyses against natural products that did not induce dauer indicated that dauer-triggering natural products accumulated in the cuticle of the pharnyx. Subsequent transport of these molecules to amphid neurons signaled entry into the dauer state. These studies provide cogent evidence supporting the roles of the glycosylated fatty acid daumone and related ascarosides and the ubiquitous plant flavone apigenin as chemical cues regulating C. elegans development.

Figure 1

Screening for dauer induction. a) A white light image depicting C. elegans induced into dauer state by 4. Relative activity of dauer-inducing probes at b) 1 μM or c) 50 μM. Assays were developed by treating 200−300 worms as egg, during hatching, or in L1 stage with the ascribed compound in a volume of 200 μL of M9 media for 20 min. The worms were then washed with fresh media to clear unabsorbed probes, transferred to blank NG agar plates, and incubated for 52−72 h at 23 °C. The percentage of dauer worms was determined from the average of five assays of 200−300 worms. Each screen was conducted with a solvent control at 0−2% dauer formation. Dauer states were counted in agar based upon their characteristic morphology as apparent in panel a and verified by counting the worms after treatment with 1% (w/v) aqueous SDS solution. The average and deviation of both plate and SDS-treated larvae are presented.

Figure 2

Histological localization of fluorescent natural product analogs in live wild-type adult C. elegans variety Bristol strain N2. a) Daumone analogs 4 (red) and 5 (green) colocalize in adult C. elegans as given by appearance of yellow from the mixing of red and green. Both b) 4 and c) 5 concentrated within buccal cavity cuticle (bcc), procorpus (p), isthmus (i), pharyngeal sieve (ps), and grinder (g) of the cuticle of the pharynx and the first intestinal cell (Int1). Control experiments as given by the treatment of adult worms with d) solvent or e) compound 12 followed by washing with media returned worms with only autofluorescent cells within the gut (af) and the first intestinal cell (Int1). f) Apigenin analog 13 also localizes within the cuticle of the pharynx (bcc, p, ps, i, and g) and the first intestinal cell (Int1). g) The two toxic probes 16 and 17 do not target the cuticle of the pharynx with uptake apparent throughout the majority of the worm and modest concentration within the gonad (go) and basal lamina (bl) of the pharynx. h) The localization of amphotericin analog 17 was comparable to that of 16 bearing a similar dispersion throughout the worm and modest concentration in the gonad (go) and basal lamina (bl) of the pharynx. i) Blue fluorescent 19 (false colored in red) and green fluorescent daumone 5 (in green) colocalize within the cuticle of the pharynx. The more active ascaroside 19 is also observed in an amphid intermediate neuron (AINR) on the right side of the worm. j) When treated for a shorter period of 10 min, ascaroside 19 is observed within both right and left amphid neurons (ANR and ANL) near the posterior to the terminal bulb of pharynx and grinder (g). Pretreatment of worms with 18 blocks the uptake of both k) 5 and l) 19. Images were collected after treating adult worms with ascribed compound in M9 media for 1 h and were then washing with M9 media to clear each probe from the digestive track, unless noted otherwise. Blue fluorescence was collected by excitation at 377 ± 50 nm and emission at 447 ± 60 nm. Green fluorescence was collected by excitation at 500 ± 24 nm and emission at 542 ± 27 nm.

Figure 3

Life cycle analysis. a) Embryos treated with 25 μM 4 in M9 media. b) A worm hatching after being treated with 25 μM 4 in M9 media during embryogenesis. This worm grows into c) a dauer worm and exits dauer as d) an L4 worm. e) Embryos treated with 50 μM 5 in M9 media. f) A worm hatched in 50 μM 5 in M9 media after being treated during embryogenesis with 25 μM 4 in M9 media. This worm grows into g) a dauer state and exits dauer as h) an L4 worm. i) Embryos treated with 50 μM 13 in M9 media. j) A worm treated with 50 μM 13 in M9 in L1 progresses into k) a dauer state and exits as l) an L4 worm. m) Embryos treated with 10 μM 19 in M9 media. n) A worm treated with 10 μM 19 in M9 in L1 enters into o) a dauer state and exits as p) anL4 worm. q) Embryos treated with 50 μM control 12 in M9 media. r) A worm treated with 50 μM of control 12 in M9 in L1 enters into s) a dauer state and exits as t) an L4 worm. Unless otherwise noted, the worm in panels b–d, f–g, j–l, n–o, and r–t were treated at L1 with the ascribed compound in M9 media, washed three times with an equivalent volume of M9 media, and then transported to M9 agar for entry into the dauer state and subsequent return to L4. Blue fluorescence was collected by excitation at 377 ± 50 nm and emission at 447 ± 60 nm, and green was collected by excitation at 500 ± 24 nm and emission at 542 ± 27 nm. Abbreviations: af, autofluorescent gut cells; bcc, buccal cavity cuticle; g, grinder; i, isthmus; Int1, first intestinal cell; ld, lipid droplets; n1–n6, neurons; p, procorpus; ps, pharyngeal sieve; pc, posterior cell.

Figure 4

Dauer-inducing natural products block the staining of amphid neurons. a) The uptake of FITC in an adult hermaphrodite. Two-color images indicate that treatment of worms with b) 4, c) 13, or d) 19 blocks the uptake of FITC. Worms treated first with FITC and then with e) 1, f) 4, g) 13, or h) 19 are not capable of expelling FITC from their amphid neurons. Blue fluorescence was collected by excitation at 377 ± 50 nm and emission at 447 ± 60 nm, and green was collected by excitation at 500 ± 24 nm and emission at 542 ± 27 nm.

Scheme 1

Structures and syntheses of fluorescent probes 4 and 5 from daumone 1 and 19 from ascaroside 18.

Scheme 2

Structures and syntheses of fluorescent probes 13 from apigenin 14, 16 from mycophenolic acid 15, and amphotericin probe 17.