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First published online 3 May 2006
doi: 10.1242/dev.02392

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Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans

ENS, Biologie cellulaire de la synapse, Paris, F-75005 France; Inserm, U789, Paris, F-75005, France.

View larger version (153K): [in a new window]   Fig. 1. DMPP is lethal at the L2/L3 molt and induces aberrant L3 cuticle synthesis. (A) Fraction of wild-type larvae reaching adulthood when grown on increasing concentrations of DMPP (n50 worms). Experiments were performed at 20°C. (B) Representative example of an animal exposed to 0.75 mM DMPP which dies as an early L3 larvae, just after shedding the L2 cuticle (arrow). Scale bar: 0.1 mm. (C-E) Electron microscopy (EM) pictures after chemical fixation of young L3 larvae. Scale bar: 5 µm. Worms grown on DMPP (D,E) have irregular shapes when compared with the control (C). (F,G) Electron microscopy pictures of young L3 larvae cuticle after high-pressure freezing fixation. Scale bar: 250 nm. (F) Control animals grown on standard plates show a typical mono-layered L3 cuticle (Cut) covering a thin epidermis (Ep) and underlying body-wall muscles (Mus). (G) Animals grown on DMPP have an aberrant L3 cuticle with several layers of heterogeneous aspects and fibrillar structures (arrow).

View larger version (26K): [in a new window]   Fig. 2. DMPP is toxic during the L2 stage by uncoupling cell divisions and differentiation events from the molt cycle. (A) Toxicity period assayed by transfer experiments of wild-type animals. Each dot represents the surviving fraction of a synchronous population (n25 worms) transferred at a given time from standard to DMPP plates (black) or from DMPP to standard plates (gray). (B,C) Exposure to DMPP delays L2 development. (B) L2 development was divided in five stages based on seam cell (SC) divisions and anchor cell (AC) differentiation (1, undivided SC; 2, SC divided once; 3, SC divided twice; 4, anterior SC fused to hyp7, no Pcdh-3::GFP expressed in AC; 5, Pcdh-3::GFP expressed in AC). (C) The proportion of worms belonging to these five stages was scored every 2 hours during the L2 stage (n>20). Bars represent the mean of two independent experiments (exp 1: 30, 32, 34, 36 hours; exp 2: 26, 28, 32, 34, 36 hours). (D) DMPP does not affect the timing of L1/L2 and L2/L3 molts. Each dot represents the percentage of worms pumping at a given time (n>25 individuals). (E) Schematic representation of worm development at high DMPP concentration (0.75 mM). L2 development, represented here by seam cell divisions, is delayed while the molt cycle is not disrupted (hatched bars). Uncoupling these developmental events is lethal at the L2/L3 molt.

View larger version (43K): [in a new window]   Fig. 3. Forcing development through an L2 arrest suppresses DMPP-induced lethality. (A) Worms grown on a low amount (80 µg) of heat-killed bacteria arrest development as L2 larvae. Bars represent the % of larvae at each developmental stage, mean of two independent experiments. (B,C) DIC pictures of a mid-L2 larva grown on live E. coli (B) and an arrested L2 larva grown on 80 µg heat-killed E. coli (C). Arrested L2 larvae show a seam cell pattern typical of a worm having completed the two successive L2 divisions without extensive anterior sister cell migration. There is high storage droplet content in the epidermis (C). Scale bar: 10 µm. (D) Schematic representation of worm development on low amount of heat-killed E. coli as schematized in Fig. 2D. (E) DMPP resistance of animals after L2 arrest. Both groups were grown on 0.75 mM DMPP. Worms transferred on standard plates after a 3-day arrest in L2 were partially resistant to DMPP (`3 days L2 diapause') when compared with control (`continuous development'). Error bar represents s.e.m., n=3 independent experiments.

View larger version (22K): [in a new window]   Fig. 4. UNC-63-containing nAChRs are required to implement the DMPP signal. (A) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n3). Punc-63::unc-63: extrachromosomal array carrying an unc-63 genomic fragment; Pmyo-3::unc-63: muscle specific promoter driving the expression of an unc-63 cDNA. unc-63(kr13);Punc-63::unc-63, unc-38(x38) and unc-29(e1072) are significantly less DMPP resistant than unc-63(kr13) (*: P< 0.05, Mann-Whitney test). Pmyo-3::unc-63 does not rescue unc-63(kr13) DMPP resistance while it restores wild-type locomotion and levamisole sensitivity (see Fig. S1 in the supplementary material). (B-D) unc-63 expression profile. Confocal picture of a transgenic larva expressing Punc-63::unc-63-SL2-GFP. unc-63 is expressed in body-wall muscles (B, arrow), head muscles (C, arrowhead), and in many neurons in the head (C) and the ventral cord (D, arrow). Scale bar: 20 µm. (E) unc-63(kr13) does not alter the timing of L1/L2 and L2/L3 molts. Each dot represents the percentage of worms pumping at a given time (n>25 individuals). Broken lines indicate the timing of wild-type events.

View larger version (26K): [in a new window]   Fig. 5. DMPP resistance of daf-12 and daf-9 mutants. (A) daf-12 gene structure. Mutation sites of new Mos1 alleles and alleles tested for DMPP resistance are indicated. *, STOP codon. (B) Dose-response sensitivity to DMPP of wild-type and daf-12(rh61rh411) mutants. (C) daf-12(rh61rh411) does not alter the timing of L1/L2 and L2/L3 molts. Each dot represents the percentage of worms pumping at a given time (n>25 individuals). broken lines indicate the timing of wild-type events. (D) daf-12(rh61rh411) is insensitive to DMPP-induced developmental delay. L2 development was divided into five stages based on seam cell (SC) divisions and anchor cell (AC) differentiation (see Fig. 2B). Developmental stage was monitored using DIC optics, which did not allow the discrimination between classes 3 and 4. The proportion of worms belonging to each class was scored 32 hours after egg laying (n12). Data presented are from one representative experiment out of three independent trials. (E) DMPP resistance of daf-12 and daf-9 mutants (0.75 mM DMPP). Error bars represent s.e.m. (n3). Table presenting dauer and heterochronic phenotypes [adapted, with permission, from Antebi et al. (Antebi et al., 1998)]. dtc, distal tip cell.

View larger version (13K): [in a new window]   Fig. 6. Environmental modulation of the sensitivity to DMPP. (A) DMPP resistance of `genetically starved' worms. Surviving eat-6 and pep-2 mutants on DMPP were scored 4 and 5 days after egg laying, respectively. By this time, most mutants grown on control plates had reached adulthood. (B) DMPP toxicity dose-response curves at 15, 20 and 25°C (wild-type N2 strain). (C) High pheromone concentrations induce partial DMPP resistance at 20°C. As pheromone signaling is antagonized by a food signal, animals were grown on plates lacking peptone, which slightly decreases the quantity of available food (Golden and Riddle, 1984a). Error bar represents s.e.m., n=3 independent experiments. Dauer larvae are not induced under these experimental conditions but pheromone activity was tested independently for its ability to cause dauer formation (data not shown).

View larger version (22K): [in a new window]   Fig. 7. Two independent timers can be differentially regulated at the L2 stage. Activation of nAChRs by DMPP slows developmental speed without affecting the timing of molting, hence resulting in a lethal heterochronic phenotype. The nuclear receptor DAF-12 bound to a hormonal ligand is necessary to implement the developmental effect of nAChR activation. Environmental cues that reduce the available amount of liganded-DAF-12 or that delay the L2/L3 molt protect the animals from DMPP-induced lethality (see the text for a full discussion).