Figure 1.
A genome-wide RNAi screen for scla genes yielded the nAChR gene acr-7.
(A) Experimental design of the feeding RNAi screen in liquid culture. On day 1, rrf-3 animals were synchronized, and bacteria was cultured from the Ahringer RNAi feeding library. Synchronized L1 animals were added to induced cultures on day 2 and were allowed to grow to adulthood for 3 days. Clozapine was added on day 5, and progeny were allowed to develop for 3 days. Progeny were scored for the Scla phenotype on day 8. (B1) N2 animals grew to adulthood within 3 days in the presence of 0.1% DMSO alone. (B2) 320 µM clozapine caused larval arrest in N2 animals exposed to feeding RNAi bacteria with empty vector alone. (B3) acr-7(RNAi) animals in 0.1% DMSO alone displayed normal development. (B4) acr-7(RNAi) suppressed developmental delay caused by 320 µM clozapine. (B5) The acr-7(tm863) knockout suppressed developmental delay caused by 320 µM clozapine. Note that experiments depicted in (B) were performed on NGM plates, not in liquid culture, to allow clear photographs.
Table 1.
Summary of scla genes recovered in genome-wide RNAi screen.
Figure 2.
Functional diversity of scla genes.
Genes were classified by biological process based on Gene Ontology terms from Wormbase. The percentages for each functional class of genes required for suppression of clozapine-induced larval arrest are shown.
Figure 3.
ACR-7 protein sequence alignment and acr-7 gene structure.
(A) The C. elegans ACR-7 protein and human α7-nAChR subunit CHRNA7 were aligned using ClustalW. The four putative transmembrane domains, as predicted for the human CHRNA7 protein, were boxed. nAChRs are members of the cys-loop family of ionotropic neurotransmitter receptors, and this conserved di-cysteine loop is dotted line-boxed. α-nAChR subunits contain a pair of vicinal cysteines within the Ach binding site, and these residues are marked with asterisks. The C. elegans and human proteins share 36% identity overall and 41% identity within the pore-lining transmembrane domain II (M2). (B) The acr-7 gene contains 13 exons, and the region deleted in the tm863 allele is marked with a line. tm863 is an out-of-frame deletion which removes exons 6–7 and most of exons 5 and 8. (C) The tm863 knockout is predicted to lack all four transmembrane domains and to be a null allele.
Figure 4.
Genetic and pharmacological characterization of clozapine-induced larval arrest.
(A) Clozapine induced developmental delay in wild-type C. elegans in a concentration-dependent manner. acr-7(tm863) partially suppressed this developmental delay, and the suppression was rescued by expression of ACR-7 in the mutant background. Expression was driven by a putative acr-7 promoter in the case of acr-7(tm863) mchEx40 or by the pharyngeal muscle-specific myo-2 promoter in the case of acr-7(tm863) mchEx207. Growth was measured as the percentage of different development stages 72 hours after loading synchronized L1 animals into the drug plates. Different concentrations (80, 160, 320 µM) of clozapine were dissolved in DMSO with the maximum concentration of DMSO being 0.1%. 0.1% DMSO alone was used for control wells. (B) nAChR agonists nicotine (Nico) and levamisole (Leva) induced developmental delay in a concentration-dependent manner, mimicking the effect of clozapine. (C) Nicotine-induced developmental delay is suppressed by acr-7(tm863), and suppression was partially rescued by expression of full-length ACR-7 in the mutant background. (D) Nicotine receptor antagonists d-TC (100 and 500 µM) and DHβE (100 and 500 µM) suppressed clozapine (Cloz)-induced developmental delay. d-TC or DHβE alone did not affect the growth of the animals at 100 and 500 µM.
Figure 5.
Suppression by acr-7(lf) is specific.
A series of 22 nAChR mutants were tested for suppression of clozapine-induced larval arrest, and only acr-7(lf) produced robust suppression.
Figure 6.
Ach release was not the mechanism of clozapine-induced larval arrest.
Mutations in genes required for Ach release failed to suppress clozapine-induced developmental delay. Strong loss-of-function of the choline acetyltransferase gene cha-1 or the synaptic vesicle Ach transporter gene unc-17 alone produced developmental delay. Therefore, both weak (n2411 or e113) and strong (p1152 or e245) loss-of-function alleles were tested.
Figure 7.
ACR-7 acted in the pharyngeal muscle.
(A) ACR-7 was highly expressed in the pharynx as indicated by the arrows. A, anterior; P, posterior; D, dorsal; V, ventral. Scattered expression was also observed in the tail and vulval regions. (B) Clozapine inhibited pharyngeal pumping in wild-type animals in a concentration-dependent manner. Suppression of clozapine-induced inhibition of pharyngeal pumping was seen in acr-7(tm863) animals, and rescue was observed with expression of ACR-7 in the mutant background. A putative acr-7 promoter was utilized to drive expression for the acr-7(tm863) mchEx40 strain, whereas the pharyngeal muscle-specific myo-2 promoter was utilized for the acr-7(tm863) mchEx207 strain. (C) Both nicotine and clozapine inhibited the pumping rate in N2 animals. d-TC partially blocked these effects. Importantly, d-TC alone had no effect on pumping. (D) d-TC failed to block clozapine- or nicotine-induced inhibition of pumping in acr-7(lf) animals. * P<0.05; ** P<0.01; *** P<0.0001.