Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 α- and 2 non-α-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.     

RNA interference and retinoblastoma-related genes are required for repression of endogenous siRNA targets in Caenorhabditis elegans

In Caenorhabditis elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs (siRNAs) may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Here, we present a microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1, and alg-1 and the retinoblastoma (Rb) mutant lin-35. We found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) up-regulated in the rde-4 and zfp-1 mutants and b) up-regulated in the lin-35(Rb) mutant, but not the down-regulated genes are highly represented in the set of genes with corresponding endogenous siRNAs (endo-siRNAs). Our study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. elegans.     

Caenorhabditis elegans SID-2 is required for environmental RNA interference

In plants and in the nematode Caenorhabditis elegans, an RNAi signal can trigger gene silencing in cells distant from the site where silencing is initiated. In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi.     

Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans

CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).     

Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans

The genome of Caenorhabditis elegans possesses two genes, dpy-18 and phy-2, that encode alpha subunits of the enzyme prolyl 4-hydroxylase. We have generated deletions within each gene to eliminate prolyl 4-hydroxylase activity from the animal. The dpy-18 mutant has an aberrant body morphology, consistent with a role of prolyl 4-hydroxylase in formation of the body cuticle. The phy-2 mutant is phenotypically wild type. However, the dpy-18; phy-2 double mutant is not viable, suggesting an essential role for prolyl 4-hydroxylase that is normally accomplished by either dpy-18 or phy-2. The effects of the double mutation were mimicked by small-molecule inhibitors of prolyl 4-hydroxylase, validating the genetic results and suggesting that C. elegans can serve as a model system for the discovery of new inhibitors.     

Repetitive-DNA elements are similarly distributed on Caenorhabditis elegans autosomes

The positions of approximately 4,800 individual miniature inverted-repeat transposable element (MITE)-like repeats from four families were mapped on the Caenorhabditis elegans chromosomes. These families represent 1-2% of the total sequence of the organism. The four MITE families (Cele1, Cele2, Cele14, and Cele42) displayed distinct chromosomal distribution profiles. For example, the Cele14 MITEs were observed clustering near the ends of the autosomes. In contrast, the Cele2 MITEs displayed an even distribution through the central autosome domains, with no evidence for clustering at the ends. Both the number of elements and the distribution patterns of each family were conserved on all five C. elegans autosomes. The distribution profiles indicate chromosomal polarity and suggest that the current genetic and physical maps of chromosomes II, III, and X are inverted with respect to the other chromosomes. The degree of conservation of both the number and distribution of these elements on the five autosomes suggests a role in defining specific chromosomal domains.     

Caenorhabditis elegans functional orthologue of human protein h-mucolipin-1 is required for lysosome biogenesis

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disease characterized by severe psychomotor retardation, achlorhydria, and ophthalmological abnormalities. Cells from several tissues in MLIV patients accumulate large vacuoles that are presumed to be lysosomes, but whose exact nature remains to be determined. Other defects include the deterioration of neuronal integrity in the retina and the cerebellum. MCOLN1, the gene mutated in MLIV patients, encodes a protein called h-mucolipin-1 that has six predicted transmembrane domains and functions as a Ca(2+)-permeable channel that is modulated by changes in Ca2+ concentration. CUP-5 is the Caenorhabditis elegans functional orthologue of h-mucolipin-1. Mutations in cup-5 result in the accumulation of large vacuoles in several cells, in increased cell death, and in embryonic lethality. We demonstrate here that CUP-5 functions in the biogenesis of lysosomes originating from hybrid organelles. We also show that at least two h-mucolipin family members rescue cup-5 mutant endocytic defects, indicating that there may be functional redundancy among the human proteins. Finally, we propose a model that relates the lysosome biogenesis defect in the absence of CUP-5/h-mucolipin-1 to cellular phenotypes in worms and in humans.     

Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans

Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions.The nematode Caenorhabditis elegans secretes the ascarosides, structurally diverse derivatives of the 3,6-dideoxy-l-sugar ascarylose, as chemical signals to control its development and behavior. Under favorable growth conditions, C. elegans progresses from the egg through four larval stages (L1–L4) to the reproductive adult. At high nematode population densities, however, specific ascarosides, which are together known as the dauer pheromone, trigger entry into a specialized L3 larval stage called the dauer (Fig. 1A) (1–5). Dauers have a thickened cuticle, do not feed, derive energy from fat stores, and up-regulate stress-resistance pathways (6). Certain ascarosides also influence behaviors, including male attraction to hermaphrodites, hermaphrodite attraction to males, avoidance, and aggregation (7–12). C. elegans senses the ascarosides using several classes of G protein-coupled receptors (GPCRs), which are expressed in specific chemosensory neurons (13, 14). The dauer pheromone ascarosides induce dauer formation by downregulating the insulin/insulin-like growth factor 1 (IGF-1) and TGF-β pathways, which control not only dauer development but also metabolism and lifespan in C. elegans (15).Open in a separate windowFig. 1.Ascaroside pheromones and β-oxidation enzymes implicated in their biosynthesis. (A) Dauer-inducing ascaroside pheromones. (B) Modular structure of the ascarosides. The ascarosides can be named according to the number of carbons in their side chains using the following rubric: head group-asc-(ω)(Δ)C#-terminus group. Head groups can be attached via the 4′-hydroxyl of ascarylose (e.g., indole-3-carbonyl group) or the 2′-hydroxyl of ascarylose (e.g., glucosyl group). Modifications to the terminus of the side chain include para-aminobenzoic acid and methylketone. R = H for the ω-ascarosides and R = CH₃ for the (ω-1)-ascarosides. (C) The β-oxidation enzymes implicated in ascaroside biosynthesis. The red dot tracks the position of the β-carbon during the β-oxidation cycle.All ascarosides have a simple, modular structure with a fatty acid-derived side chain (Fig. 1B). The fatty acid side chain is attached to the ascarylose sugar at either its penultimate (ω-1) or terminal (ω) carbon, and is sometimes unsaturated (Δ) at the α-β position. The ascarosides can be named according to the number of carbons in their side chains using the following rubric: head group-asc-(ω)(Δ)C#-terminus group (16). Head groups attached to the ascarylose sugar include the indole-3-carbonyl (IC) and glucosyl (Glu) modifications, and terminus groups on the fatty acid terminus include the methylketone (MK) and para-aminobenzoic acid (PABA) modifications (Fig. 1B). The dauer pheromone consists of at least five ascarosides, asc-C6-MK (C6; ascr#2), asc-ΔC9 (C9; ascr#3), asc-ωC3 (C3; ascr#5), IC-asc-C5 (C5; icas#9), and asc-ΔC7-PABA (ascr#8) (Fig. 1A) (2–5). Of the dauer pheromone ascarosides, only asc-ωC3 has a side chain that is attached to the ascarylose sugar at its ω-carbon [as opposed to its (ω-1)-carbon]. Intriguingly, this ascaroside works synergistically with the others to induce dauer formation (3), and targets a unique class of GPCRs (13, 14).Biosynthesis of the side chains of the ascarosides requires peroxisomal β-oxidation cycles that shorten the side chains by two carbons per cycle (11, 17–19). Four peroxisomal enzymes, an acyl-CoA oxidase (ACOX-1), enoyl-CoA hydratase (MAOC-1), (3R)-hydroxyacyl-CoA dehydrogenase (DHS-28), and 3-ketoacyl-CoA thiolase (DAF-22), have been implicated in the β-oxidation cycles for ascaroside biosynthesis (Fig. 1C) (11, 17–19). These enzymes are homologous to mammalian peroxisomal enzymes that process very long chain fatty acids, branched-chain fatty acids, and bile acid intermediates (20). Worms with mutations in maoc-1, dhs-28, or daf-22 do not produce any of the short-chain, dauer-inducing ascarosides, but instead accumulate ascarosides with long-chain side chains, stalled at different steps in the β-oxidation process (11, 17). This phenotype suggests the following three models for the role of β-oxidation in ascaroside biosynthesis: (i) β-Oxidation shortens long-chain ω/(ω-1)-ascarosides to short-chain ω/(ω-1)-ascarosides; (ii) β-oxidation shortens long-chain ω/(ω-1)-hydroxylated fatty acids to short-chain ω/(ω-1)-hydroxylated fatty acids, which are then attached to the ascarylose sugar; or (iii) β-oxidation shortens long-chain fatty acids to short-chain fatty acids, which are then ω/(ω-1)-hydroxylated and attached to the ascarylose sugar (Fig. S1). Worms with mutations in acox-1 have a less severe phenotype; they produce very little asc-ωC3, asc-C5, asc-ΔC7, and asc-ΔC9 and, instead, accumulate asc-ωC5 and asc-C9, implicating ACOX-1 in the β-oxidation of both ω- and (ω-1)-ascarosides (11). This result suggests that fatty acid side-chain hydroxylation precedes β-oxidation during the biosynthetic process and that the third model is not correct.Ascaroside production has been shown to be influenced by a variety of factors, including developmental stage (21), temperature (3, 19), and sex (8), although interpretation of these results is often complicated by the fact that they were obtained by changing multiple variables at the same time (21) or by using long-term, mixed-larval stage cultures (3, 19). Starvation has been suggested to increase the ratio of asc-ΔC9 to asc-ωC3 in long-term, mixed-stage cultures (11). On the other hand, starved L1 larvae, in comparison with those that are fed, have been shown to secrete a higher ratio of ascarosides with shorter, 5-carbon side chains to those with longer, 9-carbon side chains (12).Here we show that specific acyl-CoA oxidases enable C. elegans to modulate the composition of the ascaroside pheromones that it produces under different environmental conditions. Specifically, an ACOX-1 homodimer acts on the CoA-thioester of an ascaroside with a 9-carbon (ω-1)-side chain, an ACOX-1/ACOX-3 heterodimer acts on one with a 7-carbon (ω-1)-side chain, and an ACOX-2 homodimer acts on one with a 5-carbon ω-side chain. Using quantitative (q)RT-PCR, we show that acox-2 and acox-3, which act as gatekeepers for the production of shorter-chain ascarosides, are regulated by changes in temperature and food availability, leading to corresponding changes in ascaroside production. Studies of the ascaroside biosynthetic pathway and how it is modulated by different factors to control pheromone production will shed light on chemical communication in the C. elegans model system, as well as in other free-living and parasitic nematode species, which also use ascarosides as pheromones (22, 23).     

Six and Eya promote apoptosis through direct transcriptional activation of the proapoptotic BH3-only gene egl-1 in Caenorhabditis elegans

The decision of a cell to undergo programmed cell death is tightly regulated during animal development and tissue homeostasis. Here, we show that the Caenorhabditis elegans Six family homeodomain protein C. elegans homeobox (CEH-34) and the Eyes absent ortholog EYA-1 promote the programmed cell death of a specific pharyngeal neuron, the sister of the M4 motor neuron. Loss of either ceh-34 or eya-1 function causes survival of the M4 sister cell, which normally undergoes programmed cell death. CEH-34 physically interacts with the conserved EYA domain of EYA-1 in vitro. We identify an egl-1 5′ cis-regulatory element that controls the programmed cell death of the M4 sister cell and show that CEH-34 binds directly to this site. Expression of the proapoptotic gene egl-1 in the M4 sister cell requires ceh-34 and eya-1 function. We conclude that an evolutionarily conserved complex that includes CEH-34 and EYA-1 directly activates egl-1 expression through a 5′ cis-regulatory element to promote the programmed cell death of the M4 sister cell. We suggest that the regulation of apoptosis by Six and Eya family members is conserved in mammals and involved in human diseases caused by mutations in Six and Eya.     

sqv mutants of Caenorhabditis elegans are defective in vulval epithelial invagination

By screening for mutations that perturb the invagination of the vulva of the Caenorhabditis elegans hermaphrodite, we have isolated 25 mutations that define eight genes. We have named these genes sqv-1 to sqv-8 (squashed vulva). All 25 mutations cause the same vulval defect, an apparent partial collapse of the vulval invagination and an elongation of the central vulval cells. Most sqv mutations also cause an oocyte or somatic gonad defect that results in hermaphrodite sterility, and some sqv mutations cause maternal-effect lethality. We propose that the sqv genes affect a pathway common to vulval invagination, oocyte development, and embryogenesis.     

A RHO GTPase-mediated pathway is required during P cell migration in Caenorhabditis elegans

The Rho family of guanine triphosphate hydrolases controls various cellular processes, including cell migration. We describe here the demonstration of a role for a RhoA GTPase homologue during cell migration in Caenorhabditis elegans. We show that eliminating or reducing rho-1 gene function by using a dominant-negative transgene or dsRNA interference results in a severe defect in migration of hypodermal P cells to a ventral position. Biochemical and genetic data also suggest that unc-73, which encodes a Trio-like guanine nucleotide exchange factor, may act as an activator of rho-1 in the migration process. Mutations in let-502 ROCK, a homologue of a RhoA effector in mammals, also cause defects in P cell migration, suggesting that it may be one of several effectors acting downstream of rho-1 during P cell migration. Finally, we provide evidence to support the idea that other small Rac subfamily small GTPases act redundantly and in parallel to RHO-1 in this specific cell migration event.     

Polycomb-like genes are necessary for specification of dopaminergic and serotonergic neurons in Caenorhabditis elegans

The molecular mechanisms underlying the formation of neurons with defined neurotransmitters are not well understood. In this study, we demonstrate that the PcG-like genes in Caenorhabditis elegans, sop-2 and sor-3, regulate the formation of dopaminergic and serotonergic neurons and several other neuronal properties. sor-3 encodes a novel protein containing an MBT repeat, a domain that contains histone-binding activity and is present in PcG proteins SCM and Sfmbt in other organisms. We further show that mutations in sor-3 lead to ectopic expression of Hox genes and cause homeotic transformations. Specification of certain neuronal identities by these PcG-like genes appears to involve regulation of non-Hox gene targets. Our studies revealed that the PcG-like genes are crucial for coordinately regulating the expression of discrete aspects of neuronal identities in C. elegans.     

A circuit for navigation in Caenorhabditis elegans

Caenorhabditis elegans explores its environment by interrupting its forward movement with occasional turns and reversals. Turns and reversals occur at stable frequencies but irregular intervals, producing probabilistic exploratory behaviors. Here we dissect the roles of individual sensory neurons, interneurons, and motor neurons in exploratory behaviors under different conditions. After animals are removed from bacterial food, they initiate a local search behavior consisting of reversals and deep omega-shaped turns triggered by AWC olfactory neurons, ASK gustatory neurons, and AIB interneurons. Over the following 30 min, the animals disperse as reversals and omega turns are suppressed by ASI gustatory neurons and AIY interneurons. Interneurons and motor neurons downstream of AIB and AIY encode specific aspects of reversal and turn frequency, amplitude, and directionality. SMD motor neurons help encode the steep amplitude of omega turns, RIV motor neurons specify the ventral bias of turns that follow a reversal, and SMB motor neurons set the amplitude of sinusoidal movement. Many of these sensory neurons, interneurons, and motor neurons are also implicated in chemotaxis and thermotaxis. Thus, this circuit may represent a common substrate for multiple navigation behaviors.     

Caenorhabditis elegans chromatin-associated proteins SET-2 and ASH-2 are differentially required for histone H3 Lys 4 methylation in embryos and adult germ cells

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1-MLL5. The activity of SET1 and MLL proteins relies on protein-protein interactions within large multisubunit complexes that include three core components: RbBP5, Ash2L, and WDR5. It remains unclear how the composition and specificity of these complexes varies between cell types and during development. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RbBP5, Ash2L, and WDR5. Here we show that SET-2 is responsible for the majority of bulk H3K4 methylation at all developmental stages. However, SET-2 and absent, small, or homeotic discs 2 (ASH-2) are differentially required for tri- and dimethylation of H3K4 (H3K4me3 and -me2) in embryos and adult germ cells. In embryos, whereas efficient H3K4me3 requires both SET-2 and ASH-2, H3K4me2 relies mostly on ASH-2. In adult germ cells by contrast, SET-2 serves a major role whereas ASH-2 is dispensable for H3K4me3 and most H3K4me2. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function in the maintenance of a functional germ line. This study demonstrates that individual subunits of SET1-related complexes can show tissue specificity and developmental regulation and establishes C. elegans as a model to study SET1-related complexes in a multicellular organism.