Interacting Genes Required for Pharyngeal Excitation by Motor Neuron Mc in Caenorhabditis Elegans

We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.     

eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.     

EAT-20, a novel transmembrane protein with EGF motifs, is required for efficient feeding in Caenorhabditis elegans

The pharynx of Caenorhabditis elegans is a neuromuscular organ responsible for feeding, concentrating food by its pumping movement. A class of mutants, the eat mutants, are defective in this behavior. We have identified a novel eat gene, eat-20, encoding a unique transmembrane protein with three EGF motifs. Staining with a specific polyclonal antibody reveals that EAT-20 is expressed predominantly in the pharyngeal muscles and a subset of neurons. Some hypodermal cells also express EAT-20. eat-20 mutant animals are starved, have smaller brood sizes, and have prolonged egg-laying periods. The starvation apparently results from pharyngeal pumping defects, including a reduced pumping rate and "slippery pumping," in which the contents of the pharynx sometimes move rostrally. However, electrical activity of eat-20 mutants appears normal by electropharyngeogram.     

eat-11 encodes GPB-2, a Gbeta(5) ortholog that interacts with G(o)alpha and G(q)alpha to regulate C. elegans behavior

In C. elegans, a G(o)/G(q) signaling network regulates locomotion and egg laying [1-8]. Genetic analysis shows that activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is suppressed by perturbations of this network, which include loss of the GOA-1 G(o)alpha, DGK-1 diacylglycerol kinase, EAT-16 G protein gamma subunit-like (GGL)-containing RGS protein, or an unidentified protein encoded by the gene eat-11 [9]. We cloned eat-11 and report that it encodes the Gbeta(5) ortholog GPB-2. Gbeta(5) binds specifically to GGL-containing RGS proteins, and the Gbeta(5)/RGS complex can promote the GTP-hydrolyzing activity of Galpha subunits [10, 11]. However, little is known about how this interaction affects G protein signaling in vivo. In addition to EAT-16, the GGL-containing RGS protein EGL-10 participates in G(o)/G(q) signaling; EGL-10 appears to act as an RGS for the GOA-1 G(o)alpha, while EAT-16 appears to act as an RGS for the EGL-30 G(q)alpha [4, 5]. We have combined behavioral, electrophysiological, and pharmacological approaches to show that GPB-2 is a central member of the G(o)/G(q) network and that GPB-2 may interact with both the EGL-10 and EAT-16 RGS proteins to mediate the opposing activities of G(o)alpha and G(q)alpha. These interactions provide a mechanism for the modulation of behavior by antagonistic G protein networks.     

A Caenorhabditis elegans MAP kinase kinase, MEK-1, is involved in stress responses

The c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, was shown to be involved in the response to various stresses in cultured cells. However, there is little in vivo evidence indicating a role for a JNK pathway in the stress response of an organism. We identified the Caenorhabditis elegans mek-1 gene, which encodes a 347 amino acid protein highly homologous to mammalian MKK7, an activator of JNK. Mek-1 reporter fusion proteins are expressed in pharyngeal muscle, uterus, a portion of intestine, and neurons. A mek-1 deletion mutant is hypersensitive to copper and cadmium ions and to starvation. A wild-type mek-1 transgene rescued the hypersensitivity to the metal ions. Double mutants of mek-1 with an eat-5, eat-11 or eat-18 mutation, which are characterized by a limited feeding defect, showed distinct growth defects under normal conditions. Expression of an activated form of MEK-1 in the whole animal or specifically in the pharynx inhibited pharyngeal pumping. These results suggest a role for mek-1 in stress responses, with a focus in the pharynx and/or intestine.     

D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons

Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.     

Two RGS proteins that inhibit Galpha(o) and Galpha(q) signaling in C. elegans neurons require a Gbeta(5)-like subunit for function

BACKGROUND: Gbeta proteins have traditionally been thought to complex with Ggamma proteins to function as subunits of G protein heterotrimers. The divergent Gbeta(5) protein, however, can bind either Ggamma proteins or regulator of G protein signaling (RGS) proteins that contain a G gamma-like (GGL) domain. RGS proteins inhibit G protein signaling by acting as Galpha GTPase activators. While Gbeta(5) appears to bind RGS proteins in vivo, its association with Ggamma proteins in vivo has not been clearly demonstrated. It is unclear how Gbeta(5) might influence RGS activity. In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit Galpha(o) and Galpha(q) signaling, respectively. RESULTS: We knocked out the gene encoding the C. elegans Gbeta(5) ortholog, GPB-2, to determine its physiological roles in G protein signaling. The gpb-2 mutation reduces the functions of EGL-10 and EAT-16 to levels comparable to those found in egl-10 and eat-16 null mutants. gpb-2 knockout animals are viable, and exhibit no obvious defects beyond those that can be attributed to a reduction of EGL-10 or EAT-16 function. GPB-2 protein is nearly absent in eat-16; egl-10 double mutants, and EGL-10 protein is severely diminished in gpb-2 mutants. CONCLUSIONS: Gbeta(5) functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gbeta(5), these RGS proteins have little or no function. The formation of RGS-Gbeta(5) complexes is required for the expression or stability of both the RGS and Gbeta(5) proteins. Appropriate RGS-Gbeta(5) complexes regulate both Galpha(o) and Galpha(q) proteins in vivo.     

A Homolog of FHM2 Is Involved in Modulation of Excitatory Neurotransmission by Serotonin in C. elegans

The C. elegans eat-6 gene encodes a Na⁺, K⁺-ATPase α subunit and is a homolog of the familial hemiplegic migraine candidate gene FHM2. Migraine is the most common neurological disorder linked to serotonergic dysfunction. We sought to study the pathophysiological mechanisms of migraine and their relation to serotonin (5-HT) signaling using C. elegans as a genetic model. In C. elegans, exogenous 5-HT inhibits paralysis induced by the acetylcholinesterase inhibitor aldicarb. We found that the eat-6(ad467) mutation or RNAi of eat-6 increases aldicarb sensitivity and causes complete resistance to 5-HT treatment, indicating that EAT-6 is a component of the pathway that couples 5-HT signaling and ACh neurotransmission. While a postsynaptic role of EAT-6 at the bodywall NMJs has been well established, we found that EAT-6 may in addition regulate presynaptic ACh neurotransmission. We show that eat-6 is expressed in ventral cord ACh motor neurons, and that cell-specific RNAi of eat-6 in the ACh neurons leads to hypersensitivity to aldicarb. Electron microscopy showed an increased number of synaptic vesicles in the ACh neurons in the eat-6(ad467) mutant. Genetic analyses suggest that EAT-6 interacts with EGL-30 Gαq, EGL-8 phospholipase C and SLO-1 BK channel signaling to modulate ACh neurotransmission and that either reduced or excessive EAT-6 function may lead to increased ACh neurotransmission. Study of the interaction between eat-6 and 5-HT receptors revealed both stimulatory and inhibitory 5-HT inputs to the NMJs. We show that the inhibitory and stimulatory 5-HT signals arise from distinct 5-HT neurons. The role of eat-6 in modulation of excitatory neurotransmission by 5-HT may provide a genetic explanation for the therapeutic effects of the drugs targeting 5-HT receptors in the treatment of migraine patients.     

Actions of glutamate and ivermectin on the pharyngeal muscle of Ascaridia galli: a comparative study with Caenorhabditis elegans

The actions of glutamate and ivermectin were examined in the pharynx of Ascaridia galli and the results compared with those on the pharynx of Caenorhabditis elegans. In both preparations glutamate elicits a depolarization and inhibition of pharyngeal pumping, but the response of the pharynx of A. galli was much less than for C. elegans. This may be either because the pharyngeal membrane potential of the former is closely linked to the equilibrium potential for chloride ions (E(Cl)) while that of C. elegans is independent of E(Cl), or that there is a lower density of glutamate receptors on the pharyngeal muscle of A. galli compared with C. elegans. The maximum depolarization to glutamate of the pharyngeal muscle was 4.5+/-0.8 mV in A. galli while it was >25 mV in C. elegans. Picrotoxin was a weak antagonist of the glutamate response in both species. Flufenamic acid, pentobarbitone and flurazepam had no significant effect on either preparation at concentrations up to 100 microM. Three glutamate receptor agonists, ibotenate, kainate and quisqualate were all more potent than glutamate on the A. galli pharyngeal muscle. In contrast, only ibotenate was more potent than glutamate in C. elegans pharynx, the other two agonists being approximately 20 times less potent. The potency of ivermectin differed markedly between the two species, being approximately three orders of magnitude less potent on the pharynx of A. galli compared with C. elegans. This study demonstrates clear differences between the properties of the pharyngeal muscle of the two species and shows that care must be taken when extrapolating data from free-living to parasitic species of nematode.     

Regulation of intermuscular electrical coupling by the Caenorhabditis elegans innexin inx-6

The innexins represent a highly conserved protein family, the members of which make up the structural components of gap junctions in invertebrates. We have isolated and characterized a Caenorhabditis elegans gene inx-6 that encodes a new member of the innexin family required for the electrical coupling of pharyngeal muscles. inx-6(rr5) mutants complete embryogenesis without detectable abnormalities at restrictive temperature but fail to initiate postembryonic development after hatching. inx-6 is expressed in the pharynx at all larval stages, and an INX-6::GFP fusion protein showed a punctate expression pattern characteristic of gap junction proteins localized to plasma membrane plaques. Video recording and electropharyngeograms revealed that in inx-6(rr5) mutants the anterior pharyngeal (procorpus) muscles were electrically coupled to a lesser degree than the posterior metacorpus muscles, which caused a premature relaxation in the anterior pharynx and interfered with feeding. Dye-coupling experiments indicate that the gap junctions that link the procorpus to the metacorpus are functionally compromised in inx-6(rr5) mutants. We also show that another C. elegans innexin, EAT-5, can partially substitute for INX-6 function in vivo, underscoring their likely analogous function.     

Analysis of pharyngeal resistance and genioglossal EMG activity using a model of orifice flow.

A model of orifice flow has been used to analyze the relationships among pressure, flow, and genioglossal electromyographic activity in the human pharynx during inspiration. The orifice flow model permits one to assess the character of airflow (laminar or turbulent) and to estimate the cross-sectional area of the orifice from pressure and flow measurements. On the basis of other data (J. Appl. Physiol. 73: 584-590, 1992), this analysis suggests that pharyngeal airflow is turbulent. Furthermore the area of the pharynx appears to increase as flow increases, but the actual change in pharyngeal diameter necessary to fit the pressure-flow data is quite small (0.11-0.87 cm, depending on the assumptions in the model). The flow-related increase in orifice area can be attributed, in part, to the activation of the genioglossus muscle. However, other flow-related factors may also contribute to pharyngeal dilation as airflow increases. Different airway shapes (circular and elliptical) and orientations (major axis anteroposterior and lateral) were incorporated into the model calculations; these factors modify considerably the apparent efficiency of genioglossal electromyographic activity. Genioglossal muscle shortening increases pharyngeal area and reduces pharyngeal resistance more effectively when the pharynx is elliptical, with the long axis of the ellipse oriented laterally. Hence the genioglossus may operate at a significant mechanical disadvantage in those patients with obstructive sleep apnea with a small sagittally oriented pharyngeal lumen.     

Human vesicular glutamate transporters functionally complement EAT-4 in C. elegans

The vesicular glutamate transporter (VGLUT) transports glutamate into pre-synaptic vesicles. Three isoforms of VGLUT have been identified in humans, but their functional differences remain largely unknown. EAT-4 is the only homologue of human VGLUT in C. elegans. Here we report that mutants of eat-4 exhibit hyperforaging behavior and that each of the isoforms of human VGLUT functionally rescues the defects in eat-4 worms.     

Pharmacological characterization of the response of the leech pharynx to acetylcholine

In this study we document the sensitivity of the leech pharynx to acetylcholine and begin to characterize the acetylcholine receptor mediating this response by examining the effects of selective cholinergic agonists and antagonists on the contractile behavior of the pharynx. The order of potency derived from the EC50 of each agonist was (+/-)epibatidine > acetylcholine (in the presence of physostigmine) > McN A-343 > carbachol > nicotine. However, when response amplitude was considered, the order of potency to the tested agonists was (+/-)epibatidine > nicotine > McN A-343 > carbachol > acetylcholine. Acetylcholine-induced contractions of the pharynx were antagonized by d-tubocurarine, but not by alpha-bungarotoxin, alpha-conotoxin M1, or mecamylamine. Application of high concentrations of hexamethonium (1 mM) augmented the acetylcholine-induced contractions. However, this augmentation was apparently due to inhibition of acetylcholinesterase by hexamethonium. The muscarinic antagonist atropine produced complex actions and apparently acted as a mixed agonist/antagonist. Atropine by itself produced an increase in basal tonus and increased the frequency and amplitude of phasic contractions. Atropine increased the peak tension of the acetylcholine-induced response; however, it reduced the amplitude of both the acetylcholine-induced increase in basal tonus and integrated area. Based on the pharmacological profile of the pharyngeal acetylcholine response, we conclude that the acetylcholine receptor mediating the response is a nicotinic receptor. However, the responsiveness of the pharynx to muscarinic agents diverges from that of a classical nicotinic receptor.     

Regulation of the pharynx of Caenorhabditis elegans by 5-HT, octopamine, and FMRFamide-like neuropeptides.

More than fifty FMRFamide-like neuropeptides have been identified in nematodes. We addressed the role of a subset of these in the control of nematode feeding by electrophysiological recording of the activity of C. elegans pharynx. AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), AF8 (KSAYMRFamide), and GAKFIRFamide (encoded by the C. elegans genes flp-8, flp-14, flp-6, and flp-5, respectively) increased pharyngeal action potential frequency, in a manner similar to 5-HT. In contrast, SDPNFLRFamide, SADPNFLRFamide, SAEPFGTMRFamide, KPSVRFamide, APEASPFIRFamide, and AQTVRFamide (encoded by the C. elegans genes flp-1; flp-1; flp-3; flp-9; flp-13, and flp-16, respectively) inhibited the pharynx in a manner similar to octopamine. Only three of the neuropeptides had potent effects at low nanomolar concentrations, consistent with a physiological role in pharyngeal regulation. Therefore, we assessed whether these three peptides mediated their actions either directly on the pharynx or indirectly via the neural circuit controlling its activity by comparing actions between wild-type and mutants with deficits in synaptic signaling. Our data support the conclusion that AF1 and SAEPFGTMRFamide regulate the activity of the pharynx indirectly, whereas APEASPFIRFamide exerts its action directly. These results are in agreement with the expression pattern for the genes encoding the neuropeptides (Kim and Li, 1999) as both flp-8 and flp-3 are expressed in extrapharyngeal neurons, whereas flp-13 is expressed in I5, a neuron with synaptic output to the pharyngeal muscle. These results provide the first, direct, functional information on the action of neuropeptides in C. elegans. Furthermore, we provide evidence for a putative inhibitory peptidergic synapse, which is likely to have a role in the control of feeding.     

A comparison of electrically evoked and channel rhodopsin-evoked postsynaptic potentials in the pharyngeal system of Caenorhabditis elegans

Dissecting the function of neural circuits requires the capability to stimulate and record from the component neurones. Optimally, the methods employed should enable precise activation of distinct elements within the circuit and high-fidelity readout of the neuronal response. Here we compare two methods for neural stimulation in the pharyngeal system of Caenorhabditis elegans by evoking postsynaptic potentials (PSPs) either by electrical stimulation or by expression of the channelrhodopsin [ChR2(gf)] in cholinergic neurones of the pharyngeal circuit. Using a dissection that isolates the pharynx and its embedded neural system of 20 neurones permits analysis of the neurotransmitter pathways within this microcircuit. We describe protocols for selective electrically evoked or ChR2-mediated cholinergic synaptic events in this circuit. The latter was achieved by generating strains, punc-17::ChR2(gf);yfp, that express ChR2(gf) in cholinergic neurones. PSPs evoked by both electrical and light stimulation exhibited a rapid time-course and were blocked by cholinergic receptor antagonists and rapidly reversed on cessation of the stimulus. Electrically evoked PSPs were also reduced in a hypomorphic mutant for the synaptic vesicle acetylcholine transporter, unc-17, further indicating they are nicotinic cholinergic PSPs. The pharyngeal nervous system is exquisitely sensitive to both electrical and light activation. For the latter, short light pulses of 200 μs delivered to punc-17::ChR2(gf);yfp are capable of generating full muscle action potentials. We conclude that the application of optogenetic approaches to the C. elegans isolated pharynx preparation opens the way for a precise molecular dissection of synaptic events in the pharyngeal microcircuit by providing a molecular and system level analysis of the synapses that control the feeding behaviour of C. elegans.     

avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.

Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin's effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr-15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr-15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate-gated chloride channel subunits. An avr-15-encoded subunit forms a homomeric channel that is ivermectin-sensitive and glutamate-gated. These results indicate that: (i) an ivermectin-sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate-gated chloride channels in essential excitable cells such as those of the pharynx.     

Postsynaptic neuroligin enhances presynaptic inputs at neuronal nicotinic synapses

Neuroligins are cell adhesion molecules that interact with neurexins on adjacent cells to promote glutamatergic and GABAergic synapse formation in culture. We show here that neuroligin enhances nicotinic synapses on neurons in culture, increasing synaptic input. When neuroligin is overexpressed in neurons, the extracellular domain induces presynaptic specializations in adjacent cholinergic neurons as visualized by SV2 puncta. The intracellular domain is required to translate the SV2 puncta into synaptic input as reflected by increases in the frequency of spontaneous mini-synaptic currents. The PDZ-binding motif of neuroligin is not needed for these effects. Together, the extracellular and proximal intracellular domains of neuroligin are sufficient to induce presynaptic specializations, align them over postsynaptic receptor clusters, and increase synaptic function. Manipulation of endogenous neuroligin with beta-neurexin-expressing cells confirms its presence; repressing function with dominant negative constructs and inhibitory shRNA shows that endogenous neuroligin helps confer functionality on existing nicotinic synaptic contacts. Endogenous neuroligin does not appear to be required, however, for initial formation of the contacts, suggesting that other components under these conditions can also initiate synapse formation. The results indicate that postsynaptic neuroligin is important for functional nicotinic synapses on neurons and that the effects achieved will likely depend on neuroligin levels.     

Functions of the Caenorhabditis elegans regulatory myosin light chain genes mlc-1 and mlc-2.

Caenorhabditis elegans contains two muscle regulatory myosin light chain genes, mlc-1 and mlc-2. To determine their in vivo roles, we identified deletions that eliminate each gene individually and both genes in combination. Functions of mlc-1 are redundant to those of mlc-2 in both body-wall and pharyngeal muscle. mlc-1(0) mutants are wild type, but mlc-1(0) mlc-2(0) double mutants arrest as incompletely elongated L1 larvae, having both pharyngeal and body-wall muscle defects. Transgenic copies of either mlc-1(+) or mlc-2(+) rescue all defects of mlc-1(0) mlc-2(0) double mutants. mlc-2 is redundant to mlc-1 in body-wall muscle, but mlc-2 performs a nearly essential role in the pharynx. Approximately 90% of mlc-2(0) hermaphrodites arrest as L1 larvae due to pharyngeal muscle defects. Lethality of mlc-2(0) mutants is sex specific, with mlc-2(0) males being essentially wild type. Four observations suggest that hermaphrodite-specific lethality of mlc-2(0) mutants results from insufficient expression of the X-linked mlc-1(+) gene in the pharynx. First, mlc-1(0) mlc-2(0) double mutants are fully penetrant L1 lethals in both hermaphrodites and males. Second, in situ localization of mlc mRNAs demonstrates that both mlc-1 and mlc-2 are expressed in the pharynx. Third, transgenic copies of either mlc-1(+) or mlc-2(+) rescue the pharyngeal defects of mlc-1(0) mlc-2(0) hermaphrodites. Fourth, a mutation of the dosage compensation gene sdc-3 suppresses hermaphrodite-specific lethality of mlc-2(0) mutants.