Ventral Cord Motor Neurons Research Articles

The recurrent pathogenic Pro890Leu substitution in CLTC causes a generalized defect in synaptic transmission in Caenorhabditis elegans.

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders.

The Conserved ASCL1/MASH-1 Ortholog HLH-3 Specifies Sex-Specific Ventral Cord Motor Neuron Fate in Caenorhabditis elegans.

Neural specification is regulated by one or many transcription factors that control expression of effector genes that mediate function and determine neuronal type. Here we identify a novel role for one conserved proneural factor, the bHLH protein HLH-3, implicated in the specification of sex-specific ventral cord motor neurons in C. elegans. Proneural genes act in early stages of neurogenesis in early progenitors, but here, we demonstrate a later role for hlh-3. First, we document that differentiation of the ventral cord type C motor neuron class (VC) within their neuron class, is dynamic in time and space. Expression of VC class-specific and subclass-specific identity genes is distinct through development and is dependent on the VC position along the A-P axis and their proximity to the vulva. Our characterization of the expression of VC class and VC subclass-specific differentiation markers in the absence of hlh-3 function reveals that VC fate specification, differentiation, and morphology requires hlh-3 function. Finally, we conclude that hlh-3 cell-autonomously specifies VC cell fate.

Diversification of C. elegans Motor Neuron Identity via Selective Effector Gene Repression

A common organizational feature of nervous systems is the existence of groups of neurons that sharecommon traits but can be divided into individual subtypes based on anatomical or molecular features. Weelucidate the mechanistic basis of neuronal diversification processes in the context of C.elegans ventral cord motor neurons that share common traitsthatare directly activated by the terminal selector UNC-3. Diversification of motor neurons into different classes, each characterized by unique patterns of effector gene expression, iscontrolled by distinct combinations of phylogenetically conserved, class-specific transcriptional repressors. These repressors are continuously required in postmitotic neurons to prevent UNC-3, which is active in all neuron classes, from activating class-specific effector genes in specific motor neuron subsets via discrete cis-regulatory elements. The strategy of antagonizing the activity of broadly acting terminal selectors of neuron identity in a subtype-specific fashion may constitute a general principle of neuron subtype diversification.

STR-33, a Novel G Protein-coupled Receptor That Regulates Locomotion and Egg Laying in Caenorhabditis elegans

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

A minimal model of C. elegans forward locomotion: the larval L1 circuit

The nematode Caernohabditis elegans possesses one of the simplest central nervous systems, and by far the best characterized. In the adult, the nervous system consists of a mere 302 neurons, of which the majority are located in the head, and the rest are distributed in the pharynx, along the ventral cord or in small ganglia. In particular, the local control (along the body) of the worm’s sinuous undulations is instantiated by 8 classes of ventral cord motor neurons. But of those, only 3 classes exist as the larva hatches out of the egg (the so called L1 stage in development). This appears quite remarkable, given the similar repertoire of behaviors in the larva, as compared to the fully developed adult. Here, we ask what minimal design principles must apply for the L1 circuit to generate robust forward locomotion. Robustness is taken to mean effective locomotion in different physical environments (e.g. high viscosity agar gels versus water) [1,2]. To address this challenge, we adopt an integrated neuromechanical model of forward locomotion in the adult that consists of the ventral cord nervous system, muscles, a body and a physical environment [2], and adapt the model nervous system and muscles to match the L1. In particular, we assume the forward locomotion control in the L1 consists of repeating units, each consisting only of a pair of neurons: one excitatory DB and one inhibitory DD. DB neurons are modeled as bistable elements [2] innervating dorsal muscles, and DD neurons are modeled as linear elements inhibiting ventral muscles. High level control is modeled with an on/off AVB unit that drive all B-neurons. The rhythm is generated entirely on the dorsal side and is achieved by a closed-loop proprioceptive feedback (via postulated stretch sensitivity of DB neurons). Inhibitory neurons receive synaptic inputs from the local DB neuron and are therefore enslaved to its oscillations. The integrated model successfully generates and propagates coordinated undulations on agar and liquid model environments. In the model, both of these behaviors are achieved by a single (i.e., fixedparameter) model worm. The model makes a number of relatively easily testable predictions: 1) L1 larva have a minimal, reflex-driven mechanism to control body undulations. 2) Inhibitory D-neurons are essential for forward locomotion in the L1. 3) Muscle properties must differ substantially between the ventral and dorsal side (specifically, ventral but not dorsal muscles must be spontaneously active).

Coexpressed D1- and D2-Like Dopamine Receptors Antagonistically Modulate Acetylcholine Release in Caenorhabditis elegans

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1- and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1- and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans.

Localisation of serotonin and dopamine in Haemonchus contortus

Serotonin and dopamine play important roles in the biology of nematodes where they exert their effect on feeding, locomotion and reproductive behavior. Haemonchus contortus, a parasitic nematode which infects small ruminants, is responsible for considerable economic losses in agriculture. In the current study we have mapped the localisation of these two neurotransmitters in this parasite using immuno-staining. Serotonin localised in amphidial and pharyngeal neurons in both adult female and male worms. Serotonin was also found in ray sensory neurons as well as in a few ventral cord motor neurons exclusively in adult males. Surprisingly, dopamine was only detected in the neuronal commissures linking the lateral and sub-lateral nerve cords in both sexes. We also studied the effect of these two molecules on female adult worms in vitro. Serotonin mainly inhibited movement whereas dopamine had a profound paralytic effect on the mid-body of the worms.

CWN-1 functions with DSH-2 to regulate C. elegans asymmetric neuroblast division in a β-catenin independent Wnt pathway

In Caenorhabditis elegans, Wnt signaling regulates many asymmetric cell divisions. During embryogenesis, the C. elegans Dishevelled (Dsh) homolog, DSH-2, regulates asymmetric neuroblast division of the ABpl/rpppa blast cell. Dsh is a key intracellular component of both β-catenin dependent and β-catenin independent Wnt pathways. In C. elegans, most of the well-characterized asymmetric cell divisions regulated by Wnts are dependent on β-catenin. In the ABpl/rpppa neuroblast division, however, we determined that DSH-2 regulates cell polarity through a β-catenin independent Wnt pathway. We also established that the C. elegans Wnt homolog, cwn-1, functions to regulate asymmetric division of the ABpl/rpppa blast cell. Our results indicated that cwn-1 does not act alone in this process, and it functions with another redundant ligand that appears not to be a Wnt. Finally, we show widespread requirements for DSH-2 during embryogenesis in the generation of many other neurons. In particular, DSH-2 function is necessary for the correct production of the embryonic ventral cord motor neurons. This study demonstrates a role for DSH-2 and Wnt signaling in neuronal specification during C. elegans embryogenesis.

The Caenorhabditis elegans AMP-activated Protein Kinase AAK-2 Is Phosphorylated by LKB1 and Is Required for Resistance to Oxidative Stress and for Normal Motility and Foraging Behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.

UNC-1 Regulates Gap Junctions Important to Locomotion in C. elegans

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

Heterotrimeric G proteins in C. elegans

Heterotrimeric G proteins, composed of alpha, beta, and gamma subunits, are able to transduce signals from membrane receptors to a wide variety of intracellular effectors. In this role, G proteins effectively function as dimers since the signal is communicated either by the G alpha subunit or the stable G betagamma complex. When inactive, G alpha-GDP associates with G betagamma and the cytoplasmic portion of the receptor. Ligand activation of the receptor stimulates an exchange of GTP for GDP resulting in the active signaling molecules G alpha-GTP and free G betagamma, either of which can interact with effectors. Hydrolysis of GTP restores G alpha-GDP, which then reassociates with G betagamma and receptor to terminate signaling. The rate of G protein activation can be enhanced by the guanine-nucleotide exchange factor, RIC-8, while the rate of GTP hydrolysis can be enhanced by RGS proteins such as EGL-10 and EAT-16. Evidence for a receptor-independent G-protein-signaling pathway has been demonstrated in C. elegans early embryogenesis. In this pathway, the G alpha subunits GOA-1 and GPA-16 are apparently activated by the non-transmembrane proteins GPR-1, GPR-2, and RIC-8, and negatively regulated by RGS-7. The C. elegans genome encodes 21 G alpha, 2 G beta and 2 G gamma subunits. The alpha subunits include one ortholog of each mammalian G alpha family: GSA-1 (Gs), GOA-1 (Gi/o), EGL-30 (Gq) and GPA-12 (G12). The remaining C. elegans alpha subunits (GPA-1, GPA-2, GPA-3, GPA-4, GPA-5, GPA-6, GPA-7, GPA-8, GPA-9, GPA-10, GPA-11, GPA-13, GPA-14, GPA-15, GPA-16, GPA-17 and ODR-3) are most similar to the Gi/o family, but do not share sufficient homology to allow classification. The conserved G alpha subunits, with the exception of GPA-12, are expressed broadly while 14 of the new G alpha genes are expressed in subsets of chemosensory neurons. Consistent with their expression patterns, the conserved C. elegans alpha subunits, GSA-1, GOA-1 and EGL-30 are involved in diverse and fundamental aspects of development and behavior. GOA-1 acts redundantly with GPA-16 in positioning of the mitotic spindle in early embryos. EGL-30 and GSA-1 are required for viability starting from the first larval stage. In addition to their roles in development and behaviors such as egg laying and locomotion, the EGL-30, GSA-1 and GOA-1 pathways interact in a network to regulate acetylcholine release by the ventral cord motor neurons. EGL-30 provides the core signals for vesicle release, GOA-1 negatively regulates the EGL-30 pathway, and GSA-1 modulates this pathway, perhaps by providing positional cues. Constitutively activated GPA-12 affects pharyngeal pumping. The G alpha subunits unique to C. elegans are primarily involved in chemosensation. The G beta subunit, GPB-1, as well as the G gamma subunit, GPC-2, appear to function along with the alpha subunits in the classic G protein heterotrimer. The remaining G beta subunit, GPB-2, is thought to regulate the function of certain RGS proteins, while the remaining G gamma subunit, GPC-1, has a restricted role in chemosensation. The functional difference for most G protein pathways in C. elegans, therefore, resides in the alpha subunit. Many cells in C. elegans express multiple G alpha subunits, and multiple G protein pathways are known to function in specific cell types. For example, Go, Gq and Gs-mediated signaling occurs in the ventral cord motor neurons. Similarly, certain amphid neurons use multiple G protein pathways to both positively and negatively regulate chemosensation. C. elegans thus provides a powerful model for the study of interactions between and regulation of G protein signaling.

Identification and molecular characterization of the G alpha12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans.

G alpha 12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. Here, we identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in Caenorhabditis elegans (CeRhoGEF), which functions downstream of gpa-12, the C. elegans homologue of G alpha 12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology-pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1-RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of G alpha 12 or G alpha 13 through their RGS domains. We demonstrated by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an AIF4- activation-dependent manner and confirmed that the Dbl homology-pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the G alpha 12-RhoGEF pathway in C. elegans. Expression of DsRed or GFP under the control of the promoter of CeRhoGEF or gpa-12 revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for CeRhoGEF and gpa-12 resulted in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the G alpha 12/13-RhoGEF pathway is likely to be involved in embryonic development and neuronal function in C. elegans.

Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities.

The Cubitus interruptus (Ci) and Gli proteins are transcription factors that mediate responses to Hedgehog proteins (Hh) in flies and vertebrates, respectively. During development of the Drosophila wing, Ci transduces the Hh signal and regulates transcription of different target genes at different locations. In vertebrates, the three Gli proteins are expressed in overlapping domains and are partially redundant. To assess how the vertebrate Glis correlate with Drosophila Ci, we expressed each in Drosophila and monitored their behaviors and activities. We found that each Gli has distinct activities that are equivalent to portions of the regulatory arsenal of Ci. Gli2 and Gli1 have activator functions that depend on Hh. Gli2 and Gli3 are proteolyzed to produce a repressor form able to inhibit hh expression. However, while Gli3 repressor activity is regulated by Hh, Gli2 repressor activity is not. These observations suggest that the separate activator and repressor functions of Ci are unevenly partitioned among the three Glis, yielding proteins with related yet distinct properties.

Serotonin inhibition of synaptic transmission: Galpha(0) decreases the abundance of UNC-13 at release sites.

We show that serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and we describe a signaling pathway that mediates this effect. Release of acetylcholine from motor neurons was assayed by measuring the sensitivity of intact animals to the acetylcholinesterase inhibitor aldicarb. By this assay, exogenous serotonin inhibited acetylcholine release, whereas serotonin antagonists stimulated release. The effects of serotonin on synaptic transmission were mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 G(alpha)0 accumulated abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites.

The C. elegans gene pag-3 is homologous to the zinc finger proto-oncogene gfi-1

Mutations in the Caenorhabditis elegans gene pag-3 result in misexpression of touch receptor-specific genes in the BDU interneurons and in motility defects. We cloned pag-3 and found that the gene encodes a C2H2-type zinc finger protein related to the mammalian GFI-1 protein. Sequencing of the three pag-3 alleles showed that two apparent null alleles encode a nonsense mutation before the zinc fingers and a missense mutation in the fourth zinc finger that changes a coordinating histidine to a tyrosine. The third allele contains a nonsense mutation in the N-terminal region but is not a null allele. Northern analysis showed that a single pag-3 transcript of about 1.6 kb is present in embryos and L1, L2 and L3 larvae. pag-3 message levels were about twofold higher in pag-3 mutants than in wild-type animals, which suggested that pag-3 may negatively regulate its own expression. pag-3lacZ fusion genes were expressed in the BDU interneurons, the touch neurons, 11 VA and 11 VB ventral cord motor neurons, two AVF interneurons and in unidentified neurons of the retrovesicular ganglion. The BDU neurons and the ALM touch neurons are lineal sister cells in the AB.a lineage and the VA and VB motor neurons are lineal sister cells in the AB.p lineage. The VA motor neurons are required for backward movement and the VB motor neurons are required for forward movement. Mosaic analysis showed that the wild-type pag-3 gene is required in the AB.p lineage for coordinated movement and in the AB.a lineage to suppress touch neuron gene expression in the BDU neurons. Because pag-3 is expressed in both the BDU neurons and in the touch neurons, another protein(s) not expressed in the touch neurons may interact with pag-3 to repress touch neuron gene expression in the BDU neurons. Alternatively, another protein in the touch receptor cells may inactivate PAG-3 and allow expression of the touch receptor program. These results show that pag-3 is a temporally regulated gene that is expressed early in development and functions in multiple types of neurons. They also strongly suggest that the PAG3 protein is a DNA-binding protein with properties similar to the mammalian proto-oncogene product GFI-1.

Selective expression of the tba-1 α tubulin gene in a set of mechanosensory and motor neurons during the development of Caenorhabditis elegans

In the nematode Caenorhabditis elegans, a monoclonal antibody 3A5 raised against Drosophila α tubulins selectively stains the nervous system immuno-cytochemically. Direct screening of a C. elegans cDNA expression library with 3A5 has allowed cloning of the tba-1 ( t u b ulin a lpha- 1 ) gene from C. elegans. The corresponding genomic DNA encodes a protein of 449 amino acid residues that has a high homology with the vertebrate α tubulins but a lower homology with yeast α tubulins. Interestingly, the carboxyl-terminus sequence EEEGEEY (Glu-Glu-Glu-Gly-Glu-Glu-Tyr) of the nematode tba-1 encoded isotype is identical to these residues in human, mouse, rat, pig and chicken α-1 tubulin isotypes that are expressed in the brain. Temporal and spatial expression studies of the tba-1 gene using Northern blot analysis and tba-1:: lacZ fusion gene expression analysis during embryonic and the postembryonic development of C. elegans reveal that the tba-1 tubulin is preferentially expressed in the nematode nervous system, especially in a set of mechanosensory neurons and a set of ventral cord motor neurons (DA, DB, VA, and VB) during embryonic and postembryonic development. Our results indicate an inter-species conservation of the α tubulin carboxyl-terminal domain in functionally related brain specific isotypes from metazoans as divergent as mammals and nematodes. These results also suggest specificity of the individual α tubulin isotypes during neural development.

Dominant unc-37 mutations suppress the movement defect of a homeodomain mutation in unc-4, a neural specificity gene in Caenorhabditis elegans.

The unc-4 gene of Caenorhabditis elegans encodes a homeodomain protein that defines synaptic input to ventral cord motor neurons. unc-4 mutants are unable to crawl backward because VA motor neurons are miswired with synaptic connections normally reserved for their sister cells, the VB motor neurons. These changes in connectivity are not accompanied by any visible effects upon neuronal morphology, which suggests that unc-4 regulates synaptic specificity but not axonal guidance or outgrowth. In an effort to identify other genes in the unc-4 pathway, we have devised a selection scheme for rare mutations that suppress the Unc-4 phenotype. We have isolated four, dominant, extragenic, allele-specific suppressors of unc-4(e2322ts), a temperature sensitive allele with a point mutation in the unc-4 homeodomain. Our data indicate that these suppressors are gain-of-function mutations in the previously identified unc-37 gene. We show that the loss-of-function mutation unc-37(e262) phenocopies the Unc-4 movement defect but does not prevent unc-4 expression or alter VA motor neuron morphology. These findings suggest that unc-37 functions with unc-4 to specify synaptic input to the VA motor neurons. We propose that unc-37 may be regulated by unc-4. Alternatively, unc-37 may encode a gene product that interacts with the unc-4 homeodomain.

The C. elegans unc-18 gene encodes a protein expressed in motor neurons

The C. elegans unc-18 gene is required to maintain normal acetylcholine levels. We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids. The protein shows sequence similarity with elements of the secretory pathway in the yeast S. cerevisiae. Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all ventral cord motor neurons in situ. These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons.