The C. elegans touch response facilitates escape from predacious fungi

Predator-prey interactions are vital determinants in the natural selection of behavioral traits. Gentle touch to the anterior half of the body of Caenorhabditis elegans elicits an escape response in which the animal quickly reverses and suppresses exploratory head movements [1, 2]. Here, we investigate the ecological significance of the touch response in predator-prey interactions between C. elegans and predacious fungi that catch nematodes using constricting hyphal rings. We show that the constricting rings of Drechslerella doedycoides catch early larval stages with a diameter similar to the trap opening. There is a delay between the ring entry and ring closure, which allows the animal to withdraw from the trap before being caught. Mutants that fail to suppress head movements in response to touch are caught more efficiently than the wild-type. This demonstrates that the coordination of motor programs allows C. elegans to smoothly retract from a fungal noose and evade capture. Our results suggest that selective pressures imposed by predacious fungi have shaped the evolution of C. elegans escape behavior.     

Semaphorin 1a and semaphorin 1b are required for correct epidermal cell positioning and adhesion during morphogenesis in C. elegans

The semaphorin family comprises secreted and transmembrane proteins involved in axon guidance and cell migration. We have isolated and characterized deletion mutants of C. elegans semaphorin 1a (Ce-sema-1a or smp-1) and semaphorin 1b (Ce-sema-1b or smp-2) genes. Both mutants exhibit defects in epidermal functions. For example, the R1.a-derived ray precursor cells frequently fail to change anterior/posterior positions completely relative to their sister tail lateral epidermal precursor cell R1.p, causing ray 1 to be formed anterior to its normal position next to ray 2. The ray cells, which normally separate from the lateral tail seam cell (SET) at the end of L4 stage, remains connected to the SET cell even in adult mutant males. The ray 1 defects are partially penetrant in each single Ce-sema-1 mutant at 20 degrees C, but are greatly enhanced in Ce-sema-1 double mutants, suggesting that Ce-Sema-1a and Ce-Sema-1b function in parallel to regulate ray 1 position. Both mutants also have defects in other aspects of epidermal functions, including head and tail epidermal morphogenesis and touch cell axon migration, whereas, smp-1 mutants alone have defects in defecation and brood size. A feature of smp-1 mutants that is shared with mutants of mab-20 (which encodes Sema-2a) is the abnormal perdurance of contacts between epidermal cells.     

Multiple ephrins control cell organization in C. elegans using kinase-dependent and -independent functions of the VAB-1 Eph receptor

Eph receptor (EphR) tyrosine kinases and their ephrin ligands mediate direct cell-to-cell signaling. The C. elegans genome encodes four potential GPI-modified ephrins (EFN-1 to -4) and one EphR (VAB-1). Single and multiple ephrin mutants reveal functions for EFN-1, EFN-2, and EFN-3 in epidermal cell organization that, in aggregate, mirror those of VAB-1. Ephrin mutants have defects in head morphology and enclosure of the embryo by the epidermis and identify ephrin-EphR signaling functions involved in aligning and fusing tail and head epidermal cells, respectively. Biochemical analyses indicate that EFN-1, EFN-2, and EFN-3 jointly activate the VAB-1 tyrosine kinase in vivo. Mutant phenotypes and expression pattern analysis suggest that multiple ephrins are involved in distinct aspects of kinase-dependent and kinase-independent VAB-1 signaling required for proper cell organization during development in C. elegans.     

Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2(RO54)). Tdc2(RO54) flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine beta-hydroxylase gene (TbetaH(nM18)) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TbetaH(nM18) flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes.     

LIM homeobox gene-dependent expression of biogenic amine receptors in restricted regions of the C. elegans nervous system

Biogenic amines regulate a variety of behaviors. Their functions are predominantly mediated through G-protein-coupled 7-transmembrane domain receptors (GPCR), 16 of which are predicted to exist in the genome sequence of the nematode Caenorhabditis elegans. We describe here the expression pattern of several of these aminergic receptors, including two serotonin receptors (ser-1 and ser-4), one tyramine receptor (ser-2), and two dopamine receptors (dop-1 and dop-2). Moreover, we describe distinct but partially overlapping expression patterns of different splice forms of the ser-2 tyramine receptor locus. We find that each of the aminergic receptor genes is expressed in restricted regions of the nervous system and that many of them reveal significant overlap with the expression of regulatory factors of the LIM homeobox (Lhx) gene family. We demonstrate that the expression of several of the biogenic amine receptors is abrogated in specific cell types in Lhx gene mutants, thus establishing a role for these Lhx genes in regulating aspects of neurotransmission. We extend these findings with other cell fate markers and show that the lim-4 Lhx gene is required for several but not all aspects of RID motor neuron differentiation and that the lim-6 Lhx gene is required for specific aspects of RIS interneuron differentiation. We also use aminergic receptor gfp reporter fusions as tools to visualize the anatomy of specific neurons in Lhx mutant backgrounds and find that the development of the elaborate dendritic branching pattern of the PVD harsh touch sensory neuron requires the mec-3 Lhx gene. Lastly, we analyze a mutant allele of the ser-2 tyramine receptor, a target of the ttx-3 Lhx gene in the AIY interneuron class. ser-2 mutants display none of the defects previously shown to be associated with loss of AIY function.     

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.     

Genes for neurotransmitter synthesis, storage, and uptake

We found that the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) (EC 1.14.16.2), dopamine beta-hydroxylase (EC 1.14.17.1), and phenylethanolamine N-methyltransferase (EC 2.1.1.28) share similar protein domains in their primary structures and that they share common gene coding sequences. In a recent report we also demonstrated that antiserums directed against choline acetyltransferase (EC 2.3.1.6), glutamic acid decarboxylase (EC 4.1.1.15), and TH cause specific complement-mediated lysis of cholinergic, gamma-aminobutyric acid-ergic, and dopaminergic subpopulations of synaptosomes, respectively. This interaction of specific antibodies to the specific subpopulation of synaptosomal membrane, e.g., recognition of antibody to TH to only the dopaminergic subpopulation of synaptosomal membrane protein, indicates that the neurotransmitter enzyme and membrane protein of its own synaptosomes may also share common protein domains. Therefore, we postulate that the specific neurotransmitter biosynthetic enzyme and a certain membrane protein of the nerve endings may share similar gene coding sequences, and that expression of these proteins may determine the phenotype of the neuron.     

MIG-13 controls anteroposterior cell migration by interacting with UNC-71/ADM-1 and SRC-1 in Caenorhabditis elegans

The transmembrane protein MIG-13 is a key regulator required for anterior migration of neural cells in Caenorhabditis elegans, but the signaling mechanisms involved remain unknown. Here, we isolated a suppressor mutation in the unc-71/adm-1 gene, which rescued the AVM neuron migration defect in mig-13 mutants. Genetic analyses revealed that UNC-71 at least partly acts downstream of MIG-13 and has an inhibitory effect on the anterior cell migration. The unc-71 mutation also rescued the anterior migration defect of AVM neuron in src-1 mutants. These findings suggest that MIG-13 controls anteroposterior cell migration by interacting with UNC-71 and SRC-1 in C. elegans.     

The neuroethology of C. elegans escape

Escape behaviors are crucial to survive predator encounters. Touch to the head of Caenorhabditis elegans induces an escape response where the animal rapidly backs away from the stimulus and suppresses foraging head movements. The coordination of head and body movements facilitates escape from predacious fungi that cohabitate with nematodes in organic debris. An appreciation of the natural habitat of laboratory organisms, like C. elegans, enables a comprehensive neuroethological analysis of behavior. In this review we discuss the neuronal mechanisms and the ecological significance of the C. elegans touch response.     

The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.     

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD-2) cloned from the C-1300 tumor. 8Br-cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta-hydroxylase activity. In contrast, catechol O-methyltransferase, monoamine oxidase and aromatic-l -amino-acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta-hydroxylase were similar with respect to dose response relationships and with respect to time course of onset. Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20-1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta-hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD-2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta-hydroxylase activity. The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta-hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta-hydroxylase in adrenergic cells.     

Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule

The Malpighian (renal)tubule of Drosophila melanogaster is a useful model forstudying epithelial transport. The purpose of this study was toidentify factors responsible for modulating transepithelial chlorideconductance in isolated tubules. I have found that tyrosine and severalof its metabolites cause an increase in chloride conductance. The mostpotent of these agonists is tyramine, which is active at low nanomolarconcentrations; the pharmacology of this response matches that of thepreviously published cloned insect tyramine receptor. In addition, thetubule appears capable of synthesizing tyramine from applied tyrosine,as shown by direct measurement of tyrosine decarboxylase activity.Immunohistochemical staining of tubules with an antibody againsttyramine indicates that the principal cells are the sites of tyramineproduction, whereas previous characterization of the regulation ofchloride conductance suggests that tyramine acts on the stellate cells. This is the first demonstration of a physiological role for an insecttyramine receptor.

     

Genes Affecting Sensitivity to Serotonin in Caenorhabditis Elegans

Regulating the response of a postsynaptic cell to neurotransmitter is an important mechanism for controlling synaptic strength, a process critical to learning. We have begun to define and characterize genes that may control sensitivity to the neurotransmitter serotonin in the nematode Caenorhabditis elegans by identifying serotonin-hypersensitive mutants. We reported previously that mutations in the gene unc-2, which encodes a putative calcium channel subunit, result in hypersensitivity to serotonin. Here we report that mutants defective in the unc-36 gene, which encodes a homologue of a calcium channel auxiliary subunit, are also serotonin-hypersensitive. Moreover, the unc-36 gene appears to be required in the same cells as unc-2 for control of the same behaviors. Mutations in several other genes, including unc-8, unc-10, unc-20, unc-35, unc-75, unc-77, and snt-1 also result in hypersensitivity to serotonin. Several of these mutations have previously been shown to confer resistance to acetylcholinesterase inhibitors, suggesting that they may affect acetylcholine release. Moreover, we found that mutations that decrease acetylcholine synthesis cause defective egg-laying and serotonin hypersensitivity. Thus, acetylcholine appears to negatively regulate the response to serotonin and may participate in the process of serotonin desensitization.     

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal pumping and foraging behavior in Caenorhabditis elegans

Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical Ki values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC50 values. Tyramine, but not octopamine, also elevates Ca2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonin-dependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.     

Functional phenotypic rescue of Caenorhabditis elegans neuroligin-deficient mutants by the human and rat NLGN1 genes

Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C) and worm NLG-1 (R437C) proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X) and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA), both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1) or pan-muscular (myo-3) specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders.     

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.     

The Caenorhabditis elegans spalt-like gene sem-4 restricts touch cell fate by repressing the selector Hox gene egl-5 and the effector gene mec-3

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3. A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which we find is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; we show that a subset also express egl-5. We report: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. We conclude that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes, that sem-4 indirectly modulates mec-3 expression through its repression of egl-5 and that this negative regulation is required for proper determination of neuronal fates. We suggest that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes.