Chemotactic behavior of mutants of the nematodeCaenorhabditis elegans that are defective in osmotic avoidance

Summary Wild-typeC. elegans and seven derivative strains previously selected for absence of the wild-type tendency to avoid highly concentrated solutions (Culotti and Russell, 1978) were tested for responsiveness in 11 assays of chemotaxis, including 6 attractant and 3 repellent stimuli. The two strains altered in the geneosm-1 (P808 and P816) did not respond in any test except, possibly, one or two weak responses. Strain P801 responded to one attractant and two repellents. Strain P802 made moderately strong responses to most stimuli and avoided CO₂ in phosphate buffer as strongly as the wild-type. Of particular interest, this strain avoided OH^– which is attractive to wild-type. Strain P821 avoided CO₂ in phosphate buffer weakly, if at all, but did respond to the attractants Na⁺ and Cl^–. Conversely, strains P813 and P811 made little if any response to any attractant but did respond to the two strong repellents. Taken together with other results, these findings suggest that the osmotic response has more gene requirements in common with both attractive and repellent chemical stimuli than with thermal or mechanical stimuli. In addition, they indicate that the known chemical stimuli and the osmotic stimulus are probably mediated by at least 9 different receptors.This research was supported by the National Science Foundation and the National Institutes of Health. I would also like to thank Ms. Georgann Hardin, Mr. Kirt Rusenko, and Ms. Deborah Higgins for their assistance in carrying out the experiments. Drs. J.G. Culotti and R.L. Russell generously provided the mutant strains they had isolated.     

Molecular neurogenetics of chemotaxis and thermotaxis in the nematode Caenorhabditis elegans

Chemotaxis and thermotaxis in Caenorhabditis elegans are based on the chemical senses (smell and taste) and the thermal sense, respectively, which are important for the life of the animal. Laser ablation experiments have allowed identification of sensory neurons and some interneurons required for these senses. Many mutants that exhibit various abnormalies have been isolated and analyzed. These studies have predicted novel signaling pathways whose components include a putative odorant specific transmembrane receptor (ODR-10) and a cyclic nucleotide-gated channel (TAX-4/TAX-2) functioning in taste and thermosensation as well as in smell. The emerging picture of the mechanisms of sensory transduction in C. elegans seems to be basically similar to what is known of visual and olfactory sensory transduction in vertebrates. Thus, molecular and cellular analyses of chemotaxis and thermotaxis in C. elegans have proved useful and will continue to provide significant implications for the molecular basis of sensory systems in higher animals.     

In search of new mutants in cell-signaling systems of the nematodeCaenorhabditis elegans

Development of multicellular organisms is controlled mainly by cell-signaling systems. In this review I first discuss methods of genetic analysis and properties of mutants of cell-signaling systems in general and in the nematodeC. elegans. Then, I describe two of our approaches to isolating new mutants in cell-signaling ofC. elegans. The first approach is to select for mutants that have the same visible phenotype as those in known cell-signaling genes. In a survey of larval lethal mutations we found that there are quite a few mutants in which the inner surface of the body wall is detached from the outer surface of the intestine. Some of them map in genes that are known to act in cell-signaling systems in vulval induction or sex myoblast migration, which are not essential to the growth and survival ofC. elegans. Therefore, we think many of the mutations of the above phenotype disrupt cell-signaling in an unidentified essential function, and also cell-signaling in the non-essential functions. The second approach is to isolate mutants resistant to a drug expected to disturb cell-signaling. As the drug we have chosen sodium fluoride, which depletes calcium ion, activates G-proteins and inactivates some phosphatases. The mutants are grouped into two classes (three and two genes, respectively) according to degree of fluoride-resistance and growth rate of larvae. Although there is so far no direct evidence that these mutants are related to cell-signaling, they show complex epistasis that can be explained by a model consisting of a cell-signaling pathway.     

Salmonella typhimurium mutants generally defective in chemotaxis.

The mutations of eight chemotaxis-deficient strains of Salmonella typhimurium, including five new mutants in strain LT2, were mapped by P22 transduction in relation to various fla mot deletions in S. abortus-equi. Seven recessive che mutations mapped between motB and flaC: three, all nontumbling, the che region I, adjacent to motB, and four, including one ever-tumbling, in che region II, adjacent to flaC. Mutant che-107, never-tumbling and dominant to wild type, mapped at flaAII, other mutations of which cause either absence of flagella or lack of locomotor function. We surmise that gene flaAII specifies a protein that polymerizes to form an essential component of the basal apparatus (so that absence of gene product prevents formation of flagela); that a component built up from certain mutationally altered proteins cannot transmit (or generate) active rotation of the hook and flagellum, and so causes the Mot (paralysis) phenyotype; and that a component built up from protein with the che-107 alteration permits only counterclockwise rotation, so that the tumble, normally produced by transient clockwise rotation, cannot be effected.     

Egg-laying defective mutants of the nematode Caenorhabditis elegans

We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl. for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new components of the neural circuitry that control egg laying.     

Temperature-sensitive zygote defective mutants of Caenorhabditis elegans

Three genetically complementing temperature-sensitive mutants of Caenorhabditis elegans have been studied. Each of the three mutants has two critical times of temperature sensitivity and two distinctive corresponding phenotypes. Exposure to high temperature during gonadogenesis blocks the production of zygotes. Exposure of adults to high temperature interrupts embryogenesis of the zygotes being produced. Each of the mutants carries an autosomal mutation with a maternal effect. These mutants indicate that the individual temperature-sensitive functions are required at least twice during development and that early embryogenesis is dependent on the contribution of these functions from the maternal gonad.     

Appetitive response of the nematodeCaenorhabditis elegans to oxygen

Summary Using a technique of recording the behavior of individual nematodes during exposure to various solutions, it was demonstrated thatC. elegans made more reversal behaviors after transfer to solutions of lower oxygen tension than higher. The response was stronger after the first hour in the apparatus than initially. This change was not dependent on reduced oxygen availability during the initial period. Starvation is the most likely cause of this change. A variety of mutant strains ofC. elegans that are defective in response to most known chemotactic stimuli, including two strains that have been shown to be severely abnormal in the ciliated endings of all sensory neurons of the worm's snout, all responded to changes in oxygen tension. This observation suggests that oxygen is sensed internally rather than by specialized peripheral receptor cells.I thank Deborah Higgins for technical assistance. This work was supported by a grant awarded by the Biomedical Research Support Grant Program of the National Institutes of Health.     

Responses of the nematodeCaenorhabditis elegans to controlled chemical stimulation

Summary A new method is described for studying the behavioral responses of nematodes to controlled chemical stimulation. The worm is held by the tail with a suction pipet. Behavior is recorded by an array of light sensors connected to a multichannel recorder. Several types of behavior can be detected in addition to the normal backward propagating waves of about 2 Hz that propel untethered worms forward. The most dramatic of these is the reversal bout, consisting of forward propagating waves of about 0.7 Hz, that propel untethered worms backward. The latter waves are easily distinguished from the former by the large amplitude motion caused by the fact that they contain a sharper bend at the tail. This technique was used to demonstrate that a purely temporal change in chemical stimulation can cause a large change in the probability of occurrrence of a reversal bout. These altered probabilities adapt back to the basal level in about one minute. Increased probabilities adapt faster than decreased. Stronger stimulation causes slower adaptation. Since the reversal bout is associated with changes in direction of locomotion, these observations suggest that klinokinesis with adaptation plays a role in the movement of nematodes in chemical gradients.I would like to thank Deborah Higgins for technical assistance. This work was supported by Grant RR 07024q3 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health and by the National Science Foundation under Grant GB-43561.     

Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants

The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.     

Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans

In complex neural circuits of the brain, massive information is processed with neuronal communication through synaptic transmissions. It is thus fundamental to delineate information flows encoded by various kinds of transmissions. Here, we show that glutamate signals from two distinct sensory neurons bidirectionally affect the same postsynaptic interneuron, thereby producing the opposite behaviours. EAT-4/VGLUT (vesicular glutamate transporter)-dependent glutamate signals from AFD thermosensory neurons inhibit the postsynaptic AIY interneurons through activation of GLC-3/GluCl inhibitory glutamate receptor and behaviourally drive migration towards colder temperature. By contrast, EAT-4-dependent glutamate signals from AWC thermosensory neurons stimulate the AIY neurons to induce migration towards warmer temperature. Alteration of the strength of AFD and AWC signals led to significant changes of AIY activity, resulting in drastic modulation of behaviour. We thus provide an important insight on information processing, in which two glutamate transmissions encoding opposite information flows regulate neural activities to produce a large spectrum of behavioural outputs.     

C. elegans feeding defective mutants have shorter body lengths and increased autophagy

Background  

Mutations that cause feeding defects in the nematode C. elegans are known to increase life span. Here we show that feeding defective mutants also have a second general trait in common, namely that they are small.     

Isolation and characterization of Enterobacter cloacae mutants which are defective in chemotaxis toward inorganic phosphate.

Enterobacter cloacae IFO3320 is attracted to Pi when cells are starved for Pi. Two Tn1737KH-induced mutants, which were constitutive for alkaline phosphatase, failed to exhibit Pi taxis even under conditions of Pi limitation. Both of the mutant strains exhibited normal chemotactic responses to peptone, suggesting that they are specifically defective in Pi taxis. Cloning and sequence analysis showed that the TN1737KH insertions were located in either the pstA or pstB genes which encode the channel-forming proteins of the Pi-specific transport (Pst) system in E. cloacae. These results suggest that the E. cloacae Pst system is required for Pi chemoreception.     

A steep thermal gradient thermotaxis assay for the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans with its well-described nervous system is one of the multicellular organisms of choice to study thermotaxis. The neuronal circuitry for thermosensation has been analyzed at the level of individual cells. Two methods have previously been described to study the behavior of C. elegans with respect to temperature: 1) isothermal tracking assays and 2) linear thermal gradients (Hedgecock and Russell, 1975). Here we present a short linear thermal gradient assay which is faster and which allows statistical evaluation of different populations using a thermotaxis index. Thin agar plates are used on which a temperature gradient from about 10 degrees to 30 degrees is induced over the distance of about 5 cm. The short linear thermal gradient uses inexpensive materials so that multiple tests can be performed in parallel in a short period of time.     

Caenorhabditis elegans mutants having altered preference of chemotaxis behavior during simultaneous presentation of two chemoattractants

Upon presentation of two distinct chemoattractants such as sodium acetate and diacetyl simultaneously, the nematode Caenorhabditis elegans was preferentially attracted by one of these chemoattractants. We isolated two mutants having altered preference of chemotaxis behavior toward simultaneous presentation of sodium acetate and diacetyl. The chep-1(qr1) (CHEmosensory Preference) mutant preferred sodium acetate to diacetyl, while the chep-2(qr2) mutant preferred diacetyl to sodium acetate in simultaneous presentation of these chemoattractants. The chemotaxis behavior of chep-2(qr2) mutant in simultaneous presentation suggests a function of chep-2 gene products within the chemosensory informational integration pathway as well as in the chemosensory pathway.     

Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

Summary From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man.We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable.The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.