Iino Y et al. (2000) Japanese Worm Meeting "RNAi screen for genes required for germline development in C. elegans"

Iino Y, Hanazawa M, Ueno N, Kohara Y, Mochii M

[Japanese Worm Meeting, 2000]

The germ cells play important roles in transmission of parental genetic information to the next generation and have various characteristics that somatic cells do not have. To identify genes giving such unique characteristics to germ cells, we screened arrayed cDNAs by using subtractive differential hybridization and then employed a reverse-genetic approach. cDNA made from the glp-4 (bn2ts) animals raised at the restrictive temperature, which lack germline nuclei, and from wild type embryos, which carry maternal messages, were subtracted from a cDNA pool made from the glp-1 (q231ts) mutant transferred to the restrictive temperature, which has a number of meiotic germ nuclei. This subtraction gave the 'forward-subtracted' cDNA pool expected to include germline-specific cDNA species. A reverse subtraction was also performed to make the 'reverse-subtracted' cDNA pool for comparison. Amplified cDNA from 7584 independent genes arrayed on a nylon membrane at high density were hybridized with these probes. Roughly 300 clones with significantly stronger signals against the forward-subtracted cDNA pool were selected as candidates to be analyzed further. Since the results of in situ hybridization for a subset of candidate clones confirmed successful subtractive screening, the functions of the candidates were subsequently assessed using the RNAi technique. We have so far analyzed 168 clones and identified 34 clones that showed a range of sterility. Nine of these showed fertilization or ovulation defects and one showed a germline tumor-like phenotype. The others showed abnormal morphology of the gonad and analyses of them are in progress. These results demonstrate the efficacy of the screening strategy utilizing the EST library and the RNAi technique. This screening will be completed within this year and the results will be posted on internet.

Iino Y et al. (2000) Japanese Worm Meeting "Involvement of the insulin-like signaling pathway in the associative learning in chemotactic behavior"

Aihara S, Iino Y

[Japanese Worm Meeting, 2000]

We have previously reported that worms starved for several hours in the presence of NaCl learn to avoid NaCl. This change in chemotactic behavior has characteristics of an associative learning, because both presence of NaCl and absence of food are required to induce the behavioral change. Here we report that DAF-2, the insulin receptor-like molecule, is involved in this form of behavioral plasticity. Several mutants of daf-2, including the reference allele e1370, showed severe defects in this form of learning. The daf-2 mutants are known to have a Daf-c (constitutive dauer formation) phenotype and are long-lived. The hx546 mutant of age-1, which encodes a PI-3-kinase acting downstream of DAF-2, also showed a defect in learning. Acting further downstream, daf-16 encodes a forkhead transcription factor and is negatively regulated by daf-2 and age-1 in both the dauer formation and longevity pathways. The daf-16 mutants, including a null allele, had defects in learning that are milder than either the daf-2 or the daf-16; daf-2 mutants. This result suggests that DAF-16 is not the sole downstream effector of DAF-2 for this phenotype, as has already been proposed for the dauer formation pathway. Other daf mutants were also examined to see whether the same signals that regulate dauer formation affect the chemotaxis learning. The daf-1, -4, -11, -12 and -21 mutants were examined at 25C, where all these mutants show the Daf-c phenotype except for daf-12, which is dauer-defective. They showed either no defects or defects milder than daf-2(e1370), suggesting non-parallel relationships between regulation of dauer formation and that of chemotaxis learning. To gain more insights into how daf-2 affects learning, temperature-shift experiments were performed using the daf-2(e1370) temperature-sensitive allele. This mutant show normal learning at 15C but has a severe defect at 25C as described above. e1370 animals conditioned at 15C and tested at 25C showed a defect, which was more severe than the animals conditioned at 25C and tested at 15C. These results suggest that an insulin-like ligand is involved in the plasticity of chemotactic behavior. Based on the result of the temperature-shift experiments, we consider two possibilities: 1) DAF-2 acts in the 'read-out of memory' phase of learning; 2) DAF-2 is required simply for the avoidance behavior.

Iino Y et al. (2000) Japanese Worm Meeting "The Ras-MAPK signal transduction pathway plays multiple roles in chemotaxis to odorants"

Hirotsu T, Iino Y

[Japanese Worm Meeting, 2000]

We have previously reported that the Ras-MAPK pathway functions in mature olfactory neurons. Immunohistochemical study has suggested that the pathway is activated by odorant stimulus. Our further analysis indicated that the pathway might have multiple functions in olfaction as described below. In the immunofluorescence analysis, the activation of MAPK was induced in the AWB neurons, as well as in AWC, by exposure to high concentration (10-2 dilution) of isoamylalcohol for 10 seconds. Interestingly, the activation of MAPK in AWC was shut off by longer exposure (for 5min) to 10-2 isoamylalcohol, while the activation in AWB was sustained. On the contrary, the staining of activated MAPK was detected in AWC, but not in AWB, after application of lower concentration (10-4 dilution) of isoamylalcohol. The AWB neurons are known to mediate avoidance of volatile odorants. Our immunofluorescence analysis indicated the possibility that high concentration of isoamylalcohol might induce avoidance behavior. Indeed, worms show avoidance behavior when large amount of undiluted isoamylalcohol is presented. The let-60(gf), lin-45(lf) and mek-2(0) mutants exhibited defects in this behavior. Thus the Ras-MAPK pathway is also important for avoidance of odorants. We also observed that the odr-3, tax-2, tax-4 and osm-9 mutants had deficits in this avoidance behavior, suggesting that these components known to mediate chemotaxis to odorants are also involved in perception of highly concentrated isoamylalcohol. When wild-type worms are pre-exposed to isoamylalcohol at 10-4 dilution for 5min, they avoid isoamylalcohol at the normally attractive concentration (10-2). This behavioral plasticity is also manifested in chemotaxis to benzaldehyde (sensed by AWC), pyrazine (AWA), and trimethylthiazole (AWA & AWC). The let-60(lf) , let-60(gf) and mek-2(0) mutants showed defects in this behavior. The Ras-MAPK pathway may also play roles in this behavioral plasticity.

Iino Y et al. (2000) West Coast Worm Meeting "The ut236 mutant in C. elegans has defects in the interaction of two sensory signals and an associative learning."

Iino Y, Ishihara T, Katsura I

[West Coast Worm Meeting, 2000]

Among many environmental stimuli, C. elegans seems to respond to only a few stimuli simultaneously. To elucidate how the neural circuit processes many sensory signals to respond properly, we have designed a new assay system to analyze interaction between two responses: chemotaxis to odorants and avoidance from Cu2+ ion. In this assay, the behavior depends on the concentration of both stimuli, which are sensed by different neurons. Our observation suggests that these two responses repress each other. We can argue that this mutual interaction takes place in a defined part of the neural circuit, which consists of about 10 pairs of neurons. One mutant, ut236, has defects in this interaction. It prefers to avoid Cu2+ ion rather than to go to odorants, while its dose responses to the odorants and Cu2+ ion are indistinguishable from those of the wild type. Positional cloning revealed that the ut236 gene encodes a novel secretory protein (C36B7.7) with an LDL receptor ligand binding motif. Expression studies with a functional GFP fusion gene and by immunostaining show that it is expressed in a few sensory neurons and AIY neurons. By using cell type specific promoter, we found that the expression of C36B7.7 in AIY or ASE is sufficient for the rescue of this phenotype. Also, by using a heatshock promoter construct, it is shown that the expression of C36B7.7 in embryonic stage is not sufficient for the rescue but the expression in the late larval or adult stage is. These results suggest that C36B7.7 is necessary for the neuronal function of these neurons, but is not for the development of the nervous system. The ut236 mutant seems to have another defect in an associative learning by paired presentation of NaCl and starvation. This phenotype can be also rescued by the C36B7.7 transgene. Therefore, we postulate that this novel secretory protein is involved in the modification of sensory signals in the neural circuit to regulate their preference in chemical stimuli.

Iino Y et al. (2000) Japanese Worm Meeting "The ut236 mutant in C. elegans has defects in the interaction of two sensory signals and associative learning."

Ishihara T, Katsura I, Iino Y

[Japanese Worm Meeting, 2000]

Among many environmental stimuli, C. elegans seems to respond to only a few stimuli simultaneously. To elucidate how the neural circuit processes many sensory signals to respond properly, we have designed a new assay system to analyze interaction between two responses: chemotaxis to odorants and avoidance from Cu2+ ion. In this assay, the behavior depends on the concentration of both stimuli, which are sensed by different neurons. Our observation suggests that these two responses repress each other. We can argue that this mutual interaction takes place in a defined part of the neural circuit, which consists of about 10 pairs of neurons. One mutant, ut236, has defects in this interaction. It prefers to avoid Cu2+ ion rather than to go to odorants, while its dose responses to the odorants and Cu2+ ion are indistinguishable from those of the wild type. Positional cloning revealed that the ut236 gene encodes a novel secretory protein (C36B7.7) with an LDL receptor ligand binding motif. Expression studies with a functional GFP fusion gene and by immunostaining show that it is expressed in a few sensory neurons and AIY neurons. By using cell type specific promoter, we found that the expression of C36B7.7 in AIY or ASE is sufficient for the rescue of this phenotype. Also, by using a heatshock promoter construct, it is shown that the expression of C36B7.7 in embryonic stage is not sufficient for the rescue but the expression in the late larval or adult stage is. These results suggest that C36B7.7 is necessary for the neuronal function of these neurons, but is not for the development of the nervous system. The ut236 mutant seems to have another defect in an associative learning by paired presentation of NaCl and starvation. This phenotype can be also rescued by the C36B7.7 transgene. Therefore, we postulate that the ut236 gene product is involved in the modification of sensory signals in the neural circuit to regulate their preference in chemical stimuli.

Sato, Manami et al. (2015) International Worm Meeting "Expression pattern of C. elegans Y RNA homologs."

Ushida, Chisato, Kihara, Shinya, Sato, Manami

[International Worm Meeting, 2015]

Y RNA is a small structured ncRNA of about 100 nt in length. This RNA binds to Ro60 protein, which is a target of autoimmune disease antibody in patients with systemic lupus erythematosus and Sjogren's syndrome. Several lines of evidence suggest that the role of Y RNA and Ro60 function in the quality control of structured ncRNAs in cells under stress conditions. It is also indicated that vertebrate Y RNAs function in the initiation of DNA replication without Ro60. However, the molecular mechanisms of these functions and the contribution of Ro60/Y RNP to the autoimmune disease are still unclear. C. elegans genome encodes one Ro60 homolog (ROP-1) and 19 Y RNA homologs (1 CeY RNA and 18 sbRNAs). Other animals also have several Y RNA homologs, but C. elegans is the first example which has more than 5 Y RNA homologs encoded in the genome. Here we show the expression pattern and the cellular localization of these Y RNA homologs in C. elegans examined by the RNA fluorescent in situ hybridization (RNA-FISH). The signals of 14 homologs were detected in the intestinal cytoplasm. The signals of two other homologs were detected in the germ cytoplasm. The remaining three could not be detected, probably because they present in too low abundance to be detected by RNA-FISH. All 19 Y RNA homologs have the structural elements required for the binding of ROP-1. In other organisms, Ro60 binding stabilizes Y RNAs in cells. To know whether C. elegans Y RNA homologs also stabilized by the presence of ROP-1, we examined RNA-FISH of the Y RNA homologs against a mutant strain MQ470, which has a transposon insertion in the middle of the ROP-1 gene and lacks ROP-1 proteins in the cell. As expected, all Y RNAs examined so far decreased extensively. These were confirmed by northern hybridization. The results suggest that several C. elegans Y RNA homologs are expressed in a tissue-specific manner and most Y RNA homologs are stabilized by ROP-1 binding as well as those in other organisms.

Tomioka, M. et al. (2017) International Worm Meeting "A versatile insulin-like signaling regulates taste avoidance learning."

Tomioka, M., Iino, Y.

[International Worm Meeting, 2017]

During life, animals experience a myriad of life-threatening events and cope with them by physiological and behavioral responses. We have found that an alternative splicing (AS) event in the insulin receptor gene, daf-2, has strong impacts on several biological processes such as development, lifespan and behavior in C. elegans. DAF-2a, which is produced by skipping of a cassette exon mainly resides in the cell body to control transcription of genes related to morphogenesis and stress responses, whereas a cassette exon-inclusion isoform, DAF-2c, is translocated to neuronal axons and regulates learning of the salt concentrations experienced under starvation, which we call taste avoidance learning. This AS event appears to balance physiological and behavioral responses to cope with a variety of life-threatening conditions. Among 40 insulin-like peptides in C. elegans, an insulin-like peptide, INS-1, plays an important role in taste avoidance learning. Mutants of INS-1 show a marked defect in taste avoidance learning. INS-1 is secreted from sensory neurons and interneurons that have synaptic inputs to the gustatory neuron ASER and acts on the axonal DAF-2 isoform, DAF-2c, in ASER in the learning. We demonstrated that the INS-1-to-DAF-2c signal during salt chemotaxis, but not during starvation conditioning, is required for starvation-induced salt avoidance. On the other hand, the cell body isoform DAF-2a may control learning during salt conditioning through the FOXO transcription factor DAF-16. Our study proposes a model in which the versatile insulin-like signaling acting at different subcellular sites regulates taste avoidance learning in C. elegans.

Ohno, H. et al. (2015) International Worm Meeting "In vivo imaging of diacylglycerol signaling in a taste receptor neuron involved in salt-concentration memory."

Iino, Y., Ohno, H.

[International Worm Meeting, 2015]

Adaptation to environmental conditions by modifying behavior often involves simple forms of learning and memory. We have previously reported that C. elegans worms can memorize ambient salt concentrations and are attracted to the previous salt concentration at which they have been fed, whereas they avoid it if they have been starved (Kunitomo et al., Nat. Commun., 2013; Ohno et al., Science, 2014). This behavioral plasticity has been suggested to be regulated by the diacylglycerol (DAG)/protein kinase C (PKC) pathway acting in a taste receptor neuron, ASER. Genetic manipulations that activate or inactivate this pathway in ASER result in worm migration to higher or lower salt concentrations, respectively (Adachi et al., Genetics, 2010; Kunitomo et al., Nat. Commun., 2013). To explore the role of DAG signaling in salt concentration memory, we have monitored the abundance of DAG in ASER of living worms. When the external salt concentration is increased or decreased from the concentration that has been experienced with food, DAG level is reduced or elevated, respectively, in presynaptic regions or regions adjacent to presynapses, where a novel protein kinase C (nPKC)-epsilon/eta ortholog TTX-4 (a.k.a. PKC-1) is localized. These DAG responses are largely diminished after starvation. These results raise the possibility that the activity of DAG signaling in the presynaptic region of ASER encodes the differences between past and present salt concentrations and thereby forms an integral component of the food-associated memory.

Takashi Kanoh et al. (2003) International Worm Meeting "Genetic, pharmacological and behavioral analysis of hab-1 mutant"

Yukari Suzuki, Kiyoshi Kunoh, Takashi Kanoh, Toshihiro Sassa, Hiroyuki Fujimoto, Ryuji Hosono

[International Worm Meeting, 2003]

The C.elegans hab-1 gene mutants are habituated more slowly and are more rapidly recovered from habituation than wild-type animals (1). We further tested another integrative behavior of hab-1 mutants. Brockie et al., (2) developed a simple maze assay that animals at one pole navigate through the repellent CuSO4 to the attractant diacetyl. No hab-1(cn308) mutant was able to arrive to the target at 24 hr, though 40 % of wild-type animals reached the target within 4 hr. Wild-type and hab-1 animals were equally repelled by CuSO4 and were equally attracted to diacetyl when measured in the absence of CuSO4. These results suggest that the hab-1 mutants are impaired the ability to tail effectively their movement to complex stimuli. Wild-type animals accumulate in the center of NaCl under well-fed condition but avoid NaCl after conditioning with NaCl in the presence of food (3). In contrast, hab-1 mutants accumulated to NaCl in the presence of food but did not show response to NaCl, that is they neither avoided nor attracted to NaCl in the absence of food. These results also suggest that the hab-1 gene is essential for general learning behavior. The hab-1 mutant is partial resistance to aldicarb, one of AChE inhibitors, suggesting that the gene functions at the presynaptic terminal. And further, the mutant shows short life-span and hypersensitivity to volatile anesthetics, halothane. These phenotypes are similar to the mutant phenotype of gas-1 encoding the subunit of the NADH:ubiquinone-oxidoreductase. To elucidate further the function of hab-1, we are now in progress of cloning the gene. 1. Xu, X., Sassa,T., Kunoh, K. and Hosono, R. (2002) J.Neurogenetics 16: 29-44; 2. Brockie, P.J., Mellem J.M., Hills. T., Madsen, D.M. and Maricq, A.V. (2001) Neuron 31: 617-630; 3. Saeki, S., Yamamoto, M. and Iino, Y. (2001) J.Exp. Biol. 204:1757-1764.

Nancy L Price et al. (2003) International Worm Meeting "The utilization of Caenorhabditis briggsae as a host model to study bacterial pathogenesis"

Ann M Rose, Nancy L Price

[International Worm Meeting, 2003]

Caenorhabditis elegans has now been established as a host model for studies of infectivity by bacterial pathogens including Pseudomonas aeruginosa, Salmonella typhimurium, Burkholderia pseudomallei, and Enterococcus faecalis. However, virulence determinants of bacterial pathogens are regulated by temperature and environmental conditions, thereby limiting the use of C. elegans which cannot survive in temperatures higher than 25oC. This study shows that the related species, C. briggsae survives better than C. elegans on bacterial culture media at higher temperatures and describes the effects on C. briggsae of mammalian enteric pathogens, primarily Yersinia enterocolitica. C. briggsae grown on Y. enterocolitica accumulated bacteria in the gastrointestinal tract of the worm, resulting in decreased life-span and progeny fitness in a depleted-calcium environment. The mechanisms of the interaction are as yet unknown as both virulent and avirulent strains of Y. enterocolitica displayed the similar results. Investigations were undertaken to examine whether the shortened life span could be attributed to starvation, toxicity, or possible infection. Starvation effects was determined not to be the sole cause of pre-mature worm death as C. briggsae grown on Y. enterocolitica survived several days longer than starved worms. A bacterial mixing experiment using both Y. enterocolitica and E. coli OP50 shortened the worm life span compared to feeding on Y. enterocolitica alone, suggesting a possible toxic effect. However worms feeding on Y. enterocolitica and later shifted to E. coli OP50 resulted in reversion of the survival curve to that of worms feeding on E. coli OP50 alone, indicating that the detrimental effect of Y. enterocolitica was reversible. Fluorescent labeling of bacterial strains with a lacZ-GFP reporter gene demonstrated that Y. enterocolitica, but not E. coli OP50, is retained in the gut, a result which is compatible with a molecular interaction between Y. enterocolitica and nematode cellular components. These findings suggest that Y. enterocolitica may cause an infection within the nematode gastrointestinal tract and provide an assay for genetic dissection of the molecular basis of pathogenesis.