Kohji Miyahara et al. (2003) International Worm Meeting "Role of RhoGAP in behavioral plasticity ."

Eiko Tsuchiya, Masayuki Gosho, Kohji Miyahara

[International Worm Meeting, 2003]

In order to identify genes that associate with behavioral plasticity in C. elegans, we screened for mutants that are defective in adaptation or saturation to odorants. A screen was conducted to identify mutants that did not fail to chemotax toward a point source of odorant in a field of same odorant. From approximately 4000 EMS mutagenized genomes, we discovered 12 mutants that exhibited defects in adaptation or saturation to the AWC-sensed odorants. We cloned one mutant by transgene rescue of the mutant phenotype and found it to encode the GTPase activated protein for Rho (RhoGAP). RhoGAPs are negative regulators of Rho-GTPase signaling pathways related to actin cytoskeleton dynamics, cell proliferation, and differentiation. Our results may suggest that Rho-GTPase is associated with the mechanism of olfactory adaptation or saturation. We are currently making GFP constructs to identify the location of RhoGAP expression and are sequencing this mutation in order to understand the molecular basis of this mutant phenotype.

Kohji Miyahara et al. (2005) International Worm Meeting "Rho GTPase signaling in olfactory adaptation."

Eiko Tsuchiya, Sadaaki Tanaka, Hiroyuki Arai, Masayuki Gosho, Dai Hirata, Kohji Miyahara, Takao Inoue, Hiroyuki Kobuna

[International Worm Meeting, 2005]

Olfactory adaptation, a component of behavioral plasticity, and the signaling pathway(s) that control it are at the center of our interest. In order to identify gene(s) associated with olfactory adaptation, we screened for mutants defective in adaptation to odorants. Out of 4,000 EMS-mutagenized animals, we discovered 11 mutants that exhibiting defects in adaptation to the AWC-sensed odorants. One of the mutants turned out to contain an altered version of GEI-1, that encodes Rho-GAP (GTPase-activated protein), suggesting this protein plays an important role in adaptation. To investigate whether Rho-GTPases act in this behavior, we examined olfactory adaptation of transgenic animals expressing dominant active/negative forms of Rho-GTPases (RhoA, Cdc42, and Rac1). The dominant active RHO-1/RhoA inhibited normal olfactory adaptation similarly to the gei-1 mutant. Furthermore, the genetic analyses indicate that RHO-1 acts downstream of GEI-1 in olfactory adaptation.

Isao Katsura et al. (2002) Japanese Worm Meeting "Synthetic Dauer-constitutive Phenotype: a Paradigm of Genetic Interaction"

Takeshi Ishihara, Kohji Miyahara, Norio Suzuki, Tomoko Yabe, Kiyotaka Ohkura, Isao Katsura

[Japanese Worm Meeting, 2002]

We are studying the synthetic dauer-constitutive (Sdf) phenotype of C. elegans to learn how genes interact each other within and between the pathways of dauer larva regulation. Although the combination of mutations for this phenotype gave only limited information on the mechanism, suppression of the Sdf phenotype as well as signal-monitoring with daf-7::GFP yielded substantial information on the function of relevant genes in relation to the four dauer-regulatory pathways (TGF-beta, insulin receptor, cGMP and P450) and the four types of sensory neurons (ASI, ADF, ASG and ASJ) involved in dauer regulation. Some combinations of unc-31(e169), unc-3(e151), osm-1(p808), egl-4(n478), aex-3(sa5), tax-2(p671), tax-4(p678), tax-6(p675), flr-1(ut11) and unc-104(e1265) show strong Sdf phenotypes, but the dauer-regulatory pathway(s) affected by the mutations differ depending on the mutations and the combinations. Although unc-31 gene is thought to affect the daf-2(insulin receptor) pathway by suppression experiments (e.g., M. Ailion & JH Thomas, 2000), it also acts upstream of daf-7(TGF-beta), because the expression of daf-7::GFP in ASI neurons is suppressed in the double mutants, unc-31;unc-3 and unc-31;osm-1, but not in the single mutants unc-31, unc-3 and osm-1. Some mutations such as tax-2(p671), osm-1(p808), and unc-3(e151) are both Sdf mutations and suppressors of Sdf mutations, depending on the combination of mutations. The ambivalent nature of tax-2 may reflect the opposing roles of ASI (dauer-inhibitory) and ASJ (dauer-promoting) neurons in which tax-2 is expressed. We can deduce a parallel pathway model for the complex interaction of unc-3(e151), osm-1(p808), tax-2(p671), and flr-1(ut11), using Boolean algebra or switching theory. However, for the moment, we have to assume synthetic suppression to satisfy all the experimental data. Geneticists utilize switching theory for cascades and pathways, in which genes are arranged in series. The extension of such theory to parallel pathways may be useful for the studies of gene interaction in the post-genomic era, although we have to overcome some difficulties such as theoretical indefiniteness and lack of experimental data.

Kohtaroh Motoshige et al. (2006) East Asia C. elegans Meeting "The Ndr kinase SAX-1 is essential for olfactory adaptation"

Daichi Komiyama, Dai Hirata, Satoshi Itakura, Masayuki Gosho, Kohtaroh Motoshige, Kohji Miyahara, Kazunori Kume

[East Asia C. elegans Meeting, 2006]

In order to identify the genes associated with behavioral plasticity in sensory neurons, we screened for the mutants that are defective in adaptation/saturation to odorants. A screen was conducted to identify the mutants that did not fail to chemotaxis toward a point source of odorant in a field of same odorant. From approximately 8000 EMS mutagenized animals, we isolated 11 mutants that exhibited defects in adaptation/saturation to the AWC-sensed odorants and 1 mutant that exhibited defect in adaptation to the AWA-sensed odorants. We cloned the Ndr kinase family sax-1 gene that suppressed the phenotype of one of the mutants. Genetic analysis indicated that the mutated gene is identical with the sax-1 gene. These results suggest that SAX-1 is essential for olfactory adaptation/saturation.

Yasumi Ohshima et al. (2006) East Asia C. elegans Meeting "Food, feeding, digestion and defecation of the worm"

Yasumi Ohshima, Yusuke Kaku, Yuta Yoshidome, Yuji Osako, Katsuhiro Kunigo, Akiko Nakasone, Kohji Miyahara, Tomonori Nakamura

[East Asia C. elegans Meeting, 2006]

Food or nutrition is vital for the life of C. elegans as for other animals, and should affect growth, body size and activity of the worm. In the laboratory, worms usually feed on OP50, a uracil-requiring strain of E. coli, which is a good food for worms in the sense that it supports rapid growth and production of many progeny. However, E. coli is an enteric bacterium that may not be abundant in soil which is said to be the natural habitat of the worm. Avery & Shtonda (2003) identified from soil samples more than 10 bacterial species that supported growth of the worm. Yet, the entire image of the natural food of the worm is not clear. Although the mechanisms of feeding and defecation of E. coli have been extensively studied, much is still to be solved, for example, the mechanisms of food recognition, rate and target of defecation. Furthermore, virtually nothing is known on the mechanisms of digestion of food and absorption of nutrients in the gut of the worm. In mammals, many factors which seem to be involved in digestion or absorption are known, but there is little genetics in any animal. Thus, we have begun studies on some of these, and plan to ask further, as follows. 1) Heat-killed E. coli is not likely to support growth of C. elegans. 2) We are studying whether selected microorganisms are good food or not. For example, yeast S. cerevisiae probably does not support growth to adults. 3) We are trying to identify live or dead microorganisms in the gut of marked worms that were put into a soil sample, by DNA sequencing. 4) Several artificial microspheres of various size or chemical nature are used to get clues to food recognition, feeding and defecation. 5) We are trying to analyze the time course of feeding, digestion and defecation of GFP-marked E. coli. 6) We have found many C. elegans genes possibly involved in digestion or absorption based on amino acid sequence homology with those in mammals. We have obtained KO mutants for some of them from Dr. S. Mitani for reverse genetics. 7) We have begun isolation of mutants on digestion or absorption. The screening at present uses GFP-marked E. coli as food. Two candidate mutants were obtained that retain more GFP fluorescence in the gut without little change in the pumping cycle.

Satoshi Itakura et al. (2006) East Asia C. elegans Meeting "Role of Rho GTPase signaling in olfactory adaptation"

Dai Hirata, Sadaaki Tanaka, Kazunori Kume, Takao Inoue, Hiroyuki Arai, Hiroyuki Kobuna, Kohji Miyahara, Kohtaroh Motoshige, Satoshi Itakura, Masayuki Gosho

[East Asia C. elegans Meeting, 2006]

We are interested in olfactory adaptation, a component of behavioral plasticity. To investigate the gene(s) involved in olfactory adaptation, we screened for the mutants defective in adaptation to odorants. From out of 4,000 EMS-mutagenized animals, we isolated 11 mutants exhibiting defects in adaptation to the AWC-sensed odorants. One of the mutants turned out to contain a mutation in gei-1, that encodes Rho-GAP (GTPase-activated protein). To investigate whether Rho-GTPase(s) acts in this behavior, we examined olfactory adaptation of transgenic animals expressing dominant active/negative forms of Rho-GTPase family. The dominant active RHO-1 inhibited normal olfactory adaptation as the gei-1 mutant. Furthermore, the genetic analyses indicate that RHO-1 acts downstream of GEI-1 in olfactory adaptation.

Satoshi Itakura et al. (2007) International Worm Meeting "Role of Rho GTPase signaling in olfactory adaptation."

Sadaaki Tanaka, Masayuki Gosho, Hiroyuki Arai, Hiroyuki Kobuna, Satoshi Itakura, Kotaro Motoshige, Dai Hirata, Kazunori Kume, Kohji Miyahara, Takao Inoue

[International Worm Meeting, 2007]

We are interested in olfactory adaptation, a component of behavioral plasticity. To investigate the gene(s) involved in olfactory adaptation, we screened for the mutants defective in adaptation to odorants. From out of 4,000 EMS-mutagenized animals, we isolated 11 mutants exhibiting defects in adaptation to the AWC-sensed odorants. One of the mutants turned out to contain a mutation in gei-1, that encodes Rho-GAP (GTPase-activated protein). To investigate whether Rho-GTPase(s) acts in this behavior, we examined olfactory adaptation of transgenic animals expressing dominant active/negative forms of Rho-GTPase family. The dominant active RHO-1 inhibited normal olfactory adaptation as the gei-1 mutant. Furthermore, the genetic analyses indicate that RHO-1 acts downstream of GEI-1 in olfactory adaptation.

Noelle L'Etoile et al. (2001) International C. elegans Meeting "The role of a cGMP dependent protein kinase in olfactory adaptation"

Noelle L'Etoile, Cori Bargmann

[International C. elegans Meeting, 2001]

C. elegans can accurately discriminate among a wide variety of volatile chemosensory cues. At least five classes of odors are detected by the two AWC sensory neurons. The signaling cascade in the AWC neurons is thought to be initiated by odorant binding to a G-protein coupled receptor, which activates one or more G alphas, this is postulated to lead to increased cGMP levels either by stimulating a guanylyl cyclase or inhibiting a phosphodiesterase. cGMP gates a cation channel comprised of the two subunits, TAX-4 and TAX-2. Though each AWC neuron responds to at least two volatile compounds, adaptation to each odorant occurs independently. We discovered that ky95 , an allele of a cGMP dependent protein kinase, is required for adaptation to the AWC-sensed odorants benzaldehyde, butanone and isoamyl alcohol. This gene is allelic with egl-4/odr-9/chb-1 (Fujiwara and McIntire pers. comm). We have rescued the adaptation defects of the null egl-4(n479) with a cGMP dependent protein kinase cDNA expressed solely within the sensory neurons AWC, AWA, AWB, ADF and ASH. Although some of the other neurons could participate in adaptation, we suggest that olfactory adaptation occurs within the AWC sensory neuron itself. Expression of the kinase under a heat-shock promoter was used to show that the kinase is required in the adult for adaptation and not during development. We have tentatively identified the beta subunit of the cGMP-gated cation channel, TAX-2, as a target of phosphorylation: at short time points animals expressing an unphosphorylatable TAX-2 channel exhibit impaired adaptation. This raises the question of how adaptation can be both odorant specific yet result from regulation of the TAX-2/TAX-4 channel that is responsive to all three odorants. We are testing models that may explain this paradox. Another potential target for phosphorylation may be the Smad transcription factor DAF-3. A daf-3 mutation is epistatic to the null egl-4(n479) mutation for adaptation at long time points. This result suggests that the signaling cascade at the cilia eventually alters gene expression in the nucleus consistent with other suggestions (Miyahara et al. 1999 International Worm Meeting abstract 593). We propose that the cGMP dependent protein kinase is an integrator of olfactory signaling. Once a threshold of cGMP concentration or duration is met, a cohort of proteins within the cell alters its odor-responsiveness in an odorant specific manner.