H. STRUCTURAL BIOLOGY CENTER
H-c. Multicellular Organization Laboratory - Isao Katsura Group

RESEARCH ACTIVITIES

(1) Analysis of synthetic dauer-constitutive mutants in the nematode Caenorhabditis elegans

Tomoko Yabe, Kotaro Kimura, Takeshi Ishihara and Isao Katsura

--If newly hatched larvae of C. elegans are in a crowded state and given a limited food supply, they sense the environmental signal of pheromone and food with a head sensory organ called amphid, and deviate from the normal life cycle, ending in non-feeding larvae called dauer larvae. Since the assay of dauer larva formation is much less time-consuming than behavioral assays such as chemotaxis assays, we are analyzing the pathway of sensory signals in the amphid neural circuit by detecting dauer larva formation as the output. We found that mutations in more than 50 known genes show synthetic dauer-constitutive (synDaf) phenotypes, i.e., they induce dauer larva formation in certain mutant backgrounds, regardless of the environmental conditions. The synthetic nature of the phenotype, we think, is based on the pathway of sensory signals. Namely, the signals are transmitted through parallel routes, and therefore two mutations are required to block them. We are determining the combinations of mutations for the synDaf phenotype and the pattern of suppression of the synDaf phenotype by various suppressor mutations. In this way we hope to elucidate the pathway of sensory signals for dauer regulation.
--Furthermore, to identify new genes required for the sensory signal transduction, we have isolated and mapped 44 mutations that show the synDaf phenotype in the unc-31(e169) background, where unc-31 gene encodes CAPS protein, which acts in secretion from dense core vesicles. Eight of the mutations map in 4 known genes, but most of the remaining 36 mutations, which map at least in 13 genes, seem to be located in novel genes, which we named sdf genes. Of these genes, sdf-9, sdf-13, and sdf-14 have been cloned. sdf-9 gene encoded a protein tyrosine phosphatase-like molecule, was expressed in a pair of neuron-associated cells called XXXL/R, and regulated dauer larva formation in the steroid hormone signaling pathway (Ohkura, K. et al.: Development 130, 3237-3248, 2003). sdf-13 encoded a homologue of the transcription factors Tbx2 andTbx3, was expressed in AWB, AWC, and ASJ sensory neurons as well as many pharyngeal neurons, and controlled olfactory adaptation in AWC and dauer larva formation in cells other than AWC (possibly ASJ)¹⁾. sdf-14 gene was the same as mrp-1 gene, which was formerly identified by its homology to multidrug resistance-associated protein genes in mammals. A functional sdf-14::GFP fusion gene was expressed in many tissues including neurons, pharyngeal muscles and intestinal cells. Experiments with extrinsic cell-specific promoters revealed that expression in two, but not one, of the three tissues rescue the mutant phenotype efficiently. Interestingly, human MRP1 could substitute for C. elegans MRP-1 in dauer larva regulation, and an inhibitor of the MRP1 export activity impaired this function, showing that the export activity is required for normal dauer larva regulation.
--In the year 2004, we found by epistatis analysis that sdf-14 acts neither in the cGMP nor TGF-β signaling pathway among the four signaling pathways involved in dauer larva regulation. The result is consistent with that of the cell-specific expression experiments, because genes in these two pathways act only in neurons. We found that sdf-14 mutations strongly enhance the dauer-constitutive phenotype of the daf-2(e1370) mutation, which confers resistance to many environmental stresses. Furthermore, sodium arsenite, which is a substrate of human MRP1, as well as high temperature (27℃) enhanced the dauer larva formation of the unc-31(e169) mutant, which is known to block the daf-2 insulin signaling pathway. Thus, sdf-14 gene may be involved in heat and heavy metal stress responses. The proof of this possibility as well as its relation to dauer larva formation remains to be studied.

(2) C. elegans mutants in the “associative" learning with odorants and food

Ichiro Torayama, Hiroshi Ichijo, Kotaro Kimura, Takeshi Ishihara and Isao Katsura

--The nematode C. elegans provides a good system for the study of learning with a combination of two stimuli. However, the mechanism of this learning looks different from that of classical conditioning, because (a) the unconditioned stimulus is usually limited to food or starvation, and because (b) the learning is efficient, if the conditioned stimulus is presented at the same time as but not before the unconditioned stimulus. To elucidate the molecular mechanism of such “associative" learning in C. elegans, we are isolating and characterizing mutants that show abnormality in the learning with butanone and food/starvation. It is known that butanone attracts wild-type animals without conditioning. Conditioning with butanone and starvation decreased the efficiency of chemotaxis to butanone, while conditioning with butanone and food increased it. We isolated mutants in these behaviors, some of which showed decrease in the efficiency of chemotaxis after conditioning with food and butanone, while others are attracted efficiently by butanone only after conditioning with food and butanone. Of those mutants, ut305 and ut306, which belong to the former category, have been studied in detail. ut305, but not ut306, showed abnormality in adaptation to isoamyl alcohol and benzaldehyde, which are sensed by the same type of sensory neurons (AWC) as butanone. ut305 showed inefficient chemotaxis to butanone at low concentration, although it showed normal chemotaxis at the concentration used in the learning assay. ut305 gene encoded a novel protein containing predicted transmembrane domains and showing limited homology to the Drosophila Raw protein.
--In the year 2004, we examined the rescue of ut305 mutant phenotypes by expressing the wild-type ut305 gene in various cells, using extrinsic promoters. The mutant phenotypes were rescued, if the wild-type ut305 gene was expressed in neurons including AWC sensory neurons, although we formerly detected the fluorescence of a functional ut305::GFP fusion gene in AIA interneurons and many pharyngeal neurons, but not in AWC neurons. Furthermore, str-2::GFP fusion gene, which is normally expressed in only one of the AWCL/R neurons, was not expressed in either of them in the ut305 mutant, while it was expressed in both of them when the wild-type ut305 gene was overexpressed in AWC neurons. These results indicated that ut305 gene acts in AWC neurons for the learning with butanone and food, for the adaptation to benzaldehyde and isoamyl alcohol and for the regulation of str-2 expression. We are now making specific antibodies to the ut305 protein to examine whether the ut305 protein is present in AWC neurons. We are also determining the position of ut305 gene in the cascade of the regulation of str-2 expression, in which Ca²⁺ signaling and the ASK1 MAP kinase cascade are involved. We also cloned ut306 gene and analyzed the mutant phenotypes. The study will be continued in 2005.

(3) Molecular genetic studies on sensory integration and behavioral plasticity in C. elegans

Takeshi Ishihara, Yuichi Iino¹, Akiko Mohri², Ikue Mori², Keiko Gengyo-Ando³, Shohei Mitani³ and Isao Katsura (¹Molecular Genetics Laboratory, University of Tokyo, ²Division of Biological Science, Nagoya University, ³Department of Physiology, Tokyo Women's Medical University School of Medicine)

--Animals receive environmental cues, select and integrate necessary information, and make proper responses, while all these steps can be modified by experience or memory. In C. elegans, many behavioral mutants defective in chemotaxis and thermotaxis, for instance, have been isolated and analyzed, and the molecular mechanisms of sensation have been elucidated. On this basis and as a next step, we are analyzing mutants that show abnormality in the learning and selection (evaluation) of sensory signals, to elucidate novel mechanisms of higher order sensory signal processing.
--C. elegans shows avoidance of copper ion and chemotaxis to odorants by receiving these stimuli with different sensory neurons in the head. We developed a behavioral assay for the interaction of two sensory signals: aversive copper ion and attractive odorant, diacetyl. Wild-type animals change their preference between the responses, depending on the relative concentration of copper ion and odorants. On the basis of the C. elegans neural circuitry, the result suggests that the two sensory signals interact with each other in a neural circuit consisting of about 10 pairs of neurons. While well-fed animals are usually used for this assay, we found that animals starved for 5 hours tend to prefer chemotaxis to odorants. The change is due to the desensitization of copper ion avoidance by starvation, and can be suppressed by serotonin, which mimics the effect of food. This desensitization is advantageous in natural environment, because starved animals can search for food over a wider area.
--To elucidate the mechanism of sensory integration in the neuronal circuit, we are isolating and analyzing mutants that show abnormality in this assay. The hen-1 mutants showed much weaker tendency to cross the Cu²⁺ barrier when migrating toward attractive odorants than the wild type, although that the hen-1 mutants had defects neither in the chemotaxis toward the attractive odorant nor in the avoidance of Cu²⁺ ion per se.
--To elucidate molecular mechanisms for the sensory integration, we cloned the hen-1 gene and found that it encodes a secretory protein with an LDL receptor ligand binding domain, LDLa. This domain in HEN-1 is most similar to that domain of Drosophila signaling molecule Jeb, which regulates migration and differentiation of visceral mesodermal precursor cells. Immunostaining by using antibody against recombinant HEN-1 protein revealed that the gene product is localized in the axon and cell body of each one pair of sensory and inter-neurons. The localization in the axon was abolished in unc-104 (kinesin KIF1A homologue) mutants, which show defects in the transport of synaptic vesicles. Expression studies with various promoters showed that this gene acts non-cell-autonomously in the mature nervous system.
--The hen-1 mutants also show abnormality in learning by paired presentation of starvation and NaCl (collaboration with Dr. Iino, University of Tokyo) and by paired presentation of starvation and temperature (collaboration with Ms Mohri and Dr. Mori, Nagoya University). Wild-type animals show chemotaxis to NaCl under a well-fed condition, although they avoid NaCl after conditioned with starvation and NaCl. The hen-1 mutants show a weaker behavioral change than the wild type after the conditioning, although they show normal chemotaxis to NaCl under a well-fed condition. Wild-type animals prefer the cultivation temperature under a well-fed condition, while they avoid that temperature after conditioned in the absence of food at the same cultivation temperature. Although the hen-1 mutants show normal thermotaxis under a well-fed condition, they do not avoid the cultivation temperature after conditioned in the absence of food. Since starvation was used to induce plasticity in both learning assays, we analyzed whether hen-1 animals can sense starvation, but we could not find any abnormality in the behavior after simple starvation. These results indicate that the hen-1 show defects in the behavioral plasticity after paired presentation of starvation and NaCl or starvation and temperature, although it responds normally to each of these stimuli.
--Molecular genetic analyses of HEN-1 suggest that HEN-1 functions as a neuronal modulator for sensory integration and learning. To elucidate the molecular mechanisms of this neuromodulation, we started investigating the protein interacting with the HEN-1 protein. First, we developed a binding assay for identification of receptors for HEN-1. By using a HEN-1-alkaline phosphatase fusion protein as a ligand, which was expressed by HEK293 cells, we found that HEN-1 specifically binds a subpopulation of the primary culture cells in C. elegans, suggesting that receptors for HEN-1 exist in these cells.
--Recently, Jeb in Drosophila was reported to regulate development of mesodermal cells through the receptor tyrosine kinase DAlk, which is a homologue of human proto-oncogene Alk. Since the LDLa domain in Jeb is similar to that in HEN-1, we started to analyze the scd-2 gene, which encodes a receptor tyrosine kinase similar to DAlk. Expression analyses by using an scd-2 promoter-GFP construct suggested that SCD-2 is expressed in several sensory neurons and interneurons. scd-2 mutants showed the same behavioral defects as hen-1 mutants in paradigms for sensory integration and learning. The expression of SCD-2 in scd-2 mutants driven by neuron specific promoters and a heatshock promoter suggested that SCD-2 functions in the mature nervous system, like HEN-1. These results suggest that SCD-2 may be a receptor for HEN-1. Determination of the cells where SCD-2 functions for sensory integration and learning may reveal the center of informational processing in C. elegans.

(4) Genetic analysis of plasticity of avoidance behaviors in C. elegans

Kotaro Kimura and Isao Katsura

--C. elegans avoid many toxic and/or hazardous signals, such as high osmotic strength, acidic pH and SDS, as well as several volatile and soluble small compounds. In general, preceded stimulation(s) of animal's sensory neuron lead to a reduction in the magnitude of its response (adaptation), and C. elegans has been shown to exhibit adaptation to attractive odors or to body touch after pre-exposure to the stimulus. However, plasticity of the avoidance behaviors of C. elegans to the signals mentioned above is poorly understood. We wondered whether the avoidance behaviors of C. elegans are modulated by preceded experience of the same stimulus.
--We found that pre-exposure to 2-nonanone, one of the repellent odors, enhances the avoidance behavior of the animals to the odor. For example, the avoidance behavior to 10% (v/v) 2-nonanone of the wild-type animals was significantly enhanced after pre-exposure. The magnitude of the enhanced avoidance behavior was comparable to the behavior to 30% 2-nonanone of previously unexposed animals, suggesting that pre-exposure may enhance the sensitivity to 2-nonanone by about 3-fold.
--Enhancement after pre-exposure was also observed for 1-octanol, another repellent odor, but not for osmolarity as the repellent stimulus, suggesting that only certain type(s) of repellent stimuli trigger this phenomenon, and that it requires specific molecular and/or neuronal mechanisms.
--We are interested in the sensitization because of the following reasons: (1) Not all types of the repellent stimuli trigger the sensitization. (2) Sensitization to repellent odors and tastes has not been reported in C. elegans to our knowledge. (3) The molecular mechanisms of sensitization to odor, taste or even light in other animals are poorly understood. We believe that genetic analysis of the sensitization may reveal novel molecular mechanism of regulation of signal sensation.

(5) Class 1 flr mutants of the nematode Caenorhabditis elegans

Yuri Kobayashi, Kotaro Kimura, Takeshi Ishihara and Isao Katsura

--Class 1 flr mutants of C. elegans, which map in flr-1, flr-3 and flr-4, were originally isolated by resistance to 0.4 mg/ml NaF (Katsura, I. et al.: Genetics 136, 145-154, 1994). They also show many other phenotypes including slow growth, short defecation cycle periods, frequent skip of the expulsion step of defecation, synthetic abnormality in dauer larva formation, weak tendency to stay on food, and hypersensitivity to serotonin. The flr-1 gene encodes an ion channel belonging to the DEG/ENaC (C. elegans degenerins and mammalian amiloride-sensitive epithelial sodium channels) superfamily, while flr-4 and flr-3 code for a novel Ser/Thr protein kinase and a kinase-like molecule, respectively, both having a hydrophobic domain on the carboxyl terminus. A functional flr-1::GFP fusion gene is expressed only in the intestinal cells from the comma stage of embryos to the adult stage, while a functional flr-4::GFP is expressed in the intestinal cells from the 1.5-fold stage, in the isthmus of the pharynx from the 3-fold stage and in a pair of head neurons called AUA from L1 larvae to adults. Moreover, the expression of various flr-3::lacZ and flr-3::GFP fusion genes is detected only in the intestine. Temperature-shift experiments of a flr-4(ts) mutant showed that the activity of FLR-4 is required at the time of defecation assay (i.e., young adults) for normal defecation cycle periods. We therefore think that class 1 flr genes constitute a regulatory system that acts in the differentiated intestinal cells.
--In 2004, we confirmed using an extrinsic promoter that expression of the wild-type flr-4 gene in the intestine of flr-4 mutants is sufficient for the wild-type phenotypes in growth, defecation cycle periods, expulsion, and dauer regulation³⁾. This is consistent with our old experiments showing that killing of AUA neurons in wild-type animals and flr-4 mutant animals does not change their defecation phenotypes. Thus, we could show that the three class 1 flr genes act in the intestinal cells to regulate various functions. We are studying how they interact with one another.

(6) Class 2 flr mutants of the nematode Caenorhabditis elegans

Akane Oishi, Kotaro Kimura, Takeshi Ishihara and Isao Katsura

--Class 2 flr mutations were isolated mostly as suppressors of the slow growth or serotonin-hypersensitivity of class 1 flr mutations. Besides these phenotypes, they also suppress the dauer larva formation abnormality and weak tendency to stay on food, but not the defecation abnormalities or strong fluoride-resistance. By themselves, class 2 flr mutations show the phenotypes of weak resistance to NaF and short average longevity as compared with wild-type animals. The phenotypes suggest two possibilities on the relationship between class 1 and class 2 flr genes. (a) Class 2 flr genes may act downstream of the class 1 regulatory pathway. At the downstream, the regulatory pathway bifurcates into two branches, the growth/dauer branch and the defecation branch, while class 2 genes act in the former branch and not the latter. (b) Class 2 flr genes may act antagonistically to class 1 genes, while the threshold of the phenotypes is different between the growth/dauer phenotypes and the defecation phenotypes.
--Class 2 mutations map in four genes, flr-2, flr-5, flr-6 and flr-7, of which only flr-2 has been cloned. flr-2 encodes a secretory protein belonging to the gremlin/DAN/cerberus family. A functional flr-2::GFP fusion gene was expressed in some neurons in the head and the tail as well as many pharyngeal neurons. FLR-2::alkaline phosphatase fusion protein molecules bound specifically to the intracellular compartment of a limited number of C. elegans primary culture cells. We therefore screened for C. elegans cDNA clones whose expression in COS cells enabled the cells to bind to FLR-2::alkaline phosphatase. By screening 264 pools of cDNA, where one pool consists of about 1000 clones, we obtained a single positive clone (ZK20.1) encoding a secretory protein.
--In 2004, we carried out cell-specific expression experiments, using extrinsic promoters. Although a functional flr-2::GFP fusion gene was expressed only in neurons, expression of the wild-type flr-2 gene in the body wall muscle and intestine, respectively, rescued the flr-2 mutant phenotype, as assayed by the recovery of the slow growth phenotype of flr-2; flr-1 double mutants. Rescue with neuronal promoters was successful, only when we used low concentrations of the construct DNA for transformation. Thus, it seems that although FLR-2 is expressed and secreted by some neurons under natural conditions, body wall muscles and intestinal cells also have the ability to secrete it. Besides these experiments, we are working on the functional interaction between flr-2 and ZK20.1, by isolating a deletion mutant in the ZK20.1 gene and looking for its phenotype.

PUBLICATIONS

Papers
1. Miyahara, K., Suzuki, N., Ishihara, T., Tsuchiya, E. and Katsura, I. (2004). TBX2/TBX3 transcriptional factor homologue controls olfactory adaptation in Caenorhabditis elegans. J. Neurobiol. 58, 392-402.
2. Kimura, K.D., Miyawaki, A., Matsumoto, K. and Mori, I. (2004). The C. elegans thermosensory neuron AFD responds to warming. Curr. Biol. 14, 1291-1295.
3. Take-uchi, M., Kobayashi, Y., Kimura, K., Ishihara, T. and Katsura, I. (2005). FLR-4, a novel Serine/Threonine protein kinase, regulates defecation rhythm in Caenorhabditis elegans. Mol. Biol. Cell, 16, 1355-1365.

Reviews
4. 石原 健（2004）「線虫学習行動の分子遺伝学:連合学習制御遺伝子」蛋白質・核酸・酵素49, 450-455.
5. 桂　勲（2005）「線虫C. elegansの発生・分化研究とゲノム情報」実験医学増刊23 (1), 157-163.

ORAL PRESENTATIONS

1. Ishihara, T., Iino, Y., and Katsura, I. Receptor tyrosine kinase, SCD-2, regulates sensory integration and learning. East Asia C.elegans Meeting. Awaji Island, June-July, 2004.
2. Kimura K. and Katsura I. Another type of behaviORAL PRESENTATIONS plasticity with repellent stimuli. East Asia C.elegans Meeting, Awaji Island, June-July, 2004.
3. Torayama, I., Ishihara T., and Katsura I. Isolation and analysis of mutants defective in olfactory learning in C.elegans. East Asia C.elegans Meeting, Awaji Island, June-July, 2004.
4. Ishihara, T., Iino, Y., and Katsura, I. Receptor tyrosine kinase, SCD-2, regulates sensory integration and learning in C.elegans. The 2004 Meeting on Axon Guidance and Neural Plasticity, Cold Spring Harbor, September, 2004.
5. 石原 健「線虫における感覚情報処理の分子機構」特定領域「神経回路」冬のシンポジウム、東京都、2004年1月
6. 石原健、池田大祐、田畑孝、飯野雄一、桂勲「線虫C.elegansにおける感覚情報の統合と連合学習の分子機構」第27回日本分子生物学会年会、神戸市、2004年12月

POSTER PRESENTATIONS

1.大石あかね、武内昌哉、石原 健、桂 勲「線虫C.elegansにおいてクラス1フッ素イオン耐性変異体の成長速度を制御するクラス2遺伝子群の解析」第27回日本分子生物学会年会、神戸市、2004年12月
2.虎山一郎、木村幸太郎、石原 健、桂 勲「線虫C.elegansの学習行動変異体の解析」第27回日本分子生物学会年会、神戸市、2004年12月

EDUCATION

1. Dr. I. Katsura gave a lecture at Kyoto University, Graduate School of Science, June 2004 (in Japanese).
2. Dr. I. Katsura gave a lecture at Kyushu University Graduate School, Faculty of Sciences, June 2004 (in Japanese).
3. Dr. I. Katsura was invited to give a seminar on “Molecular biological analysis of the behavior of the nematode C. elegans" at Kyushu University Graduate School, Faculty of Sciences, June 2004 (in Japanese).
4. Dr. I. Katsura gave a lecture at the University of Tokyo, Graduate School of Arts and Sciences, July 2004 (in Japanese).

SOCIAL CONTRIBUTION AND OTHERS

1. 特願2004-314935「ヒトMRP1阻害剤のスクリーニング法」発明者:矢部智子,桂　勲,石原 健,鈴木教郎,出願人:大学共同利用機関法人情報・システム研究機構.
2. Dr. I. Katsura served as one of the associate editors of the journal “Genes to Cells".
3. 毛利秀雄,勝見允行,木村武二,中西剋爾,守 隆夫,高橋正征,桂@勲,他12名「高等学校 生物II」三省堂2004.