Ujisawa, Tomoyo et al. (2013) International Worm Meeting "Photo and pheromone sensoryneuron regulates temperature experience-dependent cold tolerance."

Ohta, Akane, Sonoda, Satoru, Ishiwari, Tomohiro, Ujisawa, Tomoyo, Kuhara, Atsushi

[International Worm Meeting, 2013]

Animals have habituation mechanism against temperature changes. We are using temperature experience-dependent cold tolerance of C. elegans, as a model to study its molecular mechanism. After cultivation at 20 deg C, wild-type animals can not survive at 2 deg C. By contrast, after cultivation at 15 deg C, most of animals can survive at 2 deg C. To investigate whether cold tolerance is established in the specific developmental stage, we used temperature shift experiments using larvae. We found that a temperature-experience of larval stages did not affect the cold tolerance in adult animal. We next used temperature shift experiments at adult stage. Only three hours after the cultivation temperature was changed from 20 to 15 deg C, the cold tolerances were established. These indicate that temperature experience in formation of cold tolerance can be replaced for three hours. To determine the cells and genes for temperature experience-dependent cold tolerance, we utilized various mutants. tax-4 mutant defective in cGMP-gated channel showed abnormal cold tolerance. This abnormality is rescued by specific expression of tax-4 cDNA in a single sensoryneuron ASJ, known as a photo and pheromone sensoryneuron. We found that ASJ responds to temperature stimuli by calcium imaging analysis using genetically encodable calcium indicator, cameleone. Defects in known-thermotaxis neural circuit did not affect the cold-tolerance. These results suggest that ASJ is involved in the cold-tolerance. We measured the cold tolerance of the mutants defective in photo signal transduction of ASJ. The mutants impairing trimetric G protein alpha subunit, guanylyl cyclase or phosphodiesterase involved in photo signal transduction in ASJ showed abnormalities of cold tolerance. lite-1 mutant lacking photoreceptor protein did not show abnormal cold tolerance, suggesting that temperature is received by other receptor. Altogether, molecular and physiological analysis demonstrated that photo- and pheromone-sensing neuron regulates temperature experience-dependent cold tolerance through G protein-coupled pathway.

Ohnishi, Kohei et al. (2017) International Worm Meeting "Screening for temperature sensor in ASJ sensoryneuron regulating cold tolerance."

Ohta, Akane, Ujisawa, Tomoyo, Ohnishi, Kohei, Miura, Toru, Kuhara, Atsushi

[International Worm Meeting, 2017]

Temperature is important environmental information for biological reaction. Animals have acclimation mechanism to environmental temperature changes. C. elegans has cultivation temperature-dependent cold tolerance. Wild-type animals cultivated at 20-degree died after 2-degree cold stimuli. By contrast, 15-degree cultivated-animals can survive after 2-degree. We are using the cold tolerance as a model for studying temperature signaling in animal. Previously reported, ASJ sensory neuron that is known as light and pheromone-sensing neuron senses temperature through trimeric G-protein pathway, which is required for photo signaling (Ujisawa et al., PLOS ONE , 2016). However, photoreceptor LITE-1 was not involved in cold tolerance and temperature sensation in ASJ(Ohta, Ujisawa et al., Nature commun, 2014). To identify the temperature sensing receptor upstream of trimeric G protein in ASJ, we are screening G protein-coupled receptor (GPCR). Mutant animals defective in G protein signaling in ASJ showed abnormal increase of cold tolerance after 20-degree cultivation. We performed exhaustive RNAi screening for about 1000 GPCR genes, as a result, about 40 gene-knocked down animals respectively showed strong abnormality in cold tolerance. We observed expression patterns of these GPCR genes by using GFP, and at least 14 genes including srg-37, str-45, str-92, srd-48, srd-19, srh-244 and srt-72 were expressed in ASJ sensoryneurons. We constructed knockout mutants of respective GPCR genes by using CRISPR/Cas9 system, and analyzed cold tolerance. However, these mutants did not show abnormal cold tolerance. We are therefore constructing mutant animals defective in multiple GPCR genes, and measuring cold tolerance of these mutants. So far, we have constructed the septuple mutant, and we are measuring their cold tolerance.

Sonoda, Satoru et al. (2015) International Worm Meeting "Cold habituation is regulated by the tissue network including neuron and sperm."

Sonoda, Satoru, Ohta, Akane, Ujisawa, Tomoyo, Kuhara, Atsushi

[International Worm Meeting, 2015]

Temperature is a critical stimulus and causes biochemical impact in animals. We are studying habituation mechanism to environmental temperature changes in C. elgans. After cultivation at 20 degree, wild-type were destroyed by cold stimuli. In contrast, after cultivation at 15 degree, most of animals can survive. We recently found that this cold habituation is regulated by ASJ temperature sensing-neuron, which releases insulin that is received by intestine and neuron (Ohta, Ujisawa et al., Nature commun, 2014). To identify novel mechanisms of cold habituation, we analyzed mutants impairing genes isolated from our DNA microarray analysis. We found that sperm genes as well as neuron genes affect cold habituation. Sperm mutants, such as gsp-3, gsp-4, ife-1 and fem-1, showed abnormal enhancement of cold habituation. Also, laser ablation of sperm in wild-type induced similar abnormality in cold habituation. Our genetic epistasis analysis and quantitative PCR analysis suggested that sperm affects cold habituation in downstream of insulin signaling of intestine. We unexpectedly found that abnormal cold habituation of gsp-4 was strongly suppressed by the mutations in temperature signaling of ASJ neuron. This speculates that sperm affects neuronal activity of ASJ temperature sensing-neuron. We therefore monitored neuronal activity of ASJ in gsp-4 mutant, by using calcium imaging under temperature changes. Temperature stimuli-dependent calcium concentration changes in ASJ of gsp-4 was lower than that in wild-type. These results hypothesized that sperm affects temperature signaling in ASJ through any feedback system such as secretory signaling. To investigate what molecules are reacquired for the secretory systems, we focused on hormonal signaling. Our DNA microarray analysis identified that the expression levels of 58 genes encoding nuclear hormone receptor (nhr) were significantly changed by temperature stimuli. We found that nhr-88 and nhr-114 mutants showed abnormal cold habituation. We are now using genetic epistasis analysis and calcium imaging of ASJ to investigate whether these mutations affect temperature signaling in ASJ.

Ohta, Akane et al. (2013) International Worm Meeting "Temperature experience-inducing cold tolerance is regulated by insulin signaling in intestine and neuron."

Ohta, Akane, Kobayashi, Yuko, Nakamoto, Hayato, Ujisawa, Tomoyo, Sonoda, Satoru, Kuhara, Atsushi

[International Worm Meeting, 2013]

Temperature is critical environmental stimuli and cause biochemical changes. Animals therefore can respond and habituate to the changes in ambient temperature. We are studying about molecular mechanism underlying temperature experience-dependent cold tolerance in C. elegans. 20 deg C-cultivated animals were died by cold stimuli 2 deg C, whereas 15 deg C-cultivated animals can survive at 2 deg C. Mutants defective in DAF-2/insulin receptor and its downstream molecules showed abnormal cold tolerance. DAF-2 is the sole insulin receptor in C. elegans, while there are about 40 ligands for insulin receptor. We have found that at least three insulin, DAF-28, INS-6 and INS-1, are essential for cold tolerance. Two insulin, DAF-28 and INS-6, are both positive agonists and work redundantly for DAF-2/insulin receptor in cold tolerance. Genetic epistasis analysis indicated that INS-1/insulin genetically inhibits DAF-2/insulin receptor through negative regulation of DAF-6/insulin. Abnormal cold tolerance in daf-28 mutant was strongly rescued by specific expression of daf-28 cDNA in ASJ sensoryneuron that is known as a light and pheromone-sensing neuron. By contrast, abnormality of daf-28 mutant was partially rescued by expressing daf-28 cDNA in other sensoryneurons. These suggest that DAF-28/insulin functions cell non-autonomously, but releasing DAF-28 from ASJ is essential for temperature experience-dependent cold tolerance. Unexpectedly, cell specific rescue experiments revealed that DAF-2/insulin receptor in both intestine and neuron is required for cold tolerance. These results suggest that a single sensoryneuron ASJ regulates temperature experience-dependent cold tolerance through insulin signaling in the intestine and neuron. Since abnormal cold tolerance of daf-2 mutant was suppressed by mutation in transcriptional factor FOXO/DAF-16, we are investigating downstream molecules of insulin signaling for cold tolerance by DNA microarray analysis.

Okahata, M et al. (2015) International Worm Meeting "Genetic analysis of natural variations for cold habituation in C. elegans."

Okahata, M, Toyoda, A, Kuhara, A, Ohta, A, Minakuchi, Y

[International Worm Meeting, 2015]

Temperature is one of the essential environmental stimuli for living and proliferation of organisms on the earth. We are studying about natural variation of genes involved in temperature response by utilizing cultivation temperature shift assay in cold habituation (Ujisawa et al., Protocol Exchange, 2014). Most of 25 degree-grown wild-type N2 animals isolated from Bristol can not survive at 2 degree, whereas 15 degree-grown N2 animals can survive at 2 degree. We previously reported that only three hours after cultivation temperature shift from 25 to 15 degC, the cold habituation was newly established (Ohta, Ujisawa et al., Nature commun, 2014). This phenomenon is regulated by CREB in nervous system (Ohta, Ioroi et al., this meeting). We found that various wild-type strains isolated from various areas exhibited different cold habituation phenotype. Natural variations, CB4856 isolated from Hawaii and CB4853 isolated from California, slowly habituated to new temperature. By contrast, AB1 isolated from Australia rapidly habituated to new temperature. To identify the responsible gene polymorphisms for these variations, we used next generation DNA sequencer (NGS) and SNP analysis. We are mapping these polymorphisms by analyzing the recombinants between AB1 and CB4853, or between AB1 and CB4856. So far, one of the responsible polymorphisms was mapped onto chromosome I.We also found that natural variation strain CB4854 can not survive at 2 degree after cultivation at 17 degree, while N2 can survive at 2 degree after cultivation at 17 degree. Responsible gene polymorphism have been mapped onto -1.603~1.73cM region on chromosome X, by the next generation DNA sequencer and SNP analysis.

Maruo, Ayana et al. (2017) International Worm Meeting "Single cell transcriptome analysis of ASJ thermosensory neuron regulating cold tolerance."

Takagaki, Natsune, Maruo, Ayana, Ohta, Akane, Kuhara, Atsushi

[International Worm Meeting, 2017]

ASJ sensory neuron that has been known as a light and pheromone sensing neuron senses temperature and regulates cold tolerance through insulin signaling (Ohta, Ujisawa et al., Nature commun, 2014). Cold tolerance in C. elegans is a phenomenon that 20 deg C-cultivated animals cannot survive at 2 deg C, whereas 15 deg C-cultivated animals can survive at 2 deg C. Our previous analyses suggest that cold tolerance is regulated by tissues network containing neurons, intestine and sperm, in which a feedback regulation from sperm to ASJ thermosensory neuron is essential (Sonoda, et al., Cell Reports, 2016). However, we have not known thermo-sensing molecules on ASJ and how ASJ is controlled by sperm signaling remain poorly understood. To identify new genes involved in temperature sensation and temperature tolerance, we conducted RNA-sequencing analysis comparing mRNA expression levels between ASJ and ASE gustatory neuron by using single cell RNA-sequencing analysis (Sugi & Ohtani, BBRC 2014). mRNAs were extracted from animals that were cultivated at constant temperature (15 deg C, 20 deg C, or 25 deg C) or cultivated under temperature-shifted conditions (15 deg C to 25 deg C or 25 deg C to 15 deg C). By comparing gene expression levels between ASJ and ASE in 20 deg C cultivated-animals, we found the expression levels of ~600 genes such as neuropeptides, neuropeptide receptors, insulin were altered. We measured cold tolerance phenotypes of about fifty mutants defective in those genes, and found that mutants defective in avr-15 encoding glutamate-gated chloride channel and clh-1 encoding CLC-type chloride channels showed abnormal cold tolerance.

Mizuno, S. et al. (2019) International Worm Meeting "Genetic analysis underlying positive regulation of cold tolerance."

Kuhara, A., Minakuchi, Y., Takagaki, N., Toyoda, A., Ohta, A., Mizuno, S.

[International Worm Meeting, 2019]

Animals have been evolved under dramatically altering temperature condition on the earth by adapting to environmental temperature changes. We are studying fundamental mechanisms underlying temperature response of animal by utilizing cold tolerance of nematode C. elegans, as a model. Wild-type animals can survive at 2?after cultivation at 15?, while animals cultivated at 25?can not survive at 2?. Cold tolerance is regulated by ASJ and ADL sensoryneurons, wchich sense temperature and negatively regulate cold tolerant status through insulin and hormonal signaling (Ohta et al., Nature Commun, 2014; Ujisawa et al., PNAS, 2018; Okahata et al., Science Advances, 2019). Although, the mechanism for positive regulation in cold tolerance have been remaining mostly unknown. Here we show that progresses of two studies for identification of molecules involved in positive regulation of cold tolerance. (1) We isolated novel cold tolerance mutant KHR018 that shows abnormal decrease in cold tolerance. By using deep DNA sequencer, we decoded genome DNA sequence of KHR018 mutant. Based on this information, its responsible gene was mapped on the middle of chromosome X and norrowed down to 15 genes. (2) By transcriptome analysis with cultivation temperature changes, the expression of rsbp-1gene was strongly altered under temperature stimuli. rsbp-1gene encodes RGS binding protein homologues to human RGS7BP and RGS9BP. RGS is a negative regulator of trimeric G protein signaling. We found that rsbp-1mutant showed decrease of cold tolerance. RSBP-1::GFP reporter was expressed in the head neurons, pharyngeal muscle and HSN neuron, as previously reported. We are now introducing a series of cell-specific rescue experiments to determine cell(s) responsible for abnormal cold tolerance in rsbp-1 mutant.

Iseki, Toshihiro et al. (2017) International Worm Meeting "Feedback system between sperm and temperature sensing-neuron, and isolation of novel genes in cold tolerance."

Iseki, Toshihiro, Takagaki, Natsune, Okahata, Misaki, Kuhara, Atsushi, Sonoda, Satoru, Ohta, Akane

[International Worm Meeting, 2017]

Temperature tolerance in animals is important mechanism for survival and proliferation. To investigate temperature tolerance, we are using cold tolerance. 20 deg C-cultivated animals did not survival at 2 deg C. In contrast, 15 deg C cultivated animals can survive. Previously reported, this cold tolerance is regulated by ASJ temperature sensing-neuron, which releases insulin that is received by intestine and neuron (Ohta, Ujisawa et al., Nature commun, 2014). (1) To identify genes in downstream of insulin-signaling, we performed DNA microarray analysis. We found that expressions of sperm genes were changed in insulin receptor mutants, and sperm mutants showed abnormal cold tolerance. Unexpectedly, genetic epistasis analysis suggested that abnormal cold tolerance of sperm mutant was suppressed by the mutations in ASJ temperature sensing-neuron. Calcium imaging analysis showed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4, and rescued by expressing gsp-4 gene in sperm. Thus, we propose a novel feedback between sperm and ASJ temperature sensing-neuron in cold tolerance (Sonoda et al., Cell reports, 2016). To investigate what molecules are required for the tissue network between sperm and ASJ neuron, we are analyzing genes encoding secretory signaling such as nuclear hormone receptor (nhr) and other receptors. So far, some nhr mutants showed abnormal cold tolerance. (2) To isolate more genes involved in cold tolerance. By the DNA micro array analysis in this study, the expression level of Y38H8A.3 gene was strongly changed, and Y38H8A.3(gk749) mutant showed defect in cold acclimation. However, another null mutant showed normal phenotype. We then speculated that background mutation excepting Y38H8A.3 causes this abnormality. To identify the responsible gene of abnormal cold acclimation, we decoded whole genome DNA sequence of Y38H8A.3(gk749) mutant by next generation sequencer (NGS). As a result, the candidate genes were narrowed down to only 14 genes. We are performing SNP analysis of these mutations based on whole genome DNA sequence of this strain.

Okahata, Misaki et al. (2017) International Worm Meeting "Natural variations of cold acclimation and analysis of KQT potassium channel in cold acclimation."

Kuhara, Atsushi, Ohta, Akane, Toyoda, Atsushi, Minakuchi, Yohei, Okahata, Misaki

[International Worm Meeting, 2017]

Temperature is one of the most critical environmental factors in the earth. Organisms have mechanisms to acclimate to environmental temperature changes. We performed two approaches to ravel the cold acclimation mechanisms in animals. 1. Genetic analysis of natural variants showing various cold acclimation We are studying about temperature acclimation by using C. elegans isolated from twelve various areas. 15 degree-cultivated N2 from Bristol can survive at 2 degree, while 25 degree-cultivated N2 can not survive at 2 degree. We previously reported that only 3 hours after cultivation temperature-shift from 25 to 15 degree, cold acclimation was newly established (Ohta, Ujisawa et al., Nature commun, 2014). We measured cold acclimation phenotypes in twelve natural variants. AB1 isolated from Australia can rapidly acclimate to new temperature, whereas CB4856 isolated from Hawaii can slowly acclimate to new temperature. In order to identify the responsible gene polymorphism involved in temperature acclimation, we used next generation sequencer and SNP analysis. As a results, the gene responsible for cold acclimation of AB1 was mapped between 1.1~5.1cM. 2. KQT-type potassium channel involve in cold acclimation We used DNA micro array analysis to identify new gene involved in cold acclimation. 79 genes were changed their expression level when 23 degree cultivated N2 transferred to 17 degree for four hours. We analyzed cold acclimation phenotype in 15 mutants. As a result, kqt-2 mutant defective in KQT-type potassium channel showed abnormal cold acclimation. The expression of KQT-2 was observed in ASK and ADL head sensory neurons, intestine, and vulval muscle. In order to identify the tissue where kqt-2 regulates cold acclimation, cell specific rescue experiments were performed. The abnormal cold acclimation phenotype of kqt-2 mutant was rescued by expressing kqt-2cDNA in sensoryneuron but not intestine. C. elegans has three kinds of KQT-type potassium channels. Among them, kqt-3 mutant showed abnormal cold acclimation, which was opposite phenotype to that of kqt-2.

Fujita, Mayu et al. (2017) International Worm Meeting "Isolation of genes required for cold acclimation."

Toyoda, Atsushi, Sakai, Shiori, Okahata, Misaki, Ohta, Akane, Fujita, Mayu, Kuhara, Atsushi, Minakuchi, Yohei

[International Worm Meeting, 2017]

Animals can sense and memorize environmental temperature to survive and proliferate in variety of temperature condition. In this study, we are studying about molecular mechanisms for temperature acclimation and its memory by using cold acclimation. Wild-type animals can not survive at 2 degree after cultivation at 25 degree, whereas they can survive at 2 degree after cultivation at 15 degree. After 25 degree-cultivated animals were transferred to and stayed at 15 degree for 3 hours, these animals can survive at 2 degree, as previously reported (Ohta, Ujisawa et al., Nature commun, 2014). This phenomenon is regulated by CREB that is a transcription factor in mammalian memory, suggesting that gene expression is involved in the regulation of cold acclimation or temperature memory (Fujii et al. this meeting). (1) We quantified gene expression-changes depending on temperature shift, by using RNA sequencing analysis with next generation sequencer. We collected mRNA of the animals cultivated at various temperature conditions. By the RNA sequencing analysis, expression levels of about 950 genes were strongly changed, in which mutants of about 520 genes were available in the stock centers. So far, abnormal cold acclimation was observed in hacd-1 mutant defective in HADH, 3-hydroxyacyl-CoenzymeA dehydrogenase. HADH is involved in beta-oxidation regulating fatty acid metabolism in a mitochondrial matrix. GFP reporter analysis indicated that hacd-1 was expressed in neurons and intestine. We found that abnormal cold acclimation of hacd-1 mutant was rescued by the expression of hacd-1cDNA in neurons and intestine. We are planning to determine which neuron is required for hacd-1-dependent cold acclimation. (2) We also used DNA microarray analysis on temperature shift to identify more genes involved in cold acclimation. We found that RB2549 sms-3(ok3540) showed abnormal cold acclimation. When 25 degree-cultivated animals transferred to 15 degree and stayed for 3 hours, RB2549 mutant can survive at 2 degree, while wild-type can not survive at 2 degree. However, another null mutant sms-3(tm4022) did not show abnormal phenotype. Therefore, we hypothesized that background mutation(s) excepting sms-3 mutation in RB2575 causes abnormal cold acclimation. To identify the responsible gene, we decoded whole genome DNA sequences by using next generation sequencer (NGS). So far, the candidate responsible genes were narrowed down to 28 genes. We are isolating recombinant strains, and are introducing SNP analysis to determine the responsible gene for abnormal cold acclimation of RB2575 strain.