Sonoda, Satoru et al. (2013) International Worm Meeting "Isolating genes for temperature experience-dependent cold tolerance."

Uehara, Yushuke, Kinoshita, Yukari, Ohta, Akane, Endo, Mikiko, Kuhara, Atsushi, Sonoda, Satoru, Furukawa, Shoko

[International Worm Meeting, 2013]

We are utilizing temperature experience-dependent cold tolerance as a model for studying temperature sensation and habituation. After cultivation at 20 deg C, wild-type were destroyed by cold stimuli. In contrast, after cultivation at 15 deg C, most of animals can survive. To isolate genes involved in the cold tolerance, we are using four approaches, (1) DNA microarray, (2) Natural variation, (3) Artificial evolution and (4) EMS-mutagenesis. (1) We tested cold tolerance of mutants defective in genes isolated from DNA microarray analysis (Ajilent array). Several genes such as protein protease PP1 and laminin are involved in cold tolerance. Since PP1/GSP-3 is involved in sperm development and motility, and mutants impairing sperm genes showed abnormal cold tolerance. We are now investigating the relationship between sperm genes for cold tolerance and known-cold tolerance signaling pathway, such as insulin signaling and G protein-coupled temperature signaling in ASJ neuron. (2) Natural C. elegans isolated from various area showed variety of cold tolerance. Responsible gene for natural variation between Bristol N2 and California CB4854 are genetically mapped on X-chromosome. Using deep DNA sequencer and SNP analysis, candidates of responsible genes are narrowed down to ~20 genes. (3) C. elegans has strong advantage for artificial evolution analysis, since life cycle is short and strains can be preserved at -80 deg C. We are maintaining wild-type at 15 or 23 deg C for gradual accumulation of mutations. So far, 87 generations are frozen, and we found that cold tolerance was notably changed at 61 generation. We are planning to decode whole genome by using deep DNA sequencer. (4) Through 2000 genomes screen by using EMS, we isolated 10 cold tolerance mutations. One of these has been mapped on X-chromosome.

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.

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.

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.

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.

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.

Tania Del Rio-Albrechtsen et al. (2005) International Worm Meeting "Two novel dominant alleles of lin-41 cause retarded male tail morphogenesis."

Shu-Yi Chiou, David H A Fitch, Tania Del Rio-Albrechtsen, Karin Kiontke

[International Worm Meeting, 2005]

The C. elegans male tail tip is an excellent model to study the mechanisms that govern morphogenesis, in which cells change shape in a well-timed and coordinated process. The tail tip is pointed in larvae, and constructed of four hypodermal cells. In males, these cells coordinately fuse, become round and retract during L4, generating the rounded, "peloderan" tail of the adult. Several mutations disrupt this process such that tail tip morphogenesis is delayed or fails to occur. We have discovered that two different mutations in a complementation group previously called lep-1 are novel semidominant alleles of the heterochronic gene, lin-41. These alleles, bx37 and bx42, cause retarded development in the male tail. Specifically, the male tail tip in these mutants does not retract, and the pointed shape of the larval tail tip is retained in the adult, a phenotype called "leptoderan" or Lep. These data suggest a role for lin-41 in the temporal regulation of male tail tip morphogenesis. LIN-41 regulates the temporal fates of hypodermal cells by inhibiting the translation of mRNAs required for adult fate (Slack et al., 2000, Mol. Cell 5:659-669). LIN-41 has two major domains: the NHL domain, possibly required for blocking translation of target mRNAs (Sonoda and Wharton, 2001, Genes Dev. 15:762-773), and an RBCC domain (RING-finger, B-box, and coiled-coil). Null or reduced-function alleles of lin-41 cause precocious transformations of hypodermal seam cell fates, producing adult fates (alae) at the third larval molt (Slack et al., 2000). The retarded Lep phenotype of bx37 and bx42 is consistent with LIN-41 acting as a developmental switch with the potential to cause both precocious and retarded development. Both Lep alleles of lin-41 are semidominant, highly penetrant, and temperature sensitive. Unlike the reduced-function mutations of lin-41 that cause precocious defects, bx37 and bx42 do not map in the NHL domain. The more copies there are of either allele, the more retarded is the phenotype; the Lep phenotype is not due to haploinsufficiency (deficiency heterozygotes are wild type). We present a model for the regulation of LIN-41 in male tail tip development. According to our molecular phylogeny (see abstract by Kiontke et al.), several changes between leptoderan and peloderan tails have occurred during the evolution of species related to Caenorhabditis. We speculate that changes in the heterochronic pathway, perhaps in lin-41, could be involved in the evolution of male tail tip diversity.