Tetsuro Toyoda et al. (2004) East Asia Worm Meeting "C. elegans RNAi phenome database (A preview)"

Tetsuro Toyoda, Yoshiki Mochizuki, Atsushi Shimizu, Naoko Iida, Chie Hayakawa, Rika Maruyama, Naoki Okamoto, Akihiko Konagaya

[East Asia Worm Meeting, 2004]

Toyoda H et al. (2000) J Biol Chem "Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo."

Kinoshita-Toyoda A, Toyoda H, Selleck SB

[J Biol Chem, 2000]

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.

Takagaki, N. et al. (2019) International Worm Meeting "Mechanoreceptor-mediated temperature sensation in cold tolerance."

Toyoda, A., Kuhara, A., Fujiwara, Y., Ohnishi, K., Takagaki, N., Ohta, A., Minakuchi, Y.

[International Worm Meeting, 2019]

Tolerance to external temperature is essential for surviving and proliferating under continual environmental temperature changes.We are studying about molecular mechanism of cold tolerance.Wild-type animals can not survive at 2 degree after cultivation at 20-25 degree, however, 15 degree-cultivated animals can survive at 2 degree. We show here that mechanoreceptor DEG-1, Degenerin/ Epithelial Sodium Channel (DEG/ENaC) type, has a role to play as temperature receptor in cold tolerance. Xanthine dehydrogenase (XDH-1) regulates cold tolerance in AIN and AVJ interneurons, and these neuronal activities in response to temperature were abnormal in xdh-1 mutants. AIN and AVJ interneurons mainly connect with mechanoreceptor-expressing neurons containing the ASG sensory neuron, while also the genetic epistasis analysis demonstrated that mechanoreceptor deg-1regulates cold tolerance upstream of xdh-1gene. Ca2+ imaging revealed that the ASG sensory neuron showed decreased-response to temperature through unfunctional DEG-1.Furthermore, the ASE, a non-thermosensitive gustatory neuron, acquired thermosensitivity by expressing DEG-1 ectopically. In addition, two-electrode voltage-clamp recording by using Xenopus oocytes demonstrated thermal stimulus evoked internally-directed current in the oocytes expressed with DEG-1 or its human homolog MDEG, suggesting that DEG-1 and MDEG have the ability to sense temperature as temperature-sensitive channels. Altogether, DEG-1, a DEG/ENaC type mechanoreceptor acting as a temperature receptor regulates cold tolerance.

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.

Bulik DA et al. (2000) Proc Natl Acad Sci U S A "sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis."

Esko JD, Toyoda H, Robbins PW, Selleck SB, Kinoshita-Toyoda A, Bulik DA, Waldrip WR, Wei G

[Proc Natl Acad Sci U S A, 2000]

sqv (squashed vulva) genes comprise a set of eight independent loci in Caenorhabditis elegans required zygotically for the invagination of vulval epithelial cells and maternally for normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 suggested a role for the encoded proteins in glycolipid or glycoprotein biosynthesis. Using a combination of in vitro analysis of SQV enzymatic activities, sqv(+)-mediated rescue of vertebrate cell lines, and biochemical characterization of sqv mutants, we show that sqv-3, -7, and -8 all affect the biosynthesis of glycosaminoglycans and therefore compromise the function of one specific class of glycoconjugates, proteoglycans. These findings establish the importance of proteoglycans and their associated glycosaminoglycans in epithelial morphogenesis and patterning during C. elegans development.

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.

Okahata M et al. (2016) J Comp Physiol B "Natural variations of cold tolerance and temperature acclimation in Caenorhabditis elegans."

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

[J Comp Physiol B, 2016]

Temperature is critical for the survival and proliferation of animals, which must be adapted to cope with environmental temperature changes. In this study, we demonstrated natural variations in the phenotypes of temperature tolerance and temperature acclimation of the nematode Caenorhabditis elegans, and we decoded whole genome sequence of six natural variations, which enabled us to map responsible gene polymorphisms onto specific chromosomal regions. The C. elegans laboratory strain, N2, survives at 2C after cultivation at 15C but is unable to survive at 2C after cultivation at 20 or 25C. This cultivation-temperature-dependent cold tolerance occurs within a few hours after the temperature shift and is termed cold acclimation. We measured the cold tolerance and cold acclimation phenotypes of many natural variants isolated from various areas. CB4854 showed weaker cold tolerance associated with gene polymorphisms on the sex chromosome decoded by whole genome sequencing. Variable cold acclimation phenotypes were exhibited in twelvenatural isolates and the large difference was seen between CB4856 and AB1 strains. CB4856, isolated from Hawaii, acclimated slowly to a new temperature, whereas AB1, isolated from Australia, acclimated rapidly. By the whole genome sequencing analysis, two different polymorphisms responsible for the accelerated cold acclimation in AB1 were mapped to specific chromosomal regions.

Takagaki N et al. (2020) EMBO Rep "The mechanoreceptor DEG-1 regulates cold tolerance in Caenorhabditis elegans."

Kuhara A, Ohnishi K, Ohta A, Toyoda A, Minakuchi Y, Fujiwara Y, Kawanabe A, Takagaki N

[EMBO Rep, 2020]

Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH-1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor-expressing ASG. Ca²⁺ imaging revealed that ASG neurons respond to warm temperature via the mechanoreceptor DEG-1, a degenerin/epithelial Na⁺ channel (DEG/ENaC), which in turn affects downstream AIN and AVJ circuits. Ectopic expression of DEG-1 in the ASE gustatory neuron results in the acquisition of warm sensitivity, while electrophysiological analysis revealed that DEG-1 and human MDEG1 were involved in warm sensation. Taken together, these results suggest that cold tolerance is regulated by mechanoreceptor-mediated circuit calculation.

Arai R et al. (2017) Sci Rep "Reduction in chromosome mobility accompanies nuclear organization during early embryogenesis in Caenorhabditis elegans."

Arai R, Sato Y, Nabeshima K, Kimura H, Kimura A, Minakuchi Y, Sugawara T, Toyoda A

[Sci Rep, 2017]

In differentiated cells, chromosomes are packed inside the cell nucleus in an organised fashion. In contrast, little is known about how chromosomes are packed in undifferentiated cells and how nuclear organization changes during development. To assess changes in nuclear organization during the earliest stages of development, we quantified the mobility of a pair of homologous chromosomal loci in the interphase nuclei of Caenorhabditis elegans embryos. The distribution of distances between homologous loci was consistent with a random distribution up to the 8-cell stage but not at later stages. The mobility of the loci was significantly reduced from the 2-cell to the 48-cell stage. Nuclear foci corresponding to epigenetic marks as well as heterochromatin and the nucleolus also appeared around the 8-cell stage. We propose that the earliest global transformation in nuclear organization occurs at the 8-cell stage during C. elegans embryogenesis.

Berninsone P et al. (2000) East Coast Worm Meeting "SQV-7, a protein involved in vulval invagination and embryonic development, transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose"

Hwang H-Y, Horvitz R, Berninsone P, Hirschberg C

[East Coast Worm Meeting, 2000]

Mutations in eight sqv (squashed vulva) genes (sqv-1 to 8) cause defects in vulval invagination and early embryonic development (1). SQV-1 and SQV-4 are similar to enzymes involved in nucleotide sugar metabolism (2), while SQV-3 and SQV-8 are similar to glycosyltransferases (3). SQV-7 is a multi-transmembrane protein resembling Golgi membrane nucleotide sugar transporters (4); these proteins specifically translocate nucleotide sugars from the cytosol into the lumen of the endoplasmic reticulum and Golgi apparatus where they are used as sugar donors by glycosyltransferases (4). Translocation is essential for glycosylation: yeast, protozoa and mammalian cell mutants defective in this function have a severe deficiency of the corresponding sugar in their glycoconjugates (4). Because GDP-mannose and UDP-glucose are the only nucleotide sugars transported into S.cerevisiae Golgi and ER vesicles in vivo and in vitro (5), this organism was used for expression of SQV-7 to determine its substrate specificity. We found that SQV-7 transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose in vitro, the first protein known to transport the former two nucleotide sugars. All three nucleotide derivatives are competitive, alternate, non-cooperative substrates, which suggests there is a single active site for all three substrates on SQV-7. Expression of the two mutant sqv-7 missense alleles results in significant reduction of the three transport activities. We hypothetize that in these mutants the biosynthesis of chondroitin, the major glycosaminoglycan in C. elegans (6), is more likely to be impaired than that of heparan sulfate since chondroitin polymerization requires both UDP-glucuronic acid and UDP-N-acetylgalactosamine while heparan sulfate polymerization does not require UDP-N-acetylgalactosamine. (1)Herman T. et al (1999) PNAS 96:968 (2)Hwang H. et al.(2000) East Coast C.elegans Meeting (3)Herman T. and Horvitz H. R. (1999) PNAS 96: 974 (4)Hirschberg C. et al (1998) Annu. Rev. Biochem.67: 49 (5)Berninsone P. et al (1997) J. Biol. Chem. 272: 12616 (6)Toyoda H. et al (2000) J. Biol. Chem. 275: 2269