Shuichi Onami et al. (2004) East Asia Worm Meeting "Quantitative cell division pattern analysis on RNAi embryos"

Yoko Nagai, Mitsuru Urai, Shuichi Onami, Eriko Masuda, Shugo Hamahashi, Yoko Suzuki

[East Asia Worm Meeting, 2004]

Aiming to understand roles and mechanisms of temporal and spatial dynamics of cellular structure during embryogenesis, we are analyzing four-dimensional (three-dimensions plus time) cell division pattern of RNAi embryos using image processing-based measurement system. L3-L4 stage worms were placed onto plates containing seeded bacteria expressing dsRNA (provided from J. Ahringer through UK MRC HGMP Resource Centre) and were incubated for 40-48 h at 22C. The worm was bisected and an embryo was obtained just after the sperm entry. Development of the embryo was recorded at 22C by 4D DIC microscope system with 66 different focal planes at a distance of 0.5 (mu, Greek)m between two focal planes, at intervals of 40 sec for 2 h. The recorded images were processed by our measurement system (Onami et al. 2001), which detects regions corresponding to nucleus in each image using image processing-based algorithm, groups regions that represent the same nucleus at each time point and connects those groups that represent the same nucleus in adjacent time points. The system outputs cell division pattern that includes 3D positions of nucleus at each time point and their lineage from 1 cell stage to 24 cell stage. All 129 genes on chromosome I whose RNAi phenotypes were reported as 100% embryonic lethal (Fraser et al. 2000) were analyzed. Four features of division pattern, i.e., life time of cell, symmetry of cell cycle, distance between cells and angle made up of 3 cells, were mathematically defined and evaluated for each RNAi embryo based on the measured cell division pattern. Clustering analysis was applied to those features and several genes whose RNAi exerts an interesting cell division pattern were identified; the interesting patterns include normal division pattern up to 24 cell but resulting in embryonic lethal, normal cell division timing but abnormal cell arrangement and slow cell division timing but normal cell arrangement. We are currently analyzing 100% embryonic lethal genes on chromosome III. Detailed analysis of cell division pattern will be presented.

Koji Kyoda et al. (2005) International Worm Meeting "Computational analysis of cell division patterns during early embryogenesis"

Taeko Oguro, Eriko Masuda, Shugo Hamahashi, Yoko Nagai, Koji Kyoda, Mitsuru Urai, Shuichi Onami

[International Worm Meeting, 2005]

To understand the mechanisms of spatiotemporal cell positioning during embryogenesis, we are measuring and analyzing quantitative cell division patterns of wild-type and RNAi-treated embryos. Early development for 85 wild-type embryos and 604 RNAi-treated embryos (245 genes on the chromosome I and III) whose phenotypes were reported as 100% embryonic lethal (Fraser et al. 2000; Kamath et al. 2003) was recorded by 4D DIC microscope system with 66 focal planes at a distance of 0.5m between adjacent planes, at intervals of 40 seconds for 2 hours. The recorded images were processed by our cell lineage acquisition system (Onami et al. 2001), which detects nuclear regions in each image by image-processing, and then groups regions that correspond to the same nucleus at each time point and connects those groups that correspond to the same nucleus in adjacent time points by using tracking algorithm. Produced cell division pattern consists of 3D positions of nucleus at each time point and their connections from 1-cell stage up to 24-cell stage. To explore function of genes during early embryogenesis, we are developing bioinformatics methods for analyzing these quantitative cell division pattern data. To facilitate the analysis, we developed systematic method to quantify various features of cell division pattern, such as timing of cell division, direction of cell division, relative position between nuclei and cell movement. These quantified features were analyzed by using multivariate analysis methods, such as clustering and pair-wise correlations methods. We also developed methods for finding RNAi phenotypes on various quantified features and those for investigating properties of each quantified feature, such as normality of cell cycle duration. Quantitative analyses of cell division patterns using these methods are in progress.

Park, Seungmee et al. (2021) International Worm Meeting "DIP-2 and SAX-2 play synergistic roles to maintain C. elegans neuronal morphology"

Park, Seungmee, Chisholm, Andrew, Jin, Yishi, Colavita, Antonio

[International Worm Meeting, 2021]

Many C. elegans neurons project monopolar or bipolar neurites. However, it is unknown how the stereotypical morphology established during development is maintained throughout life. Several factors have been found to regulate the morphology of various types of neurons. Among these, DIP-2 (Disco-interacting-protein 2) (Noblett et al 2018) and SAX-2 (sensory axon guidance defect) (Zallen et al 1999) are critical for the maintenance of neuronal morphology. DIP-2 represses ectopic neurite outgrowth from neuronal soma, and suppresses injury-induced axon regrowth. SAX-2 also represses ectopic neurite outgrowth. Both proteins are expressed in the nervous system, and act cell-autonomously to repress ectopic neurites in neurons. DIP-2 is a member of a conserved family of proteins implicated in acyl-CoA metabolism. Our genetic studies between dip-2 and the Kennedy phospholipid synthetic pathway show that DIP-2 acts downstream of the phospholipid pathway. SAX-2 is a member of the Fry/Furry family of proteins implicated in cytoskeletal dynamics (Nagai et al., 2013). By knock-in of fluorescent protein tag to endogenous locus, we show that DIP-2 displays diffuse cytosolic expression in neurons, whereas SAX-2 localizes to cytoplasmic puncta whose identity is currently being investigated. To understand potential interactions between these pathways influencing neuronal morphology, we have analyzed dip-2 and sax-2 double mutants and found that these animals display synergistic defects in neuronal morphology, as well as synergistic developmental and reproductive defects. Exploiting these synergistic defects, we conducted genetic screens and isolated multiple suppressors of the phenotypes of dip-2, sax-2, or both. We are currently mapping these suppressors to better understand how the DIP-2 and SAX-2 pathways act to maintain neuronal morphology.

Yoko Honda et al. (2007) International Worm Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment (ICE)-First."

Yoko Honda, Masashi Tanaka, Noriaki Ishioka, Akira Higashibata, Shuji Honda

[International Worm Meeting, 2007]

. Yoko Honda1, Akira Higashibata2, Noriaki Ishioka2, Masashi Tanaka1, Shuji Honda1. 1) Genomics for Longevity & Health, Tokyo Metropolitan Institute of Gerontology(TMIG), Tokyo, Japan; 2) Japan Aerospace Exploration Agency(JAXA), Tsukuba, Japan. As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International Caenorhabditis elegans Experiment (ICE)-First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan and powerful genetic system. Recently, Morley et al. reported that Huntington''s-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants (2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. We also examined the effects of space flight on the gene expression by DNA microarray analysis and real time PCR. The expression of more than ten genes specifically for nervous systems was suppressed in space-flown animals, suggesting inactivation of nervous functions in space. Next, effects of inactivation of these genes on lifespan were tested by RNAi in the laboratory. 1) Honda S et al.Oxygen-dependent perturbation of life span and aging rate in the nematode. J Gerontol. 48:B57-61. 1993 2) James F et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002.