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.

Shuichi Onami et al. (2002) Japanese Worm Meeting "Cell Lineage Analysis using Automatic Cell Lineage Extraction System"

Eriko Masuda, Shinichi Shibata, Yoshiko Kitamura, Shuichi Onami, Shugo Hamahashi

[Japanese Worm Meeting, 2002]

We developed a system that automatically acquires cell lineage from the 1-cell stage up to the 24-cell stage. The system utilizes a set of 4D Nomarski DIC microscope images of embryo consisting of 55 focal plane images at each 45 seconds. An image-processing algorithm detects regions of cell nucleus in each image, and tracking these regions creates the cell lineage. The cell lineage consists of the three-dimensional positions of nuclei at each time point and their lineage. In order to start systematic cell lineage analysis, we accelerated the system by transplanting to a newly assembled PC cluster machine (48 x Pentium4 - 2.0GHz), and automating the file reformatting processes, which needed complicated human intervention in the previous system. The total execution time was shortened from 9.5 hours to 5.5 hours. As the first step of systematic cell lineage analysis, 4D microscope image sets of 30 wildtypes and 15 par-1 mutants were recorded. Cell lineage studies of these individuals and other strains will be presented.

Shuichi Onami et al. (2001) International C. elegans Meeting "Automatic cell lineage acquisition system and analysis of early embryogenesis"

Satoru Miyano, Shuichi Onami, Shugo Hamahashi, Hiroaki Kitano, Masao Nagasaki

[International C. elegans Meeting, 2001]

We have developed a system that automatically acquires cell lineages of C. elegans from the 1-cell stage up to approximately the 25-cell stage. The system utilizes a set of 4D Nomarski DIC microscope images of C. elegans embryo consisting of more than 50 focal plane images at each minute for about 2 hours. An image-processing algorithm, utilizing the image entropy, detects the region of cell nucleus in each image, and 3D nucleus regions, each of which is a complete set of nucleus regions that represent the same nucleus at the same time point, are made. Each pair of 3D nucleus regions is then connected, if they represent the same nucleus and their time points are consecutive, and the cell lineage is created based on these connections. The resulting cell lineage consists of the three-dimensional positions of nuclei at each time point and their lineage. The system utilizes our Beowulf PC cluster, made up of 32 PC, to execute all the above processes and can deduce the cell lineage within 9 hours. We also developed a software package that three-dimensionally visualizes the resulting lineage data, which may help three-dimensional understanding of nucleus movement and division. Moreover, with this package, lineages of two different individuals - e.g. wild-type and mutant - can be visualized on the same screen. The cell lineages of 10 individual N2 worms were deduced, which were quite similar to each other and to the Sulston's cell lineage. We are establishing a standard wild-type cell lineage, which describes the mean value of nucleus position at each time point together with some statistical data, such as the variance, error distribution, etc. The lineages of par-1 (b274) mutants were analyzed and the difference from wild-type was recognized as reported previously. Encouraged by the performance of our system, we have started systematic cell lineage analysis of knock-out animals. Studies of nucleus movement will also be presented.

Shuichi Onami et al. (2003) International Worm Meeting "Analysis of early embryonic cell lineage of RNAi-treated embryos using automatic cell lineage acquisition system"

Yoko Suzuki, Eriko Masuda, Shinichi Yamaguchi, Shugo Hamahashi, Shuichi Onami, Mitsuru Urai

[International Worm Meeting, 2003]

Aiming to understand the roles and the mechanisms of temporal and spatial dynamics of cellular structure during embryogenesis, we are analyzing early embryonic cell lineages of RNAi-treated embryos.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. Then, 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 56 different focal planes at a distance of 0.5 m between two focal planes, at intervals of 40 sec for 2 h. The recorded images were processed by our automatic cell lineage acquisition system (Onami et al. 2001; Hamahashi et al. this meeting), which detects nucleus regions in each image, groups regions that represent the same nucleus at each time point, connects those groups that represent the same nucleus in adjacent time points and outputs cell lineage that consists of 3D positions of nucleus at each time point and their lineage from 1 cell stage to 24 cell stage.First, cell lineage was measured for 20 individual wild-type worms. The shape of the resulted lineage was similar to that of Sulston et al.'s. Variation of the lineage among different embryos was rather small, e.g. the standard deviation for cell-cycle length of each cell was less than 6 min (less than 2.5 min in most cases). Thus, we established a standard wild-type cell lineage that consists of the mean and the SD for each cell-cycle length and direction of each cell division.Then, cell lineage of RNAi-treated embryos was measured. Genes on the chromosome I whose RNAi phenotypes were reported as 100% embryonic lethal (Fraser et al. 2000) were chosen as targets. All 18 genes with 3-letter gene name (dad-1, dhc-1, eft-2, gsk-1, hlh-2, hmp-1, lam-1, mei-1, mei-2, mel-26, mex-1, rba-1, par-6, pfn-1, pop-1, rba-2, tba-1 and tba-2) and 15 out of 229 hypothetical genes have been examined. Unexpectedly, more than 25% (5/18) genes did not show 100% embryonic lethality in our experiments. Cell lineage measurement for the remaining genes is in progress. Detailed analysis of measured cell lineages will be presented.

Fujita, Masashi et al. (2009) International Worm Meeting "Quantitative measurements and computer simulations of C. elegans embryonic cell morphology."

Fujita, Masashi, Onami, Shuichi

[International Worm Meeting, 2009]

Cells in a developing embryo have various shapes, which imply the diversity of their internal mechanics and external microenvironments. Therefore, cataloging cell shapes and providing mechanical explanations to them will significantly deepen our insight into animal development. Since quantitative description of cell morphology is a prerequisite for such study, we developed a method for imaging and digitizing embryonic cell shapes. GFP-tagged plasma membrane is recorded using 4D microscopy and processed through semi-automated image analysis. Each cell is color-coded and transformed into a polyhedral model, which is readily applicable to computational analyses. An index of shape skewness was computed for each cell, which captured eccentricity of elongated EMS in the four-cell stage. Interestingly, this analysis also showed that EMS becomes spherical again in the six-cell stage. The quantitative description of embryo allows us comparison with theoretical predictions by physical models. As the first step, we focused on shape of cell boundary in the two-cell stage embryo. In the case of two bound soap bubbles, the smaller bubble always bulges toward the larger one because of its higher internal pressure. However, embryos do not behave in that way. The boundary between the AB and P1 cells is flat just after the first cleavage, then larger AB bulges toward smaller P1. Theoretically, this can be explained by a gradual increase in cortical tension of AB. Since the major source of cortical tension is considered to be contractile activity of actomyosin, the role of actin filaments was examined using cytoskeletal inhibitors. Treatments with cytochalasin D or latrunculin A showed that actin filaments affect boundary shape but not the sole determinant of it. Other factors are to be explored by further studies.

Fujita, Masashi et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Surface tension is the primary determinant of cell shape in the two-cell stage embryo"

Fujita, Masashi, Onami, Shuichi

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The shape of cells in tissues and embryos bears a striking resemblance to soap bubbles. Since surface tension governs the shape of soap bubbles, it has also been considered as the major determinant of the cell shape. However, evidence of this hypothesissurface tension modelbeyond the superficial similarity is still scarce. The two-cell stage embryo, which might be the simplest cell aggregate, would serve as an ideal system for a rigorous test of the model. In that stage, the larger blastomere (AB) always bulges toward the smaller one (P 1 ). This is intriguing because soap bubbles behave in the opposite way. We found that the surface tension model is not only able to explain this phenomenon but also consistent with its molecular and physical properties. Cytoskeletal inhibitors and a ts mutant showed importan ce of actomyosin on the bulge formation. NMY-2::GFP showed cortical localization and was significantly more abundant on the cortex of AB than that of P 1 . A UV-irradiation experiment confirmed the bulges dependency on AB. These results indicate that the driving force of the bulge formation is a stronger cortical contractility of AB. Computer simulation showed that higher surface tension of AB can form a bulge. Moreover, the simulated shape had close resemblance to a real embryo. Essential predictions of the model were experimentally tested. It predicts that the immediate cause of the bulge is higher pressure inside AB. This prediction was verified by cytoplasmic stream after laser fusion of the cells. Another important prediction is shrinkage of the surface area of AB, which reflects the cortical contractility. This was also confirmed by quantification of the cell geometry. These evidences strongly support the idea that surface tension is the primary factor that determ ines the cell shape of embryo.

Onoue, Shiori et al. (2021) International Worm Meeting "Individual behavioral differences in C. elegans"

Onoue, Shiori, Onami, Shuichi

[International Worm Meeting, 2021]

C. elegans is attracted or repelled by many odorants. The strength of the response varies depending on the odorant and its concentration. However, the response is not 100% even for a strong odorant. For example, isoamyl alcohol, which is one of the strongest attractants for C. elegans, attracts more than 90% of the worms but does not about the remaining 10% in the typical experimental settings. Besides, individual worms behave differently even when they have the same genetic background. It remains unknown in detail how such individual differences in behaviors are created. This study aims to elucidate the unknown mechanisms that make individual behavioral differences in a "homogeneous" group. To quantitatively analyze individual worms' behavior, we developed a system that records worms' behaviors in a 6 cm plate every 0.04 s by using a digital 4K single-lens reflex camera and quantitates the behaviors by using DeepLabCut, a deep learning-based animal tracking software tool. As a first trial, we examined chemotaxis behaviors for isoamyl alcohol by using this system. Our preliminary result showed that the majority of worms (~95%) migrated to the attractant, but a few of them (<5%) were not attracted and continued to move over a wide area in the plate. We plan to investigate the mechanisms that make such individual behavioral differences by combining data-driven and experimental analyses.

Itoga, Hiroya et al. (2021) International Worm Meeting "Worm Developmental Dynamics Database 2 - an open database with visualization for biological dynamics of large-scale RNAi experiments on C. elegans embryos"

Itoga, Hiroya, Onami, Shuichi, Kyoda, Koji

[International Worm Meeting, 2021]

Collections of images and quantitative, numerical information about morphological dynamics support understanding the molecular mechanisms of development. Recently we conducted RNAi experiments targeting all 351 essential embryonic genes reported in the previous genome-wide RNAi screening for C. elegans. We recorded three-dimensional time-lapse, i.e., 4D differential interference contrast (DIC) microscopy images of developing embryos at 66 consecutive focal planes spaced at 0.5 um for 2 hours at 20 seconds intervals. We obtained a collection that includes 33 sets of quantitative data, such as 3D coordinate values of nuclear regions and their dynamics from 1 to 8-cell stage, for wild-type embryos and 1,142 sets of quantitative data for RNAi embryos corresponding to 263 essential embryonic genes. We detected over 26,000 phenotypic alterations for 421 phenotypic characters, such as cell division timing and division axis orientation from the collection (Kyoda et al., bioRxiv 2020). To make the collection openly available, we have developed Worm Developmental Dynamics Database 2 (WDDD2; https://wddd.riken.jp/), as the successor of Worm Developmental Dynamics Database (WDDD; http://so.qbic.riken.jp/wddd/), with a completely new implementation using modern web frameworks such as Django and Three.js. WDDD consists of images and quantitative data of 50 sets of wild-type embryos and 136 sets of RNAi embryos corresponding to 72 of the 97 essential embryonic genes on chromosome III, with sparser time intervals, but WDDD2 needs to handle ten times more or over. The database shows nuclear division dynamics information interactively as a combination of 4D DIC images and 4D visualization of quantitative data. The system displays the region of the nuclei and the center of gravity of them as wireframes in 3D space, and the user can select to view the data at a specific time or all time points simultaneously. The related cell images are displayed next to the wireframe. The image and wireframe's viewpoint can be manipulated synchronized but freely by pointing device movements to compare them to each other. The 4D DIC images in the original microscopy manufacturer's format are openly available by download from the database. The quantitative data are also openly downloadable in the unified format for biological dynamics, BDML/BD5 (Kyoda et al., 2015; 2020). We plan to develop Web APIs in the future to use images, quantitative data, and phenotypic characters for analysis without download.

Takayama, Jun et al. (2021) MicroPubl Biol

Tajima, Tatsuya, Takayama, Jun, Onami, Shuichi, Nishimura, Hitoshi

[MicroPubl Biol, 2021]

Gamete fusion is a pivotal step during fertilization to create an organism of the next generation. In C. elegans, since oocytes have no thick egg coat like the zona pellucida, perhaps spermatozoa directly bind to and fuse with the oocyte plasma membrane (PM). Thus, C. elegans is an excellent model to investigate how a spermatozoon and an oocyte fuse together.

Tohsato, Yukako et al. (2015) International Worm Meeting "Resource for computational phenotype screening on nuclear division dynamics in C. elegans RNAi embryos, extracted from Phenobank."

Kyoda, Koji, Onami, Shuichi, Tohsato, Yukako, Okada, Hatsumi

[International Worm Meeting, 2015]

Along with the recent popularization of live-imaging technologies, various types of microscopy images are becoming available in public databases. Quantitative data such as positions and shapes of nuclei and cells, and their temporal changes obtained from these images include biomechanical information that is important for understanding biological phenomena as dynamical systems. Such quantitative data can be used in various kinds of computational analysis including phenotype analysis and mathematical modeling. Here, we established a new resource of quantitative data on nuclear division dynamics in C. elegans. Quantitative data for RNAi embryos were extracted from two-dimensional time-lapse DIC microscopy images in the Phenobank using our newly developed automated image processing system. Our resource consists of 1,330 sets of quantitative data for RNAi embryos. These datasets include three sets for each of 439 genes, which correspond to approximately 90% of all embryonic lethal genes. Each data contains coordinate values of the outlines of nuclear regions and their dynamics. All data were verified through manual error correction, and annotated with cell name and anterior-posterior axis. To demonstrate the value of our resource, we carried out a computational phenotype analysis on the female pronuclear migration in RNAi embryos. In wild-type embryos, the female pronucleus moves slowly at first and then accelerates just before meeting with the male pronucleus. The maximum migration speed (MMS) of the female pronucleus was calculated based on the moving distance of the centroid of nuclear region along the anterior-posterior axis. We found that four genes reproducibly exhibited faster MMS than in wild-type embryos. Ten genes reproducibly exhibited remarkably slower MMS. Our independent RNAi experiments confirmed that one of these four genes, sds-22, exhibited significantly faster MMS, and six of those ten genes, ima-3, cap-1, F44B9.8, rsa-1, cye-1, and spd-5, exhibited significantly slower MMS. All the quantitative data will be available in BDML format from SSBD database (http://ssbd.qbic.riken.jp).