Green, Rebecca A. et al. (2013) International Worm Meeting "The midbody ring, not the midbody microtubules, dictates abscission patterning in vivo."

Desai, Arshad, Audhya, Anjon, Green, Rebecca A., Lewellyn, Lindsay, Oegema, Karen, Mayers, Jonathan

[International Worm Meeting, 2013]

During cytokinesis, the cell must transition from constriction phase into abscission phase, by patterning the abscission components at the appropriate spatiotemporal location. Two structural features within the intercellular bridge are poised to serve as guidance cues to promote this process - the midbody microtubules and the midbody ring. It has been widely anticipated that the midbody microtubules are required-since key abscission determinants localize to this structure. In this study, we find that the midbody microtubules are dispensable for abscission in vivo and provide the first detailed analysis of the steps that occure during abscission phase in an intact organism. We define three temporally distinct and assayable abscission steps in vivo in the C. elegans embryo: cytoplasmic isolation, membrane shedding onset and midbody/midbody ring release; ESCRT assemblies are required only for the final step. We find that midbody microtubules are not required for any step, and that key abscission determinants are recruited to the abscission site in the absence of midbody microtubules. Furthermore, we show that the midbody ring component, the septins, is important for cytoplasmic isolation and is essential for midbody release. These results suggest that the midbody ring orchestrates the constriction to abscission phase transition in vivo.

Green, Rebecca A. et al. (2009) International Worm Meeting "A High-Content Screen to Functionally Classify Proteins Required for Cell Viability and Division."

Desai, Arshad, Audhya, Anjon, Green, Rebecca A., Oegema, Karen

[International Worm Meeting, 2009]

Systems biology analyzes the role of molecular assemblies within functional networks. The success of this approach relies upon the availability of systematic and functional information about the components of these molecular assemblies. RNA-mediated interference (RNAi) is currently the method of choice for linking genes to their cellular functions. In addition to relatively ''low content'' single reporter assays, RNAi has been used to perform high-content microscopy-based assays, integrating spatial and/or temporal information. This approach has been used to characterize the set of genes required for cell viability and division in C. elegans. Of the 20,000 genes, ~10% are required for embryo production or viability; these 2000 genes were previously screened by filming the first two embryonic divisions following individual RNAi depletion. This screen provided high quality data that allowed for the functional classification of ~400 genes, however it did not provide high quality functional information on the ~560 genes whose inhibition blocks embryo production (the sterile collection). Here, we functionally characterize the sterile collection by performing a second high-content screen. We use two-color fluorescence confocal microscopy to examine gonad structure in anesthetized worms after individual depletion of each of the 560 sterile gene products. The gonad-morphology data was analyzed by binary scoring for 94 potential defects. A clustering algorithm was used to group genes with similar phenotypic profiles, and phenotypes within the major clusters were then re-examined by eye for accuracy and identification of subclasses within each broad phenotypic group. Our live-imaging data, annotation and analysis will be integrated with the data from the prior DIC imaging based screen of the embryonic lethal collection in an online database that will be made available upon publication. Our analysis placed genes into ~20 broad classes that could be partitioned into ~100 different phenotypic sub-classes, which typically corresponded to the subunits of a specific protein complex. In addition to characterizing the 390 genes in the sterile collection, for which there was some prior functional information, our screen has placed the ~50 unknown and 120 previously uncharacterized genes in the sterile collection into functional groups. Cumulatively, our data doubles the number of genes for which we have high quality systematic functional information and provides an important platform for systems biology based analysis of the pathways contributing to embryo production and development.

Green, Rebecca et al. (2019) International Worm Meeting "4D High Content Imaging and Automated Phenotypic Profiling of C. elegans Embryogenesis."

Green, Rebecca, Ochoa, Stacy, Chow, Tiffany, Hendel, Jeff, Zhao, Zhiling, Khaliullin, Renat, Desai, Arshad, Oegema, Karen, Wang, Shaohe

[International Worm Meeting, 2019]

An important challenge is to functionally classify the ~2000 genes (>1400 conserved) that control cell-fate specification and morphogenesis during embryogenesis. Here, we perform a 4D high-content screen by filming embryogenesis using two custom-engineered C. elegans reporter strains, following individual RNAi-based knockdown (>20,000 individual movies). We monitor (1) changes in cell fate specification, by dynamically tracking fluorescently labeled endoderm, mesoderm and ectoderm nuclei, and (2) morphogenic changes during epithelial and neuronal development by monitoring tissue position and tissue shape. Consistent and timely analysis of 20,000 movies requires automation, however, the range and complexity of 4D developmental phenotypes are not easily captured by existing automated methods. To address this challenge, we manually curated a pilot set of 500 genes (>7000 movies) and used this reference to guide the development of custom automated analysis algorithms; this effort ensured that our final automated analysis method captured observed phenotypes across a spectrum of developmental defects. For each RNAi condition, our automated analysis yields phenotypic signatures consisting of >100 continuous parameters. To evaluate the phenotypic similarity between RNAi conditions, we measure the distance between phenotypes in continuous space. To correct for the fact that a strict measure of Euclidean distance penalizes genes with more severe phenotypes, we measure the angle between the average phenotypes for the two conditions (phenotypic angle of deviation; PAD). Finally, we optimized the set of parameters used for automated comparison by assessing performance of the algorithm on a manually-annotated set of phenotypic groups. Our resulting automated method effectively identifies genes whose knockdown leads to similar phenotypes; this allows partitioning of genes into functional groups that are predicted to reflect developmental pathways and will yield a systems-level view of embryonic development. This work represents the first fully automated high-content screen of an intact developing organism and is the most complex morphological profiling effort to date.

Green, Rebecca A. et al. (2011) International Worm Meeting "A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a ComplexTissue."

Schedl, Tim, Audhya, Anjon, Niessen, Sherry, Desai, Arshad, Swathi, Arur, Laband, Kimberley, Piano, Fabio, Mayers, Jonathan, Green, Rebecca A., Wang, Shaohe, Fridolfsson, Heidi, Gunsalus, Kristin, Schulman, Monty, Oegema, Karen, Kao, Huey-Ling, Starr, Daniel, Schloissnig, Siegfried, Hyman, Anthony

[International Worm Meeting, 2011]

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach- profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for 106 uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes- pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Green, Rebecca et al. (2015) International Worm Meeting "­Decoding Embryonic Developmental Pathways Using 4D-High Content Imaging of C. elegans embryos."

Desai, Arshad, Biggs, Ronald, Oegema, Karen, Zhao, Zhiling, Wang, Shaohe, Ochoa, Stacy, Green, Rebecca, Gerson-Gurwitz, Adina, Khaliullin, Renat

[International Worm Meeting, 2015]

­Embryogenesis is a complex process requiring coordination of cell division, signaling, migration, differentiation, and death. Systematically defining the genetic pathways that drive these morphogenetic events during embryogenesis is an important current challenge. Our goal is to construct a comprehensive functional network map of essential developmental genes for the model metazoan, C. elegans. To this end, we have developed a 4D-high-content screening based approach to functionally classify ~2600 developmental genes, using two-specifically engineered marker strains that readout defects in (1) germ layer specification and positioning and (2) cell shape changes and cell migration during morphogenesis. Following RNAi of targets, we image C. elegans embryos throughout the developmental time course (~10hrs) using a CV1000 spinning disk confocal high-content imaging system, which enables collection of developmental data for 50-100 embryos in a single experiment. To date, we have completed a pilot set of >500 genes. Among these, we have recovered expected phenotypes for well described developmental genes as well as severe developmental phenotypes for many uncharacterized genes, validating our overall experimental approach. This pilot data set is being used to develop custom data management algorithms (cropping, orienting, and indexing embryos) and data analysis protocols, including: manual and automated scoring of phenotypic features (Imaris and custom). Using this approach, each individual embryogenesis movie is scored and genes are clustered according to phenotypic profiles. When complete, this will be the first systems-level view of embryonic development in a complex multicellular organism. We anticipate such an effort will translate to higher organisms and help reveal the genetic basis for congenital defects, such as neural tube, craniofacial, and ventral body wall closure abnormalities.

Rebecca A Green et al. (2007) International Worm Meeting "Screening the C. elegans sterile collection to identify novel cytokinesis genes."

Rebecca A Green, Arshad Desai, Karen Oegema, Anjon Audhya

[International Worm Meeting, 2007]

The final stage of cell division is called cytokinesis, in which a single cell is split into two individual cells via the action of the acto-myosin contractile ring. Previous large scale RNAi-based screens have identified the set of genes required for cytokinesis. However, it is likely that many proteins required for abscission, the process that completes cell division, may not be identified by this approach, due to a prior requirement in the cellularization of oocytes in the syncytial gonad. We hypothesize that the well-defined class of sterile genes represents an untapped resource to identify novel components required for cytokinesis, and specifically abscission. In this work we have screened through the sterile collection of 562 genes, including approximately 170 previously uncharacterized genes, using a strain that expresses GFP::PH, to mark the plasma membrane, and mCherry::Histone 2B to visualize chromosomes. Following RNAi, Z-sections of anesthetized adult worm gonads were acquired, using spinning disk confocal microscopy, and phenotypes were scored. Novel genes that resulted in defined cellularization defects are currently being characterized. Preliminary analysis suggests several novel cytokinesis genes may be among the unknown genes in the screen. For example, depletion of unknown #193 yielded a striking multinucleated gonad phenotype and partial depletions produced multinucleated embryos. Furthermore, analysis of a GFP-tagged version of this protein revealed co-localization with known contractile ring components, suggesting this novel gene product may be required for cell division. To test unknowns for specific defects in abscission and determine when the two half-cells become limited to cytoplasmic movement, we have developed a photo-activation based assay that monitors the diffusion of a caged carboxy-q-rhodamine labeled dextran during the first embryonic division. Together, these approaches are likely to uncover novel abscission genes and aid in our understanding of the final step in cell division.

Takahashi A et al. (2014) Biosci Biotechnol Biochem "Green tea aroma fraction reduces -amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human -amyloid minigene."

Takahashi A, Hasegawa T, Watanabe T, Saito M, Suganuma M, Fujita T

[Biosci Biotechnol Biochem, 2014]

Green tea is a popular world-wide beverage with health benefits that include preventive effects on cancer as well as cardiovascular, liver and Alzheimer's diseases (AD). This study will examine the preventive effects on AD of a unique aroma of Japanese green tea. First, a transgenic Caenorhabditis elegans (C. elegans) CL4176 expressing human -amyloid peptide (A) was used as a model of AD. A hexane extract of processed green tea was further fractionated into volatile and non-volatile fractions, named roasty aroma and green tea aroma fractions depending on their aroma, by microscale distillation. Both hexane extract and green tea aroma fraction were found to inhibit A-induced paralysis, while only green tea aroma fraction extended lifespan in CL4176. We also found that green tea aroma fraction has antioxidant activity. This paper indicates that the green tea aroma fraction is an additional component for prevention of AD.

Gutierrez, Edgar et al. (2011) International Worm Meeting "Microfluidic vacuum actuated trap for gentle immobilization and high-resolution microscopy of C. elegans across developmental stages."

Green, Rebecca, Oegema, Karen, Gutierrez, Edgar, Groisman, Alex

[International Worm Meeting, 2011]

A major challenge for studying dynamic developmental processes in C. elegans has been the logistical difficulties associated with long-term high-resolution imaging of worms. Worms cannot be anesthetized over long time periods, ingest the immobilization beads when anesthesia is avoided, and their immobilization with the existing trapping techniques is incomplete, making it very difficult to track single animal throughout the course of development. Here we present a technique that uses a micro-machined elastomer chip that is pushed against a thin layer of a silicone gel (~20 mm) on a glass coverslip by the application of vacuum to trap worms between the elastomer and gel. Loading the device is simple and minimizes the loss of specially treated animals. Worms are transferred, one-by-one, to a ~5 ml droplet on the coverslip which is directly applied to the device. The device is compatible with short working distance microscope objectives (<200 mm) and has an array of continuously perfused micro-compartments with individual worms sequestered in them. The pushing force is widely adjustable by varying the level of vacuum, making the device suitable for a variety of worm sizes and enabling a user-defined degree of immobilization. A high level of vacuum is applied to completely immobilize worms during short intervals of high-resolution imaging; the vacuum is reduced between imaging to allow worms to feed and develop. Upon reduction of vacuum pressure, the worms recover their normal behavior and pharynx contractions, while staying confined in their micro-compartments. Thus multiple worms can be tracked in parallel during a long (hours to days) time course. Worms can also be easily recovered from the device into a minimal volume of buffer (<200 ml) for further propagation, by separating the elastomer chip from the gel substrate. In pilot experiments, we have successfully used this device and technique to immobilize and image worms at different developmental stages over several days, without loss of viability. Specifically, we have imaged the developmental time course of the gonad region, using DIC and two-color fluorescence confocal microscopy. This innovation makes it feasible to perform long-term high-resolution imaging on living worms, allowing for direct observation and characterization of dynamic developmental processes.

Fei T et al. (2017) Exp Gerontol "The anti-aging and anti-oxidation effects of tea water extract in Caenorhabditis elegans."

Fei T, Xu J, Huang F, Zhou Y, Yang P, Fei J, Xie T

[Exp Gerontol, 2017]

Tea includes puer tea, black tea, green tea and many others. By using model organism Caenorhabditis elegans, the anti-aging and anti-oxidation effects of tea water extract were systemically examined in this study. We found that water extract of puer tea, black tea and green tea all increased the lifespan of worms, postponed A-induced progressive paralysis in Alzheimer's disease transgenic worms, and improved the tolerance of worms to the oxidative stress induced by heavy metal Cr(6+). Moreover, the anti-oxidation effects of tea water extract at low concentration were different among 4 kinds of brands of green tea. The underlying mechanisms were further explored using genetically manipulated-mutant worms. The anti-oxidative stress effects of green tea water extract depend on the dietary restriction and germline signaling pathways, but not the FOXO and mitochondrial respiratory chain signals. Therefore, tea water extract provides benefits of anti-aging, anti-AD and anti-oxidation.

Gutierrez, Edgar et al. (2013) International Worm Meeting "Microfluidic devices for longitudinal imaging of gently immobilized worms and live imaging of early embryos during acute drug treatment."

Gutierrez, Edgar, Oegema, Karen, Green, Rebecca, Encalada, Sandra, Groisman, Alex

[International Worm Meeting, 2013]

A major challenge for studying dynamic developmental processes in C. elegans is long-term high-resolution imaging. We have used a micro-machined elastomer chip that is pushed against a cover glass by the application of vacuum to trap worms between the chip and the cover glass. The pushing force is widely adjustable by varying the level of vacuum, enabling a user-defined degree of immobilization for a variety of worm sizes. Loading the device is simple and minimizes the loss of worms. Worms can be easily recovered from the device by separating the elastomer chip from the substrate. A high level of vacuum is applied to completely immobilize worms during short intervals of high-resolution imaging; the vacuum is reduced between the imaging to allow worms to feed and develop. Upon reduction of the vacuum, the worms recover their normal behavior. We applied the device to image the vesicular transport in neurons of adult worms and the developmental time course of the gonad region, using DIC and confocal microscopy. We have performed longitudinal imaging over a 48 hour time interval, with the immobilization remaining fully functional and with worms developing from larvae to young adults. Small molecule inhibitors are a valuable tool for the analysis of fundamental cellular functions and an entry point for the development of therapeutic agents. We identified a gene whose inhibition renders the C. elegans eggshell permeable and built a microdevice for in situ worm dissection and high resolution imaging of embryos. The microdevice is made of hard plastics and has a rectangular well with an array of microwells on the bottom. After the dissection of worms, the fragile embryos are gently swept towards the microwell array, where they are protected from flow by the microwell walls. Permeable embryos are acutely exposed to drugs after the existing medium is aspirated and a medium with the drug is dispensed into the well with a pipette.