Rougvie, Ann (2021) International Worm Meeting "CGC and tribute to Don Moerman"

Rougvie, Ann

[International Worm Meeting, 2021]

Don Moerman et al. (2003) International Worm Meeting "Expression of Promoter-GFP Constructs in C. elegans"

David L Baillie, Francis Ouellette, Sheldon McKay, Zhongying Zhao, Steve Jones, Allan Mah, Don Moerman, Marco Marra, Domena Tu, Erik Sonnhammer

[International Worm Meeting, 2003]

We are examining expression profiles for 2000 C. elegans genes with human orthologs (see McKay et al. abstract) or which have been requested from the CE Knockout Project. Our aim is to develop understanding of human genes of unknown function. Analyzing human orthologs in C. elegans will enable us to quickly determine temporal, spatial and level of gene expression. We have generated promoter-GFP transgenic strains for 400 human orthologs which we are analyzing by green fluorescent protein (GFP) microscopy. We use PCR stitching to fuse potential promoter-containing regions of genomic DNA with a promoterless GFP expression cassette. Identifying target regions and designing suitable PCR primers were automated using custom software developed to integrate the C. elegans AceDB genomic database (wormbase) and other data sources with the primer design and validation programs primer3 and e-PCR to facilitate high throughput generation of constructs. Primers were designed for more than 15,000 genes, with most of the remainder having unsuitable upstream regions. Simple arrays of promoter-GFP construct and dpy-5(+) DNA was injected into Dpy-5 hermaphrodites. Wild-type progeny were analyzed for their GFP-expression patterns. About 73% of the 400 strains show expression and about 49% of these express only in one tissue type. Gut is the most common tissue for expressed genes with 38% of the GFP expressing constructs. 47% of the constructs that express in gut do not express in any other tissue. Effectively all tissues express more than one construct and most tissues have at least one construct that does not express in any other tissue. For example: muscle (17% expression and 7% only in muscle); pharynx (22% and 1.3%); and nerve cord (9% and 1.3%). 39% of the expressing transgenics show embryonic expression (7% do not show expression at any developmental stage other than embryonic). Usually promoter-GFP constructs show mosaic expression patterns which makes analysis more difficult especially in embryo; therefore, we are using X-irradiation to induce stably expressing lines. So far, we have stable lines for 30 promoter-GFP constructs. Data will be made publicly available at a website. Funded by Genome BC and Genome Canada.

Zhou H et al. (2018) Chemosphere "Trans-/multi-generational effects of deoxynivalenol on Caenorhabditis elegans."

Tang L, Qian H, Xue KS, Williams PL, Zhou H, Sun X, Wang JS

[Chemosphere, 2018]

Deoxynivalenol (DON, vomitoxin) is one of the most widely distributed trichothecene mycotoxins commonly found in cereal food and feeds. Significant acute and potential chronic toxic effects of DON have been observed in animals and human populations. However, potential adverse effects associated with DON exposure across multiple generations have not been extensively investigated. In this study, Caenorhabditis elegans (C.elegans) were used to evaluate the trans-/multi-generational toxicities of DON via 3 physiological endpoints: growth, brood size, and feeding ability. DON concentration at higher than 100g/mL significantly inhibited growth, decreased brood size, and reduced food intake in a concentration-dependent manner. Gradual decline in DON-induced impairments was observed in the filial generations when only the parental generation was exposed. However, greater damages in filial generations were found as compared to the parental generation (p<0.01) with all generations continuously exposed to DON. Overall, the endpoints of brood size and food intake were more sensitive for both trans- and multi-generational toxic effects of DON. Additionally, the interactions between concentrations and generations significantly influence the response of C.elegans to DON exposure, based on a mixed-effect model with multi-level analysis. Taken together, our results demonstrated that DON exposure produced significant trans-/multi-generational toxic effects on C.elegans, which may serve as a model organism to explore molecular mechanisms of long-term adverse health effects of DON.

Di R et al. (2018) Toxins (Basel) "Transcriptome Analysis of C. elegans Reveals Novel Targets for DON Cytotoxicity."

Lawton MA, Di R, Zhang H

[Toxins (Basel), 2018]

Deoxynivalenol (DON) is a mycotoxin produced by <i>Fusarium</i> spp. that causes <i>Fusarium</i> head blight (FHB) disease in cereal crops. Ingestion of food contaminated with DON poses serious human health complications. However, the DON cytotoxicity has been mostly deduced from animal studies. In this study, we used the nematode <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) as a tractable animal model to dissect the toxic effect of DON. Our results indicate that DON reduces the fecundity and lifespan of <i>C. elegans</i>. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that DON upregulates innate immunity-related genes including <i>C17H12.8</i> and <i>K08D8.5</i> encoding PMK-1 (mitogen activated protein kinase-1)-regulated immune effectors, and <i>F35E12.5</i> encoding a CUB-like domain-containing protein. Furthermore, our RNAseq data demonstrate that out of ~17,000 <i>C. elegans</i> genes, 313 are upregulated and 166 were downregulated by DON treatment. Among the DON-upregulated genes, several are <i>ugt</i> genes encoding UDP-glucuronosyl transferase (UGTs) which are known to be involved in chemical detoxification. The three upregulated genes, <i>F52F10.4</i> (<i>oac-32</i>), <i>C10H11.6</i> (<i>ugt-26</i>) and <i>C10H11.4</i> (<i>ugt-28</i>) encoding the O-acyltransferase homolog, UGT26 and UGT 28, respectively, are shown to contribute to DON tolerance by a RNAi bacterial feeding experiment. The results of this study provide insights to the targets of DON cytotoxicity and potential mitigation measures.

Gowrinathan Y et al. (2011) Food Addit Contam Part A Chem Anal Control Expo Risk Assess "Toxicity assay for deoxynivalenol using Caenorhabditis elegans."

Pacan JC, Gowrinathan Y, Sabour PM, Zhou T, Hawke A

[Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2011]

The progeny production and development rates of the nematode Caenorhabditis elegans when treated with deoxynivalenol (DON) were examined. Both purified DON and a crude extract from Fusarium graminearum cultured on rice were tested on C. elegans wild-type (Bristol N2) and a mutant strain (AU1). Significant effects (Tukey-HSD, p&lt;0.05) on brood size and the rate of larval development from egg to adulthood were observed. Both N2 and AU1 strains showed lower rates of development and smaller brood sizes when exposed to purified DON at concentrations of 500 and 1000 g ml(-1) When they were exposed to crude extract containing 250 g ml(-1) DON, the inhibition of egg hatching and a greatly reduced development rate were observed. The results suggest that selection of a more sensitive C. elegans mutant strain could be used as a suitable animal model for conducting DON toxicity assays.

Yang Y et al. (2018) Toxicol Sci "Transcription factor FOXO3a is a negative regulator of cytotoxicity of Fusarium mycotoxin in GES-1 cells."

Liao Y, Humpf HU, Liu N, Wu Y, Su X, Yang Y, Xu H, Yu S, Gong Y, Wu A, De Saeger S, Wang P

[Toxicol Sci, 2018]

Molecular mechanism and key factors responsible for cytotoxicity against mycotoxin deoxynivalenol (DON) from Fusarium pathogens are rarely elucidated. In this study, rapid increases of ROS were first observed in human gastric epithelial (GES-1) cells under DON exposure. Mitochondrial DNA damage, impaired respiratory chain and decreased oxygen consumption rate(OCR) values, as well as G2/M cell cycle arrest and apoptosis, were also detected. Via combinatorial approaches of a large-scale microarray of differentially expressed genes, high content and RNAi analysis, a transcription factor of Forkhead box O3 (FOXO3a) was found with crucial functionalities, regulated some apoptotic genes associated with mitochondrial toxicity and cell death after activation by nuclear translocation. Namely, knockdown of FOXO3a decreased the cytotoxicity of DON to GES-1 cells. Moreover, knockdown of the FOXO ortholog DAF16 in Caenorhabditis elegans increased the resistance to DON-induced cytotoxicity. Simultaneously, the signaling pathway of ROS/JNK/FOXO3a of DON -induced cytotoxicity was newly proposed. In total, FOXO3a via ROS/JNK/FOXO3a plays a critical role to function as negative regulator associating with DON-induced cytotoxicity, with the potential extending to other substances.

Edgley, Mark et al. (2011) International Worm Meeting "Towards a mutation in every gene: A report from the front lines."

Barstead, Robert, Mitani, Shohei, Edgley, Mark, Moerman, Don

[International Worm Meeting, 2011]

Currently the combined efforts of the Barstead, Mitani and Moerman labs have identified over 7,000 deletions in 6,013 genes. About 1/4 of these mutants are inviable as homozygotes. Most of the mutations identified to date have been isolated using the PCR deletion detection protocol but we are also using Comparative Genome Hybridization (CGH) and massively parallel short read sequencing technologies to identify alterations. While the facilities are largely request driven, we have made a concerted effort to identify mutations in a number of large critical genes families, including transcription factors (TF's) and kinases. To date we have identified mutations in close to 900 of the 935 TF's and 381 of the 416 annotated kinases. The efforts of our three facilities when combined with efforts from the community and other more recent results from Deep Sequencing bring the total numbers of genes with either a deletion, a stop codon or a splicing defect to 8,000 genes.

Warner, Adam D. et al. (2009) International Worm Meeting "Screening for essential muscle genes using high throughput RNAi identifies a copine involved in early sarcomere assembly."

Rogalski, Teresa, Somasiri, Aruna, Warner, Adam D., Moerman, Don

[International Worm Meeting, 2009]

The sarcomere is an impressively organized array of interdigitated actin and myosin filaments, anchored by dense body and M-line attachment complexes, respectively, to the sarcolemma of a muscle cell. Prominent proteins in Z-disc analogs and homologs known as dense bodies are required for body wall muscle in C. elegans to develop and function properly. Without these essential attachment complex proteins such as a and b-integrin (PAT-2, PAT-3), vinculin (DEB-1), kindlin (UNC-112), PINCH (UNC-97), ILK (PAT-4), and actopaxin (PAT-6), embryos arrest at the two-fold stage of embryonic development1. Early work on b-integrin/PAT-32 and kindlin/UNC-1123 in the worm first implicated these proteins as being essential in muscle, and the worm has continued to be a valuable model organism for identification and study of novel essential muscle genes. Focusing on the paralyzed and arrested at two-fold stage (Pat) phenotype that occurs with loss of essential muscle genes in worms, we have carried out a muscle transcriptome-wide RNAi screen to identify genes with essential roles in muscle. Four genes without a previously annotated Pat phenotype have been uncovered. One of the positive hits from our screen is F31D5.3, which codes for a transmembrane protein with a C-terminal integrin-like A domain, and bears homology to the copine family of proteins in higher organisms including humans. To date, very little research has been carried out on copines in other organisms, although some copines have been found present in human heart and skeletal muscle4. We have received a gene knockout for F31D5.3 (gk266) from the C. elegans Gene Knockout Consortium (Vancouver) that is lethal, arresting at the two-fold stage of embryogenesis just as our aforementioned RNAi study found. Furthermore, when F31D5.3 RNAi is administered to worms after they hatch from embryos, myofilaments in body wall muscle become severely disorganized. Lastly, expression data from SAGE experiments (Moerman lab, unpublished) shows highly elevated expression of F31D5.3 in embryonic body wall muscle compared to other tissue types, and a ~3.5 fold enrichment in expression levels compared with whole embryos. Further experiments are ongoing to identify where F31D5.3 is localized in body wall muscle, and to attempt placing F31D5.3 in the sarcomere assembly pathway. 1Williams BD and Waterston RH, (1994). JCB: 124(4):475-90. 2Gettner et al (1995). JCB: 129(4):1127-41. 3Rogalski et al (2000). JCB: 150(1):253-64. 4Cowland et al (2003). J. Leukocyte Biology: 74(3) 379-88.

Qadota, Hiroshi et al. (2009) International Worm Meeting "UNC-112 is a novel interactor with the cytoplasmic tail of PAT-3/b-integrin in C. elegans muscle."

Qadota, Hiroshi, Moerman, Don, Benian, Guy M.

[International Worm Meeting, 2009]

Myofilaments within C. elegans body wall muscle cells are organized and anchored to the muscle cell membrane by dense bodies and M-lines. These structures are both analogous and homologous to vertebrate focal adhesion plaques. To clarify the molecular mechanisms regulating these integrin mediated attachment structures, we are investigating protein-protein interactions between integrin-associated proteins using a two hybrid approach. From the yeast two hybrid screening with PAT-3/b-integrin cytoplasmic tail, we identified UNC-112, a FERM family protein located at dense bodies and M-lines. From the data of domain mapping experiments, we showed that binding of UNC-112 to PAT-3 cytoplasmic tail requires full-length UNC-112. We confirmed this interaction by GST-PAT-3 cytoplasmic tail pulldown of UNC-112 from a yeast lysate expressing HA-tagged UNC-112 or a C. elegans lysate. In embryos, it has been reported that PAT-3 is essential for UNC-112 localization to attachment structures (Rogalski et al., 2000 JCB 150: 253-264). We also showed that in adult muscle cells pat-3 loss-of-function causes diffuse localization of UNC-112 by using an attenuated RNAi method (Qadota et al., 2008 JMB 383: 747-752). We already reported that the UNC-112 N-terminal half binds to PAT-4/ILK. To clarify the function of UNC-112 C-terminal half, we screened a yeast two hybrid library to look for binding proteins to UNC-112 C-terminal half and isolated cDNA clones of UNC-112 lacking the C-terminal region. We have confirmed the interaction between UNC-112 N-terminus and UNC-112 C-terminus using purified proteins. This interaction suggests a comformational change of UNC-112 between closed and open states. We hypothesize that this UNC-112 conformational change might be regulated by binding of PAT-4 to UNC-112 N-terminus, resulting in ability to bind to the PAT-3 cytoplasmic tail. This hypothesis is supported by a previous report that in pat-4 mutant embryos UNC-112 cannot localize at attachment structures (Mackinnon et al., 2002 Current Biology 12: 787-797). We are conducting in vitro and in vivo experiments to obtain more evidence to prove this hypothesis.

Farris, Lily et al. (2009) International Worm Meeting "Open Science in Practice: C. elegans Researchers and Their Use of Open Access Resources."

Marden, Emily, Levy, Ed, Farris, Lily, Moerman, Don

[International Worm Meeting, 2009]

As integrated GE3LS (genomics, ethics, environment, economics, law and society) researchers with the C. elegans Gene Knockout Consortium we are exploring how publicly available data is accessed and used and how this process informs scientific advancement. Our survey asked researchers about their scientific resource use, sharing of information, and handling of information with proprietary potential. The results from this survey provide additional understanding of the relationship between the researcher''s use and exchange of information and their scientific innovation. Methods We invited researchers currently using C. elegans as a model organism who were 19 years of age or older to complete an online survey (www.wormsurvey.com) to provide information about their current use and exchange of research materials. Compiling a list of C. elegans researchers from the list of registered researchers in WormBase, we contacted a random sample of respondents five times in order to unsure higher response rates. The survey was fielded in October, 2008. We also requested that researchers circulate the survey to all members of their research team including graduate students, technicians, and research assistants who conduct any C. elegans related research. Preliminary Results Of the 349 respondents who conducted research with C. elegans, the majority supported the work of C. elegans Gene Consortium and supported the statement that their research would function differently if this resource was not available. Most respondents used WormBase on a daily or weekly basis and few respondents held patents. Overall, C. elegans researchers use and access a variety of resources with unrestricted access using these resources to produce both basic science and commercial innovation.