Cipriani, Patricia Giselle et al. (2009) International Worm Meeting "A Genetic Interaction Map of Early Embryogenesis in C. elegans."

Kao, Huey-Ling, Cipriani, Patricia Giselle, Piano, Fabio, Gunsalus, Kristin, White, Amelia

[International Worm Meeting, 2009]

Close to 1,000 genes have been identified as necessary for proper early embryonic processes in C. elegans. Yet many more genes are known to be expressed that could contribute to these processes. To uncover the structure of the underlying genetic networks during early embryogenesis and to identify new components and pathways that participate in these key events, we set up a system to systematically test for enhancing and suppressing interactions using RNAi. We have used 24 available temperature sensitive (ts) alleles whose strong loss-of-function phenotype affects the early embryo. To select the genes to test against these mutants, we used a bioinformatics approach to enrich for genes that are likely to play a role in early embryogenesis, but have not been identified by feeding RNAi screens. In addition, we enriched our list for homologs of human disease genes. Overall, we have RNAi-tested an average of 500 genes against each ts allele. We have acquired and analyzed over 100,000 RNAi experiments and identified over 560 high confidence genetic interactions. On average, we found ~18 enhancers and ~5 suppressors per essential gene. The combined genetic network connects all the tested essential genes through at least one non-essential gene. Analyzing each set of interactions separately, the network of suppressor interactions is less connected than the enhancer network, in addition to the lower number of interactions overall. Interestingly, many of our positive interaction pairs include at least one human disease gene ortholog that had not been known to work in the embryo before, opening new ways to study these genes. To investigate the interaction network more closely, we used the network navigation tool N-Browse to help navigate these data and integrate them with other available functional data. These analyses revealed that most of the observed genetic interactions we identified were not previously predicted or known and, as expected, they do not overlap with the available protein-protein interaction data. The preliminary analysis of our data has revealed new functions of previously uncharacterized genes, uncovered pathways previously not known to operate in the embryo, and shown connections between pathways that are potentially buffering each other. Some of these new pathways implicate functions such as RNA turnover as potential phenotypic capacitors in the early embryo. In addition, we see peroxisome biogenesis as a critical function not previously identified in early embryogenesis. We have confirmed some of our results from high-throughput screening by detailed phenotypic analysis and quantification, and we have also made substantial progress on the automatic scoring of the images produced.

Cipriani, Patricia G. et al. (2013) International Worm Meeting "A genome-wide network of genetic interactions in embryonic development."

Erickson, Katherine, Cipriani, Patricia G., White, Amelia, Piano, Fabio, Gunsalus, Kristin, Kao, Huey-Ling, Reboul, Jerome, Munarriz, Eliana, Lucas, Jessica, Chatterjee, Indrani

[International Worm Meeting, 2013]

The phenotypes manifested by genetic alleles are influenced by the genetic background in which they reside. Yet, we still have a very limited understanding of how genetic interactions (GIs) influence animal development. The goal of our project is to use genome-wide screens to identify all enhancing and suppressing GIs for a set of strains harboring temperature sensitive (ts) mutations in 24 essential embryonic genes. We have completed over three million primary GI assays and secondary screening of putative suppressors, and we have archived in a database all experimental metadata and images, along with quantitative scoring results from an automated phenotypic scoring algorithm we developed (DevStaR). DevStaR combines computer vision and machine learning methods to count different developmental stages in mixed populations of animals. Using these results we have developed a quantitative phenotypic "GI score" based on the multiplicative model of independence: if the effects of perturbing two genes are independent, then their combined effects should not deviate from the product of their individual effects. GI scores for individual experimental replicates correlate positively with semi-quantitative manual estimates of interaction strength. Using manual inspection as a reference, we devised criteria to combine GI scores across replicates that reliably detect suppressing interactions. We then generated final interaction scores that reflect both strength and reproducibility, which we used to define ~800 high-confidence and ~750 intermediate-confidence suppressing interactions. Based on comparisons with manual scoring, we estimate the false discovery rates in these two sets as 2% and 10%, respectively. The resulting GI network provides the first genome-wide map of suppressing genetic interactions for the embryo based on quantitative phenotypic analysis of viability.

Cipriani, Patricia et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A network of suppressors and enhancers of early embryogenesis in C. elegans"

Munarriz, Eliana, Amelia, White, Cipriani, Patricia, Julie, Young, Huey-Ling, Kao, Erickson, Katherine, Piano, Fabio, Gunsalus, Kris C

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

To uncover the structure of the underlying genetic networks during early embryogenesis and to identify new components and pathways that participate in these processes, we set up a system to systematically test for enhancing and suppressing interactions using RNAi. We are using 24 available temperature sensitive (ts) alleles whose strong loss-of-function phenotype affects the early embryo and genome-wide RNAi. We conducted a pilot study with an RNAi test set of about 2000 genes. Of this set ~ 400 genes composed a constant set that was tested against all strains, and ~100 genes were specific for each mutant. Several bioinformatics criteria were used in the selection process of this group of genes to try to increase the likelihood of finding positive interactions. From this initial screen, we identified 463 enhancing and 133 suppressing high confidence genetic interactions. Most of these interactions were not previously predicted or known. We are currently expanding the number of interactions in the second stage of this project and we will report on the progress of this screen as well as on the automatic scoring of the images produced.

Cipriani, Patricia Giselle et al. (2021) International Worm Meeting "LOTUS-domain containing proteins recruit C. elegans Vasa to germline granules and control the formation and size of the condensates"

Gan, Hin Hark, Fahs, Hala, Chung, George, Gunsalus, Kristin, Chen, Jiaxuan, Zinno, John, Duchaine, Thomas, Selbach, Matthias, Piano, Fabio, Mayya, Vinay, Gutwein, Michelle, Cipriani, Patricia Giselle, Bay, Olivia

[International Worm Meeting, 2021]

Limkain, Oskar and TUDOR 5 and 7 are LOTUS-domain containing proteins that have been described as important regulators of germline development and nucleators of ribonucleoprotein complexes in the germ cells of many metazoans. LOTUS-domain proteins have not been previously described in the C. elegans germ line. MIP-1 and MIP-2 are two previously uncharacterized paralogs that contain two LOTUS domains and extensive intrinsically disordered regions. Using co-immunoprecipitation, we found these proteins to be strong interactors of MEG-3, another intrinsically disordered protein that is a core component of embryonic P granules, and we named them MEG-3 Interacting Proteins 1 and 2. Fluorescently tagged and deletion alleles of the MIPs generated by CRIPSR-Cas9 show that the MIPs are constitutive components of germline granules in both embryos and the germ line. The simultaneous depletion of MIPs produces temperature sensitive sterility, a strong mortal germ line phenotype, and defects that affect all major developmental switches in germline development, including germline stem cell maintenance, the progression of cells through meiosis, and gametogenesis. The MIPs are jointly required for the condensation of other core P granule components, including MEG-3, the C. elegans Vasa homolog GLH-1, and PGL proteins. Interestingly, loss of function of either MIP individually affects the distribution and the size of remaining granules in the germ line in complementary ways. In oocytes, mip-1 deletion produces larger granules that are mostly cytoplasmic and mip-2 deletion produces smaller granules mostly attached to the nuclear membrane. Further biochemical and yeast-two hybrid analyses have shown that the MIPs physically interact with each other by forming homo- and heterodimers, and they directly bind GLH-1 through their N-terminal region, which contains the LOTUS domains. We propose that these proteins act as organizing centers in ribonucleoprotein networks and form a scaffold that helps recruit and balance essential RNA processing machinery within germline granules to regulate key developmental transitions in the germline.

Chen, Jia-Xuan et al. (2015) International Worm Meeting "In vivo protein interaction mapping in C. elegans embryos."

Polanowska, Jolanta, Cipriani, Patricia, Mecenas, Desirea, Gunsalus, Kristin, Selbach, Matthias, Chen, Jia-Xuan, Piano, Fabio

[International Worm Meeting, 2015]

Mapping protein-protein interactions in vivo is instrumental in deciphering the molecular mechanisms underlying animal development. We have developed a new method combining in vivo expressed GFP-tagged proteins with label-free quantitative proteomics to identify protein-protein interactions in developing C. elegans embryos. This strategy is generic and can in principle be used with any GFP-tagged protein. To test our approach we focused on eight proteins involved in essential biological processes during embryogenesis and built a pilot embryo in vivo interaction map comprising 559 interactions among 472 proteins. This network captures known biology and is highly enriched in functionally relevant interactions. We further show the utility of the map by searching for new regulators of P granule formation during embryogenesis. We discovered the worm-specific protein GEI-12 as a novel interaction partner of the DYRK kinase MBK-2 and as an important regulator of P granule dynamics and germline maintenance. This leads us to propose a hypothetical model in which the phosphorylation state of GEI-12 regulates P granule assembly and disassembly during early embryogenesis. Additionally, GEI-12 also induces granule formation in mammalian cells, suggesting that a common mechanism of ribonucleoprotein granule assembly exists in worms and humans. Our results show that in vivo interactome mapping is a powerful and versatile approach that provides unique insights into animal development.

Cipriani, P.G. et al. (2011) International Worm Meeting "A Genome-wide analysis of genetic interactions in early embryogenesis in C. elegans."

Gunsalus, K. C., Lucas, J., Munarriz, E., Kao, HL., Guzman, S., Piano, F., Young, J., Erickson, K., White, A., Cipriani, P.G.

[International Worm Meeting, 2011]

Genetic interaction studies have been useful in showing buffering between molecular pathways and predict function of non-lethal genes in many organisms. We undertook a large RNA interference (RNAi) based genome-wide screen to find genetic interactions (enhancers and suppressors) for genes involved in embryogenesis in C. elegans. To this end we have used 26 available temperature sensitive (ts) alleles of 24 essential genes (dhc-1, div-1, dnc-1, emb-8, emb-27, emb-30, glp-1, hcp-6, lin-5, mat-1, mbk-2, mel-26, par-1, par-2, par-4, pod-2, rme-8, spd-5, spn-4, tba-1, zen-4, zyg-1, zyg-8, zyg-9), whose strong loss-of-function phenotype affects the early embryo. We have developed an image acquisition system (1) and have archived over 3 million images from all RNAi experiments. To provide a rapid quantitative output, we have developed an image analysis system (DevStaR)(2). DevStaR produces an automatic classification of the developmental stages of C. elegans animals from a population of mixed stages, and achieves near real-time scoring of image data in a fully automated manner. The output is equivalent to the quantification of progeny survival in every experiment (See poster by White et al). Current analysis of the data shows that the distribution of the quantitative phenotypes allows us to compare the results of the ts mutant alleles and the controls, and to select potential positive genetic interactions for each ts allele with high accuracy. We will present data showing the trends of how genetic interaction networks are built around this set of essential genes. We are using these results to construct a comprehensive network of genetic interactions in early embryogenesis in C. elegans. 1. Cipriani P and Piano F. RNAi based High-throughput genetic interaction screening. 2011. Methods in Cell Biology. Elsevier. in press. 2.White A, Cipriani P, et al. 2010. Rapid and accurate developmental stage recognition of C. elegans from high-throughput image data. 2010 IEEE Conference on Computer Vision and Pattern Recognition. pp 3089-3096.

White, Amelia G et al. (2011) International Worm Meeting "Rapid and accurate phenotyping of embryonic lethality via image analysis of C. elegans developmental stages from high-throughput image data."

Sontag, Eduardo, Munarriz, Eliana, Cipriani, Patricia G, Kao, Huey-Ling, Piano, Fabio, Gunsalus, Kristin C, Paaby, Annalise, Geiger, Davi, White, Amelia G

[International Worm Meeting, 2011]

We present an automated image analysis system (DevStaR) for quantitative phenotyping of C. elegans embryonic lethality and sterility phenotypes. Our image analysis system counts each developmental stage in an image of a C. elegans population, allowing efficient high throughput calculation of C. elegans viability phenotypes. DevStaR is an object recognition machine comprising several hierarchical layers that build successively more sophisticated representations of the objects (developmental stages) to be classified. The algorithm segments the objects, decomposes the objects into parts, extracts features from these parts, and classifies them using an SVM (support Vector Machine) and global shape information. This enables correct classifications in the presence of complicated occlusions and deformations of the animals. Features of the classified objects are then used to obtain a count of each developmental stage. We are currently using this system to analyze phenotypic data from C. elegans high-throughput genetic screens, and have processed over one million images for lab users so far. Validation of DevStaR measurements will be shown by comparing DevStaR output to both manual counting of developmental stages and manual scores of quantitative phenotypes. DevStaR can provide an accurate measurement of quantitative phenotype and is comparable to manual scoring. DevStaR has been used to score a C. elegans genome wide RNAi screen with up to 30 repeats per clone tested at up to 5 temperatures per clone. The screen consists of over 600,000 images each scored by DevStaR, Analysis of these data illustrate the convenience of DevStaR scoring and the use of a quantitative phenotype. Our system overcomes a previous bottleneck in image analysis by achieving near real-time scoring of image data in a fully automated manner. Our system reduces the need for human evaluation of images and provides rapid quantitative output that is not feasible at high throughput by manual scoring.

Zahreddine Fahs, Hala et al. (2021) International Worm Meeting "Multi-species nematode screening uncovers a new broad-spectrum class of anthelmintic compounds targeting mitochondrial lipid metabolism"

Gopinadhan, Suma, Refai, Fathima, White, Robert, Pearson, Yanthe, Kremb, Stephan, Butterfoss, Glenn, Gunsalus, Kristin C., Zahreddine Fahs, Hala, Xie, Xin, Cipriani, Patricia, Page, Anthony, Piano, Fabio

[International Worm Meeting, 2021]

Parasitic worms infect more than 1.5 billion people and cause significant losses in livestock and crops. New anthelmintic drugs are urgently needed, as resistance to the existing drugs is emerging. We built a high-throughput (HTP) and high-content robotic screening platform to identify bioactive small molecules and their molecular targets, focusing on novel disease therapeutics and broad spectrum anthelmintics. The platform is capable of screening compound collections and genetic libraries in model organisms and mammalian cells in a fully integrated manner. To find novel anthelmintics we screened a ~2300 compound library containing most of the FDA-approved approved drugs, plus natural products and bioactive compounds for broad spectrum toxic effects on two distantly related free living nematodes C. elegans and P. pacificus as model animals while having low toxicity on human cell lines. The screen confirmed the effects of most known anthelmintics on these nematode models. Additionally, among a set of new compounds, we found six natural lipid molecules that exhibit broad-spectrum anthelmintic activity. These novel compounds kill C. elegans and P. pacificus when applied at various developmental stages, including the dauer and embryonic stages, in a dose-dependent manner. Importantly, these compounds cause mortality in all three veterinary parasite species we tested: the hookworm Haemonchus contortus (ruminants), Teladorsagia circumcincta (sheep and goat) and Heligmosomoides polygyrus (rodents). Further characterization of their mode of action using C. elegans revealed defects associated with mitochondrial function and lipid metabolism. The lethality phenotype in C. elegans is partially rescued by knocking down the enzymes acs-2, cpt-4, dif-1 and cpt-2, which facilitates mitochondrial lipid transfer, while drug inhibitors of CPT1 and CPT2 enhance the phenotype, suggesting that these compounds perturb specifically fatty acid oxidation in nematodes. These compounds constitute a new class of broad-spectrum anthelmintics targeting lipid metabolism.

Joseph Gallagher et al. (2006) European Worm Meeting "Development and Optimisation of Mos1 for Gene Tagging in C. elegans"

Joseph Gallagher, Patricia Kuwabara

[European Worm Meeting, 2006]

Joseph Gallagher and Patricia Kuwabara. The Drosophila transposon, Mos1, has been used successfully to generate insertional mutants in C. elegans1. Mos1 can be mobilised in C. elegans worms, which carry two independent extrachromosal arrays, one encoding the Mos1 transposase under the control of a heat shock promoter, and the other carrying copies of the Mos1 transposase substrate. Heat induction of the transposase leads to the mobilisation of the transposon and its integration within the C.elegans genome. Stable Mos1 insertions are detected by PCR after animals have been clonally passaged for a number of generations. As a member of the NemaGENETAG Consortium, which is funded through EU fp6, we are working with our partners to optimise the already existing Mos1 tools and to develop new strains to facilitate the high-throughput screening of C. elegans Mos1 insertional mutants. The generation of C. elegans transposon tagged genes will enhance our understanding of gene function, especially those that are associated with human disease, and provide an invaluable resource to the research community.. Here, we present our preliminary results, which were obtained after screening a library consisting of almost 1000 strains. So far, from this and subsequent screens, over 300 Mos1 strains have been identified by PCR. Each of these strains has been frozen at 80?C in triplicate, test thawed and re-tested to validate the presence of a Mos1 insertion. Many of the characterised strains carry multiple copies of Mos1 inserted at different sites, so the actual number of tagged loci is greater than the number of strains frozen. Steps are now being taken to improve the rate of transposition and the recovery of mutants. 1.. Bessereau, J. L. et al. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70-4 (2001).

Peter I. Joyce et al. (2006) European Worm Meeting "The Atypical Calpains of C. elegans"

Patricia E. Kuwabara, Peter I. Joyce

[European Worm Meeting, 2006]

Peter I. Joyce and Patricia E. Kuwabara . Regulated proteolysis of receptors, cytoskeletal proteins and transcription factors is an important process that modulates cell growth and development. A family of calcium-regulated thiol proteases, known as calpains, has been shown to perform such processes in mammals, and other eukaryotes. Loss of function mutations in calpains are linked to certain pathological conditions including limb-girdle muscular dystrophy 2A (LGMD2A); whereas, gain of function mutations are associated with the formation of cataracts. . We are investigating the biochemical and functional role of atypical calpains in C. elegans. We have identified seven atypical calpains and eight calpain-like sequences within the C. elegans genome. To gain an understanding of their possible roles in C. elegans development we have created calpain promoter transcriptional expression constructs for the atypical calpains 1 to 7 using monomeric RFP. The transcriptional expression patterns were analysed by co-localisation with the following tissue specific GFP markers: unc-119::GFP (all neurons), unc-47::GFP (GABAergic neurons within the ventral nerve cord), myo-3::GFP (body wall muscle), exc::GFP (excretory cell) and seam::GFP (seam cells). Five out of the seven atypical calpains show promoter transcriptional expression activity; clp-1, clp-2, clp-4, tra-3 and clp-7. Two of the atypical calpain promoter constructs fail to show detectable transcriptional expression activity; clp-3 (lacks all catalytic residues) and clp-6 (possible pseudogene). clp-1 shows the highest expression throughout the animal, specifically in neurons and body wall muscle. . In parallel with expression studies we have taken a global approach into studying calpain function using RNAi, calpain mutant analysis and ectopic over-expression. Preliminary studies have revealed no obvious effects. Because calpains require calcium for proteolytic activity, we are investigating ways in which calcium levels can be perturbed within C. elegans, as a way to activate calpains ectopically. We are also undertaking biochemical studies to characterise the proteolytic properties of the atypical calpains.