Labocha, Marta K et al. (2011) International Worm Meeting "Quantitative epistasis map of genes regulating sex ratio."

Aleman-Meza, Boanerges, Zhong, Weiwei, Labocha, Marta K, Luo, Wenshan

[International Worm Meeting, 2011]

Caenorhabditis elegans has two sexes, hermaphrodites (XX) and males (XO). Males are very rare among the self-progeny of wild-type hermaphrodites (1:500 to 1:2000). Several genetic pathways are known to affect sex ratios, including meiotic chromosome segregation, dosage compensation and sex determination pathways. When inactivated by RNAi, over 100 genes were found to give the high incidence of males (him) phenotype (WormBase WS212). Yet, the functional mechanisms and pathway information for the majority of these genes remain unknown. Therefore, we decided to conduct a quantitative epistasis study to uncover the genetic pathways and networks of these him genes. To enable a high-throughput quantitative screen, we first developed a computer vision system to automatically measure C. elegans sex ratios. Using a motorized stage and a camera attached to the microscope, the system scans Petri plates and records images of animals. The system analyzes morphological features of the animals and outputs the numbers of adult males and hermaphrodites on each plate. To identify pairwise interactions among genes, we inactivate each gene by RNAi or mutation, and measure the sex ratio of these single mutants using the automatic phenotyping system. We use these single mutant data to compute an expected sex ratio for the double mutant assuming that the two genes function independently. We then inactivate two genes by applying RNAi on mutants and examine whether the observed double mutant sex ratio is significantly different than the expected value. An enhancing interaction is identified if the observed phenotype is more severe than the expected value, and a suppressing interaction is denoted if the observed phenotype is milder. In a pilot study, we selected mutants of five meiosis genes (brc-1, brd-1, him-3, him-5, and zhp-3) and screened them for possible interactions with 124 genes that gave the him phenotype. From over 600 gene pairs screened, we discovered over 100 genetic interactions. Among them, 55% interactions were suppressing, and 45% were enhancing. The majority (over 97%) of these interactions are novel ones that have not been reported before. These data suggest that our quantitative epistasis approach is a powerful tool to identify genetic interactions. 44 of the 124 him genes have homozygous viable mutants. We are now in the process of composing the 44x124 gene interaction map. We will report our results at the meeting.

Toch, Katarzyna et al. (2021) International Worm Meeting "Sign and reciprocal sign epistasis across different environments"

Labocha, Marta, Buczek, Mateusz, Toch, Katarzyna

[International Worm Meeting, 2021]

Epistasis is a phenomenon of different loci interacting with each other, ergo having non-independent effect. But even though genetic interactions are widely spread across genomes their importance is still being questioned. Depending on the perspective, epistasis is said to either play an essential role in both applied (animals and plants breeding, forensics, personalized medicine) and evolutionary biology (emergence of sex, speciation, shaping evolutionary landscapes) or to be seen as a noise of linear genes effect. In general, epistasis can be either positive, when double mutant has higher than expected fitness, or negative, when the fitness is lower than expected. Sign and reciprocal sign epistasis are peculiar cases of genetic interactions, where one or both mutations have effect opposite from both mutations combined. We have tested over 1000 pairwise interactions in standard conditions and in three different environmental stressors: heat shock (37°C), oxidative (H2O2) and mutagenic (MMS) stress. We have found that sign epistasis is a prevalent event; 40-78% of all significant interactions are in fact sign epistasis. What is even more interesting, we were able to detect few examples of reciprocal sign epistasis, however only in harsh environments; one in MMS treatment and four in H2O2 treatment. All of them were the examples of positive reciprocal sign epistasis - despite the deleterious effect of both single mutations, double mutant exhibited higher than expected fitness (higher than animals without any mutations). Both sign and reciprocal sign epistasis are a game changer when it comes to predicting fitness landscapes. As much as sign epistasis is able to slow down the process of reaching fitness optimum, reciprocal sign epistasis can actually block it, unless e.g. two mutations occur simultaneously. Our findings are notably important, since they point to the importance of considering environment in studying genetics and evolutionary scenarios.

Toch, Katarzyna et al. (2019) International Worm Meeting "Dynamics of epistatic interactions in Caenorhabditis elegans under heat shock stress."

Labocha, Marta K., Toch, Katarzyna, Buczek, Mateusz

[International Worm Meeting, 2019]

Epistasis is a phenomenon of different loci interacting with each other. It is known for having an effect on evolution of sex and recombination, complex traits development, speciation and fitness landscape. What is more, it has been recently acknowledged for its important role in evolution of microbial drug resistance, disease diagnosis and forensic analysis. High-throughput studies on epistasis uncover new genes which might be involved in certain genetic pathways and point to the functions of studied genes. However, most such studies are performed in standard laboratory conditions, and change in the environment can drastically affect the epistatic landscape - in new environment new interactions are uncovered and some of the already known interactions are lost from the sight. Studies on how epistatic interactions are changing under different conditions are still rare, especially in multicellular organisms. Here, using RNA interference method we screened over 2000 gene-gene interactions in Caenorhabditis elegans in both standard and heat-shock conditions and calculated their interaction scores (S-scores). We found that epistasis tend to be more positive in stressful environment (mean S-score 0.032 in standard versus 0.241 in heat shock). In heat shock we discovered 79% more supressing than enhancing interactions, whereas in standard conditions we found the same amount of each. We propose that observed phenomenon might have an effect on the strength of selection, when organism is facing unfavourable environment.

Gasienica, Paulina et al. (2021) International Worm Meeting "Genes essentiality in various genetic backgrounds"

Zajac, Kamila, Toch, Katarzyna, Labocha, Marta, Gasienica, Paulina

[International Worm Meeting, 2021]

Essential genes can be defined as the ones which are crucial for organism survival and reproduction. They are responsible for the functions which are most important for the organism fitness pointing out to the most fundamental biological processes. They play a significant role in evolution, as their loss leads to the organism death or inability to reproduce. But this simple definition is not sufficient as gene essentiality is context dependent and vary with genetic and environmental background. So, the same mutation might lead to cell/organism death or severe sickness in some but not all genetic and environmental contexts. Most studies on gene essentiality were done in one genetic background. That means, that many genes essential in other genetic contexts were probably unidentified. Although the number of studies analysing gene essentiality in different genetic and environmental contexts is increasing, they are mainly done with unicellular organisms or cell lines. There is noticeable underrepresentation of such studies in multicellular, whole organisms. In this study we aim to uncover context dependence of gene essentiality in model multicellular organism, Caenorhabditis elegans. We show how essentiality of genes is changing depending on genetic context by using three different wild type worm isolates: N2, LKC34, and MY16. Our set of targeted genes included 100 essential and 200 nonessential genes based on DEG database (www.essentialgene.org). Each query gene was inactivated by RNA interference (RNAi) and mutant fitness was measured quantitatively as the rate of bacterial food consumption. We identified new essential genes indispensable in i) all three (22), ii) two (10), and iii) only one (11) genetic background. From previously identified N2 essential genes, we confirmed their essentiality in two additional backgrounds (68 genes), or one of them (9 cases). As we see even in a study encompassing only three strains, and 300 genes, we can observe substantial context dependence of gene essentiality. This have very important practical consequences as well. When working on gene therapies researchers should consider that different strategies might be needed depending on genetic background in which they will be used.

Marta Artal-Sanz et al. (2006) European Worm Meeting "Lysosomal Biogenesis and Function is Critical for Necrotic Cell Death in C. elegans"

Chrysanthi Samara, Nektarios Tavernarakis, Marta Artal-Sanz, Popi Syntichaki

[European Worm Meeting, 2006]

Marta Artal-Sanz, Chrysanthi Samara, Popi Syntichaki and Nektarios Tavernarakis. Necrotic cell death is defined by distinctive morphological characteristics, displayed by dying cells. The cellular events that transpire during necrosis to generate these necrotic traits are poorly understood. Recent studies in our lab show that cytoplasmic acidification develops during necrosis and is required for cell death in C. elegans. However the origin of cytoplasmic acidification remains elusive. We are investigating the role of lysosomes in necrotic cell death. We observed that alkalization of endosomal and lysosomal compartments ameliorates necrotic cell death triggered by diverse stimuli. In addition, mutations in genes that result in altered lysosomal biogenesis and function markedly affect neuronal necrosis. We used a genetically encoded fluorescent marker to follow lysosome fate during neurodegeneration in vivo. Strikingly, we find that lysosomes fuse and localize exclusively around a swollen nucleus. In advanced stages of cell death, the nucleus condenses and migrates towards the periphery of the cell, while GFP-labeled lysosomal membranes fade, indicating lysosomal rupture. Our findings demonstrate a prominent role for lysosomes in cellular destruction during necrotic cell death that is likely conserved in metazoans.

Eva Brozova et al. (2006) European Worm Meeting "NHR-40, a Caenorhabditis elegans Supplementary Nuclear Receptor Regulates the Development of Embryos and L1 Larvae"

Katerina Simeckova, Zdenek Kostrouch, Eva Brozova, Marta Kostrouchova

[European Worm Meeting, 2006]

Eva Brozova1, Katerina Simeckova1, Zdenek Kostrouch2, Marta Kostrouchova1 In this study, we functionally characterized NHR-40, a member of an extended group of supplementary nuclear receptors (Robisnon-Rechavi et al, J. Mol. Evol. 2005, 60: 577-586). We show that nhr-40 is expressed in 3 isoforms that are regulated by two promoters organized in tandem. The function of NHR-40 was studied by RNA interference, by overexpression of nhr-40 and using a mutant strain, RB840, which carries a deletion in the first intron. We show that NHR-40 regulates embryonic and early larval development. This phenotype is connected with defective movement and muscle cell development. This adds NHR-40 to the growing list of nematode NHRs involved in the regulation of development.. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi, M.W. Krause for support and advice, The C.elegans Gene Knockout Consortium for preparation of the deletion strain RB840. The work was supported by grants 303/03/1115 and 301/ 05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Katerina Simeckova et al. (2006) European Worm Meeting "NHR-60, a Caenorhabditis elegans Supplementary Nuclear Receptor residing at the Nuclear Periphery, Regulates Embryonic Development in Connection with Acyl-Coenzyme a Binding Protein"

Zdenek Kostrouch, Eva Brozova, Marta Kostrouchova, Jaroslav Vohanka, Katerina Simeckova, Michal Pohludka

[European Worm Meeting, 2006]

Katerina Simeckova1, Eva Brozova1, Jaroslav Vohanka1, Michal Pohludka2, Zdenek Kostrouch2, Marta Kostrouchova1 . NHR-60 is a Caenorhabditis elegans supplementary nuclear receptor that belongs to a subset of 18 of the 284 nuclear hormone receptors characterized by the P-box sequence CNGCKT. We show that NHR-60 is expressed ubiquitously and is predominantly localized at the nuclear periphery. The expression of NHR-60 is accented in seam cells, gland cells and in the germline. In seam cells, the expression of NHR-60 is dependent on CHR3 (NHR-23). Our research also focused on the possible interaction of Acyl-CoA-binding protein, which is known to interact with several nuclear hormone receptors, and NHR-60. The interaction in vivo between these two proteins was confirmed by co-immunoprecipitation and co-localization using confocal microscopy. acbp-1 inhibition by RNAi induced developmental defects similar to nhr-60 RNAi. These results indicate that regulation of development by nuclear hormone receptor NHR-60 is functionally linked to Acyl-CoA-binding protein and may constitute a connection with fat metabolism. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi and M.W. Krause for support and advice. The work was supported by grants 303/03/1115 and 301/05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Petr Liby et al. (2006) European Worm Meeting "Inhibition of bir-1, the Homologue of Human Survivin, Induces Changes of Expression of Developmentally Active Collagen Genes in L1 Larval Stage"

Marta Kostrouchova, Eva Brozova, Marketa Kostrouchova, Michal Pohludka, Jaroslav Vohanka, Petr Liby, Zdenek Kostrouch

[European Worm Meeting, 2006]

Petr Liby1, Marketa Kostrouchova1, Michal Pohludka1, Jaroslav Vohanka2, Eva Brozova2, Marta Kostrouchova2 and Zdenek Kostrouch1. BIR-1 is a member of the Inhibitors of Apoptosis protein family that is involved in the regulation of microtubule organization, condensation of chromosomes and cell division in C. elegans as well as in vertebrates. In C. elegans, bir-1 is organized in an operon together with SKIP, a transcription and splicing cofactor. bir-1 inhibition induces developmental defects including dpy and egl phenotypes. Here we studied the effect of bir-1 inhibition by RNAi in L1 larval stage using whole genome microarrays (Affymetrix). Microarrays detected changes in expression of several developmentally active collagen genes in bir-1 inhibited larvae. We further characterized the effect of bir-1 inhibition on selected collagen gene expression and induction of dpy phenotype. Acknowledgement: We thank Drs. A. Fire for GFP construct, vectors and host used in RNAi and M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Jaroslav Vohanka et al. (2006) European Worm Meeting "An Expression Study of Seven Putative Nuclear Receptors Organized in a Cluster on Chromosome V of Caenorhabditis elegans"

Marta Kostrouchova, Zdenek Kostrouch, Jaroslav Vohanka

[European Worm Meeting, 2006]

Jaroslav Vohanka1, Zdenek Kostrouch2 and Marta Kostrouchova1. Chromosome V contains a group of seven sequences recognized by the computer program GeneFinder as potential nuclear hormone receptors (NHRs). In order to identify whether genes from this cluster may be functional NHRs, we studied their expression during C. elegans development. RT-PCR identifies transcripts of all seven genes. The expression of all members of this cluster starts in embryos. Six of these genes are expressed throughout development while one member of this cluster is only expressed in embryos and early larval stages. We prepared constructs of GFP fusion genes containing various regions of genomic sequences. At least 2 different fragments containing putative promoters were prepared for each gene of the cluster; one consisting of approximately 500 bp and a second of approximately 2000 bp. The longer constructs contain parts of the coding sequences of the preceding genes. Our results show that members of this gene cluster have a diverse expression pattern and are likely to be functional NHRs. Acknowledgement: We thank Drs. A. Fire for GFP constructs, vectors and host used in RNAi, M.W. Krause for support and advice. The work was supported by grants 303/03/1115 and 301/ 05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Marta Maria Gaglia et al. (2006) European Worm Meeting "Transcriptional Changes Underlying Chemosensory Regulation of Lifespan"

Cynthia Kenyon, Marta Maria Gaglia

[European Worm Meeting, 2006]

Marta Maria Gaglia and Cynthia Kenyon. Mutations in the insulin/IGF-1-like receptor daf-2 alone can double the lifespan of C.elegans, suggesting that aging is not just a default state, but instead a genetically regulated process. However, it is not clear what physiological processes regulate the daf-2 pathway. Interestingly, mutants with altered chemosensation also have an extended lifespan (Apfeld and Kenyon, 1999). Both daf-2 and chemosensory mutant longevity are dependent on the activity of the FOXO transcription factor DAF-16. This suggests that external inputs through the chemosensory system may regulate the insulin pathway and in turn affect lifespan. Further studies have identified some of the cellular components of the sensory pathway of lifespan control (Alcedo and Kenyon, 2004). However, the molecular mechanism by which the chemosensory system modulates lifespan remains still largely unknown. To begin to elucidate the mechanisms of chemosensory regulation of lifespan, we have performed genome-wide expression analysis with microarrays on the long-lived chemosensory mutant daf-10. We are in the process of characterizing the genes whose expression was altered with mutants and RNA interference in sensitized backgrounds. In particular, we are interested in those genes that affect lifespan without causing a chemosensory defect, as they may act downstream of the sensory neurons. In addition we are comparing the expression changes to those occurring in daf-2 mutants, to determine how the two pathways interact to modulate lifespan.