O'Donnell, Mike et al. (2015) International Worm Meeting "Rictor limits temperature-dependent dauer formation by controlling intestine-neuron signaling."

Kammenga, Jan, Sengupta, Piali, O'Donnell, Mike

[International Worm Meeting, 2015]

Plasticity in developmental trajectory is a common strategy employed by multicellular animals to cope with stressful environments. A striking example of this is the nematode alternative larval morph - the long-lived, stress-resistant dauer larva. In C. elegans, when food is limiting and population density is high, an instructive pheromone-derived signal results in decreased TGFbeta and insulin signaling to promote dauer formation. For over thirty years, it has been appreciated that temperature acts in addition to pheromone to promote dauer formation. Despite this, little is known about the mechanisms by which the temperature signal is detected, transduced, and integrated to enhance dauer entry. Interestingly, it has previously been shown that at high temperatures of 27degC, dauer entry can occur independent of pheromone and in the presence of abundant food (Ailion et. al., 2000). Here I show that the temperature-dependent dauer entry phenotype varies in global C. elegans isolates. Variation between two strains, N2 and CB4856, is in part due to coding changes in the rict-1/Rictor gene, a component of the Target of Rapamycin Complex 2 (TORC2). The CB4856 variant is more derived compared to the ancestral rict-1 sequence, suggesting that the N2 allele has not arisen due to domestication. In N2, RICT-1/Rictor acts in the intestine in a pathway involving akt-1 and daf-16, but not akt-2, to prevent dauer entry at high temperatures. Dauer entry at high temperatures reflects a balance between pro-growth insulin signaling and pro-dauer neuropeptide signaling. Mutations in the egl-21 carboxypeptidase partially suppress rict-1 mutations suggesting that RICT-1 acts upstream of neuropeptide signaling to prevent dauer entry. These data suggest that an intestine-neuron circuit, regulated by the TORC2 pathway, determines sensitivity to high-temperature-induced dauer entry in C. elegans. .

Sterken, Mark et al. (2021) International Worm Meeting "Genetic background-dependent expression of clec-62 in Caenorhabditis elegans"

Sterken, Mark, Kammenga, Jan, Steeghs, Maarten, Riksen, Joost

[International Worm Meeting, 2021]

The pursuit of genes underlying quantitative traits is often accompanied by creating introgression lines (ILs). ILs consist of a single genetic locus introgressed in an otherwise homogeneous background. We previously generated a genome-covering IL panel by introgression of segments of the strain N2 in a CB4856 genetic background. This population complements a previously created IL panel with CB4856 segments in an N2 genetic background. Combining both panels allow the study of the effect of the genetic background at a higher resolution than previously possible with chromosome substitution strains. We exposed 150 strains of a N2xCB4856 recombinant inbred line population and the two IL panels to a normal growth regime (20oC on standard NGM plates). After 48 hours of growth, we collected the animals, flash froze them and proceeded to isolate RNA. Via qPCR, we measured the expression of clec-62, finding that only in the N2-background population QTL were detected. In other words, no QTL were detected in the RIL and reciprocal IL population. Thereby we show the strong role of the genetic background on trait variation. Here we present a case of either closely-linked loci that were only segregated in one population, or an epistatic interaction depending on the genetic background. We are currently in the process of building a double introgression panel to experimentally test the effect of two combined backgrounds.

Wang, Ziyi et al. (2013) International Worm Meeting "Genetic basis underlying differential susceptibility to bacteria in Caenorhabditis elegans."

Kammenga, Jan, Wang, Ziyi, Snoek, L. Basten, Herman, Michael

[International Worm Meeting, 2013]

Although Escherichia coli OP50 is the standard lab food for C. elegans, it is unlikely to be part of its natural diet. In fact, the bacterial environment C. elegans naturally encounter could be quite heterogeneous. In the wild, animals must defend against potential pathogens within mixed food sources. One such potential pathogen is Stenotrophomonas maltophilia, a ubiquitous bacterium that has been isolated in association with nematodes from natural environments, but can also cause nosocomial infection in humans, especially immune-compromised individuals. For instance, our lab isolate, S. maltophilia JCMS, was found surprisingly pathogenic to C. elegans in that even daf-2 mutants that are otherwise long-lived in any other bacteria tested to date, are as susceptible as wildtype animals. More interestingly, we found N2 (Bristol, England) and CB4856 (Hawaii) respond to E. coli OP50 and S. maltophilia JCMS differently and observed a significant genotype by environment (GxE) interaction, which has been thought to be a major mechanism that maintains genetic variation in natural populations. To understand the evolution of C. elegans responses to a heterogeneous bacterial diet it is crucial to identify the underlying genetic variation responsible for differential susceptibility and to characterize the corresponding molecular mechanism. We chose to take a quantitative genetic approach to this question and have mapped quantitative trait loci (QTL) in a N2xCB4856 recombinant inbred panel for post-reproductive lifespan on E. coli OP50 and S. maltophilia JCMS. We identified a novel QTL on LGX that contributes to the GxE interaction. We then used introgression lines that contain distinct lengths of chromosome X within this QTL region from CB4856 in an otherwise N2 genomic background for fine mapping. We confirmed and narrowed down the position of this locus to a relatively small region that contains reasonable number of candidate genes for further functional tests. We are currently endeavoring to pinpoint the causal genes and will report our progress.

Herman, Michael A. et al. (2009) International Worm Meeting "Identification of QTL involved in the C. elegans response to soil bacteria."

Kammenga, Jan E., Snoek, Basten, Herman, Michael A., Wang, Ziyi

[International Worm Meeting, 2009]

We are investigating the genetic basis of nematode interactions with soil bacteria. To this end, we have isolated soil bacteria in association with native soil nematodes from grassland soils. We are using C. elegans to model the interactions of native soil nematodes with soil bacteria and have identified C. elegans genes induced when worms are grown on various soil bacteria. We have used available mutants to demonstrate that the functions of many of these genes are important for life history traits, including fitness and lifespan (Coolon et al., submitted). However, these experiments were not designed to identify natural variation in genes that may be able to respond to selection pressures presented by soil bacteria. To discover such genes we have taken a quantitative genetic approach to identify quantitative trait loci (QTL) that underlie C. elegans responses to soil bacteria. For this experiment we chose to investigate C. elegans response to Stenotrophmonas maltophila, a ubiquitous bacterium that we isolated in association with an Oscheius sp. from grassland soils. S. maltophila can cause nosocommial infections in immuno-compromised individuals, thus is also of interest to understanding conserved innate immune responses that could affect human health. We have observed that exposure to S. maltophila shortens lifespan in both wild-type and daf-2 animals (see abstract by Vinod et al.), suggesting the involvement of other, perhaps uncharacterized, genes and innate immunity pathways. We used a set of recombinant inbred lines (RILs) generated by crosses between N2 and CB4856 (Li et al., 2006) to map QTL associated with lifespan in the presence of both E. coli and S. maltophila. We scored survivorship in the RILs and performed three replications of 10 animals each. Heritability of average lifespan was high on both E. coli and S. maltophila; 75 and 73 percent, respectively, indicating a large genetic component in the response. We were able to map QTL on LG I, II, IV and X and are now using near isogenic lines (NILs) to refine the positions of these QTL to identify the genes responsible. We will report our progress on these experiments. Reference: Li et al. 2006, PLoS Gen 2, e222.

Elvin, Mark et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A food consumption assay to enable super high throughput RNAi"

Frejno, Martin, Snoek, Basten, Kammenga, Jan, Poulin, Gino, Elvin, Mark

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

Performing RNAi by feeding was an important step that made loss of function analysis truly high throughput. However, the bottleneck of this technology remains the scoring. Different approaches have been used to bypass the tedious task of sitting at the microscope, but many of these are either expensive or of a lower throughput. Here we will present a strategy that is based on how much bacteria is eaten by the worms in function of the time, which is an indication of the worms fitness. The RNAi is performed in liquid, in a 96-well format and analysed using a plate reader with a 600 nm filter. Readings are taken everyday for a week and a curve of OD in function of the time is derived for every experiment. Here, we s how that this method can be used to distinguish between different worm strains, e.g. lin-15A, and lin-15B; and detect synthetic interactions e.g. lin-15AB. We have also tested whether the lack of RNAi sensitivity observed in CB4856 (Hawaii) compared to N2 (Bristol) could be detected. This effect has been largely attributed to polymorphisms in ppw-1. We have used a panel of 12 RNAi feeding clones, and found that Hawaii is rather resilient to most RNAi clones, but not so to sumo-1. Therefore, we can distinguish Bristol from Hawaii using this assay. Then, using 50 Recombinant Inbred Lines (RILs) between Hawaii and Bristol, we tested which of these are sensitive to RNAi and performed QTL analysis. We have found that even though most can fall into one or the other category a number of RILs are hypersensitive or nearly totally resistant to RNAi. Interestingly, we have found a RIL that phenotypically behaves as Hawaii (i.e. is RNAi insensitive), but is genotypically Bristol for ppw -1. Therefore, we have established a new method that significantly improves the throughput of RNAi in liquid, since reading a 96-well plate takes seconds. Additionally, we demonstrate that RNAi sensitivity is a complex trait that can be quantified using our food consumption assay.

Orbidans, Helen E. et al. (2013) International Worm Meeting "Widespread pleiotropic Bateson-Dobzhansky-Muller incompatibilities between C. elegans isolates."

Harvey, Simon C., Snoek, L. Basten, Kammenga, Jan E., Stastna, Jana, Orbidans, Helen E.

[International Worm Meeting, 2013]

Bateson-Dobzhansky-Muller (BDM) incompatibilities are a result of deleterious interactions between alleles that are neutral or advantageous in their own genetic backgrounds. Both outbreeding depression and a specific incompatibility causing embryonic lethality have been identified within C. elegans. We therefore hypothesised that alleles producing BDM incompatibilities would also be present. Identifying the underlying loci producing such negative epistatic effects within a species is important as it will allow comparison to the loci and alleles that generate isolation between species, i.e. it addresses the role of BDM incompatibilities in driving speciation. To identify genomic regions showing BDM incompatibilities we undertook screens for regions that disrupted the normal process of egg-laying, a complex, highly regulated and coordinated phenotype. Screens were undertaken in recombinant inbred lines (RILs) and a genome-wide panel of nearly isogenic lines (NILs) both produced from the isolates CB4856 and N2. These RIL and NIL analyses identify a number of quantitative trait loci (QTLs) that show synthetic effects on egg-laying, i.e. the disruption in egg-laying is not seen in the parental isolates and is a consequence of negative interactions between CB4856 and N2 alleles. Analysis of these QTLs shows that they also affect other life history traits, affecting lifespan and the internal hatching of progeny (bagging). Further analysis indicates that this approach identifies only a subset of the incompatibilities between CB4856 and N2, that these incompatibilities are a consequence of complex interactions between multiple loci, and that they interact with the stress response. LBS and JK were funded by NWO-ALW NEMADAPT (project 855.01.151) and the Graduate School Production Ecology & Resource Conservation.

Snoek, L. Basten et al. (2009) International Worm Meeting "Genome-wide evidence for genetic robustness of the alternative splicing machinery in C. elegans."

Snoek, L. Basten, Jansen, Ritsert, Riksen, Joost A.G., Li, Yang, Breitling, Rainer, Kammenga, Jan E.

[International Worm Meeting, 2009]

Alternative splicing is considered a major mechanism for creating multicellular diversity from a limited repertoire of genes. Here, we studied genetic variation in alternative splicing patterns in a recombinant inbred population of C. elegans, using whole-genome tiling arrays. This experiment allowed us to detect heritable differences in gene expression with exquisite sensitivity and resolution. Nonetheless, we find only a relatively small number of examples of heritable variation in alternative splicing patterns. This is in striking contrast to earlier observations in humans, which showed much less genetic robustness. This observation points to a profound difference in the regulation of the alternative splicing machinery, which parallels the differences in cellular diversity and developmental flexibility in the two species. Nevertheless some striking examples were found of heritable variation in alternative splicing patterns. We detected 382 genes with substantial heritable variation for at least one exon of which the large majority of eQTLs lead to a consistent differential expression across all exons of the affected gene. Some cases show evidence for a necessary refinement of existing gene definitions, predominantly by expanding known exons. We will present genome-wide proof for earlier hypotheses that in C. elegans the alternative splicing machinery exhibits a general genetic robustness, and only a minor fraction of genes shows heritable variation in splicing forms and relative abundance. Furthermore we will show some of these genes showing heritable variation in alternative splicing.

Sterken, Mark G. et al. (2015) International Worm Meeting "Transcriptional regulation in stress recovery of Caenorhabditis elegans ."

Sterken, Mark G., Bevers, Roel P.J., Kammenga, Jan E., Riksen, Joost A.G., Snoek, L. Basten

[International Worm Meeting, 2015]

C. elegans serves as an important model for integrative analysis of stress response. Analyzing the transcriptional regulation of stress response and subsequent recovery provides insight into the mechanisms of fitness optimization. Although C. elegans stress responses are well studied, the recovery from stress is poorly understood. We investigated the transcriptional response over the course of a heat-shock experiment in fully sequenced recombinant inbred lines (RILs). This allowed us to identify regulatory loci for stress recovery.The RILs, derived from a cross between N2 and CB4856, were exposed to heat-shock after which they were allowed to recover. In both environments, and in a control environment (no heat stress), genome-wide expression levels were measured. We then performed expression quantitative trait loci (eQTL) mapping across these three environments to identify regulatory loci that contribute to the recovery response.Across all RILs the largest difference in gene expression was found between control and heat-shock. Recovering populations showed patterns of gene expression that were in-between the control and heat-shock. Some RILs had a more control-like gene expression phenotype while other RILs were more heat-shock-like, thus allowing for mapping of loci regulating genetic variation in recovery. We found that genetic variation in gene expression was largely independent of the environment. More specifically, approximately 50% of the eQTL were similar in location and effect over the three environments; therefore did not display interaction with the environment. Only 1% of the genes with an eQTL in all environments displayed environment-specific interaction. The remainder of the eQTL with an environment-specific interaction were specifically detected in one environment.In conclusion, the combination of natural variation and gene expression over the course of a stress response allows for mapping loci underlying regulation in this response. The genes with an eQTL displaying environment interaction are specific for one environment. Incorporation of environment interactions facilitates specifying groups of related eQTL and thereby allows for detailed eQTL network analysis.

Bunte, Myrna et al. (2021) International Worm Meeting "Unravelling the identity of phosphorylcholine-transferring enzymes in C. elegans"

Riksen, Joost, Patrian, Marta, Garcia Perez, Elena, Wilbers, Ruud, Kammenga, Jan, Bunte, Myrna, Schots, Arjen

[International Worm Meeting, 2021]

The attachment of phosphorylcholine on carbohydrates (PC-glycans) is a common modification for nematodes including C. elegans. The presence of PC appears to be important for the development of nematodes and for immunomodulation by parasitic nematodes. PC-glycoprotein ES-62 of Acanthoceilonema vitae directs immune cells towards an anti-inflammatory phenotype and this immunomodulatory property appears to be dependent on the presence of PC-glycans. This makes glycoproteins with PC-glycans interesting therapeutic agents to combat immune disorders such as rheumatoid arthritis, systemic lupus erythematosus and asthma. However, the biosynthetic pathway of PC-glycans is not completely understood, where especially the identity of the PC-transferring enzyme, or PC-transferase, is not fully elucidated. There are strong indications suggesting that fukutin-related genes potentially encode for this PC-transferase. In this study, we examined whether four selected fukutin-related genes of C. elegans (W02B3.4, T07A5.1, T07D3.4 and Y22D7AL.11) are involved in the biosynthetic pathway of PC glycans. With CRISPR/Cas9 technology we created C. elegans knock-out lines for each fukutin-related gene, but no significant reduction of PC was observed. Interestingly, one mutant line of W02B3.4 showed an increase of PC due to a potentially introduced signal peptide, indicating that the W02B3.4 gene could encode for a PC-transferase. Currently, we are combining mutant lines into double/triple/quadruple knock-out lines, which may provide more insight whether these fukutin-related genes encode for PC-transferases. These findings will contribute to our understanding of the pathway for PC-glycan biosynthesis, offering potential opportunities for design and synthesis of PC-glycan therapeutics.

Snoek, L. Basten et al. (2009) International Worm Meeting "Natural variation reveals the autophagy gene unc-51 as a key connection between sex and death in C. elegans."

Vinuela, Ana, Kammenga, Jan E., Doroszuk, Agnieszka, Snoek, L. Basten, Riksen, Joost A.G., Gutteling, Evert W.

[International Worm Meeting, 2009]

The cost of reproduction, i.e. a decreased lifespan due to reproduction, is a crucial trade-off which drives life-history evolution in nearly all species, including humans. A key question in evolutionary and aging research is how this trade-off is regulated in natural populations. Although some induced mutations affecting lifespan also alter the reproductive state, detailed knowledge about the genetic control of the trade-off between lifespan and offspring in natural populations is scant. We combined life-history analysis and mapping of global gene transcription profiles in a C. elegans population of recombinant inbred lines (RILs) obtained from a cross between wildtypes N2 and CB4856. We found a trade-off between lifespan and offspring across the RILs and detected 27 gene transcripts that were highly correlated to both traits in an opposite way. These genes had 20% more eQTLs, i.e. were more regulated, than the less correlated genes. Using a candidate gene approach we show that knocking-down one of these genes, the autophagy gene unc-51, with RNAi increased offspring with 30% while decreasing lifespan with 15%. By introgressing a trans-acting CB4856 locus into N2 we show that the RNAi effect on the trade-off was modulated. Together our results demonstrate that the trade-off between lifespan and offspring can be highly regulated early in life and suggest that natural variation can tune C. elegans life-history by regulating unc-51 transcription during the L3 stage. Interestingly, the results also indicate that RNAi phenotypes depend strongly on the genetic background due to epistatic interactions and trans-acting loci.