Kerkhof, E. et al. (2013) International Worm Meeting "An integrative D. discoideum, C. elegans and D. rerio approach to assess developmental and reproductive toxicity."

Teunis, M., Pieters, R., Kerkhof, E., Croes, C., Lokman, C., Woollard, A., Wildwater, M., Bosch, R.

[International Worm Meeting, 2013]

An important goal of toxicity testing is to assess and identify health risks associated with exposure to existing and newly introduced chemicals. Of particular importance is developmental and reproductive toxicity (DART), because of the long-term and delayed characteristics of the toxicity. DART testing often involves time- and money-consuming animal studies. Much research activity has been focused on development of alternative test strategies in non-animal systems like single cells, or in non-mammalian multicellular organisms, but these studies showed that data often do not provide a complete picture of DART. We use the combinatorial power of three distinct non-mammalian organisms (D. rerio, C. elegans and D. discoideum) and embryonic stem cells (ES). Developmental pathways in these test systems are well defined but not yet properly related to DART. Combinatorial bioinformatics and laboratory approaches are used to assess inter-species conservation of molecular pathways involved in DART. DART datasets are analysed for affected molecular pathways/profiles, indicative of the functionality of key developmental and reproductive processes such as cell migration, differentiation and pattern formation. Conservation of those pathways in our test systems are assessed. This survey will illuminate conserved molecular mechanisms/pathways that are crucial for DART assessment and lead to identification of proper biomarkers. Our combinatorial approach will support the elucidation of novel DART testing pipelines in non-mammalian organism based systems.

Kerkhof, Engelien et al. (2015) International Worm Meeting "C. elegans as robust, high throughput in vivo system for hazard assessment."

Lokman, Christien, Wildwater, Marjolein, Pieters, Raymond, Kerkhof, Engelien

[International Worm Meeting, 2015]

Innovative toxicity test systems that enable identification of adverse outcome pathways (AOP) relevant for humans, are highly interesting to allow improved hazard identification and characterization. The nematode Caenorhabditis elegans (C. elegans) has been thoroughly established as a developmental biological test system and shows a high level of conserved molecular pathways and cellular mechanisms compared to man. The behavior of the 959 somatic cells of which the hermaphrodite nematode exists have been mapped in detail. The nematode transparency, short turnover time, small size, conserved organs, high throughput screening ability and the available technological resources allow rapid, reproducible testing of compounds and easy identification of AOPs and molecular initiating events (MIE). Furthermore, in the view of developmental and reproductive toxicology (DART) which is amongst the most difficult toxicological outcomes to assess, and which requires the highest numbers of vertebrates for testing, C. elegans can be a suitable alternative test system as it has a full life cycle. We have tested 22 well documented and well characterized DART compounds from the ECHA, Staatscourant and EPA list and show that the C. elegans test model has a predictive value of ~80% for DART related compounds, indicating the suitability of the C. elegans test system for DART assessment. To understand AOPs underlying DART toxicity in C. elegans, a RNA-sequencing approach was followed. RNA sequencing showed alterations in gene expression profiles of genes orthologous to humans. Data analysis consisted of community based profiling and a posttranscriptional gene silencing approach (RNAi). Two individual genome wide RNAi libraries are available in C. elegans that allow down regulation of gene expression of most genes in the C. elegans genome. If particular up regulated genes (identified by RNA-sequencing) are critically responsible for DART effects, posttranscriptional gene silencing by RNAi should reduce the DART effects, thus enabling the identification of critical AOP genes. Alternatively, community based alignment of DART responses enables the identification of global gene expression patterns responsive to DART effects.

Louter - van de Haar, J. et al. (2017) International Worm Meeting "Combinatorial model organism strategy to predict developmental and reproductive toxicology (DART)."

Dirks, R.P., Pijnenburg, D., Kerkhof, E., Yebra - Pimental, E., Louter - van de Haar, J., Woollard, A., Whale, G.F., Pears, C., Spaink, H.P., Racz, P.I., Pieters, R., Maxwell, S., Ruijtenbeek, R., Rooseboom, M., Smulders, C., Currie, R., Warren, E.

[International Worm Meeting, 2017]

There is an increasing requirement for innovative toxicity test systems that are translational to humans yet allow reduction, refinement and replacement (3Rs) of animal use. It has been shown that "stand alone test models" are not sufficient for hazard assessment and combinatorial testing with multiple 3R models (especially for developmental and reproductive toxicology (DART)) is required to improve potential hazard identification. We introduce a combinatorial strategy using zebrafish (Danio rerio) larvae, nematodes (Caenorhabditis elegans) and social amoebae (Dictyostelium discoideum) as an innovative toxicity test system to enable identification of adverse outcome pathways (AOP) for human hazard identification and characterization. The selected test systems allow high-throughput screening and facilitate rapid, reproducible testing of compounds both on an organismal phenotypic level as well as on a molecular level (transcriptomics and activity profiling of kinases). We have tested 42 DART positive compounds and each of the species showed high sensitivity and specificity levels indicating high predictive power. Of 42 DART positives only 1 compound was missed by all three test systems. Nine compounds were used in a detailed molecular proof of principle study to investigate molecular effects of DART chemicals in the test systems. Overlapping molecular responses of DART compounds could be identified within species amongst the selected set of DART compounds and also across species. Toxicogenomic profiling and hazard assessment revealed that the individual species are promising predictors for DART with clear added value.

Xiong, H. et al. (2017) International Worm Meeting "Identification of optimized C. elegans strains for chemical toxicity assessment assays."

Xiong, H., Woollard, A., Pears, C.

[International Worm Meeting, 2017]

Newly developed chemical compounds to be released into the environment, including those used by agrochemical, cosmetic, petrochemical and pharmaceutical industries, must be subjected to stringent regulatory toxicity assessments. Currently, the potential of each chemical to induce developmental and reproductive toxicity (DART) is routinely assessed over at least two generations of rats or rabbits. Such assays require large numbers of animals, making these assessments expensive (in terms of both animals and chemicals) and time-consuming (representing an opportunity cost). We have established a simple and fast DART prediction assay using C. elegans to screen for high risk DART compounds early in the product development pipeline, which can then be eliminated from animal testing. An adaptation of the assay allows exposure to volatile compounds or those with low solubility. However, the worm's robust cuticle presents a significant barrier to chemical uptake, increasing the amount of chemical required and reducing the value of the assay for regulatory assessment. We took advantage of available mutants with altered cuticle properties to identify sensitized strains more suitable for toxicity assays. We tested agmo-1, bus-5, bus-8, bus-16 and bus-17 for chemical permeability of their cuticle, setting this advantage in terms of chemical sensitivity against a possible reduction in physiological fitness. These criteria narrowed the candidate group down to three bus mutant strains, which were subjected to toxicity assays using a collection of hydrophilic and hydrophobic chemicals present in agrochemicals, cosmetic products, petrochemicals and pharmaceuticals, and known to cause DART in mammals. All three bus mutant strains are sensitized to chemical exposure compared to wildtype worms across a range of chemicals. With regard to our evaluation criteria and all toxicity assays, we identified bus-5 as the ideal sensitized strain for chemical toxicity assessments that is capable of detecting DART effects at lower chemical levels, including some masked in wildtype worms, thus minimizing the probability of a false-negative outcome.

Froehlich, Jonathan et al. (2021) International Worm Meeting "Parallel genetics of regulatory sequences using induced genome editing"

Herzog, Margareta, Uyar, Bora, Rajewsky, Nikolaus, Akalin, Altuna, Theil, Kathrin, Froehlich, Jonathan, Glazar, Petar

[International Worm Meeting, 2021]

Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. The functions of regulatory elements must ultimately be understood within their genomic environment and developmental- or tissue-specific contexts. Here, we used induced Cas9 expression and multiplexed guide RNAs in C. elegans to create hundreds of mutations in enhancers, promoters and 3′ UTRs of 16 genes in parallel. We then analyzed the resulting complex populations by either selecting for phenotypic traits or reporter expression, or by DNA- or RNA amplicon sequencing of bulk samples. We developed a software pipeline, crispr-DART, to analyze targeted sequencing and describe the characteristics of >12,000 dsDNA break-induced indel mutations. We also analyzed the lin-41 3′ UTR and found that the two let-7 miRNA binding sites can function independently and that one of the sites is more important for mRNA repression and phenotype. In summary, our approach enables highly parallelized functional analysis of regulatory sequences in vivo.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.

Kiontke, Karin C. et al. (2009) International Worm Meeting "New Caenorhabditis species: phylogeny and evolution."

Fitch, David H. A., Felix, Marie-Anne, Kiontke, Karin C.

[International Worm Meeting, 2009]

Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and seven from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last few years. There is no indication that we are even close to knowing all species in this genus.