Singh, Komudi et al. (2011) International Worm Meeting "Notch signaling regulates chemosensation and behavioral adaptation to stress."

Singh, Komudi, Hart, Anne C.

[International Worm Meeting, 2011]

A role for Notch signaling in development is well established, but several studies now support a role for Notch signaling in the adult nervous system. The molecular mechanisms and targets critical for Notch function in neuronal signaling and behavior are unclear. OSM-11 is one of the DOS (Delta and OSM) motif proteins that act with DSL domain ligands to activate LIN-12 Notch receptors in vulval cell fate specification (Komatsu et al., 2008). Loss of osm-11 causes defective osmotic avoidance (Wheeler & Thomas, 2006) and, as we report here, impairs avoidance of 1-octanol and increases dispersal due to increased locomotion speed and decreased spontaneous reversal rates. The role of osm-11 and Notch pathway genes in octanol avoidance response was addressed in depth and suggested a redundant role for lin-12 and glp-1 Notch receptors in this chemosensory behavior. Furthermore, dissection of cellular site of action for Notch receptors suggested that the two receptors functioned in different/non-overlapping population of neurons. Studies in the past have shown that increased external osmolarity (hyperosmotic stress) leads to increased synthesis of the osmolyte glycerol in adult C. elegans (Lamitina, et al 2006). Under these unfavorable conditions, animals might also be expected to alter their behavior. We find that adaptation to hyperosmolarity impairs octanol response and increases dispersal, which are defects observed in osm-11 animals. Additionally, we find that the physiological and behavioral consequences of hyperosmotic stress adaptation are genetically separable. The role of Notch pathway genes in these potentially adaptive changes is under investigation.

Weber, Katherine P. et al. (2011) International Worm Meeting "High throughput identification of mutations affecting aggregation using a novel Illumina sequencing analysis pipeline."

Baier, Felix, Weber, Katherine P., de Bono, Mario, De, Subhajyoti

[International Worm Meeting, 2011]

The standard C. elegans reference strain, N2, feeds in isolation, but wild-caught C. elegans aggregate on food or after starvation. Failure of N2 to aggregate is due to a gain-of-function mutation in the neuropeptide receptor npr-1: when this gene is knocked out N2 strongly aggregate. To identify molecules that promote or inihibt aggregation we mutagenized, in a series of independent screens, npr-1(null) or N2 animals and isolated mutants that suppressed or enhanced aggregation. This approach has been very fruitful: we have identified a number of genes involved in aggregation behavior. However, traditional genetic mapping and gene cloning is time-consuming, and recent work from other groups has shown that Illumina sequencing can be used to more rapidly identify mutations (Sarin et al. (2008); Doitsidou et al. (2010); Zuryn et al. (2010)). To increase the throughput of aggregation gene cloning, we have established a pipeline to sequence more than 200 uncloned modifiers of aggregation using Illumina technology. Many of these mutants have been mapped roughly (usually to ~1 Mb) so examining their genome sequences allows us to rapidly identify the lesion responsible for their phenotype. We have adapted a protocol from Dan Turner at the Sanger Institute to make amplificaton-free libraries and sequenced these on both the Illumina Genome Analyzer II (GAIIX) and on the Illumina HiSeq machine. The HiSeq has increased our output approximately six-fold. We have also assembled a bioinformatics pipeline to analyse the gigabases of data generated. Our downstream analysis can be completed on an entire flow cell (7 lanes of sequence) in approximately 2 days: it uses the BWA (Burrows Wheeler Algorithm) program to align filtered sequence reads to the reference genome, further filters the alignment output for high confidence polymorphisms, and then implements a custom pipeline to highlight which of these introduce exonic and regulatory changes, indicate genes affected and predict specific coding changes.

Kim, Woojin et al. (2017) International Worm Meeting "OrthoList 2.0: an update and further analysis of human and C. elegans gene conservation."

Greenwald, Iva, Shaye, Daniel, Kim, Woojin

[International Worm Meeting, 2017]

C. elegans is an important model for genetic studies relevant to human biology and disease. Therefore, it is critical to have a complete and up-to-date reference of genes whose functions are most likely to be conserved between C. elegans and humans. We previously assessed homology between C. elegans and humans by performing a meta-analysis of results from four orthology-prediction methods (Ensembl Compara1, OrthoMCL2, InParanoid3 and Homologene4), and defined a compendium of 7,663 C. elegans genes (~40% of the protein-coding genome) with clear human orthologs, which we called "OrthoList"5. We subsequently released an online version (Shaye, et. al, WBG 2013) to enhance the accessibility and utility of OrthoList. To update OrthoList, we analyzed the most recent results from the four original orthology-prediction programs, which are based on newer sequence data and updates to the prediction algorithms, as well as from two additional programs (OMA6 and OrthoInspector7). Updates to the four original programs added 383 new genes (~5.0%), and, surprisingly, removed 581 genes (~7.6%) from OrthoList 1.0. Results from the two new programs only added 115 (~1.5%) new genes to OrthoList, and re-introduced 70 of the 581 that were lost due to updates to the four original programs (bringing the total loss of genes from OrthoList 1.0 to 511, or ~6.7%). These results suggest that querying new methods does not greatly change the number of genes predicted to be conserved between humans and C. elegans. Moreover, our analysis of the changes (additions and losses) between OrthoList 1.0 and 2.0 highlights the importance of regularly assessing orthology, rather than relying on a single "snapshot" in time, or a single program, for determining the most complete list of genes conserved between C. elegans and human. We are updating the online OrthoList, and enhancing functionality by streamlining the user interface and adding the ability to search based on protein domains. 1) Vilella, et al., 2009. 2) Li, et al., 2003. 3) Ostlund, et al., 2010. 4) Wheeler, et al., 2007. 5) Shaye and Greenwald, 2011. 6) Altenhoff, et al., 2011. 7) Linard, et al., 2011. 8) Lanjuin, et al., 2003.

Nathalie Pujol et al. (2007) International Worm Meeting "Genetic dissection of the transcriptional response to wounding and fungal infection."

Andrew CHISHOLM, Nathalie Pujol, Daniel Wong, Jonathan Ewbank, C. Leopold Kurz, Sophie Cypowyj

[International Worm Meeting, 2007]

Drechmeria coniospora infection leads to the rapid up-regulation of certain nlp and cnc/caenacin genes that can be visualized using reporter genes (see abstract by Zugasti et al.). Infection involves a breach of the cuticle and hypodermal cells, so we tested the effect of wounding worms. A needle prick results in a rapid but transient increase in pnlp-29::gfp expression, readily detectable by microscopy and by the UBI Biosort within one hour. We also used a laser to provoke internal injury and, under these sterile conditions, saw similar rapid and large increases in pnlp-29::gfp expression. High salt also provoked a high level of pnlp-29::gfp expression. Certain mutants, including dpy-9 and osm-11, but not dpy-13 or dpy-17, have an elevated level of GDPH-1 and therefore an elevated level of intracellular glycerol and a higher capacity to resist osmotic stress (Lamitina et al. PNAS 2006; Wheeler & Thomas Genetics 2006). The former mutants also showed a high level of pnlp-29::gfp expression, while the latter did not. On the other hand, pgdph-1::gfp that is induced by salt (Lamitina et al) was not induced by infection or injury, suggesting that the response to osmotic stress is distinct, even if we found that full expression of both nlp-29 and gdph-1 requires the GATA factor ELT-3. We performed a screen for mutants exhibiting abnormal nlp-29 up-regulation upon infection. nipi-1 and nipi-2 (no induction of peptide after Drechmeria infection) block the response to infection and injury, but show a normal response to osmotic stress. They also block the induction of a pcnc-2::gfp reporter normally provoked by fungal infection (see Zugasti et al.). The nipi-3 mutant, on the other hand, has no effect on the induction of the pcnc-2::gfp reporter and only affects the infection-induced up-regulation of nlp-29. We are currently SNP mapping nipi-1 and have narrowed its position to a small region on LGIV close to dpy-9. We have identified NIPI-2 (see Ziegler et al.) and found NIPI-3 to be a protein kinase. Its catalytic domain is similar to the human Tribbles homologue 1 that functions in p38 signalling. Evidence suggests that NIPI-3 acts upstream of TIR-1, conceivably occupying an equivalent function to UNC-43, but in a pathway that responds specifically to infection.

Volkers, R. J. M. et al. (2013) International Worm Meeting "Gene-environment interactions drive genomic and transcriptomic diversity in wild Caenorhabditis elegans populations."

Chen, W., Schulenburg, H., van Hellenberg hubar, C. J., Coopman, R., Sterken, M. G., Kammenga, J. E., Volkers, R. J. M., Braekman, B. P., Snoek, L. B.

[International Worm Meeting, 2013]

RJMV and LBS contributed equally to this work. Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research in the nematode C. elegans has been instrumental for unravelling many genotype-phenotype relations. But almost all studies, including forward and reverse genetic screens, are dominated by investigations in one canonical single strain. In order to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map, it is important to study these relations in wild populations. Results: We used multiple wild strains freshly isolated from local habitats to investigate the gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness and behavioural traits. These showed a direct link with the original habitat of the strains. The separation between the isolation sites was prevalent on all chromosomes, chr. V contributing the most. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Moreover, we show that genomic and transcriptomic diversity was driven by genes involved in gene-environment interactions, like c-type lectins and fbox genes. Conclusions: Importantly, where wild C. elegans strains show a broad range of genotype-phenotype relations the widely studied canonical genotype N2 covers only a diminutive part of the myriad of genotype-phenotype relations which are present in the wild. Funding: RJMV and LBS: NWO-ALW (project 855.01.151) NEMADAPT, RC and BPB: ESF-EEFG (09-EuroEEFG-FP-002 / G.0998.10N) NEMADAPT. JEK: ERASysbio-plus ZonMW project GRAPPLE (project nr. 90201066). MGS: Graduate School Production Ecology & Resource Conservation. WC and HS: NEMADAPT (DFG grant SCHU 1415/11-1).

Nicholas Duclos et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identifying Genes Required for Signaling via the CAM-1 Receptor Tyrosine Kinase"

Nicholas Duclos, Andres Maricq, Michael Francis, Penelope Brockie

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Signaling at synapses requires that cells within the nervous system adopt the correct cell identity. Thus, both pre- and post-synaptic cells must express a specific compliment of molecules and establish the appropriate synaptic connections. We would like to understand how the C. elegans Ror receptor tyrosine kinase (RTK), CAM-1, contributes to these processes. Ror RTKs are highly expressed in the vertebrate nervous system as well as other tissues, yet the mechanism of signaling by these RTKs remains unclear. cam-1 encodes the only Ror RTK in C. elegans and is normally expressed in both the nervous system and in body wall muscles. In neurons, it has been associated with EGL-20, a Wnt signaling molecule, in regulating nervous system development (Forrester et al.). At the neuromuscular junction, it has been implicated in the stabilization and localization of the acetylcholine receptor subunit ACR-16 (Francis et al.). Overexpression of CAM-1 in either muscles or neurons produces dramatic overgrowth of cellular processes. In order to identify gene products that regulate Ror RTKs, we have initiated a forward screen for modifiers of CAM-1 function that takes advantage of this overexpression phenotype. Overexpression of CAM-1 in the command interneurons produces a dramatic change in cell morphology that is easily identifiable by the presence of numerous highly branched ectopic neuronal outgrowths. To identify genes that function in the same pathway as cam-1, we are screening for suppressors of this phenotype. We expect to find genes that encode upstream regulators of CAM-1 signaling, such as potential ligands, as well as downstream components of the CAM-1 signaling pathway. The identification of these genes will yield valuable insights in to the mechanisms of Ror RTK signaling. Francis MM, Evans SP, Jensen M, Madsen DM, Mancuso J, Norman KR, Maricq AV. The Ror Receptor Tyrosine Kinase CAM-1 Is Required for ACR-16-Mediated Synaptic Transmission at the C. elegans Neuromuscular Junction. Neuron 46: 581-594Forrester WC, Kim C, Garriga G. The C. elegans Ror RTK CAM-1 collaborates with EGL-20/Wnt to regulate cell migration. 168: 1951-1962

Wang, Lin et al. (2017) International Worm Meeting "Dissecting the roles of the ADAM10 metalloprotease SUP-17 in the BMP signaling pathway in Caenorhabditis elegans."

Liu, Jun, Liu, Zhiyu, Shi, Herong, Wang, Lin

[International Worm Meeting, 2017]

BMPs (Bone Morphogenetic Proteins) belong to the TGFbeta superfamily of ligands, and mediate a highly conserved signal transduction cascade. Upon ligand binding, type II receptors phosphorylate type I receptors, which in turns phosphorylate R-Smads (receptor regulated Smads). Phosphorylated R-Smads then complex with co-Smad (common mediator smad) and enter the nucleus to regulate gene transcription with different co-factors. BMPs play important roles in developmental and physiological processes. Malfunction of the pathway in humans can cause various disorders, such as skeleton diseases, heart diseases and cancers. So it is critical to strictly regulate the level of BMP signaling spatiotemporally. Using a highly specific genetic screen, we have identified several modulators of the BMP pathway in C. elegans. These include the Neogenin homolog UNC-40 [1], two paralogous tetraspanin proteins, TSP-12 and TSP-14, and an ADAM10 metalloproteinase (A Disintegrin and Metalloproteinase 10) SUP-17 [2]. We use the CRISPR/Cas9 system and tagged the endogenous TSP-12, SUP-17 and UNC-40 proteins with different fluorescence tags. We found that TSP-12 is localized to the cell surface and intracellular vesicles, and that TSP-12 can bind to SUP-17 and promote its cell surface localization. We have genetic evidence and preliminary biochemical evidence showing that UNC-40 is one, but not the only, substrate of SUP-17 in BMP signaling. We are currently identifying additional substrate(s) of SUP-17 in BMP signaling. Our work highlights the importance of intracellular signaling and protease processing in the regulation of BMP signaling. [1] Tian C, Shi H, Xiong S, Hu F, Xiong WC, Liu J. The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans. Development. 2013;140(19):4070-80. doi: 10.1242/dev.099838. PubMed PMID: 24004951; PubMed Central PMCID: PMCPMC3775419. [2] Wang L, Liu Z, Shi H, Liu J. Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans. PLoS Genet. 2017;13(1):e1006568. doi: 10.1371/journal.pgen.1006568. PubMed PMID: 28068334; PubMed Central PMCID: PMCPMC5261805.