Tenenhaus T et al. (1997) International C. elegans Meeting "GENETIC REQUIREMENTS FOR PIE-1 EXPRESSION AND RNA POLYMERASE II INHIBITION IN THE EMBRYONIC GERM LINEAGE OF C. ELEGANS"

Tenenhaus T, Seydoux GC

[International C. elegans Meeting, 1997]

The germline blastomeres (P1-P4) fail to transcribe many messenger RNAs expressed in somatic cells(1). This deficiency in mRNA transcription requires the germline factor PIE-1(1,2), and correlates with the absence of a particular phosphoepitope on RNA polymerase II (RNAP II-H5) (see abstract by Dunn and Seydoux). To begin to understand the genetic requirements for the repression of RNA polymerase II activity in the germ lineage, we have analysed pre-existing embryonic lethal mutations for the expression of PIE-1 and RNAP II-H5 by immunohistochemistry. We find that many mutations (par-1, par-2, par-3, par-4, par-6, cib-1, mes-1, emb-1, emb-6, emb-8, emb-16, emb-20, emb-21, emb-25, emb-27, emb-30, zyg-1, zyg-2, zyg-9) result in the complete or progressive loss of PIE-1 expression in the germ lineage. In all cases, these mutations cause inappropriate expression of RNAP II-H5 in the germ lineage, suggesting that PIE-1 is required in each germline blastomere to repress RNAP II activity. We have also found two mutations (mex-1, mex-3) that cause ectopic expression of PIE-1 in somatic blastomeres; preliminary results suggests that these mutations also affect RNAP II-H5 expression, suggesting that RNAP II-H5 expression is sensitive to variations in PIE-1 levels. Further analysis of these and other mutations will test the hypothesis that PIE-1 is necessary and sufficient to repress RNA polymerase II phosphorylation, and will identify loci required to restrict PIE-1-dependent repression of transcription to the germ lineage. We thank the CGC for strains and Craig Mello, Charlotte Schubert, Jim Priess and Steve Warren for antibodies. 1. Seydoux et al. (1996). Nature 382, 713-716. 2. Mello et al. (1996). Nature 382, 710-712.

Mihail Sarov et al. (2007) International Worm Meeting "Recombineering based high throughput protein tagging in C. elegans."

Mihail Sarov, Francis Stewart, Anthony Hyman

[International Worm Meeting, 2007]

The methods for protein tagging in C. elegans so far have been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. The recent generation of a genomic fosmid library[1] opened the possibility for direct modification of any gene of interest in its native genomic environment by recombineering in E. coli. We recently published a method for precise in vivo engineering of large genomic clones into GFP tagged transgenes for ballistic transformation[2]. Using such transgenes, we reproduce known and document new expression patterns. We further demonstrate that the tagged protein can complement the function of its endogenous counterpart. Applying the latest developments of the recombineering field and introducing several innovations of our own we have now streamlined the protocol to allow rapid 96 well format liquid culture processing in a continuous recombineering pipeline that takes just four days. This unprecedented throughput and the achieved economy of scale allow us to rapidly produce thousands of tagged transgenes. Our method has become the basis for a large-scale project aimed at comprehensive description of the expression pattern (through fluorescent reporters) and the DNA binding sites (through affinity tag based chromatin immunoprecipitation) of more than 400 C. elegans transcription factor genes (as part of the NIH funded ModENCODE program). At the meeting we will discus the advantages of a distributed, community based collaboration model that will bring this technology to any worm lab and will make the genome wide protein tagging a feasible goal. 1. Perkins, J., K. Wong, R. Warren, J.E. Schein, J. Stott, R. Holt, S. Jones, M.A. Marra, and D. Moerman. 2005. A Caenorhabditis elegans fosmid library. In 15th International C. elegans Conference, University of California, Los Angeles. 2. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A.A. Hyman, and A.F. Stewart. 2006. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods 3: 839-844.

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.