Priyadarshini, Monika et al. (2021) International Worm Meeting "Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C. elegans"

Ni, Julie Z., Gu, Sam G., Priyadarshini, Monika, Vargas-Velazquez, Amhed M., Froekjaer-Jensen, Christian

[International Worm Meeting, 2021]

Short guide RNAs target Cas proteins to specific genomic loci enabling efficient gene editing in animals and plants. We have developed a conceptually similar method, called piRNA interference (piRNAi), where 21nt 'guide' piRNAs target the PRG-1 Argonaute to specific mRNAs for transcriptional and post-transcriptional gene silencing of up to four genes simultaneously. Six guide piRNAs are encoded in short synthetic gene fragments and expressed from simple extrachromosomal arrays without cloning. We test specific models for piRNA function, targeting requirements, evolutionary conservation, and inherited silencing using piRNAi. First, we demonstrate that endogenous genes (him-5, him-8, spe-8, and spe-12) are not protected from piRNA-mediated silencing. piRNAi can silence a ubiquitously expressed gfp in the female and male germline, with silencing persisting through embryogenesis until approximately the 100-cell stage. Silencing requires the piRNA pathway (prg-1 and prde-1), but H3K9 methyltransferases (set-25, set-32, met-2), P-granule components (pgl-1, mut-7, znfx-1), and the nuclear RNAi pathway (nrde-1, nrde-2, nrde-3, hrde-1) are not required. Second, piRNAi silences genes robustly when targeting exons but not introns or untranslated regions. Furthermore, piRNA silencing tolerates 1-2 mismatches in the seed region and up to four mismatches overall. Third, we confirm that the piRNA pathway is evolutionarily conserved by silencing a him-5 homolog in C. briggsae using C. elegans piRNA transcriptional units. Finally, we use piRNAi to develop a new assay for studying transgenerational silencing. Previous studies have demonstrated that piRNAs can semi-permanently silence gfp (RNAe or paramutation). We identify two novel genes, him-5 and him-8, that show four and six generations of hrde-1 dependent heritable silencing, respectively, but no paramutation. piRNAi should be a useful tool in C. elegans for transient gene silencing in the germline and may be particularly suitable for studying piRNA biology and heritable epigenetic silencing.

Julien Mouysset et al. (2006) European Worm Meeting "A Conserved Role of C. elegans CDC-48 in ER-Associated Protein Degradation"

Julien Mouysset, Thorsten Hoppe, Christian Kahler

[European Worm Meeting, 2006]

Julien Mouysset, Christian Khler and Thorsten Hoppe. Protein degradation mediated by the ubiquitin/proteasome system is essential for the elimination of misfolded proteins from the endoplasmic reticulum (ER) to adapt to ER stress. It has been reported that the AAA ATPase p97/VCP/CDC48 is required in this pathway for protein dislocation across the ER membrane and subsequent ubiquitin dependent degradation by the 26S proteasome in the cytosol. Throughout ER-associated protein degradation, p97 cooperates with a binary Ufd1/Npl4-complex. In Caenorhabditis elegans two homologs of p97, designated CDC-48.1 and CDC-48.2, exist. Our results indicate that both p97 homologs interact with UFD-1/NPL-4 in a similar CDC-48UFD-1/NPL-4 complex. RNAi mediated depletion of the corresponding genes induces ER stress resulting in hypersensitivity to conditions which induce increased levels of unfolded proteins in the ER lumen. Together, these data suggest an evolutionarily conserved retro-translocation machinery at the endoplasmic reticulum.

Jacques Pecreaux et al. (2006) European Worm Meeting "Mechanical Oscillations during Asymmetric Spindle Positioning Require a Threshold Number of Active Cortical Force Generators"

Jonathon Howard, Frank Julicher, Stephan W. Grill, Anthony A. Hyman, Karsten Kruse, Jens-Christian Roper, Jacques Pecreaux

[European Worm Meeting, 2006]

Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.

Kamat, Shaunak et al. (2013) International Worm Meeting "Modulation of mec-10(d)-induced necrosis by ER chaperone NRA-2."

Driscoll, Monica, Yeola, Shrutika, Bianchi, Laura, Kamat, Shaunak

[International Worm Meeting, 2013]

ER chaperones play a major role in cellular homeostasis by regulating the assembly of polypeptides, intracellular Ca2+ signaling, and degradation of misfolded proteins. Here we report on an ER - resident chaperone NRA-2 that modulates hyper-activated ion channel-induced necrosis by regulating surface expression of a death-inducing DEG/ENaC channel family member subunit MEC-10(d). nra-2 was previously identified in a screen for nicotinic acetylcholine receptor (nAChR) - interacting proteins and was found to regulate the subunit composition of the AChR receptor in C. elegans muscle1. We found that loss of function of nra-2 led to a significant increase in mec-10(d) - induced necrosis in C. elegans touch receptor neurons (TRNs), and this enhancement was rescued by TRN-specific transgenic expression of nra-2. We observed that loss of nra-2 led to a significant increase in surface localization of MEC-10(d)::GFP and a significant decrease in ER localization. Electrophysiological experiments in Xenopus oocytes revealed that NRA-2 suppresses amiloride-sensitive currents induced by hyperactivated MEC channels. Our study suggests a role for NRA-2 as an ER quality control protein that inhibits surface expression of mutant MEC-10(d) channels and is thus neuroprotective against hyperactivated ion channel-induced necrosis. 1. Ruta B. Almedom, Jana F. Liewald, Guillermina Hernando, Christian Schultheis, Diego Rayes, Jie Pan, Thorsten Schedletzky, Harald Hutter, Cecilia Bouzat, and Alexander Gottschalk. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse. The EMBO Journal, 28(17):2636-2649, July 2009.

Karen Erbguth et al. (2009) European Worm Neurobiology Meeting "Applying and combining optogenetic and imaging tools in the C.elegans nervous system."

Alexander Gottschalk, Karen Erbguth, Martin Brauner

[European Worm Neurobiology Meeting, 2009]

Stimuli are evaluated and an animal responds to them with an appropriate behavior. How behavior is guided by neuronal circuits is one of the most fascinating questions approached by neuroscience today. C. elegans provides a good model system for the investigation of neuronal networks, due to its compact nervous system and rather easy access to genetic manipulation. Most physical connections between neurons have been characterized using EM reconstruction, a big step towards an extensive elucidation of the functional connections between these neurons, e.g. by mutant studies, electrophysiology or calcium imaging, and linking them to the animals. behavior. Here we want to combine the method of calcium imaging with activity-regulating light-activated ion channels. This should provide a basic and fast method in order to facilitate an easy and widespread investigation of neuronal networks, which ideally can also be correlated to behavior. Depolarizing or hyperpolarizing neurons with light-sensitive proteins, namely channelrhodopsin or halorhodopsin, and expressing genetically encoded Ca2+ sensors (GECIs) like G-CaMPs or cameleons postsynaptically to monitor the resulting effect on neuronal activity, could be the easiest and minimally invasive way towards this. Here, different approaches in spatial and spectral separation are presented, to overcome the major limitation in combining these tools, i.e. the overlapping excitation spectra. The challenge of specific expression also is addressed within our group (see posters by Christian Schultheis and Cornelia Schmitt). To be able to test our systems in terms of functionality and to have a behavioral readout as a control, we are currently applying our techniques on testing paradigms like mini-networks consisting of just two neurons.

Blaxter, Mark et al. (2015) International Worm Meeting "The Caenorhabditis Genomes Project."

Koutsovoulos, Georgios, Chandrasekar, Sinduja, Stevens, Lewis, Blaxter, Mark

[International Worm Meeting, 2015]

The sequencing of the genome of Caenorhabditis elegans remains one of the milestones of modern biology, and this genome sequence is the essential backdrop to a vast body of work on this key model organism. "Nothing in biology makes sense except in the light of evolution" (Dobzhansky) and thus it is clear that complete understanding of C. elegans will only be achieved when it is placed in an evolutionary context. While several additional Caenorhabditis genomes have been published or made available, a recent surge in the number of available species in culture makes the determination of the genomes of all the species in the genus a timely and rewarding project.We have initiated the Caenorhabditis Genomes Project. From material supplied by collaborators we have so far generated raw Illumina short-insert data for sixteen species. Where possible we have also generated mixed stage stranded RNASeq data for annotation. The data are being made publicly available as early as possible (warts-and-all) through a dedicated genome website at htttp://caenorhabditis.bio.ed.ac.uk, and completed genomes and annotations will be deposited in WormBase as mature assemblies emerge. We welcome additional collaborators to the CGP, whether to assemble new genomes or to delve into the evolutionary history of favourite gene sets and systems.Species sequenced thus far in Edinburgh: Caenorhabditis afra, Caenorhabditis castelli, Caenorhabditis doughertyi, Caenorhabditis guadeloupensis, Caenorhabditis macrosperma, Caenorhabditis nouraguensis, Caenorhabditis plicata, Caenorhabditis virilis, Caenorhabditis wallacei, Caenorhabditis sp. 1, Caenorhabditis sp. 5, Caenorhabditis sp. 21, Caenorhabditis sp. 26, Caenorhabditis sp. 31, Caenorhabditis sp. 32, Caenorhabditis sp. 38, Caenorhabditis sp. 39, Caenorhabditis sp. 40, Caenorhabditis sp. 43.[Samples have been supplied by Aurelien Richaud, Marie-Anne Felix, Christian Braendle, Michael Alion, Piero Lamelza].

Monique van der Voet et al. (2006) European Worm Meeting "The Function of LIN-5 in Chromosome Segregation and Asymmetric Division"

Matilde Galli, Christian Berends, Monique van der Voet, Justin Peters, Sander Van Den Heuvel, Mike Boxem, Marjolein Wildwater, Adri Thomas, Inge The

[European Worm Meeting, 2006]

Monique van der Voet1, Marjolein Wildwater1, Mike Boxem, Justin Peters1, Christian Berends1, Matilde Galli1, Inge The1, Adri Thomas1 and Sander van den Heuvel. The position of the spindle apparatus determines the plane of cell division. During asymmetric division, proper positioning of the spindle is needed in the generation of daughter cells that differ in size, cytoplasmic determinants and developmental fate. We have focused our attention on the C. elegans gene lin-5 to identify novel regulatory mechanisms and molecules that control spindle-mediated functions.. Our previous studies demonstrated that lin-5 is generally required for chromosome segregation and spindle positioning during meiotic and mitotic M phase. lin-5 encodes a coiled-coil protein that localizes to the cell cortex as well as spindle asters, and recruits the G protein regulators GPR-1/GPR-2 to the same locations. Results from our lab as well as others support that LIN-5 and GPR act in concert with the GOA-1/GPA-16 G?i/o subunits of heterotrimeric G proteins in an evolutionarily conserved pathway for spindle positioning. Many aspects of this process remain poorly understood. In particular, it is unclear how polarity determinants at the cortex affect LIN-5/GPR/G?i/o function to create asymmetry in the pulling forces that act on the spindle asters. In addition, the downstream targets of the LIN-5/GPR/G?i/o pathway remain unknown. To better understand the molecular mechanisms of these processes, we continue to use a combined genetic and biochemical approach. We have identified novel candidate partners of LIN-5 through immunoprecipitation from embryonic lysates followed by mass spectrometry. In addition, we identified multiple residues of LIN-5 and GPR that are phosphorylated in vivo. We are currently testing the in vivo relevance of these interactions and modifications and hope to present our results at the meeting.

John Reece-Hoyes et al. (2006) European Worm Meeting "wTF2.0: A Fundamental First Step for Mapping the Caenorhabditis elegans Transcription Factor Localizome"

Jane Shingles, A.J. Marian Walhout, Denis Dupuy, Bart Deplancke, Ian Hope, John Reece-Hoyes, Marc Vidal, Christian Grove

[European Worm Meeting, 2006]

John Reece-Hoyes1, Bart Deplancke2, Jane Shingles1, Christian Grove2, Denis Dupuy3, A.J. Marian Walhout2, Marc Vidal3 and Ian Hope1. The Localization of Expression Mapping Project (LEMP) aims to use cloned C.elegans promoter fragments to generate a genome-wide set of expression patterns termed the Localizome. The Promoterome currently contains promoter fragments of up to 2kb from about 6,500 of the approximately 20,000 identified genes in the C.elegans genome. Using the Multisite Gateway system these promoter fragments are re-cloned upstream of GFP within an unc-119 rescue vector and the resulting constructs used to transform unc-119 mutant nematodes by a modified ballistic DNA transfer technique. The current focus of the LEMP is on C.elegans transcription factor genes with the view that these expression pattern data will be essential for mapping transcription regulatory networks. While C.elegans gene models have undergone continuous refinement, the extant lists of predicted C.elegans transcription factors (collectively referred to as wTF1.0) were in need of updating. We have identified a compendium of 934 transcription factor genes (referred to as wTF2.0) by interrogating Wormbase version 140 for proteins with appropriate Gene Ontology (GO) terms and then manually removed false positives (369) and added false negatives (373). The results of various bioinformatic analyses using wTF2.0 as well as how the list can be used to investigate regulatory networks will be discussed. We expect wTF2.0 to be a dynamic resource due to regular updating of the C.elegans genome annotation as well as changes in the functional annotations of protein domains. wTF2.0 will be accessible to the scientific community via an online interface where input is possible. wTF2.0 provides a starting point to decipher the transcription regulatory networks that control metazoan development and function.

Michael M Francis et al. (2005) International Worm Meeting "The Ror Receptor Tyrosine Kinase CAM-1 is Required for ACR-16 Mediated Synaptic Transmission at the C. elegans Neuromuscular Junction."

David Madsen, Andres Villu Maricq, Michael Jensen, Michael M Francis

[International Worm Meeting, 2005]

Communication between cells of the nervous system involves a diverse array of synaptic proteins whose expression must be temporally and spatially regulated. For example, many neurons express a variety of functionally distinct post-synaptic receptors that must be segregated to specific synapses and subdomains within synapses for proper signaling. How are these receptors differentially regulated? We have begun to examine this problem at the neuromuscular junction (NMJ) of the nematode Caenorhabditis elegans (C. elegans). The C. elegans NMJ is amenable to both electrophysiological and genetic analysis, and at least 3 distinct subtypes of post-synaptic receptors are expressed in C. elegans body wall muscle: a single subtype of GABA receptor and at least 2 subtypes of ionotropic acetylcholine receptor (AChR). The 2 AChR subtypes present at the NMJ can be distinguished pharmacologically on the basis of their differential sensitivity to the cholinergic agonists levamisole and nicotine. Recently, we identified the acr-16 gene as encoding an essential component of nicotine-sensitive receptors (Jensen et al., West Coast C. elegans Meeting, 2004) and have found that the Ror receptor tyrosine kinase CAM-1 is selectively required for normal ACR-16 mediated synaptic currents. The synaptic currents observed in cam-1;acr-16 double mutants phenocopy the currents observed in acr-16 single mutants, suggesting that these genes function in the same genetic pathway. Moreover, the localization of ACR-16 receptors is altered in the body wall muscles of cam-1 mutants. We propose that CAM-1 selectively regulates ACR-16 receptors by stabilizing this receptor subtype in the post-synaptic receptor field. The presence of regulatory proteins such as CAM-1 may allow for independent control of distinct receptor populations at the neuromuscular junction.

Yang, Bing et al. (2015) International Worm Meeting "Identifying genes involved in H3K9me2 regulation in C. elegans."

Xu, Xia, Maine, Eleanor, Yang, Bing

[International Worm Meeting, 2015]

Histones can receive many different modifications, and the presence/absence of certain modifications is implicated in transcriptional control and chromosomal events. One modification, H3K9me2, has been found to be associated with unpaired chromatin during meiosis in different organisms, including C. elegans [1-3]. For example, in the male C. elelgans germline, the single X chromosome is unpaired during pachytene and is detected as a single strong focus of H3K9me2 by immuno-labeling. This interesting phenomenon led to the question of how H3K9me2 marks are specifically targeted to their genomic sites.In order to address this question, we are identifying H3K9me2 methyltransferase (MET-2) co-factors. To do so, we generated antibody against MET-2 and epitope-tagged met-2 transgenes. Using these reagents, we performed immunoprecipitation and tandem MS sequencing to recover potential co-factors. Currently, we are testing a list of potential interactors by two different approaches. First, we utilize RNAi to knock down or a mutation to knock out the candidate gene product and then examine the H3K9me2 distribution pattern in the male germline. Second, we perform reverse co-immunoprecipitation by using antibody against the candidate gene product or epitope-tagged transgene product generated either by Mos1-mediated single copy insertion [4] or CRISPR-Cas9 [5] methods. Among the list of candidate genes that have been tested so far, we have identified several genes whose activity impacts H3K9me2 deposition.References: 1. Shiu, P.K., et al., Cell, 2001. 107(7): 905-16. 2. Kelly, W.G., et al., Development, 2002. 129(2): 479-92. 3. Turner, J.M., et al., Nat Genet, 2005. 37(1): 41-7. 4. Frokjaer-Jensen, C., et al., Nat Meth, 2014. 11(5): 529-534. 5. Ran, F.A., et al., Cell. 154(6): p. 1380-1389.