Castillo-Quan, Jorge et al. (2021) International Worm Meeting "KLF Transcription Factors Regulate SKN-1 Activity in C. elegans"

Moroz, Natalie, Barrett, Julia, Castillo-Quan, Jorge, Blackwell, T. Keith, Carroll, Allison, Gilmore, Katherine, Johnson, Emma

[International Worm Meeting, 2021]

SKN-1/Nrf (NF-E2 related factor) is a transcription factor that regulates redox regulators, such as glutathione S-transferase, and lipid metabolism in Caenorhabditis elegans (C. elegans). SKN-1 counters damage caused by reactive oxygen species, which is often implicated in age-associated diseases such as Alzheimer's disease, and arteriolosclerosis. Recently, SKN-1 was shown to mediate fat accumulation and oxidative stress resistance in worms missing germline stem cells (GSC(-)). The mechanism by which this occurs is still unknown. A preliminary genome-wide screen identified Kruppel-like family of transcription factors (KLF) as possible mediators of SKN-1, under GSC(-) conditions. KLF proteins have roles in adipogenesis and autophagy, suggesting they may mediate SKN-1 regulated fat accumulation and oxidative stress resistance in GSC(-) worms. To evaluate the role of KLF transcription factors, we assessed the role of klf-1 and klf-2 in SKN-1 activation, SKN-1 mediated stress resistance, lipid metabolism, and longevity. We found that the knockdown of klf-1, under GSC (-) conditions, decreased SKN-1 activity, specifically the SKN-1c isoform, eliminated stress resistance, reduced lipid accumulation, and eliminated lifespan extension. klf-2 had less of an effect on SKN-1 however the knockdown of klf-2 surprisingly increased lipid accumulation under both basal and GSC(-) conditions suggesting that while klf-1 positively regulates SKN-1c, klf-2 may negatively regulate SKN-1c, possibly through lipids.

Ben Lehner et al. (2006) European Worm Meeting "Systematic Mapping of Genetic Interactions in C. elegans Suggests a New Paradigm for Human Genetic Disease"

Ben Lehner, Angelo Fortunato, Julia Tischler, Andrew G. Fraser, Catriona Crombie

[European Worm Meeting, 2006]

Ben Lehner, Catriona Crombie, Julia Tischler, Angelo Fortunato and Andrew G. Fraser Most heritable traits, including disease susceptibility, are affected by the interactions between multiple genes. However, we still understand very little about how genes interact since only a minute fraction of possible genetic interactions have been explored experimentally. To begin to address this, we are using RNA interference to identify genetic interactions in C. elegans, focussing on genes in signalling pathways that are mutated in human diseases. We tested ~65,000 pairs of genes for possible interactions and identify ~350 genetic interactions. This is the first systematically constructed genetic interaction map for any animal. We successfully rediscover most components of previously known signalling pathways; furthermore, we verify 9 novel modulators of EGF signalling. Crucially, our dataset also provides the first insight into the global structure of animal genetic interaction maps. Most strikingly, we identify a class of highly connected ''hub'' genes: inactivation of these genes greatly enhances phenotypes resulting from mutations in many different pathways. These hub genes all encode chromatin regulators, and their activity as genetic hubs appears conserved across metazoans. We propose that these genes function as general buffers of genetic variation and that these hub genes will act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.

Barrett, Alec et al. (2021) International Worm Meeting "Integrating bulk and single cell transcriptomics for accurate detection of tissue-specific gene expression"

Li, Mingfeng, Barrett, Alec, Weinreb, Alexis, Hammarlund, Marc, McWhirter, Rebecca, Hobert, Oliver, Miller III, David M., Sestan, Nenad, Taylor, Seth R., Varol, Erdem

[International Worm Meeting, 2021]

Advances in RNA-seq for bulk and single cell (sc) approaches have produced increasingly fine dissections of the C. elegans transcriptome. Although both techniques can yield transcriptomes for individual cell types, each comes with strengths and weaknesses. scRNA-Seq affords high resolution, but suffers from dropout, leading to false negatives. Bulk sequencing detects more genes, but suffers from contaminating cell types, resulting in false positives. In this work we integrated these orthogonal approaches to improve the accuracy of both methods. We used bulk samples collected for specific neuron types and sc datasets for all C. elegans neurons and additional non-neuronal cells (1). We used sc data to estimate contamination in each bulk sample, and developed novel methods for removing these gene counts. In one approach we used linear histogram matching to scale sc counts, and subtracted putative contamination using data from non-neuronal clusters. In another approach we used bootstrapping to estimate gene level contributions from target and contaminating tissues in sc data and apply them to bulk counts, providing a bootstrap sample distribution of corrected expression data. We assessed these approaches in two ways: 1) Measuring improvements in calling genes with known expression in all neurons; 2) Examining effects on eliminating genes expressed exclusively in contaminating tissues. We found that our analysis reduced false positives, while maintaining robust true positive detection, thus offering a unique strategy for utilizing complementary bulk and sc RNA-Seq data sets to enhance the accuracy of cell-specific expression profiling data. 1. Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al. Molecular topography of an entire nervous system. bioRxiv. 2020:2020.12.15.422897.

Hallam SJ et al. (1997) International C. elegans Meeting "syd-1 May Regulate Type D Neuron Synaptic Specificity"

Jin YS, Hallam SJ

[International C. elegans Meeting, 1997]

Previously we reported the isolation of syd-1, a gene that appears to control the synaptic specificity of type D GABAergic neurons1. In syd-1(ju2) mutants DDs innervate dorsal body wall muscles in early L1 stage. In older larval and adult stages VDs make fewer synapses to ventral body wall muscles. The overall cell morphology of D neurons in syd-1 mutants appears normal. syd-1 mutants coil ventrally when backing and also exhibit mild defects in egg laying. We cloned syd-1 by germline transformation rescue. The predicted gene product for syd-1 bears sequence homology to GTPase activating proteins (GAPs), such as p50rho, N-Chimaerin and BCR. However, the GAP domain of syd-1 appears to be divergent3. Compared with recent structural studies of the p50rhoGAP domain, a conserved Arg85 residue, implicated in binding G proteins and enhancing their intrinsic GTPase activity, has been replaced with Val in SYD-1, suggesting that syd-1 may define a novel family of GAPs. The GAP domains of N-Chimaerin and BCR regulate the small GTPases Rac and Cdc42Hs, both of which have been implicated in the clustering of integrins and membrane ruffling2. The sequence similarity between syd-1, BCR and N-Chimaerin hints at the possibility that SYD-1 may regulate the the subcellular organization of actin in the D neurons. We are in the process of identifying the molecular lesions in syd-1 alleles and determining the cells in which it is expressed . 1 S. Hallam and Y. Jin (1996) WBG vol 14, no.5. 2 C.D. Nobes and A. Hall (1995) Cell 81, 53-63. 3 T. Barrett et, al., (1996) Nature 385, 458-61

Bo Wang et al. (2006) European Worm Meeting "Multi-Parameter Axial Profiling of Transgenic C. elegans Expressing Fluorescent Proteins from Various Cell-Specific, Tissue Specific and Developmentally Regulated Promoters"

Rock Pulak, Yanping Zhang, Mariya Lomakina, Bo Wang, Julia Thompson, Bruce Holcombe, Michael Herman

[European Worm Meeting, 2006]

Bo Wang1, Julia Thompson1, Yanping Zhang2, Michael Herman2, Mariya Lomakina1, Bruce Holcombe1, Rock Pulak1 . The COPAS Biosort instrument automates the analysis, sorting, and dispensing of all stages of C. elegans, measuring the animals size and the intensity of expressed fluorescent markers. Once analyzed, animals can be selected according to user defined criteria, and then dispensed into multi-well plates for high throughput screening or collected in bulk for further analysis. With this technology, time required for large scale screening for certain changes in the optical properties of the animals, such as changes in the levels of expression of a fluorescent protein, can be dramatically reduced and human error minimized. Recent enhancements to an add-on module, called the Profiler II, have been tested for its ability to collect positional information of fluorescent expression. The instrument can simultaneously collect fluorescence information in three separate regions of the spectrum. Here we show that the instrument can analyze multi-colored transgenic animals and can be used to compare the amounts and relative positions of expression of two or three different colors of fluorescence. Furthermore, this technology can be used to screen for independent changes in the intensity or position of each reporter protein. We have tested various transgenic animals expressing green, yellow and/or red fluorescing proteins from a collection of promoters that include myo-2, str-1, egl-17, mab-5, and various others, separately and in certain combinations. We present some proof of principle examples of how these could be used in genetic screens.

Julia Tischler et al. (2006) European Worm Meeting "Analysing the Redundancy of Duplicated Genes and the Evolutionary Conservation of Genetic Interactions Using Combinatorial RNAi in C. elegans"

Andrew G Fraser, Julia Tischler, Ben Lehner

[European Worm Meeting, 2006]

Julia Tischler, Ben Lehner and Andrew G Fraser Systematic analyses of loss-of-function phenotypes have been carried out for almost all genes in S. cerevisiae, C. elegans, and D. melanogaster, and there are major efforts to make a comprehensive collection of mouse knockouts. While such studies greatly expand our knowledge of single gene function, they do not address redundancy in genetic networks, nor do they attempt to identify genetic interactions. Developing tools for the systematic mapping of genetic interactions is thus a key step for exploring the relationship between genotype and phenotype. We thus sought to establish protocols for targeting multiple genes simultaneously by RNA interference (RNAi) in C. elegans to provide a platform for the systematic identification of genetic interactions in this key animal model system.. We set up conditions for RNAi that allow us to target multiple genes in the same animal (combinatorial RNAi) in a high throughput setting and to detect the great majority of previously known synthetic genetic interactions. We then used this assay to test the redundant functions of genes that have been duplicated in the genome of C. elegans since divergence from either S. cerevisiae or D. melanogaster, and identified 16 pairs of duplicated genes that are at least partially functionally redundant. Intriguingly, 14 of these redundant gene pairs were duplicated before the split of C. elegans and C. briggsae 80-110 million years ago. Our data provide the first systematic investigation into the redundancy of duplicated genes in any organism and strongly support population genetics models, which suggest that redundancy can be maintained over substantial periods of evolutionary time.. Furthermore, we set out to test whether systematically compiled yeast genetic interaction data can predict genetic interactions in the worm. We will present these data.

Julia Grabitzki et al. (2006) European Worm Meeting "Identification of O-linked N-acetylglucosamine Modified Proteins in Caenorhabditis elegans"

Rudolf Geyer, Brigitte Schmitz, Michael Ahrend, Julia Grabitzki, Gunter Lochnit

[European Worm Meeting, 2006]

Julia Grabitzki1, Michael Ahrend2, Brigitte Schmitz2, Rudolf Geyer1 and Gnter Lochnit1. The posttranslational modification N-acetylglucosamine O-glycosidically linked (O-GlcNAc) to serine and threonine residues of proteins has been shown to be ubiquitous amongst eukaryotic proteins of the nucleus, cytoskeleton, cytoplasm, and has also been detected on cytosolic tails of membrane proteins [1]. O-GlcNAcylated proteins can form reversible multimeric complexes with other polypeptides or structures. The modification is often accompanied by phosphorylation/ dephosphorylation. O-GlcNAc can act either simultaneously or in a reciprocal fashion with phosphorylation. According to the Yin-Yang hypothesis, the phosphorylation/ dephosphorylation regulates O-GlcNAc-modified protein function (z.B. signal transduction and protein-protein interaction) in concert with phosphorylation [2-4]. The addition of O-GlcNAc to and the removal from the protein backbone is dynamic with rapid cycling in response to cellular signals or cellular stages.. Despite the fact, that Caenorhabiditis elegans is the best studied model organism, there have been no studies on O-GlcNAcylation in this organism so far. Therefore, to elucidate the role of O-GlcNAcylation, we investigated the proteome of a C. elegans mixed-stage population by two-dimensional gelelectrophoresis and subsequent western-blotting with the O-GlcNAc-specific antibody CTD 110.6 for the occurrence of this modification and identified the modified proteins by mass-spectrometry. We detected and identify several O-GlcNAc-modified proteins in C. elegans. Most of the identified proteins are involved in metabolic pathways. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic occurrence of the O-GlcNAc modification.. References:. [1]. Rex-Mathes, M., J. Koch, Werner, S., Griffith, L. S and B. Schmitz. 2002. Methods Mol Biol 194: 73-87.. [2] Zachara, N.E. and G.W. Hart, Chem Rev, 2002. 102(2): p.431-8.. [3]. Griffith, L. S. and B. Schmitz. 1999. Eur J Biochem 262(3): 824-31.. [4] Wells, L. and G. W. Hart. 2003. FEBS Lett 546(1): 154-8.

Brown, Andre et al. (2017) International Worm Meeting "A common format for worm tracking data."

Lee, Chee Wai, Hokanson, Jim, Kerr, Rex, Currie, Michael, Brown, Andre

[International Worm Meeting, 2017]

Many labs, including ours, have built a wide variety of worm trackers. These have a wide range of capabilities, from high-resolution imaging of single animals during calcium imaging, to very low-resolution imaging of animals as points. This diversity of capability enables the C. elegans community to address a wide range of problems at an appropriate scale. Most of these trackers also produce some data that is very similar to that of other trackers: animal position or spine, for example. Unfortunately, each tracker uses its own format to store data, so that any later analysis, despite being general in nature, cannot be performed on data from different machines. As the volume of tracking data grows, and the variety of downstream analysis methods expands, this limitation will pose an increasingly large barrier to replication of and extension of existing work across different labs. To address this issue, we have defined the Worm Common Object Notation, a set of rules for how to write tracking data in the ubiquitous JSON format, so that it can be easily shared between labs. To facilitate easy adoption of WCON, we have further written software in a variety of languages that will read or write data in WCON format. So far, we have implementations in Python, Scala, Matlab, and Julia, and wrapper libraries for Octave, R, and Java to use one of the main implementations. Additionally, the Tracker Commons project of which WCON is a part contains a small but rapidly growing set of pre-packaged analysis tools for routine manipulation of worm tracking data. We will also maintain a list of other WCON-compatible analysis tools as they become available. If you are involved in worm tracking, we invite you to adopt WCON and help make C. elegans behavioral data widely accessible. WCON is developed under the open source Tracker Commons project of the OpenWorm Foundation. We invite contributions and improvements!

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Gunter Lochnit, Rudolf Geyer, Michael Ahrend, Julia Grabitzki

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.

Varol, Erdem et al. (2021) International Worm Meeting "A computational approach linking neuron-specific gene expression with connectivity"

Miller, David, Reilly, Molly, Taylor, Seth, Paninski, Liam, Poff, Abigail, Varol, Erdem, Hammarlund, Marc, Vidal, Berta, Litwin-Kumar, Ashok, Basavaraju, Manasa, Tavazoie, Saeed, Xu, Chuan, Cros, Cyril, Barrett, Alec, Cook, Steven, Sestan, Nenad, Rafi, Ibnul, Glenwinkel, Lori, Oikonomou, Panos, Weinreb, Alexis, Yemini, Eviatar, Hobert, Oliver, Santpere, Gabriel, Abrams, Alexander, McWhirter, Rebecca

[International Worm Meeting, 2021]

There is strong prior evidence for genetic encoding of synaptogenesis, axon guidance, and synaptic pruning in neural circuits. Despite these foundational observations, the transcriptional codes that drive neural connectivity have not been elucidated. The C. elegans nervous system is a particularly useful model for studying the interplay between genetics and connectivity since its wiring diagram is highly stereotyped and uniquely well-defined by electron microscopy. Furthermore, recent evidence in C. elegans has suggested that a unique combination of transcription factors specifies each of the 118 neuron classes[1]. Motivated by evidence for the stereotypy of neural circuits and for the genetic encoding of neural identity, we introduce a novel statistical technique, termed Network Differential Gene expression analysis (nDGE), to test the hypotheses that neuron-specific gene expression dictates connectivity. Specifically, we test the hypothesis that pre-synaptic neural identity is defined by a "key" gene combination whose post-synaptic targets are determined by a "lock" gene combination. For our approach, we utilize neuron-specific gene expression profiles from the CeNGEN project[2] to investigate transcriptional codes for connectivity in the nerve ring[3]. We hypothesize that the expression of specific cell adhesion molecules (CAM) among synaptically-connected neurons drives synaptic maintenance in the mature nervous system. We posit that CAMs mediating synaptic stability would be more highly expressed in synaptically-connected neurons than in adjacent neurons with membrane contacts but no synapses. Thus, for each neuron, we compare the expression of all possible combinations of pairs of CAMs in the neuron and its synaptic partners relative to the neuron and its non-synaptic adjacent neurons. Two independent comparisons are generated, one for presynaptic neurons and a second result for postsynaptic neurons. Our nDGE analysis reveals that specific combinations of CAMs are correlated with connectivity in different subsets of neurons and thus provides a uniquely comprehensive road map for investigating the genetic blueprint for the nerve ring wiring diagram. Open source software of Network Differential Gene Expression (nDGE) is publicly available at https://github.com/cengenproject/connectivity_analysis along with a vignette showcasing the CAM results. 1. Reilly, M. B., Cros, C., Varol, E., Yemini, E., & Hobert, O. (2020). Unique homeobox codes delineate all the neuron classes of C. elegans. Nature, 584(7822), 595-601. 2. Taylor, S. R., Santpere, G., Weinreb, A., Barrett, A., Reilly, M. B., Xu, C. Varol, E., ... & Miller, D. M. (2020). Molecular topography of an entire nervous system. bioRxiv. 3. Cook, S. J.,... & Emmons, S. W. (2019). Whole-animal connectomes of both Caenorhabditis elegans sexes. Nature, 571(7763), 63-71.