SN Parrish et al. (1999) International C. elegans Meeting "Investigating the fates of trigger RNAs and target transcripts in RNA-mediated interference"

AZ Fire, SN Parrish

[International C. elegans Meeting, 1999]

We seek to explore the molecular mechanisms responsible for RNA-mediated genetic interference (RNAi). In nematodes, introduction of double-stranded RNA corresponding to a segment of an endogenous genetic locus can result in specific silencing of that locus, essentially producing a knock out phenotype [1]. To date, evidence indicates that this interference reflects a post-transcriptional mechanism, resulting in the loss of the endogenous transcript [2]. Only a few molecules of dsRNA are required per cell to mediate interference, suggesting either an amplification or catalytic aspect of the process [1]. To gain an understanding of the mechanism of RNAi, we are examining the fates of the two key players in this pathway, the endogenous target RNA and the dsRNA effector molecule. First, we are attempting to follow alterations in the endogenous transcripts after the introduction of dsRNA. As a start, we are trying to map possible cleavage events or potential chemical modifications through primer extension and RT PCR of the target transcript. In a complementary set of experiments, we are also examining potential changes in the dsRNA triggering molecule. Through the characterization of the target and effector RNA molecules, we hope to acquire some insight into the mechanism of RNA-triggered silencing. With this knowledge, in conjunction with genetic identification of components in the pathway, it may be possible to unravel the events and intermediates essential for RNAi. 1. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 2. Montgomery, Xu, and Fire. PNAS 95, 15502

Hung, Wesley L. et al. (2019) International Worm Meeting "A compact circuit of interneurons coordinates two central pattern generators to control forward movement."

McWhirter, Rebecca, Wang, Ying, Hung, Wesley L., Meng, Jun, Chang, Maggie M., Sathaseevan, Anson, Luo, Linjiao, Lu, Yangning, Miller III, David M., Zhen, Mei

[International Worm Meeting, 2019]

In C. elegans, there are two central pattern generators (CPGs) that contribute to forward movement - the head CPG that controls the head swing, through currently unidentified neurons, and the body CPGs, which resides in the B-type motor neurons (Xu et al., 2017). The frequency and amplitude of the head swing and body undulation are tightly coupled to allow smooth, sinusoidal forward movement. We show here that the descending interneurons, AVG and RIF, play a critical role in two aspects of forward movement: forward speed modulation and head-body coordination. While AVG is not essential for locomotion, the loss of AVG results in animals with reduced forward speed and an increased tendency to remain in a pausing/resting state. Conversely, optogenetic activation of AVG alone rapidly increases forward velocity. This effect requires gap junction-mediated activation of RIFL/R, which subsequently activates the premotor interneurons AVBL/R to increase activity of the forward movement-driving B motor neurons. When head swinging is inhibited, body undulation is decreased. Conversely, increased head swinging frequency leads to increased body undulation frequency to potentiate higher forward velocity. This suggests communication between the head and body CPGs. Our preliminary results suggest that AVG may also be required to coordinate the head and body CPGs. Activation of AVG was sufficient to drive body bends even when head swinging was inhibited. Increased head swinging is not able change body undulation when AVG is ablated. We propose that the descending interneuron circuit (AVG-RIF-AVB) permits generation of adaptive forward movement by modulating forward speed and linking the head and body CPGs. Xu, T. et. al. PNAS May 8, 2018 115 (19) E4493-E4502

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Stephen J. Nurrish, Emma Hiley, Rachel McMullan, Paul Morrison

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

William M Winston et al. (2001) International C. elegans Meeting "A Screen for Mutations that Enhance and Suppress Systemic RNAi"

Christina Molodowitch, William M Winston, Craig P Hunter

[International C. elegans Meeting, 2001]

One of the hallmarks of RNAi in C. elegans is the systemic effect: injecting gene specific dsRNA into one tissue interferes with the expression of that gene in other tissues (Fire, A. et. al, 1998). In order to elucidate the mechanisms of systemic RNAi, we have developed an assay that has allowed us to identify mutants that are specifically suppressed in their ability to execute systemic RNAi, but are still able to maintain cell autonomous RNAi. This assay has also been used to identify mutants that are apparently enhanced for RNAi. We have screened approximately 600,000 genomes in search of suppressor mutants and approximately 100,000 genomes for enhancer mutants. Towards our goal of identifying the genes necessary for systemic RNAi, we are placing the mutations into complementation groups, mapping representative mutants to linkage groups, and characterizing the gene and tissue specificity of the suppressor mutants. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 19;391(6669):806-11

de Vries CJ et al. (1998) European Worm Meeting "A cytosolic dynein heavy chain is required for sensory neuron structure and function."

Wicks SR, Plasterk RHA, de Vries CJ

[European Worm Meeting, 1998]

We have developed two new assays of nematode chemotaxis (see abstract Stephen Wicks). The first gene identified and cloned using these assays was a cytosolic isoform (1B subfamily) of the dynein heavy chain. Complementation studies show that this dynein heavy chain is the CHE-3 protein. Previously, this isoform of the dynein heavy chain was shown to be expressed during cilia outgrowth in sea urchins (Gibbons, 1994), and involved in the organisation of the Golgi-apparatus in non-ciliated human cell lines (Vaisberg, 1996). GFP expression studies indicate that che-3 is expressed exclusively in all ciliated sensory neurons in the worm. The highest levels of che-3 expression are correlated with outgrowth of cilia. To visualise the structure of ciliated endings we used two GFP markers of amphid and phasmid integrity (GPA-13::GFP and GPA-15::GFP, as integrated transgenes, gifts of Gert Jansen). These markers indicate that the amphid and phasmid cilia are malformed in the worm, and that the degree of disorganisation appears to increase as the animal ages. These results suggest that dynein heavy chain has a function in cilia outgrowth in C. elegans, but has no role in the organisation of the Golgi apparatus in the worm. Gibbons, B.H., Asai, D.J., Wen-Jing, Y., Hays, T.S., and Gibbons, I.R. (1994). Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins. Mol. Biol. Cell 5, 57-70. Vaisberg, E.A., Grissom, P.M., and McIntosh, J.R. (1996). Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J. Cell Biol. 133, 831-842.

Rauthan, M. et al. (2017) International Worm Meeting "MicroRNA regulation of nicotine-dependent behavior in C. elegans."

Gong, J., Rauthan, M., Wescott, S., Ronan, E.A., Liu, J., Xu, X.Z.S.

[International Worm Meeting, 2017]

Tobacco smoking is the leading cause of preventable death in developed nations. Nicotine is the principle addictive substance in cigarettes. Chronic exposure to nicotine up-regulates nAChRs and is thought to play a critical role in the primary steps of nicotine dependence, but the underlying mechanisms are not well understood. We have previously developed a C. elegans model of nicotine dependent behavior, and shown that the nAChR gene acr-15 is required for acute response to nicotine (Feng et al., 2006). Here we identify a key role for microRNA in regulating nicotine-dependent behavior by modulating nAChR expression in C. elegans. Specifically, we show that chronic nicotine treatment down-regulates microRNA machinery, leading to up-regulation of another nAChR gene that is specifically required for nicotine withdrawal behavior. This effect is mediated by a microRNA that recognizes the 3'UTR of nAChR transcripts. These observations uncover an interesting phenomenon that different nAChRs mediate distinct aspects of nicotine dependence in C. elegans. Our results reveal a functional link between nicotine, microRNA, nAChRs, and nicotine-dependent behavior. Reference(s): 1. Feng, Z., Li, W., Ward, A., Piggott, B.J., Larkspur, E., Sternberg, P.W., and Xu, X.Z.S. (2006). A C. elegans model of nicotine-dependent behavior: regulation by TRP family channels. Cell 127, 621-633.

Ward, Alex et al. (2009) International Worm Meeting "Eyeless but not blind: Phototransduction in C. elegans."

Ward, Alex, Inada, Hitoshi, Nishio, Nana, Yu, Yong, Xie, Zhixiong, Xu, X.Z. Shawn, Decaluwe, Brandon, Liu, Jie, Mori, Ikue, Gao, Jingwei, Kang, Lijun, Ma, Di

[International Worm Meeting, 2009]

It has long been assumed that the nematode C. elegans lacks the sense of light, mainly because it lives in the soil and does not have eyes. However, we have recently reported the surprising observation that C. elegans in fact possesses a simple visual system and engages in phototaxis behavior that is mediated by photoreceptor cells and light-sensitive channels [1]. Here we elucidate the phototransduction cascade in C. elegans photoreceptor cells through a combination of electrophysiological and behavioral analysis. As is the case with vertebrate photoreceptor cell rods and cones, C. elegans phototransduction is also mediated by G signaling and cGMP-sensitive CNG channels. Interestingly, instead of signaling through phosphodiesterases (PDEs), light-activated G proteins appear to be coupled to guanylate cyclases that produce cGMP, thereby resulting in opening of CNG channels. Our studies identify a new sensory modality in C. elegans and suggest that animals living in dark environments (e.g. soil and caves) may not be presumed to be blind. Our data also reveal a surprising conservation in phototransduction between vertebrates and C. elegans, indicating that C. elegans represents a powerful genetic model for the study of phototransduction. [1] Ward, A.*, Liu, J.*, Feng, Z., and Xu, X.Z.S. (2008) Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nature Neuroscience 11, 916-22 *co-first authors.

A Fire et al. (1999) International C. elegans Meeting "Structural requirements for the trigger in RNA-mediated interference"

A Fire, J Fleenor, S Parrish

[International C. elegans Meeting, 1999]

This poster will describe in vivo interference activities for a series of modified double-stranded RNAs that are based on segments of the genes unc-22 and gfp . Double stranded RNAs have been shown to act as potent inducers of gene-specific molecular silencing in C. elegans [a]. This process apparently reflects a well conserved control mechanism: recent reports have confirmed the effectiveness of dsRNA-triggered interference mechanisms in a variety of additional species including plant, insect, and protozoan systems [b-e]. By characterizing structural requirements (on the triggering side) for these two well defined segments in C. elegans , we hope to illuminate general features of the interference mechanism. Our experiments involve a variety of manipulations to produce dsRNAs with sequence or chemical alterations, followed by injection into C. elegans and assays for genetic interference. The manipulations are designed to address the following questions: 1. How precise and extensive are requirements for homology with the target gene? 2. What features distinguish the incoming RNA as "foreign"? 3. What chemical groups on the incoming RNA participate in interference? 4. Do the incoming sense and antisense strands have distinct roles in triggering interference? a. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 b. Waterhouse, Graham, Wang, PNAS 95, 13959 c. Ngô, Tschudi, Gull, Ullu, PNAS 95, 14687 d. Kennerdell and Carthew, Cell 95, 1017 e. Misquitta and Paterson, PNAS 96, 1451

Ralph Menzel et al. (2006) European Worm Meeting "PCB52 Induced Gene Expression in the Nematode Caenorhabditis elegans"

Shuang Li, Stephen Sturzenbaum, Hui Ling Yeo, Ralph Menzel, Jana Kulas

[European Worm Meeting, 2006]

Ralph Menzel1, Jana Kulas1, Hui Ling Yeo1, Shuang Li1, and Stephen Strzenbaum2. Polychlorinated biphenyls (PCBs) are ubiquitous organic chemicals which can affect numerous biological responses. Because PCBs are toxic to man, recalcitrance to degradation and persist in the environment, they are listed in the inventory of priority pollutants compiled by numerous Environmental Protection Agencies.. Here we present an investigation of PCB52, an ortho-substituted, non-coplanar 2,2,5,5-tetrachlorbiphenyl. PCB52 affected the reproductive capacity of C. elegans in a strict concentration dependent manner, thereby highlighting its toxicological significance. Based on the calculated EC20 (5 mg/L), whole genomic DNA microarray experiments were performed to identify differentially expressed genes. Using a two-fold increase/decrease cut-off, 1158 genes were up-regulated and 560 down-regulated by PCB52. Up-regulated genes constituted family members of small heat shock proteins, chemoreceptors, nuclear hormone receptors, lipases as well as cytochromes P450 (CYP) and short-chain dehydrogenases/reductases (SDR), which are known to be involved in the biotransformation of persistent organic pollutants. Significant enriched classes of down-regulated genes are involved in organelle biogenesis, cell integrity and further general physiological processes, such as macromolecule metabolism and nucleotide binding.. The induction of five highly differentially regulated gene classes was confirmed by semi-quantitative RT-PCR experiments. Using a multiple RNAi approach the consequences of gene knockdown of single genes as well as entire gene subfamilies was determined. The efficiency of RNAi was proven by RT-PCR. Whilst GST and SDR gene expression knockdown intensify the toxicity of PCB52 (reflected in a reduced reproductive output) the knockdown of several CYP forms increased the reproductive capacity. This may indicate that the lack of CYP gene expression prevents the generation of toxic metabolites.

M Wayne Davis et al. (2005) International Worm Meeting "High-throughput SNP mapping primers and techniques"

M Wayne Davis, Tracey Harrach, Erik M Jorgensen, Marc Hammarlund, Shawn Olsen, Patrick Hullett

[International Worm Meeting, 2005]

Single nucleotide polymorphism (SNP) mapping against CB4856 has quickly become an indispensable technique in the worm field since its introduction by Stephen Wicks et al. in 2001 (Nat Genet 2001 28: 160-164). Because SNPs are present every few thousand base pairs, SNP mapping allows mapping to extremely narrow intervals. Because mapping is done in an essentially wild-type background, very subtle or complex phenotypes can be mapped that could not be mapped using visible mutations. Typically, SNPs that alter a restriction enzyme recognition site (snip-SNPs) have been detected by running digested PCR products on agarose gels. This technique is reliable and readily accessible to any molecular biology lab; however, it requires substantial hands-on time for each reaction, as each SNP typically requires different PCR cycling and restriction digestion conditions. A number of methods have been published that provide automated analysis of SNP genotypes, including florescence polarization (Swan et al. 2002 Genome Res 12: 1100-1105) or capillary electrophoresis (Zipperlen et al. 2005 Genome Biol 6: R19). Although these methods have the advantage of reducing hands-on time for determining SNP genotypes, they require investment in expensive equipment that is not common in many molecular biology labs. In order to provide a more efficient detection protocol for snip-SNPs, we have developed a set of 48 primer pairs that detect SNPs evenly spaced across the C. elegans genome, and that work under identical PCR and restriction digestion conditions. We present a scheme using these reagents to quickly and reliably map mutations using high-throughput equipment and techniques. We have used our SNP mapping techniques to map many phenotypically subtle or genetically complex mutants, including a subtle behavioral defect resulting from three independent mutations. In fact our techniques are simple and relatively inexpensive enough to have been used successfully by undergraduates in a teaching laboratory setting.