Crittenden, Sarah L. et al. (2013) International Worm Meeting "Progress on developing the Q system to study GSCs and their control."

Crittenden, Sarah L., Kimble, Judith, Mohanty, Ipsita

[International Worm Meeting, 2013]

The Q system (Potter et al., 2010) permits inducible gene expression in C. elegans (Wei et al., 2012) and is similar in principal to the widely used GAL4-UAS system. The QF transcriptional activator directs transcription via an upstream activating sequence (QUAS). But in addition, another protein, QS, can inhibit QF activation and QS repression can be relieved by a small molecule, quinic acid or QA. QA is non-toxic and can be fed to worms (Wei et al., 2012). This inducible system has the potential to control target genes in both space and time. A chemically inducible method to control gene expression in specific cells at specific times would be tremendously useful for analyses of germline stem cells (GSCs) and their niche, the somatic distal tip cell (DTC). To this end, we are generating mosSCI insertion transgenes that rely on DTC-specific and GSC-specific regulatory sequences to drive QF expression. We are also generating a QUAS driven nuclear GFP (fused to H2B). Once we have the QF/QUAS pair working well for spatial regulation, we will add the QS/QA pair for temporal regulation. Preliminary experiments are promising and results will be shared at the meeting.

She, Xingyu et al. (2015) International Worm Meeting "Applying a Spatial-Temporal Controlled Gene Expression System to Aging Research."

She, Xingyu, Hansen, Malene

[International Worm Meeting, 2015]

C. elegans has been an effective model organism for identifying genes with important physiological functions, including in aging. However, to fully dissect gene function an effective spatial-temporal controlled gene expression system is needed. In C. elegans, several protocols have been put forward for this purpose, including driving temporal gene expression using heat shock inducible promoters (Bacaj et al. 2007), and achieving tissue-specific gene expression using site-specific recombination methods (Cre-loxP, FLP-FRT). However, none of these systems have been used for simultaneous temporal- and spatial control and it is uncertain how applicable they would be for aging studies (see Xu & Kim, 2011). While several chemicals (e.g., tetracycline, RU486) are utilized to induce gene expression in other systems, such protocols are not available in C. elegans. However, the Shen lab recently adapted the Q system to C. elegans, a system in which inducible gene expression is obtained by the chemical quinic acid (Wei et al. 2013). This binary system, originally discovered in the fungus Neurospora crassa, is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through the timing of quinic acid supplementation, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF (Wei et al. 2013). We are interested in applying the Q system for our research on genes with roles in aging. Towards this goal, we are focusing on three main aspects. First, we have tested the effects of quinic acid and the Q system on C. elegans lifespan and stress resistance and detected no apparent alterations on longevity or fitness, suggesting that the Q system is suitable for aging studies. Second, we are driving the elements of the Q system with tissue-specific promoters to test inducibility in major tissue types of adult animals. Preliminary results indicate that the Q system can be used to induce expression in selected tissues, including neurons and muscle. Third, we are creating a library of Gateway-compatible plasmids that contain the various elements of the Q system and tissue-specific promoters to expedite the generation of Q system transgenic strains for future gene-specific projects. We will present our current progress towards applying the Q system as a powerful tool for spatial- and temporal analysis of gene function in adult C. elegans. This work will also be discussed at the "Spatial and temporal analysis of gene function in C. elegans" workshop.

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."

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

[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

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

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

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

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

J Fleenor, S Parrish, A Fire

[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

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

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

[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.

Zhang, Qing et al. (2011) International Worm Meeting "Dissecting the molecular pathway underlying IFT modeling."

Hu, Jinghua, zhang, yuxia, Wei, Qing, Zhang, Qing

[International Worm Meeting, 2011]

Cilia act as motile or sensory devices on the surfaces of most eukaryotic cells, and cilia dysfunction results in a variety of severe human pathologies, collectively termed ciliopathies. Phylogenetically conserved intraflagellar transport (IFT) machinery, which is composed of IFT-A and IFT-B subcomplexes, mediates the bidirectional movement of IFT cargos that are required for the biogenesis, maintenance, and signaling of cilia. However, an understanding of how IFT particles are assembled at the ciliary base and turned around at the ciliary tip remains elusive. Our previous data suggested that IFT-B component DYF-2 is the key factor involved in regulating IFT modeling at both the ciliary base and ciliary tip. From a mutagenesis screen for C. elegans mutants with defective IFT modeling at the ciliary tip, we identified jhu616, which encodes a mutant DYF-2 with a G361R alteration in the conserved WD40 domain. Further studies indicated that DYF-2 and BBS (Bardet-Biedl syndrome) proteins coordinate IFT assembly/reassembly at the ciliary base/tip (See Wei Q. et al's abstract from the Hu lab). To further dissecting the molecular pathway underlying IFT modeling, we performed a suppressor screen of the jhu616 allele. Dye-filling (Dyf) assay was used to assess cilia integrity. jhu616 animals are 100% Dyf. In ~300,000 haploids screened, we retrieved 7 independent strains that could rescue the Dyf phenotype of jhu616 allele. Sequencing results showed that no new mutations were introduced into dyf-2 locus, indicating that the suppressors contain mutations in other players involved in IFT modeling. We are mapping the suppressors and looking forward to fully dissecting the molecular pathway underlying IFT modeling and ciliogenesis.

She, X. et al. (2017) International Worm Meeting "Applying the Q system for spatiotemporal gene expression in adult C. elegans."

Hansen, M., She, X.

[International Worm Meeting, 2017]

While C. elegans has long been on the leading edge of aging research, a spatial-temporal controlled gene expression system is currently lacking to fully understand tissue- and adult-specific functions of genes modulating lifespan and healthspan in this organism. The Shen lab recently adapted the so-called Q-system for inducible gene expression in a subset of C. elegans neurons (Wei et al., Nature Methods, 2013). Originally discovered in the fungus Neurospora crassa, this binary system is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through quinic-acid supplementation to the media plates, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF. Here, we further developed the Q-system and tested it for its usability in C. elegans aging studies. First, we drove the elements of the Q-system with tissue-specific promoters expressing in several major tissue types to show the inducibility of the system in adult animals. Our results indicate that the Q-system can be used to induce expression in such tissues. Second, we tested the effects of quinic acid and Q-system components on C. elegans lifespan and stress resistance and detected no apparent effects on longevity or fitness, suggesting that the Q-system can be used for lifespan studies. We will present current progress towards applying the Q-system as a powerful system for spatial- and temporal analysis of gene function in adult C. elegans.