Fire A et al. (1994) Worm Breeder's Gazette "Vectorology I: New lacZ Vectors (Building a Better Gene Trap)."

Fire A, Xu SQ

[Worm Breeder's Gazette, 1994]

Vectorology I: New lacZ vectors ("building a better gene trap") Andrew Fire and SiQun Xu Carnegie Institution of Washington, Baltimore, Md 21210

Niethammer P (2014) Dev Cell "Stress heals."

Niethammer P

[Dev Cell, 2014]

Reactive oxygen species (ROS) are generated as a response to cellular stress and regulate processes including cellular signaling and wound healing. In this issue of Developmental Cell, Xu and Chisholm (2014) demonstrate that mitochondrial ROS are required for proper wound healing in Caenorhabditis elegans through controlling the redox state of actin regulators.

Bonini NM (2001) Dev Cell "Stores to die for."

Bonini NM

[Dev Cell, 2001]

Genes that regulate apoptosis are well defined. In contrast, it has not been clear what genes are central to necrotic cell loss. In the September 27th issue of Neuron, Xu et al. (2001) report a critical role for genes that regulate storage and release of Ca2+ from the endoplasmic reticulum as important to necrotic-like cellular degeneration in Caenorhabditis elegans.

Fire A et al. (1998) Nature "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans."

Fire A, Mello CC, Kostas SA, Montgomery MK, Driver SE, Xu SQ

[Nature, 1998]

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.AD - Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA. fire@mail1.ciwemb.eduFAU - Fire, AAU - Fire AFAU - Xu, SAU - Xu SFAU - Montgomery, M KAU - Montgomery MKFAU - Kostas, S AAU - Kostas SAFAU - Driver, S EAU - Driver SEFAU - Mello, C CAU - Mello CCLA - engPT - Journal ArticleCY - ENGLANDTA - NatureJID - 0410462RN - 0 (Calmodulin-Binding Proteins)RN - 0 (Helminth Proteins)RN - 0 (Muscle Proteins)RN - 0 (RNA, Antisense)RN - 0 (RNA, Double-Stranded)RN - 0 (twitchin)SB - IM

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

Yee, Callista et al. (2021) International Worm Meeting "Coordinated regulation of synaptic genes during development: a tale of a transcription factor and an mRNA export complex"

Xu, Rita, Boyle, Alan, Xiang, Yang K, Shen, Kang, Matus, David Q, Yee, Callista, Medwig-Kinney, Taylor N

[International Worm Meeting, 2021]

Synapses are assembled during neuronal development and consist of a pre- and postsynapse, which are built from hundreds of proteins. While the molecular composition and architecture of pre- and postsynapses has been widely explored, much less is known about how synaptogenesis is regulated at the level of gene expression. Is there a mechanism that coordinates the expression of functionally related proteins such that they are ready to assemble into higher order structures concomitantly? In order to identify new players in presynaptic gene expression, our lab has conducted genetic screens and identified mutations that affect two subunits of the THO Complex (THOC), an RNA-binding complex implicated in mRNA export (Maeder et al., 2018). We have previously shown that in dopaminergic neurons, THOC is the primary machinery used for the export of synaptic transcripts. Mutation of THOC results in retention of these synaptic transcripts in the nucleus, while non-synaptic transcripts are largely unaffected and are exported normally. To date, it remains unclear how THOC is able to select such a specific set of targets for RNA export. Are there proteins that interact with THOC to instruct this behavior? Mass spectrometry studies conducted in mammalian systems revealed a novel interaction between EVI1/egl-43 and THOC (Ivanchoko et al., 2019). EGL-43 possesses 6 zinc fingers, all of which are highly homologous to the zinc fingers of EVI1. We performed ATAC-seq on sorted worm neuronal nuclei and identified putative regulatory regions of synaptic genes. Interestingly, we found that many of these regions contained the consensus DNA sequence that is recognized by EVI1. Using CRISPR/Cas9, we have tested the requirement of some of these binding sites and find that they are required for normal presynaptic gene expression. Depletion of EGL-43 through RNAi or auxin-mediated degredation similarly resulted in loss of synaptic markers in PDE. Using single molecule pulldown, we were able to detect weak but significant binding between EGL-43 and THOC. Taken together, our data suggests that EGL-43 could potentially be a link between THOC and its synaptic targets. References: Maeder et al., 2018, Cell 174, 1436-1449 Ivanchoko et al., 2019, Nucleic Acids Research 47, 1225-1238

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

Voia, Anna et al. (2022) MicroPubl Biol

Labbe, Jean-Claude, Voia, Anna, Poupart, Vincent

[MicroPubl Biol, 2022]

Plants of the Mimosa genus are studied and used for their bioactive properties. Among bioactive phytochemicals are quercetin and myricetin, which have been demonstrated to act as antioxidants in many contexts (Taheri et al. 2020; Xu et al. 2019), including in C. elegans (Buchter et al. 2013; Grnz et al. 2012; Sugawara and Sakamoto 2020). Other phytochemicals from these plants, such as the triterpenoid phytosterol lupeol, have been shown to have antioxidant properties but have not been as extensively characterized in model organisms (Liu et al. 2021; Shai et al. 2009). Here we employed the nematode C. elegans to assess whether lupeol elicits antioxidant response in vivo . Using reporter assays for oxidative stress, we find that treatment of animals with lupeol rescues some of the effects resulting from treatment with the prooxidant paraquat. Our results demonstrate that lupeol displays antioxidant properties in vivo in C. elegans .

Barbagallo B et al. (2010) J Neurosci "A dominant mutation in a neuronal acetylcholine receptor subunit leads to motor neuron degeneration in Caenorhabditis elegans."

Francis MM, Climer J, Barbagallo B, Prescott HA, Boyle P

[J Neurosci, 2010]

Inappropriate or excessive activation of ionotropic receptors can have dramatic consequences for neuronal function and, in many instances, leads to cell death. In Caenorhabditis elegans, nicotinic acetylcholine receptor (nAChR) subunits are highly expressed in a neural circuit that controls movement. Here, we show that heteromeric nAChRs containing the acr-2 subunit are diffusely localized in the processes of excitatory motor neurons and act to modulate motor neuron activity. Excessive signaling through these receptors leads to cell-autonomous degeneration of cholinergic motor neurons and paralysis. C. elegans double mutants lacking calreticulin and calnexin-two genes previously implicated in the cellular events leading to necrotic-like cell death (Xu et al. 2001)-are resistant to nAChR-mediated toxicity and possess normal numbers of motor neuron cell bodies. Nonetheless, excess nAChR activation leads to progressive destabilization of the motor neuron processes and, ultimately, paralysis in these animals. Our results provide new evidence that chronic activation of ionotropic receptors can have devastating degenerative effects in neurons and reveal that ion channel-mediated toxicity may have distinct consequences in neuronal cell bodies and processes.

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