Zheng Y et al. (1998) West Coast Worm Meeting "TURNING ON NEURONS: A NEW GENETIC TOOL FOR STUDYING NEURONAL CIRCUIT FUNCTION IN C. ELEGANS"

Brockie PJ, Maricq AV, Zheng Y

[West Coast Worm Meeting, 1998]

One of the best described neuronal circuits in C. elegans is the touch withdrawal circuit (Chalfie et al.; Kaplan et al.). Laser or genetic ablation of the "command" interneurons has identified the contribution of specific neurons to the control of locomotion. Genetic analysis has identified genes, such as the glr-1 glutamate receptor, that are required for withdrawal from tactile stimuli sensed by ASH. Further analysis of this neuronal circuit awaits more refined electrophysiological recording techniques, as well as genetic techniques, to modify neuronal function. We have now developed a novel genetic approach to activate neurons. The technique permits perturbing a circuit by activation. In conjunction with ablation data, our approach provides insights into how simple circuits can control complicated behaviors. Recently, Zuo et al. discovered that a defect in the d2 glutamate receptor caused the Lurcher mutation in mice. These mice are ataxic and exhibit apoptotic death of cerebellar neurons--presumably secondary to a ligand-independent activation of the receptor. The mutation in d2 causes an alanine to threonine change in transmembrane III of the glutamate receptor. This alanine residue is found in all identified glutamate receptors from worms to mammals. To modify neuronal function in C. elegans, we generated a transgenic strain that expresses a lurcher variant form of Glr-1 [Glr-1(A/T)]. Transgenic worms that express Glr-1(A/T) are severely impaired in locomotion. Rather than moving forward with infrequent backward interruptions, the glr-1(lurcher) worms ratchet rapidly back and forth and are incapable of normal forward locomotion. The neurons that express Glr-1(A/T) are not dead, but presumably are excessively activated. Our results suggest that the command interneuron circuitry acts also as a clock that regulates the relative time that a worm spends moving forwards or backwards. Depolarization of the circuit increases the clock frequency. How this occurs is unclear. Perhaps the circuitry can be driven into bistable states controlling forward and backward movement. Depolarization hastens the switch from one state to the other. Glr-1(A/T) activates specific neurons and results in a marked increase in reversals of movement. This strain will enable genetic screens to identify interacting genes that could act to regulate clock frequency. We are screening for modifiers of this activating mutation. We expect to identify both enhancers and suppressors. Increased reversals may represent enhancers of neurotransmitter release or postsynaptic response. We hope to also identify mutations in this sensitized background that cause excitotoxicity of the postsynaptic neurons. Suppressors of the lurching phenotype may modify the electrical activity of the presynaptic neurons, decrease synaptic release by the presynaptic neurons, or reduce the synaptic response of the postsynaptic neurons.

Y Zheng et al. (1999) International C. elegans Meeting "glr-3 Encodes a non-NMDA Type Glutamate Receptor that is Widely Expressed in the Nervous System of C. elegans"

AV Maricq, Y Zheng

[International C. elegans Meeting, 1999]

We have shown that a defect in a glutamate receptor subunit, glr-1 , differentially affects the transmission of sensory input from the polymodal sensory neuron ASH (see also Hart et al, 1995). Mutants are defective in the backing response to tactile stimuli, but have normal responses to osmotic stimuli. One possible explanation for this interesting phenotype is that other glutamate receptors are required for the proper transmission of sensory input from ASH. One non-NMDA subtype of glutamate receptor that we have identified is glr-3 . We are most interested in glutamate receptors found in the command interneurons. Based on transgenic strains that express fusions of glr-3 with GFP, we have shown that glr-3 is expressed in the command interneurons as well as many other neurons, including neurons in the ventral cord. We have isolated a full-length cDNA for glr-3 that encodes a 932 a.a. receptor with 4 predicted transmembrane (TM) domains. GLR-3 has many of the topological features of glutamate receptors and shows strong conservation in the predicted TM domains. It has some unusual features however, including a KG sequence in the highly conserved residues, (Q/R Q), that are found in TM II of most non-NMDA receptors. The GFP expression data obtained from transgenic strains shows that glr-3 is expressed in some of the same command interneurons that express glr-1 , glr-2 , glr-4 , nmr-1 , and nmr-2 . To evaluate the contribution of this receptor to behavior we have intiated experiments designed to generate a null mutation. We have identified two strains that contain a Tc1 transposable element in the glr-3 gene: one inserted in an intron, the other in an exon. We are now in the process of generating deletion mutations by imprecise excision of these Tc1 elements. We will examine the behavior of these mutants as well as the behavior of strains with combinations of receptor mutations. How does glr-3 contribute to the function of these neurons? To begin to address this question we will determine the subcellular location of this receptor subunit. In particular, we are interested in determining whether glr-3 co-localizes with any of the other receptor subunits. This localization data will help predict which receptor subunits form functional heteromeric receptors.

Rui Wang et al. (2005) International Worm Meeting "Identifying genes required for the function of glutamatergic synapses"

Rui Wang, Andres Maricq, Yi Zheng, Penelope Brockie

[International Worm Meeting, 2005]

Glutamatergic neurotransmission accounts for the vast majority of excitatory synaptic signaling in the vertebrate central nervous system. An important problem in neurobiology is to understand how glutamatergic synapses are formed and regulated. Recently, molecules required for activity of ionotropic glutamate receptors (iGluRs) have been identified, shedding more light on the mechanisms underlying the regulation of iGluRs. However, we still have much to learn regarding how iGluRs are synthesized, assembled, localized to the surface of appropriate synapses, and how receptor function is regulated once at the synapse. To address these questions, we have undertaken a genetic screen for modifiers of a dominantly active variant of the GLR-1 iGluR subunit in C. elegans. GLR-1 is expressed in many neurons, including the command interneurons that control locomotion. Introducing an alanine (A) to threonine (T) mutation in a highly conserved site of GLR-1 causes transgenic lurcher worms that express the GLR-1(A/T) variant to rapidly switch between forward and backward movement (Zheng et al., 1999). This dramatic phenotype provided a tool used in a suppressor screen to identify genes required for GLR-1 function. Thus, the sol-1 (suppressor of lurcher) gene was isolated (Zheng et al., 2004). In an attempt to enhance the suppressor screen, we have engineered a second mutation, glutamine (Q) to tyrosine (Y), in the GLR-1(A/T) variant. The GLR-1(Q/Y) mutation inhibits receptor desensitization (Brockie et al., 2001). Transgenic worms that express GLR-1(Q/Y; A/T) have a super-lurcher phenotype, switching between forward and backward movement at a higher frequency than transgenic GLR-1(A/T) worms. We have also tagged GLR-1(Q/Y; A/T) with GFP to facilitate the characterization of genes identified in the suppressor screen. We have screened approximately 15,000 mutagenized GLR-1 (A/T; Q/Y) haploid genomes and isolated 5 candidate suppressors of the super-lurcher locomotion phenotype. We are currently mapping and cloning these genes. Brockie et al. (2001) Neuron 31: 617-30 Zheng et al. (1999) Neuron 24: 347-61 Zheng et al. (2004) Nature 427: 451-7

Sato, Manami et al. (2015) International Worm Meeting "Expression pattern of C. elegans Y RNA homologs."

Ushida, Chisato, Kihara, Shinya, Sato, Manami

[International Worm Meeting, 2015]

Y RNA is a small structured ncRNA of about 100 nt in length. This RNA binds to Ro60 protein, which is a target of autoimmune disease antibody in patients with systemic lupus erythematosus and Sjogren's syndrome. Several lines of evidence suggest that the role of Y RNA and Ro60 function in the quality control of structured ncRNAs in cells under stress conditions. It is also indicated that vertebrate Y RNAs function in the initiation of DNA replication without Ro60. However, the molecular mechanisms of these functions and the contribution of Ro60/Y RNP to the autoimmune disease are still unclear. C. elegans genome encodes one Ro60 homolog (ROP-1) and 19 Y RNA homologs (1 CeY RNA and 18 sbRNAs). Other animals also have several Y RNA homologs, but C. elegans is the first example which has more than 5 Y RNA homologs encoded in the genome. Here we show the expression pattern and the cellular localization of these Y RNA homologs in C. elegans examined by the RNA fluorescent in situ hybridization (RNA-FISH). The signals of 14 homologs were detected in the intestinal cytoplasm. The signals of two other homologs were detected in the germ cytoplasm. The remaining three could not be detected, probably because they present in too low abundance to be detected by RNA-FISH. All 19 Y RNA homologs have the structural elements required for the binding of ROP-1. In other organisms, Ro60 binding stabilizes Y RNAs in cells. To know whether C. elegans Y RNA homologs also stabilized by the presence of ROP-1, we examined RNA-FISH of the Y RNA homologs against a mutant strain MQ470, which has a transposon insertion in the middle of the ROP-1 gene and lacks ROP-1 proteins in the cell. As expected, all Y RNAs examined so far decreased extensively. These were confirmed by northern hybridization. The results suggest that several C. elegans Y RNA homologs are expressed in a tissue-specific manner and most Y RNA homologs are stabilized by ROP-1 binding as well as those in other organisms.

Nancy L Price et al. (2003) International Worm Meeting "The utilization of Caenorhabditis briggsae as a host model to study bacterial pathogenesis"

Ann M Rose, Nancy L Price

[International Worm Meeting, 2003]

Caenorhabditis elegans has now been established as a host model for studies of infectivity by bacterial pathogens including Pseudomonas aeruginosa, Salmonella typhimurium, Burkholderia pseudomallei, and Enterococcus faecalis. However, virulence determinants of bacterial pathogens are regulated by temperature and environmental conditions, thereby limiting the use of C. elegans which cannot survive in temperatures higher than 25oC. This study shows that the related species, C. briggsae survives better than C. elegans on bacterial culture media at higher temperatures and describes the effects on C. briggsae of mammalian enteric pathogens, primarily Yersinia enterocolitica. C. briggsae grown on Y. enterocolitica accumulated bacteria in the gastrointestinal tract of the worm, resulting in decreased life-span and progeny fitness in a depleted-calcium environment. The mechanisms of the interaction are as yet unknown as both virulent and avirulent strains of Y. enterocolitica displayed the similar results. Investigations were undertaken to examine whether the shortened life span could be attributed to starvation, toxicity, or possible infection. Starvation effects was determined not to be the sole cause of pre-mature worm death as C. briggsae grown on Y. enterocolitica survived several days longer than starved worms. A bacterial mixing experiment using both Y. enterocolitica and E. coli OP50 shortened the worm life span compared to feeding on Y. enterocolitica alone, suggesting a possible toxic effect. However worms feeding on Y. enterocolitica and later shifted to E. coli OP50 resulted in reversion of the survival curve to that of worms feeding on E. coli OP50 alone, indicating that the detrimental effect of Y. enterocolitica was reversible. Fluorescent labeling of bacterial strains with a lacZ-GFP reporter gene demonstrated that Y. enterocolitica, but not E. coli OP50, is retained in the gut, a result which is compatible with a molecular interaction between Y. enterocolitica and nematode cellular components. These findings suggest that Y. enterocolitica may cause an infection within the nematode gastrointestinal tract and provide an assay for genetic dissection of the molecular basis of pathogenesis.

Mellem JE et al. (1998) West Coast Worm Meeting "ELECTROPHYSIOLOGICAL ANALYSIS OF NEURONAL CIRCUITRY SUBSERVING LOCOMOTION."

Mellem JE, Zheng Y, Brockie PJ, Maricq AV

[West Coast Worm Meeting, 1998]

Neuronal circuits have been described that transduce mechanosensation into movement (Chalfie et al., Wicks and Rankin). In all instances, coordinated movement requires the command interneurons AVA, AVB, AVD, AVE, and PVC. Our goal is to undertake an electrophysiological analysis of this circuit. We would like to understand at the neuronal level the following observations: 1) Genetic analysis of the touch response has revealed that Glr-1, a non-NMDA-type ionotropic glutamate receptor, is expressed in the command interneurons. Mutations in this receptor disrupt the normal withdrawal response to tactile stimuli delivered to the nose of the worm (Maricq et al.; Hart et al.). How is signaling from the sensory neuron ASH to the command interneurons disrupted? 2) NMDA-type ionotropic glutamate receptors, Nmr-1 and Nmr-2, are also expressed in the command interneurons (see abstract by Brockie et al.). A deletion mutation of nmr-1 disrupts a clock-like pattern generator that controls roaming behavior in the worm. How do NMDA receptors contribute to membrane excitability and synaptic function? 3) Using a novel genetic technique, we believe that we can depolarize neurons in this circuitand perhaps any circuit (see abstract by Zheng et al.). The phenotypic consequences are striking, not immediately predicted, and not well understood. The worms constantly reverse direction. Are these neurons chronically depolarized? How does depolarization modulate reversal behavior? To understand how simple circuits work, we are undertaking a systematic electrophysiological analysis of the command interneuron circuitry. Our initial efforts will be directed towards evaluating the intrinsic membrane properties of each neuron. We will also develop methods to evaluate synaptic function in this circuit. How is touch or osmotic information transmitted to these neurons? How is this information processed by the simple circuit? How do specific mutations influence synaptic transmission? We have modified the dissection preparation first described by the Lockery Lab and are now able to isolate all of the neurons that comprise the command interneurons. We will be presenting details of the dissection procedure as well as our approach to electrophysiological analysis of touch response circuitry.

Michael Muller et al. (2006) European Worm Meeting "A Knock-Out of the Small Non-Coding Y RNA in C. elegans"

Michael Muller, Michael Jantsch, Gunter Steiner, Verena Jantsch

[European Worm Meeting, 2006]

Michael Mller1, Verena Jantsch2, Michael Jantsch2, Gnter Steiner1. Ro ribonucleoproteins (RNPs) are small cytoplasmic particles of unknown function that have been found in all eukaryotes except yeasts. The core structure of human Ro RNPs is composed of one molecule of Y RNA to which the 60 kD Ro protein (Ro60) and La protein are stably bound. Ro RNPs are predominant target structures in the autoimmune diseases Systemic Lupus Erythematosus and Sjgrens syndrome and autoantibodies to Ro60 and La, can be frequently found in the sera of patients suffering from these diseases. The Ro60 protein is highly conserved in higher eukaryotes and the phenotype of a disruption of the gene encoding the C. elegans Ro60 homologue (rop-1) was characterized (Labb et al. Genetics 1999, PNAS 2000). A decrease in Y RNA levels and a higher frequency of misfolded ribosome-associated 5s rRNAs was observed in rop-1 mutant worms. Rop-1 mutants were also shown to be defective in the formation of dauer larvae. Recently the structure of Ro60 was established (Stein et al. Cell 2005) and it was shown that the Y RNA binding site of Ro60 overlaps with the binding site for misfolded RNAs. These results suggest a regulatory role the Y RNA for the binding of misfolded RNAs to Ro60.. In humans and most other vertebrates four different Y RNAs are expressed, while in the nematode C. elegans only one Y RNA species has been found. The C. elegans Y RNA is highly conserved in its structure and most closely related to human Y3 RNA. Yet it differs from other eukaryotic Y RNAs as it is missing a 3 extension to which La is binding (van Horn et al. RNA 1995). To learn more about the function of Y RNAs, we took advantage of the fact that the C. elegans genome contains only a single Y RNA gene (yrn-1). We generated a knock-out line of the C. elegans Y RNA by the method of gene targeting by biolistic transformation. Yrn-1 is located within an intron of an uncharacterized, protein-coding gene. We modified a method for gene disruption (Berezikov et al. Nucleic Acids Res. 2004) in our approach, as we deleted yrn-1 in our knock-out construct rather than disrupting the locus, which could have resulted in a double knock-out.. Yrn-1 mutant worms do not display any obvious morphological phenotype. However, preliminary results indicate that Y RNA deficient worms show a delayed response to chemoattractants. Future experiments are planned to elucidate the role of the Y RNA in damage repair upon UV induced damage, in coping with various forms of stress and in the dauer formation pathway.

Sophie Jarriault et al. (2007) International Worm Meeting "The Y to PDA cell identity change in C. elegans : an in vivo model to study cell plasticity."

Yannick Schwab, Iva Greenwald, Sophie JARRIAULT

[International Worm Meeting, 2007]

The question of trans-differentiation or how a commited cell can change its identity has important implications ranging from organ regeneration to cancer. The lineage of the nematode C. elegans has identified a few cells that change their fates as the worm develops (1, 2), but this interesting observation has never been studied further. We are interested in understanding the molecular events underlying the Y to PDA cell transformation in C. elegans. Y is an epithelial cell that is part of the rectum in young larvae. It subsequentely moves anteriorly and becomes PDA, a motor-neuron which has a characteristic axonal projection . We have developed a set of useful molecular markers that allow to track the Y to PDA transformation and characterised the steps involved in this process, which we timed precisely with respect to the development of the somatic gonad. We have characterised the epithelial features of the Y cell at the ultrastructural level by electron-microscopy and we have examined the expression of cell fate markers in both cells. In addition, we showed that Y''s identity change does not require genes known to affect the developmental timing in C. elegans and we found that cell fusion, a possible mechanism invoked for trans-differentiation in vertebrates, does not contribute to the Y to PDA transformation. Finally, we have examined the importance of cell to cell interactions for the Y to PDA transformation by cell ablation experiments and studies of mutants affecting it, and what makes the Y cell competent to change its identity. We believe that this system is a powerful model to study cell plasticity in vivo, and we will discuss the results of our findings at the meeting. 1. J. E. Sulston, H. R. Horvitz, Dev Biol 56, 110-56 (Mar, 1977). 2. J. E. Sulston, J. G. White, Dev Biol 78, 577-97 (Aug, 1980).

Rui Wang et al. (2007) International Worm Meeting "The TARP-like proteins, STG-1 and STG-2, differentially affect signaling via the GLR-1 glutamate receptor subunit in C. elegans."

Penelope J. Brockie, Andres V. Maricq, Jerry E. Mellem, David M. Madsen, Craig S. Walker, Michael M. Francis, Rui Wang

[International Worm Meeting, 2007]

Understanding how functional nervous systems are established and maintained is a fundamental problem in neuroscience. Glutamate is an essential neurotransmitter found in all well-characterized nervous systems and is used to transmit excitatory information at synapses. There are at least three classes of ionotropic glutamate receptors (iGluRs), AMPA, kainate and NMDA that mediate rapid glutamatergic signaling. In vertebrates, members of the AMPA class are of particular interest because of their relevance to neurological disorders and cellular models of learning. We would like to understand the molecular mechanisms that regulate the function of this important class of iGluRs. Using genetic, behavioral and electrophysiological analyses we have begun to unravel the molecular requirements of functional glutamatergic synapses. In vertebrates, the trafficking, localization and function of AMPA receptors are partially dependent on a family of Transmembrane AMPA receptor Regulatory Proteins (TARPs), but little is known about how TARPs regulate synaptic function and their relevance to behavior. The C. elegans</I> AMPA receptor subunit GLR-1 requires two auxiliary subunits for function, SOL-1 (a CUB-domain transmembrane protein) and the TARP-like protein STG-1 (1, 2). Using knockout techniques and sensitized genetic screens, we have now identified a second TARP-like protein, STG-2, that is essential for glutamatergic signaling in the worm. We have shown that STG-1 and STG-2 modify GLR-1 function, are differentially expressed in neurons, and help tailor glutamatergic neurotransmission to specific avoidance behaviors. Similar to the loss of SOL-1, mutations in both stg-1</I> and stg-2</I> result in behavioral and electrophysiological defects that phenocopy the loss of GLR-1. Interestingly, mutating either stg-1</I> or stg-2</I> alone results in different phenotypes suggesting that STG-1 and STG-2 have both overlapping and distinct roles in regulating AMPA receptor function and thus worm behavior. 1. Zheng, Y., et al. (2004). Nature</I> 427: 451-7 2. Walker, C.S., et al. (2006). PNAS</I> 103(28): 10781-6.

Valeria Pavet et al. (2008) European Worm Meeting "Deciphering epithelial cell plasticity in C. elegans"

Iva Greenwald, Nadege Vaucamps, Sophie JARRIAULT, Valeria Pavet, Yannick Schwab

[European Worm Meeting, 2008]

Understanding how a differentiated cell is able to change its identity. and give rise to a different kind of differentiated cell - process called. transdifferentiation (or TD)- is a challenging task, and the results. obtained will have a profound impact in both basic and clinical science.. In C. elegans, an epithelial cell named "Y" is born in the embryo and is. one of the six epithelial cells that form the rectum in a young larva.. During the second larval stage, Y detaches from the rectum, migrates. anteriorly and becomes a motor-neuron named "PDA", without cell division.. We have shown that the Y-to-PDA conversion represents a bona fide TD. event. Indeed, we have demonstrated that Y presents exclusively. epithelial features while being a part of the rectum, and that these. characteristics are lost and replaced by purely neuronal ones after its. TD into PDA. We have performed an initial characterization of this. process, using genetics and cell ablation to explore factors pertaining. to competence, lineage, and local environment. We found that. transdifferentiation does not depend on fusion with a neighbouring cell. or require migration of Y away from the rectum; that other rectal. epithelial cells are not competent to transdifferentiate; and that. transdifferentiation requires the EGL-5 and SEM-4 transcription factors. and LIN-12/Notch signalling. In order to identify and characterize the. genes and the genetic cascade(s) involved in each step of the Y-to-PDA. TD, we have performed a forward genetic screen for mutants that do not. have a PDA neuron. The mutants obtained fall into two clearly. distinguishable morphological classes: mutants having a persistent. epithelial Y cell that did not transdifferentiate and mutants where an. epithelial Y cell is no longer recognizable. We have started to map and. characterize mutants belonging to the two morphological groups and use. them to examine the intermediate steps of TD. We believe that this system. will allow us to identify the mechanisms underlying this TD event and to. propose a model of how cell plasticity proceeds.