Liu, Y. et al. (2013) International Worm Meeting "Suppression of Cell Cycle Defects through Knockdown of Tumor Suppressor Genes."

Saito, M., Tobin, D., Liu, Y.

[International Worm Meeting, 2013]

Defects in cell cycle control can be damaging or even fatal to an organism. Cell cycle control processes have many fundamental similarities between C. elegans and humans. Since the loss of cell cycle regulation is a hallmark of cancer, understanding the function of genes that control the cell cycle in C. elegans will expedite the development of cancer detection and treatment methods for human patients. Our laboratory's goal is to characterize the normal function of putative tumor suppressor genes, previously identified in a genome wide screen for animals defective in maintaining cell cycle quiescence (see abstract by Tobin et al). C. elegans Cyclin D(cyd-1) mutants animals arrest in larval development with fewer intestinal cells than wild-type animals. RNAi was used to knockdown specific genes, to test whether or not cell cycle defects of the cyd-1(lf) animals could be suppressed. Suppression of cyd-1(lf) would indicate that the tumor suppressor gene functions downstream of or in parallel to cyd-1. Several RNAi clones were found to suppress intestinal cell division defects, such as ubc-25, C56G2.1, B0393.6 and cdc-14. Suppression of cyd-1 by B0393.6 was novel and interesting given the particular genetic interactions of B0393.1. cdc-14, ubc-25, and lin-35 all suppress cell division by inhibiting CDK-2/CYE-1 activity through distinct mechanisms. This was determined using genetic enhancement tests, for example cdc-14(lf) with ubc-25(lf) is additive. Interestingly, B0393.6 knockdown combined with mutation of any of these three pathways causes an additive effect on intestinal cell number, indicating that B0393.6 is part of a previously uncharacterized G1/S inhibitory pathway. Further studies will be conducted to uncover the mechanism by which B0393.6 promotes cell cycle quiescence.

Liu, Y. et al. (2017) International Worm Meeting "The lysosomal endoribonuclease RNST-2 degrades ribosomal RNA to support embryonic and larval development in C. elegans."

Liu, Y., Wang, X., Zou, W.

[International Worm Meeting, 2017]

Lysosomes degrade cargoes derived from endocytosis, autophagy and phagocytosis and the resulting catabolites are transported out of lysosomes and re-utilized in cellular metabolism. Thus, lysosomes play important roles in cell homeostasis. We isolated a recessive mutant qx245 from an EMS screen, which contains enlarged lysosomes in hypodermal and sheath cells. By SNP mapping and whole genome sequencing, we found that qx245 affects rnst-2, which encodes a RNase T2 family protein. RNST-2 is widely expressed and localizes to lysosomes. Loss of RNST-2 function leads to accumulation of rRNA, ribosomal proteins and autophagic cargoes in enlarged lysosomes and these phenotypes were partially suppressed by autophagy-defective mutations. This indicates that rRNA and ribosomal proteins accumulated in rnst-2 lysosomes are delivered through autophagy. rnst-2(qx245) mutants are defective in embryonic and larval development. Importantly, loss of both rnst-2 and de novo synthesis of pyrimidine leads to almost 100% embryonic lethality, which can be rescued by addition of uridine and cytidine. These data suggest that lysosomal degradation of rRNA by RNST-2 provides pyrimidine required for embryogenesis. Our data reveal the essential role of autophagy- and lysosome-dependent degradation of ribosomal RNA in animal development.

Liu Y et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "The Design of the C. elegans Male Spicule Circuit Determines the Behavior Pattern to Ensure Effective Copulation"

Garcia L, Liu Y, LeBoeuf B

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

We study the C. elegans male copulation, a goal-oriented behavior, to understand what principles nature uses to design sensory-motor circuits to be effective in accomplishing their functions. The male mates by inserting his spicules into the hermaphrodite vulva to facilitate sperm transfer. To breach the vulva, the male repetitively prods spicules at the vulva (caused by rhythmic shallow contractions of the spicule "protractor" muscles), until partial spicule insertion triggers full penetration (caused by tonic "protractors" contraction). We ask how the male circuit coordinates repetitive spicule insertion attempts while maintaining precise contact between the male cloaca and the vulva, and how the circuit regulates the transition between rhythmic shallow contraction and sustained contraction. We identified the circuit that coordinates the rhythmic "protractor" contraction and sustained vulva contact. Once sense the vulva, the cholinergic post-cloacal sensilla neurons induce rhythmic spicule movement not by directly innervating the "protractors", but by stimulating adjacent "oblique" muscles. Contraction of the "obliques" induces "protractors" activity via gap junctions, and also simultaneously facilitates vulva contact, as it changes the tail curvature to press the male cloaca against the vulva. Rhythmic "protractor" contraction and vulva contact are "hinged" by gap junctions, so they are temporally coupled to optimize their effectiveness. We also found that the circuit uses a "coincidence detector" mechanism to ensure the male fully inserts spicules only when the vulva is partially penetrated. The threshold of inducing sustained "protractor" contraction is high. So for "protractors" to sustain contraction, they have to integrate signals from at least two circuit components: removal of the negative input from the hook neurons during vulva contact; and positive input from cholinergic proprioceptive SPC neurons that directly synapse the "protractors". The ERG-like K+ channel UNC-103 is used to set up the "coincidence detector", as removing it increase the probability of premature spicule protraction if the circuit receives fewer than normal inputs.

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.

Kim, E. et al. (2017) International Worm Meeting "Genetic screens for identification of the telomerase RNA component in C. elegans."

Lee, J., Kim, E.

[International Worm Meeting, 2017]

In the linear chromosome replication, DNA polymerase cannot complete the replication of chromosome ends, thus cells are faced with 'end replication problem'. Shortened chromosome ends can be considered as DNA damage, and chromosome fusion may occur through DNA repair pathways. Therefore, eukaryotic cells, which have linear chromosomes, have specific repetitive sequences at the end of chromosomes to protect them: 'telomeres'. Telomeres can be maintained by telomerase, a reverse transcriptase conserved in different organisms, from ciliates to human. The telomerase holoenzyme is composed of telomerase catalytic subunit, RNA component that acts as a template for lengthening of 3' single-stranded telomeric DNA, and several accessary proteins. Caenorhabditis elegans also has telomerase for telomere maintenance, but only the gene encoding telomerase catalytic subunit, trt-1, has been identified. As the telomerase RNA component of C. elegans has not been identified, we are performing experiments to identify it with two approaches: by candidate approach based on telomere template sequences using informatics, and by the forward genetic screen used in identification of telomerase catalytic subunit, trt-1 (1). We found several mutant lines that showed weak telomere signals in telomere dot blot. We will confirm telomere defects of these lines and identify corresponding genes. 1. Meier B, Clejan I, Liu Y, Lowden M, Gartner A, Hodgkin J, et al. (2006) trt-1 Is the Caenorhabditis elegans Catalytic Subunit of Telomerase. PLoS Genet 2(2): e18.

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.

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).

Sellegounder, D. et al. (2019) International Worm Meeting "Neuronal GPCR NPR-8 regulates C. elegans defense against pathogen infection."

Sun, J., Sellegounder, D., Liu, Y.

[International Worm Meeting, 2019]

Increasing evidence indicates that the nervous system regulates infection-triggered host defenses, including behavioral and immune responses. However, the precise mechanisms of such regulation are not well understood. Using the survival and behavioral assays, we demonstrate that NPR-8, a G protein-coupled receptor related to mammalian neuropeptide Y receptors, negatively regulates Caenorhabditis elegans defense against pathogen infection by suppressing the expression of cuticular collagen genes. The nematode cuticle is an exoskeleton composed predominantly of cross-linked collagens that form the barrier between the animal and its environment. We further show that the defense activity of NPR-8 is confined to amphid sensory neurons AWB, ASJ, and AWC. This suggests that the nervous system regulates the dynamics of cuticle structure in response to pathogen infection. It is generally believed that physical barrier defenses are not a response to infections, but are part of the body's basic innate defense against pathogens. Our results challenge this view by indicating not only that C. elegans cuticle structure dynamically changes in response to pathogen infection, but also that the nervous system regulates the cuticle barrier defense. Because collagen production is important for lifespan assurance, neural regulation of collagens required for host defense against pathogen attack could also be essential for resisting other environmental assaults and might play a role in general longevity control.

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.