Salzberg, Y. et al. (2019) International Worm Meeting "Sex and the Circuitry: The ubiquitin and netrin pathways interact to pattern sex-specific dimorphic circuits."

Oren-Suissa, M., Salzberg, Y.

[International Worm Meeting, 2019]

Sexual dimorphism in brain structure and function is evident across phyla, but little is known about the mechanisms that establish dimorphic circuits. We have recently shown that synaptic elimination plays a central role in generating sexually dimorphic circuits- synapses between sex-shared neurons are removed dimorphically during development to accommodate for sex-specific behaviors. Here we show that sex-specific synapse elimination is mediated by the ubiquitin-proteasome system (UPS). First, dimorphic synapse pruning in phasmid sensory neurons fails to occur under genetic or pharmacological UPS inhibition. Cell-specific rescue experiments place UPS activity in the presynaptic cell PHB. To address UPS specificity we screened E3 ligases and identified the conserved F-box protein SEL-10/FBW7 as required for dimorphic synapse pruning. Rescue experiments and protein localization studies show that SEL-10 functions in PHB in the synaptic region. In a search for potential synaptic substrates we found that the UNC-6/netrin receptor UNC-40/DCC contains a SEL-10 phosphodegron binding site (CPD) and is indeed degraded when co-expressed with SEL-10 in culture. unc-6 has been recently shown to be required for maintenance of the male-specific PHB-AVG connection (Weinberg et al., 2018), but it remained unclear how synapses are eliminated in hermaphrodites in the absence of unc-6. We find that sel-10 acts genetically downstream of unc-6 but upstream of unc-40, indicating a role in regulating this ligand-receptor interaction. We generated a putative undegradable unc-40 gain-of-function allele by mutating the UNC-40 CPD. In unc-40(gof) animals, dimorphic synapses were retained in both sexes, implying that SEL-10- UNC-40 binding is necessary for sex-specific synapse removal. Surprisingly, dimorphic synapses were retained in unc-40(gof) animals even in unc-6- null background, suggesting that unc-6 functions here to protect unc-40 from degradation rather than strictly activate it. We propose that inhibition of UNC-40 by SEL-10 promotes PHB-AVG synapse removal in hermaphrodites, and that UNC-6 protects UNC-40 from SEL-10 action in males. Altogether, we show how sexual identity intersects with signaling pathways and the UPS to specify synapse elimination.

Salzberg, Y. et al. (2013) International Worm Meeting "MNR-1/menorin, a novel skin-derived cue, controls arborization of sensory dendrites in C. elegans."

Buelow, H., Diaz-Balzac, C.A., Ramirez, N., Attreed, M., Kaprielian, Z., Tecle, E., Salzberg, Y.

[International Worm Meeting, 2013]

Sensory dendrites depend on cues from their environment to pattern their growth and direct them towards their correct target tissues. Yet, little is known about dendrite-substrate interactions during dendrite morphogenesis. Here we describe MNR-1/menorin (W01F3.1), which is part of the completely unstudied, conserved Fam151 family of proteins, and is expressed in the hypodermis to control the elaboration of the 'menorah'-like dendrites of the mechanosensory PVD and FLP neurons in C. elegans. We provide biochemical and genetic evidence that MNR-1 acts as a contact-dependent or short-range cue, in concert with the neural cell adhesion molecule SAX-7/L1CAM in the hypodermis, and through the neuronal transmembrane receptor DMA-1 on sensory dendrites. Our data describes a previously unknown pathway that provides spatial information from the skin substrate to non-autonomously pattern sensory dendrite development.

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.

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).

Pechuk, V. et al. (2019) International Worm Meeting "Deconstructing the sexually dimorphic circuit for chemical avoidance."

Oren-Suissa, M., Salzberg, Y., Pechuk, V.

[International Worm Meeting, 2019]

Sexually reproducing animals display sexually dimorphic behaviors, geared towards reproductive success. Are there differences in the way the two sexes interpret and respond to the same environmental sensory input? To address this question we analyzed the worm's avoidance response to hazardous conditions. C. elegans detects aversive stimuli by integrating sensory information from the polymodal nociceptive ASH neurons in the head and antagonistic cues from the polymodal tail neurons PHA and PHB, to generate an avoidance behavior. The recent full mapping of the male connectome (wormwiring.org) suggests that the sex-shared neurons in the circuit for chemical avoidance are connected differently in the two sexes. Using the tail-drop assay, which measures antagonistic responses of the tail neurons to amphid activation, we examined the behavioral responses to the aversive stimulus SDS and found sexually dimorphic dose-dependent responses. Hermaphrodites responded to the drop assay across all SDS doses, yet males responded significantly lower at lower doses. The behavioral differences and the suggested anatomical maps prompted us to functionally deconstruct the chemical avoidance circuit. Particularly, we wish to distinguish between dimorphism at the sensory cell level and changes at the connectivity level. To explore the connectivity, we activated ASH optogenetically, thus bypassing the sensory input level, and tested the locomotory responses in the two sexes using a multi-worm tracker. Hermaphrodites responded with a reversal at lower LED intensity compared to males, in agreement with the tail-drop assay results. To examine sexual dimorphism at the sensory cell level, we performed calcium imaging of ASH using microfluidic chips while delivering a chemical stimulus. We found that sensory neurons respond similarly in the two sexes. Our results show that the circuit for chemical avoidance produces sexually dimorphic behaviors, which are mediated mainly by differences in the connections between sex-shared neurons and potentially by variations at the level of sensory cells.

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.

Chiba, T. et al. (2019) International Worm Meeting "Expression pattern of C. elegans Y RNA homologs and two different RNAs from cel-7."

Huanyu, F., Kihara, S., Ushida, C., Sato, M., Chiba, T.

[International Worm Meeting, 2019]

Ro60/Y RNP was found as an autoantigen from human autoimmune disease patients with systemic lupus erythematosus and Sjogren's syndrome. The function was suggested to be the quality control of small structured ncRNAs such as 5S rRNA and U2 snRNA. Interestingly, Ro60/Y RNP was shown to be involved in the stress response of cells and bodies. However, the molecular mechanism of Ro60/Y RNP function and the relation between the function and the stress response are not yet clear. C. elegans has a Ro60 homolog, ROP-1, and 19 Y RNA homologs. Previously we reported the expression pattern and the subcellular localization of Y RNA homologs analyzed by FISH experiments using the antisense RNA probes. Because some RNAs are highly homologous to the others, they were difficult to distinguish in FISH. Therefore, the expressions of Y RNA homologs were determined by northern hybridization using the oligonucleotide DNA probes that had the antisense sequence of the loop region, which was unique in each Y RNA homolog. Total RNA from embryo, each larval stage worms and adult worms were blotted onto the membrane. The results were compared with the FISH experiment data. In most cases, the results of northern hybridization and FISH were consistently elucidated. Intriguingly, two bands of different size in northern hybridization were detected with the Cel-7 antisense oligo DNA probe. The RNAs were designated as Cel-7l (longer one) and Cel-7s (shorter one). Cel-7l increased as the developmental stage of worms proceeded but Cel-7s was detected most in embryo and adult. In addition, Cel-7s decreased in two different rop-1 mutants, but Cel-7l was not affected by the decrease of ROP-1. These indicated that Cel-7l RNA and Cel-7s RNA have alternative functions.

D Combes et al. (1999) International C. elegans Meeting "Acetylcholinesterase genes in C. elegans.II. Tandem organization of the third and fourth genes."

Y Fedon, M Arpagaus, M Grauso, E Culetto, D Combes, J -P Toutant

[International C. elegans Meeting, 1999]

In an attempt to clone ace-3, a sequence (clone 48) was obtained by RT-PCR on total RNAs from C. elegans ace-1 null mutants. This clone hybridized to YACs Y48B6 and Y59C8 indicating a site on chromosome II near unc-52, the expected location of ace-3. Blast examination of genomic sequences covering this region in C. briggsae showed that two sequences on chromosome II in this species were related to clone 48. One was the homologous of clone 48 and the other had also the characteristics of an ace gene. Those two sequences were located in very close proximity on chromosome II with only 372 bp between the stop of the upstream ORF and the ATG of the downstream ORF. A similar tandem organization of these two ace genes was also found in C. elegans by PCR (with 356 bp between the two ORFs and 200 bp separating the polyadenylation site of the 5' gene and the trans-splicing site of the 3' gene). Due to the close proximity of the two genes on chromosome II it was not possible to decide which sequence corresponded to ace-3 (defined as encoding AChE of class C) and those genes were provisionally named ace-x (upstream ORF) and ace-y (downstream ORF). Using RT-PCR, both genes were shown to be transcribed in both species. In both C.elegans and C. briggsae, ace-x contains 16 introns and ace-y 7 introns. We found FGQSAG (ace-x) and VGESAG (ace-y) for sequences around the active serine: both sequences suggest a low catalytic activity. The C-terminal sequences are of the H type suggesting that ACE-X and ACE-Y are glycolipid-anchored membrane proteins. We then sequenced ace-x and ace-y in the mutant ace-3 (strain dc2) in order to identify ace-3. We found that the only change was a deletion (580 nt-long) covering the non coding region between ace-x and ace-y as well as two small portions of coding sequence including the stop codon of ace-x and the initiator ATG of ace-y. The deletion did not shift the reading frame, so that a single long ORF was present in the mutant dc2. A single large mRNA was found by RT-PCR in dc2 instead of two distinct mRNAs in N2 strain. It is likely that the folding of the large encoded protein prevents catalytic activity of ace-x and ace-y. Thus the dc2 mutant did not allow the identification of ace-x or ace-y to ace-3.