Mangone, M. et al. (2011) International Worm Meeting "A 3'UTR clone library of the nematode C. elegans: a resource for 3'UTR biology."

Piano, F., Gunsalus, K.C., Lucas, J.M., Mangone, M., Gutwein, M.R., Mecenas, D.

[International Worm Meeting, 2011]

Metazoan messenger RNAs contain poorly defined cis-acting elements within their 3' untranslated regions (3'UTRs) that modulate gene expression at the post-transcriptional level. These elements play important roles in development, metabolism, and their dysfunction are associated with disease states such as autism, depression, diabetes, Alzheimer's and cancer. Using a combination of deep sequencing, high-throughput 3'RACE, and manual curation of public datasets, we have recently reported a 3'UTR encyclopedia defining ~26,000 3'UTRs for ~75% of protein-coding genes in the C. elegans WS190 genome. This study identified 3'end-processing elements, evolutionarily conserved blocks, predicted miRNA target binding sites, and alternative termination sites within thousands of 3'UTRs. The surprising complexity revealed by these data suggests that 3'UTR-mediated post-transcriptional gene regulation is prevalent on a genome-wide scale (1). To build a comprehensive resource for 3'UTR biology we have been constructing a library of 3'UTR clones using modular vectors that facilitate downstream in vivo functional analyses. We have completed our first pass of directed 3'UTR cloning obtaining at least one 3'UTR isoform for ~15,000 genes annotated in WS190, and we are currently isolating and deep-sequencing 3'UTR isoforms from our last batch of ~6,000 3'UTR minipools. These clones will be made available to the community through distributors and through our website UTRome.org (2). In addition, we have developed a suite of destination vectors to adapt the 3'UTRome to perform large-scale RNAi screens by feeding, to study the contribution to gene expression provided by different 3'UTR isoforms in vivo using dual-colors reporters, and to facilitate the introduction of 3'UTRs into worms either by injection or ballistic transformation. Moreover, we are now preparing entry vectors that will allow the studying of the contribution of 3'UTR isoforms to mRNAs localization using a MS2-derived system compatible with MosSCI technology. In conclusion, we have provided the first whole transcriptome-level description of the 3'UTRome in any organism with single-nucleotide resolution. Furthermore, our 3'UTR clone library provides a powerful tool to probe 3'UTR biology at a systems-level scale. Our work will help to better understand 3'UTR biology and push forward the study of important cis-regulatory elements in the genome. 1M. Mangone et al., The landscape of C. elegans 3'UTRs. Science 329, 432 (2010). 2M. Mangone et al., UTRome.org: a platform for 3'UTR biology in C. elegans. Nucleic Acids Res 36, D57 (2008).

Fradin, H. et al. (2015) International Worm Meeting "Genome sequence of a parthenogen closely related to Caenorhabditis reveals fusion of all ancestral chromosomes."

Fitch, D.H.A., Fradin, H., Baugh, L.R., Gunsalus, K., Zegar, C.M., Lucas, J., Piano, F., Gutwein, M.R., Kovtun, M., Corcoran, D.L., Kiontke, K.

[International Worm Meeting, 2015]

Parthenogenesis is an asexual mode of reproduction in which an embryo develops from an unfertilized egg. The transition from sexual reproduction to parthenogenesis occurred repeatedly in nematodes. An excellent model for understanding the evolution of parthenogenesis is provided by Diploscapter sp. 1, which belongs to the asexual lineage closest to C. elegans. We report a D. sp. 1 genome sequence of 165 Mb assembled de novo using Illumina short-insert libraries and larger PacBio reads. We find 28,028 protein-coding genes using RNA-seq transcriptomic data and in silico gene predictions. Validation of the completeness of the genome and gene content using a set of conserved eukaryotic genes and independently assembled transcriptome data shows that the assembly is of high quality. Microscopy reveals a single pair of homologous chromosomes in D. sp. 1 and in two other close species in which males seem to be absent. This was previously described in the parthenogenetic D. coronatus and is rare in eukaryotes. The closest gonochoristic relatives of Diploscapter have six or seven chromosomes. We find that scaffolds in the D. sp. 1 genome each have strong homology to a specific Caenorhabditis chromosome, consistent with the high macro-synteny generally observed in nematodes. As we note no large-scale loss of genes in D. sp. 1, we hypothesize that the whole set of ancestral chromosomes fused. We find evidence of this fusion event in our assembly and we can hypothesize in which spatial order the ancestral chromosomes fused. C. elegans mutant strains with telomere maintenance defects often display chromosome fusions. Two genes involved in telomere maintenance-mrt-1 and pot-1-are not detected in D. sp. 1. This suggests a model for the evolution of a fused chromosome in D. sp. 1 through altered telomere maintenance. Our genome sequences collapse into two divergent haplotypes. However, 10 % are homozygous and found on regions homologous to C. elegans chromosomes I and X. Microscopy and genomic features support a model of oocyte maturation with only one meiotic division that maintains diploidy and heterozygosity. Our study shows that evolution from sexual to asexual reproduction can be accompanied by dramatic changes in genome structure. It establishes D. sp. 1 and its relatives as an outstanding lineage for studying transition to parthenogenesis.

Harris TW et al. (2000) J Cell Biol "Mutations in synaptojanin disrupt synaptic vesicle recycling."

Harris TW, Hartweig EA, Jorgensen EM, Horvitz HR

[J Cell Biol, 2000]

Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in the budding of synaptic vesicles from the plasma membrane, in the uncoating of vesicles after fission, in the recovery of vesicles from endosomes, and in the tethering of vesicles to the cytoskeleton. Thus, these results confirm studies of the mouse synaptojanin 1 mutants, which exhibit defects in the uncoating of synaptic vesicles (Cremona, O., G. Di Paolo, M.R. Wenk, A. Luthi, W.T. Kim, K. Takei, L. Daniell, Y. Nemoto, S.B. Shears, R.A. Flavell, D.A. McCormick, and P. De Camilli. 1999. Cell. 99:179-188), and further demonstrate that synaptojanin facilitates multiple steps of synaptic vesicle recycling.

Tsai MC et al. (2007) J Cell Biol "Microtubules are involved in anterior-posterior axis formation in C. elegans embryos."

Tsai MC, Ahringer J

[J Cell Biol, 2007]

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O''Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.

Michelle Keaney et al. (2001) International C. elegans Meeting "Optimising dietary restriction-mediated lifespan extension"

Michelle Keaney, David Gems

[International C. elegans Meeting, 2001]

A number of reports have shown that dietary restriction (DR) results in increased lifespan in C. elegans, with the magnitude of increase depending upon the method used (1-4). How DR extends lifespan is unknown, but one possibility is that it down-regulates the insulin/IGF (IIS) signalling pathway which regulates ageing. Work to date is both consistent with (5) and contradictory to (4) this hypothesis. We are testing four methods used previously to exert DR: diluted monoxenic liquid culture (1), reduced nutrient NGM (2), axenic culture (3), and the use of eat mutations (4). We are determining which method can be optimised to (a) give the greatest magnitude of lifespan extension; (b) can be made quantitative (so the degree of DR imposed can be varied); and (c) is easy to perform. We are also seeking to maximise DR-mediated lifespan extension such that further reduction of dietary intake would lead to malnutrition and premature death. Our aim is to use an optimised DR protocol to understand the mode of action of DR, and its relationship to IIS, reproduction and oxidative damage. (1) Klass, M.R. Mech. Ageing Dev. 6 : 413 (1977). (2) Hosono, R. et al. Exp. Geront. 24 : 251 (1989). (3) Vanfleteren, J. R. and De Vreese, A. FASEB J. 9 : 1355 (1995). (4) Lakowski, B. and Hekimi, S. PNAS USA 95 : 13091 (1998). (5) Apfeld, J. and Kenyon, C. Nature 402 : 804 (1999).

Sanders, Tom et al. (2015) International Worm Meeting "Nonlinear sensory integration in C. elegans: a computational model."

Cohen, Netta, Ghosh, D.D., Sanders, Tom, Nitabach, M.N.

[International Worm Meeting, 2015]

Animal survival depends on a combination of often conflicting demands such as foraging and evading of dangers. To navigate effectively in such unknown and changing conditions, animals must continuously integrate over a variety of sensory cues, and adapt their decision making strategy in a context dependent manner. Here, we examine the neural control of a sensory integration task in the nematode C. elegans. The task involves an ASH-triggered aversive response to high osmolarity fructose and an AWA-triggered attractive response to diacetyl [1]. In the assay, worms are placed in the center of a ring of fructose; two drops of diacetyl are located outside the ring. We present a computational model, consisting of point worms, situated in a virtual arena that closely mimics this experimental assay, and endowed with a sensory motor pathway of two sensory neurons, a neural integration pathway and two motor programs (pirouettes and steering). A monoamine (PDF-2 and tyramine) modulation circuit involving RIM and ASH is overlaid on the synaptic circuit, in line with molecular data [1]. Model parameters were constrained by behavioral data for wild type and mutant animals for a range of stimulus concentrations. Based on our simulation results, we reject a null hypothesis of a linear sensory integration mechanism in RIM and present results that are consistent with the data for a sensory "coincidence detector" like process in RIM.[1] Ghosh, D.D., Sanders, T., Hong, S., Chase, D.L., Cohen, N., Koelle, M.R., and Nitabach, M.N. "Neuroendocrine reinforcement of a dynamic multisensory decision." International C. elegans meeting.

Masumi Shimada et al. (2003) International Worm Meeting "Possible roles of MOE-1/OMA-1 CCCH-type zinc-finger family proteins in C. elegans oogenesis and embryogenesis"

Hiroyuki Kawahara, Masumi Shimada

[International Worm Meeting, 2003]

Oocyte maturation is an important prerequisite for the production of progeny. Although several germ-line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation had not been fully investigated. Using a multiple RNAi technique, we have identified three redundant CCCH-type zinc-finger genes, named by us moe-1, -2 and moe-3, as a group related by functions and nucleotide sequence1. Similar results were reported by Detwiler et al.(2001), and they gave the names MOE-1 and MOE-2 to OMA-1 and OMA-2, respectively2. We and Detwiler et al. have found that they have overlapping functions that are crucial for oocyte maturation; i.e. simultaneous removal of these proteins by RNAi induces arrest and expansion of growing oocytes. The results of our in situ hybridization have revealed that each of the moe/oma transcripts is expressed from the distal to proximal region of the gonad, while their corresponding proteins accumulate specifically in the cytoplasm of growing oocytes as well as on P granules. Thus, these gene products participate in processes in the final step of the meiotic cell cycle control, a novel function for CCCH-type zinc-finger family proteins thus far discovered. Although MOE-2/OMA-2 protein is rapidly removed from P granules after fertilization, we found that MOE-2/OMA-2 associated with the centrosome-peripheral structure in dividing blastomeres. Our results suggest that moe/oma gene products are unique multifunctional proteins in terms of their redundancy and characteristic behavior during the course of oocyte maturation and subsequent embryogenesis. 1 Shimada, M., Kawahara, H., and Doi, H. (2002). Genes Cells 7, 933-947. 2 Detwiler, M.R., Reuben, M., Li, X., Rogers, E., and Lin, R. (2001). Dev. Cell 1, 187-199.

Hunter-Ensor M et al. (2000) East Coast Worm Meeting "Histamine: a possible neuromodulator in C. elegans"

Horvitz R, Hunter-Ensor M

[East Coast Worm Meeting, 2000]

Many invertebrates use neuromodulators such as histamine to achieve behavioral flexibility. Histidine decarboxylase is a biosynthetic enzyme that synthesizes histamine from the amino acid histidine. We have identified a gene that encodes a putative L-aromatic amino acid decarboxylase (adc-1) similar to human and Drosophila melanogaster histidine decarboxylases. To identify adc-1 expressing cells we generated several Padc-1::gfp transcriptional fusions. We observed GFP expression in the AIA interneurons and the HSN neurons, as well as in a neuron within the ventral ganglion. Using antibodies against histamine, we identified histamine-like immunoreactivity in adult C. elegans. This histamine-staining appears to colocalize with the anti-GFP staining of Padc-1::gfp expressing worms. We will generate antibodies against bacterially-expressed ADC-1 fusion proteins to aid in our identification of adc-1 expressing cells. Because ADC-1 is similar to dopa decarboxylase, a biosynthetic enzyme required for serotonin and dopamine synthesis, we tested whether adc-1 mutants are defective in dopamine or serotonin biosynthesis. Immunocytochemistry with an anti-serotonin antibody demonstrated that adc-1 worms synthesize serotonin. Dopamine levels, which we assayed using the method of formaldehyde-induced fluorescence, also appeared normal in adc-1 animals. To analyze the function of adc-1 we obtained three independent deletion mutations in the C. elegans adc-1 gene. These deletions, which were generously provided by M. Dong and M.R. Koelle, perturb coordination between the head and body during backward movement. Specifically, while wild-type worms move backwards in a smooth sinusoidal wave, adc-1 mutants wag their noses back and forth during reversals. We plan to define the neural circuit involved in this behavior and to determine if histamine is synthesized by ADC-1.

Richardson, Claire E. et al. (2009) International Worm Meeting "The IRE-1 Branch of the Unfolded Protein Response Functions in C. elegans Pathogen Resistance During Larval Development."

Kim, Dennis H., Kooistra, Tristan, Richardson, Claire E.

[International Worm Meeting, 2009]

The Unfolded Protein Response (UPR), a mechanism to sense and respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), has an important role in development and stress resistance from C. elegans to mammals. We asked if the UPR functions in C. elegans immune defense against the bacterial pathogen Pseudomonas aeruginosa. We found that the conserved IRE-1-XBP-1 branch of the UPR is required specifically for immunity during larval development. IRE-1 is an endoribonuclease that activates the transcription factor XBP-1 by splicing a short exon from its mRNA. Using quantitative PCR, we show that the relative amount of IRE-1-spliced xbp-1 transcript increases after pathogen exposure. Furthermore, a fluorescent reporter for IRE-1 activation is induced after exposure to Pseudomonas specifically at the site of infection - the intestine. We analyzed the relationship between the IRE-1 branch of the UPR and the PMK-1 pathway, which is known to promote C. elegans immunity, and found that IRE-1 activation in response to Pseudomonas is downstream of the PMK-1 pathway. The IRE-1-XBP-1 branch of the UPR has been recently shown to be required for cellular defense against pore-forming toxins, also functioning downstream of PMK-1 (Bischoff et al, 2008). Our data point to a more general role for the UPR in innate immunity against infection. Since PMK-1 pathway activates transcription of putative pathogen resistance proteins, we suggest that the IRE-1 branch of the UPR promotes pathogen resistance by accommodating a PMK-1-driven influx of pathogen resistance proteins to the secretory pathway. Bischof, L.J., Kao, C.-Y., Los, F.C.O., Gonzalez, M.R., Shen, Z., Briggs, S.P., van der Goot, F.G., Aroian, R.V. (2008) Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLOS pathogens, 4, 1-11.

Doi M et al. (2000) Japanese Worm Meeting "The defecation gene aex-1 may regulate a retrograde signaling pathway at neuromuscular junctions."

Iwasaki K, Doi M

[Japanese Worm Meeting, 2000]

Communication between pre- and postsynaptic cells is crucial for synaptic transmission regulation. In one regulatory mechanism, retrograde signaling, signals from postsynaptic cells control synaptic connectivity and transmission of presynaptic cells. Although there is evidence that retrograde signaling occurs at neuromuscular junctions in C. elegans 1), its mechanism is unknown. Here we report that the defecation gene aex-1 may be a regulatory component of C. elegans retrograde signaling. aex-1 mutants show phenotypes similar to those in aex-3 mutants, which were consistent with presynaptic defects. These phenotypes include defecation defects, reduced male mating, and resistance to the acetylcholinesterase inhibitor aldicarb. Based on these observations we initially hypothesized that aex-1 encodes a component which functions at presynaptic terminals. To determine the molecular function of AEX-1, we cloned and characterized aex-1. aex-1 encodes a novel protein of 1027 amino acids whose C-terminus is similar to the second C2 domain of the mouse protein Munc-13-4 and the human protein BAP-3. Using GFP fusion constructs we unexpectedly found that aex-1 was expressed primarily in body wall muscles. Furthermore, aex-1 expression driven by the muscle-specific unc-54 promoter conferred hypersensitivity to aldicarb but did not change sensitivity to the acetylcholine agonist levamisole. This suggests that AEX-1 affects presynaptic activities at neuromuscular junctions. To investigate whether AEX-1 regulates retrograde signaling, we generated egl-30(gf);aex-1 double mutants and tested whether or not hyperexcitation of ventral cord motor neurons suppresses the aldicarb-resistance phenotype of aex-1 mutants. egl-30(gf) expression in ventral cord motor neurons confers hypersentivity to aldicarb2). Our prediction was that if aex-1 functions downstream of ventral cord motor neurons then the egl-30(gf) mutation would not suppress the aex-1 phenotype. However, the egl-30(gf) mutation could suppress the aldicarb-resistance phenotype of aex-1 mutants, suggesting that aex-1 acts upstream of the egl-30(gf) mutation. This observation is consistent with the hypothesis that AEX-1 regulates retrograde signaling at neuromuscular junctions. 1) Zhao, H. and Nonet, M. L. Development 127 1253-1266 (2000) 2) Lackner, M.R. et al., Neuron 24 335-346 (1999)