Charlesworth AG et al. (2019) Cell "Next-Gen Learning: The C.elegans Approach."

Claycomb JM, Seroussi U, Charlesworth AG

[Cell, 2019]

In this issue, Moore etal. and Posner etal., provide evidence for how the activity of the nervous system in C.elegans results in gene expression changes in the germline to pass on parental experiences and learned behavior to their progeny.

Moore RS et al. (2021) STAR Protoc "Protocol for transgenerational learned pathogen avoidance behavior assays in Caenorhabditis elegans."

Moore RS, Murphy CT, Kaletsky R

[STAR Protoc, 2021]

Animal experiences, including learned behaviors, can be passed down to several generations of progeny in a phenomenon known as transgenerational epigenetic inheritance. Yet, little is known regarding the molecular mechanisms regulating physiologically relevant transgenerational memories. Here, we present a method for <i>Caenorhabditis elegans</i> in which worms learn to avoid the pathogen <i>Pseudomonas aeruginosa</i> (PA14). Unlike previous protocols, this training paradigm, either using PA14 lawns or through exposure to a PA14 small RNA (P11), induces memory in four generations of progeny. For complete details on the use and execution of this protocol, please refer to Moore etal. (2019) and Kaletsky etal. (2020).

Lim, Yunki et al. (2021) International Worm Meeting "High-throughput phenotypic screening to identify neurotoxic chemicals causing neuro-degeneration"

Moore, Jacob, Hegarty, Evan, Kagias, Konstantinos, Mondal, Sudip, Lim, Yunki, Laing, Adam, Ben-Yakar, Adela

[International Worm Meeting, 2021]

Direct exposure of humans to different chemicals becomes more prevalent as society is progressing further into industrialization. In addition, an increasing number of medical drugs are being developed at different non-clinical and clinical stages aiming to treat a variety of conditions. In order to protect the public from potential deleterious effects of these chemicals, toxicology analysis is necessary to ensure early identification of toxic effects. The most common type of chemically-induced toxicity is neurotoxicity. Therefore, screening for chemical compounds, which can cause specific neuronal damage, can help identify toxic chemicals and potentially help elucidate specific neurodegeneration mechanisms, which can lead to the development of novel targeted therapeutic approaches. Current neurotoxicity assays rely mainly on mammalian models' mortality tests and are associated with high screening costs and long experimental times. To overcome these limitations, we developed a high throughput in vivo neurotoxicity assay using C. elegans. We screened animals treated with a number of well-characterized reference chemicals using Newormics' proprietary microfluidic device (vivoChip), which enabled us to perform high-resolution imaging and multi-parametric structural analysis of GFP-labeled neurons in a high-throughput manner. We characterized the chemical-induced neurotoxicity in the dopaminergic, cholinergic, GABAergic, and serotonergic neurons generating a complete set of imaging data for each reference chemical. Semi-automatic analysis of this dataset identified the cellular and sub-cellular neuronal defects and created neuron-specific degeneration metrics for the reference chemicals. We hope to gain valuable insights into potential mechanisms of action for these neurotoxic compounds while at the same time progressing towards a more complete screen through an increasing number of chemicals using our high-content and high-throughput system.

Reboul J et al. (2001) Nat Genet "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans."

Doucette-Stamm L, Lamesch PE, Reboul J, Temple GF, Hartley JL, Brasch MA, Hill DE, Vaglio P, Thierry-Mieg N, Shin-i T, Lee H, Moore T, Vandenhaute J, Kohara Y, Vidal M, Jackson C, Thierry-Mieg J, Tzellas N, Thierry-Mieg D, Hitti J

[Nat Genet, 2001]

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.AD - Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.FAU - Reboul, JAU - Reboul JFAU - Vaglio, PAU - Vaglio PFAU - Tzellas, NAU - Tzellas NFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Moore, TAU - Moore TFAU - Jackson, CAU - Jackson CFAU - Shin-i, TAU - Shin-i TFAU - Kohara, YAU - Kohara YFAU - Thierry-Mieg, DAU - Thierry-Mieg DFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Lee, HAU - Lee HFAU - Hitti, JAU - Hitti JFAU - Doucette-Stamm, LAU - Doucette-Stamm LFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Vandenhaute, JAU - Vandenhaute JFAU - Lamesch, P EAU - Lamesch PEFAU - Hill, D EAU - Hill DEFAU - Vidal, MAU - Vidal MLA - engID - R21 CA81658 A 01/CA/NCIID - RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - United StatesTA - Nat GenetJID - 9216904SB - IM

Ouyang YB et al. (1998) J Biol Chem "Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans."

Moore KL, Ouyang YB

[J Biol Chem, 1998]

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.

Joshua Snow et al. (2001) International C. elegans Meeting "Functional analysis of potential E2 ubiquitin conjugating enzymes using RNAi."

Rhonda Moore, Lynn Boyd, Claudia Morales, Joshua Snow

[International C. elegans Meeting, 2001]

We are investigating how genes predicted to be involved protein degradation effect embryogenesis in Caenorhabditis elegans . Within the cell, protein degradation is primarily accomplished through the ubiquitin-proteasome pathway. Studies in other systems show that E2 and E3 enzymes work in tandem to attach ubiquitin to a specific protein substrate, thereby condemning the substrate to degradation by the proteasome. We have identified 26 potential E2 genes within the completed genome of C. elegans . We are assessing the function of these genes through the use of RNAi-mediated interference (RNAi). E3 ligases are less conserved and more numerous than E2s. One class of E3 enzymes contains proteins with RING finger domains. We have previously identified 112 genes containing a RING finger in the C. elegans database. Four of the RING finger proteins were found to be required for embryogenesis (Moore, ECWM 2000, 154). By comparing E2 RNAi phenotypes with the RING finger mutant phenotypes, we hope to determine which E2 ubiquitin-conjugating enzymes partner with specific RING finger proteins. One of the four essential RING finger containing genes is par-2 , a gene involved in establishing anterior-posterior polarity in the embryo. PAR-2 protein is localized asymmetrically to the posterior cortex in embryos. In order to understand if protein degradation is involved in PAR-2 localization, we are using a transgenic strain expressing PAR-2:GFP to observe PAR-2 localization in E2 RNAi embryos.

Redo Riveiro, Alba et al. (2013) International Worm Meeting "egl-46 is a novel BAG cell fate modulator."

Pocock, Roger, Redo Riveiro, Alba

[International Worm Meeting, 2013]

Genetic studies in C. elegans attempt to identify molecular components required for neural circuits to function correctly. This study focuses on BAG neurons, which have the major role in oxygen (O2) and carbon dioxide (CO2) sensing. Development of BAG neurons is controlled by the conserved transcription factors ETS-5 (Brandt, Aziz-Zaman et al. 2012) and EGL-13 (Petersen et al. 2013-in press). However the effect of ets-5 and egl-13 loss of function mutants does not affect all BAG terminal fate markers. Therefore, other genes must act in parallel to these factors to specify BAG fate. In a classical forward genetic screen, we identified egl-46 as a BAG fate modulator. EGL-46 is a zinc finger transcription factor that regulates terminal cell divisions in the Q lineage where egl-46 mutants undergo extra rounds of terminal Q cell divisions (Wu, Duggan et al. 2001). The egl-46 mammalian ortholog Insm1 has a similar function in determining cell fates. Insm1 has been implicated in the development of pancreas (Farkas, Haffner et al. 2008), cortex and hindbrain (Jacob, Storm et al. 2009). Insm1 mutant mice present a thicker layer of proliferative progenitors and a thinner neuro-basal layer in the olfactory ephitelium (Rosenbaum, Duggan et al. 2011). Here we show that egl-46 mutants have defects in the expression of specific terminal markers in the BAG neurons. We have rescued these defects by transgenically expressing a fosmid containing the egl-46 locus. Currently, we are performing genetic interaction studies with BAG-fate regulators like ets-5 and egl-13.

Beisswanger R et al. (1998) Proc Natl Acad Sci U S A "Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2."

Beisswanger R, Corbeil D, Ashman K, Vannier C, Thiele C, Kellner R, Niehrs C, Dohrmann U, Huttner WB

[Proc Natl Acad Sci U S A, 1998]

Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. &amp; Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

JC Bettinger et al. (1999) International C. elegans Meeting "Behavioral Effects of Exposure to Ethanol"

SL McIntire, JC Bettinger

[International C. elegans Meeting, 1999]

Exposure to ethanol interferes with complex behaviors in many model systems, but it has been difficult to correlate effects of ethanol on behavior with observations of its effects on specific molecular targets. Recently, studies of Drosophila demonstrated a link between ethanol sensitivity and a learning pathway 1 : A screen for mutations that cause flies to be hypersensitive to the effects of ethanol on postural control yielded an allele of amnesiac , a putative neuropeptide known to be involved in learning 2 . After exposure to ethanol, C. elegans display uncoordinated movement (characterized by a decreased amplitude of the sine waveform and lethargy), and decreased rate of pumping and egg-laying (SLM, unpublished observations). After several hours of exposure, worms develop an acute tolerance to ethanol, and recover to resemble untreated controls. We are interested in determining whether or not exposure to and development of tolerance to ethanol alter any of the more complex behaviors exhibited by worms, including chemotaxis. Our preliminary experiments on the effect of ethanol on chemotaxis suggest that brief or prolonged exposure to moderate concentrations of ethanol (100-200 mM) does not prevent chemotaxis to the volatile odorant benzaldehyde. With extended exposure, worms become insensitive to chemoattractants in a process termed adaptation. Worms that have adapted to a particular chemoattractant will not climb a gradient of that chemoattractant 3 . Given that worms are able to chemotax in the presence of ethanol, we can test the effect of ethanol on adaptation. We are determining whether or not exposure to low-to-moderate concentrations of ethanol interferes with the process of adaptation to benzaldehyde and other odorants. 1 Moore, M.S.; DeZazzo, J.; Luk, A.Y.; Tully, T.; Singh, C.M. and Heberlein, U. (1998). Cell 93: 997-1007. 2 Feany, M.B. and Quinn, W.G. (1995). Science 268: 869-873. 3 Colbert, H.A. and Bargmann, C.I. (1995). Neuron 14: 803-812.

Husson, Steven J. et al. (2011) International Worm Meeting "C. elegans mutants defective in neuropeptide amidation enzymes are abnormal in egg-laying behavior."

Landuyt, Bart, Schoofs, Liliane, Gottschalk, Alexander, Horvitz, H.Robert, Temmerman, Liesbet, Husson, Steven J., Ringstad, Niels, Meelkop, Ellen

[International Worm Meeting, 2011]

Egg laying has mainly been studied at the behavioral, neuronal and neurochemical levels, but little is known about the biochemical control of the relevant neuropeptidergic signaling systems. Biosynthesis of endogenous peptides requires processing enzymes, such as proprotein convertase 2, which is encoded by egl-3 (1, 2), and a carboxypeptidase encoded by egl-21 (3, 4). Mutants defective in these genes have egg-laying defects, consistent with the finding that FMRFamide-like peptides (FLPs) have been linked to egg laying behavior. C. elegans enzymes that carry out the last step in the production of biologically active peptides, the carboxy-terminal amidation reaction, have not been characterized. This multistep reaction involves hydroxylation of the glycine a-carbon by a peptidyl-a-hydroxylating monooxygenase (PHM), followed by a cleavage reaction performed by peptidyl a-hydroxyglycine a-amidating lyase (PAL) to generate a glyoxylate molecule and the a-amidated peptide. In vertebrates, both enzymatic activities responsible for the carboxyterminal amidation reaction are contained in one bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM). By contrast, invertebrates generally express two separate enzymes encoded by two different genes. Here we report the identification and characterization of C. elegans amidating enzymes using bioinformatics to identify candidate genes and mass spectrometry to compare the neuropeptides in wild-type and newly generated mutants. Mutants lacking a functional PHM displayed an altered neuropeptide profile, showed impaired egg laying behavior and had a decreased brood size. Interestingly, PHM mutants still displayed fully processed amidated neuropeptides, probably as a result of the presence of a bifunctional PAM, the main amidating enzyme in vertebrates. Our data indicate the existence of a robust complementation system for the amidation reaction of neuropeptides in nematodes and suggest the involvement of amidated neuropeptides in egg laying. (1) S. J. Husson et al., J. Neurochem. 98, 1999 (2006); (2) J. Kass et al., J. Neurosci. 21, 9265 (2001); (3) S. J. Husson et al., J. Neurochem. 102, 246 (2007); (4) T. C. Jacob, J. M. Kaplan, J. Neurosci. 23, 2122 (2003).