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).

Rebecca M Fox et al. (2005) International Worm Meeting "MAPCeL: Microarray Profiling C. elegans Cells"

Thomas Brodigan, Rebecca M Fox, Michael Krause, Kellen Olszewski, Susan J Barlow, Joan McDermott, W Clay Spencer, David M Miller, Stephen E Von Stetina

[International Worm Meeting, 2005]

Individual C. elegans cells can be easily visualized in vivo with GFP reporters but, owing to their small size, they are generally inaccessible for molecular analysis. We have now developed new methods for the isolation and gene expression profiling of GFP marked C. elegans cells (MAPCeL Microarray Profiling C. elegans Cells). In this strategy, GFP cells are isolated by FACS from in vitro cultures and RNA extracted for application to the C. elegans Affymetrix array. To demonstrate the utility of our approach, we have profiled cells in the embryonic motor circuit; we have identified genes that are differentially expressed in body muscle cells and in each of the three classes of embryonic ventral cord motor neurons (DA, DB, DD). Microarray data in each case were validated by strong correlation with genes known to be expressed in these cells as well as with GFP reporters that we constructed (~80%). For example, our MAPCeL data from inhibitory DD motor neurons confirms enrichment of transcripts required for the GABA phenotype (i.e. unc-25, unc-30, unc-47, snf-11). Conversely, the excitatory DA and DB motor neurons express cholinergic genes (unc-17, cha-1) as well as specific transcription factors that regulate cholinergic signaling (unc-3, unc-4). These data can now be used to identify novel genes with key roles in the locomotory circuit. For example, we have shown that DA motor neurons express a surprisingly diverse array of G-protein coupled receptors with the potential to modulate excitatory activity in response to neuropeptides as well as classical neurotransmitters. MAPCeL data from body muscle cells lead to the discovery that ACR-16 is a component of the levamisole-insensitive nicotinic ACh receptor (See Touroutine et al., this meeting). Finally, by identifying cohorts of genes with common expression patterns we can now search for cis-regulatory elements necessary for cell-specific expression. For example, we have identified enhancer element candidate motifs in the body wall muscle and DA motor neuron datasets (see Olszewski et al, this meeting). We conclude that MAPCeL can be used to generate reliable profiles of cell specific gene expression in the motor circuit and that these data will be valuable resources for correlating gene expression with function.1.Fox, RM, Von Stetina, SE, Barlow, SJ, Shaffer, C, Olszewski, KL, Moore, JH, Dupuy, D, Vidal, M, Miller, DM III. A gene expression fingerprint of C. elegans Embryonic motor neurons. BMC Genomics 2005, 6:42.

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.

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.

Lees, Hayley et al. (2013) International Worm Meeting "Investigating the role of the wrn-1 helicase in the nematode worm, C. elegans."

Cox, Lynne, Lees, Hayley, Woollard, Alison

[International Worm Meeting, 2013]

In order to ameliorate the deleterious consequences of old age, the biology of human ageing must be understood. The study of human progeroid disorders which recapitulate many of the features of normal ageing have helped to contribute to our understanding of normal human ageing. Werner syndrome is a canonical progeroid disorder, caused by mutation of the WRN gene. WRN encodes both RecQ helicase and exonuclease activities, and is known to participate in DNA replication, repair, recombination and telomere maintenance. In addition, although many interacting partners have been identified, the exact molecular functions of the WRN gene remain largely unknown. In order to dissect the roles of WRN helicase in ageing, we use mutants of the WRN homologue and interacting partners in the nematode worm, C. elegans. Reduction of function by RNA interference of the C. elegans WRN homologue wrn-1 leads to ageing phenotypes and shortened lifespan 1. We have shown that mutation of wrn-1 leads to genomic instability: interestingly, this phenotype is enhanced in a mutant cep-1 background (the C. elegans p53 homologue). Notably, lifespan also shows significant modulation, while brood size remains unchanged from that of either single mutant. Therefore we suggest that cep-1 status has a significant effect upon the role of wrn-1 helicase in longevity and germline maintenance in worms. References 1.Lee, SJ; Yook, JS; Han, SM; Koo, HS. A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint. Development, 2004. 131(11): p. 2565-2575.

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.

Onken, Brian D. et al. (2011) International Worm Meeting "Insulin Signaling and Dietary Restriction Differentially Regulate Glucose Metabolism to Impact C. elegans Healthspan."

Onken, Brian D., Driscoll, Monica

[International Worm Meeting, 2011]

A major goal of aging research is to understand the underlying relationship between nutritional intake, metabolism, and healthy aging. Low-glycemic index diets have been shown to reduce risk of age-related metabolic diseases such as diabetes and cardiovascular disease, and reduced caloric intake via dietary restriction increases healthspan across species. One potential approach for supporting healthy aging is via interventions that engage healthspan-promoting metabolism. In Caenorhabditis elegans, adding excess glucose to the growth medium shortens lifespan [1, 2], while inhibiting the glycolytic enzyme hexokinase with the glucose analog 2-deoxyglucose increases lifespan [1]. We have shown that disrupting genes encoding two other glycolytic enzymes that catalyze unidirectional, irreversible reactions lengthens C. elegans median lifespan, induces large gains in youthful locomotory ability, and triggers a fluorescent biomarker that distinguishes a healthy metabolic state. Conversely, disrupting counterpart gluconeogenic genes decreases nematode healthspan. In investigating potential longevity-related pathways that might impinge upon glucose metabolism, we found that disrupting glycolytic genes increases healthspan through the FOXO transcription factor DAF-16, which is also required for the increased lifespan seen with lowered levels of insulin signaling, and which is downregulated by increased glucose availability [2]. Strikingly, we also found that gluconeogenic activity is absolutely and specifically required for increased healthspan under dietary restriction. These results provide evidence for an intriguing new paradigm: breakdown of glucose via glycolysis negatively impacts healthy aging through insulin signaling and DAF-16, while dietary restriction engages the reciprocal gluconeogenic pathway to promote healthspan. Our observations support that healthspan might be optimized via dietary, pharmacological, or genetic interventions that increase gluconeogenic activity or decrease glycolysis. 1. Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, et al. (2007). Cell Metab 6: 280-293. 2. Lee SJ, Murhpy CT, Kenyon C (2009). Cell Metab 10: 379-391.