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.

McCright, Samuel et al. (2015) International Worm Meeting "An INX-16 mediated calcium wave is required for normal NLP-40 release."

Moore, Gabriel, McCright, Samuel, Peters, Maureen, Learman, Lisa, Ayoub, Christopher

[International Worm Meeting, 2015]

The C. elegans digestive motor program occurs every 45-55 seconds and consists of three sequential muscle contractions. This cycle's timekeeping mechanism resides in the intestine. Calcium release in the most posterior intestinal cells initiates a posterior to anterior calcium wave. This wave triggers the release of signals that directly or indirectly control the motor program's contractions. The intestinal wave's rapid progression depends on intercellular communication via gap junction connections. Innexin-16 (INX-16) is a subunit of these intestinal gap junctions. Loss-of-function inx-16 mutation causes slowed calcium waves, demonstrating that INX-16 facilitates intestinal calcium wave propagation. In the absence of INX-16 the posterior intestine loses its pacemaker status since aberrant wave initiation sites are common. The abnormal inx-16 waves cause dramatic defects in the motor steps, though the effect on each contraction varies. Posterior body contraction frequency is normal, yet the directionality is often altered to reflect the intestinal calcium wave orientation. Enteric muscle contractions are absent in most motor programs. This disparity in inx-16's effects on the downstream muscle contractions suggests that these signaling pathways are differentially sensitive to the remaining intestinal calcium flux in the mutants. We have been investigating what aspect(s) of the signaling pathway between the intestine and the enteric muscles is defective in the inx-16 mutant. We propose that inx-16's loss of enteric muscle contraction is due to faulty release of the neuropeptide NLP-40. NLP-40 co-localizes to synaptotagmin-2 tagged vesicles and is required to activate the neurons, AVL and DVB, that control enteric muscle contraction. These neurons subsequently release GABA, an excitatory neurotransmitter for enteric muscles. Optogenetic stimulation of GABAergic neurons can restore normal enteric muscle contraction frequencies in inx-16 animals. This finding demonstrates that inx-16's enteric muscles can respond to GABA properly and that AVL and DVB are capable of GABA release. To analyze if NLP-40 is released from the intestine, NLP-40::YFP levels were quantified in the intestine and coelomocytes of normal and mutant worms. A significant decrease in coelomocyte fluorescence was observed in the inx-16 mutant. Lastly, the localization of vesicle fusion proteins and the NLP-40 receptor, AEX-2, are being examined. The reduced NLP-40 levels in inx-16 mutants suggests that enteric muscle contraction fails due to ineffective fusion of NLP-40 containing dense core vesicles. Our findings suggest that inx-16 intestinal calcium waves cannot trigger this particular calcium stimulated event.

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).

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.

Schulze, Jens et al. (2011) International Worm Meeting "Evolution of embryonic development in nematodes."

Schulze, Jens, Schierenberg, Einhard

[International Worm Meeting, 2011]

Nematodes are well suited for a comparative study of early embryogenesis. Analyzing development of a single model system like Caenorhabditis elegans does not shed any light on the degree of evolutionary modifications within the taxon Nematoda. For better understanding evolution of development among nematodes including the identification of plesiomorphic and apomorphic characters, we compared early embryogenesis of representatives from all 12 nematode clades (phylogeny after Holterman et al., 2006; Mol. Biol. Evol. 23:1792-1800). Our data reveal that embryogenesis is unexpectedly variable with floating transitions that can be interpreted as frozen images of evolutionary change. Particularly, members of clade 1 and 2 differ massively from the standard C. elegans. Nevertheless, some basic developmental similarities appear to be common among all nematodes, e.g. the general existence of at least partial cell lineages and the influence of Polarity Organizing Centers (POCs). These POCs are required for generating a linear sequence of cells along the a-p axis which constitute the ventral midline. Depending on phylogenetic position fewer or more cells of this midline divide into left and right daughters this way establishing bilateral symmetry within individual lineages. Our studies reveal the stepwise emergence of founder cells and dramatic fate shifts during evolution. The comparison between embryogenesis of the basal nematode Tobrilus stefanskii and the tardigrade Hypsibius dujardini (Gabriel et al., 2007; Dev. Biol. 312, 545-59) with respect to the early cell division pattern revealed surprising similarities between these two. This may be considered as embryological support for the Ecdysozoa hypothesis.

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.

Kato Y (1996) Worm Breeder's Gazette "IMMUNE DEFENSE OF NEMATODES: Novel members of ASABF family, and their homologs in C. elegans."

Kato Y

[Worm Breeder's Gazette, 1996]

ASABF is an antibacterial peptide in the body fluid of the intestinal parasitic nematode Ascaris suum [Moore et al., wbg 13(2), 25; Kato, wbg 14(2), 32]. The mature ASABF-alpha (previously designated as ASABF) of 71 residues is processed from the 93-residue precursor deduced from a cDNA clone. A C-terminally flanked four-residue peptide is thought to be removed in addition to the elimination of a putative signal peptide by the processing (Kato and Komatsu, J. Biol. Chem., in press). A cDNA for ASABF-alpha was cloned using three-step PCR amplification; (1) RT-PCR using the poly(A)+ RNA isolated from the body walls of the adult female A. suum as templates, (2) 5'-RACE using SL1 primer, and (3) 3'-RACE. In each step, the PCR-products were cloned into the pGEM-T vector. Recently, further analyses of the clones revealed three novel cDNA coding quite similar to the amino acid sequence of ASABF-alpha. They were designated as ASABF-beta, gamma, and delta. The mRNAs for these four ASABFs (alpha-delta) were detected in an individual using RT-PCR with specific primers. These results suggest that ASABF forms a multigene family. However, only the alpha and delta products have been so far detected in the body fluid. The physiological importance of this family formation is under examination. Furthermore, the ASABF homologs in C. elegans were searched. BLAST data base searches revealed significant sequence identity with a deduced protein from the cDNA sequence, yk150c7, in the cDNA catalog by Prof. Yuji Kohara. In addition, the protein deduced from the putative gene, T22H6.5, was found to be a protein most similar to both ASABF and yk150c7 using the MPsrch data base search. The transcript for T22H6.5 was detected using RT-PCR with total RNA from mix-stage worms as templates. The sequencing of the PCR-product revealed that the splicing deduced by GeneFinder occurred. The analyses of the gene regulation of T22H6.5 is currently progressing. Acknowledgement- We thank Prof. Yuji Kohara (National Institute of Genetics) for permission to cite yk150c7 from his unpublished data.