Hutter, Harald et al. (2009) International Worm Meeting "GExplore: Multi-gene data mining for worm geneticists."

Chen, Nansheng, Hutter, Harald, Ng, Man-Ping

[International Worm Meeting, 2009]

The C. elegans genome contains some 20,000 genes, the majority of which has not been functionally characterized. Numerous genome wide data sets providing hints for gene function exist, yet mining those data to retrieve potential candidate genes for a particular function remains a challenge. Data most relevant for gene/protein function are - phenotype and expression (if known) - homology to other proteins (which might have been characterized already) - protein domains, which are frequently diagnostic for a biochemical function GExplore provides a simple database interface for worm biologists/geneticists for data mining at the gene/protein function level to help in experimental planning. It combines data downloaded from Wormbase with selected data sets related to gene expression or function, like SAGE data sets containing stage and tissue specific expression profiles. Data are preprocessed and organized for simple searches. The interface allows individual or combinatorial searches for proteins with certain domains, expression in certain tissues and/or certain phenotypes. Genes in a given genetic or physical interval can be listed to provide a convenient way to screen for candidate genes in mapping/cloning experiments. Information about available alleles can be displayed to simplify experimental planning. Literature related to entire lists of genes can be quickly retrieved and surveyed. GExplore operates on a local database and has fast response times, which is essential for exploratory searches. The interface is simple yet flexible enough to accommodate the addition of more data sets in the future. Since it is mainly based on data extracted from Wormbase it can be easily updated as new stable releases become available. GExplore is hosted under: http://genome.sfu.ca/gexplore/.

Li, Tie-Mei et al. (2013) International Worm Meeting "Absolute Quantitation of C. elegans Dafachronic Acids during Development and in Daf Mutants."

Lei, Xiaoguang, Li, Xiangke, Chen, Jie, Li, Tie-Mei, Dong, Meng-Qiu, Ding, Xiao-Jun, Chen, She

[International Worm Meeting, 2013]

Under favorable conditions, C. elegans larvae grow into reproductive adults after a series of molting cycles. When the environmental condition is harsh, they arrest as dauer larvae. The steroid hormone dafachronic acid (DA) has been shown to be critical for the suppression of dauer arrest. To understand the regulation of endogenous DA levels during development, we set out to develop a sensitive and accurate quantitation method of DAs by the use of LC-MS. We blocked the DA carboxylate group with 2-picolylamine, resulting a 100-fold increase in MS signal. Different MS methods, including Selective Reaction Monitoring (SRM) on a triple-quadruple mass spectrometer and targeted-MS2 or Selected Ion Monitoring (SIM) on a Q-Exactive orbitrap mass spectrometer, were compared for DA quantification. The SIM method consistently exhibited the lowest S/N ratio, thanks to its high mass accuracy and high resolving power. Overall, the method allowed us to detect and quantify the presence of as low as 0.5 ng of DA in 1 mL of packed worms. Using the LC-MS method established as above, we measured the DA levels at different developmental stages of N2 worms. The results showed that DA increased from 0.05 ng/mg total proteins in the L1 stage to 0.8 ng/mg total proteins in the L2 stage and remained high in the L3 stage before dropped to below 0.1 ng/mg total proteins in the L4 and the young adult stages. In Daf-c mutants representing diminished insulin, TGF-b or cGMP signaling (daf-2, daf-7 and daf-11 mutants) the DA levels remained low at the L2d stage (below 14% of that in WT L2s). Consistent with a previous study [1], exogenous supplement of DA rescued the Daf-c phenotype of these Daf-c mutants. In summary, we have developed an accurate, simple and robust method to measure the absolute quantity of DA in C. elegans. This method enabled us to obtain direct biochemical evidence that sufficient DA is essential for L2 larvae to enter reproductive development, and place insulin, TGF-b, and cGMP signaling squarely upstream of DA synthesis. References: 1.Motola, D. L. et al., Cell 124, 1209-23 (2006).

Dwyer, Donard et al. (2017) International Worm Meeting "Protophenotypes in C. elegans relevant for schizophrenia and major depression."

Aamodt, Eric, Dagenhardt, Julie, Dwyer, Donard

[International Worm Meeting, 2017]

Efforts to deconvolute the complexity of genetic contributions to psychiatric illnesses such as schizophrenia and major depressive disorder (MDD) have led to the concept of endophenotypes - heritable traits that reflect intermediary pathological processes of a complex disorder. We have coined the term 'protophenotypes' to describe endophenotypes that have been conserved across species during evolution. The goal of our studies is to characterize the genetics and pharmacology of protophenotypes in C. elegans related to pre-pulse inhibition and startle defects in schizophrenia, social withdrawal (a prominent feature of both schizophrenia and MDD) and avolition/anhedonia, which is characteristic of various affective disorders in man. To model pre-pulse inhibition we evaluated touch suppression of pharyngeal pumping in various mutant strains and with pharmacological agents. To gain insight into social withdrawal, we studied aggregation in social feeding strains in the absence and presence of drugs. Lastly, we developed a model of avolition/anhedonia or diminished motivation based on defects in insulin/IGF-1 signaling, exposure to DMSO and food deprivation. In this model, we observed "suicidal" worms that failed to seek food or escape, but remained in place until they died, despite preservation of responsiveness to external stimuli. Genetic and pharmacological studies revealed that dopamine affects touch suppression via a primitive counter-circuit that presages the arrangement of dopaminergic pathways in man controlling emotions and logical thought. We found that serotonin regulated social feeding, which was inhibited by 5-HT2 receptor antagonists. This finding was consistent with serotonergic defects in MDD and schizophrenia. Studies of the third protophenotype related to diminished motivation revealed that defects in insulin receptor signaling (DAF-2) and proteins that regulate insulin secretion (UNC-64, but not UNC-31) produced rapid immobility in animals subjected to food deprivation in the presence of 1% DMSO. The immobility state in daf-2(lf) mutants was maintained by elevated serotonergic and muscarinic cholinergic signaling. Immobility was reversed with antidepressants (e.g., amitriptyline, imipramine and amoxapine) and antipsychotics (e.g., clozapine and olanzapine, but not haloperidol) that decrease immobility in the forced swimming test - an established model of depression in man. These findings may help to explain the 2-3-fold increase in MDD in patients with diabetes. Finally, this work may establish mechanisms for how fundamental behaviors conserved through evolution contribute to the emergence of psychiatric illness in man.

Kim WJ et al. (1991) International C. elegans Meeting "IVERMECTIN RESISTANCE IN C. ELEGANS"

Kim WJ, Johnson CD

[International C. elegans Meeting, 1991]

Ivermectin is a potent anthelmintic that prevents growth of C. elegans by blocking pumping of the pharynx (first noted by M. Chalfie; Avery and Horvitz, (1990) J. Exp. Zool. 253; 263-270; L. Avery pers. comm.). Animals resistant to ivermectin are easily selected by growing eggs on ivermectin containing media. Our experiments are designed to evaluate the incidence of spontaneous and induced resistance to various concentrations of ivermectin and to analyze the phenotypes of ivermectin resistant mutants. The results have implications for ivermectin-use programs and may provide tools for the isolation and identification of the ivermectin receptor. Recent experiments have focussed in two areas: detailed examination of the effects of ivermectin near the threshold for growth inhibition and genetic analysis of high-level resistant mutants. Near the threshold, ca. 2.0 ng/ml, the response of individual larvae appears to be 'all or none': Some individuals arrest growth immediately upon hatching; others grow, at normal or near normal rates, to healthy, fertile, adults. Slow growing or sick animals are less frequently observed. Further, the progeny of many, apparently resistant, animals respond to ivermectin exactly as wild type animals. The incidence of this 'non-heritable' resistance, to 5 ng/ml ivermectin, is ca. 1 in 500 eggs, 20X the incidence of heritable resistance. The incidence of heritable, but not of non-heritable, resistance is increased in strains with active transposons (TR403, RW7097) or after EMS treatment. Resistance to 100 ng/ml ivermectin is very rare -- less than 1 in 10( 9) (spontaneous) and ca. one in 30 x 10(6) genomes (after EMS). Most ( perhaps all) high level resistance requires multiple mutations. To date, we have identified 5 genes, including two X-linked uncs, which contribute mutations conferring this phenotype.

Mark Merris et al. (2001) International C. elegans Meeting "STEROL FUNCTIONS IN C. ELEGANS"

Mark Merris, William G Wadsworth, John Lenard

[International C. elegans Meeting, 2001]

C. elegans is known to require dietary sterol, because it is unable to synthesize the sterol ring. The most commonly used sterol, cholesterol, can undergo extensive enzymatic modification in C. elegans to form unique sterols of unknown function*. The existence of this pathway, and the fact that only minute amounts of sterol are required, suggests that these compounds may have specific, hormone-like actions, perhaps mediated by one or more of the 270 nuclear receptors identified from the C. elegans genome. We are characterizing cholesterol deprivation in C. elegans, in order to identify essential genes in the cholesterol utilization pathway. We report the following observations: (1) Successful reproduction requires cholesterol. Egg-laying is reduced in the first generation of cholesterol deprivation (CF1), and completely abolished in the second (CF2). The presence of 10 ng/ ml of cholesterol in the medium in CF2 results in greatly delayed production of approximately half the normal number of progeny. The presence of 30 ng/ml results in the production of the normal number of progeny, with 1-2 days delay compared with higher concentrations. There are no significant differences in rate or number of progeny using varying amounts of cholesterol from 100 ng/ml to 10 mg/ml. (2) Normal growth requires cholesterol. Animals in CF1 are smaller than controls starting on day 4 at 20o. CF2 animals arrest at various larval stages, apparently reflecting the point at which cholesterol is required, but the stored cholesterol is exhausted. (3) A wide variety of sterols can substitute for cholesterol, but vertebrate or insect steroid hormones cannot. (4) Uptake is often rate-limiting for sterol function. This is evidenced by an initially slowed rate of growth on high-cholesterol medium following cholesterol deprivation, and by the observation that addition of detergent is necessary for effective utilization of certain sterols. Microinjection studies are in progress in order to distinguish sterol uptake from downstream effects. Microscopic studies using the cholesterol-specific stain filipin revealed that cholesterol accumulates in five specific cells in both larval and adult hermaphrodites. These include two amphid socket cells, two phasmid socket cells and the excretory gland cell, which contains secretory granules and makes connections with both the excretory canal and the pharyngeal nerve ring. These observations, taken together, suggest a hormonal role for one or more sterol metabolites. *Chitwood, D.J. 1999. Crit. Rev. Bichem. Mol. Biol. 34: 273-284.

Do Hyun Kim et al. (2006) East Asia C. elegans Meeting "The Study of Dolichol Phosphate Mannose Synthase I (DPM1) Homolog Y66H1A.2 in C.elegans"

Do Hyun Kim, Jin Won Cho

[East Asia C. elegans Meeting, 2006]

Dolichol phosphate mannose synthase is one of the multiple enzymes involved in N-glycan assembly in ER. It catalyses the synthesis of dolichylphosphatemannose(dol-P-Man) from GDP-mannose and dolichylphosphate. This enzyme is composed of a catalytic subunit DPM1 and regulatory subunits DPM2, DPM3. It was reported that partial deficiency in human DPM biosynthesis causes CDG(Congenital disorders of glycosylation) type Ie. We found that dpm1 homolog(y66h1a.2) in C.elegans was mainly expressed in hypodermis and intestine. Functional block of dpm1 homolog by RNA interference caused developmental delay, formation of huge embryos or unfertilized oocytes, abnormal cleavage of embryos, egg-laying defect, and enlargement of intestinal lumen. The intestinal lumen enlargement was defined to be associated to dysfuntion of digestion. we confirmed problems on ovary occurs when the funtion of DPM1 is inhibited ; by mating wild type male and hemaphrodite, they could not produce any progenies. These results indicates that C.elegans DPM1 have important roles involved development.

Holden, Kyle et al. (2013) International Worm Meeting "The E3 Ligase LIN-23/bTRCP Influences SKN-1/NRF2 Activity and Reduces Toxic Proteins in daf-2 Mutants."

Ghazi, Arjumand, Holden, Kyle, Balasubramani, Mani, Schrieber, Emmanuel

[International Worm Meeting, 2013]

Protein homeostasis is vital to an organism's ability to respond to environmental stress and to cope with the changing internal landscape associated with aging. Critical to this homeostasis are proteasomal E3 Ligases that control the ubiquitination of substrates. LIN-23, an F-Box Adaptor protein of the SCF E3 complex, is known to regulate cell-cycle progression during development as well as determination of adult lifespan in daf-2 mutants. LIN-23 is homologous to human bTRCP that regulates the transcription factor NRF-2. The worm homolog of NRF-2, SKN-1, is a key mediator of oxidative stress response and is essential for the longevity of daf-2 mutants. We provide evidence that LIN-23/bTRCP influences SKN-1/NRF2. LIN-23/bTRCP is required for the oxidative stress resistance of daf-2 mutants, and reduction of LIN-23 results in decreased expression of SKN-1/NRF2 target genes that help combat oxidative stress. Paradoxically, this reduced SKN-1/NRF2 activity is accompanied with increased intestinal nuclear localization, suggesting a complex regulatory relationship. To map the proteome governed by LIN-23, we used iTRAQ followed by LC-MS/MS and identified 223 proteins that are consistently stabilized following lin-23 inactivation in daf-2 mutants. We found this group to be enriched for the canonical {DSG(x)nG} as well as a non-canonical LIN-23/bTRCP phosphodegron motif {(DDG(x)nG)}. These proteins are down-regulated in long-lived worms, and reduction of their function increases the lifespan of wild-type animals. The levels of 96 proteins were lowered following LIN-23 inactivation, including known targets of SKN-1/NRF2. We tested the functional relevance of these longevity-promoting genes in daf-2 mutants. Our studies show that LIN-23/bTRCP and SKN-1/NRF2 share an evolutionarily conserved relationship that determines stress-resistance and lifespan in worms, and that LIN-23/bTRCP performs degradative as well as non-degradative functions to influence aging. The LIN-23/bTRCP substrates we identified can provide valuable information about aging as well as other LIN-23/bTRCP-mediated processes.

Nettell JJ et al. (1998) European Worm Meeting "EXPRESSION PATTERN OF C. ELEGANS RH (RHESUS) HOMOLOGUES -AN ATTEMPT TO ASSIGN FUNCTION TO HUMAN RH PROTEINS"

Kuwabara PE, Tanner MJ, Nettell JJ

[European Worm Meeting, 1998]

Rh antigens are highly immunogenic in man and are important in clinical haematology and blood transfusions. They form the major cause of haemolytic disease of the newborn in caucasian populations. The antigens are expressed on two 30kDa non-glycosylated membrane proteins (Rh30) and complex with a glycoprotein (Rhgp) of very close homology. The function of these erythroid specific proteins is unknown. In an attempt to elucidate the function of Rh we have been studying two recently discovered Rhgp homologues in the nematode C. elegans (CERH1/2). We have begun to localise the expression of both genes by constructing GFP-tagged promoter vectors containing a nuclear localisation signal (NLS). The gonad of N2 hermaphrodite C. elegans was injected and double rol-6/GFP transformed worms were visualised by laser confocal microscopy. GFP fluorescence, resulting from CERH promoter activity, indicated that expression predominates in seam cells and in some of the large polyploid nuclei of the intestine. The results also suggest CERH1 and CERH2 co-localise in these cell types. Further work should confirm this co-localisation and determine protein targeting by protein-GFP fusion constructs. Immunocytochemistry, using prepared anti-CERH peptide antibodies, will further investigate such expression patterns.

Friedman LC et al. (2000) Midwest Worm Meeting "Sexual dimorphism of organ polarity in gonad formation"

Markussen F-H, Friedman LC, Siegfried KR, Kroll-Conner PL, Mathies LD, Kimble JE

[Midwest Worm Meeting, 2000]

When the L1 larva hatches, the gonad primordium consists of 4 cells arranged with two-fold rotational symmetry in both sexes. Two of these cells, Z1 and Z4, are somatic and their development determine the shape of the organ. During L1, cell divisions generate 8 somatic cells in hermaphrodite gonads and 6 in males. In the hermaphrodite, the symmetrical arrangement of somatic cells is maintained during L1. The symmetry is continued throughout later development, producing the two-armed gonad of hermaphrodites. In males, symmetry is broken during L1, and most somatic cells coalesce in an asymmetric cluster at the anterior of the developing gonad. This event underlies the asymmetrical one-armed gonad of males. We have set out to dissect the genetic and cellular basis of sexual dimorphism during early gonadogenesis. To this end, we have isolated two mutants with defects primarily in male gonadogenesis. One mutant, tentatively called man-1 for male asymmetric gonad defective, fails to break gonad symmetry in L1 males. Another similar mutant has defects in early male gonadogenesis. We will report our progress in characterizing these mutants and our efforts to describe cellular anatomy of the gonad primordium at a molecular level.

Markella Katidou et al. (2008) European Worm Meeting "Characterization of rig-6, a member of the contactin subfamily of the immunoglobulin superfamily in C. elegans"

Nektarios Tavernarakis, Domna Karagogeos, Markella Katidou

[European Worm Meeting, 2008]

The Immunoglobulin Superfamily (IgSF) represents one of the largest. families of proteins evolutionary conserved from worm to man. Members of. the IgSF have been known to regulate numerous processes during development,. playing a leading part in cell-cell recognition and communication. The. contactin subfamily of the IgSF consists of glycosylphosphatidylinositol. (GPI) anchored glycoproteins, known to be involved in axon growth and. guidance, neuronal migration, axon fasciculation and myelination. Moreover,. the Drosophila ortholog is required for septate junction organization and. epithelial integrity. To gain insight into the function of contactins, we. are characterizing RIG-6 (C33F10.5), the only C. elegans member of the. contactin subfamily. Aiming to determine the rig-6 spatiotemporal. expression pattern, we have generated a full-length, GFP reporter fusion. encompassing both the promoter and the complete coding region of the gene.. rig-6 is expressed in head neurons, ventral cord motorneurons, in muscle. cells and hypodermis. Its expression starts from embryonic stages, and it. is maintained through adulthood. To further investigate the role of RIG-6. we are using rig-6 deletion mutants and RNAi knockdown to characterize. effects on neuronal development and axonal migration.