Page K E et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Genes Affecting Aluminium Toxicity in Caenorhabditis elegans"

McCrohan C R, Page K E, White K N, Lithgow G J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Aluminium (Al) is a highly abundant crustal metal with known toxic effects in multiple biological systems. Using the nematode C. elegans we have found a set of genetic modulators of Al toxicity. C. elegans is widely used for toxicity and aging studies due to its small size, large progeny numbers, short lifespan, and simple methods of genetic manipulation. This makes C. elegans a good model to study the toxicity mechanism of Al in animals. The exposures were carried out on agar plates, with Al in the form of Al(NO3)3, mixed with a concentrated Escherichia coli food source. Here we show that a selection of genes involved in the mechanisms of Al toxicity and/or metabolism have potential relevance in the metallostasis (metal homeostasis) of other metals, or are key regulators of known stress resistance pathways. Al negatively affects C. elegans developmental progression from 3 microM Al, fertility at 30 microM Al, and body size from 1.9 mM Al. The developmental delay phenotype caused by Al exposure can be passed on to the un-exposed next generation. At 4.8mM exposures Al affects the levels of Al and other elements in C. elegans; shown using ICP-OES. We are also investigating the effect of Al exposure to the course of normal aging in C. elegans.

Shelli N Williams et al. (2002) Mid-west Worm Meeting "Cytoskeletal defects associated with NUD-1 knockdown in early embryogenesis of C. elegans"

Shelli N Williams, Kim A Caldwell, Guy A Caldwell

[Mid-west Worm Meeting, 2002]

Dawe et al. Dev Genes Evol. (2001) 211:434-441.

Page, Kathryn E. et al. (2009) International Worm Meeting "Exploring Aluminium Toxicity in C. elegans."

Page, Kathryn E., McCrohan, Catherine R., White, Keith N., Lithgow, Gordon J.

[International Worm Meeting, 2009]

Aluminium (Al) is a highly abundant crustal metal with known toxic effects in multiple biological systems. Caenorhabditis elegans (C. elegans) is widely used for toxicity and aging studies due to its small size, large progeny numbers, short lifespan, and simple methods of genetic manipulation. C. elegans is a good model to study the toxic mechanism of Al in animals, and has been widely used for the study of environmental metal toxicology. Here we show Al effects fertility, and development progression, and that these phenotypes are transferable to the progeny of exposed worms. The exposures were carried out on agar plates with the Al mixed with concentrated OP50 which was then fed to the worms. Al negatively affects C. elegans developmental progression at 0.1 to 300 mM Al food concentration, and fertility at 1 mM Al food concentration. The developmental delay phenotype caused by Al exposure can be passed on to the next generation. It is possible that the mechanism of Al toxicity is similar to that of other toxic metals.

Richard S Allan et al. (2005) International Worm Meeting "High-resolution, digitally-encoded phenotypic analysis of gonadogenesis in C. elegans"

E Jane Albert Hubbard, Richard S Allan, Philip MacMenamin, Kristin C Gunsalus, Fabio Piano, Krishna Vijayendran

[International Worm Meeting, 2005]

A fundamental premise of genetic analysis is that genes with similar loss-of-function phenotypes likely act in similar developmental processes. Extending this concept to RNAi-based genome-scale functional characterization, Piano et al. (2002) defined 47 embryonic phenotypic characters in C. elegans and generated a digital phenotypic signature for each RNAi experiment. Genes conferring similar signatures were clustered (phenoclusters), placing previously uncharacterized genes in clusters with genes of known function. The primary data (high-magnification time-lapse movies), scoring criteria and results are publicly available and can be analyzed and queried using additional tools (e.g. Phenoblast) (Gunsalus et al., 2004; http://www.RNAi.org).We have applied this approach to gonadogenesis and germline development in C. elegans. In previous large-scale RNAi screens, over 800 genes are reported to confer a sterile (Ste) or sterile progeny (Stp) phenotype, based on low-magnification screening (Gonczy et al., 2000; Fraser et al. 2001; Piano et al., 2001; Maeda et al., 2001; Hanazawa et al. 2001; Piano et al., 2002; Kamath et al., 2003; Simmer et al., 2003). A subset of these have been examined under high magnification (Maeda et al., 2001; Hanazawa et al., 2001; Colaiacovo et al., 2002), but these data are not available in a systematic digital format amenable to analyses such as phenoclustering or Phenoblast, nor to phenotypic search criteria.We developed and are implementing a web-based digital scoring system (currently 262 characters) for high-magnification phenotypes in adult hermaphrodites. Characters include both quantitative and qualitative features of the gonad, germ line and gametes as well as several gonad-related characters. We generate a series of digital Z-plane images at high magnification from each RNAi experiment. All data will be made publicly available on the RNAi.org website. Our test set is 147 Stp genes and 260 Ste genes conferring a wide range of RNAi-induced defects.

Page, Kathryn E. et al. (2011) International Worm Meeting "Aluminium Toxicity and Elemental Composition Changes in Caenorhabditis elegans."

Killilea, David W., Lithgow, Gordon J., McCrohan, Catherine R., White, Keith N., Page, Kathryn E.

[International Worm Meeting, 2011]

Environmental metal contamination events such as the alumina sludge spill in Hungary last year highlight the importance of understanding metal toxicity and predicting detrimental effects. Aluminium (Al) is highly abundant in our environment, but can elicit a variety of toxic responses. Here we present the first characterization of the effects of Al exposure in the nematode worm Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole body metal homeostasis (metallostasis) following Al exposure. Lifespan was decreased in worms exposed to low levels of Al, but was unexpectedly increased when the Al concentration reached higher levels. Interestingly, this bi-phasic phenotype was only observed when Al exposure occurred during development, as adult worm lifespan was unaffected by Al at similar exposures. Al negatively affected C. elegans developmental progression and self-fertility when exposed to as little as 30mM Al, and reduced body size when exposed to 1.9 mM Al. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Widespread changes to the elemental content of adult nematodes was observed when chronically exposed to Al from L1. Specifically we saw increased Ba, Cr, Cu, and Fe content, and a reduction in Ca levels. Similar changes have been noted in human Al toxicity, suggesting that C. elegans may be exploited to understand the mechanisms of Al toxicity in human tissue. Elemental homeostasis is obviously highly regulated, and here we see that changing the levels of only one element can alter the whole elemental profile. This raises the question of how genes affect these elemental profiles. Here we determine how gene knockdown can alter metallostasis and elemental profiles in C. elegans. It is notable that these genetic modulators of elemental profiles are not only important for metallostasis, but are key regulators of known stress response mechanisms. Knock-down of these gene products alters the elemental profile of the worm, which suggests novel mechanisms of action for these genes in metal toxicity and general stress response.

Saleel Raut et al. (2008) C.elegans Neuronal Development Meeting "HLH-3, a C. elegans Achaete/Scute protein, has a role in the axonal pathfinding of hermaphrodite-specific motor neuron (HSN) that is independent of UNC-40."

Aixa Alfonso, Saleel Raut

[C.elegans Neuronal Development Meeting, 2008]

The basic helix loop helix protein, HLH-3, is a member of the achaete/scute family in C. elegans hlh-3 mutants have an egg-laying defective (Egl) phenotype. We have shown (Doonan et al., 2008 submitted) that the mutant phenotype results from abnormal axonal pathfinding in hermaphrodite-specific motor neurons (HSNs) that fail to innervate the vulval muscles. The NETRIN/UNC-6 receptor UNC-40/DCC is necessary for the guidance of the HSN axons (Garriga et al.,1993; Chan et al., 1996; Gitai et al., 2003; Adler et al., 2006). We hypothesized that mutant animals are not uncoordinated (Unc). Currently we are looking at SYG-1, a receptor on HSN''s that binds with the guidepost signal SYG-2 for proper synapse formation (Shen et al., 2004), as a possible target of HLH-3.

Richard Allan et al. (2006) Development & Evolution Meeting "Genome-scale High-content Phenotypic Analysis of Sterility in C. elegans"

Fabio Piano, Kristin Gunsalus, Krishna Vijayendran, E. Jane Hubbard, Philip MacMenamin, Richard Allan

[Development & Evolution Meeting, 2006]

Genes with similar loss-of-function phenotypes often act in similar developmental processes. Coupled with genome-scale RNAi studies, this premise suggests that starting from RNAi-induced phenotypes, previously uncharacterized genes required for specific processes can be identified by grouping them with genes of known function that confer similar RNAi phenotypes. Successfully using this strategy for analysis of the early embryo, Piano et al. (2002) defined 47 discrete characters to characterize complex cell biological and developmental phenotypes resulting from RNAi of genes required for embryogenesis. Phenotypic signatures - combinations of explicit outcomes for each character - were generated for each RNAi experiment, and genes were clustered by phenotypic similarity (phenoclusters). These data provided hypotheses about the function of previously uncharacterized genes. Primary data, scoring criteria and results are publicly available in RNAiDB (http://www.RNAi.org) and can be analyzed and queried using additional tools (e.g. PhenoBlast; Gunsalus et al., 2004). We have applied the same approach to RNAi-induced sterility. In previous large-scale screens, over 800 genes conferred a sterile (Ste) or sterile progeny (Stp) phenotype, based on scoring of brood size (Gonczy et al., 2000; Fraser et al. 2001; Piano et al., 2000; Maeda et al., 2001; Hanazawa et al. 2001; Piano et al., 2002; Kamath et al., 2003; Simmer et al., 2003). To characterize specific mophological defects underlying the observed sterility, we have developed within RNAiDB a scoring system for ~300 discrete phenotypic features visible at high-magnification in adult hermaphrodites, providing data amenable to phenoclustering and PhenoBlast. As a pilot study, we have chosen a set of genes previously identified as Stp and/or Ste by RNAi (Fraser et al. 2001; Kamath et al., 2003) that confer a wide range of RNAi-induced defects. Analysis of phenotypic signatures (~100 characters) and phenoclustering are in progress. Data (Z-plane stacks) will be available at RNAiDB, together with the phenotypic scoring results. The scoring system and analysis tools will also be made available for general use.

Tsai-Fang Huang et al. (2005) International Worm Meeting "A potential transcription factor controls the timing of DTC dorsal turn by regulating unc-5 expression."

Tsai-Fang Huang, Yi-Chun Wu

[International Worm Meeting, 2005]

During the larval development of a C. elegans hermaphrodite, a pair of distal tip cells (DTCs) lead the extension of two gonadal arms through three sequential phases of migration. They first move along the ventral side to the ends of the animal (phase 1), then make a dorsal turn (phase 2) and finally move along the dorsal side back to the center (phase 3). We have isolated the mutation tp3 with aberrant DTC migration. A time-course analysis showed that the tp3 DTC had an early dorsal turn. We have cloned the gene, tentatively named as mig-x here, defined by the tp3 mutation. It encodes a protein with a nuclear localization signal and five zinc fingers, suggesting that it may function as a transcription factor to regulate DTC migration. Previous studies showed the dorsal turn of DTC requires UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40 (Chan et al., 1996; Hedgecock et al., 1990; Ishii et al., 1992; Leung-Hagesteijn et al., 1992) and that the transcriptional upregulation of unc-5 in DTCs is required and sufficient for the onset of the dorsal turn (Su et al., 2000). Interestingly, premature unc-5 expression was observed in the DTC undergoing a precocious dorsal turn in tp3 mutants. Therefore, mig-x may negatively regulate unc-5 expression to control the proper timing of the dorsal turn. We are testing if unc-5 may be the direct transcriptional target of MIG-X. Intriguingly, unc-5;tp3, unc-6;tp3 and unc-40;tp3 double mutants exhibited a synthetic phenotype of prolonged longitudinal migration without phase-2 or -3. This defect is similar to that of daf-12(rh84) mutants (Antebi et al., 1998). daf-12 encodes a nuclear hormone receptor and is required for unc-5 expression (Antebi et al., 2000; Snow et al., 2000; Su et al., 2000). We are testing if mig-x expression may depend on daf-12. To explore the expression pattern of mig-x, we made a mig-x::gfp translational fusion construct, which fully rescued the tp3 phenotype. The MIG-X fusion protein was present in DTC but not muscle cells, consistent with the model that mig-x acts in DTC to control DTC migration. In addition, MIG-X::GFP is observed in the P-lineage derived cells, hypodermal cells, and a few cells in the tail. We also generated MIG-X antibodies and are investigating the expression pattern of the endogenous mig-x gene. References: Antebi et al. (1998) Development 125, 1191-205; Antebi et al. (2000) Genes & Development 14, 1512-1527; Chan et al. (1996) Cell 87, 187-95; Hedgecock et al. (1990) Neuron 4, 61-85; Ishii et al. (1992) Neuron 9, 873-81; Leung-Hagesteijn et al. (1992) Cell 71, 289-99; Snow et al. (2000) Biochimica et Biophysica Acta - Gene Structure & Expression 1494, 104-116; Su et al. (2000) Development 127, 585-94.

Malek Jundi et al. (2004) East Coast Worm Meeting "Cytological screening of the germ lines of sterile mutants for meiotic defects"

Malek Jundi, Monique C Zetka

[East Coast Worm Meeting, 2004]

Extensive screening for mutations in essential genes has been carried out for defined chromosomal regions on LG I (Linkage Group; Rose et al. 1980: Howell et al. 1987; Howell and Rose 1990; McKim et al. 1992; Johnsen et al. 2000), LG II (Sigurdson et al. 1984), LG IV (Rogalski et al. 1982; Rogalski and Baillie 1985; Clark et al. 1988; Clark and Baillie 1992), LG V (Rosenbluth et al. 1988; Johnsen and Baillie 1991) and LG X (Meneely and Herman 1979, 1981). A subset of the alleles in this mutant library result in sterility and are likely to have identified genes essential for the development of the gonad and the germ line. Because the germ line of the nematode presents an orderly and predictable progression of nuclei through the various stages of proliferation and meiosis, the function of the wild-type gene can be inferred by examining the DAPI-stained nuclei of sterile animals for cytologically visible defects. We are screening a collection of 138 previously uncharacterized sterile mutants originating from these analyses for the presence of univalents or chromosome fragments at diakinesis, which is a general indicator of defects in upstream processes like meiotic cell-cycle progression, chromosome pairing, synapsis, recombination, and chromosome morphogenesis. To date, we have identified four sterile mutations that result in a high frequency of univalents at diakinesis and that have associated defects in conserved meiotic processes. Supported by a grant from the Canadian Institutes of Health Research (CIHR)

Jon Pierce-Shimomura et al. (2002) Japanese Worm Meeting "Behavioral responses to ethanol are mediated by a BK potassium channel in C. elegans"

Antonello Bonci, Steve McIntire, Hongkyun Kim, Jon Pierce-Shimomura, Tod R Thiele, Andrew Davies

[Japanese Worm Meeting, 2002]

We screened for mutant C. elegans that are resistant to intoxication by ethanol. We found that strong resistance is conferred by mutations in the gene slo-1 , which encodes a highly conserved ortholog of the human large conductance calcium-activated potassium (BK) channel 1-3 . Prevous work has shown effects of ethanol on vertebrate BK channels in vitro 4-9 . We hypothesized that intoxication in C. elegans may be primarily mediated by ethanol activating the SLO-1 channel; thus strong resistance to intoxication by ethanol results when the SLO-1 channel is absent. To test this idea, we first performed in vivo whole-cell voltage-clamp recordings of dopamine neurons to identify a SLO-1 current that was present in neurons in wild-type worms but absent in slo-1 mutants. Next, we found that ethanol selectively and reversibly increased the amplitude of the outward-rectifying current in neurons of wild-type animals at physiologically relevant concentrations (20 & 100 mM). Ethanol had no effect, however, on currents in neurons of slo-1 mutants. To determine whether ethanol can activate SLO-1 directly in the absence of cytosolic factors, we recorded the single-channel activity of SLO-1 in patches excised from dopamine neurons. Ethanol reversibly increased the probability of channel opening. Thus, we have demonstrated a direct relationship between the SLO-1 channel, a biochemical effect of ethanol and a genetic effect on behavior 1 Wei et al (1996) Neuropharm; 2 Wang et al (2001) Neuron; 3 Chu et al (1998) Mol Pharmacol; 4 Dopico et al (1998) J Pharmacol Exp Ther; 5 Dopico et al (1996) Mol Pharmacol; 6 Dopico et al (1999) J Physiol; 7 Gruss et al (2001) Eur J Neurosci; 8 Jakab et al (1997) J Membr Biol; 9 Walters et al (2000) Am J Physiol Cell Physiol