Kathleen Morgan et al. (2007) International Worm Meeting "Modeling environmentally-induced sporadic ALS."

Kathleen Morgan, Annette Estevez, Miguel Estevez

[International Worm Meeting, 2007]

Exposure to high levels of selenium (Se) has been associated with increased risk of developing the motor neuron disease Amyotrophic Lateral Sclerosis (ALS) in human epidemiologic studies and motor neuron degeneration resembling ALS in livestock animals exposed to high levels of Se under laboratory conditions. Similarly, in C. elegans supplementation of NGM plates with sodium selenite (NaSe) induces a progressive paralysis involving motor neuron degeneration. Assessment of motor neuron morphology in Se-treated animals expressing a pan-neuronal GFP (Punc-119::gfp, Maduro and Pilgrim 1995), demonstrated classical signs of neuronal injury including: neuronal swelling, loss of the nucleus cytoplasm boundary, loss of chromatin staining, axonal blebbing/beading along the ventral cord, and altered mitochondrial morphology. The morphologic changes observed do not phenocopy the changes described in the mec neurodegeneration pathway since it involves much less swelling and is not genetically suppressible by cad-1(rf), vha-2(rf), or crt-1(rf) (Bianchi, et al. 2004). In addition, the Se-induced neurodegenerative changes do not phenocopy hypoxic changes in that pharyngeal and body wall muscles seem to be spared (Scott, et al., 2003). In order to determine what signaling pathways might be involved in this process we have screened the major stress-activated growth factor pathways in C. elegans for effects on Se-induced neurodegeneration. These studies have found that reduced signaling through the TGF-<font face=symbol>b</font> cascade powerfully sensitizes animals to Se-neurotoxicity. In contrast, decreased signaling through the insulin signaling pathway (Tissenbaum and Ruvkun, 1998), in daf-2(rf) and age-1(rf), resulted in significant resistance to Se-neurotoxicity. These insulin-pathway effects on Se-neurotoxicity appear to involve alterations in neuroprotective gene expression since reduction-of-function in daf-16(m26 and mgDf50), a gene encoding a transcriptional effector of insulin signaling, conferred increased sensitivity to Se exposure. Since the insulin cascade regulates expression of various antioxidant enzymes these results suggested that high environmental Se induces neuronal injury through increasing oxidative stress. Consistent with this hypothesis both glutathione and quercetin significantly reduced the emergence of paralysis under conditions of high environmental Se. Collectively, these results show that Se-neurotoxicity in C. elegans involves features associated with ALS pathology including: motor neuron degeneration, increased oxidative stress, mitochondrial injury, and DNA damage.

Kathleen L Morgan et al. (2005) International Worm Meeting "Developing a model of Amyotrophic Lateral Sclerosis (ALS) using Selenium toxicity in C. elegans"

Kathleen L Morgan, Annette Estevez, Miguel Estevez, Robin H Cowie

[International Worm Meeting, 2005]

Selenium (Se) is an essential nutrient which is utilized by glutathione peroxidase in the scavenging of free radicals. In humans, excess Se is toxic causing nausea, weakness, diarrhea, damage to the nervous system, and rarely death. Environmental exposure to high Se levels in a small Italian city lead to a 40-fold increase of amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disorder that affects motor neurons in the spinal cord. We are using Se toxicity in the worm to study and develop a model of ALS. Previous studies in our lab have shown that Se toxicity in worms causes embryonic lethality, larval arrest, and in adults, reduced fertility, paralysis and death. Using the adult endpoint of paralysis and death, various concentrations of seleno-L-methionine (SeMet) were used to generate a toxicity curve for N2s exposed to SeMet. Adult animals were plated on a SeMet concentration that caused paralysis and death within 48-72 hours. Before paralysis and death, animals expressing the pan-neuronal marker unc-119::GFP treated with SeMet were examined and shown to have blebbing along the ventral cord, neuronal swelling, and altered nuclear staining that was not present in untreated controls. Examination of muscle fibers using myo-3::GFP animals showed that muscle fibers were unaffected by SeMet treatment. Taken together, these data support using Se toxicity as a model for ALS. Currently, we are screening known growth factor and signal transduction mutants for resistance to selenium toxicity. In addition, we have also generated a number of selenium resistant mutants by EMS mutagenesis and are in the process of genetically characterizing them.

Robin H Cowie et al. (2003) International Worm Meeting "Selenium toxicity in C. elegans leads to paralysis, developmental arrest, and reduced fertility"

Annette Estevez, Miguel Estevez, Robin H Cowie

[International Worm Meeting, 2003]

Although selenium(Se) is an essential micronutrient required for normal antioxidant activity, endocrine function, and immune system function, the difference between healthy levels of selenium intake and toxic levels is quite narrow. At high concentrations Se can lead to diverse pathologic consequences including motor neuron degeneration indistinguishable from polio and Amyotrophic Lateral Sclerosis (ALS), reproductive failure, and congenital abnormalities (especially of the CNS). Although the toxic effects of Se have been known for many years its role in processes other than free radical scavenging and thyroid hormone synthesis remain unclear despite the large number of identified selenoproteins. Because of the association of high environmental Se with ALS, and reproductive failure in wildlife we have begun to develop a genetic approach to identify the pathological processes it causes. The effects of selenium exposure on C. elegans have not previously been characterized. A baseline selenium concentration generating observable toxicity in populations of N2 was defined using a range of seleno-L-methionine (SeMet) concentrations in the growth media. (Note: Sodium selenite was originally used and was also toxic, but bacterial metabolism of the compound led to emission of a volatile sulfurous smelling compound that caused mucosal irritation in lab members. Dont try this at home!). To develop an appropriate screening tool with which to select selenium resistant strains we standardized four classes of toxicity assays which measure acute toxicity, developmental arrest/egg viability, and fertility. Our studies have shown that SeMet is directly toxic to neurons and leads to axonal blebbing, loss of neuronal cell body morphology, and paralysis over a 48-hour period. In embryos, SeMet induces congenital malformations and developmental arrest. Embryos that do manage to hatch often have bizarre malformations of the head. SeMet also reduces the amount of eggs produced per animal. These toxic responses are similar to those seen in wildlife populations exposed to Se contaminated water. Several mutations that affect sensitivity to Se toxicity have been isolated and are being characterized.

Jianghua Yin et al. (2007) International Worm Meeting "Regualtion and function of a sma-9 target gene."

Cathy SavageDunn, Ling Yu, Jianghua Yin

[International Worm Meeting, 2007]

BMP/TGF<font face=symbol>b</font> signaling cascades regulate many aspects of development and differentiation in both vertebrates and invertebrates. In the well-characterized Drosophila Dpp signaling cascade, upon Dpp ligand binding, type I receptor phosphorylates the intracellular signal transducer Mad (R-Smads), which then forms a complex with the Co-Smad Medea (Med). The Smad protein complex will translocate into the nucleus, recruiting transcription cofactor Schnurri (Shn) and inhibiting the brinker gene transcription, which in turn negatively regulates the expression of most Dpp target genes. In Caenorhabditis elegans, the Schnurri homolog SMA-9 is also found to function as a transcription cofactor in the DBL-1 pathway, a BMP/TGF<font face=symbol>b</font>-related signaling cascade, regulating body size and male tail development. The molecular mechanisms of SMA-9 function are still unknown. Study of the sma-9 target genes will help us to illustrate the conserved role of Schnurri/SMA-9 protein function in invertebrates. The T27F2.4 gene, a bZip transcription factor, is found to be highly upregulated in sma-9 mutant background, according to the microarray analysis and real-time PCR data. Meanwhile, RNAi experiment show that in vivo, knockdown of T27F2.4 gene will cause long body size phenotype. Recently, we found that the downstream region of this gene contains a putative silencer element (SE) that is essential for mediating the Dpp-dependent repression of brinker gene transcription in Drosophila via the Shn/Mad/Med complex. This SE also functions similarly in vertebrates. We hypothesize that the regulation of T27F2.4 gene expression and tissue specificity are also mediated through this conserved mechanism. Now we are using GFP reporter constructs containing the SE element to test our hypothesis. Meanwhile, examining how T27F2.4 gene functions in body size development is another goal of our studies.

Yang, Ya-Luen et al. (2013) International Worm Meeting "Sestrin confers the regulations of muscle aging and lifespan in Caenorhabditis elegans."

Yang, Ya-Luen, Chu, I-Hua, Liou, Bang-Yu, Loh, Kah-Sin, Chen, Huan-Da, Chen, Chang-Shi, Kuo, Cheng-Ju

[International Worm Meeting, 2013]

Aging is associated with the progressive decline in body function and physiology that is shared by all multicellular organisms. The free radical theory argues that the accumulation of damage caused by reactive oxygen species (ROS) over the time is the cause of aging. Sestrins are evolutionarily conserved in metazoans and are required for ROS clearance. However, whether sestrins per se regulate longevity in multicellular organisms is still unclear. Here, we report that SESN-1, the only Sestrin ortholog in Caenorhabditis elegans, is a positive regulator of lifespan. C. elegans sesn-1 mutants exhibit shorter lifespans, hypersensitivity to oxidative stress, and premature aging in muscle. When sesn-1 gene was overexpressed and rescued, the lifespan of sesn-1 mutant worms could be extended. These findings imply that SESN-1 is required to protect against general life stressors, is important in regulation of lifespan and healthspan late in life, and might play a key role in muscle integrity.

Rayka Yokoo et al. (2007) International Worm Meeting "A novel component of the meiotic crossover machinery in C. elegans."

Anne Villeneuve, Kentaro Nabeshima, Gregory Chin, Rayka Yokoo

[International Worm Meeting, 2007]

Proper segregation of homologous chromosomes during meiosis relies on the formation of crossovers between paired homologs, resulting in a structure known as a chiasma. We are investigating the identity and function of a new component of the crossover recombination machinery defined by the me13 mutation. me13 mutant hermaphrodites produce a high incidence of males and dead embryos reflecting missegregation of the X chromosome and autosomes at meiosis. DAPI staining of diakinetic chromosomes in the me13 mutant reveals univalent rather than the wildtype bivalent structures, suggesting a lack of chiasmata, and indicating a defect in meiotic prophase. FISH experiments indicate that homologous chromosomes are appropriately paired in the me13 mutant. In addition, immunofluorescence experiments visualizing synaptonemal complex components SYP-1 and HIM-3 indicate that homologs are appropriately synapsed. These experiments indicate that the defect may be in recombination per se. Experiments assessing the response to ionizing radiation suggest that the me13 mutant is not specifically defective either in the formation of double strand breaks (DSBs) at the initiation step of recombination or in DNA repair per se. Furthermore, immunofluorescence staining of RAD-51, a protein that binds DSBs, indicates DSBs are made in the me13 mutant. RAD-51 foci diminish by late pachytene, but redistribution of HTP-1 and SYP-1, currently hypothesized to correlate with crossover events, does not occur. Together these results suggest that DSBs are repaired but by a pathway that does not result in interhomolog crossover. Whereas many features of the me13 mutant phenotype resemble defects caused by known mutations in him-14, msh-5, and zhp-3, genes that function in converting processed double strand breaks into interhomolog crossovers, we have mapped the me13 mutation to a region on the left arm of chromosome III that does not contain any previously known meiotic recombination genes. We will report progress in identifying the molecular defect and analyzing the role of me13 in the recombination process.

Edwards, Tyson et al. (2015) International Worm Meeting "Dopamine induces circling locomotion in C. elegans."

Edwards, Tyson, Bai, Jihong, Han, Bicheng

[International Worm Meeting, 2015]

Navigational circling bias occurs in various animal species, including humans1, and has been linked to dopamine signaling 2,3. We have observed a unique circling behavior in wild type worms exposed to low concentrations of exogenous dopamine. Interestingly, this behavior is phenocopied by administration of 3,4-methylenedioxymethamphetamine (MDMA, a.k.a. "ecstasy")4. We find that dopamine induces ventrally-directed circles in a dose-dependent manner. We hypothesize that distinct neurons in the locomotory circuit have differing sensitivities to dopamine, resulting in asymmetric neurotransmission that biases locomotion. Indeed, laser ablation of a subset of inhibitory motor neurons induces circling5. This subset of motor neurons may be sensitized by expression of an inhibitory dopamine receptor. Dopamine mutants that lack the canonical D2-like receptor dop-3 retain the ability to circle, suggesting that a novel receptor may be involved. We are working to identify the putative receptor, and to implement calcium imaging and electrophysiological methods to determine the circuits that mediate circling locomotion. Overall, this work hopes to shed light on the mechanisms by which neuromodulators target specific circuits to alter behavior.1. Souman, J. L., Frissen, I., Sreenivasa, M. N. & Ernst, M. O. Walking Straight into Circles. Curr. Biol. 19, 1538-1542 (2009).2. Bracha, H. S., Shults, C., Glick, S. D. & Kleinman, J. E. Spontaneous asymmetric circling behavior in hemi-parkinsonism; a human equivalent of the lesioned-circling rodent behavior. Life Sci. 40, 1127-1130 (1987).3. Mohr, C., Landis, T., Bracha, H. S., Fathi, M. & Brugger, P. Human locomotion: levodopa keeps you straight. Neurosci. Lett. 339, 115-118 (2003).4. Schreiber, M. A. & McIntire, S. L. A Caenorhabditis elegans p38 MAP kinase pathway mutant protects from dopamine, methamphetamine, and MDMA toxicity. Neurosci. Lett. 498, 99-103 (2011).5. Donnelly, J. L. et al. Monoaminergic Orchestration of Motor Programs in a Complex C. elegans Behavior. PLoS Biol 11, e1001529 (2013).

Dalfo, Diana et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Genomic-scale RNAi screen identifies a new role for TGFbeta signaling in germline proliferation"

Dalfo, Diana, Hubbard, E Jane A

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Cell proliferation is a key step in germline development that requires precise spatial and temporal regulation to assure a reproductively competent individual. GLP-1/Notch signaling is important for maintaining a proliferating population of germ cells: loss of glp-1 activity causes all germ cells to differentiate. We have shown that Notch-independent signaling pathways such as insulin/IGF contribute to the robust proliferation of the larval germ line that is required for optimal fecundity. To identify additional genes and pathways required for robust larval germline proliferation, we performed a genome-scale post-embryonic RNAi modifier screen. We developed a genetic strategy that allowed us to identify genes required for the developmental control of germline proliferat ion/differentiation, rather than the many genes required for cell proliferation per se. We identified genes that cover a broad spectrum of families and functions that give us a more extensive knowledge of the control of germline proliferation. Of particular interest, we found a novel dauer-independent role for TGF signaling to promote germline proliferation.

R Boswell et al. (1999) International C. elegans Meeting "Involvement of mag-1, a homolog of Drosophila posterior group gene mago nashi, in hermaphrodite germ-line sex determination"

B Wood, R Boswell

[International C. elegans Meeting, 1999]

In D. melanogaster , the posterior group gene mago nashi is involved in oocyte patterning during oogenesis, including dorso-ventral determination as well as posterior germ plasm assembly. The mag-1 gene is the most convincing homolog of a Drosophila posterior group gene in C. elegans , since it shares 80% identity and 88% similarity with mago nashi and can functionally replace it in flies (1). In attempts to understand the possible function(s) of mag-1 in worms, we have found using RNA-mediated interference that mag-1(RNAi) animals display a masculinization-of-germ-line (Mog) phenotype in hermaphrodites, suggesting a role in hermaphrodite germ-line sex determination. Epistasis analysis showed that mag-1 acts upstream of fog-2 , which promotes temporary masculinization of the hermaphrodite germ line during the L4 stage. This result suggests that mag-1 is required for maintaining oogenesis in adult hermaphrodites, possibly by negatively regulating fog-2 activity. The mag-1 gene also appears to act upstream of the gld-1 gene. Consistent with these results, ectopic sperm production caused by mag-1(RNAi) was found to depend on the fog-1 and fem-1 , -2 and -3 genes, which act downstream of fog-2 and gld-1 in the germ-line sex determination pathway. In addition to germ-line masculinization, mag-1(RNAi) caused lethality of progeny embryos when the Mog phenotype was suppressed in a fog-2(lf) background, suggesting an essential role for mag-1 during embryogenesis. These embryos were arrested during morphogenesis with an apparent elongation defect. The expression pattern of JAM-1(MH27)::GFP, which localizes to adherens junctions, showed that hypodermis is disorganized in these embryos. Embryonic expression of the mag-1 gene prior to and during morphogenesis is consistent with a role in embryogenesis. 1. Newmark, P. A., Mohr, S. E., Gong, L. and Boswell, R. E. (1997). mago nashi mediates the posterior follicle cell-to-oocyte signal to organize axis formation in Drosophila. Development 124: 3197-3207.

A. Shorto et al. (2006) European Worm Meeting "Dauer Larvae Development and Fitness: Does it All Depend on your Environment?"

A. Shorto, S. C. Harvey, M. E. Viney.

[European Worm Meeting, 2006]

?. A. Shorto, S. C. Harvey and M. E. Viney. Natural isolates (including N2 and DR1350) of the free-living nematode Caenorhabditis elegans vary in their phenotypic plasticity of dauer larvae development. For example, some lines appear to be highly sensitive to dauer inducing conditions while others are less so. We have sought to investigate the causes and consequences of this plasticity.. To determine the fitness consequences of this plasticity of dauer development we have investigated how lifespan, total fecundity, reproductive schedule and population growth vary in N2 and DR1350 and in N2 x DR1350 recombinant inbred lines (RILs). We have found that the plasticity of dauer larvae development is positively correlated with the population growth rate (as measured by population size after 8 days of growth). Differences in population growth appear not to be dependent on lifetime fecundity or reproductive schedule (number of eggs laid per day) per se, but rather due to how the reproductive schedule changes in response to reduced food availability. Overall, these results suggest that there may be different reproduction and dauer formation strategies in response to environmental change.