Yukiko Yonezawa et al. (2007) International Worm Meeting "Role of the type-b insulin-like genes on larval diapause and adult lifespan in Caenorhabditis elegans."

Yukiko Yonezawa, Yasuo Kimura, Kenji Gouda, Tsuyoshi Kawano

[International Worm Meeting, 2007]

In Caenorhabditis elegans, the insulin/insulin-like growth factor-I signaling (IIS) pathway regulates larval diapause, adult lifespan, fat metabolism, and stress-resistance. C. elegans has 38 putative insulin-like peptides which are grouped into type-<font face=symbol>a</font> , -<font face=symbol>b</font> , and -<font face=symbol>g</font> according to their predicted disulfide bond pattern.1) The type-<font face=symbol>g</font> peptides have the same pattern as mammalian insulin family peptides. On the other hand, the type-<font face=symbol>b</font> peptides have one additional disulfide bond to the canonical three. In 2003, Murphy and co-workers reported that ins-7 categorized into the type-<font face=symbol>b</font> has relevancy on regulation of dauer larva formation and adult lifespan.2) This fact suggested that other type-<font face=symbol>b</font> genes also have physiological functions. Therefore, we attempted to investigate all of the type-<font face=symbol>b</font> genes by RNAi knockdown and/or gene-disruption. In this meeting, we will show positive/nagative regulation of larval diapause and/or adult lifespan by the type-<font face=symbol>b</font> genes. 1) Pierce, S. B., et al. (2001) Genes Dev. 15:672. 2) Murphy, C. T. et al. (2003) Nature 424:277.

Kenji Gouda et al. (2007) International Worm Meeting "Redundant function of Caenorhabditis elegans insulin-like genes in larval diapause and adult lifespan."

Keiko Gengyo-Ando, Yasuo Kimura, Kenji Gouda, Yohei Matsunaga, Tsuyoshi Kawano, Shohei MItani

[International Worm Meeting, 2007]

The insulin/insulin-like growth factor-I signaling (IIS) pathway regulates larval diapause and adult lifespan in Caenorhabditis elegans. To date, many of the 38 insulin-like genes have been identified1), and a few of the genes have been investigated to identify their physiological function by RNAi knockdown. For instance, Murphy and co-workers reported that ins-7 RNAi knockdown induces larva diapause and an extended lifespan.2) In our previous study, we disrupted Ceinsulin-1 (ins-18) and Ceinsulin-2 (ins-17), and elucidated their function on larval diapause and adult lifespan. Disruption of ins-17 and/or ins-18 reduced dauer larva formation caused by a crude extract of dauer-inducing pheromone, while the disruption showed no effect on adult lifespan. To investigate redundant function of the insulin-like genes, we disrupted the ins-7, and then established multiple-gene-disrupted animals. Disruption of ins-7 induced an extended lifespan as expected. Disruption of ins-7 and ins-17 also revealed lifespan extension, suggesting that ins-17 is not relevant to lifespan regulation. On the other hand, disruption of ins-7 and ins-18 revealed no lifespan extension, indicating that ins-18 is necessary for lifespan extension induced by the gene-disruption of ins-7. Now we are measuring lifespan of each gene-disrupted animal under daf-2(-) conditions. 1) Pierce, S. B. et al. (2001) Genes Dev. 15:672. 2) Murphy, C. T. et al. (2003) Nature 424:277.

Fiona J Thompson et al. (2002) European Worm Meeting "Genome analysis of the effect of the host immune response on Strongyloides ratti."

Fiona J Thompson, Brandi Chiapelli, Claire Murphy, Clare P Wilkes, Mark E Viney, Jim McCarter, Sandra Clifton

[European Worm Meeting, 2002]

The establishment, survival and fecundity of nematodes is dramatically affected by the host immune response. However the molecular and biochemical nature of these effects have yet to be fully explored. We propose to exploit the nematode genome sequencing projects and EST data to analyse these phenomena. To this end we have generated a number of cDNA libraries from different stages of Strongyloides ratti including those from parasitic adults which had been exposed to different degrees of immune pressure. To date 7,793 ESTs have been sequenced and the cluster analysis of these clones will be presented. In order to generate microarrays all unique clones are being amplifed by PCR and arrayed. These microarrays will be differentially probed with cDNA isolated from populations of worms from varying immune pressures. We aim to identify gene sets whose expression levels vary as a result of changes in the host immune response and as such may be the basis of the immune dependant changes in the fitness of nematode infections.

Yueyi I Liu et al. (2004) West Coast Worm Meeting "Global Identification of C. elegans Regulatory Motifs"

Stuart K Kim, Yueyi I Liu

[West Coast Worm Meeting, 2004]

Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .

Claire Lecroisey et al. (2006) European Worm Meeting "Implication of the Dense Body Protein Dyc-1 in the Dystrophin Dependent Muscle Degeneration"

Kathrin Gieseler, Laurent Segalat, Claire Lecroisey

[European Worm Meeting, 2006]

Claire Lecroisey, Kathrin Gieseler and Laurent Sgalat. The molecular mechanism underlying muscle necrosis in dystrophinopathies remains elusive. Our group addresses this question by using the genetically amenable animal model Caenorhabditis elegans, which has the same overall sarcomere composition and architecture as those of vertebrates.. In C. elegans, mutation of the dystrophin homologue, dys-1(cx18), produces a peculiar behavioural phenotype (hyperactivity, a tendency to hypercontract). In a sensitized hlh-1(cc561ts) background, which is a mild mutation of the myogenic factor MyoD, the dys-1(cx18) mutation also leads to a progressive muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to a phenotype similar to that of dys-1(cx18) mutation, which suggests that the two genes are functionally linked. Like dys-1(cx18); hlh-1(cc561ts), the double mutant dyc-1(cx32); hlh-1(cc561ts) also shows a progressive muscle degeneration. Moreover, Dyc-1 overexpression partially suppresses the dys-1(cx18); hlh-1(cc561ts) phenotype.. In the sarcomere, Dyc-1 is localized at the edge of the dense body, the nematode muscle adhesion structure functionally equivalent to vertebrate Z disc, where actin filaments are anchored. Two hybrid and immunocytochemistry experiments indicate that Dyc-1, Deb-1 (the vinculin homologue and main component of dense bodies), Zyx-1 (a focal adhesion protein), and Atn-1 (alpha-actinin homologue) may form a complex at the dense body. Our results suggest that the dense body might be the site of early pathological events occurring in the absence of dystrophin.. We are currently trying to demonstrate that dystrophin impairs the function of dense bodies proteins.

Diana David-Rus et al. (2004) East Coast Worm Meeting "Tracking the mid-life crisis of C. elegans"

Uri Einav, Patrick Nef, Eytan Domany, Peter J Schmeissner, Monica Driscoll, Diana David-Rus, Christophe Grundschober, Beate Hartmann, Garth Patterson

[East Coast Worm Meeting, 2004]

In an effort to better understand the biology of aging with an emphasis on mid-life changes that influence healthspan, we have undertaken a DNA microarray analysis of global gene expression profiles over time using Affymetrix gene chip arrays. Our experiment includes time points including the reproductive and post-reproductive periods, with a series of consecutive mid-life time points being covered. Studies in our lab and others have suggested that critical events during the mid-life of the nematode can influence the aging of this organism. For our analyses, we used supervised and unsupervised methods, including a clustering method developed in the Domany lab. Interestingly, we find a sharp change in the transcriptional levels of numerous genes at about 10 days post egg-lay. This abrupt change in gene expression on day 10 is consistent with a window of time identified previously by our lab as a time when mutations in the age-1 gene are able to delay the deterioration of muscle tissue (Herndon et al., 2002, Nature, 419: 808-14). The abrupt change also correlates with a transition point at which autofluorescent biomarkers accumulate (see abstract by Gerstbrein et al., this volume). Since two similar microarray experiments already have been performed (Lund et al., 2001, Curr Biol. 12(18): 1566-73; Murphy et al., 2003, Nature, 424(6946):277-83), we attempted a detailed cross-comparison between data from all three experiments, using non-parametric techniques. Again, a dramatic change in gene expression was observed for some genes at time points corresponding to our day 10 time point. Approximately 100 genes show significant changes in gene expression over adulthood in all three studies. These expression changes might be relevant to rapid end-stage deterioration in old nematodes.

Diana David-Rus et al. (2004) East Coast Worm Meeting "Tracking the mid-life crisis of C. elegans"

Christophe Grundschober, Garth Patterson, Uri Einav, Eytan Domany, Beate Hartmann, Patrick Nef, Peter J Schmeissner, Diana David-Rus, Monica Driscoll

[East Coast Worm Meeting, 2004]

In an effort to better understand the biology of aging with an emphasis on mid-life changes that influence healthspan, we have undertaken a DNA microarray analysis of global gene expression profiles over time using Affymetrix gene chip arrays. Our experiment includes time points including the reproductive and post-reproductive periods, with a series of consecutive mid-life time points being covered. Studies in our lab and others have suggested that critical events during the mid-life of the nematode can influence the aging of this organism. For our analyses, we used supervised and unsupervised methods, including a clustering method developed in the Domany lab. Interestingly, we find a sharp change in the transcriptional levels of numerous genes at about 10 days post egg-lay. This abrupt change in gene expression on day 10 is consistent with a window of time identified previously by our lab as a time when mutations in the age-1 gene are able to delay the deterioration of muscle tissue (Herndon et al., 2002). The abrupt change also correlates with a transition point at which autofluorescent biomarkers accumulate (see abstract by Gerstbrein et al., this volume). Since two similar microarray experiments already have been performed (Lund et al., 2001, Curr Biol. 12(18): 1566-73; Murphy et al., 2003, Nature, 424(6946):277-83), we attempted a detailed cross-comparison between data from all three experiments, using non-parametric techniques. We found approximately 100 genes showing changes in gene expression over adulthood common to all three studies. These expression changes might be relevant to rapid end-stage deterioration in old nematodes.

Diana David-Rus et al. (2005) International Worm Meeting "A search for mid-life gene expression changes that might influence healthspan"

Patrick Nef, Peter J Schmeissner, Beate Hartmann, Diana David-Rus, Monica Driscoll, Eytan Domany, Christophe Grundschober, Garth Patterson, Uri Einav

[International Worm Meeting, 2005]

Studies in our and other labs have suggested that critical events during the mid-life of the nematode can influence the aging and lifespan of this organism. In an effort to better understand the biology of aging with an emphasis on mid-life changes that influence healthspan, we performed a DNA microarray analysis of global gene expression profiles over time using Affymetrix gene chip arrays. Our experiment includes time points covering the reproductive and post-reproductive periods, with a series of consecutive mid-life time points. For our analyses, we used unsupervised methods, including a clustering method called Two-Way SPC (see. Domany et al.,Phys. Rev.Lett 76,3521). The method is actually a new approach to clustering, based on rules which can be described with help of statistical mechanics. Interestingly, we do find a sharp change in the transcriptional levels of numerous genes at about 10 days post egg-lay. This abrupt change in gene expression on day 10 of adulthood is consistent with a transition point at which potentially harmful autofluorescent biomarkers accumulate at accelerated rates (see abstract by Gerstbrein et al.) and at which age-related muscle decline may also accelerate. Since similar microarray experiments have been published (Lund et al., 2001, Curr Biol. 12(18): 1566-73; Murphy et al., 2003, Nature, 424(6946):277-83), we attempted a detailed cross-comparison between data from all three experiments, using again unsupervised, as well as supervised techniques. From the supervised technique we found approximately 100 genes that change expression during adult lifespan that are common to all three studies. We consider that these expression changes might be relevant to rapid end-stage deterioration in old nematodes.

Akwasi Anyanful et al. (2007) International Worm Meeting "Genes controlling lifespan in C. elegans mediate resistance to enteropathogenic E. coli toxicity."

Guy Benian, Akwasi Anyanful, Shantanu Bhatt, Dan Kalman

[International Worm Meeting, 2007]

Enteropathogenic E. coli (EPEC) is a gastrointestinal pathogen that kills infants in developing countries through diarrhea and dehydration. To better understand the mechanisms of EPEC virulence and host response, we developed an EPEC/C. elegans model and showed that EPEC paralyzes and kills C. elegans via a secreted toxin. However, daf-2 and age-1 mutant worms proved to be more resistant to EPEC virulence. daf-2 via age-1 prevents the translocation of daf-16 to the nucleus to up-regulate survival genes and antimicrobial factors. Thus, EPEC killing of C. elegans appears to involve inactivation of factors that permit the worm to accommodate to environmental or other stresses associated with aging. Using microarray analysis, Murphy et. al., (2003) demonstrated that 263 and 251 genes were up-regulated to promote longevity, or down-regulated to prevent life shortening by daf-16 respectively. To determine which genes are important for mediation against EPEC toxins, we inhibited in daf-2(e1370) mutants, the up-regulated genes by RNA interference and exposed these worms to EPEC. We report that RNAi against abl-1 and hsf-1 renders daf-2(e1370) mutants as sensitive to EPEC virulence as wild type. Other genes such as spp-1, aqp-1, and dod-6 rendered the daf-2 mutants partially sensitive. We are currently studying the mechanisms underlying the sensitivity of abl-1 and hsf-1 mutants. We further determined whether specific tissues act as signaling centers by expressing DAF-16 under tissue specific promoters in the daf-2;daf-16 mutants and exposing the worms to EPEC virulence. Our analyses show that expression of DAF-16 under a neuronal promoter elicited the strongest resistance to EPEC. Together, our findings suggest that contact with EPEC initiates a protective neuronal response that may decrease daf-2 activity leading to the transcription of antimicrobial genes to facilitate survival.

Soltesz, Z. et al. (2011) International Worm Meeting "Carbon dioxide avoidance is mediated by a diverse set of sensory neurons in C. elegans."

Soltesz, Z., de Bono, M., Bretscher, A. J.

[International Worm Meeting, 2011]

Monitoring carbon dioxide levels has a twofold importance for many living organisms: CO2 can act as a sensory cue for food or other animals, while regulating internal CO2 levels is an important part of homeostasis. C. elegans relies on diffusion for gas exchange, and avoids CO2 levels as low as 1%. We are interested in the neural and molecular mechanisms underlying the C. elegans CO2 avoidance behaviour. Mutants defective in the tax-4 or tax-2 genes, which encode the a and b subunits, respectively, of a cGMP-gated ion channel, showed reduced CO2 avoidance in behavioural assays1,2. By expressing tax-2 cDNA from neuron-specific promoters in tax-2 mutants to rescue the avoidance behaviour, and by imaging neurons using the genetically encoded calcium indicator YC3.60, we have shown that sensory neurons previously implicated in oxygen, temperature, and salt-sensing, including BAG, AFD and ASE, are CO2 sensors as well3. We have observed both persistent and transient cell-intrinsic calcium-responses in several sensory neurons, suggesting that CO2 stimuli could modulate neural activity in C. elegans in a complex manner. We are therefore investigating how CO2 stimuli can affect neural processing in downstream neurons. 1 Andrew Jonathan Bretscher, Karl Emanuel Busch, and Mario de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. PNAS 105(23):8044-8049 2 Elissa A. Hallem and Paul W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. PNAS 105(23):8038-8043 3 Andrew Jonathan Bretscher, Eiji Kodama-Namba, Karl Emanuel Busch, Robin Joseph Murphy, Zoltan Soltesz, Patrick Laurent and Mario de Bono, Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior. Neuron 69(6):1099-1113.