Jeb Gaudet et al. (2001) International C. elegans Meeting "Specification of Organ Identity by the C. elegans FOXA homologue PHA-4."

Jeb Gaudet, Michael Horner, Susan E Mango

[International C. elegans Meeting, 2001]

During development, cells acquire distinct cell fates in response to a host of regulators, many of which encode transcription factors. A longstanding issue has been the nature of the target genes controlled by these transcriptional regulators: do they activate a few genes at the top of a hierarchy, or control many genes that function at multiple stages throughout development? This question has been difficult to resolve because few direct targets have been identified. To address how transcription factors influence cell fate decisions, we are studying the role of the fork head box protein PHA-4 during pharynx development. In embryos that lack pha-4 activity, cells that would normally become part of the pharynx develop as ectoderm instead. Conversely, ectopic expression of pha-4 produces excess pharyngeal cells at the expense of other cell types. Because pha-4 normally functions in pharyngeal cells irrespective of the cells' lineage or cell type, we propose that pha-4 endows cells with pharyngeal organ identity. We have used a microarray chip approach to identify genes selectively expressed in the pharynx and have analyzed those genes to determine which are direct PHA-4 targets. We took advantage of maternal effect mutants that produce embryos with excess (par-1) or no (skn-1) pharyngeal cells to identify candidate pharyngeal genes. Included among our positive clones were most (>70%) genes known to be expressed in the pharynx, indicating that our approach worked well. Three lines of evidence suggest that PHA-4 directly activates most or all pharyngeally-expressed genes. First, we examined the pharynx-specific expression of a random set of microarray positives and found that the expression of each depended on one or more predicted PHA-4 binding sites. Second, PHA-4 is able to bind these sites in vitro. Third, temperature-shift studies indicate that PHA-4 is required throughout development, consistent with a model in which PHA-4 regulates both early and late acting genes. Our analysis also showed that the affinity of PHA-4 for different binding sites regulates the relative time of onset of the different target genes. When a PHA-4 binding site is converted by a single base-pair to a higher affinity site, expression initiates earlier. Conversely, a single base-pair mutation of a PHA-4 recognition sequence to one with lower affinity leads to a delay in the onset of expression. The affinity of PHA-4 binding in vitro matches the behavior of these reporter constructs in vivo. These findings suggest that modulating the affinity of PHA-4 binding sites within a promoter provides the embryo with a subtle mechanism to coordinate expression temporally throughout a developing organ. Since direct target genes of mammalian FOXA proteins include genes expressed early (e.g. endoderm) or late (e.g. liver) during vertebrate development, we suggest that global regulation of transcription within the foregut may be a common feature of this family of developmental regulators. We are indebted to Stuart Kim, Rebecca Begley, Carrie van Doren, Kyle Duke, and Min Jiang, who performed the microarray hybridization experiments.

James Lund et al. (2001) International C. elegans Meeting "Global profile of gene expression during aging"

Kyle Duke, James Lund, Patricia Tedesco, Stuart K Kim, Thomas E Johnson

[International C. elegans Meeting, 2001]

Aging is a physiological phenomenon characteristic of metazoan life. As animals age, they suffer a broad functional decline and an exponentially increasing probability of death. Numerous gerontogene mutants have been identified in the nematode Caenorhabditis elegans. These mutants suggest several models by which aging may be slowed and life span prolonged. This study of global transcription during the chronological aging of the worm is a first step to testing several of these models including the stress response, metabolic rate and programmed aging models. DNA microarrays can be used to profile the expression levels of a large number of genes in parallel. Using our array, which contains a PCR product representing every C. elegans gene, we are profiling expression patterns during the normal aging process. We isolated RNA from synchronized cultures of sterile animals at various ages from young adult to old age. We hybridized labeled cDNA made from this RNA to DNA microarrays, and find that 212 (p<.001) genes show statistically significant changes with chronological age. This is a relatively small number of genes relative the number with significant changes during development, those specific for male and hermaphrodite developmental programs, and those involved in reproduction. We find that transposons show progressively increasing transcript levels as the worms age, and several gene classes including histone and mitochondrial genes exhibit progressive down-regulation as the worms age. Many oocyte genes maintain transcript levels past the reproductive period, declining only in old worms. This global transcript profile is the first detailed and complete molecular profile of aging in the worm.

Ellen Nollen et al. (2001) International C. elegans Meeting "BAG1, AN HSP70 CO-CHAPERONE INVOLVED IN CELL GROWTH AND CELL STRESS"

Ellen Nollen, Richard I Morimoto, Frederic Gabriel, Kyle Duke, Jim Morley, Stuart Kim, Sanjeev Satyal

[International C. elegans Meeting, 2001]

Bag1 is a multifunctional protein that interacts with a variety of partner proteins. In mammalian cells, overexpression of Bag1 influences different signal transduction pathways, which in general leads to prevention of apoptosis or inhibition of cell cycle arrest. Interaction partners of Bag1 include Bcl-2, Raf-1 kinase, hormone receptors, and Hsp70. Bag1 enhances the anti-apoptotic function of Bcl-2, stimulates of the Raf-1 kinase activity, and inhibits Hsp70 chaperone activities. We are using C. elegans as a model to investigate the function of Bag1 during development and survival of a complex multicellular organism. We have created and analyzed a deletion mutant of Bag1 (F57B10.11). Null mutants appear similar to the wild type N2 strain with respect to viability, anatomy, timing of development, and life span. This indicates that Bag1 is not an essential gene and, moreover that Bag1 is not essential for Hsp70 activity under physiological conditions. Interestingly, the Bag1 null hermaphrodites show a 20% increase in the number of progeny compared to wild type N2 worms. Given the effect of overexpression of Bag1 on signal transduction pathways in mammalian cells, often reflected in changes in transcriptional activities, we asked whether phenotypic changes in C.elegans caused by deletion of Bag1 could be explained by changes in the mRNA expression profile. We are currently analyzing the mRNA expression profile of adult Bag1 null worms, compared to adult wild type N2 worms, using full genome microarrays. We will report on our progress on the microarray analysis and on the characterization of the phenotype of the Bag1 deletion strain.

JaeYoung Kwon et al. (2002) West Coast Worm Meeting "Microarray analysis of ethanol effects in the nematode Caenorhabditis elegans identifies ethanol-sensitive genes"

Mingi Hong, Stuart Kim, Minsung Choi, JaeYoung Kwon, Kyle Duke, Junho Lee, Seol hee Im

[West Coast Worm Meeting, 2002]

In this report, we wanted to use a model organism and a genomic approach to study the gene network induced by ethanol stress. C. elegans shows responses to acute ethanol exposure that are similar to those observed in humans, mice, and Drosophila, namely hyperactivity followed by uncoordination and sedation. Therefore, we wanted to identify genes that show a difference in expression on ethanol exposure, and to functionally link those genes into an ethanol-induced gene network. After studying the changes in expression profile, further studies can be carried out more easily to elucidate the functions of individual genes in the response to ethanol. After analyszing the expression profiles of all C. elegans ORFs by 15 sets of microarray expeirments, we classified genes affected by ehtanol into five groups. Class I consists of genes whose trascription is induced early and maintained at high level. Class II consists of genes that show transcritional responses late. Class III include genes whose transcription is induced at early time points and then returns back to normal after longer exposure. Class IV genes are those whose transcription is repressed at early time points, and returns back to normal after longer exposure to ethanol. Class V consists of genes whose transcription is repressed at late time points. Genes in Class II and IV may represent those involved in adaptation to ethanol stress. The genes in class III and V may represent physiological consequences of ethanol exposure. We are most interested in genes in class I because these genes may represent those directly involved in ethanol response in C. elegans. These genes include heat shock protein genes, a glutamate receptor gene, a lipase homolog, and a few novel genes. Among ten probable glutamate receptor genes in C. elegans, glr-2 is the only gene to be induced by ethanol. We confirmed the microarray result by time-course Northern analysis. We propose that glr-2 is an ethanol-specific response gene, because we found no significant changes in the glr-2 transcription level in other microarray experiments when examined with 'History' option in the Stanford microarray database . The T28C12.4 gene encodes a novel protein with limited homology to human neuroligin. This gene was expressed in hypodermal cells and found to be specific to ethanol stress since it did not respond to heat shock, nor to high salt. We will present expression patterns of other genes and more updated results.

Stanley I Shyn et al. (2001) International C. elegans Meeting "A genomic approach to understanding the actions of fluoxetine"

Rajesh Ranganathan, Stuart K Kim, Eric Miska, William R Schafer, Kyle Duke, Bob Horvitz, Stanley I Shyn

[International C. elegans Meeting, 2001]

Re-uptake blockers are a class of antidepressants proposed to achieve their clinical effects in people by inhibiting presynaptic norepinephrine and/or serotonin re-uptake transporters, which normally function to terminate transmitter signaling at synapses. It has never adequately been explained, however, why therapeutic relief of depression takes weeks when this particular action of re-uptake blockers occurs within hours of drug administration. 1 A requirement for some form of adaptation at synapses is a frequently cited possibility, but recent work has also highlighted that there are a myriad of non-classical targets for re-uptake blockers, such as EAG-like K + -channels (blocked by imipramine) 2 and novel membrane-spanning proteins typified by NRF-6 and NDG-4, identified as fluoxetine targets in a recent screen in worms 3 . With the recent discovery of mutants in mod-5 , a gene encoding the C. elegans serotonin re-uptake transporter, 4 a new approach to tackling this long-standing question has emerged. Utilizing the DNA microarray facility at Stuart Kim's laboratory in Stanford, we have begun work on a gene chip project to perform a four-way comparison between N2 and mod-5(n3314) worms ( n3314 is a 1.6 kb deletion in the mod-5 genomic locus), each on and off fluoxetine. We hope to understand what baseline transcriptional differences exist between the two genotypes, how similar the effects of chronic fluoxetine in N2 are to the effects of a complete absence of MOD-5 in mod-5(n3314) worms, and to identify which gene expression changes induced by fluoxetine are MOD-5-dependent and which are MOD-5-independent. A preliminary experiment is expected to be completed by the end of April, 2001. 1 Schafer WR, Cell 98 :551-554 2 Weinshenker D et al . J Neurosci 19 :9831-9840 3 Choy RK et al . Mol Cell 4 :143-152 4 Ranganathan R and Horvitz HR, 1999 International Worm Meeting, abstract 70

Jae Young Kwon et al. (2001) International C. elegans Meeting "Microarray Analysis of Ethanol-treated Worms"

Kyle Duke, Seol hee Im, Junho Lee, Jae Young Kwon, Min Sung Choi, Mingi Hong, Stuart K Kim

[International C. elegans Meeting, 2001]

We used cDNA microarray analysis to identify genes that are either up- or down-regulated on varied time-lengths of exposure to 7 vol% ethanol. Several gene families, including heat shock protein families and gene families of known or unknown function, were up- or down-regulated by ethanol. These ethanol-effected genes were further examined by Northern analysis. Northern results of a total of 100 genes - 50 each of genes either up- or down-regulated by 6 hour exposure to ethanol - showed 95% consistency with microarray results. By analyzing microarray data according to varied lengths of exposure to ethanol, we were able to classify the effected genes largely into 5 groups. Class I genes show rapid increase in gene expression. Class II genes show a steady increase, and Class III genes show an increase in expression and then fall back to normal. Class IV genes show reduced expression and then go back to normal, and Class V genes show a steady decrease in expression. We are currently studying genes that belong to each group by Northern analysis and expression studies. Also, we are functionally analyzing some candidate genes using previously isolated mutants. Through these results, we are planning to identify genes that are regulated by ethanol, and study their inter-relationships.

Stuart K Kim et al. (2001) International C. elegans Meeting "A gene expression map for C. elegans"

Stewart Scherer, George S Davidson, Stuart K Kim, Jim Lund, Brian N Wylie, Min Jiang, Kyle Duke, Moni Kiraly

[International C. elegans Meeting, 2001]

The completion of the C. elegans genome sequence has identified nearly all of the genes in the genome (19,282 genes), but the function for most of these genes remains mysterious. A scant 6% of them have been studied using classical genetic or biochemical approaches (1135 genes), and only about 53% show homology to genes in other organisms (10,303 genes). The next challenge is to develop high-throughput, functional genomics procedures to study many genes in parallel in order to elucidate gene function on a global scale. One such approach is to use DNA microarrays to assay the relative expression of nearly every gene in the genome between two samples. Knowing when, where and under which conditions a particular gene is expressed can reveal the function of that gene. A consortium of laboratories has used C. elegans DNA microarrays to profile gene expression changes in a wide variety of experiments. In each experiment, RNA from one sample was used to generate Cy3-labelled cDNA, and RNA from another sample was used to prepare Cy5-labelled cDNA. The two cDNA probes were simultaneously hybridized to a single DNA microarray and the ratio of the Cy3 to Cy5 hybridization intensities was measured, revealing which genes were relatively enriched in either RNA sample. Thirty different laboratories have collectively performed 553 experiments using these C. elegans DNA microarrays, including 179 experiments with microarrays containing 11,917 genes (63% of the genome) and 370 experiments using microarrays that have 17,817 genes (94% of the genome). The experiments compare RNA between mutant and wild-type strains, or between worms grown under different conditions. Many experiments have been done to date, including experiments on wild-type development, heat shock, Ras signaling, aging, the dauer stage, sex regulation and germ line gene expression. Individual microarray experiments reveal sets of genes that change in one mutant strain or growth condition. We combine the data from all of the experiments to assemble a gene expression database, and then used this database to group together co-regulated genes and visualize them using a three dimensional expression map. By matching the expression profile of an unknown gene to those of genes with known functions, the expression map can be used to ascribe functions to the large fraction of genes in the genome whose functions were previously unknown.

Tom Blumenthal et al. (2001) International C. elegans Meeting "A Global Analysis of the Caenorhabditis elegans Operons"

Alessandro Guffanti, Chris Link, Stuart Kim, Donald Evans, Kyle Duke, Daniel Lawson, Jean Thierry-Mieg, Tom Blumenthal, Danielle Thierry-Mieg

[International C. elegans Meeting, 2001]

Unlike genomes of other animals, the genomes of Caenorhabditis elegans and its close relatives contain numerous polycistronic transcription units similar to bacterial operons. The pre-mRNAs from these operons are processed into monocistronic mRNAs by 3' end formation and a specialized form of trans-splicing using the spliced leader, SL2. In order to determine how widespread operons are in the C. elegans genome, we have probed microarrays containing 17,817 predicted and known genes with a probe specific for mRNAs containing the SL2 sequence along with a control probe to polyA+ RNA. There are 1215 genes that form a separate group with high SL2/polyA+ ratios at the 99.9% level of confidence, and 86% of these appear to be downstream genes in operons, based on their genomic arrangements. We integrated this list with a list of ~400 genes in known operons and/or with confirmed SL2-containing cDNA clones. Of the genes previously confirmed to be SL2 trans-spliced, 91% are contained on the list of operons based on the microarray experiment, and almost all are in downstream positions. We estimate the genome contains ~850 operons, of which we provide experimental support for 807. On average, the operons contain 2.6 genes, and the longest contain seven genes. At least 13% of C. elegans genes are transcribed polycistronically. Operons are relatively common on chromosomes I and III, but much rarer on V and especially X. The average spacing between the polyA site of the upstream gene and the trans-splice site of the downstream gene is 126 bp. Most of the operons uncovered in this analysis have not been reported previously, and we can cite numerous new examples of co-transcription of genes encoding apparently functionally related proteins. We suggest the possibility that close inspection of the operon list could reveal heretofore unknown protein relationships.

Sandrine Fraboulet et al. (2009) European Worm Neurobiology Meeting "Chemical rescue of paralysis in a transgenic model of Caenorhabditis elegans overexpressing human Beta-amyloid"

David I C Scopes, Andrew K Jones, David B Sattelle, Aileen Moloney, Christopher D Link, Steven D Buckingham, Mark J Treherne, Hozefa Amijee, Sandrine Fraboulet

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC. Numerous studies support a role for aggregated Ab as a mediator of the toxicity that underlies Alzheimer.s disease (AD) and other diseases such as Inclusion Body myositis (IBM), an acquired muscle disease affecting people over 50 years old. In this pathology, muscle weakness and degeneration is accompanied by chronic muscle inflammation. Remarkably, despite the inflammation component of the disease, IBM patients are only poorly responsive to anti-inflammatory drugs suggesting that inflammation per se may not be the primary cause of the pathology. We have used a C. elegans transgenic line over-expressing human amyloid 1-42 peptide in the muscles as a model with which to test the efficiency of new compounds on in vivo amyloid toxicity. This C. elegans line becomes paralyzed shortly after the induction of amyloid expression in the muscles, which makes it an easily-recordable phenotype useful for exploring candidate treatments to alleviate the paralysis. We report on the actions of two novel chemicals, which inhibit amyloid aggregation and partially rescue the amyloid-induced phenotype. References: Wu Y, W.Z., Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y. (2006) J. Neurosci., 26, 13102-13. Link CD, T.A., Kapulkin V, Duke K, Kim S, Fei Q, Wood DE, Sahagan BG. (2003) Neurobiol Aging., 24, 397-413. Jones, A.K., Buckingham, S.D. and Sattelle, D.B. (2005). Nat Rev Drug Discov, 4, 321-30.

MG de Bono et al. (1999) International C. elegans Meeting "The Genetics of C. elegans social behavior"

CI Bargmann, MG de Bono

[International C. elegans Meeting, 1999]

Natural isolates of C. elegans behave in one of two distinct ways on food. Solitary strains (e.g. N2) slow down in response to a bacterial lawn, disperse evenly across the lawn, and forage alone. Social strains accumulate where bacteria are thickest, aggregate together, and do not slow down on food until they join a clump. Behavioral differences between social and solitary strains are seen only in the presence of food. By a combination of genetic and molecular studies* we have shown that the behavioral differences between social and solitary wild strains are due to a single amino acid substitution in a single gene called npr-1 (pka bor-1 , and renamed by kind permission of Randy Cassada and the CGC). npr-1 encodes a G-protein coupled receptor that belongs to the neuropeptide Y family of seven transmembrane receptors. Predicted null mutations in npr-1 (neuropeptide receptor resemblence) make the solitary N2 strain take on social behavior. We are attempting to define the molecular and cellular circuitries that regulate social and solitary behaviors. We know that social behavior is not disrupted by mutations that abolish previously described sensory responses in C. elegans (e.g. che-3, che-13, odr-3, mec-3, ttx-3 ) but requires the cyclic nucleotide gated ion channel encoded by tax-2 and tax-4 , and the capsaicin receptor homolog osm-9 . tax-2 and tax-4 are expressed in ten overlapping sensory neurons. We are currently determining which of these neurons require tax-2/tax-4 activity for social behavior. To identify the circuit which represses social behavior in solitary animals, we are searching for the ligand for npr-1 . Based on homology, we predict that npr-1 will have a neuropeptide ligand. In collaboration with Marc Caron at Duke University we are attempting to set up an assay system in mammalian tissue culture cells that would allow us to test candidate ligands or C. elegans extracts for their ability to stimulate npr-1 . *de Bono and Bargmann, Cell, 94, 679-689