Clendinen, Chaevien S et al. (2011) International Worm Meeting "Mass Spectrometric Imaging and Behavioral Assays of Pristionchus pacificus."

Clendinen, Chaevien S, Menger, Robert, Ajredini, Ramadan, Edison, Arthur S, Yost, Richard, Khan, Ghulam

[International Worm Meeting, 2011]

Nematodes are one of the most numerous and diverse multicellular organisms on earth. They have a profound impact on human health, agriculture, and economy. Like many other organisms, they synthesize and use small molecules to communicate within and between their species. In Caenorhabditis elegans, ascarosides have been found to control dauer formation (Butcher, et al., 2007), mating (Srinivasan, et al., 2008), aggregation (Macosko, et al., 2009), and olfaction (Yamada, et al., 2010). Pristionchus pacificus is a useful satellite organism for comparative studies with C. elegans (Hong & Sommer, 2006). We are developing a bioassay for gender specific chemotaxis (e.g. mating behavior) in P. pacificus for activity-guided fractionation of the mating cue. The mating assays are complicated by aggregation behavior in males under some conditions. We are trying to understand the chemical and environmental factors that control these different behaviors. Several behaviors, including aggregation may be mediated or enhanced by chemical cues on the cuticle surface of the nematodes. Therefore, we are trying to develop an alternative approach to chemical ecology studies in nematodes using matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) (Garrett & Yost, 2006). This technique could potentially not only allow us to detect metabolites on the surface of nematodes, but also visualize the spatial distribution of these small molecules. Using a combination of MALDI MSI and principal component analysis (PCA), we can compare nematodes of different species and find differences in mass spectral profiles resulting from gender, developmental stage, strain or species. We are currently examining C. elegans strain N2 (a standard wild-type) and C. elegans daf-22 (dauer-defective mutants). Daf-22 mutants are unable to produce short chain ascarosides such as the dauer pheromones common in the N2 strain. Once this technique is developed by the use of C. elegans, we will begin to analyze the surface molecules of P. pacificus nematodes. Bibliography: Yamada, K., et al. (2010). Science, 329 (5999), 1647-1650. Butcher, R. et al. (2007). Nature Chemical Biology, 3 (7), 420-422. Garrett, T., & Yost, R. (2006). Analytical Chemistry, 78, 2465-2469. Hong, R., & Sommer, R. (2006). BioEssays, 28, 651-659. Macosko, E., et al. (2009). Nature (458), 1171-1175. Srinivasan, J., et al. (2008). Nature, 1115-18.

Gerami B et al. (1997) International C. elegans Meeting "MAPPING QUANTITATIVE TRAIT LOCI FOR CHEMOTAXIS: CORRESPONDENCE BETWEEN QTL AND odr CANDIDATE LOCI"

Phillips PC, Gerami B, Morphew JJ

[International C. elegans Meeting, 1997]

Understanding the genetics of complex traits is challenging because it can be difficult to separate out the effects of the numerous genes affecting the trait. C. elegans can serve as a useful model system for studying complex traits because of the wealth of genetic and functional information available. Chemotaxis provides an ideal trait for this type of study because it ecologically important to the nematodes, as well as likely to be influenced by many genes (a number of which are known). In collaboration with Thomas Johnson and David Shook [1] and Robert Shmookler Reis and Robert Ebert [2], who provided genotyped lines generated in a cross between N2 and BO, we have assayed the chemotaxic response of 180 recombinant inbred lines to benzaldehyde at two concentrations. These concentrations, 50 nl and 500 nl, respectively lead to an attraction or repulsion response. Separate analysis of the lines from the two labs yield different results. The SR lines show a QTL mapping to the fourth chromosome, whereas the TJ lines show several QTL, primarily on the X chromosome. The QTL on X map to the same locations as two mutants known to block the response to benzaldehyde, odr-1 and odr-5 [3]. We are in the process of evaluating these candidate loci as the actual QTL using backcrossing and complementation testing approaches. Our hope is to create a model system for studying a variety of quantitative genetic questions. [1] Genetics 142:801 (1996), [2] Genetics 135:1003 (1993), [3] Bargmann et al., Cell 74:515 (1993)

Christian Frokjar-Jensen et al. (2007) International Worm Meeting "Efficient low copy gene insertion at determined loci."

Blake Newman, Soren-Peter Olesen, Erik M. Jorgensen, Morten Grunnet, Wayne M Davis, Christian Frokjar-Jensen

[International Worm Meeting, 2007]

The ability to insert single copies of genes into the C.elegans genome in a controlled manner is desirable for a number of experiments; e.g. when germ line expression or near endogenous levels of protein expression is important. We have developed a new technique based on the mosTIC technique (Robert & Bessereau, 2006) and previously suggested by (Plasterk & Groenen, 1992) that allows us to insert transgenes at low copy number into two different non-coding loci on chromosome II or IV. The technique relies on the ability to create double-stranded breaks in the chromosomes by excising the Mos1 transposon. The insertion of transgenes then takes place by repairing the double stranded break through homologous recombination off an extrachromosomal array. In order to efficiently recover these events we have developed a system that utilizes a combination of positive and negative selection markers. An unc-119 rescue fragment is co-inserted into the chromosomal location for positive selection while a dominant temperature-sensitive mutant ion channel is used to select against animals still carrying the extragenic array. Animals that have inserted the gene of interest and have lost the extrachromosomal array will therefore be the only wild-type animals after shifting the animals to 25C. After mobilizing the transposon by heat shock we recover one integrant for every 100 heat-shocked adult animals in the F2 generation. For practical purposes, this is equivalent to 1 integrated line in every 5 large plates. In initial experiments we have inserted 4.2 kb of DNA. PCR confirms that the insertion occurred at the targeted location. Also, only a fraction of the integrated lines contain extraneous vector DNA, indicating mostly low or single copy insertions. Currently, we are: 1) testing if we can express transgenes in the germ line 2) determining the protein expression level from inserted transgenes 3) inserting larger transgenes and 4) testing a modified injection protocols designed to ensure single copy gene insertions. We believe the technique has a number of advantages compared to inserting genes by bombardment; 1) insertions do not disrupt coding regions, 2) different integrated lines are directly comparable, 3) there is no requirement for specialized equipment. The use of positive and negative selection markers makes the screening process easy. To facilitate use of the technique we have developed a Gateway compatible vector set for transgene insertion at the two chromosomal sites. _{Robert, V. & Bessereau, J.-L. (2007) EMBO 26: 170-83. Plasterk R.H., Groenen J.T. (1992) EMBO 11: 287-290}.

Amanda Hughes et al. (2007) International Worm Meeting "C. elegans RNA Helicase A genetically interacts with other proteins involved in RNAi."

Deborah Schmidt, Amanda Hughes, Katherine M. Walstrom

[International Worm Meeting, 2007]

RNA helicase A (RHA) is a conserved protein with roles in transcription regulation and histone modification in numerous organisms such as flies and humans. We are studying the role of C. elegans RHA (RHA-1) in transcription regulation and germline development. A null mutant, rha-1(tm329), has a temperature-dependent defect in repressive histone modifications and in germline transcriptional silencing of transgene arrays (Walstrom, Schmidt, Bean, and Kelly (2005) Mech. Dev. 122, 707). These defects in gene regulation lead to a sterile phenotype in hermaphrodite and male worms. Others have shown that rha-1 is required for RNA interference in the germline (Robert et al. (2005) Genes Dev. 19, 782-787). We found that rha-1 interacts genetically with rde-2 and mut-7. The RDE-2 and MUT-7 proteins are required for RNAi and physically associate with each other (Tops et al. (2005) NAR 33, 347). The precise roles of these two proteins and RHA-1 in RNAi are not well understood. Interestingly, rde-2;rha-1 mutants exhibit mutual repression while mut-7;rha-1 mutant hermaphrodites have an enhanced phenotype. We are further characterizing these double-mutants to determine how the germline histone modifications, germline development, and RNAi phenotypes are affected. We are particularly interested in the histone modifications, to which RNAi has been linked, because transcriptional silencing is an important process required for early germline development in many organisms.

Matthew Felsen et al. (2008) Development & Evolution Meeting "C. elegans RNA Helicase A Genetically Interacts with rde-2 and mut-7"

Katherine Walstrom, Amanda Hughes, Matthew Felsen, Deborah Schmidt, Anamica Bedi

[Development & Evolution Meeting, 2008]

RNA helicase A (RHA) is a conserved protein with roles in transcription regulation and histone modification in numerous organisms such as flies and humans. We are studying the role of C. elegans RHA (RHA-1) in transcription regulation and germline development. A null mutant, rha-1(tm329), has a temperature-dependent defect in germline transcriptional silencing, and these defects lead to a sterile phenotype with few germ cells produced in hermaphrodite and male worms (Walstrom, Schmidt, Bean, and Kelly (2005) Mech. Dev. 122, 707). Others have shown that rha-1 is required for RNA interference in the germline (Robert et al. (2005) Genes Dev. 19, 782-787). We found that rha-1 interacts genetically with rde-2 and mut-7. The RDE-2 and MUT-7 proteins are required for RNAi and physically associate with each other (Tops et al. (2005) NAR 33, 347). The precise roles of these two proteins and RHA-1 in RNAi are not well understood. Interestingly, rde-2;rha-1 mutants exhibit mutual repression. At 25 degC, rde-2 hermaphrodites are sterile due to nonfertile sperm and Emo oocytes, but the rde-2;rha-1 mutants have mostly fertile oocytes. The mut-7;rha-1 mutant hermaphrodites have an enhanced phenotype in that they are infertile due to defective sperm at a lower temperature than either single mutant. Using real-time RT-PCR, we are measuring levels of specific small RNAs in all these mutants. Our preliminary results indicate that rha-1 mutants have an increased level (2-4X) of some small RNAs while rde-2 and mut-7 mutants have decreased levels of the same small RNAs. The mutual suppression of the rde-2;rha-1 mutants could be explained by their near wild-type levels of small RNAs. We plan to expand these experiments to measure small RNA levels in both hermaphrodites and males.

Emily A Bates et al. (2005) International Worm Meeting "Differential contributions of C. elegans histone deacetylases to huntingtin polyglutamine toxicity"

Johnathan Whetstine, Martin Victor, Emily A Bates, Anne Hart, Yang Shi

[International Worm Meeting, 2005]

Expansion of a polyglutamine (polyQ) tract in the huntingtin protein causes neuronal degeneration and death in Huntington's disease, but the molecular mechanisms underlying polyQ mediated cell death are unclear. Previous data suggest that expanded polyQ tracts alter transcription by sequestering glutamine rich transcriptional regulatory proteins, thereby perturbing their function. We tested this hypothesis in a C. elegans model of polyQ toxicity. Loss of function alleles and RNAi were used to examine contributions of C. elegans CREB, CBP, and HDACs to polyQ-induced neurodegeneration. Deletion of CREB (crh-1)(Mark Alkema and Robert Horvitz) enhances polyQ toxicity in ASH sensory neurons. Loss of one copy of CBP (cbp-1) increases polyQ toxicity. Loss of function alleles and RNAi were used to reduce function of each C. elegans HDAC in a neuronal model of polyQ toxicity. Knockdown of most C. elegans HDACs increased Htn-Q150 toxicity; knockdown of hda-3 decreased polyQ toxicity. Increased expression of hda-1 in Htn-Q150 expressing neurons protects against Htn-Q150 toxicity. Neuronal expression of hda-3 restores Htn-Q150 induced degeneration and suggests that HDA-3 acts in the neurons to promote Htn-Q150 neurotoxicity. hda-3 loss of function suppresses degeneration in crh-1;Htn-Q150 animals suggesting that HDA-3 and CRH-1 directly or indirectly oppose each other in regulating the transcription of genes important for polyQ toxicity. Target genes may provide a link to previously implicated pathways underlying polyQ toxicity such as protein degradation, apoptosis, metabolic pathway, or axonal transport. hda-3 loss of function does not decrease neurodegeneration in hda-1;Htn-Q150 animals suggesting that HDA-1 and HDA-3 have different targets. These studies support the role of the CREB/CBP pathway in polyQ expansion diseases and suggest that specific HDAC inhibition as an interesting therapeutic approach.

Thomas M Ratliff et al. (2002) Mid-west Worm Meeting "EGL-27 MTA-interacting proteins are components of the CCR4-NOT complex and synMuv B pathway"

Thomas M Ratliff, Michael A Herman, Duaa H Mohammad

[Mid-west Worm Meeting, 2002]

Mutations in egl-27 cause defects in cell migrations, cell fate specification, cell fusion and morphogenesis. The amino terminal region of EGL-27 is similiar to a human metastasis-associated factor (MTA), recently identified as a co-purifying member of a human complex having ATP-dependent nucleosome-remodeling and histone deacetylase (NURD) function. Other C. elegans homologs of human NURD components--specifically, let-418 Mi-2, lin-53 RbAp48 and hda-1 HDAC--have been shown to act as synthetic multivulva (synMuv) class B genes. The synMuv class B genes function redundantly with synMuv class A genes to antagonize Ras signaling-mediated vulval induction in the vulval precursor cells. Several class B synMuv members have been identified, including those similar to the human tumor suppressor, pRB, and its human binding partners, DP and E2F. Despite a ubiquitous nuclear expression pattern that overlaps with other NURD and synMuv B members, the apparently obscure role of EGL-27 MTA in vulval induction suggests a lack of functional conservation with its human counterpart. In an effort to better understand the function of EGL-27, a two-hybrid screen was performed in yeast (two-hybrid library kindly provided by Robert Barstead). Multiple library clones were retrieved that corresponded to the mep-1 locus and two genes having similarity to components of the highly conserved CCR4-NOT complex, Cenot-2 and Cenot-3. MEP-1 is a large C2H2 zinc-finger protein with two potential homologs in Drosophila. CeNOT-2 and CeNOT-3 contain a previously unidentified domain of similarity located near their respective carboxy termini. Bacterially-expressed GST::EGL-27 interacts in vitro with CeNOT-2 and CeNOT-3 fragments corresponding to the two-hybrid library clones, thus verifying the two-hybrid interactions.

Zeiser, Eva A. et al. (2009) International Worm Meeting "MosSCI and Gateway compatible toolkit for germline expression of transgenes."

Frokjaer-Jensen, Christian, Ahringer, Julie, Jorgensen, Erik, Zeiser, Eva A.

[International Worm Meeting, 2009]

Transgene silencing in the germline has hampered research in the germline and early embryo. The creation of "complex" extrachromosomal arrays using genomic DNA and then the development of technology for low copy number insertions using microparticle bombardment finally allowed germline expression of transgenes (Mello et al., 1991; Praitis et al., 2001). However, bombardment is very labor intensive and complex extrachromosomal arrays often are silenced. Recently, the Mos1 mediated Single Copy Insertion (MosSCI) method was developed to insert single copies of transgenes into defined sites in the genome of Caenorhabditis elegans (Frokjaer-Jensen et al., 2008). The technique is based on the MosTIC technique (Robert & Bessereau, 2007). Directed integration of a single copy avoids problems like overdosage, passage related loss of transgenes and locus mediated phenotypes. It also avoids extrachromosomal array related gene silencing in the germline which makes it especially attractive for studies on the gonad, and early embryos. We are using the MultiSite Gateway system to generate transgenes for germline gene expression. This method allows easy fusion of three different sequences in a defined order (5'', middle, and 3''). We have generated a set of 5'' constructs containing the germline active promoter pmex-5 (Merritt et al., 2008) on its own or fused to the sequences of GFP, EGFP, Citrine and Tag RFP-T. Similarly, we have generated a set of 3'' constructs by fusing GFP, EGFP or Citrine to the tbb-2 3''UTR (Merritt et al., 2008). Using an appropriate combination of 5'' and 3'' constructs with a middle ORF of one''s choice allows easy generation of N- or C-terminal fluorescently tagged transgenes for expression in the germline or early embryo. We also show that the hsp-16.2 and hsp-16.41 heat shock promoters can be used for inducible germline and early embryo gene expression as single copy MosSCI generated insertions.

Yohann Duverger et al. (2007) International Worm Meeting "A semi-automated high-throughput approach to the generation of transposon insertion mutants."

Yohann Duverger, Jonathan Ewbank, Christophe Beclin, Sarah Scaglione, Dominique Brandli, Jerome Belougne

[International Worm Meeting, 2007]

To understand gene function, there is a clear need for the generation of as many stable mutant lines in as possible. Two KO projects are currently underway in North America (http://celeganskoconsortium.omrf.org/) and Japan (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) and have generated thousands of mutants. To these can be added the null alleles for several hundred genes produced using other PCR-based technologies or generated in classical genetic screens and available from the CGC. An alternative approach to the generation of mutants is via the use of transposons. Recently, a method using the mariner-like element Mos1 from Drosophila has been established (1). It was used in a pilot-scale project to generate random insertions throughout the genome (2). Following on from this manually project, as part of a collaborative effort funded by the European community (Nemagenetag; see abstract by Robert et al.), the French National Genopole network and support from Union Biometrica, we have developed a semi-automated high-throughput method for mutant production and screening. Using the UBI COPAS Biosort and a robotic system for PCR and electrophoretic analysis (using a 400-well gel format), we have developed a capacity to handle several thousand nematode strains in parallel for multiple generations (3) and have already generated more than 45,000 individual strains carrying Mos1 insertions. We will the describe method in detail, report recent improvements that have pushed our production capacity to above 5,000 mutant strains/month and explain how the method can be easily adapted to forward and reverse genetic screens. 1. Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. & Jorgensen, E. M. (2001) Nature 413, 70-4. 2. Granger, L., Martin, E. & Segalat, L. (2004) Nucleic Acids Res 32, e117. 3. Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. & Ewbank, J. J. (2007) Nucleic Acids Res 35, e11.

Valerie Robert et al. (2006) European Worm Meeting "MosTIC: A Novel Tool to Engineer the C. elegans Genome by Homologous Recombination"

Erik M. Jorgensen, Valerie ROBERT, Jean-Louis Bessereau.

[European Worm Meeting, 2006]

Valrie Robert, Erik M. Jorgensen1 and Jean-Louis Bessereau. Techniques based on homologous recombination provide a means to engineer custom mutations at specific loci and are widely used to study gene functions in most eukaryotic model organisms. Unfortunately, in Caenorhabditis elegans, homologous recombination between the genome and exogenous DNA fragments is very inefficient and only few examples of genome engineering by homologous recombination have been documented so far. We have developed a novel technique to introduce targeted gene modifications in the C.elegans genome using transgene-instructed gene conversion following Mos1 transposon excision.. Mos1 is a drosophila transposon that can be mobilized by expressing the Mos transposase in the C.elegans germline. Transgene-instructed DSB repair requires two steps: (1) generation of a DSB at the locus of interest by excision of a pre-existing Mos1 insertion and (2) repair of this DSB by gene conversion using an engineered transgene as a template. To establish this technique, we used Mos1 insertions in the unc63 and unc-5 genes. Following expression of the Mos transposase in the germline, we were able to demonstrate gene conversion events between the chromosome and a transgene containing only 3 kb of homologous sequences. This technique, that we called MosTIC (Mos1 induced transgene instructed gene conversion), allowed (i) the introduction of point mutations, (ii) the engineering of deletions, and (iii) the knock-in of gfp in the unc5 gene. All these events were recovered at a frequency ranging from from 5.10-5 to 5.10-4 per generation. Analysis of the conversion tracts indicated that 500 bp on each side of the Mos1 insertion site were copied from the transgene into the chromosome in 50 % of the MosTIC events.. MosTIC requires a Mos1 insertion in the vicinity of the genomic region to manipulate. Such insertions can be recovered in genetic screens using Mos1-mediated mutagenesis. In addition, progress of the NEMAGENETAG consortium to produce a comprehensive library of localized Mos1 insertions will provide a means to manipulate most C.elegans genes with the MosTIC technique.