Chien, Shih-Chieh et al. (2009) International Worm Meeting "Do orthologs of the yeast RAM pathway mediate Wnt signaling in neuronal polarity?"

Garriga, Gian, Chien, Shih-Chieh, Oppermann, Julie, Gurling, Mark

[International Worm Meeting, 2009]

Wnts regulate cell migration and polarity along the C. elegans A/P axis. For example, Wnts control the polarity of the mechanosensory neuron ALM1,2. Although single cwn-1, cwn-2 or egl-20 Wnt mutants display normal ALM polarity, the polarity of the ALMs is often reversed in cwn-1; cwn-2 or cwn-1; egl-20 double mutants. We find that ALM polarity also requires the Frizzled receptor MOM-5 and the Dishevelleds DSH-1 and MIG-5. While Frizzled and Dishevelled proteins mediate the effects of Wnts in many developmental contexts, how these molecules signal to control neuronal polarity is unclear. We find that the MIG-15 kinase and potential components of a MIG-15 signaling pathway might be novel Wnt effectors in neuronal polarity. MIG-15, the C. elegans ortholog of Nck-interacting kinase (NIK) in mice and Misshapen in Drosophila, was shown to function in cell migrations that also require Wnt function3. We found that mig-15 mutants exhibit a low frequency of ALM polarity defects that was enhanced by a mutation in cwn-1, suggesting that MIG-15 could mediate the effects of Wnts in ALM polarity. In S. cerevisiae, Kic1p, a distant relative of MIG-15, acts in the RAM signaling pathway to regulate polarized cell growth4. We asked whether C. elegans orthologs of RAM signaling molecules also regulate ALM polarity. Through RNAi and mutant analysis in a cwn-1 sensitized background, we identified mop-25.2 and sax-2 as regulators of ALM polarity. The gene mop-25.2 is the ortholog of yeast Hym1p, which physically interacts with Kic1p, and SAX-2 is the ortholog of the RAM scaffold protein Tao3p. SAX-2 acts in the ALM to establish its polarity. We were surprised to find that cwn-1; sax-1 mutants did not have an ALM polarity phenotype. SAX-1 is the ortholog of the NDR kinase Cbk1p and a target of the Kic1p kinase. We are currently testing whether the other C. elegans NDR kinase WTS-1 provides an overlapping function in ALM polarity in the absence of SAX-1. We are now testing whether the RAM pathway homologs act downstream of the Wnts in ALM polarity. 1Hilliard MA, Bargmann CI. (2006) Dev. Cell 10: 379-390. 2Prasad BC, Clark SG. (2006) Development 133: 1757-1766. 3Chapman JO, Li, H & Lundquist EA. (2008) Dev. Biol. 324: 245-257. 4Nelson B et al. (2003) Mol. Biol. Cell 14: 3782-3803.

Aurlie Blanc et al. (2005) International Worm Meeting "A French functional genomics platform."

Daniel Wong, Jerome Reboul, Jonathan Ewbank, Aurlie Blanc, Yohann Duverger

[International Worm Meeting, 2005]

For the last two years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates 500 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community.This French functional genomics platform has been made possible through funding from the National Genopole<sym02> network, Marseille-Nice genopole<sym02>, the CNRS and collaboration with Union Biometrica.

Yohann Duverger et al. (2007) International Worm Meeting "A French functional genomics platform."

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Sarah Scaglione, Daniel Wong

[International Worm Meeting, 2007]

For the last four years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates up to 1000 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented. This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community. This French functional genomics platform has been made possible through funding from the National Genopole&#174; network, Marseille-Nice genopole&#174;, INSERM, the CNRS and support from Union Biometrica.

May Tran et al. (2005) International Worm Meeting "RNAi knockout of the mitochondrial carrier protein ucp-4: effects on metabolism"

May Tran, Craig LaMunyon, Aidyl Gonzalez-Serricchio

[International Worm Meeting, 2005]

We are investigating the phenotype of ucp-4 RNAi knockout worms. Mitochondrial uncoupling protein (UCP) function in humans has been linked to obesity. UCPs are mitochondrial carrier proteins, and human UCP-1, -2, and -3 are protonophores, transporting protons from the intermembrane space to the matrix, bypassing ATP synthase, and thereby raising resting metabolic rate in response to certain cues. The function of human UCP-4 is not clear, but it may be a mitochondrial fatty-acid transporter, and it is a homolog to the C. elegans ucp-4 (K07B1.3). Initial RNAi experiments by Dr. Julie Ahringers lab deemed knockout worms to be wild-type. However, we wondered if the RNAi phenotype might be a subtle alteration of metabolic activity. We obtained Dr. Ahringers RNAi feeding clone from the UK HGMP Resource Centre and measured egg laying and defecation rates. Compared to control worms fed HT115 bacteria containing the empty feeding vector, ucp-4 RNAi fed young adults that were exposed beginning as L1 larvae laid eggs at a significantly slower rate, although preliminary data on defecation rate suggests no difference between these treatments. If the trends in these data hold, it would suggest that ucp-4 RNAi knockout worms are processing food at the same rate, but converting that energy into offspring at a reduced rate.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

P Askjaer et al. (2004) European Worm Meeting "IDENTIFICATION OF GENES REGULATING NUCLEAR ENVELOPE ASSEMBLY BY GENOME-WIDE RNAI"

V Galy, T Zimmermann, I Mattaj, M Gorjanacz, P Askjaer

[European Worm Meeting, 2004]

The nuclear envelope (NE) is essential for many cellular processes including regulation of gene expression, nuclear positioning and chromatin segregation during mitosis. In metazoans the NE disassembles when the cell enters mitosis and reassembles during anaphase or telophase to reestablish compartmentalization of the cell. Despite the importance of this process remarkably few genes have been identified, which are required for formation of a closed NE. One likely explanation is that overlapping and redundant pathways at least partially control NE formation: We and others (Liu et al., PNAS, 100:4598-4603) have found that while each of the two NE proteins emerin and MAN1 are dispensable in the worm co-depletion of the two proteins causes 100% embryonic lethality. To identify genes important for NE formation we have characterized an emerin deletion strain and use this strain in parallel with a control strain in a genome-wide RNAi screen to identify genes interacting with emerin. L1 larvae are incubated with RNAi-inducing bacteria from Julie Ahringer's RNAi library in 96-well plates enabling rapid screening of high numbers of samples. The individual wells are scored in three categories: wild type, adults without progeny (emb and ste), and larvae only (lva and lvl). In addition to visual analysis we have developed tools for automated scoring thereby standardizing data acquisition and providing documentation for each RNAi experiment. Data from our control strain allows us to compare our results with published data. Using a stringent threshold where only phenotypes with >80% penetrance are scored 13% of the RNAi constructs induce non-viable phenotypes, which is similar to other investigations. Furthermore, for previously identified essential genes we observe lethality or sterility in two thirds of the cases. We are currently retesting candidate genes in a secondary screen and will provide detailed information at the meeting.

Sylvia E J Fischer et al. (2002) European Worm Meeting "Characterization and identification of genes involved in transposon silencing."

Pawel Pasierbek, Nadine L Vastenhouw, Ronald H A Plasterk, Sylvia E J Fischer

[European Worm Meeting, 2002]

The transposon Tc1 is active in somatic cells of all C. elegans isolates. However, Tc1 activity in the germ line is restricted to certain strains: Tc1 is active in the germ line of Bergerac BO, but silenced in Bristol N2. Mori et al. showed that multiple genetic loci are involved in regulation of germ-line transposition (Mori et al., 1988). In an EMS screen for activation of Tc1 in the germ line of Bristol N2, 43 mutants were isolated that fall into two classes: mutants that are resistant to pos-1 RNAi, such as mut-7(pk204)(Ketting et al. 1999) and mut-14(pk738)(Tijsterman et al. 2002), and mutants that are wild type for pos-1 RNAi. The latter class consists of 20 mutants. These mutants are sensitive to RNAi directed against germline-expressed genes (pos-1, rba-2), as well as somatically-expressed genes (unc- 22). Furthermore, one of the mutants, mut-11 (pk724), was tested for gld-1 cosuppression, and found to be sensitive. Similarly, two previously isolated Tc1 mutators, mut-4(st700) and mut- 6(st702) are completely sensitive to both RNAi (Tabara et al., 1999; our data) and cosuppression (Ketting and Plasterk, 2000). RNAi-sensitive mutators do share some additional phenotypes with mut-7(pk204). Both RNAi- resistant and RNAi-sensitive mutators are thermo-sensitive sterile. DAPI-staining of the gonads several mutants shows a reduction of gonad size and the presence of univalents in diakinesis. Like mut-7, most RNAi-sensitive mutators show a high incidence of males. Classical genetic mapping of the Tc1-excision phenotype has placed the mut-13(pk766) mutation in the center of chromosome I. As an alternative approach to rapidly identify genes involved in transposon silencing in C. elegans, we are performing genome-wide screens using the RNAi feeding library to inactivate each C. elegans gene (in collaboration with Julie Ahringer, Cambridge, UK). We identified three putative mutator genes on chromosome I by scoring for Tc1 excision after feeding of unc- 22::Tc1 worms.

Bettina Schulze et al. (2002) European Worm Meeting "Identification of genes involved in dauer larva morphogenesis by systematic RNA interference"

Bettina Schulze, Kaja Reisner, Stephan Froede, Ekkehard Schulze

[European Worm Meeting, 2002]

Under conditions of starvation and extreme crowding C. elegans enters the dauer larva, a duration stage alternative to the third larval stage. Dauer larva exhibit a unique morphology, an extended life span and are resistant to many environmental stresses. The decision to form dauer larva is controlled by TGF-beta and insulin signaling pathways, which are defined by a large number of daf mutants and well characterized. However, the subsequent genetic control and the molecular nature of the dramatic morphological changes characterizing dauer larva formation is largely unknown. We used systematic RNA interference (1) ( Julie Ahringer's Chromosome 1 RNAi library and further additional clones) in two different daf-c background strains (daf-2(m41) and daf-1(m40)) to identify genes specifically required for the dauer molt, dauer morphogenesis, or dauer recovery. Two genes were identified, which are essential for the survival of the dauer molt, a phenotype observed here for the first time. These are the patched related ligand ptr-23 and the G- protein coupled chemorezeptor srh-49, which both are potentially involved in signaling events. Both depletion experiments do not cause phenotypes in daf-c backgrounds at the permissive temperature or in the wild type at 25C. Morphogenetically incomplete dauer larva developed when the p66 subunit of the Mi-2 histone deacetylase complex was depleted. The Mi-2 complex has been implicated in chromatin remodeling and transcriptional repression. This suggests that global transcriptional regulation caused by histone deacetylation and chromatin remodeling is important for dauer larva formation. Depletion of the ras related protein rab-11.2 interfered with the dauer specific shrinkage of the pharynx, whereas depletion of the developmentally regulated actin gene Y71F9AL.16 resulted in dauer larva with a twisted buccal cavity. These and further observations indicate that systematic RNA interference is an efficient and promising tool to identify genes involved in dauer morphogenesis. Interestingly, we also identified genes, which were essential for hermaphrodite fertility when the animal was passed through the dauer stage, but which did not influence hermaphrodite fertility when the animal had developed as a normal L3 larva.

Gorrepati, Lakshmi et al. (2009) International Worm Meeting "Identification of cell-type specific Wnt pathway targets in C. elegans."

Gorrepati, Lakshmi, Eisenmann, David

[International Worm Meeting, 2009]

The Wnt signaling pathway is one of the key extracellular pathways involved in many developmental processes in both vertebrates and invertebrates. In the nematode C. elegans, there exists a BAR-1/b-catenin mediated Wnt signaling pathway and a divergent Wnt/b-catenin asymmetry pathway. Both are involved in cell specification, polarity and migration. We and others have shown that the BAR-1 mediated Wnt signaling pathway plays a crucial role in the specification of the vulval precursor cells (VPCs) that form the hermaphrodite vulva. Work from our lab has also implicated Wnt signaling in the specification of seam cells (Julie Gleason unpublished data). Seam cells are specialized epithelial cells that lie along the apical midline of the worm hypodermis. We propose to identify the targets of the Wnt pathway functioning in the VPCs and seam cells using Affymetrix microarray analysis. We will study the alterations in gene expression due to over-activation and under-activation of the Wnt pathway by utilizing worms expressing a heat shock inducible stable variant of BAR-1/b-catenin (over-activation), or a dominant negative variant of POP-1/TCF (under-activation). The transcript pools from the VPCs and seam cells will be specifically isolated by mRNA-tagging method. In this method, transcripts will be co-immunoprecipitated with a FLAG-tagged variant of the poly-A tail binding protein PAB-1 that is expressed only in the cell-types of interest (Roy et al., 2002 and Von Stetina et al., 2007). The specificity of the transcript pool obtained by mRNA-tagging was verified by qRT-PCR. The experimental strains showed 2 fold or greater increase in expression than control strain for seam cell and VPC specific transcripts tested. Microarray analysis will be performed on the experimental and control strains. Transcripts showing 2 fold or greater change in expression will be identified as potential Wnt targets and verified by quantitative Real-Time PCR (qRT-PCR). The targets that pass this verification screen will be further analyzed for regulatory sequences, spatial and temporal expression patterns, and mutant phenotypes by RNAi.