Caenorhabditis elegans comes of age
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A report of the European C. elegans 2008 meeting, Seville, Spain, 29 March-2 April 2008.
KeywordsTranscription Termination Fred Hutchinson Cancer Research Neuroblast Migration Adult Diapause Acetylcholine Receptor Cluster
In 1998, the publication of the genome sequence of Caenorhabditis elegans (a first for metazoans) and the discovery of RNA interference (RNAi) in this species provided a Big Bang for research using this model organism. What has happened since? Ten years later, scientists continue to squeeze the excellent experimental features of this animal to push the frontiers of knowledge forward in subjects as diverse as genomics, aging, behavior and development. Such breadth and variety was showcased at the recent European C. elegans conference http://www.upo.es/ewm2008. To take just one example, as evidenced at the meeting, genomic approaches often require validation that relies on genetics, and single-gene studies in return locate the gene of interest into larger functional networks. But the need to choose between the whole and the detail is long gone these days, because by now the 'wood' and the 'trees' are irreversibly connected.
Projects in the pipeline
High-throughput work was presented by Mihail Sarov (Max Planck Institute, Dresden, Germany), who showcased the TransgeneOme project, based on an alternative recombination method for protein tagging. The TransgeneOme aims to provide an open resource of tagged fosmid transgenes for C. elegans at a genome-wide scale. The project is rolling and is initially focusing on proteins involved in transcriptional regulation as part of an international network, the modENCODE project http://www.modENCODE.org, that aims to identify all functional DNA sequence elements in the C. elegans genome. Importantly, the TransgeneOme project is open for other protein sets from C. elegans to be included in their pipeline.
Mark Edgley (University of British Columbia, Vancouver, Canada) reported on progress from the C. elegans Gene Knockout Consortium in Canada and the USA. As a result of the combined efforts of the Consortium, Shohei Mitani's group in Japan and the research community worldwide, approximately one-quarter (around 5,000) of all C. elegans genes has been knocked out, all of which are publicly available via the Caenorhabditis Genetics Center website http://www.cbs.umn.edu/CGC. This is a significant milestone, and is a big step toward completing knockout coverage for the entire genome. Denis Dupuy (University of Bordeaux, France) explained the tasks that lie ahead for the C. elegans Localizome project http://localizome.dfci.harvard.edu, which is based on expression-pattern studies in transgenic animals carrying gene promotor-green fluorescent protein (GFP) fusion constructs. The next big hurdle to overcome will be to expand the present-day small overlap between the interactome (1,541 proteins) and the localizome (1,610 gene promoters): only 48 gene/protein pairs are present in both datasets. RNAi-induced modification of expression patterns will be another way to expand the Localizome project.
Engineering the genome
Three presentations by members of Marcel Tijsterman's group (Hubrecht Institute, Utrecht, the Netherlands) presented different approaches to unraveling the mechanisms controlling DNA double-strand breakage (DSB) and repair in meiosis. Daphne Pontier described the generation of a double-transgenic strain that allows the induction of DSB at known genomic locations by controlling the expression of the rare-cutting DNA endonuclease I-SceI through heat shock. This strain is being used to identify novel players in DSB repair/signaling pathways in genome-wide RNAi screens, as reported by Wouter Koole. In an effort to develop an efficient gene targeting method in C. elegans, Bennie Lemmens explained how they are taking advantage of I-SceI expressed in the germline in order to favor homologous recombination between a given locus in the genome and a transgenic DNA construct flanked by I-SceI target sites (located on an extrachromosomal array) that is homologous to the genome locus of interest. Marie Gendrel (Ecole Normale Superieure, INSERM, Paris, France) presented a successful application of homologous recombination using the Mos1 transposon. Mos1-based insertional mutagenesis led to identification of LEV-9 as a protein required for acetylcholine receptor clustering during signaling at the neuromuscular junction. Subsequently, she and her colleagues exploited Mos1 using the MosTIC technique (Mos1 excision induced transgene instructed gene conversion) to knock in a T7 tag at the amino terminus of LEV-9. This tag made inmunostaining easier and helped locate LEV-9 at the neuromuscular junction.
Gene regulation in C. elegans
The recent completion of four genome sequences from different Caenorhabditis species is helping Frederick Partridge (University of Oxford, UK) to develop an intriguing project about translational frameshifting in C. elegans. This special regulatory mechanism of gene expression enables an alternative reading of the genetic code by moving the ribosome one base along the mRNA and thereby changing the frame of translation. By comparing the four Caenorhabditis genomes, Partridge has identified possible conserved frameshifting sites. The availability of multiple Caenorhabditis genomes also enables detailed analysis of cis-regulatory control regions. Using reporter gene analysis, Nuria Flames (Columbia University, New York, USA), has defined the phylogenetically conserved cis-regulatory logic of neuronal cell type specification. She defined a motif that is required and sufficient to determine gene expression in dopaminergic neurons and identified the ETS domain transcription factor AST-1 as an activator of this motif.
Andre Furger (University of Oxford, UK) highlighted the benefits of C. elegans to study transcription termination. The existence of operons facilitates the exploration of mechanisms that prevent premature transcription termination at the end of each gene within such a polycistronic unit. Analyzing the coupling of 3'-end formation and transcription termination at the end of transcription units, he and colleagues found that transcription can continue for up to 0.8 kb downstream of poly(A) sites before cleavage and transcription termination occurs. They also observed that after cleavage at poly(A) sites, low levels of transcription can continue to more than 1.5 kb downstream of the poly(A) site. Considering the compact C. elegans genome, these findings imply the existence of previously unknown overlapping transcription units.
Helge Grosshans (Friedrich Miescher Institute, Basel, Switzerland) reported a novel nuclear export route for micro-RNAs (miRNAs). In vertebrates, Exportin-5 functions as an exporter of miRNAs from the nucleus to the cytoplasm. Grosshans described how the lack of an exportin-5 ortholog in C. elegans guided their work toward the identification of a new export complex in the nematode. Interestingly, by working with a human cell line they observed how this novel nuclear export route is conserved in vertebrates. Giana Angelo (Fred Hutchinson Cancer Research Center, Seattle, USA) shocked the audience by describing a hitherto unreported adult diapause state that will certainly be deeply studied in the following years. By using a different signaling pathway from the one responsible for dauer formation, adult worms are able to survive starvation for at least 30 days and recover a functional germline when returned to food. Angelo noted that entry into this adult diapause is dependent upon the nuclear receptor NHR-49.
An old classic never dies: the genetic screen
Genetic screens have been developed from the very beginning of C. elegans research. Still, the variety of strategies and questions to be addressed seems unlimited in the minds of worm scientists. Valeria Pavet (IGBMC, Strasbourg, France) reported the characterization of a transdifferentiation event, a process that directly transforms a rectal epithelial cell (Y) into a motor neuron (PDA) without involving cell division. She and colleagues have so far recovered 17 mutations from a screen for genes affecting this transdifferentiation process. Some of those mutants display a persistent Y cell that does not transdifferentiate, others a loss of a recognizable Y cell (as visualized with cell-type specific GFP fusions). This screen will be a gold mine for elucidating the intermediate cellular steps the Y cell goes through and for finding the molecules governing the processes of transdifferentiation and cell plasticity. As exemplified by Marie Silhankova (Hubrecht Institute), genome-wide RNAi screens are extremely powerful for uncovering gene function. She and colleagues screened for enhancers of the Q neuroblasts migration defect in mutants of the intracellular protein trafficking complex termed the retromer. Q neuroblast migration is regulated by the EGL-20/Wnt signaling pathway. From the results of this screen, Silhankova reported that members of the myotubularin family of lipid phosphatases are required to produce the Wnt signal involved in the migration of the neuroblasts. This role, uncovered using an RNAi approach, was then confirmed by using 'real' mutations.
A screen-in-waiting was presented by Philippa Mitchell (University of Southampton, UK), who talked about modeling alcohol dependence and tolerance, withdrawal and relief from withdrawal behaviors in worms using ethanol concentrations that are equivalent to those with effects in humans. She and colleagues have dissected wild-type worm behaviors in great detail and are now ready to use this knowledge in screens to get at the genetics of behaviors affected by alcohol.
The early publication of a genome sequence, RNAi, and the efforts of public consortia generating powerful C. elegans research tools (plus tens of years of hard and gifted work beforehand) have transformed the worm into a 'top model' organism. Judging impact by the number of publications, the increasing number of C. elegans labs around the world and by the research presented at conferences like this one, the worm is here to stay for good - feeding biomedical research projects with vibrant and innovative ideas. It is humbling to realize though that quite a bit of the work described above was already envisaged by Sydney Brenner in the early 1960s, before both the authors of this report were born.