Functional genomic screen of cell division processes in C. elegans using RNAi: analysis of chromosome III

Pierre Gönczy, Christophe Echeverri, Karen Oegema, Alan Coulson, Steven J. M. Jones, Richard R. Copley, John Duperon, Jeff Oegema, Michael Brehm, Etienne Cassin, Eva Hannak, Matthew Kirkham, Silke Pichler, Kathrin Flohrs, Anoesjka Goessen, Sebastian Leidel, Anne-Marie Alleaume, Cécilie Martin, Nurhan Özlü, Peer Bork & Anthony A. Hyman

Rationale
The goal of this project was to conduct a functional genomic screen to identify genes required for cell division processes in metazoans. The early C. elegans embryo was chosen as a model system for the following reasons. First, cell division processes can be examined with high spatial and temporal resolution using simple time-lapse differential interference contrast (DIC) microscopy. Second, the C. elegans genome sequence is available. Third, using RNA interference (RNAi), the expression of a given gene in the embryo can be abolished in a sequence-specific manner by exposing parental germ cells to corresponding double-stranded (ds) RNA. Thus, RNAi was applied on a genomic scale (starting with chromosome III) to identify genes required for cell division processes.

Approach
Genomic sequences, rather than cDNAs, were used as template for generating dsRNAs, to ensure maximal coverage and unbiased testing of each predicted open reading frame. Primer pairs were selected using a customized algorithm, and oligonucleotides synthesized with addition of T7 and T3 promoter sequences. Corresponding pieces of wild-type genomic DNA were amplified by PCR, and separate T3 and T7 RNA transcription reactions performed. Resulting single-stranded (ss) RNAs were annealed to generate dsRNA. The quality of PCR products, ssRNAs and dsRNAs was monitored by agarose gel electrophoresis. Pairs of random dsRNAs were microinjected into wild-type hermaphrodite worms, and the resulting progeny analyzed 24 hours later using time-lapse DIC microscopy. Single embryos were filmed from shortly after fertilization until the four cell stage (~30 min recordings), capturing 1 image every 5 seconds. This "DIC assay" was optimized for detecting even minor deviation from the wild-type sequence of events. At the same time, 3 injected animals were transferred to a fresh plate, which was scored 2 and 4 days later using low magnification stereomicroscopy for the presence of progeny, and their appearance. This "progeny test" was used to score only gross deviations from wild-type development. Pairs of dsRNAs that gave rise to a phenotype in either assay were split, and each individual dsRNA tested in turn. Experiments conducted with dsRNAs corresponding to known genetic loci, and whose loss-of-function phenotype is visible by DIC microscopy in the one cell stage embryo, demonstrated that this screening strategy leads to the identifcation of over 90% of predicted genes required for specific cellular processes in the early embryo.

Outcome
2226 of the 2315 predicted open reading frames on chromosome III (~96%) were tested to date (testing of the remaining genes is underway). dsRNAs corresponding to 130 genes (~6% of distinct genes tested) gave rise to a phenotype detectable in the DIC assay. Importantly, detailed analysis of time-lapse DIC recordings placed these 130 genes into distinct phenotypic classes. In addition, dsRNAs corresponding to 139 genes gave rise to a phenotype detectable solely in the progeny test; 78 of these were associated with embryonic lethality, 48 with a larval phenotype and 13 with an adult phenotype, while dsRNAs corresponding to another 9 genes resulted in sterility of the injected animals. The list of all genes tested, that of the 278 genes associated with phenotypes, along with phenotypic descriptions (including sample time-lapse recording) are shown in this database.


Conclusions
Our results demonstrate that RNAi is a powerful reverse genetics approach which is usable on a genomic scale. Interestingly, ~46% of the genes associated with a DIC phenotype have orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well. Therefore, this work is generating a unique functional database of cell division proteins. The analysis will be continuted on a genome-wide basis in C. elegans to identify the molecular nature of additional components and thus achieve a fuller understanding of cell division processes.