BackgroundThe spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant.ResultsThe san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein.ConclusionTogether, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

Cellular and genetic analysis supports the notion that NPP-16/NUP50 and CDK-1 function to reversibly arrest prophase blastomeres in Caenorhabditis elegans embryos exposed to anoxia. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.

Background: The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O 2 ) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber.

The post-genomic era is marked by a pressing need to functionally characterize genes through understanding gene-gene interactions, as well as interactions between biological pathways. Exploiting a phenomenon known as synthetic lethality, in which simultaneous loss of two interacting genes leads to loss of viability, aids in the investigation of these interactions. Although synthetic lethal screening is a powerful technique that has been used with great success in many model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, this approach has not yet been applied in the zebrafish, Danio rerio. Recently, the zebrafish has emerged as a valuable system to model many human disease conditions; thus, the ability to conduct synthetic lethal screening using zebrafish should help to uncover many unknown disease-gene interactions. In this article, we discuss the concept of synthetic lethality and provide examples of its use in other model systems. We further discuss experimental approaches by which the concept of synthetic lethality can be applied to the zebrafish to understand the functions of specific genes.

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