We are characterizing two temperature-sensitive embryonic-lethal cell division mutants. One mutant,
or452ts has a mitotic spindle defect, and the second mutant,
or576ts has chromosome segregation and cytokinesis defects. Using DIC videomicroscopy, it appears that a bipolar spindle fails to assemble in approximately 50% of 1-cell stage
or452ts mutant embryos. These embryos fail to divide, and the result is a multinucleated embryo that dies prior to differentiation. In
or452ts embryos that do reach the 2-cell stage, 90% fail to assemble a bipolar spindle in either or both cells, and cell division fails. We have begun to characterize the spindle defects more thoroughly using fluorescence microscopy in both fixed and living cells. Immunofluorescence microscopy shows that monopolar spindles are frequently formed at the 1-cell stage, and DNA surrounds the single microtubule organizing center. Bipolar spindles are sometimes seen in
or452ts mutant embryos, but they appear less robust than in wild type.
or576ts mutant embryos fail to complete cytokinesis in approximately 50% of embryos at the 1-cell stage. The cleavage furrow ingresses nearly to completion, but later regresses. If cytokinesis succeeds, usually in one or both of the cells at the 2-cell stage, the cleavage furrow begins but will regress. By DIC microscopy, it appears that there is often a clump of non-segregated DNA in the cells prior to the failed cell division. We are now analyzing this phenotype using immunoflourescence and by imaging GFP::tubulin and GFP::histone in living mutant embryos. We have not yet identified the genes that are mutated in either strain. However, with this goal in mind, we have used meiotic recombination mapping with visible and SNP markers to position
or576ts to approximately 0.9 on LGII and
or452ts to LGIII at approximately 2.5 map units. Gonczy et. al. (Nature, vol. 408, pp. 331-336) reported that RNAi of D2045.1, which is predicted to be at 2.5 m.u. on LGIII, resulted in spindle and cell division defects in those embryos which were intact enough to view. The phenotype was observed 24 hours after injecting dsRNA. We fed D2045.1 RNA to wild-type worms and looked at early time points to see if the phenotype was similar to our mutant
or452ts. While we saw interesting early cell division defects, they were not a clear phenocopy of our mutant. However, we are also sequencing the D2045.1 gene in our mutant to see if there is a difference in the sequence from wild type. We believe characterization of these mutants may provide new insights into the important and complex processes of cell division.