While a conserved cell death execution machinery has been well characterized in C. elegans, the molecular events that trigger its activation are, for the most part, unknown. We are using the C. elegans tail spike cell as a model to further explore the upstream factors that govern a cell's decision to die. Previous studies suggested that activation of PCD in somatic cells in C. elegans is dependent upon EGL-1-mediated release of CED-4 from a mitochondrial CED-4:CED-9 complex. Tail spike cell death, while strongly dependent upon functional
ced-3 and
ced-4, is only partially dependent on functional
egl-1, and weakly affected by a
ced-9(
n1950) mutation, suggesting that an alternate sequence of events may activate the tail spike cells apoptotic machinery. We developed a
ced-3p::gfp marker to aid us in cell identification. The marker is expressed embryonically in many cells, including the tail spike cell. Intriguingly, tail spike
ced-3p::gfp expression begins 25 minutes before the cell dies. We speculate that
ced-3p::gfp expression may reflect endogenous
ced-3 expression, and that transcriptional upregulation of
ced-3 may be a key step in initiation of tail spike PCD. Regulation of caspases has been characterized at the post-translational level, but their transcriptional regulation has remained generally unaddressed. We have identified three regions of the
ced-3 promoter that are redundantly required for tail spike cell death. Deleting these sites has no effect on rescue of cell death in the pharynx. We speculate that the sites may represent binding sites for tail spike specific cell death regulators. To identify regulators of
ced-3 expression in the tail spike cell we performed a forward genetic screen. Mutagenized
ced-3 animals expressing our
ced-3p::gfp reporter were screened for the absence of reporter expression in the tail spike cell. A non-clonal screen of 32,000 mutagenized haploid genomes yielded three mutants defective in tail spike cell
ced-3p::gfp expression. At least one of these mutants results in inappropriate tail spike survival without any other obvious cell death defects, suggesting that
ced-3 upregulation may be critical for tail spike cell death. This mutant also exhibits defects in tail spike cell fusion. We speculate that caspase upregulation and tail spike cell death may be dependent upon cues originating from the completion of specific cellular events, such as fusion. Alternatively, this mutant may affect a regulator of tail spike cell fate. Mapping and further characterization of these mutants is underway.