The development of Caenorhabditis elegans involves the elimination of 131 somatic cells by programmed cell death. Most programmed cell deaths require the caspase
ced-3, a cysteine protease that presumably promotes death via the proteolysis of downstream substrates. Although most programmed cell deaths fail to occur in
ced-3 mutants, a small but detectable number of cells die in animals that completely lack
ced-3 activity, indicating that cells can activate a
ced-3-independent death program (P. Reddien, F. Xie and H.R. Horvitz, unpublished results). We are beginning to investigate the morphological and genetic characteristics of these
ced-3-independent cell deaths.To analyze the physical characteristics of
ced-3-independent cell deaths, we will use electron microscopy for ultrastructural characterizations as well as TUNEL and ANNEXIN V staining experiments to test for apoptosis-associated DNA fragmentation and cell surface markers, respectively. Furthermore, we will test the influence of mutations in other cell-death genes on
ced-3-independent killing, including genes of the core cell-autonomous execution pathway (
ced-4,
ced-9, and
egl-1), genes that function in the engulfment of dying cells (
ced-1,
ced-2,
ced-5,
ced-6,
ced-7,
ced-10, and
ced-12), and genes that encode homologs of other proteases potentially involved in cell death, such as cathepsin and calpain. We will also examine the role of other caspases in the
ced-3-independent cell deaths. The C. elegans genome contains three additional predicted caspase genes (
csp-1,
csp-2, and
csp-3) with no known functions. We are isolating deletion alleles of these csp genes to test for possible functions in promoting programmed cell death in the absence of
ced-3 activity.Through our studies, it may be possible to elucidate a genetic pathway that does not require
ced-3 activity - or perhaps caspase activity in general - for the activation of cell death in C. elegans. Programmed cell death has critical roles in the development of higher metazoans and in the elimination of damaged, virus-infected, or cancerous cells. Our analysis of a
ced-3-independent death program may define a pathway in C. elegans that will facilitate the characterization of a similar genetic pathway in vertebrates.