Genetic analysis of programmed cell death in the Caenorhabditis elegans germ line

Programmed cell death or apoptosis is a genetic program which is used in metazoans to rapidly kill and remove extra or damaged cells. The program is particularly active in the germ line where it can account for the fate of over 99.9% of the potential germ cells in some vertebrates. In the model system C. elegans, germ cell apoptosis occurs continuously throughout the adult worm's reproductive life, resulting in the demise of half the germ cells produced during oogenesis. This dissertation describes the experiments I have carried out to elucidate the regulation of programmed cell death in the C. elegans germ line. Chapter 2 describes the development of a staining protocol using Acridine Orange (AO) to specifically stain apoptotic cells in the germ line, as well as the characterization of this staining in genetic backgrounds in which apoptosis is altered. Chapter 3 describes a genetic screen that was carried out using AO to identify genes that regulate germ cell apoptosis, or result in germ cell apoptosis when mutated. At least ten genes were identified using this screen and the phenotypic characterization of several of them is described. Chapter 4 describes the cloning and characterization of gla-1/cpb-3, a gene identified in the AO screen which is predicted to encode an RNA binding protein important for oogenesis. In chapter 5 the cloning and characterization of a second gene, gla-3 is described. GLA-3 encodes a predicted CCCH zinc-finger protein with homology to proteins involved in germ cell fate determination. Chapter 6 explores the genetic regulation of DNA damage-induced germ cell apoptosis, showing that the processes of cell proliferation arrest and germ cell apoptosis in response to DNA damage are linked but genetically separable processes. In chapter 7 experiments are described that build on the results from chapter 6. The effects of DNA damage on sperm and oocytes with respect to embryonic survival are addressed in this section. In addition, transcriptional induction of the gene egl-1 is proposed as the mechanism whereby cell death is activated following DNA damage. Finally, using cDNA microarrays additional genes were identified that may be involved in the organism response to DNA damage. The work described here will help us better understand the link between germ cell development and apoptosis as well as how organisms regulate their apoptotic response to DNA damage.