Necrosis is not a chaotic uncontrolled reaction to cellular trauma, but a finely controlled cell death program. We have developed a model for analysis of genetic control of necrosis. In our model, nicotinamide induced necrosis of the uterine-vulval uv1 cells in pnc-1 animals is robustly rescued by overactivation of EGF signaling. LET-23, an EGF receptor, has many functions in C. elegans development, and two signaling pathway are known to mediate LET-23 signaling effects; one that signals through LET-60/ Ras and a second through ITR-1. LET-60 signaling downstream of LET-23 is necessary but not sufficient for rescue of necrosis and ITR-1 is neither necessary nor sufficient. These results suggest that an as yet unidentified pathway downstream of LET-23 mediates rescue in concert with the LET-60 pathway. We carried out a targeted RNAi screen and have discovered two genes, pmt-1 and cdk-2, that are required for let-23gf rescue of uv1 necrosis. PMT-1 is a phosphoethanolamine methyltransferase that catalyses the first step of the conversion of ethanolamine to phosphocholine, followed by conversion to phosphatidylcholine. To confirm the relevance of this pathway we knocked down pmt-2, downstream of pmt-1, and supplemented pmt-1 and pmt-2 RNAi animals with choline, an alternative substrate for phosphocholine synthesis. pmt-2 RNAi phenocopied pmt-1 RNAi and choline supplementation restored rescue . However, choline supplementation in a pnc-1 background did not rescue uv1 necrosis, which confirms that phosphocholine synthesis via the PMT pathway is required but not sufficient for rescue. We hypothesised that the PMT pathway is required for let-23gf uv1 rescue to maintain LET-23 cell membrane localization and activity. To test this we investigated the effect of pmt-1 and pmt-2 RNAi on two let-23gf phenotypes; excess vulval induction and excess uv1 cell specification. We found that neither pmt-1 nor pmt-2 RNAi affected either of these phenotypes, which suggests an alternative hypothesis; that phosphocholine itself is required for a LET-23 survival signal. This was supported by the failure of cept-1 or Y49A3A.1 RNAi, both genes required for phosphatidylcholine synthesis, to affect rescue.

Affiliation:
- Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA.