The C. elegans germline consists of both proliferating and differentiating cells. Proliferation is needed to increase cell number and provides a pool of cells that can enter meiosis and generate gametes. The balance between proliferation and meiotic entry needs to be tightly regulated for maintaining the reproductive fitness of the animal. In the hermaphrodite germline, a core regulatory network controls this decision. It consists of a canonical Notch signalling pathway to promote the proliferation and two redundant mRNA regulatory pathways, the GLD-1 and GLD-2 pathways, to promote meiotic entry. Superimposed on this core network is a complex set of factors. The functions of many are still poorly understood and some factors remain to be identified. Here we report our approach to identify some of those unknown factors using an RNA inference (RNAi)-based screen. RNAi was performed systematically by feeding against ~17,000 genes in the predicted C. elegans genome, and a genetic mutant background of
rrf-1;
gld-3 was used.
rrf-1-/- is a RNAi mutant that restricts RNAi to the germline enabling us to bypass the early lethal phenotype caused by some genes and be able to assay their germline RNAi phenotype in the adult.
gld-3 a component of the GLD-2 pathway, and
gld-3-/- provides a background sensitized to over-proliferation, which facilitates the identification of potential regulatory factors. We reason that, in the
gld-3-/- background, inhibition of gene activity by RNAi on genes that promote meiotic entry, and do not function within the GLD-2 pathway, would cause a failure of meiotic entry resulting in a tumorous germline. We have completed the primary screen and are currently conducting a secondary screen to eliminate false positives. We are also testing their genetic interactions with other genes involved in regulating proliferation in the germline. Several such genes have been identified and their preliminary characterization will be presented in this conference.