Screening for Negative Regulators of GLP-1/Notch Signaling in C. elegans

Caenorhabditis elegans is a 1-mm long roundworm that is used as a genetic system to dissect basic processes in animal biology. One of these processes is germ cell development, or the production of eggs and sperm through mitosis and meiosis. The C. elegans hermaphrodite produces both eggs and sperm in two bent tubes, enabling it to self-fertilize. In a wild type animal, germ cells proliferate in the distal end of the lobes and enter meiosis at the proximal end near the vulva. The cells develop with a distal to proximal polarity. The decision to switch from mitosis to meiosis is controlled by the evolutionarily conserved Notch signaling pathway. The transmembrane ligand LAG-2 localizes to the distal tip cell, which covers the distal end of the germ line. There, LAG-2 binds to GLP-1/Notch receptors located on the cellular membranes of distal germ cells, causing the intracellular region of GLP-1 (INTRA) to be cleaved and activated. INTRA migrates to germ cells' nuclei and binds to LAG-1, a transcription regulator. The binding signals the cell to proliferate, and this signal is maintained for about twenty cell diameters. Thus, LAG-2, GLP-1, and LAG-1 act to promote mitosis in germ cells. A gain of function (gf) mutation in glp-1 (oz264oz270) results in over-proliferation of germ cells and in partial gain of function phenotype. This phenotype is late onset tumorous at a high temperature (25^o). To identify genes that negatively regulate GLP-1/Notch signaling, C. elegans were screened for mutations that enhance the tumorous phenotype of glp-1 (oz264oz270) at low temperatures (15^o), where development is usually normal. The strain is randomly mutagenized with ethyl methanesulfonate (EMS) and with varying concentrations of N-nitroso-N-ethylurea (ENU). The worms are then screened for a tumorous phenotype in the F2 generation, to identify loss of function mutations. In initial screens, mutants were found that express a stronger over-proliferation phenotype than the original mutant strain.