At the distal end of the late-larval and adult C. elegans gonad, a population of proliferating germ cells serve as stem cells for potentially hundreds of gametes. As cells move proximally, away from the influence of the Distal Tip Cell (DTC), they leave the mitotic cell cycle and enter meiotic prophase. GLP-1/Notch receptors in distal germ cells are activated by a signal from the DTC, promoting the proliferative state. Gain-of-function (gf) consitutively active alleles of
glp-1 cause germ cells to remain proliferative, thereby forming a germline tumor.
gld-1 and
gld-2 function redundantly to inhibit proliferation and/or promote entry into meiosis and are negatively regulated by the
glp-1 pathway (Kadyk and Kimble, 1998; Francis et al. 1995). Animals lacking
gld-1 and
gld-2 form a germline tumor similar to that seen with a
glp-1(gf) allele. We have employed two genetic screens to identify other genes that are involved in regulating the switch from mitosis to meiosis. One screen identified mutations that enhance a weak
glp-1(gf) allele, (
oz112 oz120) . This screen identified
teg-1 ( t umorous e nhancer of g lp-1(gf) ), which encodes a 353 amino acid protein with similarity to a human protein that interacts with the T-lymphocyte receptor, CD2. The other screen identified mutations that are synthetic tumorous with
gld-2 . From this, we obtained multiple alleles of
syt-1 ( sy nthetic t umorous), which we cloned and identified as the
nos-3 gene (Kraemer et al 1999). This screen also identified another allele of
teg-1 , demonstrating that
teg-1 is synthetic tumorous with
gld-2 . Neither
teg-1 nor
nos-3 mutants are tumorous on their own. Thus,
teg-1 ,
nos-3 and
gld-1 appear to each function redundantly with
gld-2 to inhibit proliferation and/or to promote entry into meiosis. Close examination of the germline tumors in
gld-2;
teg-1 ,
gld-2;
nos-3 and
gld-2 gld-1 animals, however, revealed that none are equivalent to the
glp-1(
oz112gf) tumor, even though the mutations in these four loci appear to be null. Using two markers (anti-REC-8 that stains chromosomes of proliferative nuclei when specific staining conditions are used and anti-HIM-3 (Zetka et al 1999) that stains chromosomes of meiotic nuclei), we have shown that each of these synthetic tumors contain meiotic nuclei while the
glp-1(
oz112gf) tumors do not. These results suggest that these four genes (
gld-1 ,
gld-2 ,
teg-1 and
nos-3 ) could be involved in regulating the progression of meiotic prophase rather than entry into meiotic prophase. Thus the germline tumor would result from germ cells returning to mitosis after failing to progress through meiotic prophase, analogous to the
gld-1 single in female germ cells (Francis et al 1995). Conversely, if these genes do act to promote meiotic entry, the presence of meiotic nuclei in the tumors suggests that a loss of
gld-2 and
gld-1 (or
gld-2 and
teg-1 or
nos-3 ) is not equivalent to constitutive activation of GLP-1, indicating that there is additional genetic complexity in this process. To distinguish between these two models, we used the weak
glp-1 gf allele
ar202 . At 15C the germ lines of
glp-1(
ar202) animals are essentially wild type, while at higher temperatures animals display a 'Pro' phenotype (proliferative germ cells in the proximal end of the gonad; see abstract by Pepper, Lo and Hubbard) and a late onset tumorous phenotype (the size of the distal mitotic zone is expanded). We tested
gld-1 ,
gld-2 and
teg-1 for their ability to enhance the late onset tumorous phenotype of
glp-1(
ar202) at 15C. We reasoned that if a gene were normally required to promote entry into meiosis, loss of its activity could enhance the late-onset tumorous phenotype of
glp-1(
ar202) . Conversely, if no enhancement of
glp-1(
ar202) is seen, the gene might not be involved in regulating meiotic entry, but rather meiotic progression. Mutations in
gld-1 ,
gld-2 and
teg-1 each enhance the late onset tumorous phenotype of
glp-1(
ar202) at 15C, supporting the hypothesis that these genes regulate entry into meiosis. Furthermore, there are far more meiotic nuclei in the
gld-2 gld-1 and
gld-2;
teg-1 doubles than in the
gld-2 gld-1;
teg-1 triple mutant. This suggests that each gene functions to promote entry into meiosis, perhaps through three separate pathways. Since some evidence of meiotic nuclei still exists in the triple mutant, a possible fourth pathway could be involved.