In the C. elegans germline, GLP-1 Notch signaling promotes the stem cell fate and its loss results in a germline proliferation defective phenotype (Glp) where, on average, the single germ cell that corresponds to the progenitor for each gonad arm undergoes 2 rounds of division before prematurely entering into meiosis, and differentiating into sperm (Austin and Kimble, 1987). Downstream of GLP-1 Notch signaling, GLD-1 and GLD-2 pathways (GLD) redundantly promote germ cell meiotic entry. GLP-1 Notch signaling represses both pathways to promote the stem cell fate (Kadyk and Kimble, 1998; Hansen et al., 2004). GLD pathway single mutants exhibit essentially normal meiotic entry. A simultaneous disruption of genes from both the GLD-1 and the GLD-2 pathways results in a tumorous germline phenotype due to a defect in meiotic entry. The tumorous phenotype of GLD double mutants is epistatic to
glp-1(-), consistent with GLP-1 signaling promoting the stem cell fate by inhibiting the redundant GLD pathways. In
gld-1(-);
glp-1(-), the germline is Glp, however the single germ cell undergoes 2-3 additional rounds of division before entering into meiosis, as compared to
glp-1(-) (Kadyk and Kimble, 1998; Francis et al., 1995). We therefore expected a similar Glp phenotype in the mutant germlines of
gld-2 pathway genes, namely
gld-2 and
gld-3, when GLP-1 signaling is compromised. Here, we examined dissected young adult (8-hr post-adult molt) hermaphrodite germlines by REC-8 (progenitor zone marker), HIM-3 (meiotic prophase marker) and MSP (sperm/spermatogenesis marker). As expected, we found that germlines of both genotypes were Glp (absence of REC-8 positive nuclei and presence of HIM-3 positive nuclei). Compared with
glp-1(-) germlines, the total number of germ cells was slightly higher in
gld-2(-);
glp-1(-) germlines (Fig 1)(Kadyk and Kimble, 1998). In contrast, the total number of germ cells was significantly higher, and variable, in
gld-3(-);
glp-1(-) double mutant germlines with some of the gonads having more than 100 germ cells (Fig 1). Thus, like GLD-1, both GLD-2 and GLD-3 appear to repress the stem cell fate in the absence of GLP-1 signaling, although the mechanisms are unknown. That loss of GLD-3 results in much higher number of germ cells compared to GLD-2, once again underlines the functional non-equivalency of GLD-2 and GLD-3 (see Mohammad et al., 2018). One possibility is that GLD-4 partially compensates for GLD-2 loss, thus keeping the number of germ cells lower in
gld-2(-);
glp-1(-) germlines (Millonigg et al., 2014). It will be of interest to analyze at what point during larval development the
gld-3(-);
glp-1(-) germlines lose the stem cell pool.