Notch/GLP-1 signaling maintains germline stem cells (GSCs) in C. elegans through two essential, albeit functionally redundant target genes,
lst-1 and
sygl-1 (Kershner et al., 2014). Loss of the Notch receptor GLP-1 results in loss of GSCs and production of a few sperm ultimately leading to sterility, referred to as a Glp phenotype (Austin and Kimble, 1987). Single mutants for either
lst-1(
ok814) or
sygl-1(
tm5040) have a virtually normal germline and are fertile healthy animals; however,
lst-1(
ok814)
sygl-1(
tm5040) double mutants have a Glp phenotype that is indistinguishable from a Glp-1 null. To identify other GSC regulators, we performed a forward genetic screen for enhancers of either
lst-1(
ok814) or
sygl-1(
tm5040). Taking advantage of the fertility of
lst-1 and
sygl-1 single mutants, we mutagenized each of them and screened for
lst-1 and
sygl-1 enhancers with a Glp phenotype. Initially, we screened 8749
lst-1 and 5504
sygl-1 haploid genomes and uncovered nine
glp-1 alleles and one mutation in an unknown gene. We next modified the screen to include a Mos inserted version of
glp-1 (qSi44) in addition to endogenous
glp-1. We screened 7922 haploid genomes of
lst-1; qSi44 but found no enhancers; a screen of
sygl-1; qSi44 generated two
lst-1 alleles. Overall, we isolated nine alleles of
glp-1, two alleles of
lst-1, and one allele of an unknown gene. The nine
glp-1 alleles included three missense mutations, three nonsense mutations, one splice site mutation, one inversion in an intron, and one unknown mutation located outside the coding sequence and the adjacent region extending 2 kb upstream and 500 bp downstream. These
glp-1 mutations are all novel and provide additional reagents for analysis of Notch signaling. The two
lst-1 alleles included a nonsense mutation and a splice site mutation. The lack of
sygl-1 alleles shows that our screen was not saturated. The unknown gene maps to the right end of chromosome I, causes sterility on its own, and cloning is underway. In summary, our enhancer screen identified one additional gene critical in the pathway for GSC self-renewal.Austin and Kimble (1987) Cell, 51, 589-599Kershner et al. (2014) PNAS, 111(10), 3739-44.