Accumulation and maintenance of stem cells is crucial for proper organogenesis and tissue homeostasis. We previously showed that the conserved Target of Rapamycin (TOR) pathway (
let-363/TOR,
daf-15/RAPTOR) and its targets
rsks-1/p-70-S6-Kinase (S6K) and
ife-1/eIF-4E promote the accumulation of germline stem cells (GSCs) during larval germline development. Among these, only
rsks-1/S6K displays a synergistic interaction with
glp-1/Notch that implies its role in GSC maintenance: loss of S6K enhances and suppresses sterility associated with reduced and elevated
glp-1 activity, respectively. In this GSC maintenance role, S6K acts germline-autonomously and requires a conserved TOR phosphorylation site. The mechanism(s) underlying a role for the highly conserved S6K in cell fate regulation in vivo are unclear. To determine how
rsks-1/S6K maintains GSCs, we used both candidate and unbiased approaches. Similar to loss of
rsks-1, inhibiting Cyclin-E/CDK-2 or MAPK pathway activity enhances loss of GSCs in a mutant with reduced
glp-1 activity. We tested these and found that neither acts in a simple linear fashion with S6K to maintain GSCs. We undertook a genome-wide RNAi screen to identify genes that when knocked down by RNAi would phenocopy the genetic interaction of
rsks-1/S6K with
glp-1/Notch. We screened the Ahringer library in the reduction-of-function
glp-1(
e2141) mutant at a semi-permissive temperature and scored for enhancement of sterility. Sterility was strictly defined as =1 embryo in the uterus, and RNAi feeding was started at the L1 larval stage to bypass embryonic lethality. The strain also carried
rrf-1(
pk1417) to restrict RNAi primarily to the germ line and markers (pharyngeal mCherry, GFP embryos) to facilitate semi-automated scoring. We found 139 genes that, when depleted by RNAi, caused highly reproducible and penetrant sterility in
glp-1(
e2141). These include regulators of replication, transcription, RNA splicing, translation, ribosome biogenesis, cell cycle, development, and metabolism. Similar to loss of
rsks-1, RNAi targeting 62 of these genes caused penetrant sterility in the
glp-1 mutant but not in the wild type. These results considerably extend the set of known enhancers of
glp-1 sterility. Further, 20 of these 62 genes caused obvious defects in GSC maintenance in the
glp-1 background. Among these 20 are several genes that may act in a linear pathway with
rsks-1/S6K to promote GSC maintenance since depletion of these genes by RNAi did not further enhance the penetrance of GSC loss in the
glp-1(
e2141)
rsks-1(null) double mutant. Unexpectedly, our screen results and subsequent analysis implicate Hedgehog-like signaling in GSC maintenance, in a linear pathway with S6K.