Genotype 1 Wild-type gls-1(ef8) fbf-1(ok91)4 gls-1(ef8); fbf-1(ok91) fbf-2(q738)4 gls-1(ef8); fbf-2(q738) nos-3(q650)5 gls-1(ef8); nos-3(q650)6 puf-8(q725)7 gls-1(ef8); puf-8(q725) gls-1; fbf-1; fem-1(RNAi)8 gls-1; fbf-1; fem-2(RNAi)8 gls-1; fbf-1; fem-3(RNAi)8 fog-1(RNAi) gls-1; fbf-1 fog-2(q71)9 gls-1; fbf-1; fog-2(q71)9 gld-3(q730)10 gls-1(ef8); gld-3(q730)11 fbf-1(ok91) gld-3(q730)12 gls-1; fbf-1 gld-3(q730)13 100% 99% 99% 0% 99% 100% .99% 59% 97% 100% 42% 50% 0% 0% 0% 18% 87% n.a 100% 97% 0% 1% 1% 0% 0% 100% ,1% 41%6 3% 0% 58% 42% 4% 1% 0% 0% 0% n.a 0% 2% 0% 0% 0% 0% 1% 0% 0% 0% 0% 0% 8% 8% 96% 99% 100% 82% 13% n.a 0% 1% .200 83 570 80 2201 25 2000 44 1952 149 26 24 75 94 44 39 110 .100 519 105 determination cascade and the latter reinforces FBF activity at the level of fem-3 mRNA regulation [21,22]. Interestingly, each single mutant produces sperm and oocytes, similar to gls-1. However, PUF double mutants produce only sperm, exemplifying redundant controls in promoting oogenesis. To understand the role of gls-1 in sex determination and to test for a likely redundancy with fbf, we combined gls-1 with other sperm-to-oocyte switch defective genes and generated double mutants. We analyzed the germ lines with Nomarski and immunofluorescence microscopy to unambiguously identify the gamete fate in young adult hermaphrodites. We discovered an essential role for GLS-1 in the switch to oogenesis when FBF-1 is not expressed (Table 2); gls-1; fbf-1 double mutants are fully sterile and produced only sperm and no oocytes (Table 2; Figure 5B). In contrast, compromising gls-1 activity in a fbf-2 or puf-8 mutant background does not cause a sperm only (Mog) phenotype; sperm and oocytes were always present (Table 2). The synthetic Mog phenotype in double mutants was not enhanced or changed at elevated temperatures, which is consistent with gls-1(ef8) being a strong loss-of-function with respect to the sperm-to-oocyte switch. When we simultaneously eliminated nos-3 and gls-1 function, we Table 2. Sperm-to-Oocyte Switch Defects in gls-1 Mutants. Sp 2+Oo 2 Sp 2 only Oo 2 only n3 GLS-1 and Germline Development observed a rather complex phenotype; germ cells either failed to adopt the oocyte fate entirely (,20%) or were not able to maintain the oogenic fate and produced sperm cells in the distal arm at a much higher frequency (,20%) than gls-1(ef8) by itself (1%) (Table 2). However, the remaining 60% of gls-1; nos-3 germ lines switched to and maintained oogenesis. In summary, we find that gls-1 works genetically in parallel, or together, with fbf-1 and nos-3 to promote the sperm-to-oocyte switch in hermaphrodites. gls-1 Acts Upstream of All Known Sperm-Promoting Factors in the Germ Line To place the action of gls-1 and fbf-1 into the sex determination pathway we performed genetic epistasis experiments with key spermatogenesis promoting genes. We either generated true triple mutants or relied on feeding RNAi experiments to generate ‘‘triple mutant’’ phenotypes (Table 2). Interestingly, the synthetic Mog phenotype depended on the activity of each known fem and fog gene. All ‘‘triple mutant’’ germ lines produced either oocytes only or oocytes in addition to sperm. The mixture of sexual fates might be due to an incomplete penetrance caused by RNAi; the wild- type animals treated in parallel were fully feminized to a similar percentage as the gls-1; fbf-1 mutants (not shown). The gls-1; fbf-1; fog-2 triple mutant also displayed a mixture of cell fates, yet we observed mostly feminized germ lines (82%, Table 2; Figure 5C). Taken together, we provide evidence that gls-1 is upstream of all known spermatogenesis-promoting genes, similar to the activity of fbf-1. To determine if this masculinization defect is due to the activity of gld-3, we generated the gls-1(ef8); fbf-1(ok91) gld-3(q730) triple mutant. As expected, we were able to restore the oocyte fate in gls- 1; fbf-1 germ lines by eliminating gld-3; almost all triple mutant germ lines displayed a sperm and oocyte pattern (97%, Table 2; Figure 5D). Therefore, we conclude that the synthetic Mog phenotype of gls-1; fbf-1 germ lines depends on excess gld-3 activity (summarized in Figure 5E). GLS-1 Competes with FBF for GLD-3 Binding Given the known molecular interactions of GLS-1 and GLD-3 (Figure 3), the simplest interpretation is that GLS-1 is a modulator of the sperm-to-oocyte switch by acting antagonistically to GLD-3 and in parallel to FBF. In this scenario, the switch to oogenesis is promoted by relieving FBF from GLD-3L inhibition through GLS-1 binding. Support for this model comes from our in vitro observations that the C-terminal half of GLD-3L binds consis- tently with higher affinity to GLS-1 than the N-terminal half (Figure 5F). To test the proposed antagonistic model further we employed a competition experiment. FBF-1 was immobilized on beads and subsequently loaded with GLD-3L. Afterwards, the FBF-1/GLD-3L complex was challenged with increasing amounts of free GLS-1. GLS-1 and dissociated GLD-3L was washed away. We observed that the GLD-3 amount bound to FBF decreased with higher GLS-1 concentrations present, while FBF-1 remained stably bound to the beads (Figure 5G). Together, this data indicates that GLS-1 protein can recruit FBF-1 from the interaction with GLD-3. Therefore, GLS-1 may liberate FBF-1 for translational repression of spermatogenic factors. Maternal gls-1 Activity Prevents Progressive Germ Cell Degeneration Next, we addressed how GLS-1 influences GLD-3 activity to promote germline survival. The adult wild-type hermaphrodite contains two U-shaped gonadal arms filled with ,1000 germ cells [23]. By contrast, gls-1(RNAi) hermaphrodites and homozygote gls- 1animals grown at 20uC. 2Sp, sperm; Oo, oocytes. 3total number of germ lines scored. 4Taken from [27]. 5Taken from [13]. 6The number includes .20% fully Mog germ lines and .20% germ lines that return to spermatogenesis after producing oocytes. 7Taken from [22]. 8from gls-1(ef8)/ccIs4532; fbf-1(ok91)/mIn1g) mothers. 9from fog(q71) unc-51(e1189)/+ mothers (see strains). 10Taken from [5]. 11animals do not produce gametes. 12the amount of sperm is similar to fbf-1 single mutants, which is elevated compared to wild-type [26]. 13animals have no living progeny. doi:10.1371/journal.pgen.1000494.t002 PLoS Genetics | www.plosgenetics.org 8 May 2009 | Volume 5 | Issue 5 | e1000494