GLS-1 and Germline Development Figure 8. Models of How GLS-1 May Modulate GLD-3 in Cell Fate Decisions. (A) GLS-1 antagonizes GLD-3 in the sperm-to-oocyte switch to allow translational repression of FBF-binding elements (FBE) containing mRNAs encoding male fate factors. (B) GLS-1 synergizes with GLD-3 for germline survival and may expand its functions by bridging to GLD-4 to translationally activate mRNAs encoding survival factors; presumably by poly(A) tail elongation. doi:10.1371/journal.pgen.1000494.g008 binding protein GLD-3 to actively promote the oocyte fate and oogenesis per se. The formation of a maternal GLS-1/GLD-3 complex is required to promote germline survival during postembryonic germline development. In the latter decision, GLS-1 putatively transforms GLD-3 into a translational activator most likely by recruiting additional factors, such as the cytoplasmic poly(A) polymerase GLD-4 [24]. GLS-1 Modulates GLD-3L to Enhance the Switch to the Oocyte Fate The sperm-to-oocyte decision is remarkably complex and serves as a paradigm for post-transcriptional control mechanisms. Translational repression of fem-3 mRNA is essential for female germ cells to acquire the oocyte fate. FEM-3 is a novel protein and forces male fate development [25,26]. Translationally inactive fem- 3 mRNA is repressed by two conserved PUF proteins (FBF-1 and FBF-2) and NOS-3, which are thought to form an RNP complex [13,14]. To allow transient spermatogenesis in the hermaphrodite and continuous sperm production in the male, fbf activity is antagonized by gld-3. GLD-3 regulates the timing of the hermaphroditic sperm-to-oocyte switch, i.e. the numbers of sperm produced, and maintains the sperm fate in the male [5]. Molecularly, GLD-3L promotes the sperm fate by directly contacting the RNA-binding domain of FBF. As a consequence, FBF’s affinity to its cognate target sequence in fem-3 mRNA is weakened; this promotes fem-3 mRNA translation and delays the onset of oogenesis [5]. How is FBF activated to overcome GLD-3L inhibition to execute the sperm-to-oocyte switch at the correct time? Further- more, how is this repression maintained in the aging hermaph- rodite for continuous oocyte production? Our work on GLS-1 provides support for a model in which FBF is permanently liberated from GLD-3L repression by the formation of a GLS-1/ GLD-3L complex (Figure 8A). This interaction requires a region within GLD-3L that includes the entire minimal FBF-binding site. As the GLD-3 interaction sites in FBF and GLS-1 differ in their primary sequence, we regard GLS-1 as a molecular mimic, and not necessarily a structural mimic, of FBF. Our molecular model is based on the following findings: GLS-1 is expressed in the L4 gonad (data not shown) when oocytes are specified. gls-1 hermaphrodite mutants produce almost twice as many sperm than wild-type, which is similar to fbf-1 single mutants [27]. A synergistic loss of gls-1 and fbf-1 prevents the switch to oogenesis entirely and these masculinized germ lines depend on gld-3 activity. Furthermore, our in vitro protein binding competition results are consistent with the conclusion that GLS-1 performs its function by directly interfering with the GLD-3L/FBF interaction. FBF-1 is the only RNA-binding protein that is known to be involved in all well characterized translational regulations that affect the sperm-to-oocyte switch (see Figure 5E). For example, fbf- 1, but not fbf-2, activity is required to repress fog-2, a gene more upstream in the sex determination pathway than fem-3. However, FBF-1 requires the help of PUF-8 to repress fog-2 mRNA [22]. Similarly, FBF-1 depends on FBF-2, and presumably NOS-3, to fully repress fem-3 mRNA [13,14]. Our data are also in good agreement with a more central role attributed to fbf-1, rather than fbf-2, nos-3 and puf-8, in the sperm-to-oocyte switch (see Figure 5E). gls-1; fbf-1 double mutants fail to produce oocytes and produce sperm only, whereas gls-1; fbf-2 and gls-1; puf-8 double mutants produce sperm and oocytes. Consistent with our model of FBF liberation from GLD-3L repression by GLS-1, we find that gls-1 also acts upstream of fog-2 and requires gld-3 activity. These findings suggest that FBF-1 regulation is tightly linked to GLS-1/ GLD-3 complex activity and that all FBF-1 spermatogenetic target mRNAs may require the assistance of GLS-1 for translational repression. In summary, it seems likely that FBF-1 and GLD-3 provide the core of the switch machinery and that GLS-1, NOS- 3/Nanos, and the other PUF proteins are accessory modulators for the sperm-to-oocyte switch. Yet, it remains an open question, how GLD-3L handover from FBF-1 to GLS-1 is regulated and executed in a developmental fashion. GLS-1 indirectly promotes the switch by releasing FBF to inhibit sperm production. Yet, it remains to be determined if GLD-3 sequestration from the GLD- 3L/FBF complex may also be a way to modulate the roles of GLD-3 activity, to directly promote oogenesis through the translational regulation of yet unknown oocyte-promoting mRNAs. This scenario seems likely given their common maternal activities. The Maternal GLS-1/GLD-3 Complex Is Critical for Germline Survival We find that a reduction of maternal gls-1 or gld-3 activity in the early embryo, results in a common phenotype, i.e. the germline survival defect. A hallmark of the Gls defect is that germ cells are correctly specified in the early larvae, proliferate until the L3 stage and degenerate rather than differentiate in later development. This form of germ cell death appears to a large extent independent of CED-3 or CED-4-mediated apoptosis [5, this work]. Although a more detailed pathological analysis of this phenomenon is missing, a molecular framework for how germline survival may be achieved PLoS Genetics | www.plosgenetics.org 12 May 2009 | Volume 5 | Issue 5 | e1000494