GLS-1 and Germline Development Figure 7. Germline Survival Depends on a GLS-1/GLD-3 Complex. (A) Schematic diagram of GLD-3 domains and the position of the gld- 3(ax562ts) missense mutation. (B) Immunoblot for both GLD-3 isoforms. The gld-3(q730) allele is protein null. (C–D) Adult gld-3(ax562ts) hermaphrodites grown at restrictive temperature after being shifted during early embryogenesis (,100 cell stage). (C) Nomarski image. (D) whole mount DAPI image. No germ cells are visible in the gonadal tissue. spth, spermatheca. Arrows, sheath cell nuclei. Arrowhead, distal end. Caret, vulva. (E) GLD-3ax562 missense mutation impairs GLS-1/GLD-3L complex formation. Proteins co-expressed in insect cells. Wild-type(wt) or ax562(ax) MBP::GLD-3L isoforms are precipitated by an anti-GLD-3 N-terminal antibody whose epitope is outside the interaction domains. Asterisk, high molecular weight background band. doi:10.1371/journal.pgen.1000494.g007 (Figure 7E). A C-terminal truncation of GLS-1 missing the GLD- 3-binding region served as a negative control. These results were similar to our directed yeast 2-hybrid binding-tests (not shown), and do not depend on the temperature that cells were grown at (see Text S1). Interestingly, GLD-3ax562 bound efficiently to the positive control, GLD-2, in insect cells and yeast (not shown). We conclude that the gld-3(ax562) point mutation specifically abro- gates the interaction with GLS-1. Taken together, our analysis suggests that the maternal GLS-1/GLD-3ax562 complex is in vivo temperature sensitive and that upon dissociation of the complex, germ cells fail to survive into adulthood. Discussion In this study we addressed how the conserved RNA regulatory core machinery, i.e. GLD-3/Bic-C, FBF/Pumilio and NOS-3/ Nanos, that executes cell fate decisions, is regulated to implement organism-specific developmental germline programs. Our analysis focuses on the isolation and characterization of the novel P granule component, GLS-1, a protein important for the sperm-to-oocyte switch, proper oogenesis, embryogenesis and germline survival. We find that GLS-1 may directly manipulate the output of the RNA regulatory network by modulating GLD-3 availability and activity. Our data provides a molecular framework for how a single molecule may have evolved as a novel germplasm component to integrate germ cell viability with germ cell differentiation. The formation of a GLS-1/GLD-3 protein complex is redeployed multiple times throughout germline development (Figure 8). The formation of the zygotic GLS-1/GLD-3 complex promotes the oogenic cell fate by liberating the translational repressor FBF/Pumilio to turn off sperm promoting factors. In this scenario, GLS-1 acts as a molecular mimic of FBF/Pumilio. Possibly, this interaction may also redirect the putative RNA- PLoS Genetics | www.plosgenetics.org 11 May 2009 | Volume 5 | Issue 5 | e1000494