The GLP-1 (Notch) signaling pathway and FBF RNA-binding proteins are crucial regulators of germline mitoses and germline stem cell (GSC) proliferation. The FBF proteins are Puf translational repressors and homologs of Drosophila Pumilio. FBF is encoded by two nearly identical genes,
fbf-1 and
fbf-2 . An
fbf-1 fbf-2 double mutant is sterile and lacks GSC, but
fbf-1 or
fbf-2 single deletion mutants are similar to wild-type. Therefore, the nearly identical FBF-1 and FBF-2 proteins can promote mitosis interchangeably. Although most
fbf-1 and
fbf-2 single mutants are fertile, rare animals are sterile. Surprisingly, these rare sterile mutants have opposite defects:
fbf-1 single mutants can have a masculinized Mog germ line and make only sperm, and
fbf-2 single mutants can have a feminized Fog germ line and make only oocytes. Therefore,
fbf-1 and
fbf-2 have distinct, albeit subtle, roles in controlling germ line fates. While analyzing the individual characteristics of
fbf-1 and
fbf-2 , we uncovered two types of FBF control. First, FBF-1 protein increases in an
fbf-2 deletion mutant, and FBF-2 protein increases in an
fbf-1 deletion mutant. One simple model is that FBF directly controls its own expression. Indeed, FBF binding sites are present in both
fbf-1 and
fbf-2 3'UTRs, as assessed by both yeast three-hybrid and gel shift experiments. Therefore, in the wild-type germ line, FBF levels appear to be restricted by negative auto-regulation. We also noticed that the
fbf-2 5'flanking region possesses four consensus LAG-1 binding sites, while
fbf-1 has none. This suggested to us that
fbf-2 might be a direct target of GLP-1 signaling. We completed several experiments to test this idea. Gel shift assays demonstrate that predicted LAG-1 sites upstream of
fbf-2 can bind purified LAG-1. In contrast, an
fbf-1 region that differs by only a single base pair does not bind LAG-1. Both
fbf-2 mRNA and FBF-2 protein localize to the distal-most germ line, whereas
fbf-1 products are more broadly distributed. Moreover, the level of FBF-2, but not FBF-1, responds to changes in GLP-1 signaling. We attempted to generate multiple
fbf-2 transcriptional reporters without success. Nonetheless, our cumulative data supports the idea that GLP-1 signaling controls
fbf-2 transcription directly. We will propose a model in which GLP-1 signaling and cross-regulation between the fbf genes work together to establish the normal pattern of GSC proliferation and differentiation.