The adult Caenorhabditis elegans gonad contains proliferating and differentiating germ cells arrayed in a linear fashion from mitotic germ cells at the distal end to mature gametes at the proximal end. Germline development requires the coordinate regulation of diverse events such as cell proliferation, sex determination, meiotic progression, and gamete formation.
ego-1 was originally identified in genetic screens for enhancers of
glp-1 (Qiao et al., 1995). GLP-1 is a Notch-type receptor that mediates cell-cell interactions during somatic and germline development; in the germ line, it mediates a proliferative signal from the somatic distal tip cells to the distal germ cells. Based on genetic studies,
ego-1 gene activity promotes GLP-1-mediated signaling as well as later germline events such as meiotic progression and gametogenesis.
ego-1 mutants are sterile with defects at various developmental stages. In
ego-1 mutants, germ cells enter meiosis prematurely, consistent with a decrease in GLP-1-mediated signaling; some mitotic germ cells arrest, suggesting a mitotic progression defect; early meiotic prophase occurs slowly and oocytes contain desynapsed chromosome pairs, as if chromosome pairing/ recombination is abnormal; and oocytes are small and fail to take up yolk (Qiao et al., 1995; Smardon et al., 2000; E.M. and V.V., unpublished).
ego-1 encodes a member of the RdRP (RNA-directed RNA polymerase) family (Smardon et al., 2000) that includes Neurospora QDE-1 and Arabidopsis SDE-1/SGS-2. In addition to their developmental defects,
ego-1 mutants are defective in RNAi for a large proportion of germline-expressed genes (Smardon et al., 2000; E.M. and V. V., unpublished). Similarly, mutations in
qde-1 and
sde-1/sgs-2 disrupt some forms of RNA silencing, suggesting that RdRP-like proteins play a role in RNA silencing. We have found that expression of an integrated
pie-1:gfp:histone transgene (a kind gift of G. Seydoux and J. Austin) is resistant to RNAi in the
ego-1(-) background. Normally, expression of the
pie-1:gfp:histone transgene produces high GFP levels in the proximal germ line. When fed gfp dsRNA (using the Timmons and Fire L4440 vector/HT115 E. coli system), N2 and
ego-1(-/+) animals lose the GFP fluorescence within 24 hr whereas
ego-1(-) animals retain strong GFP fluorescence after three days. We used this construct to assay the RNAi response in
ego-1(-) progeny of
ego-1(+/-) mothers, and find that they express GFP at low levels. Therefore, the RNAi defect seems to be partially rescued by expression of maternal
ego-1(+) . We obtained evidence of weak maternal rescue using other germline-expressed genes, as well. These results suggest that the dose of functional EGO-1 protein is critical for a robust RNAi response. We are investigating the biological and biochemical role of EGO-1 using various approaches, as will be discussed. To study EGO-1 distribution in the germ line and the effects of EGO-1 overexpression, we have generated
ego-1:gfp fusion constructs and are collaborating with J. Austin (U Chicago) to generate integrated transgenes (Praitis et al., 2001). Intriguingly, based on anti-GLD-1 (Jones et al., 1996) and anti-PGL-1 (Kawasaki et al., 1998) staining, we find that P granule composition and distribution are altered in the
ego-1 germ line. To date, all known P granule-associated proteins are RNA binding proteins. We are collaborating with C. Cameron (Penn State) to test whether EGO-1 has RdRP activity and/or binds RNA in vitro . We have also begun using the yeast two-hybrid system to screen for EGO-1 interacting proteins. One possibility consistent with our current data is that EGO-1 activity promotes diverse processes in the germ line to ensure the formation of functional gametes