Program of the 2001 International Worm Meeting - Sternberg Lab ...

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17. ego-1, a link between germline
development and RNAi
Eleanor M. Maine, Valarie E.
Vought, Jamie Wasilenko.
Dept. of Biology, Syracuse University,
Syracuse, NY 13244
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2000; E.M. and V.V., unpublished).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unpublished).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
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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