84
Rapid evolution of the sex determination gene fog-2. Sudhir B. Nayak, Tim Schedl. Genetics, Washington University, St. Louis, MO.
Metazoan sex determination genes evolve rapidly. One such rapidly evolving gene, fog-2, is required for C. elegans hermaphrodite spermatogenesis.
The fog-2 gene is part of a large and highly diverged family sharing a similar structure; an N-terminal F-box domain and Cterminal
Duf38/FTH domain. In the C. elegans germline sex determination pathway, FOG-2 and GLD-1 form a ternary complex with the
tra-2 mRNA 3UTR to promote the male germ cell fate in the hermaphrodite allowing self-fertility. Here we present data that fog-2 represents
one of the most rapidly evolving C. elegans sex determination genes identified to date. The fog-2 family (F-box + Duf38/FTH) is
present in C. briggsae and also appears to be expanded, however, no clear orthologous relationships between fog-2 family members could
be unambiguously identified between C. briggsae and C. elegans using reciprocal BLAST, unlike the other sex determination genes. Analysis
of syntenic relationships surrounding fog-2 revealed a breakdown in local synteny and an absence fog-2 and duplicated family members
in the corresponding C. briggsae sequence. To resolve the evolutionary relationships between C. elegans and C. briggsase fog-2
family members we performed phylogenetic analysis using neighbor-joining, maximum parsimony, and maximum-likelihood methods. All
trees were in good agreement showing a separation between C. elegans and C. briggsae lineages. This suggests two possibilities about
the etiology of the fog-2 family. One, expansion of the fog-2 family occurred before the species split and was followed by rapid divergence
and presumably the acquisition of new roles such as fog-2 in C. elegans sex determination. Alternatively, while there was clearly a common
ancestral sequence, the majority of the expansion in the fog-2 family occurred after the species split suggesting that the germline sex
determination function of fog-2 is recently evolved. Providing support for the latter is that sequence analysis from C. elegans suggests that
duplications involving fog-2 occurred very recently. Additionally, using the yeast two-hybrid system we have identified unique sequences in the
final exon of fog-2, absent from the highly similar neighboring gene ftr-2, that are required for GLD-1 interaction and thus incorporation into the
tra-2 3UTR mRNA translational repression complex. Our data suggest that establishment of hermaphrodite spermatogenesis may fundamentally
different between C. elegans and C. briggsae. The fog-2 gene family may represent molecular evidence that, similar to parasitism, the
evolution of hermaphroditic species from ancestral gonochoristic species has arisen independently even in closely related lineages.

167
The Control of GLD-1 Accumulation and Meiotic Entry. David D. Hansen, Laura Berry, Thang Dang, Tim Schedl. Dept Genetics, Washington
Univ Sch Medicine, St Louis, MO.
The distal end of the germline consists of a population of proliferative germ cells that switch from proliferation to meiotic development as
they move proximally. The GLP-1/Notch signaling pathway regulates this switch. Previous work has shown that gld-1 is also involved in
regulating meiotic entry based on gld-1(0) partial suppression of glp-1(0) premature entry into meiosis phenotype, as well as the excess
proliferation (germline tumor) that occurs in the gld-2(0) gld-1(0) double mutant. Genetic epistasis places gld-1 and gld-2 downstream of
GLP-1/Notch signaling. We are interested in identifying additional genes involved in regulating the entry into meiosis decision, as well as
determining the regulatory relationships between known and newly identified factors. GLD-1 protein levels are very low at the most distal
end and increase proximally until reaching maximum levels approximately 20 cell diameters from the distal end, which is the approximate
location where cells first show evidence of meiotic entry. We show that the GLP-1/Notch signaling pathway is responsible for repressing
GLD-1 accumulation as GLD-1 levels are increased when signaling is eliminated. This regulation is likely not transcriptional as gld-1 RNA
in situs show roughly equivalent levels in glp-1(+) and glp-1(-) animals. We further show that ectopically increasing GLD-1 accumulation in
the distal end causes germ cells to enter meiosis earlier. These data suggest that a major mechanism by which the GLP-1/Notch signaling
pathway controls the proliferation versus meiotic development decision is by regulating the spatial pattern of GLD-1 accumulation. In an
attempt to identify other factors necessary for control of meiotic entry, we conducted genetic screens and identified mutations in the nos-3
gene, which is homologous to Drosophila Nanos. Interestingly, FBF-1 and FBF-2, two nearly identical proteins that are homologous to
Drosophila Pumilio, have also been implicated in regulation of meiotic entry by inhibiting distal GLD-1 accumulation. We show that nos-3
also affects GLD-1 accumulation, however unlike FBF, nos-3 promotes GLD-1 accumulation, a function that is redundant with gld-2. In a
gld-2; nos-3 double mutant, GLD-1 accumulation is markedly reduced. Furthermore, nos-3 suppresses the premature entry into meiosis
and high distal GLD-1 accumulation phenotypes of fbf-1 fbf-2 mutants. One model to explain these results is the GLP-1/Notch signaling
represses the activities of GLD-2 and NOS-3 in the distal end to keep GLD-1 accumulation low and allow for the stem cell population to be
maintained.

299
GLD-1 and Translational Regulation. Min-Ho Lee, Tim Schedl. Genetics, Washington University School of Medicine, St. Louis, MO 63110.
During germ line development, translational control emerges as a heavily utilized mechanism by which gene expression is regulated.
The germ line thus provides an excellent model for studying translational regulation. Recently, many RNA binding proteins have been identified
as essential factors governing germ line development and early embryogenesis. A comprehensive understanding of how these RNA
binding proteins control development remains largely unknown. GLD-1 is a germ line specific, maxi-KH motif containing RNA binding protein
that acts as a tumor suppressor and regulates multiple aspects of C. elegans germ cell development, suggesting that it regulates multiple
RNAs. The tumorous mutant phenotype, therefore, could result from mis-regulation of mRNA targets during early meiotic prophase.
GLD-1 has homologs in Drosohpila (How) and mouse/human (Quaking), therefore conserved throughout evolution. To understand how
GLD-1 is regulating mRNA targets to control germ cell development, we have identified multiple in vivo mRNA targets of GLD-1 by their
ability to interact with GLD-1 in cytosol extracts. These target mRNAs are preferentially expressed in the germline and as expected, several
of them have essential functions in oocyte differentiation and early embryogenesis. We also found that GLD-1 functions as a translational
repressor. The expression patterns of four mRNA targets (rme-2, puf-5, oma-1 and oma-2) at the level of protein as well as mRNA
are very similar to each other and quite consistent with GLD-1 acting as a translational repressor. We were also able to narrow down GLD-
1 binding sites on several targets. They are located at the 5-end, 3-end, or both ends depending on the mRNA target. In a few cases, GLD-
1 also can bind to ORF. Currently, we do not know the importance of either the position of GLD-1 binding sites or the number of GLD-1
binding sites. Initial characterization suggests that GLD-1 is likely to repress translation prior to ribosome assembly or soon after ribosome
elongation. We found that GLD-1 binds and protects T23G11.2 mRNA from Nonsense Mediated mRNA Decay (NMD), presumably by
binding and repressing translation. T23G11.2 mRNA has two small upstream open reading frames in 5-UTR that likely target the mRNA
for NMD. Interestingly, when GLD-1 mRNA targets acquire pre-mature stop codons by nonsense mutations, GLD-1 also can protect them
from NMD. For NMD to take place, the ribosome has to proceed to the pre-mature stop codon then recruit factors to degrade the mRNA.
Analysis of several mRNA targets that contain nonsense mutations suggests that, if bound to ribosome, then the ribosome cannot have
elongated to the pre-mature stop codon to trigger NMD during GLD-1 repression.

1048A
Genetic Studies of the Proliferation versus Meiotic Development Decision. Jessica Amrozowicz, Dave Hansen, Laura Berry, Daimon Simmons,
Tim Schedl. Genetics, Washington University, Saint Louis, MO.
GLP-1 is a member of the Notch family of transmembrane receptors and functions to promote germ cell proliferation in C. elegans. Activation
of the GLP-1 receptor, expressed in the germline, is spatially regulated by the transmembrane ligand, LAG-2, expressed in the distal
tip cell. Binding of LAG-2 to GLP-1 induces receptor cleavage generating GLP-1(INTRA), which is then presumed to translocate to the
nucleus, bind LAG-1, and alter the pattern of transcription. Disruption of this pathway causes all proliferating germ cells to prematurely
enter meiosis. Conversely, constitutive activation by the glp-1(oz112) gain-of-function (gf) mutation results in the formation of a germline
tumor where proliferative cells are found throughout the gonad. Two genes, gld-1 and gld-2, function redundantly downstream of GLP-1 to
inhibit proliferation and/or promote meiotic development. glp-1 signaling, in effect, inhibits both genes to promote germ cell proliferation.
We have taken a forward genetic approach to identify genes that either function downstream of GLP-1 to promote entry of germ cells into
meiosis, or that function to negatively regulate GLP-1 signaling. A number of screens have been conducted in sensitized backgrounds in
order to identify mutations that confer a synthetic tumorous (Syt) phenotype. One of the first screens utilized a weak glp-1(oz112oz120 gf)
background and identified mutations in the genes teg-1 and teg-4. gld-2; teg-1 and gld-2teg-4 are also tumorous. Interestingly, in contrast
to the gld-2gld-1; glp-1(null) triple mutant, which is tumorous, gld-2; glp-1teg-1 and gld-2teg-4; glp-1 triple mutants display the Glp premature
meiotic entry phenotype. The gld-1 pathway gene, nos-3, was used in another screen to identify genes that function in the gld-2 pathway.
13 Syt mutants were identified: 8 gld-2 alleles, 4 gld-3 alleles, and one weak glp-1(gf) allele, oz272. Another screen using a teg-1
mutant background identified two weak glp-1(gf) mutants, oz264 and oz274; molecular lesions are located in the second and third LNG
repeats, regions where other glp-1/lin-12(gf) mutations have been localized. Finally, another screen utilized the glp-1(oz264) mutant background,
which identified one Syt mutant, oz273. It appears to be associated with a Mog (masculinization of the germline) phenotype in the
glp-1(+) background. The mutation maps to LG1, suggesting that it may be a gld-1 allele, but complementation tests with gld-1(null) suggest
otherwise. Further mapping and sequencing analysis need to be conducted to identify this gene.

1053C
An update on ego-1, ego-2, and ego-3. Ying Liu1, Valarie Vought1, Xiang Yu1, Deborah Swenton1, David Hansen2, Elyse Connors1, Tim
Schedl2, Eleanor Maine1. 1) Department of Biology, Syracuse University, Syracuse, NY; 2) Department of Genetics, Washington University
Medical School, St. Louis, MO.
GLP-1 is a Notch-type receptor that mediates a proliferative signal from the somatic distal tip cells to the germ line. In the absence of
GLP-1 signaling, germ cells exit mitosis, enter meiosis, and differentiate. ego mutations were recovered as recessive enhancers of a mild
glp-1 defect in the germ line (Qiao et al. 1995). These screens were designed to identify components, regulators, and targets of the GLP-
1-mediated signaling pathway. We have previously characterized the mutant phenotype of these genes (Qiao et al. 1995; Smardon et al.
2000) and are now pursuing molecular studies.
ego-1 gene functions in germline development and RNA interference (RNAi) (Smardon et al. 2000). EGO-1 protein is a member of the
RNA-dependent RNA polymerase (RdRP) family whose members are widely implicated in post-translational gene silencing phenomena.
We are using several approaches to characterize the role of EGO-1 in germline development. Some of our results implicate EGO-1 in RNA
metabolism, consistent with its classification as a RdRP family member. To better understand EGO-1 function, we are describing its
expression pattern, screening for and analyzing molecular interactors, and testing candidate genetic interactors.
ego-3 gene function is required germline development and may be important for the onset of meiosis. Based on mapping with visible
genetic markers and single nucleotide polymorphisms (SNPs), we have localized ego-3 to a small region of chromosome V that is predicted
to encode ten genes. However, we have not been able to determine which gene is ego-3 based on existing mutations and RNAi.
Therefore, ego-3 may have unusual features that prevent it from being identified by the standard annotation programs, e.g., it may encode
a regulatory RNA rather than a protein. We are now refining the SNP map position and testing for the presence of another gene in the
region that might correspond to ego-3.
Mutations in ego-2 enhance glp-1(ts) in the embryo as well as the germ line. We have localized it to an ~130 kb region using SNP mapping.
Once we better define the region, we will test candidate genes using RNAi.

1087A
MPK-1 ERK signaling Is necessary for male germline sex determination. Mistue Ohmachui1, Min-Ho Lee1, Eric Lambie2, Ross Francis1,
Tim Schedl1. 1) Department of genetics, Washington University School of Medicine, St.Louis, MO; 2) Department of Biological Science,
Dartmouth College, Hanover, NH.
MPK-1 ERK signaling is necessary for pachytene progression and oocyte maturation (Church et al., 1994; Lee et al., 13th International
C. elegans Meeting). Strong loss-of-function(lf)/null mutations in lin-45 RAF, mek-2 MAPKK and mpk-1 ERK result in germ cells arrested in
pachytene. These pachytene arrested nuclei and surrounding plasma membranes are found in clumps, often in the center of the gonadal
tube. We call this a Pac phenotype, for pachytene arrest and clumped nuclei and membranes. A similar Pac phenotype is observed in
mutant males. Under certain partial lf conditions, where pachytene progression and oocyte differentiation is normal, we observed hermaphrodites
with feminized germlines (proximal germ cells developing as oocytes instead of sperm). These results raise the possibility
that the Pac phenotype observed in lin-45, mek-2 and mpk-1 mutants is the result of the combined effect of a sexual fate transformation followed
by pachytene arrest during oogenesis. In situ hybridization using a probe to rme-2 mRNA, which encodes the oogenesis specific
yolk receptor, was used to test this possibility. In wild-type L4 hermaphrodites, germ cells in the proximal gonad, most of which are in
pachytene, are undergoing spermatogenesis and lack rme-2 mRNA while germ cells in the proximal third of the distal gonad contain rme-
2 mRNA. By contrast, germ cells throughout the proximal gonad and into the distal gonad have rme-2 mRNA in L4 mpk-1 null hermaphrodites.
In mpk-1 null males, rme-2 mRNA is found throughout the region showing the Pac phenotype, unlike in wild-type males. Thus, mpk-
1 is required for the male germ cell fate in both hermaphrodites and males. Epistasis experiments indicate that mpk-1 is acting downstream
or in parallel to tra-2 in the sex determination pathway. Accumulation of MPK-1 and the appearance of activated MPK-1 is sexually
dimorphic. MPK-1 is found at high levels throughout the germline of wild-type adult hermaphrodites. By contrast, in wild-type males, MPK-
1 is found at lower levels and only in the distal most germ cells, up to about the distal third of the pachytene region. Activated MPK-1 is
found at high levels in the 2 to 3 most proximal oocytes and at moderate levels in the proximal half of pachytene in hermaphrodites. In
males, activated MPK-1 is only found at very low levels in the transition zone and the first few pachytene germ cells, a region where activated
MPK-1 is not observed in wild-type adult hermaphrodites. Thus, MPK-1 is activated sex specifically and to different extents in three
spatially distinct regions of the C. elegans germline.