The repression of pluripotency genes in ovarian germ cells around the time of meiotic initiation is a significant aspect of their development, and yet very little is known about this event. Dazl
(Deleted in azoospermia-like
) has been shown to be required for primordial germ cells to differentiate following their migration to the gonads. Subsequent downstream events, including down-regulation of pluripotency markers, are disrupted in the absence of Dazl
. Because Dazl
controls a broad differentiation event several days before pluripotency marker repression, this is likely an indirect effect of Dazl
function. We set out to determine what molecular regulators might mediate Dazl
's effects in the repression of Oct4
represented a good candidate for this role because it is a known direct repressor of Oct4
in somatic cells, both in vivo
and in vitro
. Here we showed that Gcnf
is expressed in ovarian germ cells at about the time that Oct4
are down-regulated in these cells. However, when we conditionally disrupted the LBD of Gcnf
in the fetus, we found that, despite its role in Oct4
repression during gastrulation, Gcnf
is not required for the regulation of Oct4
in ovarian germ cells. In addition, Gcnf
appears to be dispensable for the initiation of meiosis and oogenesis in fetal ovarian germ cells.
We have considered and ruled out several alternative explanations for our findings. One formal possibility is that ablation of Gcnf function was inefficient with our conditional allele, but the available evidence does not support this explanation. Each embryo provided a pair of ovaries, of which one ovary was used for RNA extraction and RT-PCR, while the other was processed for paraffin sectioning and immunostaining. Quantitative RT-PCR on individual mutant ovaries showed an average of less than 1.5% of exon 7 transcript remaining, indicating that exon 7 was deleted in the vast majority of germ cells.
We demonstrated that, in wild-type embryos, Gcnf
transcript is expressed in germ cells and somatic cells at E9.5, becomes nearly undetectable in both tissue types at E10.5, and is expressed specifically in germ cells at E14.5. Because we induced the exon 7 deletion at E10.5, it is theoretically possible that small amounts of intact GCNF protein expressed at E9.5 persisted in germ cells until E14.5, when Oct4
are down-regulated. However, the germ cell population expands from about 1,000 cells at E10.5 to more than 25,000 cells at E13.5 
, which would have severely diluted any remaining GCNF protein. Therefore, it seems unlikely that enough GCNF protein would remain in every germ cell to effectively carry out its function.
Another possibility is that the DNA binding domain (DBD) is translated and functional in the absence of the ligand-binding domain (LBD). Given the modular structure of nuclear receptors, including GCNF, it is quite possible that the N-terminal portion of the protein, including the DBD, is produced and properly folded. Unfortunately, we were unable to examine whether the DBD was present in ovarian germ cells due to the lack of appropriate antibodies. However, GCNF has been shown to interact with SMRT and NCoR, two well-characterized nuclear co-repressors 
. Both of these co-repressors interact specifically with the LBD of GCNF, which is typical of their interactions with other nuclear receptors as well. In addition, we demonstrated that the LBD is required for GCNF function earlier in somatic development, and that in its absence mutants display a phenotype remarkably similar to Gcnf
mutants lacking the DBD. Both mutants demonstrate a failure to turn from the lordotic to fetal position, a failure to completely close the neural tube, and truncated somitogenesis, as well as highly elevated Oct4
expression at E9.5 
. Although some of these defects can be observed in other mutants, taken together they suggest that disruption of the LBD does in fact abrogate GCNF function and that the LBD is required during Gcnf's
role in earlier development, when Gcnf
is thought to be required for posterior patterning 
. Therefore, although we cannot formally rule out the possibility, it is unlikely that the DBD represses Oct4
in the absence of the LBD during female germ cell development.
A more plausible explanation for our findings is that Oct4
are regulated by different enhancers in the germline and the gastrulating embryonic soma. Indeed, there is published evidence of such enhancer-controlled tissue specificity in the case of Oct4
. Yeom and colleagues demonstrated that there are two functionally separable enhancer elements present in the Oct4
promoter, one of which is more proximal to and the other more distal to the transcriptional start site 
. The proximal enhancer (PE) drives expression of Oct4
in the epiblast and epiblast-derived cells such as embryonal carcinoma cells. The PE is required for expression of Oct4
in the cells of the epiblast around the time of gastrulation. However, the distal enhancer (DE) is sufficient for expression of Oct4
in the pre-implantation embryo as well as in the germline, even in the absence of the PE.
It is unclear how these enhancer elements are differentially regulated, but it seems likely that different transcription factors bind to the two enhancers. GCNF is thought to perform its repressive function by competing with activating nuclear receptors for binding sites in promoters, and it has been shown to bind to response elements in the proximal promoter of Oct4
near, though not within, the PE 
. It is possible that, during gastrulation, GCNF's proximity to the PE disrupts the activity of transcriptional activators that normally bind there, resulting in the GCNF-dependent repression of Oct4
observed in gastrulating embryos and ES cells 
. In germ cells, on the other hand, GCNF may bind to the proximal promoter but may not be sufficient to repress Oct4
due to its distance from the DE. In this case, other factors, perhaps binding in or near the DE, would be responsible for repressing Oct4
in the germline.
Our experiments indicate that, although the LBD of Gcnf is required for repression of Oct4 and Nanog in somatic cells, it is not required for repression of these pluripotency factors in ovarian germ cells. The finding that pluripotency genes are differentially regulated in somatic and germ cells is intriguing, and it will be interesting to understand what factors contribute to this disparity. It may be due to differential regulation of enhancer elements in the promoters of these genes in somatic versus germ cells. Additionally, we cannot rule out the possibility that other factors act redundantly or in conjunction with Gcnf in this context. Finally, it will be important to determine what factors mediate Dazl's effects in the repression of pluripotency factors in fetal ovarian germ cells, and whether this repression of pluripotency factors is required for the initiation of meiosis and oogenesis.