The sperm and egg, or the germ cells are the specialized cells, which can transmit the genetic materials from one generation to the next in sexual reproduction. All the other cells of the body are somatic cells. This separation of germ and somatic cells is one of the oldest problems in developmental biology. In many animal groups, a specialized portion of egg cytoplasm, or germ plasm, is inherited by the cell lineage which gives rise to germ cells. This cell lineage is called germline. The germline progenitors eventually migrate into the gonads, where they differentiate as germ cells when the organisms are physically matured. Earlier investigators have demonstrated that germ plasm contains maternal factors required and sufficient for germline formation. In the fruit fly, Drosophila, this cytoplasm is histologically marked by the presence of polar granules, which act as a repository for the maternal factor required for germline formation. Our molecular screens have identified several factors stored in the polar granules. One of the factors is mitochondrial large rRNA which functions to form the germline progenitors, or pole cells. The other is nanos mRNA, which is required for pole cell differentiation.

I. Role of Mitochondrial Ribosomal RNAs in Pole Cell Formation

Ultrastructural studies have shown that the germ plasm is basically composed of polar granules and mitochondria. While the primary roles of the mitochondria are oxidative phosphorylation and biosynthesis of many metabolites, it has now become evident that they are also involved in germline formation.

In Drosophila, pole cell formation requires the function of mitochondrial ribosomal RNA in germ plasm. We have previously reported that mitochondrial large rRNA (mtlrRNA) and small rRNA (mtsrRNA) are both transported from mitochondria to polar granules. This transportation occurs during early embryogenesis, when mitochondria are tightly associated with polar granules in germ plasm, and it depends on the function of the maternally-acting gene, tudor, that is known to be required for pole cell formation. Mitochondrial rRNAs remain on the polar granules until pole cell formation and are no longer discernible on the granules within pole cells. Reduction of the extra-mitochondrial mtlrRNA amount results in the failure to form pole cells and injection of mtlrRNA is able to induce pole cells in embryos whose ability to form these cells has been abolished by uv-irradiation. These observations clearly show that the extra-mitochondrial mtlrRNA on polar granules has an essential role in pole cell formation, presumably cooperating with mtsrRNA.

Recently, we found that mitochondrial rRNAs form mitochondrial-type of ribosomes on polar granules, cooperating with mitochondrial ribosomal proteins. This suggests the possibility that the protein (s) essential for pole cell formation is produced by the mitochondrial-type of ribosomes. To address this issue, we examined the effect of Chloramphenicol and Kasugamycin on pole cell formation. Chloramphenicol and Kasugamycin are known to inhibit mitochondrial (prokaryotic)-type of translation. When these antibiotics were injected into the posterior pole region of early embryos, pole cell formation was severely affected. In contrast, Chloramphenicol and Kasugamycin treatment did not affect somatic cell formation at a dose we used. These observations strongly suggest that the mitochondrial-type of translation system must be intact for the embryos to form pole cells. The project to identify a targer mRNA for the mitochondrial-type of translation machinery is now on going.

Fig. 1 Role of mitochondrial rRNAs in pole cell formation

II. Role of Nanos protein in pole cell differentiation

Pole cells differ from the soma in regulation of mitosis and transcriptional activity. Pole cells cease mitosis at gastrulation and remain quiescent in the G2 phase of the cell cycle throughout their migration to the gonads, while somatic cells continue to proliferate during the rest of embryogenesis. Furthermore, pole cells are transcriptionally quiescent until the onset of gastrulation, although transcription is initiated in the soma during the syncytial blastoderm stage. Consistent with this, RNA polymerase II (RNAP II), but not RNA polymerase I, remains inactive in early pole cells. Thus, the ability to express zygotic mRNA-encoding genes is suppressed only in pole cells in early embryos.

Among the maternal components of germ plasm, Nanos (Nos) is essential for the germline-specific events occurring in pole cells. nos mRNA is localized in the germ plasm during oogenesis, and is translated in situ to produce Nos protein after fertilization. Nos is only transiently present in the posterior half of embryos during the preblastoderm stage, and is required there for posterior somatic patterning. Nos in the germ plasm is more stably inherited into the pole cells at the blastoderm stage, remaining detectable in these cells throughout embryogenesis. Pole cells that lack Nos (nos^- pole cells) are unable to follow normal germline development; they fail to migrate properly into the embryonic gonads, and consequently do not become functional germ cells. In nos^- pole cells, mitotic arrest at G2 phase is impaired, and they undergo premature mitosis. Furthermore, nos- pole cells fail to establish and/or maintain transcriptional quiescence, and ectopically express somatically-transcribed genes, including fushi tarazu (ftz), even-skipped (eve) and Sex-lethal (Sxl).

Nos represses translation of mRNAs with discrete RNA sequences called Nos response elements (NREs). In the pathway leading to posterior somatic patterning, Nos acts together with unlocalized Pumilio (Pum) protein to repress translation of maternal hunchback (hb) mRNA. This translational repression is mediated by binding of Pum to NREs in the 3'-untranslated region (UTR) of hb mRNA. In pole cells, Nos also acts with Pum to regulate germline-specific events. Pum, like Nos, is required in pole cells for their migration to the gonads.

Fig. 2 Somatic differentiation of pole cells lacking Nos

We found that Nos, along with Pum, represses translation of importin a2 (impa2) mRNA in early pole cells. The impa2 mRNA contains an NRE-like sequence in its 3'-UTR and encodes a Drosophila importin a homologue that plays a role in nuclear import of karyophilic proteins. We found that Nos inhibits expression of a somatically-transcribed gene, ftz, in pole cells by repressing Impa2-dependent nuclear import of a transcriptional activator for ftz, Ftz-F1. Furthermore, the expression of another somatic gene, eve, and RNA Polymerase II activity are also repressed by Nos in pole cells through its effects on Impa2-dependent nuclear import.

The above results raise the question whether the pole cells lacking Nos (nos^- pole cells) are able to differentiate into somatic cells. However, it is difficult to study their developmental fate, since Nos also represses apoptosis of pole cells, and almost all of nos^- pole cells are eliminated until at least the end of embryogenesis. To overcome this problem, we used Df (3L) H99, a deletion for three genes required for apoptosis. Introduction of the H99 deficiency results in nos- pole cells being escaped from apoptosis. We transplanted the nos^- H99^- pole cells into normal embryos and observed their behavior, and found that some of nos^- H99^- pole cells were able to differentiate as somatic cells. This suggests that pole cells have the ability to differentiate as somatic cells, but its ability is inhibited by Nanos activity. Recently, we have found that somatic differentiation of nos^- H99^- pole cells requires Impa-2 activity, suggesting that Nos inhibits somatic differentiation by repressing Impa-2 production.

Fig. 3 Transcriptome analysis of the embryonic gonads

III. Comprehensive analysis of genes expressed in Drosophila gonad

Pole cells migrating into the gonads are specified to be the primordial germ cells (PGCs). It has been believed that zygotic genes expressed in pole cells within the gonads are required for their fate specification. To explore the regulatory mechanism of germline specification, we attempted to identify genes expressed in pole cells and/or in somatic cells within the gonad by a comprehensive approach. From the embryos carrying EGFP-vasa transgene that express GFP only in pole cells, we isolated the gonads by using fluorescence-activated cell sorting (FACS), and costructed a gonad cDNA library. Each cDNA clone was sequenced from both 5’ and 3’ ends, and these Expression Sequence Tags (ESTs) were computationally condensed into sequence clusters, which were then subjected to whole-mount in situ hybridization (WISH). Approximately 20,000 of ESTs were generated, and were clustered into 2900 distinct genes. The WISH analysis identified more than 130 genes that were expressed predominantly in the gonads. In addition, we found gonad-specific splicing form in some transcripts. These transcriptome data will allow us to illustrate genetic networks governing the germline specification.
We also started to identify genes expressed predominantly in the embryonic gonads, using DNA microarray technique.

Fig. 4 Scatter plot of expressin levels in control (whole embryos) and the embryonic gonads.

IV. sva53, a Maternal Gene Required for Meiosis

It has been believed that maternal factors localized in germ plasm may ultimately trigger germline-specific events, such as meiosis. We have isolated an X-linked maternal mutation, sva53 that affects meiosis. Pole cells that were formed in the embryos derived from sva53 homozygous germline clone (sva53 pole cells) were able to develop into the oocytes, but they failed to execute meiosis. We also found that the germline-specific expression of vasa gene was severely affected in sva53 pole cells. These results suggest that the maternal factor encoded by sva53 gene may activate gene expression, which is essential for meiosis. In order to identify sva53 gene, we mapped sva53 mutation to 200 kb-genomic region of 11C by using duplications and deficiencies. Within this region, we found a gene encoding a Zn-finger transcription factor, of which mRNA is maternally supplied into embryos.

Publication List:

Tsuda, M., Sasaoka, Y., Kiso, M., Abe, K., Haraguchi, S., Kobayashi, S. and Saga, Y. (2003) Conserved role of nanos proteins in germ cell development. Science 301, 1239-1241.