DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION I

¹⁾ Graduate School of Biological Sciences, University of Tsukuba

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 others are nanos mRNA and Pgc RNA, which are both 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.

Since both mtlrRNA and mtsrRNA are major components of ribosomes within mitochondria, we speculated that these rRNAs function to form ribosomes on the polar granules. We reported that mtlrRNA and mtsrRNA are both localized in the polysomes formed on the surface of polar granules during a short period (embryonic stage 2) prior to pole cell formation (Fig. 1). Furthermore, mitochondrial ribosomal proteins (S12 and L7/L12) are enriched in germ plasm (Fig. 2). They are present in the polysomes on the polar granules as well as in mitochondria. In the polysomes around polar granules are there smaller ribosomes, of which size is almost identical to that of mitochondrial ribosomes but is smaller than that of cytosolic ones. We conclude that mitochondrial rRNAs form mitochondrial-type of ribosomes on polar granules, cooperating with mitochondrial ribosomal proteins.

Fig. 1. Presence of mitochondrial rRNAs and ribosomal proteins in the polar granule polysomes at stage 2. (A) An electron micrograph showing well developed polysomes (arrow) on the surface of polar granules. (B and C) Electron micrographs of sections hybridized with probes for mtlrRNA (B) and mtsrRNA (C) (arrowheads). (Scale bar=0.25 mm.) (D-G) Distribution of S12-EGFP (D), L7/L12-EGFP (E), L7/L12-HA (F). Signals were detected at the periphery of polar granules (arrows). mt, mitochondria; pg, polar granules. Scale bar= 0.2 mm.

Fig. 2. Distribution of mitochondrial ribosomal proteins in oocytes and early embryos. (A-H) Developmental changes in the distribution of S12-EGFP (A-D) and L7/L12-EGFP (E-H) detected by EGFP fluorescence. (Aand E) Mature oocytes, (B and F) stage-1 embryos, (C and G) stage-2 embryos, and (Dand H) stage-3 embryos. (I-L) Immunohistochemical detection of the Gal4-induced S12-EGFP (I) and L7/L12-EGFP (J) by an anti-GFP antibody and L7/L12-HA by an anti-HA antibody (K). (L) A wild-type embryo stained with anti-S12 antibody. Scale bar= 50 mm.

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 nospole 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 and their mitotic quiescence. We have reported that a regulatory target for Nos-dependent translational repression in pole cells is maternal cyclin B mRNA which contains an NRE-like sequence within its 3'-UTR. Nos, cooperating with Pum, inhibits mitosis of pole cells by repressing translation of maternal cyclin BmRNA. In contrast, pole cell migration and gene expression in pole cells are independent of the translational repression of cyclin B, suggesting the existence of another target mRNA for Nos-dependent translational repression in pole cells.

We found that Nos, along with Pum, represses translation of importina2 (impa2) mRNA in early pole cells. The impa2mRNA 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. Finally, we found that the repression of Impa2 production in pole cells is needed for proper migration of pole cells and the expression of a germline-specific marker, vasa (vas).

Publication List:

Amikura, R., K. Hanyu, M. Kashikawa and S. Kobayashi (2001) Tudor protein is essential for the localization of mitochondrial ribosomal RNAs in polar granules in germ plasm of Drosophila embryos. Mech. Dev. 107, 97-104.

Amikura, R., M. Kashikawa, A. Nakamura and S. Kobayashi (2001) Presence of mitochondrial-type ribosomes outside mitochondria in germ plasm of Drosophila embryos. Proc. Natl. Acad. Sci. USA. 98, 9133-9138.

Kashikawa, M., R. Amikura and S. Kobayashi (2001) Mitochondrial small ribosomal RNA is a component of germinal granules in Xenopus embryos. Mech. Dev., 101: 71-77.

Nakamura, A., R. Amikura, K. Hanyu and S. Kobayashi (2001) Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development.128, 3233-3242.

Sano, H., A. Nakamura and S. Kobayashi (2001) Identification of a transcriptional regulatory region for germline-specific expression of vasa gene in Drosophila melanogaster. Mech. Dev.(in press)

Sano, H., M. Mukai and S. Kobayashi (2001) Maternal Nanos and Pumilio regulate zygotic vasa expression autonomously in the germline progenitors of Drosophilaembryos. Develop. Growth & Differ. 43, 545-552.