RIKEN Center for Developmental Biology

2003 Annual Report

Germ cells are the only cell types capable of transmitting genetic information across generations, and their formation is characterized by unique developmental processes as well. In many types of animals, including the Drosophila fruit fly, the formation and differentiation of germ cells is controlled by mRNAs and proteins localized in a specific cytoplasmic region within eggs, called germ plasm. Germ plasm mRNAs are translated in a spatiotemporally regulated manner,　but the means by which the germ plasm is formed and achieves this controlled translation remain largely unknown. Akira Nakamura studies the establishment of the Drosophila germline as a model of the processes of germ plasm formation and differentiation, as well as for the insights this system can provide into the general mechanisms of mRNA localization and translation, which are important to a number of other developmental processes including asymmetric cell division, cell migration and axis formation.

Translational repression

RNA activity during Drosophila oogenesis involves a number of sequential processes. The Drosophila oocyte share cytoplasm with neighboring nurse cells via an incomplete cell membrane, allowing mRNAs and proteins from the nurse cells to be transported to the oocyte in the form of ribonucleoproteins. Following their export from the nurse cell nuclei, mRNAs are translationally repressed, or 'masked,' and transported to specified regions of the oocyte, where they establish fixed and precise localizations and regain their ability to undergo translation. In one example of this critically important regulation, the translation of the RNA for the maternal gene oskar, which has critical functions in embryonic patterning and the formation of germline cells, is repressed during its transport to the posterior pole of the oocyte. This transcript-specific repression is known to be mediated by the protein Bruno, which binds to the 3′ UTR of oskar mRNA, but the underlying mechanisms have remained obscure.

In recent work, the Nakamura lab demonstrated that an ovarian protein, Cup, is another protein required to inhibit the premature translation of oskar mRNA, and that Cup achieves this by binding to a second protein, eIF4E, a 5′ cap-binding general translation initiation factor. The binding with Cup prevents eIF4E from binding with a different partnering molecule, eIF4G, and thereby inhibits the initiation of translation. Findings that a mutant form of Cup lacking the sequence with which it binds eIF4E failed to repress oskar translation in vivo, that Cup interacts with Bruno in a yeast two-hybrid assay, and that the Cup-eIF4E complex associates with Bruno in vivo suggest that these three proteins form a complex that achieves translational repression by interactions with both the 3′ and 5′ ends of the oskar RNA. A similar model of protein interactions is observed in the translational repression of the cyclin-B1 RNA in the Xenopus African clawed frog, indicating that this paradigm of translational repression through the 5′/3′ interactions is conserved across species.

Nakamura next intends to look into the means by which the repressor effects of the eIF4E-Cup-Bruno complex are alleviated at the appropriate developmental stage, after the oskar ribonucleoprotein complex has reached and anchored to its appropriate destination at the pole of the egg.

Polar granule maintenance

Polar granules are large complexes of ribonucleoproteins that store RNAs and proteins required for the formation of germ cells in the fruit fly. A non-coding Polar granule component (Pgc) RNA has been shown to be important in the maintenance of these germ plasm organelles; in Pgc antisense knockdown embryos, germ cells are formed but subsequently degenerate, which has led to the hypothesis that Pgc serves as a form of supportive scaffold maintaining the structural integrity of polar granules. The Nakamura lab is now exploring Pgc function in more detail by isolating a complete loss-of-function mutant, allowing for more specific analyses of the gene’s role. They also plan to look for the gene’s functional domains, using Pgc orthologs from other species of Drosophila as a basis for comparison.

In other ongoing research, members of the Nakamura lab are using forward-genetics techniques to search for novel genes involved in germ cell development. Early results of those studies have revealed previously unexpected roles for a maternally supplied factor involved in lipid signaling, which the team is now working to characterize in more detail. Mutation of the underlying gene results in a phenotype similar to that of the Pgc mutant, in which germ cells fail to migrate to the embryonic gonads. Nakamura is now investigating the roles of both genes in the maintenance and migration of germ cells.

Development of the Ciona intestinalis germline

In addition to their investigation of fruit fly germline development, scientists in the Nakamura lab are also beginning investigations using the ascidian, Ciona intestinalis, a member of the chordate lineage from which all vertebrates arose. Commonly referred to as sea squirts, these animals provide a good model for studying vertebrate evolution. Soon after fertilization, the ascidian egg develops into a small free-swimming tadpole possessing a notochord, an evolutionary forebear of the spinal cord, and a rudimentary nervous system, both of which are lost when it enters its immobile adult stage.

Nakamura seeks to analyze the regulatory mechanisms of germline development in Ciona, which are interesting in that while several lines of evidence indicate that germ cells are formed from maternally derived germ plasm, it also appears that germ cells can be regenerated after metamorphosis, suggesting that two independent mechanisms may be at work. In research conducted in collaboration with Suma Aqualife Park, a local municipal aquarium, Nakamura's team will explore the genetic regulation of ascidian germline development by selecting candidate genes from EST and genome databases (a draft sequence of the Ciona genome is available), confirm the spatial and temporal patterns of their expression and characterize promoter regions and trans acting factors.