The main objective of this proposal is to provide a complete characterization of the RNA composition germ plasm of the wasp Nasonia vitripennis, and to then to begin the process of functionally testing confirmed factors localized to this organelle. Understanding the molecules that make up this structure is important since germ plasm has the unique capability to drive naive nuclei rapidly into the germline developmental program. Many of the characteristics of primordial germ cells are of general interest to both development and disease, including periods of general transcriptional quiescence, the potential for pluripotency, and trans-epithelial migratory behavior. The Nasonia germ plasm is of particular interest, as it is an unusually large, discrete ribonucleoprotein entity, in contrast tothe smaller granules characteristic of other germ plasms. In addition, the primordial germ cells of Nasonia behave differently from those of Drosophila, indicating a divergent molecular make-up of this entity in the wasp. Established techniques for isolating embryo fragments from Drosophila will be applied to Nasonia embryos. Fragments highly enriched for germ plasm material will be compared directly to corresponding fragments depleted for the material, by using quantitative RNA sequencing technologies. Those transcripts that are significantly overrepresented in the germ plasm containing fraction compared to the depleted fraction will be candidate components of the germ plasm. These will be confirmed by in situ hybridization, and by functional testing with RNAi. This study will touch on the fields of developmental biology, RNA regulation, cell biology, genomics, and evolutionary biology. This work will include the intense participation of a graduate student, who will be recruited in the coming year, and may also involve an undergraduate student. It is expected that the students will be at the early training level of experience, and the Principal Investigator will invest significant time in trainig and mentoring the recruited student(s).
The germline is made up of cells that can give rise to all cells of the body, and is essential for the continuation of generations. Understanding how these cells are made in embryonic development is important for understanding the nature and function of these cells. This proposal aims to characterize the molecular basis of germline determination in a convenient insect model, which will provide basic knowledge about how germline cells are established throughout all animals, including humans.
