Breaking Down the Balbiani Body and Germplasm: An Endogenous Structure and Function Analysis on the Buc Protein - Project Summary/Abstract: Conserved in invertebrates and vertebrates, the Balbiani body (Bb) is an aggregate of maternal RNAs, proteins, and/or organelles that in most animals is the first polarized structure in early-stage oocytes. The Bb recruits and localizes maternal factors required during early embryogenesis at the oocyte cortex, defining the vegetal pole and the animal-vegetal axis. During early embryogenesis maternal factors at the vegetal pole are transported to the blastomeres, where they will serve two key functions: maternal factors known as germplasm will specify the germline, and dorsal determinants will induce the signaling center that mediates dorsoventral axis formation. The intrinsically disordered Buc protein is the only known factor required for Bb assembly, which is hypothesized to be mediated by the self-aggregation of a prion-like domain (PrLD) within the amino terminus of the protein. There is evidence, however, that the PrLD is insufficient to ablate Bb localization and suggests that other regions of the protein contribute to Bb and germplasm assembly. Since protein organizers of other intracellular granules are predicted to be intrinsically disordered, I hypothesize that Buc protein disordered regions also mediate Bb and germplasm assembly in early-stage zebrafish oocytes. To investigate the role of Buc protein disordered regions, I will delete distinct disordered regions of the buc endogenous locus and probe for defects in Bb assembly, oocyte polarity, and germline specification in Aim 1. The Mullins Laboratory has generated a hypomorphic buc allele, which produces a truncated Buc protein that is sufficient to polarize the oocyte but fails to aggregate the germplasm in the embryo and specify the germline. buc hypomorphic mutant mothers produce viable embryos with a grandchildless phenotype. Our data suggest that a key germplasm aggregation factor is deficient that prevents wild-type Buc from rescuing the germline specification defect. Using a candidate list of factors that are sufficient to induce ectopic PGCs in the zebrafish blastoderm, Aim 2 will identify which critical factors are missing from the germplasm of buc hypomorphic mutants by overexpressing these factors in combination. Aim 2 will also investigate if the mutant Buc hypomorphic protein is insufficient to recruit these missing factors. These experiments will systematically test the function of various regions of the Buc protein in Bb assembly and germline specification, thereby performing the first endogenous structure-function analysis of the buc locus. Since protein aggregation is a feature of many neurodegenerative diseases, these studies are expected to provide insight into the aggregation properties of these deleterious protein aggregates.
