Oocyte polarity requires a Bucky ball-dependent feedback amplification loop

Abstract

In vertebrates, the first asymmetries are established along the animal-vegetal axis during oogenesis, but the underlying molecular mechanisms are poorly understood. Bucky ball (Buc) was identified in zebrafish as a novel vertebrate-specific regulator of oocyte polarity, acting through unknown molecular interactions. Here we show that endogenous Buc protein localizes to the Balbiani body, a conserved, asymmetric structure in oocytes that requires Buc for its formation. Asymmetric distribution of Buc in oocytes precedes Balbiani body formation, defining Buc as the earliest marker of oocyte polarity in zebrafish. Through a transgenic strategy, we determined that excess Buc disrupts polarity and results in supernumerary Balbiani bodies in a 3'UTR-dependent manner, and we identified roles for the buc introns in regulating Buc activity. Analyses of mosaic ovaries indicate that oocyte pattern determines the number of animal pole-specific micropylar cells that are associated with an egg via a close-range signal or direct cell contact. We demonstrate interactions between Buc protein and buc mRNA with two conserved RNA-binding proteins (RNAbps) that are localized to the Balbiani body: RNA binding protein with multiple splice isoforms 2 (Rbpms2) and Deleted in azoospermia-like (Dazl). Buc protein and buc mRNA interact with Rbpms2; buc and dazl mRNAs interact with Dazl protein. Cumulatively, these studies indicate that oocyte polarization depends on tight regulation of buc: Buc establishes oocyte polarity through interactions with RNAbps, initiating a feedback amplification mechanism in which Buc protein recruits RNAbps that in turn recruit buc and other RNAs to the Balbiani body.