Abstract The Balbiani body (Bb) is a membraneless organelle that forms in oocytes from insects to humans. In most vertebrates the Bb contains essential RNA-protein (RNP) granules, establishes oocyte polarity, and is required embryogenesis and reproduction. In mammals the Bb is associated with primordial follicle formation. However, despite its discovery in 1845, the mechanisms of Bb formation are still unknown in any species. Here, we discovered mechanisms of Bb formation in zebrafish. We first provide three lines of live-imaging, molecular, and biochemical evidence in developing ovaries in-vivo to conclude that the Bb forms a molecular condensate. We show that Bb RNPs form insoluble complexes dependently on Buc, an intrinsically disordered protein with a functional prion-like domain, which is essential for Bb formation. By using in vivo live-imaging, FRAP experiments and genetics, we reveal that Bb condensation requires the early liquid-liquid phase-transition of Buc, which later progresses to liquid-solid phase-separation. Further, we uncovered multi-step regulation by microtubules in controlling Bb condensation: first, by direct dynein-mediated trafficking of early condensing Buc granules, then by scaffolding condensed granules likely via molecular crowding, and finally by caging the mature Bb to prevent its over-growth and coarsening, likely by a Pickering effect. These regulatory steps ensure the formation of a single Bb condensate, which is essential for oocyte polarization and development. Our work resolves the origins of embryonic polarity, and identifies molecular condensation as an overlooked phenomena in the developing vertebrate ovary. While phase-separation is often viewed as a self-assembly process, our work proposes a new paradigm for the regulation of molecular condensation widely in biology.

Multi-Step Cellular Control of Molecular Condensation by Microtubules in Early Oogenesis