Establishing precise oocyte symmetry breaking and phase-separation mechanisms by in vivo quantitative live time-lapse analysis in zebrafish

Cell polarity is a major theme in biology, but underlying symmetry breaking events are mostly illusive. Oocyte animal-vegetal polarity is crucial for embryonic development and established during oogenesis by the Balbiani body (Bb), a conserved large mRNP granule. We previously established that a nuclear asymmetry at the onset of meiosis, called the chromosomal bouquet, breaks the oocyte symmetry, leading to Bb formation. In the bouquet, telomeres associate with perinuclear microtubules through Sun/KASH proteins on the nuclear envelope (NE), and rotate. They ultimately cluster to the NE pole opposing the centrosome, facilitating chromosomal pairing and recombination. Bb granules first polarize by localizing to the bouquet centrosome dependently on bouquet microtubules. Microtubules mechanistically couple Bb granule localization with telomere clustering. However, how Bb granules localize by Microtubules and precisely correlate with telomere rotation is unknown. We developed protocols for ovarian culturing and quantitative live time-lapse analysis to determine the sequence of these events and the granule localization mechanism. We detect bouquet chromosomal rotation for the first time in vertebrate oocytes in vivo, quantifying their rates. Essential for Bb formation is the intrinsically disordered protein Bucky ball (Buc) that localizes to the bouquet centrosome and drives Bb granule phase-separation. We are recording a functional transgenic Buc-GFP reporter, detecting Buc-GFP localization and condensation in symmetry breaking and in phase-separating granules. Buc-GFP fluorescence recovery after photobleaching (FRAP) analysis measures granule turnover and determines its localization mode and correlation with bouquet dynamics. Our in vivo 4-dimesnional analysis will uncover new paradigms in cellular polarization and phase-separation.