A genetic disconnection of the nuclear-cytoplasmic coordination in zebrafish oocyte symmetry breaking to analyze its contribution to oogenesis

A major question in biology is how cell polarity is established. In most vertebrates, oocyte animal-vegetal polarity is crucial for embryonic development, and is established by a structure called the Balbiani body (Bb), a conserved phase-separated mRNP granule. Loss of the Bb results in radially symmetrical eggs and embryonic lethality. How the Bb forms and is cellularly positioned is unknown. Our lab traced the symmetry breaking events that lead to Bb formation to a nuclear asymmetry that is universal in meiosis, the chromosomal bouquet. In the bouquet, telomeres are bound to Sun/KASH proteins on the nuclear envelope (NE) and associate with microtubules. Telomeres rotate to facilitate chromosomal pairing and cluster on the NE apposing the centrosome. Bb granules first localize to the bouquet centrosome dependently on bouquet microtubules to assemble the Bb, demonstrating that bouquet and Bb formation are mechanistically coupled. To study the role of this mechanistic coordination in symmetry breaking, we are genetically disconnecting nuclear from cytoplasmic events, generating loss-of-function mutations in Terb1, a meiotic protein tethering telomeres to the SUN protein, and a transgenic line over-expressing in oocytes a dominant-negative form of KASH that associates telomeres with microtubules. Both tools will disrupt bouquet formation and test its requirement for symmetry breaking and Bb formation. The roles of telomere NE-tethering in terb1 mutants, and NE-microtubule association in KASH loss-of-function will determine how each contributes to Bb granule localization. Our functional analysis will uncover the oocyte symmetry breaking mechanism and the significance of its nuclear-cytoplasmic coordination in oocyte differentiation.