PROJECT SUMMARY Asymmetric cell division (ACD) plays a critical role in fate specification and morphogenesis during development. This process is also crucial for tissue homeostasis and repair in adulthood. Dys-regulation of ACD results in developmental/intellectual disabilities and tumorigenesis, making it critical to understand the underlying cellular and molecular mechanisms. A critical aspect of ACD is the establishment of cell polarity. Studies in invertebrate systems have identified important cortical polarity regulators, which ensure proper segregation of fate determinants into two daughter cells. These studies further shed light on how distinct protein complexes establish and maintain their reciprocal cortical polarity. Despite these advances, how cortically localized proteins polarize non-cortically distributed cell fate determinants is not well understood. Research into vertebrate systems has begun to uncover new insights into the function of these evolutionarily conserved polarity regulators. Radial glia progenitors (RGPs), the principal vertebrate neural stem cells (NSCs), represent a model cell type as they predominantly undergo ACD during active neurogenesis to balance self-renewal and differentiation. Our prior work shows that in embryonic zebrafish forebrain RGPs, the key cortical polarity regulator Par-3 is critical to establish asymmetric Notch signaling activity in daughter cells. It remains unknown how Par-3 establishes such asymmetry. Recently, we uncover, for the first time to our knowledge, that Par-3 is present in the cytosol and associates with the dynein motor complex on Notch ligand-containing endosomes. Together, Par-3 and dynein are required in the mother RGP to directionally transport Notch ligand-containing endosomes to the self- renewing daughter. Additionally, we discover that cortical Par-3 domain shifts from apical at interphase toward posterior during mitosis, to align with cell division orientation along the anteroposterior embryonic axis. In this application, we wish to build upon these new findings to address the following questions: 1) how does Par-3 work together with dynein to direct polarized dynamics of Notch ligand-containing endosomes? 2) what is the dynamic relationship between cortical and cytoplasmic Par-3? 3) What mechanisms reconstruct the axis of Par-3 polarity from apicobasal during interphase to anterior-posterior during mitosis? Our central hypothesis is that both intrinsic and extrinsic mechanisms operate to reshape Par-3 cortical polarity; this cortical polarity then sets up a polarized cytoplasmic gradient of Par-3, which in turn directly facilitates endosome dynamics by activating dynein. The proposed work is expected to yield significant new insights into asymmetric division and neural stem cell fate regulation during vertebrate brain development. These findings should have a positive impact on revealing fundamental principles and laying groundwork for elucidating disease etiology and...