Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notch self-renewing and a Notch differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.