|
Fig. 1 Polarized dynamics of Notch ligand–containing endosomes during RGP division.(A) Schematic diagram of a 28-hpf embryo (dorsal view) indicating the site of anti-Delta D-Atto647N (Dld) antibody injection (left). Right: Internalized Dld (blue) imaged 2 hours after antibody injection shows punctate appearance in RGPs (membrane labeled red). FV, forebrain ventricle. (B) Time-lapse imaging of mitotic RGP showing the dynamics of labeled Dld endosomes. Membrane, nucleus, and centrosomes [green fluorescent protein (GFP)–centrin] are marked as shown. (C) Schematic diagram showing typical phases of internalized Dld dynamic patterns in (B). (D) Schematic diagrams and statistics of distribution patterns of Dld endosomes in telophase RGPs (see Materials and Methods for the quantification of asymmetry index). Dotted lines in the scatter plot denote the asymmetric threshold of |0.2|. n = 88 RGPs, from 20 embryos of six experiments. (E) Automated tracking of Dld endosomes. (E1) Plot of time-lapse data from a composite of 19 RGPs. Each dot represents a tracked endosome at a given time. Color codes for cell cycle stages. The blue vertical line denoting the midpoint between two centrosomes is used for image registration. (E2 and E3) The plot trajectory of a singly labeled Dld endosome in two RGPs. Time is registered and color-coded (anaphase, T = 0). A-P, anteroposterior; Ap-Ba, apicobasal. In all images/plots, maximum intensity projection (MIP) of 5-μm z-stacks (1-μm z-step) is shown. The time interval between each z-stack is 12 s.