Upregulation of endocytic regulators and apical membrane internalization during NB ingression. (A and B) NBs increase apical endocytosis. Ingressing live NB (cell in the center; A) after injection of the lipophilic, non-cell permeable fluorescent dye FM4-64 at 8 mM, which marks the plasma membrane and apical vesicles (endosomes). Scale bar, 5 μm. (B) Intracellular FM4-64 in late ingressing NBs (14 NBs, four embryos) and temporally matched NICs (14 cells from four embryos), normalized to the initial time point of movie recording (∼2 min after injection of the dye into the perivitelline space of the embryo). The initial mean apical area of NBs at T = 0 min was ∼19 μm². Over the course of 6 min, the average fluorescence of internalized FM4-64 increased in ingressing NBs and kept constant in NICs. T = 0 min, start of video recording. Means ± SEM. (C–F) Ingressing live NB co-expressing Dynamin::GFP and GAP43::mCherry (C and D) or Clathrin::GFP and GAP43::mCherry (E and F). Dots/arrowheads indicate the NB apical domain. T = 0 min, onset of ingression. Scale bar, 5 μm. Normalized fluorescence intensity during early and late ingression. Individual dots are averages of protein levels at the cell junctions of 13 temporally registered ingressing NBs from two embryos (D; **** Dynamin::GFP early versus late, P = 5.9 × 10^-11; GAP43::mCherry, P = 2.8 × 10^-13; KS test) or 31 temporally registered ingressing NBs from seven embryos (F; **** Clathrin::GFP, P = 1.5 X 10^-14; GAP43::mCherry, P = 1.5 X 10^-9; KS test). Bars are medians and error bars are IQR.