Neur, but not Delta, is required for removal of Crb from the NB apical membrane during ingression. (A) Endo-Crb::GFP embryos showing NBs clusters (circled) in neur-RNAi (20 NBs, two embryos) and Delta-RNAi (20 NBs, two embryos) embryos. Note: Ectopic accumulation of apical Crb and slower ingression with neur-RNAi compared with Delta-RNAi. Scale bar, 5 μm. (B and C) Total Crb::GFP (B) at apical domain (junctional and medial pools) and apical area loss (C) during ingression in neur-RNAi (20 NBs, two embryos) and Delta-RNAi (20 NBs, two embryos) embryos. Delta-RNAi was chosen as a control as it causes a neurogenic defect similar to neur-RNAi. Due to reduced levels of apical Crb in both conditions, the apical surface was traced starting at 26 μm2 (mid-ingression, T = 0 min) in both data sets; means ± SEM. (D) Amplitude and duration of apical contractions and expansions during NB ingression in neur-RNAi versus Delta-RNAi embryos. Median amplitudes for Delta-RNAi and neur-RNAi for contractions, 4.1/1.9 μm2/min, ****, P = 9.6 × 10−13; and expansions, 2.3/1.3 μm2/min, ****, P = 5.3 × 10−6 (KS test). Median durations for Delta-RNAi and neur-RNAi for contractions, 39.3/38.4 s; ns, P = 0.88; expansions, 16.6/24.1 s, ***, P = 8.2 × 10−5 (KS test); n values as in B; 230–440 events per condition. The median amplitudes of apical contractions and expansions were reduced by 54 and 41%, respectively, in NBs of neur-RNAi embryos, and the median duration of apical expansions was increased by 45% compared to Delta-depleted controls. (E and F) Crb endocytosis is reduced in SdtΔ3 and Neur-depleted embryos. Average total fluorescence of intracellular Crb per NB after ingression (stage 9) in Sdt3::GFP (63 NBs, three embryos) and SdtΔ3::GFP (94 NBs, four embryos) embryos (E). Average total fluorescence of intracellular Crb per NB after ingression (stage 9) in neur-RNAi (134 NBs, seven embryos) and control (118 NBs, six embryos) embryos (F). Scale bar, 5 μm. Means ± SD. **, P = 3 × 10−3; ****, P = 4.5 × 10−11 (two-tailed T test).
