Crb overexpression delays NB ingression. (A) Ingressing live NBs (arrowheads) expressing endo-Ecad::GFP and sqh-Sqh::mCherry in H2O injected (control) and Crb overexpression (OE) embryos. T = 0 min, onset of ingression. Scale bars, 5 μm. (B) NB ingression speed in Crb OE (51 NBs, 10 embryos) and controls (48 NBs, 6 embryos). Median values: −1.35 (control), −1.0 (Crb OE), KS test: *, P = 2.5 × 10−2; F-test (to test for differences in variance of ingression speed): ns (not significant), P = 0.07; bars are IQRs. (C) Apical area loss of NBs is slowed in Crb OE embryos. Control (blue line; 20 NBs, two embryos, 159 time points spaced by 10 s) versus Crb OE (green line; 26 NBs, five embryos, 220 time points spaced by 10 s). (D) Amplitude and duration of apical contractions and expansions of NB in Crb OE embryos (26 NBs, five embryos) versus controls (20 NBs, two embryos). Median amplitudes of expansions for control/Crb OE, 1.17/1.84 μm2/min; ***, P = 5 × 10−4; and contractions, 2.76/3.04 μm2/min; ns, P = 0.43. Median durations of expansions for control/Crb OE, 17.39/18.87 s; *, P = 0.01; and contractions, 48.64/33.06 s, **, P = 1 × 10−3 (KS test); 241–566 events per condition. (E) Ventral ectoderm in late-stage 8 control and Crb OE embryos, stained for Crb (red) and Ecad (green). Apical is up. Note: Ectopic Crb on the lateral cell membrane in Crb OE cells and loss of apical Ecad signal. Scale bars, 5 μm. Quantification of Ecad levels in ingressing NBs. N values as in D. ****, P = 9.6 × 10−67 (two-tailed T test; mean ± SD). (F) Myosin levels in NBs of Crb OE embryos. Schematic of junctional and medial myosin pools in the apical domain of ingressing NBs. Crb OE reduces junctional myosin and enhances medial myosin. N values as in D. ****, P = 8.2 × 10−136; 8.2 × 10−38 (two-tailed T test; means ± SD). (G) Representative plots showing the rate of medial myosin change (a.u./min) during ingression in a control and a Crb OE NB. Positive rates indicate medial myosin assembly; negative rates indicate disassembly. T = 0 min, onset of ingression. (H) Medial myosin assembly and disassembly rates (Amplitude) during ingression. Duration indicates total time medial myosin spent increasing/assembling or decreasing/disassembling. N values as in D. Median rates of medial myosin change of control/Crb OE for assembly, 0.07/0.1 a.u./min; ***, P = 3.0 × 10−4; and disassembly, 0.06/0.08 a.u./min; **, P = 2.6 × 10−3. Median durations for control/Crb OE for assembly, 24/28 s; ***, P = 1 × 10−3; and disassembly, 18/28 s, ****, P = 7.9 × 10−6 (KS test); 328–553 events per condition.
