Figure 6.
Endocytosis and vesicle trafficking are essential for NB ingression. (A–C) Loss of AP2 and Dyn function blocks endocytosis. (A) Whereas control NBs lose their apical domain within ∼24 min (n = 40 NBs, two embryos), knockdown of AP2α (AP2α-RNAi) results in significant delays in ingression (n = 87 NBs, four embryos) or incomplete ingression, with 24% of NBs dividing on the embryo surface (middle panels) and 14% of NBs apically constricted but failing to complete ingression (lower panels). Membrane labeled with GAP43::mCherry. Scale bar, 5 μm. (B) Quantification of ingression dynamics of NBs in AP2α-RNAi (n = 66 NBs, three embryos) versus control embryos (n = 40 NBs, two embryos). AP2α-RNAi NBs show slower apical area loss and reduced amplitude of contractions compared to control. Control cells were only tracked until their apical domain reached 9 μm2 (in contrast to ∼1.5 μm2 in other figures) as 38% of NBs in AP2α-RNAi only constricted their apices to that value, but not below. Median amplitudes of contractions for control or AP2α-RNAi, 4.1/3.0 μm2/min, ****, P = 4 × 10−6, and expansions, 1.5/1.5 μm2/min, ns, P = 0.88 (KS test). Median durations of contractions for control or AP2α-RNAi, 67.2/62.7 s; ns, P = 0.22, and expansions, 24/27.4 s, ns, P = 0.27 (KS test); 233–855 events per condition. (C) Mean apical area loss and total apical Crb levels during NB ingression in DynTS (shibire1 mutant) embryos at 22°C and 32°C (32 NBs from three embryos at 22°C and 31 NBs from two embryos at 32°C). Lower panels show cross-sections of ventral ectoderm of stage 10 embryos expressing the NB reporter HG4-1 at the permissive temperature (22°C) or restrictive temperature (32°C) where some NBs failed to ingress (arrowheads). N = 8 and 11 DynTS embryos at 22 and 32°C, respectively. Apical side, up. Scale bar, 15 μm. T = 0 min, onset of ingression; means ± SEM. (D) Rab5 activity promotes ingression. Mean apical area loss during NB ingression in embryos expressing constitutive active Rab5-CA (58 NBs, four embryos) or normal Rab5 (Rab5-WT; control; 36 NBs, three embryos). T = 0 min, onset of ingression; means ± SEM. Amplitude and duration of individual apical contractions or expansions during ingression in Rab5-WT versus Rab5-CA embryos. Median amplitude of contractions for Rab5-WT or Rab5-CA: 2.4/3.3 μm2/min; ****, P = 4.8 × 10−5. Median duration of expansions for Rab5-WT or Rab5-CA: 19/15 s (log10), **, P = 1.4 × 10−3 (KS test); 341–448 events per condition. (E) Retromer limits ingression speed. Mean apical area loss during NB ingression and total apical Crb levels in Vps26(mz) mutants (23 NBs, 5 embryos; mz indicates maternal-zygotic mutants) versus controls (59 NBs, 12 embryos). Total intracellular levels of Crb in ingressed NBs from Vps26(mz) mutants (98 NBs, seven embryos) and controls (115 NBs, five embryos). (F) ESCRT enhances loss of apical Crb and apical membrane. Mean apical area loss during NB ingression and total apical Crb levels in Hrs(mz) mutants (36 NBs, seven embryos) versus controls (40 NBs, five embryos). Total intracellular levels of Crb in ingressed NBs from Hrs(mz) mutants (103 NBs, six embryos) and controls (115 NBs, five embryos). In E and F: Snail (blue) identifies NBs. Scale bar, 5 μm. T = 0 min, onset of ingression; means ± SEM.

Endocytosis and vesicle trafficking are essential for NB ingression. (A–C) Loss of AP2 and Dyn function blocks endocytosis. (A) Whereas control NBs lose their apical domain within ∼24 min (n = 40 NBs, two embryos), knockdown of AP2α (AP2α-RNAi) results in significant delays in ingression (n = 87 NBs, four embryos) or incomplete ingression, with 24% of NBs dividing on the embryo surface (middle panels) and 14% of NBs apically constricted but failing to complete ingression (lower panels). Membrane labeled with GAP43::mCherry. Scale bar, 5 μm. (B) Quantification of ingression dynamics of NBs in AP2α-RNAi (n = 66 NBs, three embryos) versus control embryos (n = 40 NBs, two embryos). AP2α-RNAi NBs show slower apical area loss and reduced amplitude of contractions compared to control. Control cells were only tracked until their apical domain reached 9 μm2 (in contrast to ∼1.5 μm2 in other figures) as 38% of NBs in AP2α-RNAi only constricted their apices to that value, but not below. Median amplitudes of contractions for control or AP2α-RNAi, 4.1/3.0 μm2/min, ****, P = 4 × 10−6, and expansions, 1.5/1.5 μm2/min, ns, P = 0.88 (KS test). Median durations of contractions for control or AP2α-RNAi, 67.2/62.7 s; ns, P = 0.22, and expansions, 24/27.4 s, ns, P = 0.27 (KS test); 233–855 events per condition. (C) Mean apical area loss and total apical Crb levels during NB ingression in DynTS (shibire1 mutant) embryos at 22°C and 32°C (32 NBs from three embryos at 22°C and 31 NBs from two embryos at 32°C). Lower panels show cross-sections of ventral ectoderm of stage 10 embryos expressing the NB reporter HG4-1 at the permissive temperature (22°C) or restrictive temperature (32°C) where some NBs failed to ingress (arrowheads). N = 8 and 11 DynTS embryos at 22 and 32°C, respectively. Apical side, up. Scale bar, 15 μm. T = 0 min, onset of ingression; means ± SEM. (D) Rab5 activity promotes ingression. Mean apical area loss during NB ingression in embryos expressing constitutive active Rab5-CA (58 NBs, four embryos) or normal Rab5 (Rab5-WT; control; 36 NBs, three embryos). T = 0 min, onset of ingression; means ± SEM. Amplitude and duration of individual apical contractions or expansions during ingression in Rab5-WT versus Rab5-CA embryos. Median amplitude of contractions for Rab5-WT or Rab5-CA: 2.4/3.3 μm2/min; ****, P = 4.8 × 10−5. Median duration of expansions for Rab5-WT or Rab5-CA: 19/15 s (log10), **, P = 1.4 × 10−3 (KS test); 341–448 events per condition. (E) Retromer limits ingression speed. Mean apical area loss during NB ingression and total apical Crb levels in Vps26(mz) mutants (23 NBs, 5 embryos; mz indicates maternal-zygotic mutants) versus controls (59 NBs, 12 embryos). Total intracellular levels of Crb in ingressed NBs from Vps26(mz) mutants (98 NBs, seven embryos) and controls (115 NBs, five embryos). (F) ESCRT enhances loss of apical Crb and apical membrane. Mean apical area loss during NB ingression and total apical Crb levels in Hrs(mz) mutants (36 NBs, seven embryos) versus controls (40 NBs, five embryos). Total intracellular levels of Crb in ingressed NBs from Hrs(mz) mutants (103 NBs, six embryos) and controls (115 NBs, five embryos). In E and F: Snail (blue) identifies NBs. Scale bar, 5 μm. T = 0 min, onset of ingression; means ± SEM.

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