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Fig. 2
Impairment of actomyosin-driven basal constriction or proliferation does not prevent RNE formation.
(A) Confocal scan of optic cup at 30 ss stained for phalloidin. Control (left), phenotypes after 7 h of Rockout treatment: invagination defect in 30% embryos (middle), invagination defect with epithelial accumulation in 40% embryos (right), (n = approx. 30 embryos, N = 5 experiments, see Figure 2—source data 1). Arrowheads mark the basal side of the RNE. Arrow marks the epithelial accumulation outside the RNE. Asterisk marks the developing lens. Dashed lines indicate the angle of invagination. Rockout treatment started around 13–14 ss. Lookup table indicates the minimum and maximum intensity values. Scale bar = 10 µm. (B) Brightfield images of optic cup at the end of the 7 h treatment. Control (left), treated with Rockout (right). Arrow marks the epithelial accumulation outside the RNE. Scale bar = 50 µm. (C,D) Normalized average intensity distributions of phalloidin (C) and phosphomyosin (D) in the tissue volume along the apicobasal axis of the RNE at 30 ss. Mean ± SEM. Control (brown), Rockout treated (grey). Tissue sections, n = 25; N = 5 embryos each. See >Figure 2—source data 2 and 3. (E) Time-lapse of RNE morphogenesis in DMSO Control (upper) and Rockout-treated (lower) embryos expressing actin marker GFP-UtrophinCH. N = 5 embryos each. Rockout treatment was started 2 h before imaging around 13–14 ss. Imaging started at around 18 ss. Both movies were imaged simultaneously and under identical imaging conditions. Time in h:min. Frames from Video 3. Scale bar = 10 µm. (F) Invagination angle at 30 ss. Mean ± SD. The schematic shows the invagination angle as the angle held at the base of central RNE by the inner lips of the optic cup. P values for Mann-Whitney test with Control are as follows: Rockout p=0.0004, HU+Aphi p=0.0421 and for laminin a1 Mo, opo Mo and ezrin Mo p<0.0001. See Figure 2—source data 4. (G) Average basal area of RNE cells with mean ± SD. Each dot represents one embryo. P values for Mann-Whitney test with 30 ss control are as follows: 30 ss Rockout p=0.0952, 30 ss HU+Aphi p<0.0001. See Figure 2—source data 5.