Figure 4—source data 1.

Figure 4—source data 1.

(A–J) Confocal images of dorsally viewed Tg(E1-bhlhe40:GFP) embryos before (17 hpf; A) and 2.5 hr after incubation (19.5 hpf) with either DMSO (B, E, H), blebbistatin (C), paranitroblebbistatin (D), or azidoblebbistatin (Ableb) (F, G, I, J) with (G, J) or without irradiation (F, I) in the prospective RPE (F–G) or neural retina (NR) (I–J). Images in E’, F’, G’ H’, I’, and J’ are high power views of RPE morphology. Embryos were immunostained for GFP (green), β-catenin (white), and counterstained with Hoechst (blue). Note that the optic cup (OC) forms and the RPE flattens (white arrowhead in B) normally in all DMSO-treated embryos (B, E, E,’ H, H’) or in embryos incubated in Ableb without irradiation (F, F’, I, I’). In contrast, the RPE remains cuboidal (white arrowhead in C) and NR cells seem not undergo basal constriction (yellow arrowhead in C) in the presence of myosin inhibitors (C, D). Photoactivation of Ableb in the RPE prevents cell flattening (compare E’, F’, with G’) and impairs OV folding (G). When Ableb is photoactivated in the NR, folding of the OV is also impaired (J) but RPE cells undergo flattening (compare H’, I’, with J’). The number of embryos analysed and showing the illustrated phenotype is indicated on the top right corner of each panel and the average invagination angle and mean A–B on the left bottom corner. The yellow dashed line in (E, H) indicates how the invagination angle (α) was determined. (K, M) Normalized RPE height in DMSO- and Ableb-treated embryos, irradiated either in the RPE (K) or in the NR (M). (L, N) Normalized invagination angle in DMSO- and Ableb-treated embryos irradiated either in the RPE (L) or in the NR (N). Data represent mean ± SD; **p < 0.01 and ***p < 0.001. ns, non-significant. Scale bars: 50 µm in A–J and 25 µm in E’–J’.

Figure 4—source data 1.