Inhibiting Shroom3 expanded the apical domain and reduced neurogenesis in retinal neuroepithelia. (A) Schematic showing modular organization of wild-type vertebrate Shroom3 and the dominant-negative (Shrm3DN) version. (B) Localization of prom1b-GFP in non-transgenic and Shrm3DN transgenic retina following heat shock (HS). (C,D) Transmission electron micrographs of 32 hpf retinal neuroepithelial cells from (C) non-transgenic and (D) Shrm3DN transgenic retina following heat shock. The apical membrane above the electron-dense adherens junctions is highlighted (red outline). Individual cells are pseudo-colored green for contrast. (E) Confocal image (left panel) of the apical retinal surface of 32 hpf prom1b-GFP (green) transgenic embryos injected with shrm3DN:ires:mCherry plasmid (red). Segmentation of the confocal image, used for quantification, is shown in the right panel. (F) Comparison of the apical area between Shrm3DN-positive cells (gray bars, n=20 cells, three embryos) and mCherry-negative cells (black bars, n=20 cells, three embryos). (G) Schematic showing the experimental design for evaluating neurogenesis in Shroom3DN-expressing cells in wild-type host eyes. (H) Comparison of the proportion of atoh7:GFP-positive cells in mCherry-positive clusters with either hsp70:mCherry-(black bars) or hsp70:shrm3DN:ires:mCherry (gray bars)-injected plasmid (for each condition, n=10 clusters, 10 embryos). (I) Examples of clusters expressing either hsp70:mCherry (red, right) or hsp70:shrm3DN:ires:mCherry (red, left) in a atoh7:GFP background (green cells) following heat shock (HS). For F and H, error bars represent s.e.m.; P, Student’s t-test. Scale bars: 500 nm in C,D.
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