Loss of Llgl1 disrupts retinal development. (A) Phylogenetic comparison of Llgl proteins showing percentage identity (left, top) and cladogram when compared with zebrafish Llgl1 (right, top). Sequence comparison (bottom) showing conservation of serines known to be phosphorylated. (B) In situ hybridization of llgl1 mRNA in zebrafish embryos at 24, 48 and 72 hours post-fertilization (hpf). (C) Side views of morphant embryos at 36 hpf: 8 ng control+ 8ng tp53 morpholino (MO) (top); 8 ng llgl1 ATG MO (middle); 8 ng tp53 + 8ng llgl1 ATG MO (bottom). (D) Localization of Llgl1 immunoreactivity (red puncta) in 32 hpf retina of H2A-GFP (green nuclei) transgenic embryos (left) or those injected with 8 ng tp53 + 8ng llgl1 ATG MO (middle). Localization of fluorescence in 32 hpf retina of wild-type embryos injected with 100 pg GFP-Llgl1 mRNA (right). (E) Sagittal images of eyes in living embryos stained with Acridine Orange (AO) (green puncta, arrows) to label dying cells: 8 ng control+ 8ng tp53 morpholino (MO) (left); 8 ng llgl1 ATG MO (middle); 8 ng tp53 + 8ng llgl1 ATG MO (right). Average number of Acridine Orange-positive cells per eye±s.e.m. (50 μm confocal optic section) (bottom). For each condition, n=10 eyes from 10 embryos were quantified. *P<0.001 (Student’s t-test). (F) Retinal histology of eyes from 80 hpf zebrafish embryos: 8 ng control+ 8ng tp53 morpholino (MO) (left); 8 ng llgl1 ATG MO (middle); 8 ng tp53 + 8ng llgl1 ATG MO (right). (G) Retinal histology of eyes from E15.5 day mice: wild type (left panels) and Llgl1 homozygous mutant (right panels). Note the rosettes in the retinal pigment epithelium (arrow) and neural retina (arrowhead). Scale bars: 250 μm in C; 20 μm in D; 100 μm in E-G.
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