Cell fate in wild-type and outm233 retinae. (A-B) Transverse sections through the retina stained with anti-neurolin antibody (red) in wild-type (A) and outm233 (B) embryos. The ganglion cell layer and the optic nerve display grossly normal appearance at 56 hpf. Sections were counterstained with YoPro (green). (C-D). Anti-parvalbumin antibody (red) stains a subset of amacrine cells on transverse sections through the retinae of wild-type (C) and outm233 mutant (D) embryos. A displacement of some amacrine cells towards and occasionally into the photoreceptor cell layer is observed in approximately 50% of outm233 embryos (arrowhead in D). (E - F) Anti-paravalbumin and Zpr1 double immunostaining (both in green) in wild-type (E) and in outm233 mutant (F) embryos. In approximately 50% of the embryos, Zpr1-positive cells are absent in the dorsal and/or ventral regions of the photoreceptor layer (arrowheads in F). (G - H) Images of whole embryos at 42.5 hpf. Ganglion cells are visualized with brn3c-GFP transgene in wild-type (G) and outm233 mutant (H) embryos. Anterior is to the left. (I -J) The Zpr1 antibody stains red and green cones (green signal) while anti-serotonin antibody labels serotonin-positive amacrine cells (red) in wild-type (I) and outm233 mutant (J) embryos. (K-L) Anti-carbonic anhydrase antibody (red) stains Müller glia in wild-type (K) and outm233 mutant (L) embryos. Arrowhead in the inset indicates the apical processes of the Muller glia, which terminate at the outer limiting membrane. (M - N) Anti-GABA antibody (green) stains a subset of amacrine cells in wild-type (M) and outm233 mutant (N) embryos. On the majority of sections, the number of GABA-positive neurons is increased in the retinal periphery of outm233 homozygotes (arrowheads). (O) Graph showing the frequency of GABA-positive cells in the central retina. The number of cells per an arbitrary unit of distance if provided. Fewer GABA-positive cells are observed in the mutant, compared to the wild type (p < 0.01, t-test). Blue dots represent sections. As some sections have the same frequency of GABA-positive cells, the number of dots does not equal the number of sections. n ≥ 4 embryos for both wild-type and mutant. Asterisks mark the lens. (C-F) and (I-N) show retinae at 5 dpf.
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