Fig. 4: The Ush2a^delG/delG retina shows a decline in cone number, mislocalization of cone opsins, and an upregulation of the stress marker GFAP.

A Cone cell density count from four 0.016 mm² areas per retinal whole mount from P500 Ush2a^delG/delG and WT mice (one in each petal) taken at a 300 µm distance from the optic nerve (see the Method Section) and stained with PNA, showing that number of cones is significantly reduced in the Ush2a^delG/delG retina mirroring the functional decline. Shown are means ± SEM from N = 3 animals. P value: p = 0.0221 (*) by a two-tailed unpaired t-test. B Retinal sections from the indicated genotypes at P500 were co-labeled with PNA (green) and a mixture of anti-S- and M-opsins (red) antibodies. Nuclei in all sections were counterstained with DAPI (blue). White arrows show an accumulation of cone opsins in the ONL and IS. Two panels on the right side of each genotype showing 3D construction of the OS/IS and ONL/OPL areas where mislocalization of cone opsins is observed. Shown are representatives of N = 3 animals per genotype. C Total number of cones counted from the entire retinal cross sections shown in B. Also shown are the number of cones with mislocalized cone-opsins either in the IS/ONL or at the synaptic terminals. N values: WT: Presented are average cone counts from two sections per eye from five independent mice. Ush2a^delG/delG: Presented are average cone counts from three sections per eye from six independent mice. Significance was determined by a two-way ANOVA with a Bonferroni post hoc test. P values: total: p < 0.0001 (****), IS/ONL: p < 0.0001 (****), synaptic: p < 0.0001 (****). D Paraffin retinal sections from WT and Ush2a^delG/delG at P60, P200, and P500 were labeled for PNA (green) and immunolabeled either for S- or M-opsin (red) and counterstained with DAPI (blue). On the right side of each panel a 3D reconstruction of 8-12 confocal stacks (1 µm thickness) is shown, highlighting the photoreceptor cell layer. White arrows pointing to S- or M-opsin mislocalized in the IS, ONL, or synaptic terminal. E Retinal sections from WT and KI animals at P30, P180, and P360 were labeled with antibodies against GFAP. At P360, upregulation of GFAP was observed. Shown are single-plane images. OS outer segments, IS inner segments, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer.