Key features of human ESCS and the Nrl^−/− mouse model. A) Topographic maps of visual sensitivity for S cones (left panels) and L/M cones (middle panels) in a healthy subject and patients with ESCS at different ages. Normally, S-cone sensitivities are less than those of L/M cones. In the patients with ESCS, S-cone sensitivities are greater than L/M-cone sensitivities at the same loci. P1, a 13-yr-old patient, has supernormal S-cone sensitivities; in P2, at 2 ages (40 and 48 yr), S-cone sensitivities are normal or subnormal but still greater than those of colocalized L/M cones; in the 8-yr interval, a progressive loss of vision was found. Loci showing this positive (enhanced) difference are marked (+, right panels). B) Kinetic visual field extent for a large bright target (V-4e) as a function of age in 9 patients with ESCS with longitudinal measurements spanning at least a decade. A decline with age seen in a proportion of these patients is attributable to progressive retinal degeneration. Inset: kinetic fields at 2 ages in ESCS P3 illustrates loss of field extent over a 19-yr interval. C) Cross-sectional OCT scans of retinal architecture along >10 mm of the horizontal meridian through the fovea (F) of ESCS P1 (top panel) compared with a healthy subject (top panel). Outer nuclear layer (ONL) and inner segment (IS) thicknesses are labeled at right. Rectangles show temporal retinal region quantified in next panel. D) Longitudinal reflectivity profiles (LRPs) of outer retinal lamina in 2 representative healthy subjects (left panel) and 3 patients with ESCS (middle panel). Identifiable layers are labeled and colored. Among notable LRP features are thicker ONL and IS layers in these patients and abnormal structures between the IS and RPE. Quantification of IS thickness in 6 patients with patients with ESCS vs. control subjects (right panel) showed a significant difference. E) En face autofluorescence images of the central fundus of 2 patients with ESCS illustrating hyperautofluorescent features (white dots). Insets: cross-sectional images in colocalized regions show dysmorphology with intraretinal hyperreflective lesions. F) OCT of Wt and Nrl^−/− mouse retinas illustrates phenotypic changes in the Nrl^−/− retina resembling those in human ESCS disease; e.g., a hyperreflective RPE-photoreceptor interface and nuclear layer rosette formation (asterisk). ONL thicknesses are labeled at right. G) Three-dimensional spectral domain OCT of Wt and Nrl^−/− mice indicates retinal disorganization caused by rosette formation as well as an abnormal photoreceptor-RPE interaction in the Nrl^−/− retina.

RNA-Seq of Wt and Nrl^−/− retinas reveals new differentially expressed genes arising from transcriptional misregulation. A) Wt retina RNA-Seq run, represented by robust Nrl transcript detection, detected 11,677 transcripts at 1 FPKM or higher. B) Pie chart shows breakdown of GO term categories to which the transcripts are assigned, with the number of transcripts in each category indicated. C) Single-base resolution of the RNA-Seq run of Nrl^−/− retina reveals ablation of Nrl transcript detection in the regions of exon 2 and 3 where there is a neomycin cassette. D) Run detected 11,778 transcripts at 1 FPKM or higher; pie chart shows breakdown of GO term categories to which the transcripts are assigned, with the number of transcripts in each category indicated. E) RNA-Seq runs of Wt and Nrl^−/− retina are plotted to show their differential expression pattern. Plots of Log FPKM of the retinal runs of Wt and Nrl^−/− illustrate that, whereas the majority of reads fall along the line representing equal expression, a range of transcripts falls either above or below the line that represent differentially expressed transcripts. Cnga1, Esrrb, Gnat1, Kcnj14, Nr2e3, Nrl, Rho, Slc24a1, and Susd3 (arrows) are among the highest expressed transcripts in the Wt retina, whereas Clca3, Cngb3, Fabp7, Gnat2, Gnb3, Gngt2, Opn1sw, Pde6c, Pde6h, and Six6os1 (arrows) are among the highest expressed transcripts in the Nrl^−/− retina RNA-Seq run. F) RT-PCR validated differential expression patterns detected by RNA-Seq. To validate differences from RNA-Seq experiments, retinal tissue from Wt and Nrl^−/− mice was used for RT-PCR using probes against well characterized targets from previous studies as well as newly identified targets from the current RNA-Seq study. RT-PCR results validated the RNA-Seq differential expression pattern that ranged from those genes that were highly differentially expressed (Egr1, Opn1sw) to those with more subtle differential expression (Gdf11, Otx2, Thrb) and even those without a significant fold change (Crx). Blue bars indicate RT-PCR of retina; red bars, RNA-Seq of whole eye; green bars, RNA-Seq of retina. Current RNA-Seq experiment, when compared to 2 previous microarray studies looking at differential expression between Wt and Nrl^−/− retina, reveals more comprehensive and quantitative data. G) RNA-Seq data reveal 3659 unique transcripts up-regulated in the Nrl^−/−retina compared to previous data sets, indicating a considerable amount of newly differentially expressed transcripts compared to previous findings. Moreover, the bottom panel shows that whereas microarray studies can assess gross changes well (5-fold or greater), more subtle changes in differential expression are more robustly characterized using RNA-Seq. H) RNA-Seq reveals 2230 unique transcripts down-regulated in the Nrl^−/−retina compared to previous data sets. The bottom panel again highlights the greater coverage of differential expression at lower thresholds using RNA-Seq.

ESCS photoreceptors of Nrl^−/− mice display aberrant packing and OS morphology caused by buildup of material in OS heads and aberrant photoreceptor phagocytosis. Retinal whole-mount confocal microscopy displays the tight packing of Wt mouse retinal photoreceptors in 3-D space. A, B) Cone OS (S-cone opsin antibody; A) and cone sheath (peanut agglutinin; B) signals overlap and illustrate the packing of cones in normal retina. C, D) Staining of cone OS (C) and rod OS (rhodopsin C-terminal 1D4 antibody; D) reveals that the retina is fully occupied by photoreceptors, especially densely packed rods. E) SEM imaging of critical point dried Wt retina further emphasizes that photoreceptors pack tightly in the retina. F) Closer examination by SEM shows that rod photoreceptors display their characteristic cylindrical shape. In contrast, Nrl^−/− retinas exhibited disrupted photoreceptor packing with clusters of densely populated cones separated by empty patches. G, H) Cone-like OS (G) and extracellular sheath (H) signals overlap but also are absent from some retinal patches. I, J) Staining of cone OS (I) and rod OS (J) reveals only ESCS photoreceptors. K) SEM imaging of critical point dried Nrl^−/− retina shows disrupted packing of photoreceptors in the retina, with a less dense population of photoreceptors than Wt. L) Closer examination by SEM highlights abnormal OS morphology with enlarged head structures. M–R) Thin-sectioned retinas from Wt and Nrl^−/− mice were prepared for TEM imaging. M) Thin sectioning of Wt retina reveals the internal structure of photoreceptors. N) Discrete stacked discs are seen in rod photoreceptors. O) Because these samples were prepared at the peak of photoreceptor turnover, TEM imaging captures the disc shedding process and RPE mediated phagocytosis (asterisk). P) Thin sectioning of Nrl^−/− retina shows a distinctive OS disc arrangement that differs from Wt rods. Q) Discs retain some interconnections (arrow) as well as connections to the plasma membrane. R) Closer examination of ESCS photoreceptors reveals that most photoreceptors have enlarged head structures owing to buildup of material at the photoreceptor-RPE interface (asterisk), which would not occur with normal phagocytosis. Scale bars = 5 μm (A–D, G–J); 1 μm (E, F, K, L, O, R); 250 nm (M, N, P, Q).

SBF-SEM allows visualization of impaired phagocytosis present in ESCS retinal degeneration. Because photoreceptor disc phagocytosis is a dynamic process that occurs throughout the retina, SBF-SEM imaging was used to collect precise serial sections and investigate phagocytosis of shed discs. A) In Wt retina, the photoreceptor-RPE interface is clearly visible, with tight packing of rods opposed to the RPE (see Supplemental Movie S1). B) Moreover, phagosomes (asterisk) ingested by the RPE are clearly visible. C) Three-dimensional reconstructions of collected data with Reconstruct allow visualization of multiple phagosomes (red), including the one indicated by asterisk in panels A, B, throughout a RPE cell (gray, with nucleus in blue) and also reveal the tight packing of rods (green and blue) in a plane. D) In Nrl^−/− retina, the photoreceptor-RPE interface is visible, but photoreceptors (asterisks) are not as densely packed against the RPE (see Supplemental Movie S1). E) Enlarged OS head structures of ESCS photoreceptors (asterisk) are seen with less electron-dense material at the tips, indicating loss of OS material in that area. F) Resulting 3-D reconstruction illustrates these enlarged headed photoreceptors (asterisks) and their interactions with the RPE (gray, with nucleus in blue). Of note is the absence of any visible phagosomes within the modeled Nrl^−/− RPE. Scale bars = 1 μm.

Absent phagosome staining at the photoreceptor-RPE interface confirms impaired phagocytosis in Nrl^−/− mice. Absence of proper phagocytosis was confirmed by biochemical staining for phosphatidylserine (PS) found on phagosomes and its localization with shed cone disc packets. A) In Wt retina, shed cone opsin discs (red; cone opsin antibody) colocalize with phagosomes stained for PS (green; indicated by arrow) at the photoreceptor-RPE interface. (Note that not all the PS staining indicates phagosomes as PS dye also stains the RPE cell nucleus). B–D) Zoom views show a cone opsin disc (red; indicated by arrow; B), PS phagsome staining (green; indicated by arrow; C), and colocalization of the two stains (indicated by arrow; D). E) In samples of Wt mouse retina examined at the peak of phagocytosis, staining of phagosomes (red) was found at the photoreceptor-RPE interface (arrows). F) Through optical sectioning of the collected data, confocal imaging of the retina-RPE interface reveals that the PS signal is present at the photoreceptor-RPE interface. Still image of the tangential plane of these collected data shows 3 corresponding slices of data at right, indicating layers containing nuclear (blue), cone sheath (green), and phagosome (red) signals. G) In contrast, comparable Nrl^−/− retinal samples fail to exhibit staining for PS at the photoreceptor-RPE interface. H) Confocal imaging of the Nrl^−/− retina-RPE interface shows that no detectable PS signal is evident at the photoreceptor-RPE interface. Note that it appears that there is some PS staining in the Nrl^−/− retina, but it is not located at the interface. Still image of the tangential plane of the collected data shows 3 slices of data at right, indicating layers containing nuclear (blue), ESCS photoreceptor (green), and phagosome (red) signals. Staining: cone opsin for cone OS; PNA for cone sheaths; DAPI or Topro 3 nuclear stain; phosphatidylserine for phagosomes. Scale bars = 5 μm.

Wt and Nrl^−/− mouse RPE phagocytose both Wt and Nrl^−/− photoreceptor OS membranes. First, the phagocytosis assay was run with no OSs as a negative control. A–C) Wt mouse RPE cells in culture not subjected to photoreceptor challenge (A) were just washed with FITC-labeled dye (B) where only faint background fluorescence was detected; images were overlaid (C). D–F) Wt RPE cells (D) were challenged with isolated Wt photoreceptor OS membranes (E); phagocytosis of the OS membranes was seen in the overlapping images (F). G–I) Wt RPE cells (G) challenged with isolated Nrl^−/− photoreceptor OS membranes (H) phagocytosed the OS membranes, as evident in the overlapping images (I). J–L) Nrl^−/− RPE cells (J) challenged with isolated Wt photoreceptor OS membranes (K) phagocytosed the OS membranes, as evident in the overlapping image (L). M–O) Similarly, Nrl^−/− RPE (M) challenged with isolated Nrl^−/− photoreceptor OS membranes (N) phagocytosed the OS membranes, as evident in the overlapping images (O).

Transcriptional misregulation causes precocious development of cone-like cells in the Nrl^−/− retina, which are then maintained by transcriptional networks that alter key homeostatic processes. A) Increased levels of Eya1 in the Nrl^−/− retina can activate the expression of Six6, which causes retinal progenitor cell (RPC) proliferation. Commitment of these retinal cells to an early cell fate requires a premature cell cycle exit. This is mediated by altered levels of Notch and Hedgehog transcriptional networks that produce increased levels of NeuroD1 and decreased levels of Dhh in the Nrl^−/− retina. Six6 and NeuroD1 together with Six6os1, exercising a possible regulatory role on Six6, synergize to promote the S-cone fate rather than alternative early cell fates. B) Maintenance of the cone-like cells in the mature retina can be attributed to a series of genes involved in transcriptional control of retinoid metabolism, transport, cell cycling, and signal transduction. Unique transcripts identified by RNA-Seq to be ≥5-fold differentially expressed in Nrl^−/− vs. Wt mouse retina provide a resource for identifying maintenance factors required for cone cell survival and the alterations that accompany disease. Up and down arrows indicate transcripts that are up- and down-regulated, respectively, in Nrl^−/− vs. Wt retina, as determined by RNA-Seq. C) Transcriptional misregulation causes changes in the expression of key homeostatic genes involved in phagocytosis, leading to the pathological degeneration in ESCS. The most critical receptor tyrosine kinase, Mertk, involved in RPE phagocytosis is unchanged in the Nrl^−/− retina compared to Wt. However, key homeostatic genes involved in photoreceptor OS phagocytosis and toxic metabolic movement, such as Abca4, Atp8a2, Tub, Tulp1, are down-regulated in the Nrl^−/− retina compared to Wt, thus contributing to the defect in photoreceptor phagocytosis. Down arrows indicate transcripts that are down-regulated in the Nrl^−/− retina compared to Wt; the up/down arrow indicates transcripts that were unchanged in expression, as determined by RNA-Seq.