Excess cones in the retinal degeneration rd7 mouse, caused by the loss of function of orphan nuclear receptor Nr2e3, originate from early-born photoreceptor precursors

Abstract

The orphan nuclear receptor NR2E3 is a direct transcriptional target of NRL, the key basic motif leucine zipper transcription factor that dictates rod versus cone photoreceptor cell fate in the mammalian retina. The lack of NR2E3 function in humans and in retinal degeneration rd7 mutant mouse leads to increased S-cones accompanied by rod degeneration, whereas ectopic expression of Nr2e3 in the cone-only Nrl(-/-) retina generates rod-like cells that do not exhibit any visual function. Using GFP to tag the newborn rods and by 5-bromo-2'-deoxyuridine birthdating, we demonstrate that early-born post-mitotic photoreceptor precursors in the rd7 retina express cone-specific genes. Transgenic mouse studies in the rd7 background show that Nr2e3 when expressed under the control of Crx promoter can restore rod photoreceptor function and suppress cone gene expression. Furthermore, Nr2e3 expression in photoreceptor precursors committed to be rods (driven by the Nrl promoter) could completely rescue the retinal phenotype of the rd7 mice. We conclude that excess of S-cones in the rd7 retina originate from photoreceptor precursors with a 'default' fate and not from proliferation of cones and that Nr2e3 is required to suppress the expression of S-cone genes during normal rod differentiation. These studies further support the 'transcriptional dominance' model of photoreceptor cell fate determination and provide insights into the pathogenesis of retinal disease phenotypes caused by NR2E3 mutations.

Figure 1.

Expression of GFP in some but not all S-opsin+ photoreceptors of the Nrl::GFP/rd7 retina. Nrl::GFP/WT and Nrl::GFP/rd7 retinas at P21 (A and B) were immunostained with anti-S-opsin antibody (red). More S-opsin positive cells are observed in the Nrl::GFP/rd7 retina. GFP (green) and S-opsin are co-localized in the Nrl::GFP/rd7 mouse retina, but not in the Nrl::GFP/WT mouse retina. (C) Dissociated cells from P10 Nrl::GFP/rd7 mouse retina were immunolabeled with S-opsin antibody (red). Nuclei are stained with bisbenzimide (blue). Some S-opsin+ cells also express GFP (green). Arrows indicate photoreceptors with GFP and S-opsin co-localization, while arrowheads refer to photoreceptors positive only for S-opsin. OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars are indicated.

Figure 2.

Increase in cone gene expression in GFP-positive cells flow-sorted from the Nrl::GFP/rd7 retina. qPCR analysis using cDNAs generated from flow-sorted GFP+ cells of Nrl::GFP/WT and Nrl::GFP/rd7 retinas shows increased expression of cone genes and somewhat lower expression of rod genes. Expression levels are normalized to Hprt first and then compared with WT. Fold change in expression of genes (rd7 photoreceptors compared to WT) is shown in log2 scale. Error bars show standard deviation. Gene symbols are: rhodopsin (Rho), rod transducin (Gnat1), guanine nucleotide-binding protein beta 1 (Gnb1), phosphodiesterase β subunit (Pde6b), phosphodiesterase γ subunit (Pde6g), cyclic nucleotide-gated channel α-1 (Cnga1), S-opsin or blue cone opsin (Opn1sw), M- opsin or green cone opsin (Opn1mw), cone transducin (Gnat2), guanine nucleotide-binding protein beta 3 (Gnb3), phosphodiesterase 6c (Pde6c), chloride channel calcium-activated 3 (Clca3) and cone arrestin (Arr3).

Figure 3.

Generation of excess S-cones in the rd7 retina from early-born photoreceptors. (A) Immunostaining of P21 WT and rd7 retina for S-opsin (green) and BrdU (red) after a single injection of BrdU at E15.5. Rare (or no) co-localization of BrdU and S-opsin observed in the ONL of WT retina; however, in rd7 retina, co-localization is observed in the middle or inner side of the ONL as indicated by arrows. Arrowheads show S-opsin(+) cells with weak BrdU labeling. (B) Immunostaining of P21 Nrl::GFP/rd7 retina with S-opsin (blue) and BrdU (red) after a single injection of BrdU at E16.5. Arrows indicate the co-localization of GFP (green), S-opsin and BrdU. Arrowhead indicates a BrdU+ and S-opsin+ but GFP(−) photoreceptor. (C) Immunostaining of P21 WT and rd7 retina for S-opsin (green) and BrdU (red) after a single injection of BrdU at P2. No co-localization of BrdU and S-opsin is observed in the ONL of either WT or rd7 retina. Dashed lines represent the inner and outer half of the ONL. OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars are indicated.

Figure 4.

Complete suppression of cone phenotype and rescue of rod function in the Crx::Nr2e3/rd7 retina. (A) Immunohistochemistry of the P21 Crx::Nr2e3/rd7 retina using anti-S-opsin, anti-cone arrestin (rabbit) and anti-rhodopsin (mouse) antibodies. S-opsin or cone-arrestin (red) is undetectable, whereas rhodopsin (green) is expressed uniformly in outer segments as well as ONL. Nuclei are stained with bisbenzimide (blue). RPE, retinal pigment epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer. Scale bars are indicated. (B) Typical dark-adapted ERG intensity series from 3-month-old WT (left), rd7 (center) and Crx::Nr2e3/rd7 (right) mice. (C) ERG intensity–response curves of the mean dark-adapted b-wave and a-wave amplitude for each group. Vertical bars indicate ± SE. (D) Light-adapted ERG responses from 3-month-old WT (left), rd7 (center) and Crx::Nr2e3/rd7 (right) mice. (E) ERG intensity–response curves of the mean light-adapted b-wave amplitude for each group. Vertical bars indicate ±SE.

Figure 5.

Phenotype rescue by Nr2e3 expression in rod precursors of the Nrl::Nr2e3/Nrl^−/− retina. (A) Nrl::Nr2e3 construct. The mouse Nrl promoter (10) was used to drive Nr2e3 expression. (B) Southern analysis of genomic DNA from Nrl^−/− (lane 1) and Nrl::Nr2e3/Nrl^−/− (lane 2) mice. The endogenous Nr2e3 gene is represented by a 9 kb band, and the transgene by a weak 8 kb and a strong 4.9 kb band. (C) RT–PCR analysis of Nr2e3 mRNA in the Nrl::Nr2e3/Nrl^−/− retina during the developmental stages. (D) Immunoblot analysis of NR2E3 protein in the Nrl::Nr2e3/Nrl^−/− retina during development. (E) Immunohistochemistry of the Nrl::Nr2e3/Nrl^−/− retina with NR2E3 (red) and rhodopsin (green) antibodies. (F) Immunohistochemistry using the flat-mount of Nrl::Nr2e3/Nrl^−/− retina with rhodpsin (green) and S-opsin (red) antibodies. (G) Immunohistochemistry of the Nrl::Nr2e3/Nrl^−/− retina with rhodpsin (green) antibody, showing a dorsal (D) versus ventral (V) expression pattern.

Figure 6.

Phenotype rescue by Nr3e3 expression in rod precursors of the Nrl::Nr2e3/rd7 retina. (A) Immunoblot analysis of P6 retinal extracts from WT, rd7 and transgenic mice shows the expression of NR2E3 in the Nrl::Nr2e3/rd7 retina. γ-Tubulin is used as a control for protein amount. (B and C) Immunohistochemistry of WT, rd7 and Nrl::Nr2e3/rd7 retinal sections at P21 (B) and flat mounts (C) with anti-S-opsin (rabbit) antibodies. Nuclei are stained with bisbenzimide (blue). Whorls and rosettes are observed in the rd7 retina but not in the WT or transgenic retina. S-opsin staining (red) in the transgenic retina is similar to that of WT. OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer. Scale bars are indicated. (D) Typical dark- or light-adapted ERG recordings from 6-month-old WT, rd7 and Nrl::Nr2e3/rd7 mice. ERG intensity-response curves of the mean dark-adapted b-wave and a-wave amplitude for each group are shown. Vertical bars indicate ±SE.

Figure 7.

A model of photoreceptor differentiation. Pools of retinal progenitor cells (RPCs) undergo terminal mitosis at specific times during development. CRX acts as a competence or permissive factor and is expressed in all post-mitotic precursor cells (PMCs) that are committed to the photoreceptor lineage. PMCs expressing a threshold level and/or a fully transcriptionally active form of NRL, and consequently NR2E3, are directed towards a rod cell fate, whereas those with no or low NRL/NR2E3 produce cones. The expression of thyroid hormone receptor TRβ2 in the latter induces M-cone differentiation, while the remaining NRL(−) cells adopt the ‘default’ S-cone fate. In the rd7 mouse (and ESCS patients), cone genes are not completely suppressed in the NRL(+) cells due to the absence of NR2E3 function. Thus, early-born immature rods have S-cone-like characteristics and others acquire rod-like or rod-cone hybrid phenotype. Expression of NR2E3 in these cells can rescue the rod morphology and gene expression. In the Nrl−/− retina, since NRL and NR2E3 are not present, the potential rod precursors adopt the ‘default’ S-cone fate. Ectopic expression of NR2E3 in the NRL(−) cells represses cone genes and produces rod-like characteristics, whereas expression of NRL (and consequently NR2E3) generates functional rods.