Feedback induction of a photoreceptor-specific isoform of retinoid-related orphan nuclear receptor β by the rod transcription factor NRL

Abstract

Vision requires the generation of cone and rod photoreceptors that function in daylight and dim light, respectively. The neural retina leucine zipper factor (NRL) transcription factor critically controls photoreceptor fates as it stimulates rod differentiation and suppresses cone differentiation. However, the controls over NRL induction that balance rod and cone fates remain unclear. We have reported previously that the retinoid-related orphan receptor β gene (Rorb) is required for Nrl expression and other retinal functions. We show that Rorb differentially expresses two isoforms: RORβ2 in photoreceptors and RORβ1 in photoreceptors, progenitor cells, and other cell types. Deletion of RORβ2 or RORβ1 increased the cone:rod ratio ∼2-fold, whereas deletion of both isoforms in Rorb(-/-) mice produced almost exclusively cone-like cells at the expense of rods, suggesting that both isoforms induce Nrl. Electroporation of either RORβ isoform into retinal explants from Rorb(-/-) neonates reactivated Nrl and rod genes but, in Nrl(-/-) explants, failed to reactivate rod genes, indicating that NRL is the effector for both RORβ isoforms in rod differentiation. Unexpectedly, RORβ2 expression was lost in Nrl(-/-) mice. Moreover, NRL activated the RORβ2-specific promoter of Rorb, indicating that NRL activates Rorb, its own inducer gene. We suggest that feedback activation between Nrl and Rorb genes reinforces the commitment to rod differentiation.

Keywords: Cell Differentiation; Neurodevelopment; Photoreceptor; Retina; Transcription Factor.

FIGURE 1.

Differential temporal expression of RORβ isoforms. A, diagram of RORβ1 and RORβ2 isoforms showing the divergent N termini but identical DNA binding domain (DBD) and C terminus. The numbers refer to amino acid coordinates. B, expression profiles of RORβ1 and RORβ2 mRNA during retinal development, determined by RNA sequencing analysis of whole retina. RNA samples represented pooled retinas (from more than three mice) at each time point. FPKM, fragments per kilobase of transcript per million mapped reads; E13.5, embryonic day 13.5. C, expression profiles of RORβ1 and RORβ2 mRNA in rod photoreceptors, determined by RNA sequencing analysis of purified rod photoreceptors from Nrlp-GFP transgenic mice at the ages shown. The expression of NRL and NR2E3 rod factors rose over this period.

FIGURE 2.

Targeted deletion of RORβ2 and cellular expression of RORβ isoforms. A, deletion of the RORβ2-specific exon and replacement with lacZ fused at the RORβ2 ATG codon, creating the Rorb^2lacz (2z) allele. Triangle, RORβ2 promoter; loxP, residual loxP site after deletion of the ACN selection cassette. B, RT-PCR showing loss of RORβ2-specific mRNA (249-bp band) in 2z/2z mice and loss of RORβ1-specific mRNA (442-bp band) in RORβ1-deficient (1g/1g) mice. The alternatively spliced RORβ2-e* mRNA (302-bp band) was also deleted in 2z/2z mice. RORβ2-e* encodes a non-functional, 16-amino acid peptide without a DNA binding domain (24). C, Western blot analysis showing loss of RORβ2 and retention of RORβ1 protein in 2z/2z mice at the ages indicated. In 1g/1g mice, RORβ1 is lost, but RORβ2 is retained. A control blot for actin is shown below. Arrowheads, RORβ1 ∼52-kDa and RORβ2 ∼53-kDa bands. D, expression of RORβ2 in the immature photoreceptor layer (ONL) detected by histochemistry for β-galactosidase (turquoise) in +/2z mice at P7. Control +/+ mice gave no detectable β-galactosidase signal. RPE, retinal pigmented epithelium; INL, inner nuclear layer; GCL, ganglion cell layer. E, developmental expression of RORβ2 and RORβ1 in retinas monitored by, respectively, histochemistry for β-galactosidase (turquoise) in 2z/2z mice and immunohistochemistry for GFP (brown) in +/1g mice. IS/OS, inner/outer segments; OPL, outer plexiform layer; IPL, inner plexiform layer. F, RORβ1 in photoreceptor precursors detected by double fluorescence in Rorb^+/1gfp mice for GFP (turquoise) with TRβ2 (purple) for cones or NRL (purple) for rods in the outer neuroblast layer (ONBL) at the indicated ages. Scale bars = 25 μm.

FIGURE 3.

Retinal morphology and gain of cones in mice lacking RORβ isoforms. A, histological sections revealed a normal retinal structure in RORβ2-deficient (2z/2z) mice but with an ∼2-fold excess of cone nuclei. RORβ1-deficient (1g/1g) mice also displayed excess cone nuclei and, as reported, loss of horizontal and amacrine cells and disorganized inner and outer plexiform layers. The asterisks indicate the missing amacrine cell layer in the inner nuclear layer. In comparison, Rorb^−/− mice have almost exclusively cone-like cells instead of rods, lack photoreceptor segments, lack horizontal and amacrine cells, and display disorganized plexiform layers. RPE, retinal pigmented epithelium; OS, outer segment; IS, inner segment; opl, outer plexiform layer; INL, inner nuclear layer; ipl, inner plexiform layer; GCL, ganglion cell layer. B, higher magnification indicating cone nuclei (large with dispersed chromatin, arrowheads) and a representative rod nucleus (r, small with dense chromatin in +/+ mice (left panel)). Excess cone nuclei are present in 2z/2z and 1g/1g mice. Many cones in 2z/2z mice are misplaced in the inner zone of the ONL, but, in 1g/1g mice, most are in the outer zone. C, counts (mean ± S.D.) of cone and rod nuclei in 160-μm-long ONL fields showing excess cones in 2z/2z and 1g/1g genotypes (each p < 0.001 versus +/+) and an almost exclusive presence of cones in Rorb^−/− mice. D, counts (mean ± S.D.) of cone nuclei in outer ¼ and inner ¾ zones of the ONL showing substantial dispersal of cones in the inner zone of 2z/2z mice (p < 0.001 versus +/+). Rorb^−/− mice were not included in this comparison because they possess a thin ONL that cannot be compared with the ONL in the other mouse strains and because almost all cells in the ONL in Rorb^−/− mice are cone-like. Scale bars = 25 μm.

FIGURE 4.

Excess cone opsin-expressing cells in mice lacking RORβ isoforms. A, in situ hybridization revealed ∼2-fold increases in S opsin⁺ and M opsin⁺ cells, with many cones misplaced in the inner zones of the ONL in 2z/2z mice at 3 months of age. 1g/1g mice also showed increased cone opsin⁺ cells, most of which were in a normal location in the outer zone of the ONL. Stepwise deletion of additional RORβ isoforms in 2z/− or 1g/− compound heterozygotes further increased cone opsin⁺ cell numbers. In Rorb^−/− mice lacking both RORβ isoforms, cone opsins were overexpressed, and rhodopsin was almost completely lacking. RPE, retina pigment epithelium. Scale bar = 50 μm. B, analysis by qPCR of S opsin, M opsin, and rhodopsin mRNA in the retina of 3-month-old mice of the indicated genotypes.

FIGURE 5.

Electroretinogram analysis of mice lacking RORβ isoforms. A, intensity-response curves of electroretinogram responses for groups of five to eight mice (means ± S.E.) at ∼8 weeks of age. cd.s, candela second. Rod (left panel), S cone (center panel), and M cone (right panel) responses were absent in 1g/1g and −/− mice. B, representative electroretinogram traces for +/+ and 1g/1g mice. Left panel, scotopic rod responses. Center and right panels, photopic cone responses to 360- and 520-nm wavelengths that optimally activate S and M opsins, respectively. Families of traces are shown for stimuli of varying intensities.

FIGURE 6.

Induction of Nrl and rod gene expression by RORβ isoforms. A, retinal explants from +/+ or Rorb^−/− mice at P0 electroporated with RORβ1 or RORβ2 expression vectors. After 8 days in vitro, electroporated cells were identified with Tdt marker (purple), and NRL, NR2E3, and rhodopsin were detected by immunostaining (turquoise; or double-positive, whitish). Rorb^−/− explants severely lacked NRL⁺, NR2E3⁺, and rhodopsin⁺ cells, but substantial expression of these markers was recovered by electroporation of Ub/RORβ1 or Ub/RORβ2. INL, inner nuclear layer. Scale bar = 25 μm. B, similar electroporations with RORβ2 or RORβ1 (data not shown) expression vectors in Nrl^−/− explants failed to recover NR2E3 or rhodopsin expression. C, percentage of electroporated cells that express rod markers in explants from Rorb^−/− mice counted per 160-μm length of retina. p < 0.001 for Ub/RORβ1 or Ub/RORβ2, each versus Ub/empty. D, analysis by qPCR showing reinduction of rod genes (rhodopsin, Nrl, Nr2e3, Pde6b, and Mef2c) after electroporation of Ub/RORβ1 or Ub/RORβ2 in Rorb^−/− explants but not in Nrl^−/− explants. A control bipolar cell gene, Chx10, did not vary in response to RORβ isoforms in any genotype. In a control electroporation in Nrl^−/− retina, Ub/NRL expression vector reinduced mRNA for rod markers (data not shown).

FIGURE 7.

NRL-dependent expression of RORβ2 in the retina. A, Western blot analysis showing lack of RORβ2 in the retina in Nrl^−/− mice at P21. The slightly smaller RORβ1 isoform was retained, identified by comparison with 2z/2z mice that express only RORβ1. Control lanes for 1g/1g and Rorb^−/− show loss of RORβ1 and loss of all RORβ bands, respectively. NR2E3-deficient mice showed no obvious loss of RORβ2 or RORβ1. B, quantitative PCR analysis showing severe loss of RORβ2 but not RORβ1 mRNA in retinas of Nrl^−/− mice during postnatal development.

FIGURE 8.

NRL-dependent promoter in the Rorb gene. A, diagram of the Rorb gene showing RORβ1- and β2-specific promoters and NRL-bound peaks detected by ChIPseq. Location of potential NRL response element (NRE, turquoise ovals) and CRX response element (CRE, purple diamonds) in the promoter and intron around the RORβ2-specific exon with nucleotide coordinates noted relative to the ATG (+1). Location of NREs is on the basis of NRL ChIPseq peaks and CREs on a consensus TAATCC motif and location of CRX ChIPseq peaks (39). Triangle, RORβ2 mRNA 5′ end mapped by 5′ rapid amplification of cDNA ends. B, RORβ2 promoter-GFP reporter (Rorb2p-GFP) carrying promoter and intron regions with the NREs and CREs shown in A. A schematic for electroporation and culture of retinal explants is shown below. C, Rorb2p-GFP expression (turquoise) in rod precursors in electroporated retina of +/+ mice. Almost all GFP⁺ cells were NRL⁺ (purple, or whitish for double-positive). Scale bar = 20 μm. INL, inner nuclear layer. Right panel, GFP⁺ cell showing rod-like morphology. Scale bar = 5 μm. D, Rorb2p-GFP lacked activity in the retinas from Nrl^−/− pups but was reinduced by coelectroporation with the Ub/NRL expression vector. Electroporated cells were identified by the Tdt marker (purple, or whitish for double-positive). E, expression of electroporated Rorb2p-GFP (turquoise) and endogenous NR2E3 (purple) was not detected in Nrl^−/− mice, but both were recovered in the same cells (whitish) by coelectroporation of Ub/NRL. Scale bar = 20 μm.

FIGURE 9.

Model for the induction of the NRL rod differentiation factor by differentially expressed RORβ isoforms. In this proposed model, RORβ1 initiates the induction of NRL in immature precursors. NRL then exerts dual actions to stabilize the rod outcome. First, it induces RORβ2, which, in turn, reinforces NRL expression by positive feedback. Secondly, NRL induces NR2E3, which, together with NRL, activates rod genes and suppresses cone genes. Other factors not shown here, including OTX2 and CRX, probably enhance the expression of NRL and RORβ2.