Human Molecular Genetics, 2011, Vol. 20, No. 21 doi:10.1093/hmg/ddr334 Advance Access published on August 3, 2011
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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 Hong Cheng1,{, Naheed W. Khan1, Jerome E. Roger2 and Anand Swaroop1,2,∗ 1
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI, USA Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD, USA 2
Received March 29, 2011; Revised July 19, 2011; Accepted July 29, 2011
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 Nrl2/2 retina generates rod-like cells that do not exhibit any visual function. Using GFP to tag the newborn rods and by 5-bromo2′ -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.
INTRODUCTION The retina is a highly stratified sensory tissue in the central nervous system. It consists of three cellular layers containing six major types of neurons and one type of glial cell. Two types of photoreceptors (rods and cones) are exclusively located in the outer nuclear layer (ONL); in most mammals, rods greatly outnumber cones, which are primarily distributed along the outer (scleral) side of the ONL (1). Rods are highly photon sensitive and function in low light conditions, whereas cones are responsible for visual acuity and mediate daylight and color vision (2). Based on differences in visual pigments (called opsins), the human retina has three subtypes of cones ∗
(short wave-length, S; medium wave-length, M and long wave-length, L) (3,4). The mouse retina has only S and M cones, and these are spatially distributed in a ventral-to-dorsal gradient with a majority expressing both cone opsins (5,6). In the vertebrate retina, all neurons and Mu¨ller glia are generated from pool(s) of multipotent neuroepithelial progenitors with a relatively conserved birth order (7,8). In mouse retina, almost all cones are generated between embryonic day 11 (E11) and E16. On the contrary, rod genesis spans a broader temporal window that overlaps with the birth of all other neurons—from E12 to postnatal day 8 (P8)—but a majority of rods are born postnatally (P0 – P2) (9 – 11). The early-born (prenatal) rods are present primarily in the inner side of the
To whom correspondence should be addressed at: N-NRL, National Eye Institute, Bldg 6/338, MSC0610, National Institutes of Health, 6 Center Drive, Bethesda, MD 20892, USA. Tel: +1 3014355754; Fax: +1 3014801769; Email:
[email protected] † Present address: Department of Pathology and Laboratory Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.
Published by Oxford University Press 2011.
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ONL and have certain characteristics distinct from the lateborn (postnatal) rods (12). Post-mitotic photoreceptor precursors, in both rodents and primates, exhibit variable delays before expressing their respective opsin photopigments (12,13). Photoreceptor differentiation is guided by extrinsic cues and intrinsic factors (7,8). Intrinsic mechanisms seem to play a dominant role in cell fate determination (14,15). The homeobox genes, OTX2 and CRX, are key regulators of both rod and cone photoreceptor lineages (16– 18). TRb2, RORb and RXRg have essential functions in cone differentiation (19– 21). The NRL, which is expressed preferentially in rods and pineal gland, is a critical regulator of rod photoreceptor cell fate (10,22– 24). In the Nrl knockout (Nrl2/2 ) mouse, photoreceptor precursors fated to be rods instead produce functional S-cones (10,25,26). Conversely, ectopic expression of Nrl in all Crx-expressing photoreceptor precursors results in functional rods (27,28). Retinoid-related orphan nuclear receptor RORb also participates in rod differentiation acting upstream of the NRL during cell fate determination (29). Recently, BLIMP1 was identified as another regulatory factor involved in photoreceptor differentiation (30,31). The photoreceptor-specific nuclear receptor NR2E3 (also called PNR) (32,33) is a direct downstream target of the NRL (34), and functions as a co-activator of rod genes while concurrently suppressing cone gene expression (10,33,35,36). Mutations in the NR2E3 gene are associated with enhanced S-cone syndrome (ESCS) and related clinical phenotypes (37– 44). Many NR2E3 mutations alter its association with NRL and CRX (45,46). The loss of function of NR2E3, caused by the insertion of antisense L1 (47), leads to additional S-cones in the retinal degeneration 7 (rd7) mice (48,49). The phenotypic similarity of the Nrl2/2 and rd7 mice strongly suggested a critical role for NR2E3 during photoreceptor differentiation in NRL-centric gene regulatory network. It is conceivable that increased S-cones in the rd7 mice are generated from photoreceptor precursors, as in the Nrl2/2 mice. If that is the case, why are only a limited number of precursors transformed into S-cones, and not all those fated to be rods? It has been suggested that additional S-cones originate from abnormal cone proliferation due to the de-suppression of functional NR2E3 in cone precursors (49,50). Though NR2E3 is transiently expressed in both rod and cone progenitors in fish (36), its expression has so far been demonstrated only in developing and mature rods in the mammalian retina (33,35,51). We undertook this study to elucidate molecular mechanism(s) of enhanced S-cone function in the rd7 mouse and ESCS patients. Here, we report that additional S-cones in the rd7 mouse retina originate from the post-mitotic earlyborn photoreceptor precursors that are committed to rod fate and not by proliferation of cone photoreceptors. Our data suggest distinct roles of NRL and NR2E3 in establishing rod identity during retinal differentiation, provide strong evidence for S-cone being the ‘default’ fate of mammalian photoreceptor precursors and reveal insights into possible mechanism of retinal disease caused by NR2E3 mutations.
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RESULTS Loss of Nr2e3 function leads to expression of cone-specific genes in rod precursors To investigate the origin of increased S-cones in rd7 retina, we generated the Nrl::GFP/rd7 mice by breeding the rd7 mouse with the Nrl::GFP/WT transgenic line, where developing and mature rods are tagged with GFP expressed under the control of mouse Nrl promoter (10). Immunohistochemistry with anti-S-opsin antibody revealed positive staining primarily in the ventral retina at different developmental stages that were examined, and more S-opsin(+) cells were detected in the Nrl::GFP/rd7 retina than in the Nrl::GFP/WT retina (Fig. 1A and B, showing data at P21). The S-opsin and GFP signals co-localized in the Nrl::GFP/rd7 retina but not in the Nrl::GFP/WT retina. Cell counting revealed co-labeling of 60– 70% of the S-opsin-positive cells with GFP in the Nrl::GFP/rd7 retina. However, in P28 rd7 retina, the GFP signal was weaker and detectable in fewer S-opsin(+) cells that were present primarily in the middle or inner part of ONL (data not shown). Similar results were obtained with the Nrl2/2 retina, where GFP expression decreased at P28 and was undetectable at later stages (data not shown). Immunostaining of dissociated retinal cells from the Nrl::GFP/rd7 mice was performed to validate the co-localization of S-opsin and GFP in photoreceptors. Both S-opsin and GFP were co-localized in some dissociated retinal cells from P10 Nrl::GFP/rd7 mouse (Fig. 1C), but not from the Nrl::GFP/WT mouse (data not shown). Cell counting showed co-labeling of 50% of the S-opsin(+) cells with GFP. Immunoreactivity of both S-opsin and GFP is detected in the outer segments and cell bodies, respectively. Because outer segments are generally broken off from the cell bodies during dissociation and GFP signal is somewhat weaker, we were not able to detect S-opsin and GFP co-localization in retinal photoreceptors from P28 Nrl::GFP/ rd7 mice. We did not detect any precise co-localization of GFP and M-opsin in either retinal sections or dissociated retinal cells (data not shown). We then performed quantitative RT – PCR (qPCR) assays using RNA from flow-sorted GFP(+) photoreceptors of the Nrl::GFP/rd7 and Nrl::GFP/WT retina (Fig. 2). Reduced expression of rod-specific genes (Rho, Gnat1, Gnb1, Pde6b, Pde6g and Cncg) was observed with a concomitant increase in cone gene transcripts (Opnsw, Gnat2, Gnb3, Pde6c, Clca3 and Arr3, except Opnmw) in the Nrl::GFP/ rd7 samples compared with the Nrl::GFP/WT photoreceptors (Fig. 2). Since the Nrl promoter directed the expression of GFP to developing and mature rod photoreceptors in WT retina, co-localization of S-opsin and GFP, together with expression profiling of GFP(+) photoreceptors of Nrl::GFP/rd7 mice, indicates that some photoreceptor precursors abnormally express S-cone genes in the absence of NR2E3. These data are similar to those obtained with the Nrl2/2 mice, in which all photoreceptor precursors fated to be rods are transformed into S-cones in the absence of functional NRL and NR2E3 (10).
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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.
Excess of S-opsin expressing photoreceptors in the rd7 retina are predominantly generated from the early-born photoreceptor precursors Why do some, but not all, GFP(+) rod cells express S-opsin in the rd7 retina? Previous thymidine birthdating studies in mice had shown that early-born rods are located in the inner and middle part of the ONL, whereas late-born rods are distributed in the outer part of the ONL (9). As most of the excess S-opsin(+) cells are located in the inner and middle part of the ONL in the rd7 retina, we hypothesized that these ‘extra
S-cones’ were generated from photoreceptor precursors that are supposed to produce early-born (prenatal) rods. To examine this further, we performed birthdating studies by injecting a single dose of 5-bromo-2′ -deoxyuridine (BrdU) at E15.5 during the late phase of cone but an early stage of rod genesis, and then analyzed the retina at P21. In the WT retina, most solid BrdU(+) photoreceptors localized to the inner part of the ONL and did not express S-opsin (early-born rods). Fragmented BrdU signals were observed primarily in the outer part of the ONL, indicating that these
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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 nucleotidebinding protein beta 1 (Gnb1), phosphodiesterase b subunit (Pde6b), phosphodiesterase g subunit (Pde6g), cyclic nucleotide-gated channel a-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).
photoreceptors were primarily late-born rods. A few solid BrdU(+) and S-opsin(+) photoreceptors that localized to the outer part of the ONL close to the peripheral retina were late-born cones (Fig. 3A), consistent with a previous report (9). While the overall BrdU staining pattern did not change in the rd7 retina, co-localization of solid BrdU and S-opsin was observed in the inner and middle part of the ONL where rod photoreceptors are present in the WT mice (Fig. 3A). Cell counting revealed 12 + 4 solid BrdU+ cells phf21 (per high power field) in both rd7 and WT retina, with 80 + 10 S-opsin+ cells hpf21 in the rd7 mice versus 25 + 5 S-opsin+ cells hpf21 in the WT retina. Co-labeling of the solid BrdU and S-opsin signals was detected in 10 + 4 cells hpf21 in the rd7 retina compared with 3 + 2 cells hpf21 in the WT retina. To further investigate whether photoreceptors labeled with both BrdU and S-opsin are of rod origin, we performed similar BrdU birthdating in E16.5 Nrl::GFP/rd7 mice and examined the retina at P6, P10 and P21, using the Nrl::GFP/Nrl2/2 mice as control. In the Nrl::GFP/Nrl2/2 retina, both early- and late-born precursor cells gave rise to S-opsin+ photoreceptors. Immunohistochemistry of the Nrl::GFP/rd7 retina at P21 and P10 showed solid BrdUstained cells that were equally distributed above and below the middle line of the ONL, while slightly more solid BrdU-stained cells were observed in the inner part of the ONL at P6 (Fig. 3B, and data not shown). However, the total number of BrdU-positive cells was similar (13 + 4 cells hpf21). The slight difference between P6 and P21 (or P10) might indicate that cells have not finished the migration
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within the ONL before their terminal differentiation. The cells co-labeled with strong GFP, solid BrdU and S-opsin were identified in the ONL of Nrl::GFP/rd7 and Nrl::GFP/ Nrl2/2 mice at P21 (Fig. 3B, and data not shown). However, when the mice were examined at P10 and P6, the GFP signal in BrdU+/S-opsin+ cells was faint but not convincing in both Nrl::GFP/rd7 and Nrl::GFP/Nrl2/2 retina (data not shown), suggesting poor activation of Nrl promoter in the S-opsin+ cells or sequestration of GFP signal by BrdU staining. Cell counting revealed 8 + 3 cells phf21 co-labeled with GFP, BrdU and S-opsin in the Nrl::GFP/rd7 retina at P21, similar to that observed in the Nrl::GFP/ Nrl2/2 mice. We then performed similar BrdU birthdating in late developmental stages by injecting a single dose of BrdU at P2 during the peak stage of rod genesis, and analyzed the retina at P21. Solid BrdU staining was primarily observed in the outer part of the ONL and no significant fragmented BrdU signal identified because of the late proliferative stage of retinal development. Cell counting revealed 40 + 5 BrdU+ cells hpf21 in both rd7 and WT retina. S-opsin and BrdU did not co-localize in either WT or rd7 retina (Fig. 3C), further confirming that only early-born rods express S-opsin in the rd7 retina. We also performed short-term (1 h) BrdU labeling and TUNEL staining at E16, P2, P6 and P10; no obvious differences in cell proliferation and apoptosis were observed between the rd7 and WT retina (data not shown). Thus, our data show that additional S-cones in the rd7 retina are generated from photoreceptor precursors fated to be rods and not by abnormal cell proliferation or selective apoptosis.
Ectopic expression of Nr2e3 suppresses cone genes and restores rod function in the rd7 retina We had previously shown that ectopic expression of Nr2e3 driven by the Crx promoter could suppress all cone genes in the photoreceptor precursors of the Nrl2/2 retina (Crx::Nr2e3/Nrl2/2 mice), but produced only non-functional rods (52). Although the Crx::Nrl transgenic mice showed a rod-only phenotype in the WT background (27), the Nrl transgene could not fully suppress S-opsin in the rd7 background (34), indicating an important role of Nr2e3 during early developmental stage. To investigate the function of Nr2e3 in the early-born photoreceptor precursors, we mated the rd7 mice with the Crx::Nr2e3/Nrl2/2 mice to generate the Crx::Nr2e3/rd7 mice. Immunohistochemical analyses revealed that, in the Crx::Nr2e3/rd7 mice, rhodopsin was uniformly expressed in the ONL, whereas expression of cone genes (S-opsin, M-opsin, and cone arrestin) was undetectable. Whorls and rosettes that are characteristic of rd7 mice were not observed in the Crx::Nr2e3/rd7 retina in both retinal section and by flat mount analysis (Fig. 4A, and data not shown). TUNEL assays in both WT and Crx::Nr2e3/rd7 retina at E16, P2, P6 and P10 revealed no significant differences (data not shown). Therefore, as in the Crx::Nr2e3/WT retina (52), ectopically expressed NR2E3 does not appear to induce the death of photoreceptor (or those committed to cone fate) precursors.
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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(2) 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.
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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.
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Figure 5. Phenotype rescue by Nr2e3 expression in rod precursors of the Nrl::Nr2e3/Nrl2/2 retina. (A) Nrl::Nr2e3 construct. The mouse Nrl promoter (10) was used to drive Nr2e3 expression. (B) Southern analysis of genomic DNA from Nrl2/2 (lane 1) and Nrl::Nr2e3/Nrl2/2 (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/Nrl2/2 retina during the developmental stages. (D) Immunoblot analysis of NR2E3 protein in the Nrl::Nr2e3/Nrl2/2 retina during development. (E) Immunohistochemistry of the Nrl::Nr2e3/Nrl2/2 retina with NR2E3 (red) and rhodopsin (green) antibodies. (F) Immunohistochemistry using the flat-mount of Nrl::Nr2e3/Nrl2/2 retina with rhodpsin (green) and S-opsin (red) antibodies. (G) Immunohistochemistry of the Nrl::Nr2e3/Nrl2/2 retina with rhodpsin (green) antibody, showing a dorsal (D) versus ventral (V) expression pattern.
Scotopic and photopic ERGs with increasing stimulus intensities were performed at 3 months of age to evaluate rod and cone photoreceptor function in the WT (n ¼ 4), rd7 (n ¼ 6) and Crx::Nr2e3/rd7 (n ¼ 5) mice (Fig. 4B and D). Intensity-response plots of a-wave and/or b-wave as a function of stimulus intensity are shown in Figure 4C and E. Rod photoreceptor function, as measured by the
dark-adapted a-wave amplitude, was reduced by 15% at the brighter stimulus intensities in the rd7 mice, yet was restored to normal in the Crx::Nr2e3/rd7 mice (Fig. 4B and C). However, in the Crx::Nr2e3/rd7 mice, rod bipolar function (as measured by b-wave amplitude) tracked the WT amplitudes until about 22.5 log cd s m22 and was reduced by 30% of the WT mice at the brighter stimulus
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intensities. Both WT and rd7 mice had normal cone responses (Fig. 4D and E), while the Crx::Nr2e3/rd7 mice exhibited no detectable photopic response even at maximum stimulus intensity (1.09 log cd s m22). The dark-adapted ERG responses in mice from threshold to approximately 21.5 cd s m22 exclusively reflect rod photoreceptor activity. Above this range, the b-wave is influenced by the emergence of the a-wave and cone photoreceptor activity. The absence of cone responses in the Crx::Nr2e3/rd7 mice would explain the reduction in b-wave amplitude in these mice in the upper intensity range. These results suggest that NR2E3 is sufficient to suppress the cone genes in the photoreceptor precursors and prevent the degeneration of rods in rd7 mouse. Notably, oscillatory potentials (OPs) were present even at dimmer intensities in the scotopic range for the WT, rd7 and Crx::Nr2e3/rd7 mice. Although we did not filter the waveforms to extract the OPs and measure their amplitudes, we observed that the OPs were smaller in five out of six Crx::Nr2e3/rd7 mice when compared with the WT mice (see Fig. 4B). This may indicate a postreceptoral abnormality in the Crx::Nr2e3/rd7 mice. A similar OP amplitude difference was noted between the rd7 and WT mice. Nr2e3 expression in new-born photoreceptors can completely rescue the rd7 phenotype It has been argued that transient expression of Nr2e3 during an early developmental stage suppresses cone proliferation, thereby leading to excess S-cones in the rd7 mice (50). To directly evaluate this possibility, we generated Nrl::Nr2e3 transgenic mice in which expression of Nr2e3 is driven by the Nrl promoter (Fig. 5A), previously shown to drive the expression of GFP in new-born rod photoreceptors of the Nrl::GFP transgenic mice (10). To evaluate the genotype versus phenotype relationship, we first generated the Nrl::Nr2e3 transgenic mice in the rodless Nrl2/2 background, which had no Nr2e3 expression. Southern blot analysis of the Nrl::Nr2e3 transgenic mouse DNA could discriminate the endogenous Nr2e3 gene (9.0 kb band) from the transgene (a weak 8 kb and a strong 4.9 kb band) (Fig. 5B, showing the data from one founder used for breeding the Nrl::Nr2e3/rd7 mice). All seven founder mice that were examined revealed the Nr2e3 transgene DNA, transcript and protein (data not shown); four lines had higher expression level than the other three, which might reflect different transgene insertion sites. Nr2e3 mRNA peaked at P6 and then decreased after P14, while NR2E3 protein was detected at a much lower expression level in the transgenic mice compared with the WT (Fig. 5C and D, showing the data from one founder). Examination of the Nrl::Nr2e3/Nrl2/2 retina showed whorls and rosettes (data not shown). Only some of the photoreceptors expressed rhodopsin, and these cells also immunostained with NR2E3 antibody (Fig. 5E) but not with S-opsin antibody (Fig. 5F). Scotopic and photopic ERGs of the Nrl::Nr2e3/Nrl2/2 mice did not reveal a significant change compared with the Nrl2/2 mice (data not shown). Interestingly, the expression of rhodopsin was
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detected in a dorsal-to-ventral pattern, with higher levels in the dorsal retina (Fig. 5G). A similar phenotype was observed in the transgenic lines with low Nr2e3 expression, but with a less dorsal pattern (data not shown). Notably, this dorsal-to-ventral pattern was also observed in the Nrl::GFP/ WT retina during the early developmental stage (10), suggesting that NR2E3 could only contribute to fate determination in the early- but not late-born photoreceptor precursors to a rod pathway. However, due to low expression during early development, we could not directly demonstrate whether the Nr2e3 transgene was expressed only in post-mitotic cells. We then bred Nrl::Nr2e3/Nrl2/2 mice (the high Nr2e3 expressing lines) with rd7 mice to generate Nrl::Nr2e3/rd7 mice. Immunoblot analysis revealed similar expression levels of Nr2e3 in both Nrl::Nr2e3/rd7 and WT retina (Fig. 6A). We examined retinas from WT, rd7 and Nrl::Nr2e3/rd7 transgenic mice at P21 by immunohistochemistry using antibodies against S- and M-opsins. The number and distribution pattern of S- and M-opsin(+) cells in the Nrl::Nr2e3/rd7 retina appeared to be similar to those in the WT mice (Fig. 6B and C and data not shown). In addition, whorls and rosettes were observed in the rd7 but not in the WT and Nrl::Nr2e3/rd7 retina, as shown in both retinal section and flat mount (Fig. 6B and C). qRT-PCR analysis did not reveal a significant difference in the expression of rod- and cone-specific genes in the retina of WT and Nrl::Nr2e3/rd7 transgenic mice at P21 (data not shown). We conclude that the expression of functional NR2E3 in newborn photoreceptors is sufficient to restore the rod phenotype in the rd7 mice by suppressing the abnormally expressed cone genes and activation of the rod genes. Scotopic and photopic ERGs were also performed at 6 months of age to evaluate rod and cone photoreceptor function, respectively, in the WT (n ¼ 5), rd7 (n ¼ 4) and Nrl::Nr2e3/rd7 (n ¼ 4) mice (Fig. 6D). The dark-adapted aand b-wave amplitudes were reduced by 50% at the higher intensities in the rd7 mice, but they were restored to the WT level in the Nrl::Nr2e3/rd7 mice. Differences in the OPs were observed between the WT and Nrl::Nr2e3/rd7 mice as with the Crx::Nr2e3/rd7 mice. Amplitude of the conemediated b-wave in the rd7 mice was 80% of WT at the maximum stimulus intensity (1.09 log cd s m22) (53). The b-wave amplitude for the Nrl::Nr2e3/rd7 mice was comparable with that of the WT mice. The full rescue of histology and function in the Nrl::Nr2e3/rd7 mice strongly suggests that the excess S-cones in rd7 mice are primarily generated from early-born rod photoreceptors and not by cone proliferation.
DISCUSSION During retinal neurogenesis, an array of cell types are produced from multipotent neuroepithelial progenitors whose states of competence are defined by intrinsic programs and altered over time leading to restricted cell fate choice (8,54,55). The data reported here, combined with previous studies, show that both NRL and NR2E3 play distinct roles during rod photoreceptor differentiation. The NRL first
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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. g-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.
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activates NR2E3 in newborn photoreceptors that are committed to a rod fate. In these early-born rods, NR2E3 suppresses the cone pathway, and together with the NRL it reduces the competence of the early-born photoreceptor precursors, giving rise to functional rods only. In the absence of NR2E3, early-born rods cannot develop normally as S-cone genes (expressed as part of the ‘default’ program) continue to be expressed. In the late-born committed rod precursors, the NRL is sufficient to activate rod differentiation but the presence of NR2E3 is necessary to completely suppress the cone genes in rod precursors. Hence, the late-born cells may give rise to functional ‘hybrid’ rods. The ability of the NRL to drive rod photoreceptor differentiation could also be modulated by posttranslational modification such as sumoylation (56). Indeed, NRL sumoylation appears to be necessary to fully activate NR2E3 expression in late-born photoreceptors and might also regulate the inhibition of cone genes. Recently, sumoylation of NR2E3 was shown to be important for transcriptional suppression of cone gene expression (57). The strength of cone gene inhibition could be explained by the presence of NRL and NR2E3 and also by the regulation of their activity by sumoylation or other posttranslational modifications. We conclude that though NRL determines the rod fate in photoreceptor precursors, a combined action of NRL and NR2E3 is essential for stabilization of the rod fate and their functional maturation. Notably, additional S-cones in the rd7 retina maintain the normal dorsal– ventral gradient. It would therefore appear that only some of the early-born rod precursors express S-cone genes. Several possibilities exist: (i) the NRL alone can suppress S-cone genes, but in a concentration and/or an activity-dependent manner. In the Crx::Nrl/rd7 transgenic mice, ectopic expression of NRL can only partially suppress S-opsin, especially in the dorsal retina (34). Crx promoter directs the expression of NRL in all photoreceptor precursors but its activation may reflect a dorsal-to-ventral pattern (52). A similar dorsal-to-ventral expression pattern is noted for the Nrl promoter during early developmental stages in the Nrl::GFP/ WT transgenic mice (10). It is likely that high activity and/or expression levels of NRL in early-born rods in the dorsal retina and in all late-born rods globally lead to suppression of cone genes. (ii) Other cone gene regulators may play a role in maintaining the dorsal-to-ventral gradient. In the absence of NR2E3, these factors may be sufficient to suppress the expression of S-cone-specific genes but not to influence M-opsin expression. (iii) Asymmetric differentiation can also account for the observed phenotype (15,58,59). In fate mapping experiments, one could use a Cre or Flp strategy to permanently mark a lineage. A caveat of our approach is that the Nrl::GFP promoter may have variable expression in early stages of development; however, the rescue experiments (in Figs 5 and 6) provide strong evidence that Nrl+ cells are still plastic in rd7 mouse and that Nr2e3 expression stabilizes rod fate. Mutations in the human NR2E3 gene have been identified in ESCS patients, which show higher sensitivity to shortwavelength light and early onset rod degeneration (49). The phenotypic findings strongly suggest developmental defects in both rod and cone photoreceptors. Our studies provide a possible mechanism of disease pathogenesis resulting from NR2E3
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mutations. We suggest that aberrant expression or loss of NR2E3 function causes de-repression of cone genes in the developing rod photoreceptors with predominantly S-cone characteristics. It should be noted that S-opsin is the first visual pigment to appear in the human fetal retina, and S-cones account for .90% of the photoreceptors at fetal week 19, with subsequent decrease in number as development proceeds (60). We propose that many of these S-cones acquire rod phenotype upon NRL and NR2E3 expression, and that the loss of functional NR2E3 does not permit complete suppression of S-cone genes leading to ESCS. Aberrant expression of cone genes in NRL-expressing rod photoreceptors would then result in cell death. Our studies are consistent with the transcriptional dominance model (14) and support the combined role of NRL and NR2E3 in driving the rod photoreceptor fate from a postmitotic photoreceptor precursor, which otherwise would follow a ‘default’ pathway to generate S-cones. The model in Figure 7 [based on the data reported here and in refs. (34,52)] represents the scenarios in the presence or in the absence of NRL or NR2E3. The NRL primarily acts as an instructive factor and initiates the expression of genes that define rod morphology and function. However, NRL(+) cells stay in a reversible transition stage and maintain plasticity until NR2E3 is expressed. NR2E3, a downstream target of NRL, acts primarily to stabilize the rod fate by suppressing the cone genes in NRL-expressing photoreceptor precursors. NR2E3 is therefore a key factor for rod photoreceptor lineage restriction.
MATERIALS AND METHODS Generation of transgenic mice All studies involving mice were performed in accordance with institutional and federal guidelines and approved by the University Committee on Use and Care of Animals at the University of Michigan and National Eye Institute. Nrl::GFP/rd7 mice were generated by breeding rd7 (C57BL/6) mice with Nrl::GFP/WT (C57BL/6 X SJL) mice (10). The Crx::Nr2e3/rd7 mice were generated by breeding rd7 (C57BL/6) mice with Crx::Nr2e3/WT (mix background of 129X1/SvJ and C57BL/6) mice (52). The Nrl::Nr2e3/rd7 mice were generated with the same Nrl promoter used in the Nrl::GFP/WT mouse. Briefly, the eGFP gene in the Nrl::GFP construct (10) was replaced with Nr2e3 gene (52). The 4.9 kb Nrl::Nr2e3 fragment, excluding the vector backbone, was purified and injected into fertilized Nrl2/2 (mix background of 129X1/SvJ and C57BL/6J) mouse oocytes (UM transgenic core facility). Transgenic founder mice and their progeny were identified by PCR, and then confirmed by Southern blot analysis of tail DNA. Transgenic founders were bred to the Nrl2/2 mice to generate F1 progeny. The transgenic progeny was then mated to rd7 mice to generate Nrl::Nr2e3/rd7 mice. Immunohistochemistry We used 7 mm thick retinal cryosections for immunohistochemistry with the following primary antibodies: rabbit anti S-opsin polyclonal antibody (a gift from C. Craft), mouse anti-
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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 TRb2 in the latter induces M-cone differentiation, while the remaining NRL(2) 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 Nrl2/2 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(2) cells represses cone genes and produces rod-like characteristics, whereas expression of NRL (and consequently NR2E3) generates functional rods.
rhodopsin (4D2) monoclonal antibody (a gift from R. Molday) and rat anti-BrdU monoclonal antibody (BU1/75, Harlan Sera-Lab, Loughborough, UK). Fluorescent detection was performed using AlexaFluor-546 or 633 (Molecular Probes), and Texas Red (Jackson ImmunoResearch, West Grove, PA) conjugated secondary antibodies. Sections were visualized under a conventional fluorescent microscope or FV500 confocal
microscope and digitized. The detailed methods have been described previously (52). Retinal cell dissociation and Immunocytochemistry Freshly dissected retina was transferred into an eppendorf tube with 500 ml PBS. Trypsin was added at a final concentration
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of 1 mg ml21 and incubated at 378C for 5 min. The mixture was triturated 3 – 5 times with a P1000 tip to generate a singlecell suspension. Then, 50 ml of horse serum was added to inhibit trypsin, followed by the addition of DNase I (final concentration 20 mg/ml). Dissociated cells were then centrifuged at 3000g for 1 min. Cells were resuspended in PBS and fixed with 4% paraformaldehyde (PFA). Immunocytochemistry procedure was similar, except that the cells were incubated on a shaker and spun down using a centrifuge (52). qPCR analysis The protocol and primers for qPCR of rod and cone genes have been described previously (10,25,31). Briefly, total RNA was extracted from the freshly flow-sorted GFP+ retinal cells or whole retina using Trizol reagent (Invitrogen) and then treated with RNase-free DNase I before reverse transcription. Quantitative (real-time) PCR (35 cycles) was conducted with the iCycler IQ system (BioRad, Hercules, CA, USA). Hprt gene was used as an internal control. BrdU birthdating Timed pregnant females or pups received a single intraperitoneal injection of BrdU (Sigma, 0.1 mg g21 body weight) at E15, E16, E17, P2, P6 or P10. Pups were sacrificed after 1 h or at 3 weeks of age. Eyes were fixed in 4% PFA and cryosectioned. BrdU immunohistochemistry was performed after the immunostaining with other antibodies. Sections were washed in PBTx (PBS + 0.1% Triton X100) and incubated in 2.4 N HCl– PBTx at 378C for 75 min. After washing with PBTx, the sections were processed for BrdU staining (10). Cell counting All eyeballs were oriented appropriately to reflect ventral-to-dorsal expression of S-opsin in the mouse retina, and the sections were cut through the optic nerve. The area chosen for cell counting were in the middle part of ventral retina. Three sections from three different mice were used after immunohistochemistry. The criteria for solid BrdU staining included BrdU staining outlining a nucleus shape and signal over at least 70% of nucleus. Pictures taken by ×40 objective lens (high power field) were used for quantification. ERG recording ERGs were recorded as described (34,61). Briefly, animals were dark-adapted for 12 h and were prepared under dim red illumination. Animals were anesthetized with a loading dose of ketamine (93 mg kg21, IP) and xylazine (8 mg kg21, IP). Pupils were dilated with topical 1% atropine and 0.5% tropicamide. Body temperature was maintained at 378C with a heating pad. Corneal ERGs were recorded from both eyes using gold wire loops with 0.5% tetracaine topical anesthesia and a drop of 2% methylcellulose for corneal hydration. A gold wire loop placed in the mouth was used as reference, and ground electrode was on the tail. ERGs were recorded with a Ganzfeld configuration to brief xenon white flashes (1.09 log cd s m22 per flash maximum
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intensity). Responses were amplified at 10 000 gain at 0.1 –1000 Hz, and digitized at a rate of 32 kHz. A notch filter was used to remove 60 Hz line noise. Scotopic ERGs were recorded at 3 – 60 s intervals depending on the stimulus intensity and were computer averaged. Stimulus intensity was attenuated with neutral density filters over a 6.5 log unit range. Animals were then light adapted for 10 min by exposure to a white 32 cd m22 rod saturating background, and photopic ERGs were recorded for single flash white stimuli over a 2 log unit range.
ACKNOWLEDGEMENTS We are grateful to P. Raymond, P.F. Hitchcock, T. Glaser, D. Goldman, R. Koenig, M. Uhler and G.R. Dressler for stimulating discussions and/or comments on the manuscript. We acknowledge Steve Lenz (confocal facility), Thom Saunders (transgenic core), Maggie Van Keuren (transgenic core), M. Gillett, Scott Szalay and Scott Almburg (vision core, P30 EY007003) and Seid Ali for technical assistance, members of the Swaroop laboratory (specially Edwin Oh and Jessica Chang) for comments/discussions, and S. Ferrara and L. Lawrence for administrative support. Conflict of Interest statement. None declared.
FUNDING This research was supported by the intramural program of the National Eye Institute and by grants from the National Institutes of Health (EY011115), The Foundation Fighting Blindness, Research to Prevent Blindness, Organogenesis Training Program (T-32-HD007505) and Michigan Diabetes Research and Training Center.
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