Photoreceptor-specific Nuclear Receptor Research Articles

Dual role of Nr2e3 in photoreceptor development and maintenance

Nr2e3 is a photoreceptor-specific nuclear receptor believed to play a role in photoreceptor development, differentiation, and survival. Much research has focused on the interaction of Nr2e3 with other transcription factors in determining the milieu of target gene expression in photoreceptors of the neonatal and adult retina. To investigate the downstream targets of Nr2e3 and thereby shed light on the functional pathways relevant to photoreceptor development and maintenance, expression profiling was performed on retinas from two different mouse knockout lines, one containing a targeted disruption of the Nr2e3 gene ( Nr2e3−/−), the other containing a spontaneous null allele of the Nr2e3 locus ( rd7). Using whole genome microarrays, mRNA expression profiles of retinas from the two mutant strains were compared to those of wildtype C57BL/6 mice over a time course that ranged from postnatal day (p) 2 to 6 months of age (p180). Additionally, expression profiling was performed on retinal explants treated with a putative NR2E3 agonist. The molecular profiling of Nr2e3−/− and rd7/rd7 retinas identified 281 putative Nr2e3-dependent genes that were differentially expressed between wildtype and mutant retinas during at least one time point. Consistent with previous reports that Nr2e3 is necessary for the repression of cone-specific genes, increased expression of cone-specific genes was observed in the mutant samples, thereby providing proof-of-concept for the microarray screen. Further annotation of these data sets revealed ten predominant functional classes involved in the Nr2e3-mediated development and/or maintenance of photoreceptors. Interestingly, differences in the expression of Nr2e3-dependent genes exhibited two distinct temporal patterns. One group of genes showed a sustained difference in expression as compared to wildtype over the entire time course of the study, whereas a second group showed only transient differences which were largest around p10. Comparison of gene expression changes in Nr2e3−/− and rd7/rd7 retinas with those uncovered by treating retinal explants with a putative NR2E3 agonist revealed four genes that were down-regulated in mutant retinas that lack Nr2e3 function but were up-regulated in agonist-treated explants. These results strongly suggest that the four genes may be direct targets of Nr2e3. Our identification of two sets of Nr2e3-regulated genes provides further evidence of a dual role for Nr2e3 in specification of photoreceptor fate during development as well as photoreceptor maintenance in the adult.

Nr2e3 and Nrl can reprogram retinal precursors to the rod fate in Xenopus retina

Transformation of undifferentiated progenitors into specific cell types is largely dependent on temporal and spatial expression of a complex network of transcription factors. Here, we examined whether neural retina leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3 transcription factors contribute to cell fate determination. We cloned the Xenopus Nr2e3 gene and showed that its temporal and spatial expression is similar to its mammalian ortholog. We tested its in vivo function by misexpressing these transcription factors in Xenopus eye primordia, demonstrating that either human Nr2e3 or Nrl directed photoreceptor precursors to become rods at the expense of cones. Furthermore, overexpression of Xenopus Nrl dramatically increased the number of lens fibers, whereas human Nrl did not, suggesting evolutionary divergence of function of the Nrl gene family. Misexpression of Nrl and Nr2e3 together were more effective than either transcription factor alone in directing precursors to the rod fate.

Expression of immature and mature retinal cell markers in retinoblastoma

To clarify the expression of immature and mature retinal cell makers in retinoblastoma cells and to give insights into the cell origin of the retinoblastoma. Five samples from five eyes diagnosed with retinoblastomas were analysed by a standard immunohistochemistry using antibodies against Nestin and the hairy and enhancer of split mammalian homologue-1 (HES-1), both as markers for undifferentiated cells, and against Chx10, as a marker for both undifferentiated retinal cells and mature bipolar cells. Photoreceptor-specific nuclear receptor (PNR) was used as a postmitotic rod photoreceptor cell-specific marker, glial fibrillary acidic protein (GFAP) as a mature glia cell marker, and microtubule-associated protein (MAP) 2 as a mature neuronal cell marker. Nestin was detected in what were possibly Müller cells, but not in the tumour stroma. HES-1 was not detected in the retinoblastoma tissue. Chx10 was detected in one of the five samples. In this one sample, Chx10 expression was confined in a minor portion of the retinoblastoma cells. PNR was not detected in the retinoblastoma tissue. Expression of GFAP was detected only in the stromal cells of the tumour, which presumably represents reactive stromal astrocytes. In contrast, in all the samples, MAP2 was expressed in most of the retinoblastoma cells. The results of the current study support that retinoblastomas are derived from mature neural cells but do not originate from tumour stem cell(s).

The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain–hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24 hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2 dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.

Nuclear receptors of the honey bee: annotation and expression in the adult brain

The Drosophila genome encodes 18 canonical nuclear receptors. All of the Drosophila nuclear receptors are here shown to be present in the genome of the honey bee (Apis mellifera). Given that the time since divergence of the Drosophila and Apis lineages is measured in hundreds of millions of years, the identification of matched orthologous nuclear receptors in the two genomes reveals the fundamental set of nuclear receptors required to ‘make’ an endopterygote insect. The single novelty is the presence in the A. mellifera genome of a third insect gene similar to vertebrate photoreceptor-specific nuclear receptor (PNR). Phylogenetic analysis indicates that this novel gene, which we have named AmPNR-like, is a new member of the NR2 subfamily not found in the Drosophila or human genomes. This gene is expressed in the developing compound eye of the honey bee. Like their vertebrate counterparts, arthropod nuclear receptors play key roles in embryonic and postembryonic development. Studies in Drosophila have focused primarily on the role of these transcription factors in embryogenesis and metamorphosis. Examination of an expressed sequence tag library developed from the adult bee brain and analysis of transcript expression in brain using in situ hybridization and quantitative RT-PCR revealed that several members of the nuclear receptor family (AmSVP, AmUSP, AmERR, AmHr46, AmFtz-F1, and AmHnf-4) are expressed in the brain of the adult bee. Further analysis of the expression of AmUSP and AmSVP in the mushroom bodies, the major insect brain centre for learning and memory, revealed changes in transcript abundance and, in the case of AmUSP, changes in transcript localization, during the development of foraging behaviour in the adult. Study of the honey bee therefore provides a model for understanding nuclear receptor function in the adult brain.

Identification of potent agonists of photoreceptor-specific nuclear receptor (NR2E3) and preparation of a radioligand

Agonists of NR2E3 (PNR, RNR) have been identified and optimized to EC 50 < 200 nM. A tritiated analogue of one agonist was prepared to aid in the development of a binding assay.

The orphan nuclear receptor Nr2e3 plays dual functions in rod photoreceptor differentiation

During retinal development, rod and cone photoreceptors are generated from common pools of neuroepithelial progenitors. Nrl, Crx, and Nr2e3 are the key transcriptional regulators that control rod differentiation. Nr2e3 is a rod photoreceptor specific orphan nuclear receptor. However, the loss of its function results in enhanced S-cones and rod degeneration in both human (ESCS patients) and mice (rd7) retina. Nr2e3 is not detected in the Nrl / retina, which exhibits excess functional S-cones at the expense of rods. Here, we show that, using GFP to tag the newborn rod precursors, some early born rod precursors are transformed into S-opsin-positive cells in the rd7 mouse. On the other hand, forced over-expression of functional Nr2e3 in postmitotic cone precursors induces them to adopt rod pathway at the expense of cone differentiation. However, these new rod-like photoreceptors are not functional, partially due to the lack of rod transducin. The dual functions of Nr2e3 on rod and cone gene regulation are not dependent on Nrl and/or Crx but rely on its expression time and level. Thus, our in vivo studies reveal a critical role of Nr2e3 in rod photoreceptor differentiation during retinal development.

Effects of L1 retrotransposon insertion on transcript processing, localization and accumulation: lessons from the retinal degeneration 7 mouse and implications for the genomic ecology of L1 elements

The retinal degeneration 7 (rd7) mouse is a naturally occurring model of enhanced S-cone syndrome, Goldman-Favre syndrome and clumped pigmentary retinopathy in humans, allelic disorders caused by inactivation of a photoreceptor-specific nuclear hormone receptor, NR2E3. We show here that the rd7 mutation arose from the antisense insertion of a long interspersed nuclear element (LINE-1) (or L1) into exon 5 of the mouse Nr2e3 gene. L1 insertion blocks splicing of Nr2e3 intron 5 by separating an inefficient splice donor from essential splicing enhancers within exon 5, with the result that incompletely spliced transcripts accumulate to high levels at the mutant Nr2e3 locus in photoreceptor nuclei. The high efficiency of transcription through the 7 kb L1 was unexpected and led us to compare the effect on transcript abundance of sense or antisense L1 insertions in transfected cells. In a variety of sequence contexts antisense L1 insertions had little or no effect on transcript levels or the production of full-length transcripts, whereas sense L1 insertions reduced transcript levels from several-fold to more than 10-fold. A bioinformatic analysis of all mouse L1s shows a approximately 2-fold under-representation of L1s in introns when compared with bulk genomic DNA, and, within introns, a further approximately 2-fold under-representation of sense when compared with antisense L1s. Interestingly, there is no evidence for orientation-specific positive or negative selection within any subregions of the L1 element. These data suggest that L1s have evolved to present the host transcriptional machinery with a minimally disruptive profile in the antisense orientation such that antisense intronic L1s often escape purifying negative selection.

The photoreceptor-specific nuclear receptor Nr2e3 interacts with Crx and exerts opposing effects on the transcription of rod versus cone genes

Nr2e3 is an orphan nuclear receptor expressed specifically by retinal photoreceptor cells. Mutations in Nr2e3 result in syndromes characterized by excess blue cones and loss of rods: enhanced S-cone syndrome (ESCS) in humans and rd7 in mice. Using yeast two-hybrid screens with Nr2e3 as bait, the cone-rod homeobox protein Crx was identified as an interacting partner of Nr2e3. Immunoprecipitation assays confirmed this Nr2e3-Crx interaction and identified the DNA-binding domain of each protein as the interaction motif. Immunohistochemistry demonstrated that Crx and Nr2e3 are co-expressed by rod photoreceptors and their precursors. Chromatin immunoprecipitation assays on mouse retina demonstrated that Nr2e3 and Crx co-occupy the promoter/enhancer region of several rod and cone genes in the rod photoreceptor cells. The promoter/enhancer occupancy of Nr2e3 is Crx-dependent, suggesting that Nr2e3 is associated with photoreceptor gene targets by interacting with Crx. Transient transfection assays in HEK293 cells demonstrated that Nr2e3 enhances rhodopsin, but represses S- or M-cone opsin transcription when interacting with Crx. Quantitative real-time RT-PCR analysis on postnatal day 28 (P28) retina of the rd7 mouse, which lacks Nr2e3 protein, revealed an up-regulation of cone genes, but down-regulation of rod genes. Several mutant forms of human Nr2e3 identified from ESCS patients showed defects in interacting with Crx and/or in transcriptional regulatory function. Altogether, our findings suggest that Nr2e3 is a dual-function transcriptional regulator that acts in concert with Crx to promote and maintain the function of rod photoreceptors.

The Rod Photoreceptor-Specific Nuclear Receptor Nr2e3 Represses Transcription of Multiple Cone-Specific Genes

This study addresses one genetic regulatory mechanism that establishes the distinct identities of rod and cone photoreceptors. Previous work has shown that mutations in either humans or mice in the gene coding for photoreceptor-specific nuclear receptor Nr2e3 cause a progressive retinal degeneration characterized by increased numbers of short-wave cones. In the present work, we have examined the cellular and developmental pattern of Nr2e3 protein localization in mammals and fish, identified an optimal Nr2e3 DNA-binding site using cycles of binding to recombinant Nr2e3, characterized the transcriptional activity of wild type and one of the disease-associated point mutations in Nr2e3 in transfected cells, and characterized the transcriptional defects in the naturally occurring Nr2e3 mutant (rd7) mouse. These experiments indicate that in the mature vertebrate retina Nr2e3 is expressed exclusively in rods, that expression of Nr2e3 is one of the earliest events in the pathway of rod-specific photoreceptor development, and that Nr2e3 functions, either directly or indirectly, as a repressor of cone-specific genes in rod photoreceptor cells.

Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors.

NR2E3, a photoreceptor-specific orphan nuclear receptor, is believed to play a pivotal role in the differentiation of photoreceptors. Mutations in the human NR2E3 gene and its mouse ortholog are associated with enhanced S-cones and retinal degeneration. In order to gain insights into the NR2E3 function, we performed temporal and spatial expression analysis, yeast two-hybrid screening, promoter activity assays and co-immunoprecipitation studies. The Nr2e3 expression was localized preferentially to the rod, and not to the cone, photoreceptor nuclei in rodent retina. The yeast two-hybrid screening of a retinal cDNA library, using NR2E3 as the bait, identified another orphan nuclear receptor NR1D1 (Rev-erbalpha). The interaction of NR2E3 with NR1D1 was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. In transient transfection studies using HEK 293 cells, both NR2E3 and NR1D1 activated the promoters of rod phototransduction genes synergistically with neural retina leucine zipper (NRL) and cone-rod homeobox (CRX). All four proteins, NR2E3, NR1D1, NRL and CRX, could be co-immunoprecipitated from the bovine retinal nuclear extract, suggesting their existence in a multi-protein transcriptional regulatory complex in vivo. Our results demonstrate that NR2E3 is involved in regulating the expression of rod photoreceptor-specific genes and support its proposed role in transcriptional regulatory network(s) during rod differentiation.

Nuclear receptors in disease: thyroid receptor beta, peroxisome-proliferator-activated receptor gamma and orphan receptors.

The nuclear receptor superfamily comprises a group of proteins that includes the molecular targets for classical steroid hormones such as glucocorticoids, androgens and vitamin D, together with a number of so-called 'orphan' receptors whose ligands and/or function remain to be determined. Many of the world's most commonly prescribed drugs act via nuclear receptors, attesting to their importance as therapeutic targets in human disease [for example, the novel anti-diabetic thiazolidinediones rosiglitazone and pioglitazone are high-affinity ligands for peroxisome-proliferator-activated receptor gamma (PPARgamma)]. The study of transgenic mice harbouring global and tissue-specific alterations in nuclear receptor genes has greatly enhanced our understanding of the roles that these receptors play in mammalian physiology. In many cases, these findings have been complemented by the study of human subjects harbouring naturally occurring mutations within the corresponding receptor, whereas in others, such studies have served to highlight important differences that exist between human and mouse physiology especially, for example, in relation to aspects of metabolism. Here we review the diverse clinical phenotypes that have been reported in subjects found to have germline mutations in thyroid hormone receptor beta, PPARgamma, hepatocyte nuclear factor 4alpha, small heterodimer partner, steroidogenic factor 1, DAX1, photoreceptor-specific nuclear receptor and NUR-related factor 1, and consider the molecular mechanisms through which aberrant signalling by mutant receptors might contribute to the pathogenesis of the associated disorders.

A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse.

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Identification of a photoreceptor cell-specific nuclear receptor.

Nuclear receptors comprise a large and expanding family of transcription factors involved in diverse aspects of animal physiology and development, the functions of which can be modulated in a spatial and temporal manner by access to small lipophilic ligands and/or the specificity of their own localized expression. Here we report the identification of a human nuclear receptor that reveals a unique proximal box (CNGCSG) in the DNA-binding domain. The conservation of this feature in its nematode counterpart suggests the requirement for this type of P box in the genetic cascades mediated by nuclear receptors in a wide variety of animal species. The expression of this receptor, PNR (photoreceptor-specific nuclear receptor), appears strongly restricted in the retina, exclusively in photoreceptor cells. In human cell lines, PNR expression was observed in Y79 retinoblastoma along with other photoreceptor marker genes such as CRX. Among vertebrate receptors, PNR shares structural kinship with an orphan receptor TLX, and despite distinct differences in the DNA binding domain, PNR is able to recognize a subset of TLX target sequences in vitro. Analyses of the human PNR gene revealed its chromosomal position as 15q24, a site that has recently been reported as a susceptible region for retinal degeneration. These data support a role for PNR in the regulation of signalling pathways intrinsic to the photoreceptor cell function.