To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE).
Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR.
Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified.
The EyeSAGE database, combining three different gene-profiling platforms including the authors’ multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.
The retinal pigment epithelium (RPE) plays an essential role in maintaining the health of the retina. The RPE is also the site of pathologic processes in a wide variety of retinal disorders including monogenic retinal dystrophies, age-related macular degeneration, and retinal detachment. Despite intense interest in the RPE, little is known about its molecular response to ocular damage or disease. We have conducted a comprehensive analysis of changes in transcript abundance (the “genomic response”) in the murine RPE following light damage. Several dozen transcripts, many related to cell-cell signaling, show significant increases in abundance in response to bright light; transcripts encoding visual cycle proteins show a decrease in abundance. Similar changes are induced by retinal detachment. Environmental and genetic perturbations that modulate the RPE response to bright light suggest that this response is controlled by the retina. In contrast to the response to bright light, the RPE response to retinal detachment over-rides these modulatory affects.
retinal pigment epithelium; transcription; micro-array; mouse; ocular disease; light damage; retinal detachment
Separation of the neurosensory retina from the retinal pigment epithelium (RPE) yields many morphologic and functional consequences, including death of the photoreceptor cells, Müller cell hypertrophy, and inner retinal rewiring. Many of these changes are due to the separation-induced activation of specific genes. In this work, we define the gene transcription profile within the retina as a function of time after detachment. We also define the early activation of kinases that might be responsible for the detachment-induced changes in gene transcription.
Separation of the retina from the RPE was induced in Brown-Norway rats by the injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested at 1, 7, and 28 days after separation. Gene transcription profiles for each time point were determined using the Affymetrix Rat 230A gene microarray chip. Transcription levels in detached retinas were compared to those of nondetached retinas with the BRB-ArrayTools Version 3.6.0 using a random variance analysis of variance (ANOVA) model. Confirmation of the significant transcriptional changes for a subset of the genes was performed using microfluidic quantitative real-time polymerase chain reaction (qRT-PCR) assays. Kinase activation was explored using Western blot analysis to look for early phosphorylation of any of the 3 main families of mitogen-activated protein kinases (MAPK): the p38 family, the Janus kinase family, and the p42/p44 family.
Retinas separated from the RPE showed extensive alterations in their gene transcription profile. Many of these changes were initiated as early as 1 day after separation, with significant increases by 7 days. ANOVA analysis defined 144 genes that had significantly altered transcription levels as a function of time after separation when setting a false discovery rate at ≤0.1. Confirmatory RT-PCR was performed on 51 of these 144 genes. Differential transcription detected on the microarray chip was confirmed by qRT-PCR for all 51 genes. Western blot analysis showed that the p42/p44 family of MAPK was phosphorylated within 2 hours of retinal-RPE separation. This phosphorylation was detachment-induced and could be inhibited by specific inhibitors of MAPK phosphorylation.
Separation of the retina from the RPE induces significant alteration in the gene transcription profile within the retina. These profiles are not static, but change as a function of time after detachment. These gene transcription changes are preceded by the activation of the p42/p44 family of MAPK. This altered transcription may serve as the basis for many of the morphologic, biochemical, and functional changes seen within the detached retina.
The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE–specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.
The retinal pigment epithelium or RPE in the back of the eye is critical for the normal function of the retina, and its abnormalities can lead to retinal disorders such as adult-onset macular degeneration. Insights into the pathogenesis of such disorders, and potential therapies, may come from using RPE cells generated in vitro from induced pluripotent stem cells. To obtain authentic RPE cells in vitro, we need to thoroughly understand the normal process of their development in vivo. Here we find that the potent retina-inducing transcription factor PAX6 plays a critical anti-retinogenic role in the RPE of mice. But how can PAX6 be pro-retinogenic in the retina and anti-retinogenic in the RPE? To address this question, we used gene expression studies and combined them with chromatin immunoprecipitation assays, which analyze the interaction of transcription factors with chromatin in vivo. Our findings show that, in the RPE, PAX6 cooperates with either one (or both) of two related RPE transcription factors, MITF and TFEC, to suppress extracellular signals that in the normal retina induce a signaling cascade promoting retina formation. Hence, this study provides mechanistic insights into RPE development that may become important for the efficient generation of retina and RPE from induced pluripotent stem cells.
This study showed that mouse retina and retinal pigment epithelial (RPE) cells express the heme transporters FLVCR, BCRP, and PCFT. FLVCR is localized to the apical membrane, and BCRP and PCFT are localized to the basolateral membrane in RPE cells. Hemochromatosis, a genetic disease with iron overload, is associated with upregulation of FLVCR and PCFT, but with downregulation of BCRP in retina and RPE.
FLVCR, BCRP, and PCFT/HCP-1 represent the three heme transporters identified thus far in mammalian cells, but there is very little known about their expression and regulation in the retina. In this study, the expression of these transporters in mouse retina and retinal pigment epithelium (RPE) and their regulation in the iron-overload disease hemochromatosis were examined.
The expression of FLVCR, BCRP, and PCFT in mouse retina and primary mouse RPE cells was studied by RT-PCR and immunofluorescence. Polarized localization of the transporters in RPE was studied by co-localization using a specific marker of the RPE apical membrane. Uptake of heme in primary RPE cells was determined using zinc-mesoporphyrin, a fluorescent heme analogue. The regulation of heme transporters by iron overload was studied in two genetic models of hemochromatosis (HFE-null mouse and HJV-null mouse) and in two nongenetic models of iron overload (cytomegalovirus infection and treatment with ferric ammonium citrate).
All three heme transporters were expressed in the retina and RPE. In the RPE, the expression of FLVCR was restricted to the apical membrane, and the expression of BCRP and PCFT was restricted to the basolateral membrane. In all cases of iron overload, the expression of FLVCR and PCFT was upregulated and that of BCRP was downregulated.
Hemochromatosis is associated not only with excessive accumulation of free iron in the retina and RPE but also with excessive accumulation of heme. Since heme is toxic at high levels, as is free iron, heme-induced oxidative damage may also play a role in hemochromatosis-associated retinal pathology.
Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5′ promoter region, intron1 and the 3′ non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm2) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.
During development of the central nervous system, stem and progenitor cell proliferation and differentiation are controlled by complex inter- and intracellular interactions that orchestrate the precise spatiotemporal production of particular cell types. Within the embryonic retina, progenitor cells are located adjacent to the retinal pigment epithelium (RPE), which differentiates prior to the neurosensory retina and has the capacity to secrete a multitude of growth factors. We found that secreted proteinaceous factors in human prenatal RPE conditioned media (RPE CM) prolonged and enhanced the growth of human prenatal retinal neurospheres. The growth-promoting activity of RPE CM was mitogen-dependent and associated with an acute increase in transcription factor phosphorylation. Expanded populations of RPE CM-treated retinal neurospheres expressed numerous neurodevelopmental and eye specification genes and markers characteristic of neural and retinal progenitor cells, but gradually lost the potential to generate neurons upon differentiation. Misexpression of Mash1 restored the neurogenic potential of long term cultures, yielding neurons with phenotypic characteristics of multiple inner retinal cell types. Thus, a novel combination of extrinsic and intrinsic factors was required to promote both progenitor cell proliferation and neuronal multipotency in human retinal neurosphere cultures. These results support a pro-proliferative and anti-apoptotic role for RPE in human retinal development, reveal potential limitations of human retinal progenitor culture systems, and suggest a means for overcoming cell fate restriction in vitro.
We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.
gene delivery; intravitreal; retina; liposome; polymer
During mammalian eye development, the restriction of Wnt/β-catenin signaling at the junction of the neural retina and the retinal pigment epithelium in the peripheral eyecup is required for the development of the ciliary margin, a non-neural region of the eyecup that is the precursor of the ciliary body and iris of the adult eye.
To identify genes that are modulated by β-catenin activity in the embryonic retina, we performed gene expression profiling in Li+-treated retinal explants, a pharmacological model of β-catenin activation. The Li+-modulated gene data set was searched for β-catenin/T-cell specific transcription factor binding sites.
Functional annotations of this data set revealed significant enrichments for genes involved in chromatin organization, neurogenesis, and cell motion/migration. Quantitative real-time polymerase chain reaction (qRT–PCR) analysis confirmed the modulation of 12 genes in Li+-treated explants and retinas of mice with Cre-mediated induction of constitutively active β-catenin (β-catact). In situ hybridization revealed β-catenin-specific upregulation of cyclin-dependent kinase inhibitor 1A (P21) [Cdkn1a] and tumor necrosis factor receptor superfamily, member 19 (Tnfrsf19) in the developing retina consistent with the antineurogenic and proliferation changes associated with ectopic Wnt/β-catenin signaling in the eyecup.
This data set of Li+-modulated genes provides a valuable resource for characterizing the Wnt/ β-catenin regulated gene network in eyecup patterning.
Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.
The effect of the functional deletion of the RPE65 gene on the expression of molecular markers in the mutant dog retina was investigated. Some markers changed in the inner and outer retina, but the retinal structure was well preserved.
Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed.
Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies.
Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas.
The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans.
The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis. Stem Cells 2012;30:2784–2795
Retina; Neural stem cells; Hes4/XHairy2; Wnt and Hedgehog signaling; Cell cycle kinetics
Retina organ cultures can be used as a valuable tool to study retina development ex vivo. Comparison between culture methods has revealed that timing the start of the culture and the presence of the retinal pigment epithelium (RPE) are critical for the development of the rods and cones, which are the two types of photoreceptors; rods can develop in the absence of the RPE, cones cannot. One of the necessary compounds produced by the RPE and essential for cone development and survival is the chromophore 11-cis retinal. Here, we further examined rod and cone development, chromophore production by the RPE, and photoreceptor signaling to the inner retina under organ culture conditions.
Retina-RPE cultures were prepared from 7-day-old C57BL/6 pups and maintained in culture for 11 days. Rod and cone structure was analyzed by immunohistochemistry, and cell-specific mRNA expression was analyzed by quantitative real-time PCR. We quantified 11-cis retinal spectrophotometrically by measuring rhodopsin. Signal transmission in the rod pathway was studied by analyzing c-fos expression in the inner retina in response to stroboscopic illumination.
In retina-RPE cultures analyzed after 11 days in culture, rod and cone numbers exhibited a similar ratio to those observed in the intact animal. Although photoreceptor outer segments were shorter when grown ex vivo, membrane proteins, such as cone opsin and transducin, were localized appropriately to the outer segment. Relative 11-cis retinal production ex vivo plateaued after 7 days in culture, resulting in approximately 30% of the in vivo level by day 11. The retinas responded to prolonged stroboscopic illumination with the normal nuclear expression of c-fos in cells in the inner retina.
Mouse retinal structure is maintained in retina–RPE organ cultures. The RPE in organ cultures produces sufficient amounts of 11-cis retinal to promote cone development and support signal transmission in the rod pathway. Organ cultures may be a powerful low-throughput screening tool to identify novel agents to promote photoreceptor cell survival and signaling.
Embryonic and induced pluripotent stem cells (IPSCs) derived from mammalian species are valuable tools for modeling human disease, including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. Here we used transgenic common marmoset (Callithrix jacchus) and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP) reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures, we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications.
Genome-wide characterization of the retinal transcriptome is central to understanding development, physiology and disorders of the visual system. Massively parallel, short-read sequencing of mRNA libraries was used to generate an extensive map of the transcriptome of the adult, murine neural retina. RNA-seq data strongly corroborates prior transcriptome studies by microarray and SAGE. However, several novel features of the retinal transcriptome were discovered. For example, retinal disease genes were discovered to be among the most highly expressed in the transcriptome. We also demonstrate other interesting features of the retinal transcriptome, for example, that the retina appears to employ a very specific and restricted set of synaptic vesicle genes, and also that there is persistence of expression of a majority of “neurodevelopmental” genes into adulthood. Retina transcriptome studies utilizing novel sequencing methods have been highly informative and these data may also serve as a resource for the community of researchers.
retina; transcriptome; RNA-seq; mouse; alternative splicing
To understand the expression of genes involved in different complement pathways in the retina and retinal pigment epithelium (RPE)/choroid under physiologic conditions and how their expression is regulated by inflammatory cytokines.
The expression of complement components of the classical pathway (CP), mannose-binding lectin (MBL) pathway, alternative pathway (AP), and terminal pathway in the retina and RPE/choroid was determined by conventional reverse transcription polymerase chain reaction (RT–PCR). The effect of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α, 20 ng/ml), interleukin (IL)-6 (10 ng/ml), interferon-gamma (IFN-γ, 100 ng/ml) or lipopolysaccharides (LPS, 1 μg/ml) on the expression of these complement component genes was tested in vitro in primary cultured RPE cells and a microglial cell line (BV2 cells) and quantified by real-time RT–PCR.
In the CP, complements C1qb, C1r, C1s, C2, and C4 were constitutively expressed by retina and RPE/choroid. Complement factor H and factor B of the AP as well as C3 were also detected in the retinal and RPE/choroidal tissues. In the MBL pathway, low levels of mannose-binding lectin (MBL)-associated serine protease (MASP)-1 in the retina and RPE/choroid and MASP2L in the retina were detected. Other components, including mannose-binding lectin 1 (MBL1), mannose-binding lectin 2 (MBL2), complement factor I (CFI), complement component 5 (C5) and complement factor H-related protein 1 (CFHR1), were not detected in either the retina or the RPE/choroid. The expression of CP- and AP-complement component genes in RPE and microglial cells was upregulated by interferon (IFN)-γ treatment. Treatment with TNF-α selectively upregulated the expression of C1s and C3 genes but downregulated complement factor H gene expression in RPE and microglial cells. The expression of genes involved in the MBL pathway was not affected by the inflammatory cytokines tested in this study.
Retina and RPE/choroid express a variety of complement components that are involved mainly in the CP and AP. RPE and microglial cells are the main sources of retinal complement gene expression. Retinal complement gene expression is regulated by inflammatory cytokines, such as IFN-γ and TNF-α.
The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.
The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65−/− mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65−/− mice is caused by proteolytic degradation.
The eyes of three-week-old Rpe65−/− mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.
Degradation of the M-opsin protein in Rpe65−/− mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.
Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65−/− mice to some extent.
Circadian clocks are widely distributed in mammalian tissues, but little is known about the physiological functions of clocks outside the suprachiasmatic nucleus of the brain. The retina has an intrinsic circadian clock, but its importance for vision is unknown. Here we show that mice lacking Bmal1, a gene required for clock function, had abnormal retinal transcriptional responses to light and defective inner retinal electrical responses to light, but normal photoreceptor responses to light and retinas that appeared structurally normal by light and electron microscopy. We generated mice with a retina-specific genetic deletion of Bmal1, and they had defects of retinal visual physiology essentially identical to those of mice lacking Bmal1 in all tissues and lacked a circadian rhythm of inner retinal electrical responses to light. Our findings indicate that the intrinsic circadian clock of the retina regulates retinal visual processing in vivo.
The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate.
The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. It is unclear how culture conditions might alter barrier properties of isolated RPE. We examined whether retinal secretions that increase the barrier functions of tight junctions in vitro also make gene expression in general more in vivo-like.
Chick RPE from embryonic day 7 (E7) and E14 were cultured on filters. Media conditioned by organ culture of E14 neural retinas was added to the apical medium chamber. RNA was isolated to probe the chick genome on Affymetrix microarrays, and expression was compared to native E14 RPE. Expression was further analyzed by quantitative real-time PCR immunoblotting and immunocytochemistry.
More than 86% of the genes expressed in vivo were expressed in basal culture conditions, including RPE-specific markers such as RPE65 and bestrophin. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most effect on members of the claudin family. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization.
Gene expression in primary cultures of embryonic RPE resembled the native tissue, but differentiation and the levels of gene expression became more in vivo-like when elements of the retinal environment were introduced into the medium bathing the apical side of the cultures. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE.
Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells.
Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease.
Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE.
Vertebrate retinal pigment epithelium (RPE) cells are derived from the multipotent optic neuroepithelium, develop in close proximity to the retina, and are indispensible for eye organogenesis and vision. Recent advances in our understanding of RPE development provide evidence for how critical signaling factors operating in dorso-ventral and distal-proximal gradients interact with key transcription factors to specify three distinct domains in the budding optic neuroepithelium: the distal future retina, the proximal future optic stalk/optic nerve, and the dorsal future RPE. Concomitantly with domain specification, the eye primordium progresses from a vesicle to a cup, RPE pigmentation extends towards the ventral side, and the future ciliary body and iris form from the margin zone between RPE and retina. While much has been learned about the molecular networks controlling RPE cell specification, key questions concerning the cell proliferative parameters in RPE and the subsequent morphogenetic events still need to be addressed in greater detail.
Mitf/Otx/Chx10/Pax6/activin/sonic; hedgehog/fibroblast; growth; factor/neuroepithelial; domain specification/evolution
A 4 base pair deletion in a splice donor site of the Mfrp (membrane-type frizzled-related protein) gene, herein referred to as Mfrprd6/rd6, is predicted to lead to the skipping of exon 4 and photoreceptor degeneration in retinal degeneration 6 (rd6) mutant mice. Little, however, is known about the function of the protein or how the mutation causes the degenerative retinal phenotype. Here we examine ultrastructural changes in the retina of Mfrprd6/rd6 mice to determine the earliest effects of the mutation. We also extend the reported observations of the expression pattern of the dicistronic Mfrp/C1qtnf5 message and the localization of these and other retinal pigment epithelium (RPE) and retinal proteins during development and assess the ability of RPE cells to phagocytize outer segments in mutant and WT mice. At the ultrastructural level, outer segments do not develop normally in Mfrprd6/rd6 mutants. They are disorganized and become progressively shorter as mutant mice age. Additionally, there are focal areas in which there is a reduction of apical RPE microvilli. At P25, the rod ERG a-wave of Mfrprd6/rd6 mice is reduced in amplitude by ~50% as are ERG components generated by the RPE. Examination of β-catenin localization and Fos and Tcf-1 expression, intermediates of the canonical Wnt-pathway, showed they were not different between mutant and WT mice, suggesting that MFRP may operate through an alternative pathway. Finally, impaired outer segment phagocytosis was observed in Mfrprd6/rd6 mice both in standard ambient lighting conditions and with bright light exposure when compared to WT controls.
Mfrp; C1qtnf5; Retinal Pigment Epithelium; Phagocytosis; Outer Segments
Hepcidin is a hormone central to the regulation of iron homeostasis in the body. It is believed to be produced exclusively by the liver. Ferroportin, an iron exporter, is the receptor for hepcidin. This transporter/receptor is expressed in Müller cells, photoreceptor cells, and retinal pigment epithelium (RPE) within the retina. Since the retina is protected by the retinal-blood barriers, we asked whether ferroportin in the retina is regulated by hepcidin in the circulation or whether the retina produces hepcidin for regulation of its own iron homeostasis. Here we show that hepcidin is expressed robustly in Müller cells, photoreceptor cells, and RPE, closely resembling the expression pattern of ferroportin. We also show that bacterial lipopolysaccharide (LPS) is a regulator of hepcidin expression in Müller cells and RPE, both in vitro and in vivo, and that the regulation occurs at the transcriptional level. The action of LPS on hepcidin expression is mediated by the Toll-like receptor-4. The upregulation of hepcidin by LPS occurs independent of Hfe (Human leukocyte antigen-like protein involved in Fe homeostasis). The increase in hepcidin levels in retinal cells in response to LPS treatment is associated with a decrease in ferroportin levels. The LPS-induced upregulation of hepcidin and consequent downregulation of ferroportin is associated with increased oxidative stress and apoptosis within the retina in vivo. We conclude that retinal iron homeostasis may be regulated in an autonomous manner by hepcidin generated within the retina and that chronic bacterial infection/inflammation of the retina may disrupt iron homeostasis and retinal function.
hepcidin; retina; hemochromatosis; lipopolysaccharide; ferroportin; retinal pigment epithelium