Increased density of retinal pigment epithelium in cd81-/- mice

Our laboratories have focused on the role of the tetraspanin CD81 in the regulation of mitotic activity. Previously we have shown that antibodies directed against CD81 can block the proliferation of cultured retinal pigment epithelial (RPE) cells. The present study investigates the role of this protein by analyzing the structure of the adult retina in mice with a null mutation of cd81. Adult cd81(-/-) mice were produced by crossing two inbred strains, NIHS-BC/Tac and 129X1/SvJ, carrying the cd81 mutation as heterozygotes (+/-). Seven cd81(-/-) mice and 11 wildtype (cd81(+/+)) littermates were anesthetized and perfused with paraformaldehyde. The eyes were removed and processed for examination by light and electron microscopy. In general, the retinas of the cd81(-/-) mice appeared normal. However, upon close examination, there was an 18% increase in the number of RPE nuclei in the cd81(-/-) mice. The photoreceptor layer of the cd81(-/-) mice was significantly thinner than that of the wild-type mice, even though there was no difference in the total thickness of the retinas in the two groups of mice. At the electron microscopic level we did not observe any differences in cell-cell junctions in the retinas of the cd81(-/-) mice as compared to their wild-type littermates. These data support a role for CD81 controlling cell-cycle and the number of RPE nuclei in the mouse retina.     

Retinal pigment epithelial cell multinucleation in the aging eye – a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age‐related retinal degenerative disorders particularly age‐related macular degeneration. During aging RPE cells decline in number, suggesting an age‐dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose‐dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted ‘postmitotic’ status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long‐standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age‐related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice

Summary In the normal histogenesis of mouse retina localized distribution of acid phosphatase positive granules has been seen around the photoreceptor cell nuclei along the outer limiting membrane. These granules disappear during the development of the rod elements. Temporarily increased activity is also seen along the nuclei of the inner layer adjacent to and in the course of the development of the outer and the inner plexiform layers. Within the inner nuclear layer, the cells at the outer and inner rows develop localized acid phosphatase positive granules which persist in the adult retina. Ganglion cells and the layer of nerve fibres show little change. In the pigment epithelium the enzyme gradually increases. In mice, homozygous for the retinal degeneration gene, degenerating photoreceptor cell nuclei, characterized by perinuclear acid phosphatase staining, can be detected before morphological signs of degeneration. Increased frequency of such nuclei and intensity of staining are recorded with the progress of degeneration. Enzyme activity in the photoreceptor cells, within the inner nuclear layer and in the degenerating photoreceptor cell nuclei is demonstrable using naphthol substrates but not -glycerophosphate. Positive reaction with -glycerophosphate is obtained in these sites in the presence of dimethyl sulphoxide. Existence of differential permeability among the retinal lysosomes is tentatively suggested.     

Morphological effects of cadmium on proximal tubular cells in rats

Twenty-four male rats of the Wistar strain divided into four groups were injected sc with a dose of 0.8, 1.5, and 3.0 mg Cd/kg body wt as CdCl₂ in saline, and saline alone to the control rats, three times a week for 3 wk. Cadmium levels of whole kidney homogenate, supernatant (cytosol), precipitate, and metallothionein (MT) fraction were measured. Histological changes of the renal proximal tubules were investigated by optical and electron microscopy. In the kidneys, Cd levels were increased with the increment of Cd dosage; 80–90% of Cd was contained in cytosol, and 55–75% was in MT fraction. Non-MT-Cd reached a maximum in the 1.5 mg Cd group, whereas that of the 3.0 mg Cd group showed some decline. With increasing Cd doses, the size of nuclei and nucleoli in the cells of proximal tubule showed significant enlargement and also an increase in the number of nucleoli on light microscopy. At higher doses, chromatin condensation of the tubular nuclei and vacuolar degeneration of the tubular cells were evident. On electron microscopy, perichromatin granules of the proximal tubular nuclei were increased in number, especially in the rats of Cd 0.8 mg and 1.5 mg/kg groups. As the Cd doses increased, ring-shaped nucleoli were increased in number and nucleolar segregation was observed more clearly. Moreover, in the 3.0 mg/kg Cd group, nuclear indentation and nucleoli containing compact dense granules were observed. In the cytoplasm, there was an increase of lysosomes, myelin bodies, ring-shaped mitochondria, and vesiculation; ultimate changes were degeneration and cell necrosis. The injured cells were heterogenously distributed in each nephron and this heterogeneity was attributed in the difference in Cd content and cell cycle in each cell of the nephron.     

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

Background

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.

Methodology/Principal Findings

In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.

Conclusions/Significance

These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.     

Apomorphine inhibits the growth-stimulating effect of retinal pigment epithelium on scleral cells in vitro

Visual deprivation of the chicken eye causes axial elongation with high myopia. The cartilaginous layer of the myopic sclera shows an increase of mitotic activity. Previous studies reported that the in vivo administration of apomorphine, a dopamine nonselective agonist, effectively prevents visual-deprivation myopia. Because the retinal pigment epithelium (RPE) regulates growth of the sclera as we and others have shown previously, it is speculated that the RPE cells may play an important role in this preventive effect of apomorphine. In this study, to clarify the mechanism by which the administration of apomorphine inhibits the proliferation of scleral chondrocytes in vivo, we have investigated the effect of apomorphine on the proliferation of scleral chondrocytes with or without co-cultured RPE cells in vitro. We previously demonstrated that cell proliferation of scleral chondrocytes remarkably increases with co-cultured RPE cells. In this study, we found that apomorphine at concentrations of higher than 2×10⁻⁵ M dramatically reduced the growth-stimulatory effect of RPE cells on the scleral chondrocytes, whereas the inhibitory effect of apomorphine on the proliferation of scleral chondrocytes without RPE cells was very little. Our results strongly suggest that apomorphine may reduce the production and/or release of some humoral factors from RPE cells, which stimulate the growth of scleral cells. There is also a possibility that apomorphine reduces the reactivity of scleral cells to the humoral factors released from RPE cells. © 1997 John Wiley & Sons, Ltd.     

Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10‐PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL‐1β and TNF‐α , were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086–1100, 2017     

Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE‐choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two‐step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell‐extracellular matrix and/or cell–cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

The lens controls cell survival in the retina: Evidence from the blind cavefish Astyanax

The lens influences retinal growth and differentiation during vertebrate eye development but the mechanisms are not understood. The role of the lens in retinal growth and development was studied in the teleost Astyanax mexicanus, which has eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms. A lens and laminated retina initially develop in cavefish embryos, but the lens dies by apoptosis. The cavefish retina is subsequently disorganized, apoptotic cells appear, the photoreceptor layer degenerates, and retinal growth is arrested. We show here by PCNA, BrdU, and TUNEL labeling that cell proliferation continues in the adult cavefish retina but the newly born cells are removed by apoptosis. Surface fish to cavefish lens transplantation, which restores retinal growth and rod cell differentiation, abolished apoptosis in the retina but not in the RPE. Surface fish lens deletion did not cause apoptosis in the surface fish retina or affect RPE differentiation. Neither lens transplantation in cavefish nor lens deletion in surface fish affected retinal cell proliferation. We conclude that the lens acts in concert with another optic component, possibly the RPE, to promote retinal cell survival. Accordingly, deficiency in both optic structures may lead to eye degeneration in cavefish.     

CYTOKINE- AND NEUROPEPTIDE-MEDIATED DIFFERENTIATION IN RETINAL PIGMENT EPITHELIAL CELLS IN VITRO

To determine the mechanism of growth and differentiation of retinal pigment epithelial (RPE) cells it is important to understand the pathogenesis of several retinal diseases. Recently it has been reported that several cytokines and neuropeptides regulate the growth of RPE cells. In this study, the role of cytokines and neuropeptides in melanin synthesis, which is one indication of the RPE cell differentiation, was examined using chick RPE cells in vitro IL-1β, TNF-α, substance P, β-endorphin and methionine-enkephalin stimulated the melanin synthesis of RPE cells in a dose-dependent manner. The most effective concentrations of these agents on RPE cell melanin synthesis were not the same as that for RPE cell proliferation. These results indicate that cytokines and neuropeptides play an important role not only for the growth but also for the differentiation of RPE cells.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

ATP released via gap junction hemichannels from the pigment epithelium regulates neural retinal progenitor proliferation

The retinal pigment epithelium (RPE) plays an essential role in the normal development of the underlying neural retina, but the mechanisms by which this regulation occurs are largely unknown. Ca2+ transients, induced by the neurotransmitter ATP acting on purinergic receptors, both increase proliferation and stimulate DNA synthesis in neural retinal progenitor cells. Here, we show that the RPE regulates proliferation in the underlying neural retina by the release of a soluble factor and identify that factor as ATP. Further, we show that this ATP is released by efflux through gap junction connexin 43 hemichannels, the opening of which is evoked by spontaneous elevations of Ca2+ in trigger cells in the RPE. This release mechanism is localized within the RPE cells to the membranes facing the neural retina, a location ideally positioned to influence neural retinal development. ATP released from RPE hemichannels speeds both cell division and proliferation in the neural retina.     

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.     

Evidence of decreased adhesion between the neural retina and retinal pigmented epithelium of the Mitf vit (vitiligo) mutant mouse

In order for the retina to function properly, photoreceptor cell outer segments must be in contact with the adjacent retinal pigmented epithelium (RPE). A mouse model homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene manifests disruption of the outer segment/RPE interdigitation and demonstrates progressive loss of the photoreceptor cells. The mouse nevertheless has near normal levels of rhodopsin for many weeks and it is not known whether there is an in vivo loss of adhesion or whether the disruption is visible following tissue processing for histology. To assess this, a mechanical separation experiment was performed in which neural retinas were peeled free from the RPE and examined for the amount of pigment adherent to them. The peeling experiment indicated that control neural retinas retained significant amounts of adherent pigment at all ages examined. Neural retinas of mutant mice at age 2 weeks demonstrated adherent pigment, but older animals retained minimal pigment. Scanning electron microscopy indicated that the RPE cells of control mice were markedly damaged upon peeling and displayed different planes of cleavage, whereas those of mutants showed minimal cellular damage upon peeling, suggestive of decreased adhesion. A recombination experiment revealed that the mutant RPE/eyecup could reappose mutant and control retinas under in vitro conditions, suggesting that RPE fluid transport abilities were intact. The data provide the first direct experimental evidence that the Mitf^vit mutant mouse has a naturally occurring retinal detachment and hence support its value as a model for studies of retina/RPE adhesion.     

The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [3H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [3H]thymidine and [3H]ouabain. Cycling cells which had [3H]thymidine-labeled nuclei did not have notably higher labeling with [3H]ouabain. However, [3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.     

The retinal pigment epithelial cells of the adult newt and rat under conditions of in vitro organotypic culture

To understand why the retinal pigment epithelium (RPE) has different potentials for neural differentiation in lower and higher vertebrates, the RPEs of adult newts and rats were compared under similar in vitro cultivation conditions. The RPEs of both animal species were organotypically cultivated within the posterior eye wall under constant rotation in the serum medium free of growth factors. Comparison of the cell morphology, proliferation, and expression of pan-neural markers demonstrated that the RPE cells of adult newts and rats under similar in vitro conditions displayed both similarities and differemces. They were able to synthesize DNA but rarely divided mitotically. In addition, part of the RPE cells of both the newt and the rat were dislodged from the layer, migrated, and acquired a macrophage phenotype. However, the majority of the cells retained the initial morphology and remained within the layer. In several cases, these cells displayed the initial characteristics of neural differentiation, namely, expression of pan-neural proteins. The difference between the newt and rat RPE cells was in the ability of the former to generate in vitro an additional row of dedifferentiated NF-200-positive cells, characteristic of in vivo newt retinal regeneration. These data demonstrate that the RPE cells of the adult newt and rat retain the potential of manifesting neural cell traits; however, more advanced changes towards differentiation are characteristic of only the newt RPE.     

Influence of tyrosinase levels on pigment accumulation in the retinal pigment epithelium and on the uncrossed retinal projection

To study the relationship among tyrosinase activity, melanin production, and the routing of retinal ganglion cell (RGC) axons at the optic chiasm, we analysed mice with varying doses of the tyrosinase gene. These include the dark-eyed albino (Tyrc44H), a radiation-induced hypomorphic allele of tyrosinase; and transgenic mice carrying 1 or 2 alleles of a tyrosinase minigene on both wild-type (Tyr+) and albino (Tyrc) backgrounds. Melanization of the retinal pigment epithelium (RPE) occurred gradually even at <2% wild-type tyrosinase activity and was sensitive to tyrosinase activity up to <35% of wild-type levels, beyond which melanin synthesis appeared to be saturated. Overexpression of tyrosinase led to tyrosinase activity above wild type level, but did not increase melanin production. Although a loss of melanin because of a mutation in tyrosinase is associated with a decrease in the number of uncrossed fibers, elevating tyrosinase levels does not appear to cause an increase in the size of the uncrossed retinal projection. Our results suggest that replacing less than 35% of wild-type tyrosinase activity is sufficient to restore normal pigmentation of the RPE, and potentially, to allay visual defects.