Differential effects of bFGF on development of the rat retina

A variety of growth factors can influence the expression of differentiated properties by cell types of the developing retina. One unresolved question has been whether these factors can direct the differentiation pathway of uncommitted precursors or whether they act to help the expression of properties by already committed cells. To address this question we have studied the effects of basic fibroblast growth factor (bFGF) on the differentiation of ganglion cells and rod photoreceptors in explant cultures of embryonic rat retinas. Incubation of retinas in the presence of bFGF accelerated the appearance of differentiated ganglion cells and incubation in the presence of anti-bFGF antibodies delayed the appearance. bFGF had no effect on the appearance of differentiated rod photoreceptors as judged by expression of opsin, although all-trans-retinoic acid did increase the number of cells expressing opsin. bFGF inhibited the formation of rod photoreceptor rosettes suggesting that it does influence some properties of rods or the adjacent Müller glial cells. The results suggest that bFGF can alter the timing of differentiation of retinal ganglion cells but not direct their production from retinal precursors.     

Retinoic acid promotes rod photoreceptor differentiation in rat retina in vivo.

Previous studies have indicated that retinoic acid (RA) promotes rod photoreceptor differentiation in dissociated cultures of rat retina and in zebrafish embryos. To determine whether RA will have the same affect in the mammalian retina in vivo, pregnant rats were given single i.p. injections of RA on the 18th and 20th days of gestation, and the retinas of the pups were analyzed for rods. HPLC showed that i.p. injections of RA substantially increased levels of retinal RA in the embryos. Embryonic exposure to RA caused an increase in the number of cells that differentiated as rod photoreceptors. There was a comparable decrease in the number of cells that differentiated as amacrine cells. These results demonstrate that RA promotes the differentiation of rods in vivo and further support the hypothesis that differentiation of rods is normally controlled partly by the RA concentration in the developing retina or RPE.     

Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies

This study describes the identification and differentiation of neonatal rat retinal cells in monolayer cultures. A panel of monoclonal antibodies was used as a molecular probe of both cell type and developmental stage. Previously described cell-type specific monoclonal antibodies were used to label rod photoreceptors, horizontal cells, amacrine cells or ganglion cells. Two new antibodies that react with rat retina are described. The first, RET-G7, reacts with a cytoplasmic antigen of Muller glia, astrocytes and some horizontal cells. The second, RET-B2, reacts with bipolar cells and photoreceptor inner segments. Two main findings are presented. The first is that each of the major subclasses of retinal neurons have been unambiguously identified in these cultures. The morphology of some subclasses was very characteristic. All photoreceptors, as defined by reactivity with antibody RET-P1, were small spherical cells with one or fewer processes. Horizontal cells, as defined by reactivity with antibody B-1, were large with a characteristic multipolar network of processes. Bipolar and amacrine cells, on the other hand, were of similar size and could only be distinguished on the basis of immunocytochemical labeling. The second finding is that while RET-B2 antigen appeared on bipolar and photoreceptor cells after about 5 days in culture, several Muller cell and photoreceptor antigens were not expressed in monolayer cultures. The results suggest that the expression of some molecules in culture is the result of properties intrinsic to the cells whereas expression of others depends upon extrinsic factors or cell interactions that may not be present in monolayer cultures.     

Interleukin-4 blocks proliferation of retinal progenitor cells and increases rod photoreceptor differentiation through distinct signaling pathways

Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been related to the differentiation of the rodent retina in vitro, but constitutive presence of either IL-4 or of IL-4 receptor in the retina has not been reported. In this work we examined the expression of IL-4 and its specific receptor alpha subunit (IL-4Ralpha). IL-4Ralpha is expressed both in neural retina and non-neural ocular tissue, while IL-4 was found mainly in non-neural tissue. We characterized a novel trophic effect of IL-4 upon the retina. We showed that IL-4 can inhibit the proliferation of retinal cells (approximately 40%) through the cAMP-PKA pathway and associated with a reduction of cyclin D1 and increase of p27(kip1). IL-4 also promotes the differentiation of rod photoreceptors. Activation of tyrosine kinases, protein kinase C, and mitogen-activated kinases of the Erk family were required for IL-4-induced rod photoreceptor differentiation, independent of the release of other trophic factors in culture. Taken together, our results show, for the first time, that IL-4 directly modulates proliferation of retinal cells and rod photoreceptor differentiation, through distinct signaling pathways.     

Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina.

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Müller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.     

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.     

Dysmorphic photoreceptors in a P23H mutant rhodopsin model of retinitis pigmentosa are metabolically active and capable of regenerating to reverse retinal degeneration

This study evaluated the capacity of Xenopus laevis retina to regenerate photoreceptor cells after cyclic light-mediated acute rod photoreceptor degeneration in a transgenic P23H mutant rhodopsin model of retinits pigmentosa. After discontinuation of cyclic light exposure, we monitored histologic progression of retinal regeneration over a 3 week recovery period. To assess their metabolomic states, contralateral eyes were processed for computational molecular phenotyping. We found that retinal degeneration in the P23H rhodopsin mutation could be partially reversed, with regeneration of rod photoreceptors recovering normal morphology (including full-length rod outer segments) by the end of the 3 week recovery period. In contrast, retinal degeneration mediated by directly induced apoptosis did not recover in the 3 week recovery period. Dystrophic rod photoreceptors with truncated rod outer segments were identified as the likely source of rod photoreceptor regeneration in the P23H retinas. These dystrophic photoreceptors remain metabolically active despite having lost most of their outer segments.     

IGF-1 produced by cone photoreceptors regulates rod progenitor proliferation in the teleost retina

Teleost eyes grow throughout life by adding neurons and stretching extant tissue. New retinal neurons of all types are added at the ciliary margin and new rod photoreceptors are inserted throughout retina in the outer nuclear layer (ONL). New rod photoreceptors result from the division of progenitor cells located in the ONL amidst functioning rod photoreceptor cell nuclei, but it is not known how new rod addition is regulated. Previous experiments using an organotypic retinal slice preparation revealed that insulin-like growth factor 1 (IGF-1) up-regulates the division of the rod progenitor cells [Dev. Brain Res. 76 (1993) 183], but the site of IGF-1 action was unknown. Here, we show where in the retina IGF-1 is made, where IGF receptors are located, and we identify the role of IGF-1 in adult retinal rod neurogenesis with both gain-and loss-of-function experiments. We found that IGF-1 is expressed by cone photoreceptor cells and its abundance varies with a daily rhythm, being significantly higher at night. In vivo application of exogenous IGF-1 increases rod progenitor cell division, an effect that is greater at night than during the day. We also show that inhibiting the function of IGF receptors decreases proliferation of rod progenitor cells. Finally, we show that IGF receptors are located on rod progenitor cells as well as on cone and rod photoreceptors. Taken together, these data suggest that the rhythmic production and release of IGF-1 plays a role in regulating the insertion of new rod photoreceptors into the retina. The diurnal change in IGF-1 abundance and effects of exogenous IGF-1 are consistent with the previous demonstration that rod progenitor cell division is threefold greater at night than in the day [Brain Res. 673 (1995) 119; Brain Res. 712 (1996) 40]. We also show that the insertion of new rod photoreceptors at the central edge of the ciliary neurogenic zone very likely also depends on IGF-1 production by cone photoreceptors. We propose that addition of new rod photoreceptors into the functioning retina is regulated through a feedback mechanism mediated at least in part via the IGF-1 produced in the cone photoreceptors.     

Interphotoreceptor matrix in the human retina: cone-like domains surround a small population of rod photoreceptors.

The interphotoreceptor matrix (IPM) in mammalian retinas is subdivided into rod and cone specific compartments: peanut agglutinin (PNA) binding glycoconjugates are associated with cones, whereas wheat germ agglutinin (WGA) binding glycoconjugates are associated with rods. To establish the identity of a photoreceptor cell type in the human retina with rod dimensions but with a matrix domain which stains with PNA, double label studies, using PNA-ferritin to decorate the extracellular domains and immunocytochemical techniques using a rod specific anti-opsin antibody were conducted. The PNA-binding domains were observed in the cone-associated IPM as well as in the IPM surrounding a small population of rod-shaped photoreceptors. The outer segments of these rod-shaped photoreceptors showed intense labeling with a rod specific anti-opsin antibody as did all other rods which were free of PNA-labeling. A quantitative analysis of all retinal quadrants indicates that this novel rod represents approximately 0.3% of the total rod population in the human retina.     

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-β-induced apoptosis

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-β (Aβ) and the degeneration of photoreceptors. To explore the largely unknown role of Aβ in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aβ(1-42). Subretinal injection of the Aβ peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aβ did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aβ-induced photoreceptor apoptosis. Subretinal injection of Aβ was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aβ in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aβ-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aβ-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aβ-injected retinas. Our study pinpoints the roles of Aβ and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.     

Lazy eyes zebrafish mutation affects Müller glial cells,compromising photoreceptor function and causing partial blindness

A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5-day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild-type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a- and b-wave amplitudes relative to wild-type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild-type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function.     

Retinal pigment epithelial cells promote spatial reorganization and differentiation of retina photoreceptors

Retina differentiation involves the acquisition of a precise layered arrangement, with RPE cells in the first layer in intimate contact with photoreceptors in the second layer. Here, we developed an in vitro coculture model, to test the hypothesis that RPE cells play a pivotal role in organizing the spatial structure of the retina. We cocultured rat retinal neurons with ARPE-19 epithelial cells under various experimental conditions. Strikingly, when seeded over RPE cells, photoreceptors attached to their apical surfaces and proceeded with their development, including the increased synthesis of rhodopsin. Conversely, when we seeded RPE cells over neurons, the RPE cells rapidly detached photoreceptors from their substrata and positioned themselves underneath, thus restoring the normal in vivo arrangement. Treatment with the metalloproteinase inhibitor TIMP-1 blocked this reorganization, suggesting the involvement of metalloproteinases in this process. Reorganization was highly selective for photoreceptors because 98% of photoreceptors but very few amacrine neurons were found to redistribute on top of RPE cells. Interestingly, RPE cells were much more efficient than other epithelial or nonepithelial cells in promoting this reorganization. RPE cells also promoted the growth of photoreceptor axons away from them. An additional factor that contributed to the distal arrangement of photoreceptor axons was the migration of photoreceptor cell bodies along their own neurites toward the RPE cells. Our results demonstrate that RPE and photoreceptor cells interact in vitro in very specific ways. They also show that in vitro studies may provide important insights into the process of pattern formation in the retina.     

Mosaic deletion of Rb arrests rod differentiation and stimulates ectopic synaptogenesis in the mouse retina

The retinoblastoma gene (Rb) regulates neural progenitor cell proliferation and cell fate specification and differentiation. For the developing mouse retina, two distinct functions of Rb have been described: regulation of retinal progenitor cell proliferation and rod photoreceptor development. Cells that would normally become rods fail to mature and remain as immature cells in the outer nuclear layer in the adult. By using Chx10-Cre;Rb(Lox/-) mice, we generated a chimeric retina with alternating apical-basal stripes of wild-type and Rb-deficient tissue. This provides a unique model with which to study synaptogenesis at the outer plexiform layer within regions that lack differentiated rods. In regions where rods failed to differentiate, the outer plexiform layer (OPL) was disrupted. Horizontal cells formed, and their somata were appropriately aligned, but their neurites did not project laterally. Instead many horizonal cell neurites extended apically, forming ectopic synapses with photoreceptors at all levels of the outer nuclear layer. These ectopic photoreceptor terminals contained synaptic ribbons, horizontal cell processes with synaptic vesicles, and a single mitochrondrion characteristic of rod spherules. Rb-deficient bipolar cells differentiated normally, extended dendrites to the OPL, and formed synapses that were indistinguishable from adjacent wild-type cells. In contrast to OPL-positioned synapses, ectopic synapses did not contain bipolar dendrites. This finding suggests that horizontal cells and photoreceptors can form stable synapses that are devoid of bipolar dendrites outside the normal boundaries of the OPL. Finally, analysis of P4, P7, P12, and P15 retinae suggests that the apical horizontal cell processes result from their failure to establish their normal lateral projections during development.     

Inhibition of alpha(1-6)-linked fucose decreases inner retinal cells and increases photoreceptors in chicken retinal reaggregates

We investigated the developmental role of alpha(1-6)-linked fucose, applying Aleuria aurantia lectin to a specific retinal regeneration system. Thereby, dissociated retinal cells of chicken embryos reaggregate, proliferate, and differentiate in vitro into histotypical spheres, so-called retinospheroids. Under the influence of A. aurantia lectin, processes of proliferation, differentiation and histogenesis of retinospheroids were disturbed. Extending these in vitro studies, we here show that A. aurantia lectin treatment decreases cells of the inner half retina and their processes into inner plexiform layer areas, as revealed by quantitative enzyme histochemistry for butyryl- and acetylcholinesterase, and immunohistochemistry using antibodies to acetylcholinesterase, Pax-6, calbindin-D, and F11. Concomitantly, the number of rod and red/green photoreceptors dramatically increases, using the antibodies rho4D2 and CERN901 (both specific for rods) and CERN906 (specific for red/green cones). These findings show that glycoproteins exhibiting fucose in alpha(1-6)-linkage are involved in processes determining retinal cell fate, strongly shifting the relative ratio of cells of the inner towards cells of the outer retina.     

Histiotypic organization and cell differentiation in rat retinal reaggregate cultures

Reaggregate cultures have been formed from cell suspensions of neonatal rat retinas. Histological sections of the reaggregates showed evidence of lamination with central rosettes formed around a lumen, a clear neuropil layer and an outer cellular layer. Each of the major retinal cell types, except ganglion cells, could be positively identified using cell type-specific antibodies to label cryostat sections. Many of these were found to occupy positions within the reaggregates similar to those found in the intact retina. Electron microscopic observations showed abundant immature and mature synaptic endings within the neuropil layer, including a number of ribbon synapses. Examination of the rosettes showed an arrangement of Müller glia and photoreceptors that closely resembled that of the intact retina. Within the lumen of rosettes, photoreceptors were found to contain stacks of disc-like membranes bounded by a plasma membrane, analogous to immature outer segments. The photoreceptors within rosettes also underwent molecular differentiation and expressed an outer segment specific marker. The findings suggest that retinal cells have intrinsic properties that allow them to organize themselves into a correctly laminated structure and that particular cell interactions are necessary for continued differentiation of at least rod photoreceptors and Müller cells.