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.     

Basic fibroblast growth factor induces retinal pigment epithelium to generate neural retina in vitro.

During embryogenesis, the cells of the eye primordium are initially capable of giving rise to either neural retina or pigmented epithelium (PE), but become restricted to one of these potential cell fates. However, following surgical removal of the retina in embryonic chicks and larval amphibians, new neural retina is generated by the transdifferentiation, or phenotypic switching, of PE cells into neuronal progenitors. A recent study has shown that basic fibroblast growth factor (bFGF) stimulates this process in chicks in vivo. To characterize further the mechanisms by which this factor regulates the phenotype of retinal tissues, we added bFGF to enzymatically dissociated chick embryo PE. We found that bFGF stimulated proliferation and caused several morphological changes in the PE, including the loss of pigmentation; however, no transdifferentiation to neuronal phenotypes was observed. By contrast, when small sheets of PE were cultured as aggregates on a shaker device, preventing flattening and spreading on the substratum, we found that a large number of retinal progenitor cells were generated from the PE treated with bFGF. These results indicate that bFGF promotes retinal regeneration in vitro, as well as in ovo, and suggest that the ability of chick PE to undergo transdifferentiation to neuronal progenitors appears to be dependent on the physical configuration of the cells.     

Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.     

CNPase activity in the vertebrate retina, retinal pigmented epithelium, and choroid

The activity of the enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase, E.C.3.1.4.37) has been studied in the retina of three vertebrate species. Activity was highest in the goldfish, followed by Xenopus laevis and Rana pipiens. Also, high activity levels were found in goldfish retinal pigment epithelium and choroid, but not in the other two species. When added to in vitro culture systems, 2',3'-cyclic nucleotides were found to have no effect on goldfish cone retinomotor movement, but caused a marked inhibition of Rana pipiens rod outer segment disc membrane shedding. It is suggested that CNPase may play a role in cellular processes requiring membrane structural reorganization.     

Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium

The retinal pigmented epithelium (RPE) is known to be site of the primary lesion in inherited retinal dystrophy in the Royal College of Surgeons (RCS) rat, a model for retinitis pigmentosa. Although the only functional defect so far detected in these cells is their failure to efficiently phagocytose shed photoreceptor outer segment debris, the actual cause of photoreceptor cell death is still unknown. Recently the possibility of “trophic factors” important in photoreceptor survival produced by normal RPE but not by dystrophic RPE has been suggested. Hence we decided to investigate the presence and abundance of two candidate diffusible factors, the acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), as well as their high affinity cell surface receptors (FGF-R). mRNA was isolated from primary cultures of purified normal and dystrophic RPE and analyzed by PCR amplification using specific oligonucleotide primers for aFGF and bFGF: the size and abundance of amplified fragments was similar for both cell types. Also, aFGF protein, detected by immunocytochemistry using specific antisera, appeared to be present in approximately equal amounts and distributed in a similar pattern. However, scatchard analysis of radio-labelled bFGF binding to primary cultures of normal and dystrophic rat RPE revealed that dystrophic RPE possess only 29% the number of surface receptors compared to congenic normal cells. Furthermore, the level of expression of FGF-R2 mRNA, but not that of FGF-R1, was significantly different. Other parameters measured (receptor affinity, profile of ligand internalization and degradation, receptor molecular weight and mitogenic activity) did not show any significant differences between normal and dystrophic RPE. The precise role of FGF-R deficiency in the etiology of the disease hence remains to be determined, but it indicates the importance of trophic factors in the normal functioning of the retina. © 1993 Wiley-Liss, Inc.     

Formation of retinal pigment epithelium in vitro by transdifferentiation of neural retina cells.

Chick embryonic neural retina (NR) dedifferentiates in culture and can transdifferentiate spontaneously into retinal pigment epithelium (RPE). Both, primary RPE and transdifferentiated RPE (RPEt), are characterized by pigmentation, expression of RPE-specific protein, eRPEAG and lack of expression of the neural cell adhesion molecule, NCAM. In contrast, NR cells are unpigmented and express NCAM but not eRPE(AG). Functionally, both primary RPE and the RPEt cells display a pH(i) response to bFGF, which is different from that of the NR. We used these characteristics to distinguish cell types in primary cultures of chick NR and follow the changes in phenotype that occur during transdifferentiation. We show that the RPEt forms as small "islands" in the packed regions of the primary, "mother" NR cell sheets, in a stochastic process. Because of a small number of cells involved in the initiation of the transdifferentiation we refer to it as a "leader effect" to contrast it with the "community effect" which requires many competent cells to be present in a group to be able to respond to an inductive signal. The RPEt then expands centrifugally and underneath the surrounding NR sheet. To determine if the RPEt maintains its identity in isolation while displaying the RPE-typical phenotypic plasticity, we explanted the islands of RPEt and treated half of them with bFGF. The untreated RPEt maintained its closely packed, polygonal pigmented phenotype but the bFGF-treated RPEt transdifferentiated into a non-pigmented, NR-like phenotype, indicating that RPEt encompasses the full differentiation repertoire of native RPE.     

Peroxidases in the neural retina and pigment epithelium.

Peroxidase activity, assayed with 2 mM-H₂O₂ and suitable hydrogen donors (either p-phenyl-enediamine or diaminobenzidine), was demonstrated in homogenates of neural retina and pigment epithelium of both the dog and the cow. The enzyme is particle-associated in the native state, but is readily extractable by brief sonication or freeze-thawing. At optimum pH, which is between 4.0 and 4.5 for both sources, the specific activity is up to 40 times greater in pigment epithelial cells than in neural retina. Some catalase activity was detected in extracts from both bovine and canine neural retina, but catalase was essentially absent in pigment epithelium. Fractionation of bovine pigment epithelial cells showed that peroxidase activity is associated mainly with heavy organelles sedimenting at low centrifugal forces. Melanosomes, nuclei, melanolysosomes and plasma membranes were the principal organelles identified in these low speed sediments. It was not possible to separate them either by differential centrifugation or on discontinuous sucrose gradients. However, melanosomes were excluded as the only source of peroxidase activity by isolating separately the melanotic and amelanotic cell populations; equal peroxidase was found in both cell types. Since nuclei are not a likely source of this enzyme, it is suggested that most of the peroxidase activity in bovine pigment epithelial cells is localized in either the melanolysosomes, plasma membranes, or both.     

Apoptosis and differentiation in presumptive neural retina and presumptive retinal pigmented epithelium during early eye development in toad, Bufo raddei strauch

Apoptosis and differentiation in presumptive neural retina (PNR) and presumptive retinal pigmented epithelium (PRPE) were investigated during early retina development of toad, Bufo raddei Strauch. TUNEL staining was used to evaluate apoptotic cells and the immunohistochemistry was used to assess the expression levels of glial fibrillary acidic protein (GFAP), RT97 and tyrosinase (Tyr) during early eye development respectively. The density of apoptotic cells and protein expression were quantitated with Image-Pro Plus 6.0. Apoptosis was found in both PNR and PRPE and the density of apoptotic profiles in PRPE was higher than that in PNR (most P < 0.01) at the same stage during early eye development. The expression levels of GFAP and RT97 changed from low to high in PNR, but from high to low in PRPE, whereas the expression level of Tyr, was contrary to those of GFAP and RT97 in both PNR and PRPE. The point of intersection of these, increase and decrease respectively was found at 5?C6 h after formation of optic vesicle (FOV). PRPE becomes thinner than PNR, one of the reasons might be due to higher density of apoptosis in PRPE than that in PNR during early eye development. Molecular differentiation, however, occurred after the contact of the optic vesicle outer wall with the overlying ectoderm which promotes the expression of specific molecules and inhibits the expression of non-specific molecules in PNR and PRPE respectively.     

Growth arrest specific gene 1 is a positive growth regulator for the cerebellum.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595--607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated.     

Melanosome metabolism in the retinal pigmented epithelium of the opossum

Summary Melanosomal metabolism, including both formation and degradation of melanosomes, was studied in the retinal pigmented epithelium (RPE) of the adult opossum. The majority of the observations were made on a transitional zone between the tapetal and non-tapetal RPE, the region where melanosome metabolism was at its highest level. Formation of melanosomes, demonstrated ultrastructurally by the presence of stage-II and -III premelanosomes, was also examined autoradiographically following the incorporation of the melanin precursor, dihydroxyphenylalanine. The autoradiographic evidence indicated that many newly formed melanosomes were rapidly incorporated into complexes. Ultrastructural observations suggested that melanosome complexes were formed by at least two methods, via the fusion of melanosomes with phagosomes derived from outer segments of photoreceptors, or by the sequestration of melanosomes by cisternae. A central finding of this study, supported by both ultrastructural and histochemical data, is that there are specialized cellular regions that vary in melanosomal formation and lysosomal activity. Stage-II premelanosomes were observed only in the basal parts of the RPE cells, whereas stage-III and -IV melanosomes were found primarily in the apical RPE. Both ultrastructural and cytochemical observations indicated that degradation of melanosomes occurs only in the basal RPE. These findings are interpreted in terms of the expression of both tapetal and nontapetal characteristics in transitional cells. Finally, this study illustrates the role of lysosomal enzymes in shaping the pattern of pigmentation, and shows that the association of lysosomal activity with melanosomes depends on the functional state of the melanosome.This investigation was supported by National Institutes of Health research grant EY 01429 and, in part, by a Bob Hope award from Fight for Sight, Inc., New York City (to R.H. Steinberg), and a Fight for Sight, Inc. Summer Fellowship to K.G. Herman     

Functions of the growth arrest specific 1 gene in the development of the mouse embryo

The growth arrest specific 1 (gas1) gene is highly expressed in quiescent mammalian cells (Schneider et al., 1988, Cell 54, 787-793). Overexpression of gas1 in normal and some cancer cell lines could inhibit G(0)/G(1) transition. Presently, we have examined the functions of this gene in the developing mouse embryo. The spatial-temporal expression patterns for gas1 were established in 8.5- to 14.5-day-old embryos by immunohistochemical staining and in situ hybridization. Gas1 was found heterogeneously expressed in most organ systems including the brain, heart, kidney, limb, lung, and gonad. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were investigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression could not prevent G(0)/G(1) progression. It was determined that gas1 could only induce growth arrest if p53 was also coexpressed. In contrast, gas1 overexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressing cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing heart and limb cells, we did not find these cells preferentially distributed at G0/G1, as compared with Gas1-negative cells. However, in the 12.5 day heart and limb, we did find significantly more Gas1-expressing cells distributed at G0/G1 phase than Gas1-negative cells. These results implied that Gas1 alone, during the early stages of development, could not inhibit cell growth. This inhibition was only established when the embryo grew older. We have overexpressed gas1 in subconfluent embryonic limb cells to determine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the interaction of gas1 with Ap-1, NFkappaB, and c-myc responsive elements tagged with a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-1, NFkappaB, and c-myc activities. In contrast, gas1 overexpression in 12.5 day limb cells enhanced AP-1 response while it inhibited NFkappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital tissues. We overexpressed gas1 in these tissues and discovered that it promoted interdigital cell death. Our in situ hybridization studies of limb sections and micromass cultures revealed that, during the early stages of chondrogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage started to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cells overexpressing gas1/GFP could not participate in cartilage formation, unlike cells that just express the GFP reporter. We speculated that the reason gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary between chondrogenic and nonchondrogenic forming regions.     

Effects of TGF-beta on retinal pigmented epithelium in vitro

Embryonic chick retinal pigmented epithelium (RPE) has been grown on glass derivatized with covalently bound proteins of basement membrane and treated with transforming growth factor-beta (TGF-beta). In the present paper we show that over the concentration range tested (0.1-10 ng/ml) TGF-beta has no effect on RPE cell proliferation either in the presence or the absence of serum, cell motility and the organization of cytoskeleton-extracellular matrix linkage complexes with respect to their structure and presence of actin, vinculin, talin, integrin and fibronectin. The protein profiles of total cell/ECM extracts of cells grown in the presence or the absence of TGF-beta are similar although some stimulation of protein synthesis and of production of fibronectin-containing extracellular matrix has been detected.     

Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina

The retinal pigment epithelium (RPE) is unique among epithelia in that its apical surface does not face a lumen, but, instead, is specialized for interaction with the neural retina. The molecules involved in the interaction of the RPE with the neural retina are not known. We show here that the neural cell adhesion molecule (N-CAM) is found both on the apical surface of RPE in situ and on the outer segments of photoreceptors, fulfilling an important requisite for an adhesion role between both structures. Strikingly, culture of RPE results in rapid redistribution of N-CAM to the basolateral surface. This is not due to an isoform shift, since the N-CAM expressed by cultured cells (140 kD) is the same as that expressed by RPE in vivo. Rather, the reversed polarity of N-CAM appears to result from the disruption of the contact between the RPE and the photoreceptors of the neural retina. We suggest that N-CAM in RPE and photoreceptors participate in these interactions.     

Transdifferentiation of "lentoid" structures in cultures derived from pigmented epithelium was inhibited by collagen.

Cells from pigmented retina of 8- to 9-day-old chick embryos were cultured under two different conditions: on noncoated (NS) or collagen-coated (CS) substrates. Although cells on CS seemed to start dividing 2 to 3 days earlier than those on NS, their early growth rates were basically similar. Cells on CS stopped growing after attaining confluency and formed a monolayer, while cells on NS continued to grow after confluency and overlapped each other. In early growth phase, cells on both substrates became depigmented. Cells became repigmented earlier on CS than on NS. The average melanin content of cells in confluent cultures on CS was two to three times higher than that of cells on NS. By Day 30 “lentoid bodies” were formed only in cultures on NS. Immunoelectrophoretic tests showed the presence of all crystallins (α-, β-, and δ) in cultures on NS but not in cultures on CS. It is concluded that a collagen substrate inhibits “transdifferentiation” of pigmented retinal cells into lens during cell culture.