Role of Bruch's membrane in the process of metaplasia of the ocular pigmented epithelium of Xenopus laevis]

The metaplasia of pigmented epithelium into retina with the formation of nuclear and reticular layers took place in the experiments with wrapping a sheet of pigmented epithelium from tadpoles up in the Bruch's membrane of adult frogs X. laevis. The implant with de novo formed retina resembled the inverted eye. In those experiments where the Bruch's membrane of tadpoles came into contact with that of adults only depigmentation of the pigmented epithelium cells was observed. The pigmented epithelium metaplasia suggests that the Bruch's membrane is permeable not only for low molecular weight substances, but also for inducing agents. The adult Bruch's membrane serves in the process of metaplasia in the experiments described as a new and limiting system which ensures more orderly formation of new retina.     

Metaplastic transformation of the tissue of the eye in tadpoles and adult Xenopus laevis frogs]

The pigment epithelium of the tadpoles and adults X. laevis, as well as of other anurans and cyprinids, is not capable of transformation into the retina without the special influences of agents produced by the retina. When implanting a layer of pigmented epithelium of tadpoles with the Bruch's membrane into the cavity of lensless eye of a tadpole, the transformation of pigment epithelium into retina proceeded in 40% of cases and when implanting the pigment epithelium of adults without the Bruch's membrane, the transformation proceeded in 68% of cases. The lens regeneration from the cornea which proceeds simultaneously under the retina influence exerted no effect upon the metaplasia of pigmented epithelium.     

The development of differentiation of pigment epithelium in teleosts and its stimulation to metaplasia]

The time was determined when pigmented epithelium acquires stable differentiation and the possibility was investigated for pigmented epithelium to transform in retina at different developmental stages in the Issyk-kul chebatchok Leuciscus bergi (Cyprinidae). By means of implantation of a layer of pigmented epithelium in pericardium it was established that the pigmented epithelium cells acquired stable differentiation rather early. When an already pigmented layer of pigmented epithelium was implanted in the cavity of a lensless eye, its cell transformed in retina under the influence of the whole retina. Conditions of pigmented epithelium metaplasia in retina in teleosteans proved to be similar with those for frogs.     

Cellular and ultrastructural features of the adult and the embryonic eye in the marine gastropod,Ilyanassa obsoleta

Light and electron microscopic techniques show that the eye of the marine prosobranch gastropod, Ilyanassa obsoleta, is composed of an optic cavity, lens, cornea, retina, and neuropile, and is surrounded by a connective tissue capsule. The adult retina is a columnar epithelium containing three morphologically distinct cell types: photoreceptor, pigmented, and ciliated cells. The retina is continuous anteriorly with a cuboidal corneal epithelium. The neuropile, located immediately behind the retina, is composed of photoreceptor cell axons, accessory neurons, and their neurites. The embryonic eye is formed from surface ectoderm, which sinks inward as a pigmented cellular mass. At this time, the eye primordium already contains presumptive photoreceptor cells, pigmented retinal cells, and corneal cells. Several days later, just before hatching, the embryonic eye remains in intimate contact with the cerebral ganglion. It has no ciliated retinal cells, neuropile, optic nerve, or connective tissue capsule and its photoreceptor cells lack the electron-lucent vesicles and multivesicular bodies of adult photoreceptor cells. As the eye and the cerebral ganglion grow apart, the optic nerve, neuropile, and connective tissue capsule develop.     

Ultrastructural study of the retinal pigment epithelium during metamorphosis in the sea lamprey (Petromyzon marinus L.)

Summary The sequence of morphological changes in the retinal pigment epithelium during the metamorphic period of the sea lamprey Petromyzon marinus L. has been investigated using electron microscopy. At early metamorphic stages (stages I and II), photoreceptors are present in a small zone of the retina. During these stages, the lateral surface of the epithelial cells shows zonulae occludentes and adhaerentes. The degree of cell differentiation varies throughout the retinal pigment epithelium. Cells covering the differentiated photoreceptors in the central retina have phagosomes, whereas pigment granules appear only in the retinal pigment epithelium dorsal to the optic nerve head. Most epithelial cells have myeloid bodies; their morphology is more complex around the optic nerve head. At stage III, when photoreceptors develop over the whole retina, the distribution of cytoplasmic organelles is almost homogeneous in the retinal pigment epithelium. Subsequently, the basal plasma membrane of the epithelial cells becomes progressively folded and their apical processes enlarged. In addition, extensive gap junctions develop between retinal pigment cells. In late metamorphic stages, noticeable growth of myeloid bodies occurs and consequently the retinal pigment epithelium resembles that of the adult. This study also describes, for the first time, the presence of wandering phagocytes in the retinal pigment epithelium of lampreys; their role in melanosome degradation is discussed.     

THE DEMONSTRATION OF A BLOOD-OCULAR BARRIER IN THE ALBINO RAT BY MEANS OF THE INTRAVITAM DEPOSITION OF SILVER

When silver nitrate is administered to rats in their drinking water for many months, they develop a generalized argyria. In the central nervous system, the deposition of silver follows the pattern of the so called hematoencephalic barrier (Wislocki and Leduc, (2); Dempsey and Wislocki, (3)). The present observations concern the deposition of silver in the rat's eye, investigated by both light microscopy and the electron microscope. In the eye, silver is not detected in the specific neural elements of the retina. Instead, it is heavily deposited in the basement membrane of the epithelium of the ciliary processes and in Bruch's basal membrane between the choriocapillary layer and the retinal epithelium. Traces of silver are visible in the basement membranes of the retinal capillaries with the electron microscope, but cannot be identified with the light microscope. In all of these respects, the pattern of the silver resembles the mode of its deposition in the brain. The heavy accumulation of metal in Bruch's membrane and the ciliary processes is analogous to that observed in the chorioid plexuses, and the traces encountered in the walls of the retinal capillaries correspond to traces observed in the basement membranes of the cerebral capillaries. Hence, with respect to silver, the eye possesses a blood-ocular barrier similar to the hematoencephalic barrier. Silver appears to be restrained from entering the aqueous humor by a barrier in the basement membrane of the ciliary processes, from reaching the photoreceptor elements of the retina by Bruch's basal membrane, and from penetrating the inner layers of the retina by a barrier in the basement membrane surrounding the retinal capillaries.     

猫外层视网膜和脉络膜的年龄相关变化

取老年猫(12龄,2.5~3 kg)和青年猫(1~3龄,2~2.5 kg)各4只的视网膜,经4%多聚甲醛处理后用H.E染色以显示视网膜和脉络膜的结构。光学显微镜下观察感光细胞层、玻璃膜(Bruch’s membrane)结构的变化,计数色素上皮层(retinal pigment epithelium,RPE)细胞数、脉络膜毛细血管数,测量玻璃膜、脉络膜厚度,脉络膜毛细血管之间的距离。结果显示,与青年猫比较,老年猫视网膜感光细胞层结构杂乱;色素上皮细胞数显著下降;玻璃膜厚度无显著变化,出现较多碎片;脉络膜厚度明显变薄,脉络膜毛细血管数显著减小,脉络膜毛细血管之间的距离显著增大。推测老年猫脉络膜的退化可能是导致玻璃膜、色素上皮层的退化,进而导致感光细胞的功能衰退的重要原因。     

Activin signaling limits the competence for retinal regeneration from the pigmented epithelium

Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e. FGF and Pax6) have also been implicated in transdifferentiation. Therefore, we tested whether activin, a TGFbeta family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina. We have found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFbeta/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration.     

Retinal pigmented epithelium does not transdifferentiate in adult goldfish

The neural retina of adult goldfish can regenerate from an intrinsic source of proliferative neuronal progenitor cells, but it is not known whether the retina can regenerate by transdifferentiation of the retinal pigmented epithelium (RPE), a phenomenon demonstrated in adult newts. In this study, we asked whether following surgical removal of the neural retina in adult goldfish the RPE was capable of autonomously transdifferentiating and generating new neural retina. The retina was prelabeled by injecting the fluorescent dye Fluoro-Gold (FG) into the eye prior to surgical removal; this procedure ensured that residual retina was labeled with FG and could therefore be distinguished from unlabeled, regenerated retina. To examine the time course of retinal regeneration, and to identify regenerated retinal neurons, the thymidine analogue bromodeoxyuridine was injected intraocularly, and retinas were examined up to 2 months later. We found that the RPE did not transdifferentiate; instead, retinas regenerated only when pieces of residual neural retina were left intact. Under these circumstances, newly regenerated cells derived from proliferating cells intrinsic to the residual neural retina. When retinas were completely removed, as was evident from a lack of FG labeling, there was no retinal regeneration. © 1995 John Wiley & Sons, Inc.     

mosaic eyes: a zebrafish gene required in pigmented epithelium for apical localization of retinal cell division and lamination

For proper function of the retina, the correct proportions of retinal cell types must be generated, they must be organized into cell-specific laminae, and appropriate synaptic connections must be made. To understand the genetic regulation of retinal development, we have analyzed mutations in the mosaic eyes gene that disrupt retinal lamination, the localization of retinal cell divisions to the retinal pigmented epithelial surface and retinal pigmented epithelial development. Although retinal organization is severely disrupted in mosaic eyes mutants, surprisingly, retinal cell differentiation occurs. The positions of dividing cells and neurons in the brain appear normal in mosaic eyes mutants, suggesting that wild-type mosaic eyes function is specifically required for normal retinal development. We demonstrate that mosaic eyes function is required within the retinal pigmented epithelium, rather than in dividing retinal cells. This analysis reveals an interaction between the retinal pigmented epithelium and the retina that is required for retinal patterning. We suggest that wild-type mosaic eyes function is required for the retinal pigmented epithelium to signal properly to the retina.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

Implication of the hypoxia response element of the Vegf promoter in mouse models of retinal and choroidal neovascularization, but not retinal vascular development

Retinal neovascularization (NV) and macular edema, resulting from blood-retinal barrier (BRB) breakdown, are major causes of visual loss in ischemic retinopathies. Choroidal NV (CNV) occurs in diseases of the retinal pigmented epithelium/Bruch's membrane complex and is another extremely prevalent cause of visual loss. We used mice in which the hypoxia response element (HRE) is deleted from the vascular endothelial growth factor (vegf) promoter (Vegf(delta/delta) mice) to explore the role of induction of VEGF through the HRE in these disease processes. Compared to wild type (Vegf+/+) mice with oxygen-induced ischemic retinopathy (OIR) in which vegf mRNA levels were increased and prominent retinal NV and BRB breakdown occurred, Vegf(delta/delta) littermates with OIR failed to increase vegf mRNA levels in the retina and had significantly less retinal NV and BRB breakdown, but showed prominent dilation of some superficial retinal vessels. Vegf(+/delta) littermates with ischemic retinopathy developed comparable retinal NV to Vegf+/+ mice, exhibited intermediate levels of BRB breakdown, and did not show vasodilation. In a mouse model of CNV, due to laser-induced rupture of Bruch's membrane, the area of CNV at Bruch's membrane rupture sites was more than tenfold greater in Vegf+/+ mice than in Vegf(delta/delta) littermates. In contrast to these dramatic differences in pathologic ocular NV, Vegf(delta/delta) mice showed subtle differences in retinal vascular development compared to Vegf+/+ mice; it was slightly delayed, but otherwise normal. These data suggest that induction of VEGF through the HRE in its promoter is critical for retinal and CNV, but not for retinal vascular development.     

The expression of LIM‐homeobox genes,Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye‐related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM‐homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle‐like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.     

Neuronal stem/progenitor cells in the vertebrate eye

We acquire information from the outside world through our eyes which contain the retina, the photosensitive component of the central nervous system. Once the adult mammalian retina is damaged, the retinal neuronal death causes a severe loss of visual function. It has been believed that the adult mammalian retina had no regenerative capacity. However, the identification of neuronal progenitor cells in the retina sheds some light on cellular therapies for damaged retinal regeneration. In this review, we highlight three potential stem/progenitor cells in the eye, the ciliary body epithelium cells, the iris pigmented epithelium cells, and Müller glia. In order to make them prime candidates for the possible treatment of retinal diseases, it is important to understand their basic characters. In addition, we discuss the key signaling molecules that function extracellularly and determine whether neuronal progenitors remain quiescent, proliferate, or differentiate. Finally, we introduce a secreted protein, Tsukushi, which is a possible candidate as a niche molecule for retinal stem/progenitor cells.     

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

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     

Transdifferentiation Potential of Ciliary and Pigment Epithelial Cells in Lower Vertebrates and Mammals

Cellular composition of the peripheral region of the eye in amphibians and mammals as well as embryonic fissure in amphibians was studied. Different distributions of proliferating cells in retinal pigment epithelium have been revealed in adult amphibians (newt, axolotl, and Xenopus). Single cells incorporated [³H]thymidine in the newt and Xenopus; 0.4% cells, in the axolotl. An embryonic fissure was observed in the eye of the axolotl. Pigment epithelial cells in the embryonic palpebral region actively proliferated: about 20% cells incorporated [³H]thymidine. Proliferating cells were also localized in the ciliary marginal zone of the retina in all studied amphibians, particularly, in the axolotl. In newborn hamsters, [³H]thymidine-labeled cells have been revealed in the pigment epithelium as well as in the outer pigmented and inner unpigmented layers of the ciliary body. Proliferative activity of the peripheral regions of the eye is due to eye growth in adult amphibians and newborn hamsters. After retinectomy, the retina is regenerated from the cells of the growth ciliary marginal zone in all amphibians, pigment epithelial cells in the newt, and pigment epithelial cells of the embryonic fissure in the axolotl. Heterogeneous composition of the pigment epithelium in the newt and axolotl reflects high transdifferentiation potential of these regions. Structural comparison of the peripheral region of the eye in amphibians and mammals demonstrate that the ciliary body of mammals containing stem cells is homologous to the ciliary marginal zone of amphibians containing multipotent cells.     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.