Molecular cloning and characterization of chick SPACRCAN

MY-174, a monoclonal antibody that reacts with specific sialylated O-linked glycoconjugates of chick SPACR (sialoprotein associated with cones and rods), also recognizes another molecule of 300 kDa. Here, we verified that this 300-kDa molecule is chick SPACRCAN (sialoproteoglycan associated with cones and rods), another member of a novel interphotoreceptor matrix molecule family. Screening for chick SPACRCAN was carried out by plaque hybridization using a probe for chick SPACR. Specific polyclonal antibodies raised against chick SPACRCAN were used for the following experiments. To determine whether the 300-kDa molecule detected by MY-174 was identical to 300-kDa chick SPACRCAN, the migrations of these bands were examined after various glycosidase digestions. Furthermore, the expression levels were measured during retinal development and compared with those of chick SPACR. The results demonstrated that the 300-kDa molecule recognized by MY-174 was chick SPACRCAN, and we further identified it as a proteoglycan with chondroitin sulfate chains. SPACRCAN had heavily sialylated N- and O-linked glycoconjugates, and its MY-174 antigenicity was abolished by O-glycanase treatment after neuraminidase treatment, as observed for chick SPACR. During retinal development, the mRNA and core protein expression levels, MY-174 antigenicity, and hyaluronan binding ability of SPACRCAN peaked around embryonic day 17 and then gradually decreased, whereas the corresponding expression levels of SPACR simply increased, but not its hyaluronan binding ability. The MY-174 reactivity of SPACRCAN in the adult retina was decreased compared with that in the newborn retina, whereas that of SPACR was increased. The decreased hyaluronan binding of SPACR was induced by an inhibitory effect of the excess of sialic acids in the adult stage. Thus, with similar core protein structures and specific sialylated glycoconjugates but distinct chondroitin sulfate chains, SPACRCAN and SPACR may have separate roles in the retina due to their differing expression profiles during development.     

Role of calcium-dependent and calcium-independent mechanisms in the adhesion of retinal and pigment epithelium cells in the chick embryo

The mechanisms of adhesion of the retinal and pigment epithelium cells, as well of cell interaction within each of these tissues were studied during development. It was shown by means of separation of retina from pigment epithelium in different dissociation media that the adhesion of these tissues in 5-6 day old chick embryos is realized via a Ca2+-independent mechanism. The adhesion of these tissues decreases between days 7 and 16. Starting from day 16, both Ca2+-independent and Ca2+-dependent mechanisms are involved in the interaction of the retinal and pigment epithelium cells. By measuring the output of single cells into the suspension after the treatment of retina and pigment epithelium with different dissociating agents, it was shown that from the 5th day of incubation on the adhesion of pigment epithelium cells is mediated by Ca2+-dependent mechanism. In the retina three types of cells were found: interacting via Ca2+-dependent mechanism only, Ca2+-independent mechanism only, and both the mechanisms. In the course of differentiation, the numbers of the population of cells interacting only via Ca2+-dependent mechanism increase, while those of cells interacting via Ca2+-independent mechanism decrease. It is suggested that at each developmental stage those retinal cell possess Ca2+-dependent mechanism of adhesion which are closest to the definitive state.     

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Altered retinal metabolism in diabetes. II. Measurement of sodium-potassium ATPase and total sodium and potassium in individual retinal layers

Pathological changes in retinas of diabetics include specific morphological, biochemical, and functional abnormalities. As biochemical manifestations of the disease, increased sorbitol and decreased myo-inositol were found in retinas of experimentally diabetic animals. Similar alterations in polyol metabolism have been associated in nerves of diabetics with a reduction of Na+-K+-ATPase activity. To determine whether this association extends to the retinas of diabetic animals, we applied quantitative histochemical techniques to measure ATPase activities and the amounts of sodium and potassium in samples from nine individual layers of cryostat sections of rabbit retina. ATPase activities were determined fluorimetrically, and the ions were measured by atomic absorption with a carbon rod atomizer. The activity of Na+-K+-ATPase was reduced in the retinal pigmented epithelium (retinal pigment epithelium) and in selected layers of the neural retina, and total sodium in the retinal pigment epithelium layer was elevated in diabetes. The retinal pigment epithelium forms the outer component of the blood-retinal barrier and partly determines the composition of the retinal interstitial fluid. Changes in retinal pigment epithelium biochemistry and function might alter the intraretinal environment, predisposing neural retina or retinal blood vessels to disease. The morphologically and functionally well defined retinal pigment epithelium may provide a useful model for studying the pathogenesis of diabetic complications.     

Experimental uveitis induced by products of activated lymphocytes: Intraocular effects of rhodopsin-induced lymphokines

Immunization of strain 13 guinea pigs by footpad injection of bovine rhodopsin in complete Freund's adjuvant produces experimental autoimmune uveitis (retinopathy) with retinal pathology characterized by destruction of the retinal photoreceptor cell layer, as we reported previously. Since rhodopsin-induced EAU can be passively transferred by immune cells but not antirhodopsin antibody, the present studies were conducted to determine if soluble products of activated lymphocytes (PAL) could cause the retinal pathology seen in experimental uveitis. These studies, reported here, show that intravitreal injection of supernatant factors generated by guinea pig lymph node cells specifically activated in vitro with rhodopsin or purified protein derivatives of tuberculin or nonspecifically activated with the mitogen concanavalin A produce uveitis in the normal guinea pig eye. The PAL produced mononuclear cell infiltration in the vitreous and a retinopathy with loss of retinal photoreceptor cells. Inflammatory cell infiltrates were found in the retina but not found in the anterior segment of the eye. Control lymphocyte supernatants did not produce retinal pathology. In guinea pigs sensitized to rhodopsin-complete Freund's adjuvant, intravitreal injection of the PAL produced a mononuclear vitreal infiltrate, disruption of the photoreceptor cell layer, necrosis of the inner retina, and a granulomatous infiltrate in the choroid with damage to the retinal pigment epithelium. Injection of control supernatants into the rhodopsin-complete Freund's adjuvant sensitized guinea pig eye did not produce inflammation and the retinal photoreceptor cell layer remained intact. Results from our studies indicate a role for the PAL along with other, yet undefined, factors in the initiation of autoimmune uveitis and in the autoimmune pathology of the retina.     

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。     

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.     

Vertebrate features revealed in the rudimentary eye of the Pacific hagfish (Eptatretus stoutii)

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens and a retinal architecture built of three cell layers: the photoreceptors, interneurons and ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here, we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable PAX6+ ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the RH1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homologue of gnathosome retinal pigment epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina and subsets of these express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution.     

Neuron-specific TGF-beta signaling deficiency results in retinal detachment and cataracts in mice

We generated a mouse model (cKO) with a conditional deletion of TGF-beta signaling in the retinal neurons by crossing TGF-beta receptor I (TGF-beta RI) floxed mice with nestin-Cre mice. Almost all of the newborn cKO mice had retinal detachment at the retinal pigment epithelium (RPE)/photoreceptor layer junction of the neurosensory retina (NSR). The immunostaining for chondroitin-6-sulfate showed a very weak reaction in cKO mice in contrast to intense staining in the photoreceptor layer in wild-type mice. Macroscopic cataracts, in one or both eyes, were observed in 50% of the mice by 6 months of age, starting as early as the first month after birth. The cKO mouse model demonstrates that the TGF-beta signaling deficiency in retinal cells leads to decreased levels of chondroitin sulfate proteoglycan in the retinal interphotoreceptor matrix. This in turn causes retinal detachment due to the loss of adhesion of the NSR to RPE.     

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

Abstract: The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5–50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase Ftreatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-β-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-β-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-α-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.     

An Organotypic Culture of the Newt Retina together with Other Tissues of the Posterior Eye Segment as a Model for Studying the Effects of Cell Adhesion Glycoproteins

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins as well as the localization of acetylcholinesterase activity were found during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.     

Basic fibroblast growth factor induces retinal regeneration in vivo

In the present study, we have investigated the effect of basic fibroblast growth factor (bFGF) on retinal regeneration in the stage 22-24 chick embryo. The neural retina was surgically removed in ovo leaving the retinal pigment epithelium (RPE) intact and then slow-release, plastic implants containing bFGF were inserted into the eye. Light microscopic examination of eyes 7 days later revealed that bFGF induced retinal regeneration in a dose-dependent manner. The absence of the RPE in these eyes and the reversed polarity of the regenerated neural retina is consistent with the hypothesis that this process occurs by transdifferentiation of the RPE. This represents the first time that a known molecule has been shown to induce retinal regeneration in vivo.     

Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.     

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.     

Differentiation Markers of Retinal Cell Types in Studies on Vertebrate Eye Development and Regeneration

Data on the use of various immunochemical markers specifically indicating cell types of the neural retina and pigment epithelium are reviewed. It is demonstrated how this approach can be applied to the analysis of specific features of vertebrate retinal development, including the order and timing of differentiation of the main cell types, their interdependence in the course of this process, and factors controlling the latter. Problems concerning the state of differentiation and its change in the cells of retinal pigment epithelium and glial cells are discussed in respect to their analysis with the aid of specific protein markers. The current state of retina regeneration research involving the use of labeled cell sources and regenerated cells in lower vertebrates is analyzed. Problems in the search for new markers of retinal photoreceptor, macroglial, and microglial cells and their use in experiments are addressed.     

An extracellular retinol-binding glycoprotein in the rat eye—characterization,localization and biosynthesis

We have identified and partially purified interstitial retinol-binding protein (IRBP) from the subretinal space of the rat. It appeared to be glycosylated. Its apparent mol. wt was 270,000 by gel-filtration and 144,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rat IRBP cross-reacted with anti-bovine IRBP sheep and rabbit sera, bound all-trans-[15-³H] retinol and was bound by concanavalin A. IRBP was not detected in the cytosols of the neural retina or retinal pigment epithelium and choroid. This distribution was confirmed by immunocytochemistry using a fluorescence-labeled second antibody. Immunospecific fluorescence was most intense in the interphotoreceptor matrix in a 6.5 μm band adjacent to the retinal pigment epithelium. It was less intense over the remainder of the rod outer segment layer and was comparatively faint over the inner segment region. Its occurrence in the interstitial space between the photoreceptors and retinal pigment epithelium coupled with the fact it bound all-trans-[15-³H] retinol supports the concept that it may be implicated in the transport of retinoids between the retina and the retinal pigment epithelium during the visual cycle. When incubated with [³H]leucine or [³H]glucosamine, isolated retinas (but not retinal pigment epithelium and choroid) secreted labeled IRBP into the medium. This suggests that the retina plays a role in regulating the amount of IRBP in the subretinal space.     

Markers of retinal-type cell differentiation in studies of eye development and regeneration in vertebrates]

Data on the use of various immunochemical markers specifically indicating cell types of the neural retina and pigment epithelium are reviewed. It is demonstrated how this approach can be applied to the analysis of specific features of vertebrate retinal development, including the order and timing of differentiation of the main cell types, their interdependence in the course of this process, and factors controlling the latter. Problems concerning the state of differentiation and its change in the cells of retinal pigment epithelium and glial cells are discussed in respect to their analysis with the aid of specific protein markers. The current state of retina regeneration research involving the use of labelled cell sources and regenerated cells in lower vertebrates is analyzed. Problems in the search for new markers of retinal photoreceptor, macroglial, and microglial cells and their use in experiments are addressed.     

beta 1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican.

Integrins are cell-surface glycoproteins that mediate cell activities, including tissue morphogenesis, development, immune response, and cancer, through interaction with extracellular proteins. Here we report a novel means by which integrin signaling and functions are regulated. In pull-down assays and immunoprecipitation, beta(1)-integrin bound to the C-terminal domain of PG-M/versican, an extracellular chondroitin sulfate proteoglycan. This was confirmed by cell-surface binding assays. Binding was calcium- and manganese-dependent. Upon native gel electrophoresis, beta(1)-integrin comigrated with the C-terminal domain of PG-M/versican. The interaction of beta(1)-integrin with the C-terminal domain of PG-M/versican activated focal adhesion kinase, enhanced integrin expression, and promoted cell adhesion. As a result, cells expressing the C-terminal domain of PG-M/versican were resistant to free radical-induced apoptosis. As the PG-M/versican peptide used in this study does not contain the RGD consensus-binding motif for integrins, the mechanism of the observed binding represents an entirely new function.