The human retina and retinal pigment epithelium are abundant sources of vitronectin mRNA.

Vitronectin (Vn), a multifunctional plasma protein synthesized primarily in the liver, is often present as a component of the extracellular plaques and deposits that accompany various age-related human diseases. Recently, we reported that Vn is also a prominent molecular constituent of drusen, the extracellular deposits associated with age-related macular degeneration (AMD) (1). The cellular source(s) of the Vn in drusen, as well as in these other plaques and deposits, remains uncertain. In this study, we used real-time quantitative RT-PCR to measure the relative levels of Vn mRNA in the cells and tissues that lie in close proximity to drusen. The results confirm that the human liver is an abundant source of Vn mRNA. Levels of Vn mRNA in kidney, lung, and fetal or adult brain are <3% of those in liver. Remarkably, mean Vn mRNA levels in the neural retina significantly exceed those in brain and represent close to 40% of the Vn mRNA value measured in human liver. Substantial levels of Vn mRNA are also present in the adjacent retinal pigment epithelium (RPE). These results identify the neural retina, for the first time, as an abundant source of Vn mRNA. They also suggest that both the neural retina and RPE are potent biosynthetic sources of Vn in humans, and potentially significant local contributors to the Vn that accumulates in drusen.     

Differentiation of the retina and pigment epithelium in the ontogeny of the common frog. 1. The retina

Changes in the ultrastructure of the differentiating retinal cells were studied by means of electron microscopy in Rana temporaria at successive developmental stages. Common features of the onset of differentiation of the retinal cells have been shown: appearance of the granular endoplasmic reticulum elements, of the polysomes, beginning of utilization of the yolk and lipids, elimination of ovarial melanosomes. Later during the differentiation of retinal neurons the protein synthesizing machinery and Golgi complex of these cells develop markedly, the number of mitochondria increases. The differentiation of retina begins from the Müllerian cells (stage 28) which determine the direction of growth of the neuron processes. They are followed by the ganglion cells and photoreceptors (stage 29). The signs of differentiation of the inner nuclear layer neurons become apparent later, in the amacrine and horizontal cells at the same time and in the bipolars later. The main features of neuronal organization of the retina which determine the structural basis of its function of light perception are formed by stage 40.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

The ultrastructure of the epithelium of the ciliary body

Summary The fine structure of the human and rabbit ciliary body epithelium has been studied with the electron microscope, both under normal conditions and after paracentesis of the anterior chamber. The disposition of the junctional complexes in the two layers of the ciliary epithelium is described in detail. Junctional complexes appear particularly developed between the apical surfaces of the cells of the two layers, but are present, as in other monolayered epithelia, also between the lateral surfaces of adjacent cells of each layer. The junctional complexes connecting the apical surfaces of the cells of the two layers are represented by zonulae occludentes, zonulae adhaerentes and desmosomes following each other irregularly, with interposition of rounded dilatations of the intercellular space called ciliary channels. The zonulae occludentes and adhaerentes found along the lateral surfaces of the epithelial cells probably form discontinuous and overlapping fasciae. Moreover, the existence of a peculiar dove-tail arched junction called macula occludens is suggested. Few differences were encountered when comparing the arrangement of junctional complexes in the ciliary epithelium of man with that of the rabbit. Many desmosomes connecting the basal portion of lateral surfaces of the non-pigmented cells were found only in human ciliary bodies.The study of the modifications of the junctional apparatus of the ciliary epithelium following paracentesis of the anterior chamber, confirms the functional hypotheses on junctional complexes previously suggested: in particular only zonulae occludentes cause a real block of the intercellular spaces. On the basis of the present work, the close relationships between the number, kind and disposition of junctional complexes in epithelia and the functional possibilities of these epithelia are stressed.Dedicated to Professor W. Bargmann on his 60th birthday.Dr. Orzalesi is the recipient of a research grant from Ministero della Pubblica Istruzione for 1964.     

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.     

The ultrastructure of the compound eye of Munida rugosa (Crustacea: Anomura) and pigment migration during light and dark adaptation

The galatheid squat lobster, Munida rugosa, has compound eyes of the reflecting superposition type in which a distal cone cell layer and a proximal rhabdom layer are separated by an extensive clear zone. The eye is shown to have certain unique features. In all other reflecting superposition eyes, the clear zone is traversed by crystalline tracts formed by the cone cells. In M. rugosa a thin distal rhabdom thread, formed by the eighth retinula cell, connects the cones to the proximal fusiform rhabdoms. The cytoplasm of the other retinula cells also crosses the clear zone in a complex pattern. Fully light-adapted ommatidia are optically isolated by limited migrations of distal shielding pigments. A reflecting pigment multilayer lines each cone to facilitate the formation of a superposition image. This also shows a light-induced change which may limit the acceptance angle of the eye during light adaptation.     

Growth of the eye and pigment epithelium of the retina during postnatal development of rats]

By the weight changes, the rat's eye grows during the whole life, whereas its scleral sector with the area equal to that of pigment epithelium of the retina grows most intensively between the 2nd and 5th days after birth. During this period, its weight attains half of the value characteristic of the scleral sector for one year old rats. This period of growth of the scleral sector coincides with the previously established peak of proliferative activity and appearance of first binuclear cells in the pigment epithelium. From the 5th day till the 12th month after birth, the weight of scleral sector increases twice and its area 6 times. This suggests that the mechanical tension of the scleral sector walls is one of the factors of growth of this eye part. On the basis of comparison of the scleral sector growth, changes in proliferative activity and number of polyploid cells in the pigment epithelium of the central zone of fundus oculi, the following periodization of the life cycle of cell population of the pigment epithelium is proposed: (1) from birth till the 15th day--period of principal determination (the number of binuclear tetraploid cells attains 80%), (2) from the 15th day till, at least, the 5th month--period of stabilization, (3) after 5 months--period of senescence characterized by the accumulation of highly ploid tri- and tetranuclear cells; its lower limit is not clearly defined.     

Hyphal ultrastructure in fruit-body primordia of the basidiomycetesSchizophyllum commune andCoprinus cinereus

Summary Light and electron microscopic investigations of fruit-body primordia of the basidiomycetesSchizophyllum commune andCoprinus cinereus showed that hyphal fusions are common in specific areas of the fruit-body primordia of both species. In these areas conspicuous amounts of a mucilaginous substance occur between the closely packed hyphae. A possible function of this mucilage is to aid the adhesion of hyphae prior to fusion. In the fruit-body primordium ofCoprinus cinereus the dolipore/parenthesome septum is surrounded by an additional hemispherical membranous cap (outer cap). Such a cap was never found inSchizophyllum commune fruit-body primordia.     

Post-natal maturation of the retina in the albino rat. I. The pigment epithelium

The Authors studied the postnatal development of the retinal pigment epithelium in the albino rat, in order to elucidate its morphological and functional evolution, correlated to the numerous functional roles played in Vertebrates (Scheme 1). At birth, epithelial cells show few cytoplasmic organules and the apical surface provided of small depressions. From the third to the fifth postnatal day the first apical microfolds surround the depressions. From the seventh to the ninth day inner segments develop, whilst the apical surface of the epithelial cells is covered by many finger-like microfolds. During the eleventh postnatal day the buds of the outer segments and many lamellar microfolds can be demonstrated. During the sixteenth day the retina reaches its adult morphology. It is therefore well-evident that birth, similarly to many other Vertebrates, is not the last step, but only a moment, in the development of the retina: this process is completed only during postnatal life, when environmental light is able to stimulate every ocular structure.     

Epidermal ciliary ultrastructure of adult and larval sipunculids (Sipunculida)

The ultrastructure of the ciliary apparatus of multiciliated epidermal cells in larval and adult sipunculids is described and the phylogenetic implications discussed. The pelagosphera of Apionsoma misakianum has a dense cover of epidermal cilia on the head region. The cilia have a long, narrow distal part and two long ciliary rootlets, one rostrally and one vertically orientated. The adult Phascolion strombus has cilia on the nuchal organ and on the oral side of the tentacles. These cilia have a narrow distal part as in the A. misakianum larva, but the ciliary rootlets have a different structure. The first rootlet on the anterior face of the basal body is very short and small. The second, vertically orientated rootlet is long and relatively thick. The two ciliary rootlets present in the larval A. misakianum are similar to the basal metazoan type of ciliary apparatus of epidermal multiciliated cells and thus likely represent the plesiomorphic state. The minute first rootlet in the adult P. strombus is viewed as a consequence of a secondary reduction. No possible synapomorphic character with the phylogenetically troublesome Xenoturbella was found.     

The ultrastructure of the nauplius eye ofSapphirina (Crustacea: Copepoda)

Summary The ultrastructure of the specialized nauplius eye of three species of the copepod genusSapphirina was investigated. The gross morphology described earlier (Elofsson, 1966a) was confirmed. The ventral cup is covered by a red pigment and the lateral cups by a red and a black pigment. The ultrastructural configuration of the pigment granules was found to differ in the two kinds of pigment cells. The black pigment cell, moreover, contains a large number of transversely banded fibrils and is able to produce reflecting crystals. The pigment granules of the black pigment cell show a variation in electron density. An intimate connexion exists between the black pigment cell and large retinula cells in the lateral cups, indicating an exchange of material. The tapetal cells present in all three cups form crystal platelets contained in two sets of membranes. It is suggested that the ventral cup and part of the lateral cups function as thePecten-eye (Land, 1965). The rhabdomeres of the retinula cells are composed of microvilli measuring 400 Å. The orientation of these seems to exclude polarotactic behaviour. The ventral cup and the four small cells of the lateral cups contain some retinula cells with microvilli arranged parallel to the incoming light. The retinula cells further develop an intricate system of membrane-invaginations penetrating deep into the cell and associated with numerous mitochondria. Retinula cells of the ventral cup and part of the lateral cups contain clear portions filled with granular material only. Retinula and other cells contain attenuated mitochondria with parallel tubuli. The proximal lens in front of each lateral cup consists of one cell. A development from the conjunctival cells is suggested. The results are evaluated in terms of function and evolution.This work has been supported by a grant from the Swedish Natural Science Research Council (2760-2).     

The ultrastructure of the eye of the mosquitofishGambusia affinis

Summary The ultrastructure of the tissue components of the eye ofGambusia affinis, excluding the sensory cells, is described. The cornea consists of two different sections of collagenous layers of different density. The choroid includes an argentea composed of- and-melanophores, lipopterinophores and a choriocapillaris associated with the rete mirabile of the choroid body. Bruch's membrane, underlying the retinal pigment layer, can develop complex associations with fibroblasts delimiting the choriocapillaris. The outer section (stroma) of the iris includes several cell types that are not found in the inner or vitread section. In adultGambusia the lens capsule is well developed, but in twoweek-oldSarotherodon larvae the lens epithelium is covered only by a glycocalyx.     

Comparative analysis of the uptake and expression of plasmid vectors in human ciliary and retinal pigment epithelial cells in vitro.

The retinal pigment epithelium is uniquely suited to gene therapy that uses lipid-mediated DNA transfer due to its high phagocytic activity in situ. We compared the relative efficacy of phagocytosis on the uptake of labeled plasmid vectors by retinal pigment epithelial and ciliary epithelial cells in vitro. Relative levels of endocytosis were then compared with the efficiency of marker transgene expression in these cells. Human retinal pigment epithelial and ciliary epithelial cells from a single donor were isolated and expanded in vitro. Polyplex-mediated transfections were performed using a rhodamine-labeled expression vector for green fluorescent protein. Rhodamine-labeled endosomes were examined by fluorescence microscopy at different time points. Rhodamine labeling and green fluorescent protein expression were analyzed by flow cytometry 48 h after transfection. These gene transfer studies showed that expression of transgenes does occur in both human retinal pigment epithelial and ciliary epithelial cells in vitro. Endocytosis of labeled plasmid vectors occurs at a significantly higher number and density in retinal pigment epithelial cells than in ciliary epithelial cells (P < 0.04). However, the efficiency of marker transgene expression is similar in the two cell types. These studies demonstrate that the higher intrinsic phagocytic activity does not enhance the efficacy of transgene expression in retinal pigment epithelial cells in vitro. Both human retinal pigment epithelial and ciliary epithelial cells are competent recipients for lipid-mediated gene transfer, and transgene expression occurs at similar levels in both cell types.     

The intracytoplasmic channel in pigment epithelial cells of the chick retina

Summary The fine structure of pigment epithelial cells in the chick retina was studied by electron microscopy with a special attention to the intracytoplasmic channel which is considered to be an important passage of metabolites from the choroidal side to the vitreal side. The chick retina was fixed either by perfusion with glutaraldehyde followed by osmium tetroxide or by immersion in situ with osmium tetroxide before removal of the eyeball. The infoldings appearing in the basal zone of the retinal pigment epithelial cell were provided with the gear-like projection which was encountered as their bottom in many cases, suggesting selective absorption of proteins. It was noticed that certain interspaces of the infoldings were continuous to tubular elements of the agranular endoplasmic reticulum. Moreover, the tubular elements were found in association with such other cellular components as nuclear envelope, mitochondria, fuscin granules and plasma membrane surrounding the outer segment of photoreceptor. The pigment epithelial cell appeared to be continuous with the photoreceptor through the pores of their plasma membranes. The presence of a certain intracytoplasmic channel from the choroid to the photoreceptor is considered to facilitate the transport of metabolites in the pigment epithelial cell.Part of these observations was presented at the Sixth International Congress for Electron Microscopy, Kyoto in 1966.I wish to thank Prof. Gonpachiro Yasuzumi for his valuable advices and discussions through this study.