Retinal Pigmented Cells Research Articles

Isolation and Characterization of a Mouse Homolog of the X-Linked Ocular Albinism (OA1) Gene

Ocular albinism type 1 (OA1) is an X-linked human genetic disorder that affects retinal pigment cells and, to a lesser degree, neural crest-derived melanocytes. TheOA1gene is located close to the pseudoautosomal region and predicts a novel protein whose function is unknown. However, histologic studies of affected patients have suggested a potential role in melanosome biogenesis. Here we report the isolation and characterization of the mouse homolog of the humanOA1gene, termedMoa1.TwoMoa1isoforms were isolated from a melanoma cDNA library and predicted to encode proteins of 405 and 249 amino acids with six and two transmembrane-spanning regions, respectively. Interspecific backcross mapping yielded a map order and distances (cM) of cen–Moa1–3.1 ± 1.8–Piga–2.1 ± 1.5–Amel,indicating thatMoa1is located much farther away from the pseudoautosomal region than its human homolog. In adult tissues, bothMoa1isoforms were detected in the eye by Northern hybridization. In neonatal tissues,Moa1RNA was detected in both skin and eyes by Northern hybridization and was not affected by the absence of pigment in mice carrying thealbinomutation, or by the type of pigment synthesized, i.e., eumelanin vs pheomelanin, in mice carrying theblack-and-tanmutation. Expression ofMoa1RNA was not detected in embryonic tissues by Northern analysis or byin situhybridization despite the active synthesis of ocular pigment by E16.5. These results provide insight into the structure and possible function of the OA1 protein and suggest a more complex relationship between the human and mouse X chromosomes than was previously thought to exist.

Characterization of a new melanocyte-specific gene (QNR-71) expressed in v-myc-transformed quail neuroretina.

Quail neuroretina cells (QNR) infected with the v-myc-expressing retrovirus MC29 become pigmented after several passages in vitro. After differential screening of a cDNA library constructed from these cells, we have isolated a cDNA clone (QNR-71) which identifies an RNA expressed only in the pigmented layer of the retina and in the epidermis. This gene can also be induced in other cell types transformed by MC29, suggesting that QNR-71 may be regulated by the v-myc protein. Sequence analysis showed that the QNR-71 cDNA exhibits stretches of homologies with melanosomal proteins encoding genes. From bacterially expressed QNR-71 peptides we obtained rabbit antisera able to specifically recognize two proteins of 95 and 100 kDa in pigmented retinal cells, but not in the neuroretina. To study the regulation of QNR-71, we used promoter fragments linked to the CAT reporter gene, in transient co-expression assay. We observed an increase in CAT expression with a c-MYC and microphtalmia (mi) expression vectors. Both MYC and mi activate the QNR-71 promoter through direct binding to a CATGTG site present in the promoter fragment.

Light intensity and schooling behaviour in larval gulf menhaden

Schooling behaviour was examined in larval gulf menhaden Brevoortia patronus under decreasing and increasing light intensities and related to movements of retinal photoreceptors and pigment. Nearest neighbour angles and nearest neighbour distances between fish were inversely related to light intensity and were independent of the direction of intensity change. Swimming speeds of individual fish were directly related to light intensity when intensity was decreased but did not increase when intensity was raised. The changes in nearest neighbour angles and distances more closely paralleled movements of retinal pigment cells than cone cell migration, suggesting that rod photoreceptors play a greater role than cones in determining the threshold light intensity for schooling. A general model is developed relating the threshold light intensity for schooling to eye diameter for a number of different species and the implications of this relationship are discussed.

Single cell motion in aggregates of embryonic cells.

We investigate the motion of individual pigmented retinal cells in aggregates of neural retinal cells obtained from chicken embryos. Individual cells in aggregates move randomly in the absence of chemical or adhesion gradients: the power spectrum of position and velocity versus time correspond to Brownian motion and the velocities are uncorrelated in time. Thus a quasithermal model of cell migration like the extended Potts model is appropriate. We also measure cell diffusivity and the cell velocity distribution. The results support the idea that collections of embryonic cells behave as liquids with membrane fluctuations playing the role of temperature.

Isolation and in vitro propagation of human choroidal fibroblasts

Subretinal ingrowth of choroidal fibroblasts is of importance in several clinical settings such as age-related macular degeneration or after traumas such as laser treatment and infection. In the present investigation we describe a method for isolation and propagation of human choroidal fibroblasts in vitro. Outgrowth from the outseeded choroid was studied by means of phase contrast microscopy, scanning electron microscopy and by immunohistochemical methods. The secondary cultures were found to contain almost only fibroblasts (more than 95% of the cells), some contaminating retinal pigment cells were also found. Choroidal fibroblasts contribute to the fibrovascular subretinal scarring process, and our cell culturing system seems to be a suitable tool for studying factors regulating the behavior of these cells in vitro.

Quantitative comparison between differential adhesion models and cell sorting in the presence and absence of fluctuations.

We study the effect of inhibition of cell-membrane ruffling on cell sorting in experiments and simulations. We use aggregates of neural retinal and pigmented retinal cells obtained from chicken embryos and inhibit membrane ruffling by adding the drug cytochalasin B to the culture medium. Simulations using the large-Q Potts model with differential adhesivity, with the simulation temperature controlling the membrane ruffling amplitude, agree quantitatively with the experimental results for both normal and low fluctuation sorting, providing strong support for the validity of the model. In both cases, inhibition of ruffling prevents large scale cell sorting.

The origin and development of retinal pigment cells and melanophores analyzed by Xenopus black-white chimeras.

At the 16 cell stage, three kinds of borealis-laevis and eight kinds of laevis-laevis chimeric embryos were produced by replacing a particular blastomere of albino embryos of Xenopus laevis with that of wild-type embryos of X. borealis or X. laevis, and then leaving the embryos to develop into frogs. In the borealis-laevis chimera frogs, we found that all the melanized cells (retinal pigment cells and melanophores) were derived from a transplanted wild-type blastomere with a nuclear marker of X. borealis and that all the albino-mutant cells derived from the host did not become melanized. Thus, retinal pigment cells and melanophores differentiated according to their own genotype. We then examined the origin of these two types of cells, using melanin as a cell-marker in the borealis-laevis and laevis-laevis chimeras. Retinal pigment cells derive from A1 (dorso-animal) and A2 (latero-animal) blastomeres. A1 of one side contributes to retinal pigment cells in both eyes. Though the blastomeres of one side contribute to the formation of bilateral melanophores, the major contribution is to melanophores of the same side. A1, A2 and V2 (latero-vegetal) form the anterior part of the neural fold, and A2 and V2 contribute to melanophores of the head region. The most anterior part of the neural fold derived from A1 does not make a significant contribution to melanophores. Though V2 is a vegetal blastomere, it forms the anterior part of the neural fold by upward movement against the downward movement for gastrulation. A3 forms the middle and posterior parts of the neural fold and contributes to melanophores of the trunk and hindlimbs. Melanophores of hindlimbs also come from A2, A4 and V2. It is to be noted that A4 contributes to melanophores of hindlimbs, despite no apparent contribution to the neural fold. Development of the retinal pigment cells and melanophores is discussed from the point of pigmentation patterns of the chimeras.

Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development

Analysis of retinal development in Delta (Dl) temperature-sensitive mutants reveals requirements for Delta function in the specification of all retinal cells, including photoreceptors, cone cells, pigment cells and cells that make up interommatidial bristles. In situ hybridization and immunohistochemistry indicate that Delta is expressed dynamically during the specification of different cell types. Comparisons of Delta expression patterns with developmental defects in Dl mutants implies that Delta functions in a cell-nonautonomous manner in the specification of photoreceptors. Delta protein resides predominantly in subcellular vesicles located primarily at the apical ends of developing retinal cells. Localization of Delta protein in Dl and shibire tsl mutants implies that Delta is targeted to the cell surface, but is efficiently removed via endocytosis, resulting in vesicular accumulation.

Molecular cloning, expression, and localization of a brain-specific phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha) from rat brain.

We have isolated cDNA clones encoding the rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), a novel member of the membrane phosphodiesterase I gene family. PD-I alpha cDNA has a 2,655-nucleotide open reading frame encoding a polypeptide of 885 amino acids with a calculated M(r) of 101,302. Northern blot analysis revealed that PD-I alpha transcript was abundantly present in cerebrum and cerebellum while its level was quite low in other tissues. In situ hybridization analysis revealed that PD-I alpha mRNA is localized in secretory epithelial cells in the brain and the eye including choroid plexus epithelial cells, ciliary epithelial cells, iris pigment epithelial cells, and retinal pigment cells. Localization of PD-I alpha mRNA was also observed in glial cells in the molecular layer of the cerebellum. These results indicate that this protein might be involved in the synthesis of adenosine as well as in the regulation of the secretion/transport of epithelial cells and glial cells in the central nervous system.

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

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 F treatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-beta-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-beta-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-alpha-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for alpha 2,6-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.

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

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.

Cellular Immune Response to la Induction by Intraocular Gamma-Interferon

BALB/c mice and Lewis rats were injected intraocularly with recombinant gamma-interferon (IFN-gamma). The distribution of Ia antigens and cellular infiltrates was studied over a 21-day time course. In mice, Ia antigens were induced on cells in the corneal stroma, corneal endothelium, iris, ciliary body, retinal pigment epithelium and cells in the retina. No detectable cellular infiltrate accompanied class II major histocompatibility (MHC) antigen induction in mice. In rats, cells in all of the intraocular tissues mentioned above were induced to express Ia antigens. However, in distinct contrast to mice, the induction of Ia antigens on intraocular structures in rats was accompanied by intense cellular inflammation composed predominantly of polymorphonuclear cells. These findings demonstrate that aberrant Ia expression on intraocular tissues by IFN-gamma is not sufficient to elicit a cellular autoimmune response in mice. The observed species-related differences in cellular immune response to class II MHC antigen induction on intraocular tissues parallels the susceptibility of mice and rats to experimental autoimmune uveitis.

A monoclonal antibody (3G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes.

The identification of microvascular pericytes in vitro relies principally on morphological characteristics and growth dynamics, as there is a paucity of immunochemical markers for these cells. Consequently, an attempt was made to identify mAb reagents that would aid in both the rapid identification and enrichment of retinal capillary pericytes in vascular cell cultures. A panel of mAbs raised by xenogeneic immunization of mice with various tissues was screened for immunoreactivity with dissociated cultures of bovine retinal capillary pericytes. Two antibodies from the panel (3G5 and HISL-8) were seen to react with pericytes by indirect immunofluorescence. The mAb 3G5 was selected for further study. mAb 3G5 did not react with dissociated cultures of smooth muscle cells, endothelial cells, or retinal pigmented endothelial cells. The pericyte 3G5 antigen was insensitive to the action of trypsin; therefore, mAb 3G5 was used to selectively purify pericytes from trypsinized mixed retinal cell cultures by flow cytometry. 3G5+ pericytes (representing 8% of cells in a mixed retinal cell culture) were enriched at least nine-fold to represent greater than 70% of cells. The mAb 3G5 stained retinal capillaries in vivo with a fluorescence distribution consistent with pericyte staining. The 3G5 antigen of cultured pericytes was found to be a glycolipid of mobility intermediate between ganglioside markers GM1 and GM2.

Retinal Detachment After Choroidal Ischemia

Injection of a 15-micron microsphere suspension through one or two vortex veins of nine monkey eyes caused various degrees of sectorial choroidal ischemia, which were documented by fluorescein angiography and electron microscopy. The severity of the lesions to the fundus depended on the volume of microspheres injected (0.4 to 1.6 ml of a suspension of 600,000 microspheres/ml). Three hours after embolization white patches appeared in the retinal pigment epithelium as well as a posterior pole serous retinal detachment in five eyes. Delayed choroidal filling was noted in the quadrant involved, but a few choriocapillaris units slowly perfused, leading to fluorescein leakage in the serous retinal detachments. Histologic examination showed various types of damage to the retinal pigment epithelium, including vacuolization and cell membrane rupture.

Further studies on the effect of low-dose laser irradiation on cultured retinal pigment cells of the chick.

The effect of low-intensity laser irradiation on pigment cells cultured by chorioallantoic method was studied. Laser irradiation influenced the cell shapes and sizes of the pigment cells. Metabolically, this treatment increased thymidine uptake and incorporation but reduced those of leucine. The phagocytic activity as measured by latex particles uptake was not affected. It was concluded that the biological effect of laser irradiation on cell cultures depends on the dose applied, the individual tissue and other factors.

Cytochemical localization of 5'-nucleotidase activity in retinal photoreceptor cells

Further cytochemical studies on the ultrastructural localization of 5′-nucleotidase in the rat retina have revealed activity to be associated with the complex synapses formed by the rod spherules of the receptors and the bipolar and horizontal cell processes. Activity was also seen on the axolemma of receptor fibers. In the rod inner segment strong reaction product is located intracellularly. In the rod outer segment the enzyme appears to be located only on the cytoplasmic side of the disc membrane and not intradiscally. Retinal pigment cells are rich in 5′-nucleotidase. Their microvilli accompany the tips of the receptor cells and show enzyme activity in an ecto position. A role for 5′-nucleotidase is possible in the metabolism of guanylate and adenylate nucleotides both of which are important for visual transduction processes.

Supercoiling of 30-nm chromatin fibers around a nuclear axis in human retinal pigment cell nuclei: Electron microscope evidence for a chromatin macrostructure

Nucleosomes and higher-order chromatin structures have been identified in the range of resolution of 10–30 nm. No information is available on higher configurations that involve chromatin arrangements at the nuclear level. Thin sections of human retinal pigment cell nuclei reveal an orderly array of 30-nm chromatin fibers on the inner nuclear surface. The observations can be three-dimensionally explained by a coiled or a serially circular arrangement of fibers around a central axis of the nucleus. The axial chromatin orientation shows no apparent relationship to the epithelial cell polarity nor to the irregular shape of the nuclei. The described configuration, which is compatible with a chromatin structure of third order, represents the first example of chromatin architecture coherently organized at the nuclear level.

Human retinal pigment cell culture.

Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm.

Autophagocytosis of melanosomes in cultured embryonic retinal pigment cells.

Autophagocytosis not only accounts for the early pigment loss found in proliferating cultured retinal pigment cells, but also occurs in slowly growing and in non-proliferating cells. Both melanosome synthesis and destruction may take place concurrently in cells. Autophagosomes may contain both fully formed melanosomes and also premelanosomes in various stages of formation, are positive for lysosomal enzyme activity, and likely represent secondary lysosomes.

Modulation of melanosome ultrastructure in cultured embryonic pigment cells.

Melanosomes synthesized by embryonic chick retinal pigment cells in vitro are of a different size and shape than those made in ovo and in vivo. Ultrastructurally, the melanosomes made by cultured cells are small, spherical, contain an irregular internal matrix, and appear similar to the pheomelanosomes found in red hair and feathers. This modulation in melanosome ultrastructure in cultured retinal pigment cells does not appear to be caused by an irreversible change in differentiated capability but to a response of cells to the culture environment. Cells grown in embryo extract-free and serum-free defined media were found to synthesize melanosomes of normal size and shape in vitro, although at a much reduced number. The predominant melanosome type found was still the smaller, spherical variety. It appears that some still unknown factors in the culture environment have produced a shift to a different type of melanogenesis.