Immunocytochemical localization of photopigments in cephalopod retinae

Summary The photopigments, rhodopsin and retinochrome, have been localized in cephalopod retinae using light and electron microscopic immunocytochemical methods. Polyclonal antibodies prepared against squid opsin demonstrated the presence of this protein in the photoreceptor rhabdomes, Golgi zone, Golgi-associated vesicles, plasma membrane, large cytoplasmic vesicles, and axonal membranes of octopus retinae. Monoclonal anti-opsin immunostained the rhabdomes and multivesicular bodies in the photoreceptor inner segments of squid. We believe the multivesicular bodies are involved in rhabdome turnover. Polyclonal anti-retinochrome localized this photopigment to the myeloid bodies of the photoreceptor inner segments, the rhabdomes, and to the extracellular space between opposing rhabdomeres in octopus retina. The results suggest some interesting functional relationships between rhodopsin and retinochrome with regard to chromophore exchange between illuminated forms of these photopigments and chromophore addition to newly synthesized opsin.     

Expression of developmentally defined retinal phenotypes in the histogenesis of retinoblastoma.

Retinoblastoma, the most common intraocular tumor of childhood, is a malignant neoplasm that arises during retinal development. The embryonal cell target for neoplastic transformation is not yet clearly defined. To better understand the histogenetic potential of this tumor, the expression of photoreceptor and glial cell-associated proteins were examined in 22 primary retinoblastomas. Interphotoreceptor retinol-binding protein (IRBP), cone and rod opsins were selected as the photoreceptor specific proteins due to their different temporal patterns of expression during normal retinal development. Neoplastic Müller cell differentiation, and non-neoplastic reactive astrocytes were identified using cellular retinaldehyde binding-protein (CRAlBP), and glial fibrillary acidic protein (GFAP), respectively. Photoreceptor proteins were present in 16 cases and showed different cellular patterns of expression. IRBP and cone opsin were usually abundant. Although rod opsin was clearly identified in eight tumors, its expression was more restricted than either IRBP or cone opsin. This differential pattern of expression, opposite to the normal pattern of photoreceptor gene expression in the adult retina, corresponded to a marked decrease in mRNA for rod opsin. Cone opsin and IRBP colocalized in fleurettes demonstrating that neoplastic human cone cells are capable of IRBP synthesis. Müller cell differentiation was present in 12 of the 16 cases in which photoreceptor proteins were detected. In contrast, GFAP was only present in reactive, stromal astrocytes associated with blood vessels. Our data suggest that the retinoblastoma has the histogenetic potential of the immature neural retinal epithelium which can give rise to both photoreceptor and Müller cell lineages. The differential expression of cone and rod phenotypes in retinoblastoma is consistent with the "default" mechanism of cone cell differentiation.     

Fused rhabdomeres (fur) in Drosophila: an eye mutation that alters rhabdomere morphology and retinal function.

In the Drosophila compound eye, the photoreceptor cells are organized in highly precise units, the ommatidia. In each photoreceptor cell, the primary photopigment, opsin, is contained in the rhabdomere, an ordered array of densely packed microvilli. A genetic and phenotypic analysis of a new X-linked. P element-induced mutation, fur, (fused rhabdomeres) is presented. Light and electron microscope studies show that mutations at the fur locus result in the fusion of the adjacent rhabdomeres in the developing eye and the fusion takes place during the pupal stage of eye development. Electrophysiological experiments indicate that the fur mutant photoreceptors have reduced sensitivity to light and lack a PDA (prolonged depolarizing afterpotential), a response characteristic of normal photoreceptor cells. Recombination and deficiency mapping localize fur to the proximal region of the X chromosome. Reversion analysis indicates the fur mutant is the result of a P element insertion. These studies suggest that the fur locus encodes a gene that has specific roles in rhabdomere morphogenesis and retinal function.     

Domain-specific early and late function of Dpatj in Drosophila photoreceptor cells.

The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Dpatj (Drosophila homolog of mammalian Patj) is a multi-PDZ domain protein that localizes to the apical cell membrane and forms a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (Sdt). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the dpatj gene. Our genetic analysis of hypomorph, null, and RNA interference reveals distinct dual functions of Dpatj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Dpatj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Dpatj is essential for animal viability and photoreceptor morphogenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced levels of Dpatj. Dpatj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. Our study provides evidence that Dpatj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells.     

Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina

Immunohistochemistry was used to determine the distribution of Rac1, Cdc42, RhoA and RhoB GTPases during development of the chick retina. All proteins appear as early as embryonic day 5 (E5) in cells of the vitreal margin, E7–8 in cells of the inner third of the inner nuclear layer and E9–10 in photoreceptors. From E10 until hatching, RhoA, Rac1 and Cdc42 were seen in perikarya and/or processes of amacrine, ganglion cells, and photoreceptors. Rho proteins were also observed in retinal Müller cells, with different distributions. RhoB showed a transient expression, being severely down regulated after E18. The distribution pattern of Rho proteins during the development of the chick retina suggests a concerted role in the differentiation of specific cell types, and probably during synaptogenesis.     

Structural changes in retinal pigmented epithelium of Rivulus marmoratus Poey embryos during development

Summary In Rivulus marmoratus development of retinal pigmented epithelium (RPE) parallels that of retinal photoreceptors. Although structurally functional by mid-incubation the full complex structure is not achieved even when the yolk-sac is absorbed (3-days post-hatched). Melanogenesis is evident at 0.2 incubation with premelanosomes present up to three days after hatching. The distribution of junctional complexes, basal membrane foldings and coatedpits throughout development is noted. Myeloid bodies, already present at mid-incubation, appear initially as single lenticular-shaped structures which later may form whorls, or coalesce around oil droplets, glycogen clusters or phagosomes thereby giving rise to myeloid patterns characteristic of a mature RPE. The functional significance of these changes is discussed.     

S100B and S100A1 proteins in bovine retina:their calcium-dependent stimulation of a membrane-bound guanylate cyclase activity as investigated by ultracytochemistry.

The Ca2(+)-binding proteins of the EF-hand type, S100B and S100A1, were detected in the outer segment of bovine retina photoreceptors where they are localized to disc membranes, as investigated by immunofluorescence and immunogold cytochemistry. S100B and S100A1 stimulate a membrane-bound guanylate cyclase activity associated with photoreceptor disc membranes in dark-adapted retina in a Ca2(+)-dependent manner, although with different Ca2+ requirements, as investigated by an ultracytochemical approach. Other retinal cell types express S100B and S100A1 as well. S100B is detected in the outer limiting membrane, fine cell processes in the outer nuclear layer and the outer plexiform layer, cell bodies in the inner nuclear layer and the ganglion cell layer, and the inner limiting membrane, whereas S100A1 has a more discrete distribution. S100B and S100A1 also stimulate a membrane-bound guanylate cyclase activity in photoreceptor cell bodies and Muller cells, but their effect appears independent of the light- or dark-adapted state of the retina and is observed at relatively high Ca2+ concentrations. These data represent the ultrastructural counterpart of recent biochemical observations implicating S100B and, possibly, S100A1 in the Ca2(+)-dependent stimulation of a photoreceptor membrane-bound guanylate cyclase activity [T. Duda, R. M. Goraczniak and R. K. Sharma (1996) Molecular characterization of S100A1-S1000B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclast. Biochemistry 35, 6263-6266; A. Margulis, N. Pozdnyakov and A. Sitaramayya (1996) Activation of bovine photoreceptor guanylate cyclast by S100 proteins. Biochem. Biophys. Res. Commun. 218, 243-247]. Our data suggest that at least S100B may take part in the regulation of a membrane-bound guanylate cyclase-based signalling pathway in both photoreceptors and Muller cells.     

The pineal gland in wild-type and two zebrafish mutants with retinal defects

Light and transmission electron microscopy were used to characterize the ultrastructural features of the pineal glands of wild-type and two mutant zebrafish strains that have retinal defects. Particular attention was given to the pineal photoreceptors. Photoreceptors in the pineal gland appear quite similar to retinal cone photoreceptors, having many of the same structural characteristics including outer segment disk membranes often confluent with the plasma membrane, calycal processes surrounding the outer segments, and classic connecting cilia. The pineal photoreceptor terminals differ from photoreceptor terminals in the retina in that they have short synaptic ribbons and make dyad synapses which may or may not be invaginated. Pineal photoreceptors in two zebrafish mutants with abnormal retinal photoreceptors were also studied. Pineal photoreceptors in the niezerka (nie) mutant degenerate, as they do in the retina, indicating that pineal and retinal photoreceptors share at least some genes. However, the synaptic terminals of no optokinetic response c (nrc) pineal photoreceptors are normal, suggesting that this mutation is specific to the retina.     

Immunocytochemical localization of opsin in rod photoreceptors during periods of rapid disc assembly

Summary Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors ofXenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5–6 loop regions of bovine opsin. This was done on isolated retinas incubatedin vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining ofXenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the fracture-label technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.     

Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.     

Mpp4 recruits Psd95 and Veli3 towards the photoreceptor synapse

Membrane-associated guanylate kinase (MAGUK) proteins function as scaffold proteins contributing to cell polarity and organizing signal transducers at the neuronal synapse membrane. The MAGUK protein Mpp4 is located in the retinal outer plexiform layer (OPL) at the presynaptic plasma membrane and presynaptic vesicles of photoreceptors. Additionally, it is located at the outer limiting membrane (OLM) where it might be involved in OLM integrity. In Mpp4 knockout mice, loss of Mpp4 function only sporadically causes photoreceptor displacement, without changing the Crumbs (Crb) protein complex at the OLM, adherens junctions or synapse structure. Scanning laser ophthalmology revealed no retinal degeneration. The minor morphological effects suggest that Mpp4 is a candidate gene for mild retinopathies only. At the OPL, Mpp4 is essential for correct localization of Psd95 and Veli3 at the presynaptic photoreceptor membrane. Psd95 labeling is absent of presynaptic membranes in both rods and cones but still present in cone basal contacts and dendritic contacts. Total retinal Psd95 protein levels are significantly reduced which suggests Mpp4 to be involved in Psd95 turnover, whereas Veli3 proteins levels are not changed. These protein changes in the photoreceptor synapse did not result in an altered electroretinograph. These findings suggest that Mpp4 coordinates Psd95/Veli3 assembly and maintenance at synaptic membranes. Mpp4 is a critical recruitment factor to organize scaffolds at the photoreceptor synapse and is likely to be associated with synaptic plasticity and protein complex transport.     

Autoradiographic and biochemical analysis of photoreceptor membrane renewal inOctopus retina

Summary Using autoradiographic and biochemical methods, we have demonstrated the renewal of light-sensitive membranes and photopigments inOctopus visual cells. After the injection ofOctopus with [³H]leucine, electron microscope autoradiography revealed an intracellular pathway similar to that in vertebrates for the synthesis and transport of nascent protein from the inner segments to the rhabdomes. However, migration of labelled protein from synthetic sites to the light-sensitive rhabdomes took longer inOctopus than the equivalent process in vertebrates. Biochemical analysis of [³H]leucine-labelled retinas identified some of the labelled protein observed in autoradiographs of the rhabdomes as the visual pigment, rhodopsin. We have shown that retinochrome, a second photopigment in cephalopod retinas, is also renewed. Biochemical analysis 8 h after injection of [³H]leucine revealed heavy labelling of this photoprotein. Light microscope autoradiography ofOctopus retina 8 h after injection of [³H]retinol showed labelling of both the rhabdomes and the myeloid bodies of the inner segments. Biochemical data gathered 8 h after injection of [³H]retrnol indicated chromophore addition to both rhodopsin and retinochrome with retinochrome being more heavily labelled than rhodopsin. Thus, silver grains observed over the rhabdomes and inner segments could arise from one or both photopigments. These data suggest that retinal is stored in the myeloid bodies of the photoreceptor inner segments. Retinal could then be transferred, perhaps via retinochrome, to newly synthesized opsin before the visual pigment is assembled into new rhabdomeric membranes. Alternatively, retinochrome may serve to transport retinal from the myeloid bodies to the rhabdomes to regenerate rhodopsin as previously proposed.     

DPATJ plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye.

The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including DPATJ (formerly known as Discs lost). Taking advantage of a DPATJ mutant line in which only a truncated form of the protein is synthesized, we demonstrate that DPATJ is necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, DPATJ protects against light-induced photoreceptor degeneration.     

Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3

Mutations in the retinitis pigmentosa 2 (RP2) gene cause a severe form of X-linked retinal degeneration. RP2 is a ubiquitous 350 amino acid plasma membrane-associated protein, which shares homology with the tubulin-specific chaperone cofactor C. RP2 protein, like cofactor C, stimulates the GTPase activity of tubulin in combination with cofactor D. RP2 has also been shown to interact with ADP ribosylation factor-like 3 (Arl3) in a nucleotide and myristoylation-dependant manner. In this study we have examined the relationship between RP2, cofactor C and Arl3 in patient-derived cell lines and in the retina. Examination of lymphoblastoid cells from patients with an Arg120stop nonsense mutation in RP2 revealed that the expression levels of cofactor C and Arl3 were not affected by the absence of RP2. In human retina, RP2 was localized to the plasma membrane of cells throughout the retina. RP2 was present at the plasma membrane in both rod and cone photoreceptors, extending from the outer segment through the inner segment to the synaptic terminals. There was no enrichment of RP2 staining in any photoreceptor organelle. In contrast, cofactor C and Arl3 localized predominantly to the photoreceptor connecting cilium in rod and cone photoreceptors. Cofactor C was cytoplasmic in distribution, whereas Arl3 localized to other microtubule structures within all cells. Arl3 behaved as a microtubule-associated protein: it co-localized with microtubules in HeLa cells and this was enhanced following microtubule stabilization with taxol. Furthermore, Arl3 co-purified with microtubules from bovine brain. Following microtubule depolymerization with nocodazole, Arl3 relocalized to the nuclear membrane. These data suggest that RP2 functions in concert with Arl3 to link the cell membrane with the cytoskeleton in photoreceptors as part of the cell signaling or vesicular transport machinery.     

Defining the integration capacity of embryonic stem cell-derived photoreceptor precursors

Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx(+) and Rhodopsin(+) photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ESC-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ESC-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.     

TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein

We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.     

Pineal and retinal photoreceptors in embryonic Rivulus marmoratus poey

The development of pineal and retinal photoreceptors was studied in embryos of Rivulus marmoratus Poey from 0.2 to 0.7 incubation period. Photoreceptors in both these organs are developed and clearly divided into outer and inner segments at mid-incubation period, in addition retinal photoreceptors are arranged in a square mosaic. However, while retinal photoreceptors contain synaptic ribbons at this developmental stage pineal photoreceptors do not; although presynaptic electron densities are present. At 0.7 incubation period synaptic ribbons are present in basal processes of pineal photoreceptors, while those in retinal photoreceptors have lengthened. The sequence of the development of photoreceptor structure in pineal and retina is discussed.     

A phosphatase activity and a synaptic vesicle antigen in multivesicular bodies of frog retinal photoreceptor terminals

Summary Previous work has suggested that multivesicular bodies participate in endocytosis and membrane cycling at nerve terminals, including the presynaptic terminals of retinal photoreceptors. We now have found that multivesicular bodies located in the presynaptic terminals of photoreceptors in retinae ofRana pipiens show reaction product in preparations incubated to demonstrate phosphatase activity at pH 5, using cytidine monophosphate as the substrate. Evidently, multivesicular bodies in photoreceptors can possess at least some hydrolytic enzymes during their sojourn in the terminals. We have also found that the multivesicular bodies in frog retinal photoreceptor terminals stain, immunocytochemically, for the presence of SV2, an antigen of synaptic vesicles. This observation supports the suggestion that, along with the extensive, repeated reuse of membrane components for synaptic vesicle recycling, there is some incorporation of the components into structures that are potentially degradative.