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.     

Histological techniques for study of photoreceptor orientation

An histological method for the study of photoreceptor orientation in primate eyes is described. To preserve photoreceptor orientation it is necessary to protect the fragile rod and cone outer segments to the maximum extent possible from mechanical deformation and from injury by solvent extraction. To prevent mechanical deformation the eyes are freeze-dried and embedded in plastic with or without prior vapor fixation. Solvent extraction from the lipidrich outer segment is limited by avoidance or restriction of organic solvents. When large segments of primate eyes are so treated, it is possible to section the plastic blocks along the visual axis, polish the block surface, and view photoreceptor orientation by epi-illumination microscopy. In such specimens a differential orientation of photoreceptors exists with the long axis of photoreceptor inner and outer segments in line with incoming light rays.     

Photoreceptors of cubozoan jellyfish

The anatomically sophisticated visual system of the cubozoan jellyfish Carybdea marsupialis is described. Individual cubomedusae have eight complex eyes, each with a cornea, lens, and retina of ciliated photoreceptor cells, eight slit ocelli, and eight dimple ocelli. The photoreceptor cells of the complex eyes are bipolar and resemble vertebrate rod cells. Each photoreceptor has an outer cylindrical light-receptive segment that projects into a vitreous space that separates the lens and the retina, an inner segment rich in pigment granules, and a basal region housing the nucleus. The outer segment is a modified cilium with a 9 + 2 arrangement of microtubules plus stacks of membrane. These stacks of membrane form numerous discs that are oriented transversely to the long axis of the cell. The outer segment is connected to the inner segment by a slender stalk. The basal end of each photoreceptor forms an axon that projects into an underlying layer of interneurons. Each ocellus is composed of ciliated photoreceptor cells containing pigment granules. Rhodopsin-like and opsin-like proteins are found in the membrane stacks of the outer segments of the photoreceptors of the complex eyes. An ultraviolet-sensing opsin-like protein is present in the inner segments and basal regions of some of the photoreceptors of the complex eyes. Rhodopsin-like proteins are also detected in the photoreceptors of the slit ocelli. The cellular lens, composed of crystallin proteins, shows a paucity of organelles and a high concentration of homogeneous cytoplasm. Neurons expressing RFamide (Arg-Phe-amide) comprise a subset of interneurons found beneath the retinas of the complex eyes. RFamide-positive fibers extend from these neurons into the stalks of the rhopalia, eventually entering into the subumbrellar nerve ring. Vision may play a role in the navigation, feeding, and reproduction of the cubomedusae.     

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer''s solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco''s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10⁵ cells/cm². The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.     

Intra- and interspecific changes in retinal morphology among mesopelagic and demersal teleosts from the slope waters of New Zealand

Synopsis Retinae from mesopelagic teleosts with adult ranges in the shallow, mid and deep mesopelagic zones, respectively, were examined by light microscopy. Retinal characteristics were described, and photoreceptor densities, outer segment dimensions, and convergence ratios measured from transverse sections. Juveniles of all species had lower photoreceptor densities, outer segment lengths and convergence ratios than adults. In species with multiple banks of photoreceptors, additional banks were added as the retina increased in size. A positive correlation was found between the degree of retinal specialisation for vision in dim light, and the depth of occurrence. The retina of each specimen was given a rank based on log unit changes in photoreceptor density and convergence ratio, the length of photoreceptor outer segments and the presence or absence of multiple banks of photoreceptors. Higher ranks (indicating greater retinal specialisation) were found among species occurring at greater depths. Among species showing a change in depth preference with growth, there was a corresponding increase in retinal rank. It is suggested that the proposed system of ranks has application in predicting the depth of occurrence of a species with a given pattern of retinal morphology.     

A rod-dominated visual system in leptocephalus larvae of elopomorph fishes (Elopomorpha: Teleostei)

The nature and distributions of photoreceptor cell types were investigated in the retinas of 12 species (5 families) of elopomorph anguilliform leptocephalus larvae. Anti-opsin immunofluorescence, light microscopy and transmission electron microscopy (TEM) were used to assess opsin distribution across the retinas and to associate photoreceptor morphology and opsin content. Retinas of all species were immunoreactive with anti-rhodopsin throughout, while anti-cone opsin immunoreactivity was restricted only to the ventral region of the retina in all specimens. Rod and cone photoreceptors were morphologically indistinguishable at low magnifications; TEM revealed that nearly all photoreceptors had rod-like ultrastructure, with only rare examples of cone-like cells identified in the ventral retina. These results indicate a rhodopsin/rod-dominated retina in leptocephalus larvae of anguilliform eels in the teleost subdivision Elopomorpha, contrasting with the cone-dominated retinas of nearly all other species of teleost larvae. This distinctive developmental pattern shared among elopomorph larvae has important evolutionary and ecological implications, indicating a shared ancestor and/or ecological characteristics that are very different from most other teleost larvae.     

FINE STRUCTURE OF THE PINEAL ORGANS OF THE ADULT FROG, RANA PIPIENS

Frontal organs and epiphyses of the pineal system from the adult frog, Rana pipiens, were fixed in s-collidine-buffered osmium tetroxide, embedded in Epon 812, and examined by electron microscopy. Epiphyseal material was also fixed in a variety of ways and subjected to a series of cytochemical tests for light microscopy. An ultrastructure resembling that of lateral eye retina is confirmed in this species. Photoreceptor cells of the epiphysis and frontal organ display many cytological features similar to those of retinal rods and cones in the arrangement of their outer and inner segments and synaptic components. However, in these pineal organs the outer segments are disoriented relative to each other and may display a disarranged internal organization unlike normal retinal photoreceptors. Furthermore, other pineal outer segments often appear degenerate. Since immature stages in the development of new outer segments also appear to be present, adult pineal photoreceptors are probably engaged in a constant renewal of outer segment membranes. The evidence further suggests that macrophages are involved in phagocytosis of degenerated outer segments. Postulated photoreceptor activities and the possibility of secondary pineal functions, such as secretion, are discussed in view of current morphological and cytochemical findings.     

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.     

The pineal organ as a folded retina: immunocytochemical localization of opsins

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a 'folded retina'. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.     

Cone photoreceptor reflectance variation in the northern tree shrew and thirteen-lined ground squirrel

In vivo images of human cone photoreceptors have been shown to vary in their reflectance both spatially and temporally. While it is generally accepted that the unique anatomy and physiology of the photoreceptors themselves drives this behavior, the exact mechanisms have not been fully elucidated as most studies on these phenomena have been limited to the human retina. Unlike humans, animal models offer the ability to experimentally manipulate the retina and perform direct in vivo and ex vivo comparisons. The thirteen-lined ground squirrel and northern tree shrew are two emerging animal models being used in vision research. Both models feature cone-dominant retinas, overcoming a key limitation of traditional rodent models. Additionally, each possesses unique but well-documented anatomical differences in cone structure compared to human cones, which can be leveraged to further constrain theoretical models of light propagation within photoreceptors. Here we sought to characterize the spatial and temporal reflectance behavior of cones in these species. Adaptive optics scanning light ophthalmoscopy (AOSLO) was used to non-invasively image the photoreceptors of both species at 5 to 10 min intervals over the span of 18 to 25 min. The reflectance of individual cone photoreceptors was measured over time, and images at individual time points were used to assess the variability of cone reflectance across the cone mosaic. Variability in spatial and temporal photoreceptor reflectance was observed in both species, with similar behavior to that seen in human AOSLO images. Despite the unique cone structure in these animals, these data suggest a common origin of photoreceptor reflectance behavior across species. Such data may help constrain models of the cellular origins of photoreceptor reflectance signals. These animal models provide an experimental platform to further explore the morphological origins of light capture and propagation.     

Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

Localization of actin and tubulin in developing and adult mammalian photoreceptors

Summary In order to define cytoskeletal domains of the mammalian photoreceptor, actin and tubulin were localized in adult retinae of mouse and human. For light-microscopic localization, actin was labeled using fluorescent phalloidin or monoclonal antibodies against actin, and tubulin was labeled using monoclonal antibodies against alpha- and beta-tubulin in an immunocytochemical method. Actin and tubulin were also localized by ultrastructural immunocytochemistry in the mouse. Filamentous actin was present in the retina at the outer limiting membrane and in synaptic terminals, especially of the cones, while globular actin was observed additionally in the inner segments. Müller cell cytoplasm and apical microvilli at the outer limiting membrane were also labeled for filamentous actin. Alpha- and beta-tubulin were evident throughout the photoreceptors, including the inner segments, but not in the synaptic terminals or at the outer limiting membrane. In the early postnatal retina of mouse, actin and tubulin were present at the ventricular surface. This pattern changed as photoreceptors fully elongated and as synaptogenesis occurred in the outer plexiform layer.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.     

Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.     

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.     

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.     

Fine structure of the retina of black bass, Micropterus salmoides (Centrarchidae, Teleostei).

The structure of light- and dark-adapted retina of the black bass, Micropterus salmoides has been studied by light and electron microscopy. This retina lacks blood vessels at all levels. The optic fiber layer is divided into fascicles by the processes of Müller cells and the ganglion cell layer is represented by a single row of voluminous cells. The inner nuclear layer consists of two layers of horizontal cells and bipolar, amacrine and interplexiform cells. In the outer plexiform layer we observed the synaptic terminals of photoreceptor cells, rod spherules and cone pedicles and terminal processes of bipolar and horizontal cells. The spherules have a single synaptic ribbon and the pedicles possess multiple synaptic ribbons. Morphologically, we have identified three types of photoreceptors: rods, single cones and equal double cones which undergo retinomotor movements in response to changes in light conditions. The cones are arranged in a square mosaic whereas the rods are dispersed between the cones.     

PHOTORECEPTOR-PIGMENT EPITHELIAL CELL RELATIONSHIPS IN RATS WITH INHERITED RETINAL DEGENERATION : Radioautographic and Electron Microscope Evidence for a Dual Source of Extra Lamellar Material

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H³ and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.     

Inhibition of Müller glial cell division blocks regeneration of the light-damaged zebrafish retina

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.