Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina

Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones.     

Dysfunction of heterotrimeric kinesin-2 in rod photoreceptor cells and the role of opsin mislocalization in rapid cell death

Due to extensive elaboration of the photoreceptor cilium to form the outer segment, axonemal transport (IFT) in photoreceptors is extraordinarily busy, and retinal degeneration is a component of many ciliopathies. Functional loss of heterotrimeric kinesin-2, a major anterograde IFT motor, causes mislocalized opsin, followed by rapid cell death. Here, we have analyzed the nature of protein mislocalization and the requirements for the death of kinesin-2-mutant rod photoreceptors. Quantitative immuno EM showed that opsin accumulates initially within the inner segment, and then in the plasma membrane. The light-activated movement of arrestin to the outer segment is also impaired, but this defect likely results secondarily from binding to mislocalized opsin. Unlike some other retinal degenerations, neither opsin–arrestin complexes nor photoactivation were necessary for cell loss. In contrast, reduced rod opsin expression provided enhanced rod and cone photoreceptor survival and function, as measured by photoreceptor cell counts, apoptosis assays, and ERG analysis. The cell death incurred by loss of kinesin-2 function was almost completely negated by Rho^−/−. Our results indicate that mislocalization of opsin is a major cause of photoreceptor cell death from kinesin-2 dysfunction and demonstrate the importance of accumulating mislocalized protein per se, rather than specific signaling properties of opsin, stemming from photoactivation or arrestin binding.     

Characteristics of Rod Regeneration in a Novel Zebrafish Retinal Degeneration Model Using N-Methyl-N-Nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.     

Sequential genesis and determination of cone and rod photoreceptors in Xenopus

In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 227–244, 1998     

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.     

Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin- ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.     

Cone cells fail to develop normally in transgenic mice showing ablation of rod photoreceptor cells

Transgenic mice were derived containing the cytotoxic dt-α gene driven by opsin promoter sequences. Mice expressing this construct showed progressive degeneration of rod photoreceptor cells commencing at birth, with obvious depletion of such cells by postnatal day 7. Ablation of rod photoreceptor cells in the transgenic retina was accompanied by the failure of developing cone cells to elaborate outer segments, although all other aspects of cone cell cytodifferentiation appeared normal. The results suggest that the 1.0-kb opsin promoter segment contains rod cell type specificity and that cone cells require maturation of rod cells to complete the late stages of their terminal differentiation and for their maintenance and cellular integrity.     

Ciliary neurotrophic factor blocks rod photoreceptor differentiation from postmitotic precursor cells in vitro

The development of photoreceptors in the mammalian retina is thought to be controlled by extrinsic signals. We have shown previously that ciliary neurotrophic factor (CNTF) potently inhibits photoreceptor differentiation in cultures of rat retina. The present study analyzes which developmental processes are affected by CNTF. Rod differentiation as determined by opsin and recoverin immunocytochemistry was effectively blocked by CNTF and leukemia inhibitory factor, but not by other neurotrophic agents tested. CNTF did not influence proliferation, cell death, or survival, and had no effect on the downregulation of nestin immunoreactivity in progenitor cells. Opsin-positive rods could be reverted to an opsin-negative state initially, but became unresponsive to CNTF later. No compensatory increase in the number of other cell types was observed. Application of neutralizing antibodies against CNTF revealed that rod development was partially blocked by an endogenous CNTF-like molecule in control cultures. Our results suggest that CNTF can act as a specific negative regulator of rod differentiation. Its action on photoreceptor precursor cells could serve to synchronize the maturation of photoreceptors, which are born over an extended period of time. Together with other stimulatory signals, CNTF may thus control the temporally and numerically correct integration of photoreceptors into the retinal network.     

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.     

Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/-) retina, rods are converted into functional cone-like cells. The Nrl(-/-) retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/-) retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl(-/-) retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/-) retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.     

Posttranslational modifications of tubulin in teleost photoreceptor cytoskeletons

1. Posttranslational modifications of tubulin by acetylation and detyrosination have been correlated previously with microtubule stability in numerous cell types. 2. In this study, posttranslational modifications of tubulin and their regional distribution within teleost photoreceptor cones and rods are demonstrated immunohistochemically using antibodies specific for acetylated, detyrosinated, or tyrosinated tubulin. 3. Immunolocalization was carried out on isolated whole cones and mechanically detached rod and cone inner/outer segments. 4. Acetylated tubulin within rods and cones is found only in microtubules of the ciliary axoneme of the outer segment. Detyrosinated tubulin is also enriched in axonemes of both rod and cone outer segments. 5. Distributions of tyrosinated and detyrosinated cytoplasmic microtubules differ within cones and rods. In cones, detyrosinated and tyrosinated tubulins are both abundant throughout the cell body. In rods, the ellipsoid and myoid contain much more tyrosinated tubulin than detyrosinated tubulin. Comparisons between whole cones and cone fragments suggest that detyrosinated microtubules are more stable than tyrosinated microtubules in teleost photoreceptors. 6. Our findings provide further evidence that microtubules of teleost cones differ from rod microtubules in their stabilities and rapidity of turnover within the photoreceptor inner segment.     

Alpha transducin is present in blue-, green-, and red-sensitive cone photoreceptors in the human retina

Phototransduction in vertebrate rod and cone photoreceptor cells involves G protein-mediated light stimulation of cGMP hydrolysis. Enzymes of the cGMP hydrolysis cascades of rods and cones are products of different genes. Three different classes of cones in the human retina are maximally sensitive to either blue, green, or red light. Distinct opsin genes are expressed in each type of cone. The distribution of cone types in human retina was determined using anti-peptide antibodies that recognize specific amino acid sequences in green/red opsin and blue opsin. These antibodies together with an anti-peptide antibody against Tc alpha were used in double labeling experiments to demonstrate the presence of the Tc alpha peptide in all types of cones. cDNA clones corresponding to human rod and cone transducin alpha subunit (Tr alpha and Tc alpha) genes were isolated. Southern blot analyses of human genomic DNA suggest that there is only one rod T alpha gene but more than one cone T alpha gene. The multiple Tc alpha genes could be closely related genes or different Tc alpha alleles, or one could be a pseudogene.     

Time course of opsin expression in developing rod photoreceptors

We have investigated the time course of rod photoreceptor determination in the goldfish retina. Rod precursor cells located in the outer nuclear layer of the mature retina continuously generate rod photoreceptors. In this study, we asked when rod precursor cells begin to express opsin, which would signal their commitment to the rod pathway of differentiation. There are three possibilities: a rod precursor could express opsin while still mitotic, at or shortly after the terminal mitosis but before differentiation, or during differentiation. We used immunocytochemistry with antibodies against bromodeoxyuridine, BrdU (a thymidine analogue) and against opsin to determine when during the mitotic history of a cell the expression of opsin first occurred, taking a double labelled cell to be evidence of commitment to the rod cell fate. We found that the first double labelled cells appeared at 4 days after BrdU injection. The number of double labelled cells increased to peak at 10 days, and then fell. These results support the hypothesis that dividing rod precursor cells are probably multipotent stem cells not committed to the rod cell fate.     

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.