Diminished foraging performance of a mutant zebrafish with reduced population of ultraviolet cones

Ultraviolet (UV) cones are photoreceptors that sense light in the range 300–450 nm and are found in the retinas of non-mammalian vertebrates and small mammals. Despite their widespread presence across taxa, the functions that these cones exert in the lives of animals remain largely unknown. In this study, I used the zebrafish lor (lots of rods) mutant, characterized by a diminished UV cone population compared to that of wild-type zebrafish, to test whether its foraging performance differed from that of the wild-type (control). The mean location distance and angle (variables that are reliable indicators of foraging performance) at which control fish detected zooplankton prey were, on average, 24 and 90% greater than corresponding measures for lor fish. Such inferior foraging performance of the mutant could be explained by reduced contrast perception of the prey, resulting from the diminished population of UV cones and associated sensitivity. Thus, UV cones enhance the foraging performance of zebrafish, a crucial ecological function that may explain why small zooplanktivorous fishes retain UV cones throughout their lives.     

Crumbs proteins stabilize the cone mosaics of photoreceptors and improve vision in zebrafish

Although the unique organization of vertebrate cone mosaics was first described long ago,both their underlying molecular basis and physiological significance are largely unknown.Here,we demonstrate that Crumbs proteins,the key regulators of epithelial apical polarity,establish the planar cellular polarity of photoreceptors in zebrafish.Via heterophilic Crb2a-Crb2b interactions,the apicobasal polarity protein Crb2b restricts the asymmetric planar distribution of Crb2a in photoreceptors.The planar polarized Crumbs proteins thus balance intercellular adhesions and tension between photoreceptors,thereby stabilizing the geometric organization of cone mosaics.Notably,loss of Crb2b in zebrafish induces a nearsightedness-like phenotype in zebrafish accompanied by an elongated eye axis and impairs zebrafish visual perception for predation.These data reveal a detailed mechanism for cone mosaic homeostasis via previously undiscovered apical-planar polarity coordination and propose a pathogenic mechanism for nearsightedness.     

Coupling mechanical deformations and planar cell polarity to create regular patterns in the zebrafish retina

The orderly packing and precise arrangement of epithelial cells is essential to the functioning of many tissues, and refinement of this packing during development is a central theme in animal morphogenesis. The mechanisms that determine epithelial cell shape and position, however, remain incompletely understood. Here, we investigate these mechanisms in a striking example of planar order in a vertebrate epithelium: The periodic, almost crystalline distribution of cone photoreceptors in the adult teleost fish retina. Based on observations of the emergence of photoreceptor packing near the retinal margin, we propose a mathematical model in which ordered columns of cells form as a result of coupling between planar cell polarity (PCP) and anisotropic tissue-scale mechanical stresses. This model recapitulates many observed features of cone photoreceptor organization during retinal growth and regeneration. Consistent with the model's predictions, we report a planar-polarized distribution of Crumbs2a protein in cone photoreceptors in both unperturbed and regenerated tissue. We further show that the pattern perturbations predicted by the model to occur if the imposed stresses become isotropic closely resemble defects in the cone pattern in zebrafish lrp2 mutants, in which intraocular pressure is increased, resulting in altered mechanical stress and ocular enlargement. Evidence of interactions linking PCP, cell shape, and mechanical stresses has recently emerged in a number of systems, several of which show signs of columnar cell packing akin to that described here. Our results may hence have broader relevance for the organization of cells in epithelia. Whereas earlier models have allowed only for unidirectional influences between PCP and cell mechanics, the simple, phenomenological framework that we introduce here can encompass a broad range of bidirectional feedback interactions among planar polarity, shape, and stresses; our model thus represents a conceptual framework that can address many questions of importance to morphogenesis.     

Regeneration of Cone Photoreceptors when Cell Ablation Is Primarily Restricted to a Particular Cone Subtype

We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors via stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.     

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c^w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c^w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c^w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.     

Notch-Delta signaling is required for spatial patterning and Müller glia differentiation in the zebrafish retina

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, we examined retinas from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. We found that inactivating Notch-Delta signaling did not prevent differentiation of retinal neurons, but it did disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiated, but their laminar arrangement was disrupted. Photoreceptor differentiation was initiated normally, but its progression was slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors was disrupted: cones of the same spectral subtype were clumped rather than regularly spaced. In contrast to neurons, Müller glia failed to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis.     

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1^?/? zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1^?/? zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1^?/? retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1^?/? zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1^?/? retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.     

Recoverin depletion accelerates cone photoresponse recovery

The neuronal Ca²⁺-binding protein Recoverin has been shown to regulate phototransduction termination in mammalian rods. Here we identify four recoverin genes in the zebrafish genome, rcv1a, rcv1b, rcv2a and rcv2b, and investigate their role in modulating the cone phototransduction cascade. While Recoverin-1b is only found in the adult retina, the other Recoverins are expressed throughout development in all four cone types, except Recoverin-1a, which is expressed only in rods and UV cones. Applying a double flash electroretinogram (ERG) paradigm, downregulation of Recoverin-2a or 2b accelerates cone photoresponse recovery, albeit at different light intensities. Exclusive recording from UV cones via spectral ERG reveals that knockdown of Recoverin-1a alone has no effect, but Recoverin-1a/2a double-knockdowns showed an even shorter recovery time than Recoverin-2a-deficient larvae. We also showed that UV cone photoresponse kinetics depend on Recoverin-2a function via cone-specific kinase Grk7a. This is the first in vivo study demonstrating that cone opsin deactivation kinetics determine overall photoresponse shut off kinetics.     

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.     

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.     

Retinal Cone Photoreceptors of the Deer Mouse Peromyscus maniculatus: Development,Topography, Opsin Expression and Spectral Tuning

A quantitative analysis of photoreceptor properties was performed in the retina of the nocturnal deer mouse, Peromyscus maniculatus, using pigmented (wildtype) and albino animals. The aim was to establish whether the deer mouse is a more suitable model species than the house mouse for photoreceptor studies, and whether oculocutaneous albinism affects its photoreceptor properties. In retinal flatmounts, cone photoreceptors were identified by opsin immunostaining, and their numbers, spectral types, and distributions across the retina were determined. Rod photoreceptors were counted using differential interference contrast microscopy. Pigmented P. maniculatus have a rod-dominated retina with rod densities of about 450.000/mm² and cone densities of 3000 - 6500/mm². Two cone opsins, shortwave sensitive (S) and middle-to-longwave sensitive (M), are present and expressed in distinct cone types. Partial sequencing of the S opsin gene strongly supports UV sensitivity of the S cone visual pigment. The S cones constitute a 5-15% minority of the cones. Different from house mouse, S and M cone distributions do not have dorsoventral gradients, and coexpression of both opsins in single cones is exceptional (<2% of the cones). In albino P. maniculatus, rod densities are reduced by approximately 40% (270.000/mm²). Overall, cone density and the density of cones exclusively expressing S opsin are not significantly different from pigmented P. maniculatus. However, in albino retinas S opsin is coexpressed with M opsin in 60-90% of the cones and therefore the population of cones expressing only M opsin is significantly reduced to 5-25%. In conclusion, deer mouse cone properties largely conform to the general mammalian pattern, hence the deer mouse may be better suited than the house mouse for the study of certain basic cone properties, including the effects of albinism on cone opsin expression.     

Mutation of cGMP phosphodiesterase 6alpha'-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish

In mammals, the blockade of the phototransduction cascade causes loss of vision and, in some cases, degeneration of photoreceptors. However, the molecular mechanisms that link phototransduction with photoreceptor degeneration remain to be elucidated. Here, we report that a mutation in the gene encoding a central effector of the phototransduction cascade, cGMP phosphodiesterase 6alpha'-subunit (PDE6alpha'), affects not only the vision but also the survival of cone photoreceptors in zebrafish. We isolated a zebrafish mutant, called eclipse (els), which shows no visual behavior such as optokinetic response (OKR). The cloning of the els mutant gene revealed that a missense mutation occurred in the pde6alpha' gene, resulting in a change in a conserved amino acid. The PDE6 expressed in rod photoreceptors is a heterotetramer comprising two closely related similar hydrolytic alpha and beta subunits and two identical inhibitory gamma subunits, while the PDE6 expressed in cone photoreceptors consists of two homodimers of alpha' subunits, each with gamma subunits. The els mutant displays no visual response to bright light, where cones are active, but shows relatively normal OKR to dim light, where only rods function, suggesting that only the cone-specific phototransduction pathway is disrupted in the els mutant. Furthermore, in the els mutant, cones are selectively eliminated but rods are retained at the adult stage, suggesting that cones undergo a progressive degeneration in the els mutant retinas. Taken together, these data suggest that PDE6alpha' activity is important for the survival of cones in zebrafish.     

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.     

Synaptojanin 1 Is Required for Endolysosomal Trafficking of Synaptic Proteins in Cone Photoreceptor Inner Segments

Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1) is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc^a14, which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc^a14 cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc^a14 inner segment. The structure of the Endoplasmic Reticulum in nrc^a14 mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc^a14 cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc ^a14 mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.     

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background  

Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.     

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.     

Knocking out lca5 in zebrafish causes cone-rod dystrophy due to impaired outer segment protein trafficking

Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1–2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7 dpf lca5^−/− zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5^−/− zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.     

Therapeutic strategy for handling inherited retinal degenerations in a gene-independent manner using rod-derived cone viability factors

The most common hereditary retinal degeneration, retinitis pigmentosa (RP), leads to blindness by degeneration of cone photoreceptors. Meanwhile, genetic studies have shown that a significant proportion of RP genes is expressed only by rods, which raises the question of the mechanism leading to the degeneration of cones. Following the concept of sustainability factor cones, rods secrete survival factors that are necessary to maintain the cones, named Rod-derived Cone Viability Factors (RdCVFs). In patients suffering from RP, loss of rods results in the loss of RdCVFs expression and followed by cone degeneration. We have identified the bifunctional genes nucleoredoxin-like 1 and 2 that encode for, by differential splicing, a thioredoxin enzyme and a cone survival factor, respectively RdCVF and RdCVF2. The administration of these survival factors would maintain cones and central vision in most patients suffering from RP.