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.     

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.     

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.     

Light exposure during embryonic and yolk-sac alevin development of Chinook salmon Oncorhynchus tshawytscha does not alter the spectral phenotype of photoreceptors

Colour vision is mediated by the expression of different visual pigments in photoreceptors of the vertebrate retina. Each visual pigment is a complex of a protein (opsin) and a vitamin A chromophore; alterations to either component affects visual pigment absorbance and, potentially, the visual capabilities of an animal. Many species of fish undergo changes in opsin expression during retinal development. In the case of salmonid fishes the single cone photoreceptors undergo a switch in opsin expression from SWS1 (ultraviolet sensitive) to SWS2 (blue-light sensitive) starting at the yolk-sac alevin stage, around the time when they first experience light. Whether light may initiate this event or produce a plastic response in the various photoreceptors is unknown. In this study, Chinook salmon Oncorhynchus tshawytscha were exposed to light from the embryonic (5 days prior to hatching) into the yolk sac alevin (25 days post hatching) stage and the spectral phenotype of photoreceptors assessed with respect to that of unexposed controls by in situ hybridization with opsin riboprobes. Light exposure did not change the spectral phenotype of photoreceptors, their overall morphology or spatial arrangement. These results concur with those from a variety of fish species and suggest that plasticity in photoreceptor spectral phenotype via changes in opsin expression may not be a widespread occurrence among teleosts.     

Opsin expression patterns coincide with photoreceptor development during pupal development in the honey bee,Apis mellifera

The compound eyes of insects allow them to catch photons and convert the energy into electric signals. All compound eyes consist of numerous ommatidia, each comprising a fixed number of photoreceptors. Different ommatidial types are characterized by a specific set of photoreceptors differing in spectral sensitivity. In honey bees, males and females possess different ommatidial types forming distinct retinal mosaics. However, data are lacking on retinal ontogeny and the mechanisms by which the eyes are patterned. In this study, we investigated the intrinsic temporal and circadian expression patterns of the opsins that give rise to the ultraviolet, blue and green sensitive photoreceptors, as well as the morphological maturation of the retina during pupal development of honey bees. qPCR and histological labeling revealed that temporal opsin mRNA expression differs between sexes and correlates with rhabdom elongation during photoreceptor development. In the first half of the pupal stage, when the rhabdoms of the photoreceptors are still short, worker and (dorsal) drone retinae exhibit similar expression patterns with relatively high levels of UV (UVop) and only marginal levels of blue (BLop) and green (Lop1) opsin mRNA. In the second half of pupation, when photoreceptors and rhabdoms elongate, opsin expression in workers becomes dominated by Lop1 mRNA. In contrast, the dorsal drone eye shows high expression levels of UVop and BLop mRNA, whereas Lop1 mRNA level decreases. Interestingly, opsin expression levels increase up to 22-fold during early adult life. We also found evidence that opsin expression in adult bees is under the control of the endogenous clock. Our data indicate that the formation of the sex-specific retinal composition of photoreceptors takes place during the second half of the pupal development, and that opsin mRNA expression levels continue to increase in young bees, which stands in contrast to Drosophila, where the highest expression levels are found during the late pupal stage and remain constant in adults. From an evolutionary perspective, we hypothesize that the delayed retinal maturation during the early adult phase is linked to the delayed transition from indoor to outdoor activities in bees, when vision becomes important.     

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.     

The evolution of irradiance detection: melanopsin and the non-visual opsins

Circadian rhythms are endogenous 24 h cycles that persist in the absence of external time cues. These rhythms provide an internal representation of day length and optimize physiology and behaviour to the varying demands of the solar cycle. These clocks require daily adjustment to local time and the primary time cue (zeitgeber) used by most vertebrates is the daily change in the amount of environmental light (irradiance) at dawn and dusk, a process termed photoentrainment. Attempts to understand the photoreceptor mechanisms mediating non-image-forming responses to light, such as photoentrainment, have resulted in the discovery of a remarkable array of different photoreceptors and photopigment families, all of which appear to use a basic opsin/vitamin A-based photopigment biochemistry. In non-mammalian vertebrates, specialized photoreceptors are located within the pineal complex, deep brain and dermal melanophores. There is also strong evidence in fish and amphibians for the direct photic regulation of circadian clocks in multiple tissues. By contrast, mammals possess only ocular photoreceptors. However, in addition to the image-forming rods and cones of the retina, there exists a third photoreceptor system based on a subset of melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). In this review, we discuss the range of vertebrate photoreceptors and their opsin photopigments, describe the melanopsin/pRGC system in some detail and then finally consider the molecular evolution and sensory ecology of these non-image-forming photoreceptor systems.     

Immunohistochemical evidence for multiple photosystems in box jellyfish

Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment. To investigate whether multiple types of photoreceptor cells are present in the rhopalium, and whether the different eye types possess different types of photoreceptors, we have used immunohistochemistry with a range of vertebrate opsin antibodies to label the photoreceptors, and electroretinograms (ERG) to determine their spectral sensitivity. All photoreceptor cells of the two lens eyes of the box jellyfish Tripedalia cystophora and Carybdea marsupialis displayed immunoreactivity for an antibody directed against the zebrafish ultraviolet (UV) opsin, but not against any of eight other rhodopsin or cone opsin antibodies tested. In neither of the two species were the pigment cup eyes immunoreactive for any of the opsin antibodies. ERG analysis of the Carybdea lower lens eyes demonstrated a single spectral sensitivity maximum at 485 nm suggesting the presence of a single opsin type. Our data demonstrate that the lens eyes of box jellyfish utilize a single opsin and are thus color-blind, and that there is probably a different photopigment in the pigment cup eyes. The results support our hypothesis that the lens eyes and the pigment cup eyes of box jellyfish are involved in different and specific visual tasks.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

Gene Expression of the Neuropeptide-Processing Enzyme Carboxypeptidase E in Rat Photoreceptor Cells

Abstract: We characterized the spatial expression of mRNA for the enzyme Carboxypeptidase E (CPE) in the Long-Evans rat retina. CPE is involved in the processing of neuroactive peptides to a mature form. A cDNA encoding the 3' terminus of CPE mRNA was cloned by polymer-ase chain reaction amplification of rat retina single-stranded cDNA. The sequence of this cDNA was identical to a rat genomic clone for CPE and nearly identical (130/ 132 nucleotides) to a cDNA for rat brain CPE. In addition, the cDNA hybridized to a single allele on Southern blots and to a 2.1-kb mRNA on northern blots of both rat brain and retina. These data support the conclusion of others that CPE is a single-copy gene in the rat. In cell fractionation experiments, the majority of CPE mRNA fractionated with rod opsin mRNA, suggesting that CPE is expressed predominantly in rod photoreceptors. The high abundance of CPE mRNA in photoreceptors was confirmed by in situ hybridization studies, although CPE was detected at lower levels in other retinal cell types as well. The presence of abundant levels of the mRNA of a neuro-peptide-processing enzyme in photoreceptor cells suggests that photoreceptors may utilize neuropeptides for normal function.     

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.     

Developmental predetermination of the structural and molecular polarization of photoreceptor cells

Through mechanisms still unknown, the apparently homogeneous neuroepithelium of the embryonic optic cup differentiates into such divergent cell types as photoreceptors, glia, and various subsets of neurons. Questions that still remain unanswered in this field include the timing and mechanism of action of the "instructive" events directing each neuroepithelial cell to undergo the sequence of phenotypic changes necessary to develop into a specific retinal cell type. This laboratory is investigating some of these questions using cultures in which dissociated neural retina cells, obtained before the onset of overt photoreceptor differentiation, develop at low density in the absence of glia and pigment epithelium. The cultures initially are a morphologically homogeneous population of process-free, round cells. Some cells retain this morphology throughout the first week in vitro, while others develop either as photoreceptors or as multipolar neurons. Photoreceptors elongate and become very asymmetric as they do in vivo, with characteristic compartments orderly arranged along their longitudinal axis (an outer segment-like process, inner segment, cell body, and a characteristically short, single neurite). Cell polarization can also be observed in the distribution of opsin immunoreactive materials and some cytoskeletal elements. Thus, certain precursor cells present in the embryonic retina seem to be programmed to differentiate into photoreceptors even when developing in the absence of contacts with other retinal cells. However, interactions with other constituents of the retina/pigment epithelium complex are probably necessary to ensure final photoreceptor maturation, including further growth of the opsin-rich outer segment process.     

Visualization of rod photoreceptor development using GFP-transgenic zebrafish

Zebrafish retina contains five morphologically distinct classes of photoreceptors, each expressing a distinct type of opsin gene. Molecular mechanisms underlying specification of opsin expression and differentiation among the cell types are largely unknown. This is partly because mutants affected with expression of a particular class of opsin gene are difficult to find. In this study we established the transgenic lines of zebrafish carrying green fluorescent protein (GFP) gene under the 1.1-kb and 3.7-kb upstream regions of the rod-opsin gene. In transgenic fish, GFP expression initiated and proceeded in the same spatiotemporal pattern with rod-opsin gene. The retinal section from adult transgenic fish showed GFP expression throughout the rod cell layer. These results indicate that the proximal 1.1-kb region is sufficient to drive gene expression in all rod photoreceptor cells. These transgenic fish should facilitate screening of mutants affected specifically with rod-opsin expression or rod cell development by visualization of rod cells by GFP.     

Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.     

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.     

Developmental evolution of the insect retina: insights from standardized numbering of homologous photoreceptors

The canonical number of eight photoreceptors and their arrangement in the ommatidia of insect compound eyes is very conserved. However significant variations exist in selective groups, such as the Lepidoptera and Hymenoptera, which independently evolved additional photoreceptors. For this and historical reasons, heterogeneous labeling conventions have been in use for photoreceptor subtypes, despite developmentally and structurally well-defined homologies. Extending earlier efforts, we introduce a universal photoreceptor subtype classification key that relates to the Drosophila numbering system. Its application is demonstrated in major insect orders, with detailed information on the relationship to previous conventions. We then discuss new insights that result from the improved understanding of photoreceptor subtype homologies. This includes evidence of functionally imposed ground rules of differential opsin expression, the underappreciated role of R8 as ancestral color receptor, the causes and consequences of parallel R7 photoreceptor addition in Hymenoptera and Lepidoptera, and the ancestral subfunctionalization of outer photoreceptors cells, which may be only developmentally recapitulated in Drosophila. We conclude with pointing out the need for opsin expression data from a wider range of insect orders.