Vertebrate Eye Research Articles

Cataract-causing Y204X mutation of crystallin protein CRYβB1 promotes its C-terminal degradation and higher-order oligomerization

Crystallin proteins are a class of main structural proteins of the vertebrate eye lens, and their solubility and stability directly determine transparency and refractive power of the lens. Mutation in genes that encode these crystallin proteins is the most common cause for congenital cataracts. Despite extensive studies, the pathogenic and molecular mechanisms that effect congenital cataracts remain unclear. In this study, we identified a novel mutation in CRYBB1 from a congenital cataract family, and demonstrated that this mutation led to an early termination of mRNA translation, resulting in a 49-residue C-terminally truncated CRYβB1 protein. We show this mutant is susceptible to proteolysis, which allowed us to determine a 1.2-Å resolution crystal structure of CRYβB1 without the entire C-terminal domain. In this crystal lattice, we observed that two N-terminal domain monomers form a dimer that structurally resembles the WT monomer, but with different surface characteristics. Biochemical analyses and cell-based data also suggested that this mutant is significantly more liable to aggregate and degrade compared to WT CRYβB1. Taken together, our results provide an insight into the mechanism regarding how a mutant crystalin contributes to the development of congenital cataract possibly through alteration of inter-protein interactions that result in protein aggregation.

Bloodstained flowers and bloodthirsty flies.

This article is a Commentary on Heiduk et al. (2023), 239:1490–1504.

Species independence of eye lens dimensions in teleosts and elasmobranchs.

The vertebrate eye lens grows incrementally, adding layers of elongated, tightly packed lens fiber cells at the outer margin of the lens. With subsequent growth, previously-deposited fiber cells degrade, leaving a region of fully denucleated and organelle-free cells which are responsible for the high transparency and low light scattering characteristics of the lens. The objective of this study was to determine if the horizon separating the gelatinous outer cortex of the lens from its hardened interior occurred at a consistent location within the lens of several teleost and elasmobranch fish species, and could be linked to fiber cell morphology or function. A fixed ratio of 0.69±0.01 of hardened eye lens diameter (HD) to overall eye lens diameter (LD) was observed in a broad size range of Atlantic cod (Gadus morhua), haddock (Melanogrammus aeglefinus), thorny skate (Amblyraja radiata) and round ray (Rajella fyllae). The location of the hardened lens horizon was similar to that reported for optical plasticity and spherical aberration, but not that of fiber cell denucleation, suggesting that fiber cell dehydration continues after the loss of internal organelles. Our findings support a previous suggestion that the maintenance of optical quality during fish eye lens growth requires a precisely-fixed HD:LD ratio, while the ubiquity of a fixed ratio across fish taxa may suggest that many fish species possess a common refractive index profile. The linear relationship between HD and fish length should allow fish length to be backcalculated from the diameter of the isolated lens core, thus aiding research using isotope ratios of lens laminae or inner cores to reconstruct early life history events.

An Anatomy of Fungal Eye: Fungal Photoreceptors and Signalling Mechanisms.

Organisms have developed different features to capture or sense sunlight. Vertebrates have evolved specialized organs (eyes) which contain a variety of photosensor cells that help them to see the light to aid orientation. Opsins are major photoreceptors found in the vertebrate eye. Fungi, with more than five million estimated members, represent an important clade of living organisms which have important functions for the sustainability of life on our planet. Light signalling regulates a range of developmental and metabolic processes including asexual sporulation, sexual fruit body formation, pigment and carotenoid production and even production of secondary metabolites. Fungi have adopted three groups of photoreceptors: (I) blue light receptors, White Collars, vivid, cryptochromes, blue F proteins and DNA photolyases, (II) red light sensors, phytochromes and (III) green light sensors and microbial rhodopsins. Most mechanistic data were elucidated on the roles of the White Collar Complex (WCC) and the phytochromes in the fungal kingdom. The WCC acts as both photoreceptor and transcription factor by binding to target genes, whereas the phytochrome initiates a cascade of signalling by using mitogen-activated protein kinases to elicit its cellular responses. Although the mechanism of photoreception has been studied in great detail, fungal photoreception has not been compared with vertebrate vision. Therefore, this review will mainly focus on mechanistic findings derived from two model organisms, namely Aspergillus nidulans and Neurospora crassa and comparison of some mechanisms with vertebrate vision. Our focus will be on the way light signalling is translated into changes in gene expression, which influences morphogenesis and metabolism in fungi.

Fish to learn: insights into the effects of environmental chemicals on eye development and visual function in zebrafish.

Vision is the most essential sense system for the human being. Congenital visual impairment affects millions of people globally. It is increasingly realized that visual system development is an impressionable target of environmental chemicals. However, due to inaccessibility and ethical issues, the use of humans and other placental mammals is constrained, which limits our better understanding of environmental factors on ocular development and visual function in the embryonic stage. Therefore, as complementing laboratory rodents, zebrafish has been the most frequently employed to understand the effects of environmental chemicals on eye development and visual function. One of the major reasons for the increasing use of zebrafish is their polychromatic vision. Zebrafish retinas are morphologically and functionally analogous to those of mammalian, as well as evolutionary conservation among vertebrate eye. This review provides an update on harmful effects from exposure to environmental chemicals, involving metallic elements (ions), metal-derived nanoparticles, microplastics, nanoplastics, persistent organic pollutants, pesticides, and pharmaceutical pollutants on the eye development and visual function in zebrafish embryos. The collected data provide a comprehensive understanding of environmental factors on ocular development and visual function. This report highlights that zebrafish is promising as a model to identify hazardous toxicants toward eye development and is hopeful for developing preventative or postnatal therapies for human congenital visual impairment.

Eye development and developmental expression of crystallin genes in the long arm octopus, Octopus minor

The eye of a cephalopod is a well-known example of convergent evolution and resembles the vertebrate eye. Although cephalopods and vertebrates exhibit similar eye form and function, they differ in visual origin and structure. The common long-arm octopus (Octopus minor) is a good model system in evolutionary and developmental studies due to its highly centralized nervous system, shorter life cycle, and specific camera-type eyes that contribute to convergence with vertebrate eye. Lens-containing eyes represent a significant improvement of simple eye and have evolved by convergent mechanisms, a variety of lenses and corneas containing diverse crystallin. The diversity and taxon-specificity of lens crystallin is indicative of convergent evolution of crystallin roles. Previous studies have focused on morphological, ontogenetic and phylogenetic analysis of crystallin to understand the evolution of lens-containing eyes. However, little is known about the functional analysis of taxon-specific crystallin genes at the molecular level in the eye of O. minor. Using an embryonic staging system of Octopus minor as a model system, we investigated fifteen genomes and the structure of eye by immunohistochemistry, phalloidin staining and the three-dimensional structures. We also obtained the crystallin-related genes (i.e., α-, S-, and Ω-crystallin) from the transcriptome data of O. minor. Subsequent molecular phylogenetic analysis based on these genes revealed a distinct divergence pattern among the three gene classes and further suggested the evidence supporting the taxon-specific convergent evolutionary trend. We analyzed the expression pattern of crystallin genes via in situ hybridization during developmental stages. All crystallin genes are commonly expressed in the lentigenic cells of ciliary body. The α-crystallin found in cephalopods was also expressed at the peripheral region of the lens including ciliary body, suggesting a possible role in lens formation in cephalopods. This study will provide information on the eye development of O. minor and support the typical models of convergent evolution by demonstrating independent recruitment of different types of proteins to fulfill their unique visual role.

Universality of Form: The Case of Retinal Cone Photoreceptor Mosaics.

Cone photoreceptor cells are wavelength-sensitive neurons in the retinas of vertebrate eyes and are responsible for color vision. The spatial distribution of these nerve cells is commonly referred to as the cone photoreceptor mosaic. By applying the principle of maximum entropy, we demonstrate the universality of retinal cone mosaics in vertebrate eyes by examining various species, namely, rodent, dog, monkey, human, fish, and bird. We introduce a parameter called retinal temperature, which is conserved across the retinas of vertebrates. The virial equation of state for two-dimensional cellular networks, known as Lemaître's law, is also obtained as a special case of our formalism. We investigate the behavior of several artificially generated networks and the natural one of the retina concerning this universal, topological law.

Bacterial origin of a key innovation in the evolution of the vertebrate eye

The vertebrate eye was described by Charles Darwin as one of the greatest potential challenges to a theory of natural selection by stepwise evolutionary processes. While numerous evolutionary transitions that led to the vertebrate eye have been explained, some aspects appear to be vertebrate specific with no obvious metazoan precursor. One critical difference between vertebrate and invertebrate vision hinges on interphotoreceptor retinoid-binding protein (IRBP, also known as retinol-binding protein, RBP3), which enables the physical separation and specialization of cells in the vertebrate visual cycle by promoting retinoid shuttling between cell types. While IRBP has been functionally described, its evolutionary origin has remained elusive. Here, we show that IRBP arose via acquisition of novel genetic material from bacteria by interdomain horizontal gene transfer(iHGT). We demonstrate that a gene encoding a bacterial peptidase was acquired prior to the radiation of extant vertebrates >500 Mya and underwent subsequent domain duplication and neofunctionalization to give rise to vertebrate IRBP. Our phylogenomic analyses on >900 high-quality genomes across the tree of life provided the resolution to distinguish contamination in genome assemblies from true instances of horizontal acquisition of IRBP and led us to discover additional independent transfers of the same bacterial peptidase gene family into distinct eukaryotic lineages. Importantly, this work illustrates the evolutionary basis of a key transition that led to the vertebrate visual cycle and highlights the striking impact that acquisition of bacterial genes has had on vertebrate evolution.

Glial cells expressing visual cycle genes are vital for photoreceptor survival in the zebrafish pineal gland.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.

The functional anatomy of the cornea of the Shorthead lamprey, Mordacia mordax (Mordaciidae, Agnatha): a comparison between downstream and upstream migrants.

The Shorthead lamprey Mordacia mordax (Mordaciidae, Agnatha) represents one of the earliest stages in vertebrate evolution. This study investigates the ultrastructural anatomy of the cornea, iris and anterior chamber in the eyes of this species in both the downstream and upstream migrant phases of its protracted lifecycle to assess the morphological and quantitative changes associated with growth, corneal function and vision. Using light and both scanning and transmission electron microscopy, the cornea is found to be divided into dermal and scleral components separated by a mucoid layer. A range of distinguishing corneal features are compared in the two adult phases of the lifecycle including epithelial microprojections, mucus-secreting epithelial cells, the number, thickness, formation and level of branching and anastomosing of collagen lamellae, the type and distribution of vertical sutures, the structure of the mucoid layer and annular ligament and the number and distribution of a large number of basement membranes throughout the cornea. Significant differences are found between the two phases, which are thought to reflect adaptations to the variable environmental conditions encountered throughout this species' lifecycle. The study provides insights into the evolutionary pressures on extant representatives of the earliest stages in the evolution of the vertebrate eye. This article is protected by copyright. All rights reserved.

Identification of HSPA8 as an interacting partner of MAB21L2 and an important factor in eye development.

Pathogenic variants in human MAB21L2 result in microphthalmia, anophthalmia, and coloboma. The exact molecular function of MAB21L2 is currently unknown. We conducted a series of yeast two-hybrid (Y2H) experiments to determine protein interactomes of normal human and zebrafish MAB21L2/mab21l2 as well as human disease-associated variant MAB21L2-p.(Arg51Gly) using human adult retina and zebrafish embryo libraries. These screens identified klhl31, tnpo1, TNPO2/tnpo2, KLC2/klc2, and SPTBN1/sptbn1 as co-factors of MAB21L2/mab21l2. Several factors, including hspa8 and hspa5, were found to interact with MAB21L2-p.Arg51Gly but not wild-type MAB21L2/mab21l2 in Y2H screens. Further analyses via 1-by-1 Y2H assays, co-immunoprecipitation, and mass spectrometry revealed that both normal and variant MAB21L2 interact with HSPA5 and HSPA8. In situ hybridization detected co-expression of hspa5 and hspa8 with mab21l2 during eye development in zebrafish. Examination of zebrafish mutant hspa8hi138Tg identified reduced hspa8 expression associated with severe ocular developmental defects, including small eye, coloboma, and anterior segment dysgenesis. To investigate the effects of hspa8 deficiency on the mab21l2Arg51_Phe52del allele, corresponding zebrafish double mutants were generated and found to be more severely affected than single mutant lines. This study identifies heat shock proteins as interacting partners of MAB21L2/mab21l2 and suggests a role for this interaction in vertebrate eye development.

Evolution of the visual system in ray-finned fishes.

The vertebrate eye allows to capture an enormous amount of detail about the surrounding world which can only be exploited with sophisticated central information processing. Furthermore, vision is an active process due to head and eye movements that enables the animal to change the gaze and actively select objects to investigate in detail. The entire system requires a coordinated coevolution of its parts to work properly. Ray-finned fishes offer a unique opportunity to study the evolution of the visual system due to the high diversity in all of its parts. Here, we are bringing together information on retinal specializations (fovea), central visual centers (brain morphology studies), and eye movements in a large number of ray-finned fishes in a cladistic framework. The nucleus glomerulosus-inferior lobe system is well developed only in Acanthopterygii. A fovea, independent eye movements, and an enlargement of the nucleus glomerulosus-inferior lobe system coevolved at least five times independently within Acanthopterygii. This suggests that the nucleus glomerulosus-inferior lobe system is involved in advanced object recognition which is especially well developed in association with a fovea and independent eye movements. None of the non-Acanthopterygii have a fovea (except for some deep sea fish) or independent eye movements and they also lack important parts of the glomerulosus-inferior lobe system. This suggests that structures for advanced visual object recognition evolved within ray-finned fishes independent of the ones in tetrapods and non-ray-finned fishes as a result of a coevolution of retinal, central, and oculomotor structures.

Impact of α-crystallin protein loss on zebrafish lens development

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Unique horizontal gaze control in the box jellyfish, Tripedalia cystophora

All known cubozoans, box jellyfish, have a similar visual system. They possess four sensory structures called rhopalia, which carry-six eyes each. Two of these six eyes are true image-forming camera type eyes in several ways similar to vertebrate eyes. The rhopalia hang by a thin flexible stalk and in the distal end, there is a high-density crystal. In an earlier study of the Caribbean species Tripedalia cystophora, we showed that the crystals act as weights ensuring that the rhopalia are always upright no matter the orientation of the medusa and the vertical part of the visual field of the eyes thus kept relatively constant. Here we have examined the horizontal part of the visual field under different experimental conditions including different visual environments. We find that the horizontal gaze direction is largely controlled by the anatomy of the rhopalium and rhopalial stalk, similar to what has previously been shown for the vertical gaze direction. In a vertically oriented medusa, the rhopalia are kept with a 90° angle between them with the lower lens eyes (LLE) pointing inwards. This 90° shift is kept in horizontally swimming medusa, resulting in the left LLE gazing right, the right gazing left, the bottom gazing orally (backwards compared to swimming direction), and the top LLE gazing aborally (forwards compared to swimming direction). The light environment was manipulated to test if the visual input influences this seemingly strict horizontal gaze direction but even in complete darkness there is tight mechanistic control.

Diversity and evolution of optically complex eyes in a family of deep-sea fish: Ocular diverticula in barreleye spookfish (Opisthoproctidae)

Several families of mesopelagic fish have tubular eyes that are usually upwardly directed. These maximise sensitivity to dim downwelling sunlight and dorsal bioluminescence, as well as facilitating the detection of dark silhouettes above the animal. Such eyes, however, have a much-reduced field of view and will not be sensitive to, for example, lateral and ventral bioluminescent stimuli. All mesopelagic Opisthoproctidae so far examined have evolved mechanisms for extending the limited visual field of their eyes using approximately ventrolaterally directed, light-sensitive, diverticula. Some genera have small rudimentary lateral retinal areas capable of detecting only unfocussed illumination. Others have more extensive structures resulting in eyes that simultaneously focus light from above onto the main retina of the tubular eye using a lens, while diverticula produce focussed images of ventrolateral illumination using either reflection or possibly refraction. These bipartite structures represent perhaps the most optically complex of all vertebrate eyes. Here we extend the limited previous data on the ocular morphology of five Opisthoproctidae (Opisthoproctus soleatus, Winteria telescopa, Dolichopteryx longipes, Rhynchohyalus natalensis, and Bathylychnops exilis) using a combination of histology and magnetic resonance imaging and provide a preliminary description of the eyes of Macropinna microstoma. We note an increase in diverticular complexity over the life span of some species and quantify the contribution of the diverticulum to the eye’s total neural output in D. longipes and R. natalensis (25 and 20%, respectively). To help understand the evolution of Opisthoproctidae ocular diversity, a phylogeny, including all the species whose eye types are known, was reconstructed using DNA sequences from 15 mitochondrial and four nuclear genes. Mapping the different types of diverticula onto this phylogeny suggests a process of repeated evolution of complex ocular morphology from more rudimentary diverticula.

Comparative Analysis of Central Corneal Thickness in Four Fish Models.

To understand the left-right asymmetry of vertebrate eyes, this study measured the central corneal thickness (CCT) of Oryzias curvinotus, Oryzias melastigma, Oryzias latipes, and zebrafish with optical coherence tomography. The results showed that the CCTs were significant differences among different species and groups, even between the right and left eyes of each fish. The values of the CCTs (mean ± SD, μm) for the four species were 104.71 ± 14.49, 61.88 ± 8.63, 64.76 ± 10.36 and 56.96 ± 10.48, respectively. Moreover, comparing the two wild groups of O. curvinotus from Sanya on N18° and Gaoqiao on N21°, the CCT value for the low-latitude group was 104.71 ± 14.49 μm, greater than the high latitude group 76.13 ± 5.70 μm significantly (t-test, p = 0.0001). Lastly, the paired Student's t-test revealed that significant CCT differences existed between the left and right eye for all four species and groups, of which zebrafish and O. melastigma were belonging to the left thicker type in contrast to the others. This study laid a foundation for understanding the causes of the difference in CCT, and also provided possible fish models for human researches on keratomileusis, glaucoma, and other corneal diseases.

Genetic dissection of eye development – Lessons from Pax6

AbstractA paired domain and homeodomain‐containing transcription factor Pax6 is an evolutionarily conserved regulator implicated in the eye development of both vertebrates and invertebrates. Mutations of the Pax6 gene disrupt eye development in both mammals and insects while Pax6 misexpression can lead to the formation of ectopic eyes. Genetic and expression studies have indicated that the development of vertebrate eyes is guided by a conserved gene regulatory network with Pax6 being one of the key transcription factors. To define the regulatory inputs into Pax6 gene we performed a genetic dissection of its regulatory landscape. To uncover the direct target genes of Pax6 we performed chromatin immunoprecipitation (ChIP‐seq) using mouse embryonic retina as a sample. We will present our preliminary genetic data on functional validation of Pax6‐regulated enhancers.

Cell fate decisions, transcription factors and signaling during early retinal development.

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases.

Muscles that move the retina augment compound eye vision in Drosophila.

Most animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all of these species must resort to moving either their head ortheir body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye1-3. Here we show that Drosophila use their retinal muscles to smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. We show that when the retina moves, visual receptive fields shift accordingly, and that even the smallest retinal saccades activate visual neurons. Using a head-fixed behavioural paradigm, we find that Drosophila perform binocular, vergence movements of their retinas-which could enhance depth perception-when crossing gaps, and impairing the physiology of retinal motor neurons alters gap-crossing trajectories during free behaviour. That flies evolved an ability to actuate their retinas suggests that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlight a notable example of convergent evolution.

Light-induced shifts in opsin gene expression in the four-eyed fish Anableps anableps.

The development of the vertebrate eye is a complex process orchestrated by several conserved transcriptional and signaling regulators. Aside from partial or complete loss, examples of exceptional modifications to this intricate organ are scarce. The unique eye of the four-eyed fish Anableps anableps is composed of duplicated corneas and pupils, as well as specialized retina regions associated with simultaneous aerial and aquatic vision. In a previous transcriptomic study of the A. anableps developing eye we identified expression of twenty non-visual and eleven visual opsin genes. Here, we surveyed the expression territories of three non-visual melanopsins genes (opn4×1, opn4×2, opn4m3), one teleost multiple tissue opsin (tmt1b) and two visual opsins (lws and rh2-1) in dorsal and ventral retinas. Our data showed that asymmetry of non-visual opsin expression is only established after birth. During embryonic development, while inside pregnant females, the expression of opn4×1, opn4×2, and tmt1b spans the whole retina. In juvenile fish (post birth), the expression of opn4×1, opn4×2, opn4m3, and tmt1b genes becomes restricted to the ventral retina, which receives aerial light. Raising juvenile fish in clear water instead of the murky waters found in its natural habitat is sufficient to change gene expression territories of opn4×1, opn4×2, opn4m3, tmt1b, and rh2-1, demonstrating that different lighting conditions can shift opsin expression and potentially contribute to changes in spectral sensitivity in the four eyed fish.