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Zebrafish as an Experimental Model for Inherited Retinal Diseases

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Shimpei Takita and Yuko Seko

Submitted: 05 February 2024 Reviewed: 09 February 2024 Published: 27 March 2024

DOI: 10.5772/intechopen.1004858

Zebrafish Research IntechOpen
Zebrafish Research An Ever-Expanding Experimental Model Edited by Geonildo Rodrigo Disner

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Zebrafish Research - An Ever-Expanding Experimental Model [Working Title]

Dr. Geonildo Rodrigo Disner

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Abstract

Zebrafish are becoming a popular experimental animal model for vision science and human-inherited retinal diseases. In this chapter, we describe application of zebrafish for the retinitis pigmentosa (RP) caused by digenic LDL receptor-related protein 5 (LRP5) and Eyes shut homolog (EYS). RP is the most common genetic disorder in inherited retinal diseases, and EYS is one of the major causes of RP. EYS orthologs are absent in rodents but present in zebrafish. Using this advantage, we generated and analyzed the digenic eys+/−; low-density lipoprotein (LDL)-related receptor-5 (lrp5)+/− zebrafish, the same form of gene defects emerged from a human case report as a candidate of RP. The analysis discovers that retinol binding protein 1a (rbp1a) gene is remarkably downregulated and that Lrp5 protein is a strong candidate for the receptor of all-trans-retinol in the visual cycle. Furthermore, in this review, we also discuss functional roles of EYS in vertebrates with an emphasis on its possible involvement in the retinal metabolism, the visual cycle, aiming at integrating our findings with recent advances in the research field.

Keywords

  • retinitis pigmentosa
  • Eys
  • Lrp5
  • Rbp1
  • visual cycle
  • familial exudative vitreoretinopathy

1. Introduction

Humans rely heavily on vision for a lot of information. Curing or slowing the progression of the vision loss contributes significantly to medicine, as vision loss impairs quality of life significantly. Vision is an intricate biological phenomenon that involves the retina and the visual field in the brain. Vision is initiated and processed by a network of retinal cells and molecular processes within the retina, which is located at the back of the eye. The first step of vision is to detect light, i.e., photons, and convert the information to chemical information in photoreceptors within the retina (for review, see [1, 2]).

In the photoreceptors, 11-cis-retinal (11-cis RAL) forms a Schiff’s base with a lysine residue (K) in the protein opsin to form rhodopsin (or cone opsins) in darkness. 11-cis RAL is a derivative of vitamin A. Vitamin A, a fat-soluble natural compound, circulates in the body bound with retinol-binding protein 4 (RBP4) and enters the retina from the retinal pigment epithelium (RPE) through the receptor, STRA6 [3, 4]. In the context of sustained vision, which is particularly important for mesopic to photopic vision, the retinoid cycle called the “visual cycle” is the fundamental process. The visual cycle is a series of biochemical reactions between the retina and the RPE and within the retina and is essential for maintaining a constant supply of 11-cis RAL, a crucial component for phototransduction.

Both environmental and genetic factors can cause blindness. In this review, we focus primarily on genetic factors (i.e., IRDs). Among IRDs, retinitis pigmentosa (RP) and familial exudative vitreoretinopathy (FEVR) are included. In developed countries, IRD is going up in ranking as the cause of visual impairment. Although mouse genetics using the knocked-out (KO) approach is powerful to study whether a gene of interest is causative of an IRD and/or elucidating its pathogenesis and molecular functions, there are cases where the human phenotype is not recapitulated well [5, 6] or a gene of interest does not exist in mice [7, 8].

Researchers have been using zebrafish (Danio rerio) as a valuable model organism. Findings and implications extend beyond adding new knowledge to fish biology, and insightful information can come from fish (e.g., [9]). More specifically, the study of zebrafish orthologous genes associated with human inherited retinal diseases has provided insightful knowledge for uncovering the molecular mechanisms underlying visual impairment and vision [10, 11]. The eyes shut homolog (eys) gene is one of them as eys possesses striking homology to the human EYS gene implicated in various forms of photoreceptor degeneration and blindness. On the other hand, we also have to be aware that there are some differences between humans and zebrafish. For example, in zebrafish, there are four types of cones expressed and red and green cones form unique shape called double cone.

In this chapter, we aim to insightfully bridge the current knowledge gap between fish and mammals and get insight into the role of EYS in human vision that is key to photoreceptor degeneration by closely reviewing the accumulating results of eys mutant zebrafish and medaka fish as well as studies human patients are employed. We particularly provide zebrafish lrp5 and eys genetics, the visual cycle, and their implications for human vision. Through the integration of novel insights from the knockout zebrafish models and existing and novel literature, we aim to shed light on the major gene responsible for human inherited retinal diseases, bringing our current complex knowledge one step closer to unraveling the functional role(s) of EYS protein.

This review first focuses on the findings from the zebrafish low-density lipoprotein (LDL)-related receptor-5 (Lrp5) and Eys proteins and then possible EYS roles in vision. Recent advancements in genetic manipulation techniques, including CRISPR/Cas9, have enabled the creation of knockout zebrafish models more easily than before, offering a unique opportunity to investigate the functional consequences of disruption of gene(s) of interest. Although not the main topic of this review, we briefly introduce these techniques for readers who are not familiar with zebrafish.

Human LRP5 gene (https://www.ncbi.nlm.nih.gov/gene/4041; its proteins is LRP5 protein) or EYS gene (https://www.ncbi.nlm.nih.gov/gene/346007; its proteins is EYS) have one copy of zebrafish ortholog; the lrp5 gene (https://www.ncbi.nlm.nih.gov/gene/568518; its proteins is Lrp5 protein) and the eys gene (https://www.ncbi.nlm.nih.gov/gene/557044; its proteins is Eys protein), respectively. EYS gene is responsible for one of the major causes of retinitis pigmentosa in human inherited retinal diseases with significant implications for understanding its function. This research not only contributes to our fundamental understanding of the retinoid and visual cycle but also holds promise for uncovering novel therapeutic targets to combat human inherited retinal diseases. As we delve into the intricacies of the visual cycle, it becomes apparent that Lrp5 and possibly Eys too play a pivotal role in these processes. Our exploration of knockout zebrafish models targeting the genes aims to decipher the repercussions of gene disruption on the visual cycle and, by extension, on visual function. This would contribute significantly to the understanding of the basis of pathogenesis of these diseases and, more broadly, photoreceptors/RPE biology.

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2. Inherited retinal diseases (IRDs)

IRD is a group of diseases characterized by a progressive degeneration of the retina as time goes, which can lead to progressive severe vision loss or blindness. IRD primarily affects the structure and/or function of the retina and alters them [12]. This process could be during the developing of the retina (this can be syndromic that may also be critically important for normal development in tissues other than the retina) or after the completion of development of the retina (i.e., maintenance and therefore, typically nonsyndromic) depending on types of IRDs. Because phenotypes can be changed by the types of mutations in a same gene, and sometimes it is difficult to distinguish phenotypes by those typical names, it may be preferable to use the term IRD in some cases, which would include all types of blindness.

There are several different types of IRDs where photoreceptors are especially affected, among the six major retinal neuronal cell types [13]. Leber congenital amaurosis (LCA) is a congenital retinal disease that results in severe vision loss present at birth [14, 15]. LCA can be caused by mutations in genes involved in a wide variety of photoreceptor functions such as centrosomal protein 290 (CEP290), guanylate cyclase 2D, retinal (GUCY2D) and retinoid isomerohydrolase RPE65 (RPE65). In RP, rod photoreceptors, dominant in the periphery in humans, are mainly affected. On the other hand, cone photoreceptors are affected in cone-rod-dystrophy (CRD). Although there is debate about environmental and genetic factors as causes of this disease [16], in age-related macular degeneration (AMD), central vision is affected. In humans, cones are dominant in the central region, macula, and its central region is called fovea. Therefore, the cones are considered to be mainly dysfunctional in AMD. The awareness of the symptoms usually starts during young adulthood in RP and CRD. Instead, AMD mostly occurs in people with the age over 50. Stargardt disease is a rare genetic disease caused by the accumulation of fatty substances in the macula [17], and, unlike AMD, onset of this disease usually occurs before age 20.

Ciliopathies are a group of diseases that involve dysfunction of the cilium. Because the photoreceptor outer segment is one of the cilia particularly specialized for absorbing light, mutations in genes important for formation and/or maintenance of cilia can cause IRDs frequently accompanying a wide range of syndromic phenotypes such as obesity. Among them, Bardet-Biedl syndrome (BBS) is well known [18], and Meckel-Gruber syndrome (MKS) and Alström syndrome (ALMS) are also known [19, 20, 21].

Adjacent supporting cells, the retinal pigment epithelium (RPE), are crucial for maintenance of photoreceptors. Therefore, dysfunction of RPE cells can kill photoreceptors, and there are known IRDs such as RP, AMD, and CRD.

FEVR is also a group of IRDs. This disease is characterized by abnormal retinal angiogenesis and abnormal vascularization of the peripheral retina with subsequent retinal ischemia. Abnormality can be both hypovascularization and hypervascularization. Norrin cystine knot growth factor NDP (NDP) (OMIM 300658), frizzled class receptor 4 (FZD4) (OMIM 604579), LRP5 (OMIM 603506), tetraspanin 12 (TSPAN12) (OMIM 613138), zinc finger protein 408 (ZNF408) (OMIM 616454), and kinesin family member 11 (KIF11) (OMIM 148760) are the common genes that cause FEVR [22, 23]. Most of them are known to be involved in the Wnt signaling.

One thing to note about FEVR is that the phenotype of mouse models of angiogenesis including knockout mice often does not recapitulate the symptoms of human patients; in other words, the phenotypes of humans and mice are often different [24], presumably due to the difference of capillary. In relation to zebrafish, confirmative studies have been conducted for Znf408 and Fzd4 [25, 26]. In general, in many cases, with some of those exceptions, IRD-related genes are conserved between zebrafish and humans, and vision scientists use zebrafish as an appropriate animal model.

Usually, mutation(s) in a single gene account for IRD. On the other hand, it could be caused by multiple genes. For example, it is well known that digenic mutations in peripherin 2 (PRPH2) and retinal outer segment membrane protein 1 (ROM1) cause RP [27]. Likewise, heterozygous cyclic nucleotide-gated channel subunit alpha 3 (Cnga3)-null mutation exacerbates the cone dystrophy phenotype in cyclic nucleotide-gated channel subunit beta 3 (Cngb3)R403Q/R403Q mice [28]. CNAGA3 and CNGB3 proteins are known to form the tetrameric cyclic nucleotide-gated ion channel in cone photoreceptors. However, it is difficult to track multiple generations, and genetic test is limited particularly in humans.

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3. Visual cycle

Visual cycle is a retinoid metabolism that regenerates photoisomerized retinal that allows rhodopsin or cone opsin can capture light multiple times for sustained vision [29, 30]. The visual cycle takes place mainly between photoreceptors and the RPE as well as between cones and Müller cells (called cone-specific visual cycle). In darkness, 11-cis RAL (a derivative of vitamin A) forms a Schiff’s base with a lysine residue (K) in the protein opsin to form rhodopsin (or cone opsins).

Upon absorption of light, 11-cis RAL in rhodopsin or cone opsin is photoisomerized to all-trans-retinal (atRAL). atRAL is converted to all-trans-retinol (atROL) by ATP binding cassette subfamily A member 4 (ABCA4). atROL is released from photoreceptors, which is unique to vertebrate opsins and is incorporated into the RPE.

Inside the RPE, retinol binding protein 1 (RBP1) plays a role [31, 32, 33]. RBP1 binds and transports atROL to endoplasmic reticulum (ER). atROL is esterified within this RBP1-atROL complex by lecithin retinol acyltransferase (LRAT). This all-trans-retinyl ester is converted to 11-cis-retinol (11-cis-ROL) by the retinoid isomerohydrolase, RPE65. 11-cis-ROL is converted to 11-cis retinal (11-cis-RAL) by retinol dehydrogenase 5 (RDH5). The RPE turns 11-cis-RAL back to photoreceptors again by a transporter, retinaldehyde binding protein 1 (RLBP1), and inter-photoreceptor retinol binding protein (IRBP).

Retinoid metabolism crucial for sustained light absorption involves multicellular and multiple proteins. These proteins are essential and some of them show no phenotype in mice suggesting that visual cycle is conserved in vertebrates but there could be some variation among species. atROL could be carried by IRBP, however, how atROL is released from photoreceptors and is incorporated into the RPE has not been well known [34, 35].

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4. LRP5 in humans and zebrafish and its involvement in IRDs

LRP5 protein is a member of the LDL receptor superfamily conserved in vertebrates. LRP5 protein was identified in 1998 in human and mouse cDNA libraries [36, 37, 38, 39]. LRP5 is best known as a co-receptor for Wnt ligands involved in the wingless (Wnt) signaling pathway on cell membranes, which is essential for the development of vascular endothelial cells, Müller cells, and retinal interneurons [40]. LRP5 is also reported to be involved in cholesterol and glucose uptake [41, 42]. Furthermore, the possible involvement of LRP5 in cell types involved in vitamin A (i.e., atROL) is suggested based on its expression [43].

Domain structures of human LRP5 and zebrafish Lrp5 are well conserved between humans and zebrafish. Human and zebrafish LRP5 proteins consist of a signal peptide sequence, low-density lipoprotein receptor YWTD (LY; also known as β-propeller), EGF, and LDLa domains in the N-terminal extracellular domains. These domains are followed by one transmembrane domain and low complexity region in the C-terminal intracellular region. LRP5 protein seems to act as a receptor of both protein and small molecules.

In addition to our group, there are at least two groups that use lrp5 mutant zebrafish [44, 45], both of them study osteoporosis, a highly prevalent systemic skeletal disease characterized by decreased bone mass and decline of bone mineral density (BMD) resulting in compromised bone architecture, bone fragility and increased fracture risk. Cholesterol biosynthesis pathway is particularly changed in lrp5−/− skulls suggesting conservation of Lrp5 function between zebrafish and mammals.

In vision, LRP5 is a well-known causative gene of FEVR [46, 47, 48]. Hypovascularization, where peripheral vasculogenesis is affected, accompanying leakage is the typical phenotype of LRP5 mutations. This can be observed in both autosomal dominant (ad) and recessive (ar) forms [48, 49, 50]. It appears that it is an ad form (i.e., typically gain-of-function), particularly when the type of mutations is missense mutation. On the other hand, the ar form of the mutation (i.e., loss-of-function) in LRP5 is found in the pedigrees of FEVR [47, 48], and hypovascularization is the retinal phenotype of loss-of-function of Lrp5 by animal studies using mouse and rat Lrp5 knockout models [51, 52]. So far, only one mutation has been suggested to cause RP, and this is a heterozygous digenic form together with mutation in EYS [53].

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5. EYS in humans and zebrafish and its involvement in IRDs

Human eyes shut homolog (EYS) gene (Online Mendelian Inheritance in Man no. 612424), an ortholog of the Drosophila eyes shut/spacemaker (eys, https://www.ncbi.nlm.nih.gov/gene/3771890) [7, 8], is a major causative gene of nonsyndromic autosomal recessive retinitis pigmentosa (RP) [7], and cone-rod dystrophy (CRD) [54, 55]. EYS is originally identified in Drosophila which is important for maintenance of photoreceptors. And several years later human ortholog, EYS, was found to be a causative gene of RP. And in 2010 and 2012, our group and others reported that this gene is a major cause of RP in the French [56] and Japanese population [57, 58], and therefore its importance in ophthalmology globally [59].

The predominant human EYS transcript, tv1, encodes 3144 amino acids and zebrafish eys transcript encodes 2906 amino acids, and through the spices, the domain structure of the orthologous protein is conserved [60]. Eys is structurally related to Pikachurin and Agrin [61]. Characteristics of these proteins are that they consist of multiple EGF and EGF-like domains in its 5′ portion and 3′ portion consists of multiple Laminin G domains [62]. Human and zebrafish EYS proteins consist of a signal peptide sequence for secretion, epidermal growth factor (EGF), EGF-like and Ca2+ EGF domains in the N-terminal half, and laminin A G-like (LamG) and EGF domains in the C-terminal half.

This protein is present in invertebrates as well. Eys was originally identified in Drosophila that is a secreted protein filling a space in photoreceptors [63, 64]. This is in accordance with the other related proteins such as Pikachurin and Agrin that are secreted and functions outside cells [65]. In Drosophila, Eys is believed to be important for open rhabdom and other invertebrates having this structure together with prominin 1 (Prom1) [66]. Based on localization studies in zebrafish and primates including humans, this protein, Eys, is shown to be localized to ciliary pocket in both rod and cone photoreceptors [67]. In addition, EYS is also localized to cone OSs and terminals [67]. In particular, it is interesting that EYS is localized unilaterally to the edge of cone OSs, ciliary axoneme, in primates, and it looks as “whip-like” signal [68].

Our group reported that this gene is most prevalent in the Japanese population [58], and five mutations that are most frequently observed in the Japanese population (JV1, c.4957dupA and p.(Ser1653Lysfs*2); JV2, c.8868C > A and p.(Tyr2956*); JV3, c.2528G > A and p.(Gly843Glu); JV4, c.6557G > A and p.(Gly2186Glu); JV5, c.6563 T > C and p.(Ile2188Thr)) [69]. These five mutations are located from N-terminal region to C-terminal region of EYS protein. Full-length Eys is highly expressed in zebrafish photoreceptors and this expression is specific or dominant [60], and full-length EYS is expressed is expressed in human photoreceptor-like cells derived from dermal fibroblasts (our unpublished observation). It would be important to note that the evidence suggests that full-length EYS protein is crucial for preventing photoreceptors from degeneration.

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6. Available resources and genetic manipulation in zebrafish

In zebrafish, the eye starts to form around 20–24 hours post-fertilization (hpf) [70, 71]. Around this time point, zebrafish eyes are transparent allowing unique opportunities for eye formation or photoreceptor differentiation [72], which occurs around 48 hpf in the ventronasal patch [73].

We initially investigated zebrafish orthologs of human EYS and LRP5 genes. As a result of whole-genome duplication, there are two orthologs corresponding to mammals for many genes [29, 74]. Recent advance in the database has made it dramatically easier to make connections of human, mouse, and zebrafish orthologs than decades ago. Orthologs of human EYS and LRP5 genes are eys and lrp5, respectively (see also Introduction for links to the National Center for Biotechnology Information (NCBI) websites).

One more important thing to note is that there are multiple strains actively used in zebrafish; from our experience using two different strains, AB and TL, we do not observe substantial differences in molecular cloning in both strains.

We used mutant zebrafish obtained from Zebrafish International Resource Center (ZIRC) where N-ethyl-N-nitrosourea (ENU)-induced mutant zebrafish are stocked. Zfin (https://zfin.org/) allows users to look up all available mutant zebrafish for each gene. Busch Lab website (https://zmp.buschlab.org/) is also helpful to look up mutations in more detail about the ENU-induced mutant zebrafish. Once desired mutants are found, they can be obtained through ZIRC (https://zebrafish.org/home/guide.php) or the European Zebrafish Resource Center (EZRC) (https://www.ezrc.kit.edu/).

We received 50–100 of 3–5 days post-fertilization (dpf) zebrafish embryos and raised the embryos to adults. We collected tails, extracted genomic DNAs, and genotyped to screen the obtained zebrafish and about 25% of them carried the described mutation in the eys gene in a heterozygous manner. In our experience with a total of several mutant lines from the ZIRC, 25–50% of the resulting zebrafish in any strain had the exact mutation described and were not particularly troubled.

To avoid off-target effect potentially arisen from ENU-treatment, it would be ideal to cross-obtained mutant zebrafish with wild-type zebrafish for several generations to reduce the possibility of unexpected ENU-induced mutations being introduced at other loci. In our experience, this is probably sufficient for practical purposes, as the zebrafish obtained from these resource centers are of F2 generation, and ENU-induced mutations that induce point mutations are more random than CRISPR-induced off-target mutations.

Alternatively, because the CRISPR/Cas9 system has become accessible and much easier in zebrafish, researchers can generate knockout zebrafish using CRISPR/Cas9 in their own labs. We briefly explain our method here [75], which we believe is easiest. We obtain Cas9 protein (Alt-R® S.p. HiFi Cas9 Nuclease V3) [76], crRNA (Alt-R® CRISPR-Cas9 crRNA), and tracrRNA (Alt-R® CRISPR-Cas9 tracrRNA), the components necessary for CRISPR/Cas9, from Integrated DNA Technologies (IDT). We form gRNA by annealing the equimolar of custom-synthesized crRNA with universal tracrRNA using Nuclease Free Duplex Buffer (IDT), and mix the gRNA with the Cas9 protein to form ribonucleoprotein (RNP) complex. We inject the RNP complex into one-cell stage zebrafish eggs and raise them to adults and uses as founders (F0).

It is important to minimize the possibility of off-target effects when we select target sequence for knockout. We use CRISPRdirect (http://crispr.dbcls.jp/) [77]. We typically select sequences having minimal similarity to other locations in the reference sequence by choosing “Zebrafish (Danio rerio) genome, GRCz11/danRer11 (May, 2017)”. After deciding target sequence, it is important to read actual DNA sequence surrounding the target region in our own zebrafish cohort because there could be SNPs in the target region from the reference genome.

To create knock-in zebrafish is attractive. For target size with smaller than 200 nt, there are reports [78]. However, accurate visualization of the localization of proteins tagged with fluorescent proteins such as EGFP and mCherry may still be particularly challenging, as the typical size of fluorescent proteins is ~750 nt.

We also have to be aware that there are some differences between humans and zebrafish. For example, in humans, rods are dominantly located in peripheral regions while rods are uniformly distributed in zebrafish. In addition, in zebrafish, there are four types of cones expressed and red and green cones form unique shape called double cone. Retinomoter movement reflecting circadian rhythm and/light is also very unique to fish, which is not necessarily disadvantage but needs to be note [79, 80]. One more important aspect is that photoreceptors have capability to regenerate throughout life in zebrafish [81, 82, 83]. This is advantageous for the study of regenerative medicine; this may make analysis a bit more difficult when we study retinal diseases because of continuous supply of new retinal cells.

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7. Lrp5 protein and the visual cycle

EYS does not exist in rodents but does exist in zebrafish, which makes zebrafish valuable for EYS study. In humans, a case study was reported in 2017 describing digenic form of the heterozygous mutations in EYS and LRP5 genes might cause RP [53]. We took this advantage and generated mutant zebrafish, eys+/−; lrp5+/− zebrafish, having the same form of eys and lrp5 mutations to test this possibility in zebrafish. In humans, the EYS gene is a cause of autosomal recessive RP and the LRP5 gene has not been reported to be a cause of RP. From these facts, some possibilities are inferred that RP occurred by one changed gene of EYS or genetic interaction between EYS and LRP5.

We chose mutations in the C-terminal region of the Eys gene. This is because its C-terminal region is frequently mutated in the EYS patients (e.g., JV2) and other groups had tested if eys knockout is pathogenic or not, but no groups had tested if mutations in the C-terminus are pathogenic in zebrafish at that time. Likewise, we checked disease mutations in Lrp5 gene. Substitution mutation in the Lrp5 seems to cause gain of function that leads primary osteoporosis or high bone density syndrome in humans, which are particularly located in the first β-propeller domain. Based on the information, we chose nonsense mutation after the first β-propeller domain to reduce potential phenotypes in other organs. After confirming mutant zebrafish are available from ZIRC, we obtained the eyssa31957 and lrp5sa11097 from ZIRC. We crossed the zebrafish line and established the eys+/−; lrp5+/− zebrafish.

In the digenic eys+/−; lrp5+/− zebrafish, photoreceptor layer is mildly thinner than wild-type zebrafish at 14 months post-fertilization (mpf). This suggests that this form of mutation can cause RP in humans.

Then we subjected the whole eyes to an unbiased global gene expression analysis, microarray analysis, at 3.5 mpf. At this stage, zebrafish are sexually mature young adults and we expected that this stage would allow us to identify gene(s) that are primarily affected by the heterozygous digenic mutations. Indeed, retinol-binding protein 1 a (rbp1a (rbp1.1); https://www.ncbi.nlm.nih.gov/gene/791484) is remarkably downregulated in the digenic mutant eyes (less than 10% of wild-type eyes) [75]. This is remarkable in two aspects: (1) this kind of dramatic mRNA downregulation is unique to rbp1a and is not observed for other genes. (2) RBP1 protein is well-known as one of important proteins that is a transporter of atROL in the RPE as described in the above section.

This discovery highly motivated us to further investigate the reason(s) this gene is specifically downregulated in the eys+/−; lrp5+/− eye. We first confirmed that Rbp1a protein is downregulated in the eys+/−; lrp5+/− eye. We then analyzed the detailed localization of Rbp1a protein in the eye. The staining pattern is very unique and Rbp1a protein is localized to the string-like (or accessary-like) structure called microvilli of the RPE confirmed with a marker of microvilli of the RPE, Moesin (Msn). This is in good agreement with the literature where RBP1 is a transporter of atROL in the visual cycle. Of note, Rbp1a protein is concentrated in the tip of the microvilli and these most concentrated regions are Msn negative.

We then investigated localization of Lrp5 protein in the eye. Lrp5 protein colocalizes with Rbp1a protein at the most concentrated regions in the microvilli of the RPE, which is very close to the inner segment (IS) of photoreceptors in the outer retina. This is strong supporting evidence that Lrp5 protein plays a role in the visual cycle as a receptor of atROL at the tip of the microvilli in the RPE. Using in vitro purified C-terminal intracellular region of Lrp5 protein and full-length RBP1a protein involving a binding assay similar to Agrin and Lrp4 [84, 85], the direct binding of Rbp1a and Lrp5 proteins is further confirmed.

The expression level of the rbp1a gene was remarkably decreased (~10%) compared with the wild-type siblings in the lrp5−/− adult eyes. We generated rbp1a−/− zebrafish using CRISPR/Cas9 system and examined the expression level of the lrp5 gene in the rbp1a−/− eyes. Contrary to rbp1a expression, the expression level of lrp5 is not changed in the rbp1a−/− adult eyes (~142%). These genetic studies clarified that rbp1a gene plays a role downstream of lrp5 gene in the eye.

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8. Perspective: potential involvement of EYS protein in the visual cycle and/or ciliary transport

Eys was originally found in Drosophila as a secreted protein present in extracellular matrix. Now two possibilities are raised: Eys is involved in (1) visual cycle and (2) ciliary transport/OS maintenance.

(1) Visual cycle: there are studies that support the idea that Eys protein is involved in the visual cycle. First, we have shown that Lrp5 is a strong candidate for the receptor of the atROL based on the possible genetic interaction between Eys and Lrp5. In this study, genetic studies suggest that the expression level of rbp1a gene is 10.9% in the lrp5−/− eye and 6.8% in the eys+/−; lrp5+/− eye compared with wild-type eye. These observations suggest that the remarkable decrease of rbp1a expression in the eys+/−; lrp5+/− eye is not solely attributed to lrp5 gene but also from the contribution of the eys gene.

Indeed, Eys and Lrp5 proteins are structurally related to Agrin (AGRN) and LDL receptor-related protein 4 (LRP4) proteins, respectively, and AGRN interacts with LRP4 in neuromuscular junction [84, 85]. Applying this to photoreceptors and the RPE, Eys and Lrp5 could interact with each other in the interphotoreceptor matrix between photoreceptors and the RPE. Based on the above evidence and suggestions from the previous study, we proposed in 2020 that Eys could be involved in the visual cycle (Figure 1).

Figure 1.

A proposed model of LRP5 function in the visual cycle. LRP5 protein (orange in right bottom) is a strong candidate for the receptor of atROL in the visual cycle that forms a complex with RBP1 protein (the transporter of atROL; light green in right bottom) is involved in the uptake of atROL in the microvilli of the RPE (adopted from reference [75]). The flow of retinoid is shown by red arrows and the unknown possible interactions are indicated with “?” as well as possible involvement of EYS protein in the visual cycle. atROL, all-trans retinol. ABCA4, ATP binding cassette subfamily A member 4; IRBP, inter-photoreceptor retinol-binding protein (also known as RBP3, retinol-binding protein 3); LRAT, lecithin retinol acyltransferase; LRP5, LDL receptor related protein 5; RBP1, retinol binding protein 1 (also known as CRBP, cellular retinol binding protein); RDH, retinol dehydrogenase; RLBP1, retinaldehyde binding protein 1 (also known as CRALBP, cellular retinal binding protein); RPE65, retinoid isomerohydrolase RPE65; STRA6, stimulated by retinoic acid gene 6.

Studies in good line with our proposal are now emerging in both zebrafish and humans. First, human clinical trial published recently shows that administration of vitamin A worsened the photoreceptor function in the EYS patients [86]. This study is strong evidence for the possible involvement of EYS proteins in the visual cycle, particularly in the step where atROL accumulates and its dysregulation could kill photoreceptors [75]. Second, human fundus image employing artificial intelligence-guided diagnosis (machine diagnosis) shows that fundus images of EYS patients is closer to those of ABCA4 patients than those of RP1 like 1 (RP1L1) patients, a protein thought to be involved in transport of OS protein in the connecting cilia [87]. This would suggest that EYS causes a phenotype resemble to the disease caused by accumulation of retinal species (i.e., vitamin A derivatives) in photoreceptors and/or the RPE [88, 89], and therefore again supports the idea that EYS is involved in the visual cycle, particularly the conversion of atRAL to atROL and/or the clearance of atROL from the OS. Third, in zebrafish, Eys is found to be localized to the cone periciliary membrane, cone accessory outer segment (AOS), and photoreceptor ribbon synapse using immunoelectron microscope [90]. AOSs are structures described in teleost fish that extend from the ISs of cones, run along the cone OSs towards the RPE. AOSs are thought to be structural support to the cone OSs towards the RPE and are thought to be involved in the exchange of metabolites between cones and the RPE [91, 92, 93]. Indeed, numerous vesicles accumulate in AOSs in the eys knockout medaka [94]. This is in good agreement with the idea that Eys protein is involved in interaction between photoreceptors and the RPE. Fourth, early functional abnormality in cones is observed in EYS patients using 30 Hz Flicker electroretinogram (ERG) and macular cone-mediated focal ERG (fERG) [95]. This suggests that EYS is involved not only in rod function but also in cone function in humans even though it does not cause cone degeneration and therefore, the patients may be unaware of it. With respect to retinal vasculature, it is reported that EYS does not seem to cause alteration of retinal vasculature using OCT [96].

(2) Ciliary transport/OS maintenance: the speculation that Eys protein is involved in ciliary transport may have arisen from two observations in the early studies. One is that, in Eys knockout zebrafish, ciliary pocket disappears in the cones [67], and opsins are mislocalized to IS to some extent in cones in terms of cell number [94, 97]. With respect to mislocalization of OS proteins, careful examination would be necessary because it is widely known that mislocalization of OS proteins to the IS and even non-OS proteins to the OS are rather a common phenomenon frequently observed in degenerative photoreceptors [98, 99, 100, 101, 102]. With this in mind, in the eys knockout medaka, ciliary pocket is not always affected and remains observable [94]. This would raise a possibility that loss of ciliary pocket is a secondary effect rather than a primary cause of the cone degeneration in zebrafish. In addition, in both zebrafish and medaka, the affected cells appear to be sparsely distributed in both eys knockdown and knockdown larvae [94, 97]. Theoretically, if defects in ciliary transport were the direct cause of degeneration, mislocalization of OS proteins would be expected to be uniformly distributed throughout the photoreceptor cell layer as observed in defects in intraflagellar transport (IFT) proteins [103, 104]. Careful reexamination would be necessary in the future study.

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9. Future direction: to investigate possible involvement of EYS protein in the visual cycle in terms of AOS and/or other aspects

Based on the current knowledge, the most clinically important aspect of the Eys protein is whether Eys is indeed involved in the visual cycle. If Eys is involved, the search for the existence of other proteins that might be involved in the visual cycle would be a meaningful topic from both clinical and biological perspectives.

In this regard, a possible interaction between Eys and the Prom1 protein has long been proposed based on studies in Drosophila [66]. Based on the assumed EYS function, Prom1 protein appears to be an attractive candidate that functions in AOS. Since there is no genetic interaction observed between eys and two zebrafish orthologs of prominin 1 (Prom1), prom1a (https://www.ncbi.nlm.nih.gov/gene/322857) or prom1b (https://www.ncbi.nlm.nih.gov/gene/378834), in the zebrafish eye [75], it would be necessary to test the possibility of interaction between them at protein level. It should be noted, however, that Prom1 is thought to be localized to the open rim in cones, which is opposite to the AOS.

Other possibly intriguing candidate proteins are Usher proteins. It is interesting that in humans, similar cone abnormality is observed in USH2A (Usherin) patients [105, 106], and EYS-RP and USH2A-RP patients showed better-preserved rod function compared with PDE6A-RP and RPGR-RP patients in chromatic pupil campimetry [106]. These studies indicate that phenotypes are overlapped at least to some extent between EYS-RP and USH2A-RP patients. In mice, USH2a knockout mice shows slow photoreceptor degeneration and Eys does not exist in mice. This aspect could be key to elucidate functional roles of Eys in zebrafish in the context of the visual cycle, and this might be true for the ciliary transport as well. It seems reasonable to assume that atROL is transported from the OS to the RPE directly through the AOS or through the boundary between the OS and IS (connecting cilium region), calyceal processes (the structure Usher proteins are localized to and is nearly missing in mice), and AOS (see Figure 1).

In either case, AOS seems to be the crucial structural component for understanding EYS function and its possible involvement in the visual cycle. This is an interesting but unknown structure that has not been reported outside of fish, and even in fish it has been little studied. This may be an excellent opportunity to open up a new field of research. On the other hand, assuming that EYS functions in the visual cycle in humans based on previous studies, the presence of AOS seems natural, while it would be surprising if AOS is present in humans and other primates but has not been found before. Future studies on AOS are awaited.

It is a little bit surprising that photoreceptor degeneration is observed in zebrafish while this is not observed in medaka. It might be interesting that this is also observed when mutations are introduced in other loci in the medaka eys gene.

In addition, there are studies showing mislocalization of OS proteins in the IS in eys knockout zebrafish or knockdown medaka in larval stages [94, 97, 107]. This might suggest that Eys protein could also be involved in proper localization of OS proteins in the larval stage. Whether this observation is the actual primary cause of photoreceptor degeneration awaits definitive corroboration and/or further supporting evidence in the future.

In humans, cystoid macular edema (CME) is a well-known symptom frequently observed in RP patients [108, 109]. Involvement of WNT signaling is suggested in CME [110, 111], and LRP5 is one of the important WNT signaling molecules. If edema is observed in the outer retina rather than the inner retina, especially at early stages in the progression, it may suggest that both EYS and LRP5 play some roles in the retina.

There are a non-negligible number of studies claiming sector RP as one of possible phenotypes in EYS-RP patients [112, 113]. Sector RP is common in some forms of rhodopsin RP where inferior region is typically more severely affected than other regions. This could be due to higher exposure to light that can activate larger number of rhodopsin molecules [114]. In EYS patients, superotemporal region could be more likely to be affected [112, 115], or inferior retina may be more affected than superior retina [96]. Careful follow-up studies would be needed in the possible effect of regional differences on photoreceptor degeneration. In any case, it would be noteworthy to mention that vitamin A supplementation has different effect on RHO-RP patients and EYS-RP patients [86].

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10. Conclusions

In this review, we summarized utilization of zebrafish in vision science and IRDs focusing on our recent findings in the digenic eys+/−; lrp5+/− zebrafish. Our group shows that Lrp5 is a potent candidate for the receptor of atROL in the RPE, and have discussed for the first time the possible involvement of Eys protein in the visual cycle. We think that the highlights of recent studies obtained in medaka and humans that seek to understand the functional role of EYS are all in good agreement with our proposal in 2020 [75]. In this review, we have taken the discussion a bit further with the intention of contributing to the understanding of the functional role of the EYS. Because EYS is a major cause of arRP in Europe and Asia, we hope to make a significant contribution to the cure of EYS patients through basic research that seeks to understand the function of EYS, and we hope this review will help in this regard.

Acknowledgments

We would like to thank the patients involved in the course of our study. This work was supported by JSPS KAKENHI Grant Numbers 17K16995 (to S.T.), 20K18364 (to S.T.), 15H04998 (to Y.S.), and 20H03845 (to Y.S.). The zebrafish mutant lines, eyssa31957, lrp5sa11097, and prom1bsa13905 were obtained from the ZIRC.

Conflict of interest

The authors declare there is no conflict of interest or no competing interest in this study.

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Written By

Shimpei Takita and Yuko Seko

Submitted: 05 February 2024 Reviewed: 09 February 2024 Published: 27 March 2024

© The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.