|Molecular Vision 2005;
Received 28 July 2005 | Accepted 31 August 2005 | Published 13 December 2005
Expressed sequence tag analysis of zebrafish eye tissues for NEIBank
Thomas S. Vihtelic,1
James M. Fadool,2 James Gao,3
Kimberley A. Thornton,2 David R.
Hyde,1 Graeme Wistow3
1Center for Zebrafish Research, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN; 2Department of Biological Science, Florida State University, Tallahassee, FL; 3National Eye Institute, National Institutes of Health, Bethesda, MD
Correspondence to: Thomas S. Vihtelic, Center for Zebrafish Research, Galvin Life Sciences Building, University of Notre Dame, Notre Dame, IN, 46556; Phone: (574) 631-2895; FAX: (574) 631-7413; email: firstname.lastname@example.org
Purpose: To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank.
Methods: mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector. Approximately 4,000 clones from each library were sequenced and analyzed using various bioinformatics programs.
Results: The analysis yielded approximately 2,500 different gene clusters for each library. Combining data from the five libraries produced 10,392 unique gene clusters. GenBank accession numbers were identified for 37.6% (3,906) of the total gene clusters in the combined libraries and approximately 50% were linked to Unigene clusters in the current database. Several new crystallin genes, including two γN-crystallins, and a second major intrinsic protein (MIP) were identified in the lens library. In addition, a zebrafish homolog of cochlin (COCH), a gene that may play a role in the pathogenesis of human glaucoma, was identified in the anterior segment library. Surprisingly, no clear ortholog of the major retinal transcription factor Nrl was identified.
Conclusions: The zebrafish eye tissue cDNA libraries are a useful resource for comparative gene expression analysis. These libraries will complement the cDNA libraries made for the Zebrafish Gene Collection (ZGC) and provide an additional source for gene identification and characterization in the vertebrate eye.
The molecular genetic analysis of eye development and function has benefited from a variety of animal models including different mammalian, amphibian, and fish species. Zebrafish exhibit enormous potential as a model organism for the identification and functional characterization of genes that act during eye development and in the adult. A variety of chemical and insertional mutagenesis screens in zebrafish identified numerous genes that are essential for normal eye development and retinal cell survival [1-9]. Complementing this powerful forward genetic approach are an array of reverse genetic techniques that allow for the analysis of gene function in zebrafish by antisense gene knockdown, transgenesis, and high throughput target-selected gene inactivation [10-17].
Zebrafish eye structure and organization are similar to that in other vertebrate species, including human. The laminated retina is organized into three nuclear layers and is composed of six neuronal cell types and the Müller glial cells . The outermost retinal layer consists of the rod photoreceptors and four different cone cell types: the principal and accessory members of the double cones, long single cones, and the short single cones [19,20]. The photoreceptor outer segments interdigitate with processes of the retinal pigmented epithelial cells, which play a critical role for photoreceptor function and health [21,22]. The relatively large spherical zebrafish lens protrudes slightly into the anterior chamber and lies near the edge of the iris, which is composed of both pigmented and nonpigmented epithelium . The ciliary region of the zebrafish eye is located within the posterior chamber between the base of the iris and the retinal margin . In addition to the structural organization of the fish eye, many of the molecular components regulating unique physiological functions within the different eye tissues are conserved between zebrafish and humans [24-26].
Unlike humans, zebrafish retinas undergo continual neurogenesis . Adult stem cells maintained at the retinal margin within the circumferential germinal zone, continually differentiate into the different retinal cell types [28-31]. New rods also arise more centrally from a spatially distinct self-renewing population of progenitor cells maintained within the inner nuclear layer [32-35]. This persistent neurogenesis is necessary to maintain visual sensitivity during continual retinal growth and accounts for the ability of zebrafish to regenerate damaged retinal neurons [36-39]. Taken together, zebrafish provide a rich genomic resource for the identification and characterization of genes pertaining to eye development, function and human disease.
The NEIBank project was established to identify and display for public access expressed sequence tag (EST) data for eye tissues from human and various animal models [40-51]. We report the construction and sequence analysis of five different zebrafish cDNA libraries for NEIBank. These non-normalized libraries were made from adult eye tissues using lenses, retinas, tissue combinations derived from the anterior or posterior eye segments, or whole eyes. In total, 10,392 nonredundant gene clusters were identified among the five combined libraries. These collections of expressed sequence tag (EST)s derived from the zebrafish eye will further contribute to our understanding of tissue-specific gene expression and function, allow for comparative analyses of gene expression patterns and provide information for candidate gene analysis of zebrafish eye mutant phenotypes.
Animals, tissue dissections, and library designations
Eyes from adult (1-year-old) AB wild-type zebrafish raised in the University of Notre Dame and Florida State University fish facilities were used to make five cDNA libraries. A total of 250 eyes were utilized for the retina, anterior segment, and posterior segment libraries. A total of 500 lenses were used for the lens library and 230 eyes were used to make the whole-eye library. The appropriate institutional animal care and use committees approved all animal use protocols.
Prior to manipulation, the light-adapted fish were anesthetized in 0.1% 2-phenoxyethanol (Sigma, St. Louis, MO). The eyes were first divided into anterior and posterior portions by cutting just proximal to the iris along a line circling the globe parallel to the equator. The anterior segment, including the intact cornea, iris, and ciliary region, was lifted away from the posterior eyecup and separated from the lens. Similarly, retinas were dissected away from the posterior segments leaving RPE, choroid, sclera, portions of attached extraocular muscles, and the optic nerve. Thus, eye tissues were collected into five different sets that included whole eyes, lenses, anterior segments (minus lens), retinas, and posterior segments lacking retinas (Figure 1). The resulting libraries were designated whole eye (naa), lens (nab), anterior segment (nap), retina (naq), and posterior segment (nao), respectively.
Total RNA was extracted from the various eye tissues with RNAzol (Tel-Test Inc., Friendswood, TX) and poly(A)+ RNA was prepared by oligo(dT) cellulose affinity chromatography as previously described . The cDNA synthesis and directional cloning into the Sal I-Not I sites of the pCMVSport6 vector (Invitrogen, Carlsbad, CA) were also previously described .
Library sequencing and data analysis
Clones from each of the libraries were randomly selected for sequencing at the NIH Intramural Sequencing Center [45,48]. Sequence data from the libraries was processed to remove vector and other irrelevant sequences using PHRED, RepeatMasker, and CrossMatch . The insert sequences were identified by BLAST matches to the databases and grouped using GRIST . Each group of cDNA clones that were assigned to the same gene by the GRIST software application are referred to as a gene cluster. Thus, each cluster of transcript sequences potentially corresponds to one gene. Gene ontology analysis of the clones in each library was performed using GeneSifter. The ontology analyses were restricted to those clones within the different libraries with identified GenBank accession numbers.
Phylogenetic analysis of vertebrate arrestins
Vertebrate arrestin sequences were downloaded from GenBank, nucleotide sequences were translated using Sequencher; protein sequences were aligned using ClustalW . A phylogenetic tree was constructed using PIR Multiple Alignment and the Limulus visual arrestin as an outgroup. Sequences from the following were used in the analysis: human S-antigen (CAA30984), mouse S-antigen (AAA40090), rat S-antigen (CAA36076), bovine S-antigen (AAA30378), tiger salamander rod arrestin (AAF14636), leopard frog rod arrestin (AAC59750), bullfrog rod arrestin (AAC59748), medaka KfhArr-R1 (BAA21718), tiger salamander cone arrestin (AAF14637), leopard frog cone arrestin (AAC59751), bullfrog cone arrestin (AAC59749), African clawed frog cone arrestin (AAC42225), human X arrestin (AAB84302), medaka cone arrestin (BAA21719), medaka kfharrR2 (BAA82259), mouse cone arrestin 3 (AAG38954), mouse β arrestin 2 (AAH16642), human β arrestin 2 (NP_945355), human arrestin-C (AAC78395), African clawed frog S-arrestin (P51477), horseshoe crab lateral eye arrestin (P51484), pufferfish Rod2 (CR731788), pufferfish Rod1 (CAG02124), and pufferfish cone (CR687407).
Phylogenetic analysis of lens membrane proteins
Sequences for zebrafish MIP and Mp19/Lim2-related proteins were assembled from multiple overlapping sequence reads from the EST analyses. The GenBank accession numbers for the zebrafish MIP1 and MIP2 sequences are NM_001003534 and DQ003080, respectively, while the accession numbers for the zebrafish Mp19 sequences are DQ118395, DQ118396, DQ118397, DQ118398, DQ118399, and DQ118400. Human AQP0/MIP-AQP12, EMP2, and PMP22 and zebrafish EMP2 and PMP22 protein sequences were retrieved from the Entrez interface to GenBank (NCBI, NLM). Cladistic analysis used the program MEGA3 . For this procedure, gaps and missing data were handled by pair-wise deletion. Poisson correction was applied and the neighbor-joining option was used to generate trees of related sequences. Phylogeny was tested by 950 bootstrap replications.
Whole-mount tissue in situ RNA hybridization
The tissue in situ hybridizations were performed as described . Gene-specific primers were used to amplify templates for probe generation by reverse transcription PCR. PCR products were sequenced to insure their amplification accuracy and riboprobes were transcribed in vitro using the digoxygenin RNA-labeling kit (Roche, Indianapolis, IN).
Results & Discussion
Summary of the libraries
Approximately 4,000 clones from each library were randomly picked and sequenced. Each library consisted of approximately 1 million primary clones, therefore, less than 1% of the total clones were sequenced in each library. After bioinformatic analysis, each of the libraries yielded an average of 2,500 different gene clusters with about 18% of the clusters containing at least two clones (Table 1). All of the EST sequences were submitted to GenBank. Further analysis of the combined 12,417 gene clusters identified in all the libraries revealed that they corresponded to 10,392 nonredundant gene clusters (Table 1). GenBank accession numbers were identified for 3,906 of the 10,392 gene clusters (37.6%) in the combined eye tissue libraries, although approximately 50% had matching UniGene clusters. The clear identification of orthologs between zebrafish and humans using this type of analysis is difficult because the zebrafish genome sequence is not complete and annotation is not yet at the level of the human genome.
Those genes identified by GenBank accession numbers were analyzed and categorized by function (Table 2). As expected, the different genes fell into a wide variety of functional categories within each of the different ontology reports, which included gene categorizations by molecular function, cellular component or biological process. While this analysis attached putative functional roles to most genes, relatively large percentages of the gene clusters in each report (13-38%) could not be assigned to a functional category. This demonstrates the wide variety of potential gene functions associated with this large collection of eye tissue transcripts, although the ontology categories are only general guides.
The distribution of genes among the 25 zebrafish chromosomes was also determined (Table 3). Over 2,300 of the 10,392 gene clusters corresponded to definitive chromosome assignments based on alignments to the June 2004 build of the zebrafish genome using BLAT . The genes were relatively evenly distributed between the different chromosomes. Chromosome 24 was assigned the fewest genes (43), while chromosome 7 had the most assignments with 147 genes assigned to this linkage group.
Individual libraries, whole eye
Over 2,000 gene clusters comprised the whole eye library (naa) with nearly 14% of the clusters composed of two or more clones and 48.6% of the clusters corresponding to sequences with GenBank accession numbers (Table 1). As expected, this library contained abundant retinal transcripts corresponding to phototransduction genes including rhodopsin, various cGMP-specific phosphodiesterase (PDE) subunits, and both rod and cone arrestins, which are discussed below. Also, several of the most abundant lens crystallin transcripts such as γSa- and βB2-crystallin were identified (Table 4). In addition, a variety of transcriptional regulators were identified in the whole eye library. These included the paired box transcription factors pax6a and pax6b, two sine oculis homologs (six3a and six7), and neuroD. Interestingly, a possible zebrafish ortholog of human SOX22, a gene primarily expressed in developing nervous tissues including the retina, was also identified in the zebrafish whole eye library . The presence of sox22 in the whole eye library may be due to the persistent neurogenesis in the adult zebrafish retina.
Many of the abundant transcripts in the whole eye library were also identified as highly expressed genes within the different individual libraries. For example, several different claudin gene transcripts were abundant in some of the libraries. Claudins b and i were identified in the whole eye library, while these plus claudins a, e, 7, 12, and 19 were expressed in the anterior segment library. No classical claudins were identified in the lens cDNA library, but as described below, this tissue expresses several related tetraspanin proteins of the Mp19/Lim2 family. Extensive claudin gene duplications have occurred in the teleost fish lineage resulting in 56 claudin genes with 21 being specific to fish . Claudin proteins are structural components of epithelial and endothelial cell tight junctions and likely determine permeability properties by affecting intercellular complex composition [59,60]. The complexity of possible hetero-oligomerizations between the different claudin proteins combined with their potential cell- and tissue-type-specific expression patterns suggest the various claudins can function as highly selective channels. Mutations in some of the corresponding claudin genes in humans and animals are associated with diseases, such as deafness [61-63].
Transcripts corresponding to five different arrestin gene clusters were also identified in the whole eye library. Arrestins are highly conserved molecules that interact with G-protein-coupled receptors [64,65]. In the retina, arrestin binding to light-activated phosphorylated rhodopsin leads to the rapid inhibition of signaling by preventing further rhodopsin-transducin interactions. Each of the zebrafish arrestin clusters shared close to 100% identity with full-length clones in the public database. Phyletic reconstruction using Limulus arrestin as an outgroup identified two of the zebrafish sequences in the rod arrestin family (DrArr-R1 and DrArr-R2), two sequences in the cone arrestin family (DrArr3a and DrArr3b), and the fifth sequence as a β arrestin2 ortholog [66,67]. Pairwise comparisons between the two rod arrestins, or the two cone arrestins, revealed 64% identity within these two groups and 45-48% identity across the two groups.
The arrestin sequence relationships among teleost species were explored by query of the nucleotide database using the predicted zebrafish arrestin amino acid sequences. Two putative rod arrestins and a single cone arrestin cDNA were identified from a pufferfish retinal library, while the previously identified medaka cDNAs encoding two rod arrestins (KfhArr-R1 and KfhArr-R2) and one cone arrestin (KfhArr-C) were also identified [68,69]. The phylogenetic tree constructed with these and other vertebrate arrestin sequences revealed that the teleost sequences formed a separate clade within the vertebrate rod arrestin family that was composed of two branches with pufferfish, medaka and zebrafish Arr-R1 on one branch and the Arr-R2 sequences from each of these species on the other branch (data not shown). Similarly, within the vertebrate cone arrestin family, zebrafish Arr3a was most related to the pufferfish and medaka cone arrestins, while the zebrafish Arr3b sequence fell to a more basal branch of the tree (not shown). These phyletic relationships of the teleost arrestin genes are consistent with the ancient genome duplication in the ray-finned fish lineage and retention of the arrestin sequences in zebrafish .
The spatial and temporal distributions of these newly identified zebrafish arrestins were determined by whole-mount in situ RNA hybridizations of zebrafish embryos and larvae (Figure 2). All four of the arrestins were detected in the outer nuclear layer of the developing retina and in the photoreceptors of the pineal (data not shown). In the retina, the two zebrafish cone arrestins displayed overlapping expression patterns that were consistent with the pattern of cone cell differentiation [71,72]. Their expression was first evident in a small patch of cells in the ventral nasal retina (ventral patch, vp) at 2 days post-fertilization (dpf, Figure 2A). The expression of the cone arrestin genes spread to the temporal and dorsal retina at 4 dpf (Figure 2B). In comparison, rod arrestin expression also initiated in the ventral retina at 2 dpf, but was next detected in the dorsal retina at 4 dpf (Figure 2C), which is consistent with the spatial and temporal expression patterns previously reported for rhodopsin [71,72]. In contrast to the expression patterns of the rod and cone photoreceptor arrestins, β arrestin2 expression was not restricted to the retina of either zebrafish embryos or larvae.
Several of the opsin genes were also identified in the whole eye library. Although transcripts corresponding to only one of the four green opsins (MWS-2) were found in the whole-eye library, green opsin transcripts corresponding to the MWS-2 and MWS-3 genes were detected in the retinal library. In contrast, both of the red cone opsin genes (LWS-1 and LWS-2) were identified in the whole-eye library, while only LWS-1 clones were found in the retinal library. Thus, analysis of the whole-eye library will likely reveal additional cDNAs that may not be included in the selected tissue libraries, whose descriptions follow.
The lens library (nab) yielded 1,163 gene clusters from a total of 3,372 quality sequence reads with 190 (16.3%) of the clusters possessing two or more sequences. GenBank accession numbers were identified for 558 of the genes in the lens library (47.9%).
As expected, crystallin genes comprised the majority of the most abundant transcripts identified in the lens library (Table 5, Table 6). The ten most abundant transcripts corresponded to crystallin genes with 23 different crystallins among the 40 most abundant lens transcripts. Included among the zebrafish lens crystallin cDNAs identified in this library were 16 different γ-crystallins including two γN genes (zfγN1 and zfγN2) and an unusual member of the γM family, zfγMX (GenBank accession number AY738755), whose sequence has diverged significantly from the other members of the aquatic γM gene family . In addition, orthologs of the familiar β-crystallins, a divergent β-like sequence designated zfβγX (GenBank accession number DQ136043), and a second αB-crystallin (zfαB2) were also identified [73,74]. Overall, the zebrafish lens expresses a greater number of crystallin genes, particularly the γ-crystallins, relative to the lenses of terrestrial vertebrates . This large variety of γ-crystallin proteins may be required to adjust steep gradients of refractive index in the hard, highly refractive fish lens. While the air-cornea interface is responsible for most of the refraction in the eyes of terrestrial organisms, the water-cornea interface allows little bending of incident light and almost all focusing is performed by the lens [75-78]. Alternatively, the greater diversity of crystallins may be involved in preventing crystallization of the highly concentrated proteins of the fish lens.
Cystatin B, a member of the cysteine protease inhibitor superfamily, was the most highly expressed noncrystallin gene in this library (Table 5, Table 6). Although it is not yet known if the cDNA abundance is reflected at the protein level, this raises the possibility that this gene was recruited to an additional role as a taxon-specific crystallin like several enzymes in other vertebrate species .
Genes encoding several abundant structural proteins were also identified in the zebrafish lens library. The major lens membrane protein in vertebrates is the lens fiber major intrinsic protein (MIP or Aquaporin 0). cDNAs for two closely related MIP genes, one of which is novel and was named MIP2 (GenBank accession number DQ003080), were identified in the lens library. The two MIP sequences (MIP1 and MIP2), encode proteins that share 84% amino acid identity and are apparently the result of recent gene duplication (Figure 3A). Many genes in zebrafish and other teleosts are represented in duplicate because of a genome duplication that occurred in the ray-finned fish lineage , which may allow additional specialization of daughter genes.
More strikingly, the adult lens EST collection also contained clones for five different zebrafish transcripts with sequence similarity to LIM2/Mp19, the second most abundant protein in mammalian lenses (Figure 3B) [81-83]. Mammalian Mp19 (also known as Mp20) is involved in gap junction formation and the insertion of Mp19 into the lens fiber cell membrane during the formation of an inter-fiber diffusion barrier indicates it might function in cell adhesion [84-86]. While the precise role of Mp19 in the lens has not been determined, a mutation in the gene causes dominant cataracts in mouse and microphthalmia when the mutation is homozygous [87,88]. Mp19 belongs to a family of claudins that includes epithelial membrane protein 2 (EMP2) and peripheral myelin protein 22 (PMP22). Phylogenetic analysis (Figure 3B) shows the five zebrafish Mp19-like sequences all group on the Mp19 branch of the family, but show evidence of considerable sequence divergence. This may reflect specialization for roles in specific regions or developmental stages associated with zebrafish lens maturation.
Other major lens components such as the CP49 beaded filament protein (phakinin) and vimentin are also represented in this library (Table 6), although no clones for an ortholog of filensin are present. In addition, the zebrafish ortholog of the lens-specific lengsin  and cDNAs with similarity to the lens epithelium gene (LEP503) were identified within the most abundant lens transcripts. The LEP503 gene is expressed exclusively in the lens epithelial cells of mammals and may function in regulating the differentiation of the lens epithelial cells into fiber cells . The human LEP503 gene mapped to the same chromosomal region as a locus responsible for causing zonular pulverulent cataract .
Lens growth is dependent on the regulated proliferation and migration of the epithelial cells, and their differentiation into new fiber cells . Midkine-related growth factor, a heparin binding cytokine with roles in growth and migration in a variety of tissues, was highly expressed in the adult zebrafish lens along with the extracellular matrix-associated protein SPARCL1 [92,93]. While null mutants in the mouse SPARC gene displayed vacuolization of lens fiber cells and cataract formation, the related SPARCL1 (Hevin/SC1) protein may act to antagonize cell adhesion through mediation of cell matrix interactions [94,95]. pitx3, TGFβ-3, and a phosphatase and tensin homolog (pten) were also among the genes expressed in the adult zebrafish lens with putative roles in the control of cell differentiation and growth [96-100].
The anterior segment library (nap) was made from tissues that included the cornea, iris, and ciliary region, but not the lens. In total, 3,011 gene clusters were identified in this library (Table 7). Approximately 15% of the anterior segment library gene clusters contained two or more clones and GenBank accession numbers identified 1,508 (50.1%) of the total clusters.
Gelsolin was expressed at very high levels within this tissue group and likely reflects its extreme corneal-enriched expression pattern [101,102]. The corneal gelsolin may interact with actin to maintain corneal cell transparency . Both type I and type II cytokeratins and the genes encoding several different claudin proteins were also highly expressed within the tissues of this library.
The dissection method employed to collect these anterior segments resulted in the inclusion of the retinal margins. This was confirmed by the identification of photoreceptor-specific transcripts in the anterior segment library such as rhodopsin and some of the cone opsins. The circumferential germinal zone of the retinal margin in teleost fish is characterized by a population of post-embryonic stem cells, which undergo continual renewal and differentiate into the different retinal cell types [28,31]. Consequently, this region of the eye is characterized by the hierarchical expression of transcription factors that are responsible for the early specification and later differentiation of multiple retinal cell lineages . As expected, the anterior segment library contained cDNAs for several transcription factors that may play roles in neural cell genesis, including Pax6a and Pax6b, Six1 and Six3a, Sox2, and Sox4, the Distal-less homeobox gene 3 and NeuroD . Also present were transcripts corresponding to the Iroquois homeobox protein 5, POU domain class 4 and the Forkhead box C1 and D2 proteins. Other transcripts whose proteins may play roles in regulating the growth and proliferation of these adult retinal progenitor cells included two midkine-related growth factors (mdkA and mdkB) and the insulin-like growth factor binding proteins 2 and 5 (IGFBP2 and IGFBP5) [93,105].
Several zebrafish orthologs of genes associated with human eye anterior segment disease were also identified in the anterior segment cDNA library, including pitx3 and foxC1. In the eye, pitx3 expression is largely restricted to the lens, although transcripts were also detected in the zebrafish iris during development . Human congenital eye anomalies characterized by anterior segment dysgenesis are associated with mutations in PITX3, while mutations in FOXC1 cause a spectrum of anterior segment malformations that often result in glaucoma [99,100,107-109]. We also identified a coagulation factor C homolog corresponding to a full-length clone in the database whose putative encoded protein possesses 51% amino acid identity with human Cochlin (COCH), which is associated with autosomal dominant sensorineural deafness [110,111]. Clones corresponding to this zebrafish cochlin homolog also were identified in the whole eye and posterior segment libraries. Recent proteomic analysis of the trabecular meshwork (TM) from glaucoma patients and control donors identified high levels of Cochlin protein within the glaucomatous TM . Although COCH transcripts were identified by RT-PCR in both the normal and diseased TM, no protein was detectable by immunoblot of the normal tissue .
The zebrafish retina library (naq) yielded 2,835 unique gene clusters (Table 1). Nearly 23% of these clusters had corresponding GenBank accession numbers, which is less than half the percentage observed in the other libraries. In addition, fewer multiple clone gene clusters characterized the retinal library (7.9%) compared to the other libraries (14-16%). These characteristics of the retinal library may be due to specific features of the tissue, such as the complexity of the neuronal population, or the method of library construction.
The most abundant group of transcripts in this retina data set corresponds to a sequence that was initially identified with claudin g, a relationship still reflected in the current UniGene build. However, this identification was due to the presence of a chimeric clone in GenBank and these clones are not derived from the claudin g gene. These abundant cDNA clones are associated with GenBank entry BC044441, which appears to encode a highly conserved homolog of LSm3, a small protein associated with U6 small nuclear RNA; however, gaps in the current build of the zebrafish genome sequence make it difficult to positively identify this gene. Why this gene is so abundant in this retina library (and the other zebrafish eye libraries) is unknown.
Photoreceptor-enriched transcripts were among the most abundant in the retina library. These clones included the rod and cone α transducin (G protein) subunits, as well as the rod and cone PDE α and γ subunits, recoverin, arrestin 3 (X-arrestin), and phosducin (Table 8). While many, but not all, of the different opsin gene transcripts were identified in the retinal library, rhodopsin was not the most abundant transcript in this library. This may reflect the greater diversity of opsin family transcripts in the teleost retina, as compared with the rhodopsin dominated mammalian retina.
Zebrafish possess rods and four cone cell types and express nine different opsin genes . Rhodopsin is expressed in the rod photoreceptors, while each of the two members of the double cone cell pair expresses either green or red opsin. Also, the long single cones express blue opsin and the short single cones express an ultraviolet-absorbing opsin protein [19,20,71]. In addition to the intron-less rhodopsin gene , four different green (MWS-1, MWS-2, MWS-3, and MWS-4), two different red (LWS-1 and LWS-2), and single blue (SWS-2) and ultraviolet (SWS-1) opsin genes were previously identified . The cone opsin genes are expressed in overlapping temporal and spatial patterns during development and in the adult retina .
The retinal library contained the following opsin gene clones: rhodopsin, MWS-2 and MWS-3, LWS-1, SWS-1, and SWS-2. Opsin clones were also identified in the whole eye, anterior segment and RPE/choroid libraries. Clones for MWS-4 were found in the anterior segment library and a single short clone for MWS-1 was obtained from the posterior segment collection. In both cases, this distribution may reflect aspects of the regional expression of the different cone visual pigments. In the adult, MWS-1 and MWS-2 are expressed in the central and dorsal areas, while MWS-3 and MWS-4 are both expressed in the ventral and marginal retinal regions . MWS-1 and MWS-4 were the only green opsin subtypes we identified previously by hybridization screening of an adult whole eye cDNA library . Quantitative RT-PCR revealed significant expression level differences between the four green opsin genes. Thus, MWS-2 is expressed at significantly higher levels than MWS-1, MWS-3, and MWS-4 .
A zebrafish nrl ortholog was not identified in this or the other zebrafish eye libraries. In the human and mouse retina cDNA libraries, NRL was the most abundant transcription factor [41,50]. NRL is essential for normal rod cell development in these species and regulates rhodopsin transcription through interactions with CRX [115,116]. Although a zebrafish crx ortholog was identified, attempts to identify an nrl ortholog in zebrafish have failed  (personal communication, Pamela Raymond). Recently, a predicted gene for zebrafish nrl was identified in silico . Expression analysis by in situ hybridization demonstrated this gene is expressed at high levels in the developing zebrafish lens, while much lower expression was detected in the adult photoreceptors , although no ESTs for this gene were identified in the adult zebrafish lens or retina collection. Indeed, there are very few ESTs corresponding to this sequence in dbEST from any tissue or developmental stage and none are present in the large Washington University (St. Louis, MO) collection of zebrafish retina ESTs (NEIBank). Interestingly, examination of the chicken genome failed to identify a convincing ortholog of Nrl . This suggests that the prominent role for NRL in mammals is not conserved in other vertebrates and that mammalian NRL perhaps arose to provide specialized functions for a rod-dominated retina.
Over 3,000 gene clusters were identified in the posterior segment library (nao), with 46.6% of the clusters corresponding to sequences with GenBank accession numbers (Table 1). Approximately 16% (492) of these gene clusters were composed of two or more sequenced clones. As expected, hemoglobin complex genes were highly expressed within the zebrafish choroidal tissue (Table 9). Thus, ba1 globin (52% amino acid identity with the embryonic globin expressed during mouse development) and the adult α-1 globin (hbaa1) were among the most abundant transcripts identified in this library [119,120].
Although retinas were removed from the posterior segments prior to library construction, adherent photoreceptor cells were still present, as high levels of rhodopsin and the rod PDE subunit transcripts were detected in the posterior segment library (Table 9). Retinas dissected from light-adapted eyes retain some cells of the RPE and portions of the photoreceptors remain with the RPE tissue. This intimate association of the photoreceptor cells and the retinal pigmented epithelium is critical for the operation of the phototransduction cascade and maintenance of photoreceptor health . Recovery of the photosensitive rhodopsin conformation requires the formation of 11-cis-retinal from all-trans-retinal via the retinoid cycle . While the first step in this pathway, reduction of the all-trans-retinal to all-trans-retinol, occurs in the photoreceptor outer segments, the remaining biochemical reactions in this process occur in the RPE . Thus, a number of transcripts encoding proteins that are critical in this retinoid cycle were identified in the zebrafish posterior segment library. These included the interphotoreceptor retinoid-binding protein (IRBP), retinaldehyde binding protein 1 (CRALBP), cellular retinol binding protein 1 (CRBP), and retinol dehydrogenase 2 (photoreceptor-associated type II). In addition, a gene corresponding to plasma retinol binding protein (rbp4l) was also highly expressed in this library.
Many of the same genes that are abundantly expressed in the zebrafish posterior segment tissues are also highly expressed in the human RPE/choroid tissues . These genes include glyceraldehyde-3-phosphate dehydrogenase, ferritin heavy chain polypeptide, glutamine synthase (both a and b isoforms in zebrafish), and the regulator of G protein signaling 5 (RGS5). In addition, abundant transcripts corresponding to the heat shock 70 kDa protein 8 and heat shock protein 90, two constitutively expressed members of the heat shock protein family, were also detected in both the zebrafish posterior segment library and the human RPE/choroid library. The zebrafish posterior segment library also contained heat shock protein 70, heat shock protein 4 and heat shock factor 2 transcripts. Insulin-like growth factor binding protein (IGFBP) genes were also identified in both libraries. IGFBP 5 and IGFBP 7 were expressed in the human tissues, while the zebrafish posterior segment library contained cDNAs corresponding to IGFBP 1, 3, and 5. In comparison, IGFBP 2 and IGFBP 5 were identified in the zebrafish anterior segment library.
Bestrophin, an abundantly expressed gene of the adult human RPE/choroid , was not present in the zebrafish posterior segment library. Human bestrophin functions as a chloride channel and is responsible for a form of early onset macular degeneration [122-124]. Bestrophin transcripts also were not detected in the mouse RPE/choroid library, although a subsequent expression analysis and immunolocalization study identified bestrophin in the basolateral plasma membranes of early postnatal mouse RPE . This suggests a specialized role for the bestrophin protein in the RPE of mammals that may not be shared by all vertebrate species.
We described the construction and sequence analysis of five different cDNA libraries that were made from adult zebrafish whole eyes or various dissected eye tissues. The tissues included lenses, anterior segments (minus lens), retinas, and posterior segments (minus retina). In total, over 10,000 nonredundant gene clusters were identified among the different libraries. The numbers of clones in each of the different gene clusters provides a preliminary representation of relative transcript abundance within the various tissues . Unlike traditional cDNA libraries, the NEIBank libraries were neither amplified nor normalized prior to their sequence analysis. Thus, relative mRNA expression level data are also obtainable from these sequence collections.
Our analysis of the zebrafish eye libraries suggests they will be useful to identify new genes and zebrafish orthologs of human disease genes. For example, five different isoforms of Mp19 were identified in the adult lens, while mammals express only a single Mp19 gene [126,127]. In addition, two γN crystallin genes and a second αB crystallin were identified within the lens library . Similarly, the anterior segment library yielded a zebrafish ortholog of cochlin. While cochlin gene mutations were shown to cause human sensorineural deafness, recent proteomic analysis identified increased levels of the Cochlin protein within the trabecular meshwork of glaucoma patients [110,112]. Thus, the zebrafish eye EST analysis corresponded with the proteomic analysis of human eye tissues. The further characterization of these eye libraries may lead to the identification of additional disease markers. The ability to utilize a variety of reverse genetic approaches in zebrafish should facilitate the characterization of the tissue-specific functions of these molecules and suggest their potential roles in other vertebrates, such as humans.
These collections of adult zebrafish eye ESTs that were assembled for NEIBank will complement the zebrafish cDNA libraries, which were made for the Zebrafish Gene Collection (ZGC) project, such as the adult retina library and the library made from the eyes of 72 h post-fertilization larvae. Furthermore, these NEIBank cDNAs will provide a resource for gene expression analysis and will aid in the assembly and interpretation of microarrays and the selection of candidate genes during mutation analyses.
We thank the Freimann Life Sciences Center at the University of Notre Dame for zebrafish husbandry and care. Dr. Ryan Thummel provided graphics expertise for figure assembly. Jill Scarborough (UND) helped with some of the tissue dissections. KAT was supported by an HHMI fellowship in Computational Biology to FSU; TSV by NIH grant R01 EY014455.
1. Perkins BD, Nicholas CS, Baye LM, Link BA, Dowling JE. dazed gene is necessary for late cell type development and retinal cell maintenance in the zebrafish retina. Dev Dyn 2005; 233:680-94.
2. Gross JM, Perkins BD, Amsterdam A, Egana A, Darland T, Matsui JI, Sciascia S, Hopkins N, Dowling JE. Identification of zebrafish insertional mutants with defects in visual system development and function. Genetics 2005; 170:245-61.
3. Amsterdam A, Hopkins N. Retroviral-mediated insertional mutagenesis in zebrafish. Methods Cell Biol 2004; 77:3-20.
4. Brockerhoff SE, Hurley JB, Janssen-Bienhold U, Neuhauss SC, Driever W, Dowling JE. A behavioral screen for isolating zebrafish mutants with visual system defects. Proc Natl Acad Sci U S A 1995; 92:10545-9.
5. Fadool JM, Brockerhoff SE, Hyatt GA, Dowling JE. Mutations affecting eye morphology in the developing zebrafish (Danio rerio). Dev Genet 1997; 20:288-95.
6. Li L. Zebrafish mutants: behavioral genetic studies of visual system defects. Dev Dyn 2001; 221:365-72.
7. Maaswinkel H, Mason B, Li L. ENU-induced late-onset night blindness associated with rod photoreceptor cell degeneration in zebrafish. Mech Ageing Dev 2003; 124:1065-71.
8. Malicki J, Neuhauss SC, Schier AF, Solnica-Krezel L, Stemple DL, Stainier DY, Abdelilah S, Zwartkruis F, Rangini Z, Driever W. Mutations affecting development of the zebrafish retina. Development 1996; 123:263-73.
9. Vihtelic TS, Hyde DR. Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects. Vision Res 2002; 42:535-40.
10. Draper BW, McCallum CM, Stout JL, Slade AJ, Moens CB. A high-throughput method for identifying N-ethyl-N-nitrosourea (ENU)-induced point mutations in zebrafish. Methods Cell Biol 2004; 77:91-112.
11. Fadool JM. Development of a rod photoreceptor mosaic revealed in transgenic zebrafish. Dev Biol 2003; 258:277-90.
12. Kennedy BN, Vihtelic TS, Checkley L, Vaughan KT, Hyde DR. Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors. J Biol Chem 2001; 276:14037-43.
13. Linney E, Udvadia AJ. Construction and detection of fluorescent, germline transgenic zebrafish. Methods Mol Biol 2004; 254:271-88.
14. Nasevicius A, Ekker SC. Effective targeted gene 'knockdown' in zebrafish. Nat Genet 2000; 26:216-20.
15. Perkins BD, Kainz PM, O'Malley DM, Dowling JE. Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors. Vis Neurosci 2002; 19:257-64.
16. Urtishak KA, Choob M, Tian X, Sternheim N, Talbot WS, Wickstrom E, Farber SA. Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics. Dev Dyn 2003; 228:405-13.
17. Wienholds E, van Eeden F, Kosters M, Mudde J, Plasterk RH, Cuppen E. Efficient target-selected mutagenesis in zebrafish. Genome Res 2003; 13:2700-7.
18. Dowling JE. The Retina: an approachable part of the brain. Cambridge (MA): Belknap Press of Harvard University Press; 1987.
19. Raymond PA, Barthel LK, Rounsifer ME, Sullivan SA, Knight JK. Expression of rod and cone visual pigments in goldfish and zebrafish: a rhodopsin-like gene is expressed in cones. Neuron 1993; 10:1161-74.
20. Vihtelic TS, Doro CJ, Hyde DR. Cloning and characterization of six zebrafish photoreceptor opsin cDNAs and immunolocalization of their corresponding proteins. Vis Neurosci 1999; 16:571-85.
21. Bok D. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci Suppl 1993; 17:189-95.
22. Boulton M, Dayhaw-Barker P. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye 2001; 15:384-9.
23. Nicol JAC. The eyes of fishes. Oxford: Clarendon Press; 1989.
24. McMahon C, Semina EV, Link BA. Using zebrafish to study the complex genetics of glaucoma. Comp Biochem Physiol C Toxicol Pharmacol 2004; 138:343-50.
25. Goldsmith P, Harris WA. The zebrafish as a tool for understanding the biology of visual disorders. Semin Cell Dev Biol 2003; 14:11-8.
26. Barut BA, Zon LI. Realizing the potential of zebrafish as a model for human disease. Physiol Genomics 2000; 2:49-51.
27. Hitchcock PF, Raymond PA. The teleost retina as a model for developmental and regeneration biology. Zebrafish 2004; 1:257-71.
28. Hagedorn M, Fernald RD. Retinal growth and cell addition during embryogenesis in the teleost, Haplochromis burtoni. J Comp Neurol 1992; 321:193-208.
29. Meyer RL. Evidence from thymidine labeling for continuing growth of retina and tectum in juvenile goldfish. Exp Neurol 1978; 59:99-111.
30. Johns PR. Growth of the adult goldfish eye. III. Source of the new retinal cells. J Comp Neurol 1977; 176:343-57.
31. Johns PR, Easter SS Jr. Growth of the adult goldfish eye. II. Increase in retinal cell number. J Comp Neurol 1977; 176:331-41.
32. Otteson DC, D'Costa AR, Hitchcock PF. Putative stem cells and the lineage of rod photoreceptors in the mature retina of the goldfish. Dev Biol 2001; 232:62-76.
33. Julian D, Ennis K, Korenbrot JI. Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina. J Comp Neurol 1998; 394:271-82.
34. Johns PR, Fernald RD. Genesis of rods in teleost fish retina. Nature 1981; 293:141-2.
35. Johns PR. Formation of photoreceptors in larval and adult goldfish. J Neurosci 1982; 2:178-98.
36. Wu DM, Schneiderman T, Burgett J, Gokhale P, Barthel L, Raymond PA. Cones regenerate from retinal stem cells sequestered in the inner nuclear layer of adult goldfish retina. Invest Ophthalmol Vis Sci 2001; 42:2115-24.
37. Vihtelic TS, Hyde DR. Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina. J Neurobiol 2000; 44:289-307.
38. Yurco P, Cameron DA. Responses of Muller glia to retinal injury in adult zebrafish. Vision Res 2005; 45:991-1002.
39. Cameron DA. Cellular proliferation and neurogenesis in the injured retina of adult zebrafish. Vis Neurosci 2000; 17:789-97.
40. Ahmed F, Torrado M, Zinovieva RD, Senatorov VV, Wistow G, Tomarev SI. Gene expression profile of the rat eye iridocorneal angle: NEIBank expressed sequence tag analysis. Invest Ophthalmol Vis Sci 2004; 45:3081-90.
41. Ida H, Boylan SA, Weigel AL, Smit-McBride Z, Chao A, Gao J, Buchoff P, Wistow G, Hjelmeland LM. EST analysis of mouse retina and RPE/choroid cDNA libraries. Mol Vis 2004; 10:439-44 <http://www.molvis.org/molvis/v10/a55/>.
42. Ozyildirim AM, Wistow GJ, Gao J, Wang J, Dickinson DP, Frierson HF Jr, Laurie GW. The lacrimal gland transcriptome is an unusually rich source of rare and poorly characterized gene transcripts. Invest Ophthalmol Vis Sci 2005; 46:1572-80.
43. Rabinowitz YS, Dong L, Wistow G. Gene expression profile studies of human keratoconus cornea for NEIBank: a novel cornea-expressed gene and the absence of transcripts for aquaporin 5. Invest Ophthalmol Vis Sci 2005; 46:1239-46.
44. Tomarev SI, Wistow G, Raymond V, Dubois S, Malyukova I. Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci 2003; 44:2588-96.
45. Wistow G. A project for ocular bioinformatics: NEIBank. Mol Vis 2002; 8:161-3 <http://www.molvis.org/molvis/v8/a22/>.
46. Wistow G, Bernstein SL, Touchman JW, Bouffard G, Wyatt MK, Peterson K, Behal A, Gao J, Buchoff P, Smith D. Grouping and identification of sequence tags (GRIST): bioinformatics tools for the NEIBank database. Mol Vis 2002; 8:164-70 <http://www.molvis.org/molvis/v8/a23/>.
47. Wistow G, Bernstein SL, Ray S, Wyatt MK, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K. Expressed sequence tag analysis of adult human iris for the NEIBank Project: steroid-response factors and similarities with retinal pigment epithelium. Mol Vis 2002; 8:185-95 <http://www.molvis.org/molvis/v8/a25/>.
48. Wistow G, Bernstein SL, Wyatt MK, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K. Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants. Mol Vis 2002; 8:171-84 <http://www.molvis.org/molvis/v8/a24/>.
49. Wistow G, Bernstein SL, Wyatt MK, Fariss RN, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K. Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants. Mol Vis 2002; 8:205-20 <http://www.molvis.org/molvis/v8/a27/>.
50. Wistow G, Bernstein SL, Wyatt MK, Ray S, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K. Expressed sequence tag analysis of human retina for the NEIBank Project: retbindin, an abundant, novel retinal cDNA and alternative splicing of other retina-preferred gene transcripts. Mol Vis 2002; 8:196-204 <http://www.molvis.org/molvis/v8/a26/>.
51. Wistow G. The NEIBank project for ocular genomics: Data-mining gene expression in human and rodent eye tissues. Prog Retin Eye Res 2006; 25:43-77.
52. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998; 8:186-94.
53. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:4673-80.
54. Kumar S, Tamura K, Nei M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 2004; 5:150-63.
55. DeCarvalho AC, Cappendijk SL, Fadool JM. Developmental expression of the POU domain transcription factor Brn-3b (Pou4f2) in the lateral line and visual system of zebrafish. Dev Dyn 2004; 229:869-76.
56. Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002; 12:656-64.
57. Jay P, Sahly I, Goze C, Taviaux S, Poulat F, Couly G, Abitbol M, Berta P. SOX22 is a new member of the SOX gene family, mainly expressed in human nervous tissue. Hum Mol Genet 1997; 6:1069-77.
58. Loh YH, Christoffels A, Brenner S, Hunziker W, Venkatesh B. Extensive expansion of the claudin gene family in the teleost fish, Fugu rubripes. Genome Res 2004; 14:1248-57.
59. Heiskala M, Peterson PA, Yang Y. The roles of claudin superfamily proteins in paracellular transport. Traffic 2001; 2:93-8.
60. Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2001; 2:285-93.
61. Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol 1999; 147:891-903.
62. Gow A, Southwood CM, Li JS, Pariali M, Riordan GP, Brodie SE, Danias J, Bronstein JM, Kachar B, Lazzarini RA. CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice. Cell 1999; 99:649-59.
63. Wilcox ER, Burton QL, Naz S, Riazuddin S, Smith TN, Ploplis B, Belyantseva I, Ben-Yosef T, Liburd NA, Morell RJ, Kachar B, Wu DK, Griffith AJ, Riazuddin S, Friedman TB. Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29. Cell 2001; 104:165-72.
64. Hargrave PA, McDowell JH. Rhodopsin and phototransduction: a model system for G protein-linked receptors. FASEB J 1992; 6:2323-31.
65. Krupnick JG, Gurevich VV, Benovic JL. Mechanism of quenching of phototransduction. Binding competition between arrestin and transducin for phosphorhodopsin. J Biol Chem 1997; 272:18125-31.
66. Battelle BA, Andrews AW, Kempler KE, Edwards SC, Smith WC. Visual arrestin in Limulus is phosphorylated at multiple sites in the light and in the dark. Vis Neurosci 2000; 17:813-22.
67. Craft CM, Whitmore DH. The arrestin superfamily: cone arrestins are a fourth family. FEBS Lett 1995; 362:247-55.
68. Hisatomi O, Imanishi Y, Satoh T, Tokunaga F. Arrestins expressed in killifish photoreceptor cells. FEBS Lett 1997; 411:12-8.
69. Imanishi Y, Hisatomi O, Tokunaga F. Two types of arrestins expressed in medaka rod photoreceptors. FEBS Lett 1999; 462:31-6.
70. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C, Bernot A, Nicaud S, Jaffe D, Fisher S, Lutfalla G, Dossat C, Segurens B, Dasilva C, Salanoubat M, Levy M, Boudet N, Castellano S, Anthouard V, Jubin C, Castelli V, Katinka M, Vacherie B, Biemont C, Skalli Z, Cattolico L, Poulain J, De Berardinis V, Cruaud C, Duprat S, Brottier P, Coutanceau JP, Gouzy J, Parra G, Lardier G, Chapple C, McKernan KJ, McEwan P, Bosak S, Kellis M, Volff JN, Guigo R, Zody MC, Mesirov J, Lindblad-Toh K, Birren B, Nusbaum C, Kahn D, Robinson-Rechavi M, Laudet V, Schachter V, Quetier F, Saurin W, Scarpelli C, Wincker P, Lander ES, Weissenbach J, Roest Crollius H. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 2004; 431:946-57.
71. Raymond PA, Barthel LK, Curran GA. Developmental patterning of rod and cone photoreceptors in embryonic zebrafish. J Comp Neurol 1995; 359:537-50.
72. Schmitt EA, Dowling JE. Comparison of topographical patterns of ganglion and photoreceptor cell differentiation in the retina of the zebrafish, Danio rerio. J Comp Neurol 1996; 371:222-34.
73. Wistow G, Wyatt K, David L, Gao C, Bateman O, Bernstein S, Tomarev S, Segovia L, Slingsby C, Vihtelic T. gammaN-crystallin and the evolution of the betagamma-crystallin family in vertebrates. FEBS J. In press 2005.
74. Posner M, Kantorow M, Horwitz J. Cloning, sequencing and differential expression of alphaB-crystallin in the zebrafish, Danio rerio. Biochim Biophys Acta 1999; 1447:271-7.
75. Patel S, Marshall J, Fitzke FW 3rd. Refractive index of the human corneal epithelium and stroma. J Refract Surg 1995; 11:100-5.
76. Sivak JG, Howland HC, West J, Weerheim J. The eye of the hooded seal, Cystophora cristata, in air and water. J Comp Physiol [A] 1989; 165:771-7.
77. Land MF. Vision in air and water. In: Dejours P, Bolis L, Taylor CR, Weibel ER, editors. Comparative physiology: Life in water and on land. New York: Springer; 1987. p. 289-302.
78. Sivak JG. A survey of vertebrate strategies for vision in air and water. Ali MA, editor. Sensory ecology. New York: Plenum Press; 1978. p. 503-19.
79. Piatigorsky J, Wistow GJ. Enzyme/crystallins: gene sharing as an evolutionary strategy. Cell 1989; 57:197-9.
80. Amores A, Force A, Yan YL, Joly L, Amemiya C, Fritz A, Ho RK, Langeland J, Prince V, Wang YL, Westerfield M, Ekker M, Postlethwait JH. Zebrafish hox clusters and vertebrate genome evolution. Science 1998; 282:1711-4.
81. Gonen T, Grey AC, Jacobs MD, Donaldson PJ, Kistler J. MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3. BMC Cell Biol 2001; 2:17.
82. Taylor V, Welcher AA, Program AE, Suter U. Epithelial membrane protein-1, peripheral myelin protein 22, and lens membrane protein 20 define a novel gene family. J Biol Chem 1995; 270:28824-33.
83. Taylor V, Suter U. Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family. Gene 1996; 175:115-20.
84. Chen T, Li X, Yang Y, Erdene AG, Church RL. Does lens intrinsic membrane protein MP19 contain a membrane-targeting signal? Mol Vis 2003; 9:735-46 <http://www.molvis.org/molvis/v9/a88/>.
85. Grey AC, Jacobs MD, Gonen T, Kistler J, Donaldson PJ. Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier. Exp Eye Res 2003; 77:567-74.
86. Voorter CE, Kistler J, Gruijters WT, Mulders JW, Christie D, de Jong WW. Distribution of MP17 in isolated lens fibre membranes. Curr Eye Res 1989; 8:697-706.
87. Steele EC Jr, Wang JH, Lo WK, Saperstein DA, Li X, Church RL. Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant. Mol Vis 2000; 6:85-94 <http://www.molvis.org/molvis/v6/a12/>.
88. Steele EC Jr, Kerscher S, Lyon MF, Glenister PH, Favor J, Wang J, Church RL. Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3. Mol Vis 1997; 3:5 <http://www.molvis.org/molvis/v3/a5/>.
89. Wen Y, Sachs G, Athmann C. A novel lens epithelium gene, LEP503, is highly conserved in different vertebrate species and is developmentally regulated in postnatal rat lens. Exp Eye Res 2000; 70:159-68.
90. Wen Y, Ibaraki N, Reddy VN, Sachs G. Functional analysis of the promoter and chromosomal localization for human LEP503, a novel lens epithelium gene. Gene 2001; 269:61-71.
91. Zelenka PS, Gao CY, Rampalli A, Arora J, Chauthaiwale V, He HY. Cell cycle regulation in the lens: proliferation, quiescence, apoptosis and differentiation. Prog Retin Eye Res 1997; 16:303-22.
92. Girard JP, Springer TA. Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC. Immunity 1995; 2:113-23.
93. Winkler C, Schafer M, Duschl J, Schartl M, Volff JN. Functional divergence of two zebrafish midkine growth factors following fish-specific gene duplication. Genome Res 2003; 13:1067-81.
94. Gilmour DT, Lyon GJ, Carlton MB, Sanes JR, Cunningham JM, Anderson JR, Hogan BL, Evans MJ, Colledge WH. Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J 1998; 17:1860-70.
95. Sullivan MM, Sage EH. Hevin/SC1, a matricellular glycoprotein and potential tumor-suppressor of the SPARC/BM-40/Osteonectin family. Int J Biochem Cell Biol 2004; 36:991-6.
96. Dunker N, Krieglstein K. Reduced programmed cell death in the retina and defects in lens and cornea of Tgfbeta2(-/-) Tgfbeta3(-/-) double-deficient mice. Cell Tissue Res 2003; 313:1-10.
97. Groszer M, Erickson R, Scripture-Adams DD, Lesche R, Trumpp A, Zack JA, Kornblum HI, Liu X, Wu H. Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 2001; 294:2186-9.
98. Li L, Liu F, Salmonsen RA, Turner TK, Litofsky NS, Di Cristofano A, Pandolfi PP, Jones SN, Recht LD, Ross AH. PTEN in neural precursor cells: regulation of migration, apoptosis, and proliferation. Mol Cell Neurosci 2002; 20:21-9.
99. Semina EV, Ferrell RE, Mintz-Hittner HA, Bitoun P, Alward WL, Reiter RS, Funkhauser C, Daack-Hirsch S, Murray JC. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nat Genet 1998; 19:167-70.
100. Semina EV, Reiter RS, Murray JC. Isolation of a new homeobox gene belonging to the Pitx/Rieg family: expression during lens development and mapping to the aphakia region on mouse chromosome 19. Hum Mol Genet 1997; 6:2109-16.
101. Kanungo J, Swamynathan SK, Piatigorsky J. Abundant corneal gelsolin in Zebrafish and the 'four-eyed' fish, Anableps anableps: possible analogy with multifunctional lens crystallins. Exp Eye Res 2004; 79:949-56.
102. Xu YS, Kantorow M, Davis J, Piatigorsky J. Evidence for gelsolin as a corneal crystallin in zebrafish. J Biol Chem 2000; 275:24645-52.
103. Perron M, Kanekar S, Vetter ML, Harris WA. The genetic sequence of retinal development in the ciliary margin of the Xenopus eye. Dev Biol 1998; 199:185-200.
104. Hitchcock P, Kakuk-Atkins L. The basic helix-loop-helix transcription factor neuroD is expressed in the rod lineage of the teleost retina. J Comp Neurol 2004; 477:108-17.
105. Boucher SE, Hitchcock PF. Insulin-related growth factors stimulate proliferation of retinal progenitors in the goldfish. J Comp Neurol 1998; 394:386-94.
106. Shi X, Bosenko DV, Zinkevich NS, Foley S, Hyde DR, Semina EV, Vihtelic TS. Zebrafish pitx3 is necessary for normal lens and retinal development. Mech Dev 2005; 122:513-27.
107. Nishimura DY, Searby CC, Alward WL, Walton D, Craig JE, Mackey DA, Kawase K, Kanis AB, Patil SR, Stone EM, Sheffield VC. A spectrum of FOXC1 mutations suggests gene dosage as a mechanism for developmental defects of the anterior chamber of the eye. Am J Hum Genet 2001; 68:364-72.
108. Nishimura DY, Swiderski RE, Alward WL, Searby CC, Patil SR, Bennet SR, Kanis AB, Gastier JM, Stone EM, Sheffield VC. The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nat Genet 1998; 19:140-7.
109. Smith RS, Zabaleta A, Kume T, Savinova OV, Kidson SH, Martin JE, Nishimura DY, Alward WL, Hogan BL, John SW. Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum Mol Genet 2000; 9:1021-32.
110. Grabski R, Szul T, Sasaki T, Timpl R, Mayne R, Hicks B, Sztul E. Mutations in COCH that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix deposition of cochlin. Hum Genet 2003; 113:406-16.
111. Lo J, Lee S, Xu M, Liu F, Ruan H, Eun A, He Y, Ma W, Wang W, Wen Z, Peng J. 15000 unique zebrafish EST clusters and their future use in microarray for profiling gene expression patterns during embryogenesis. Genome Res 2003; 13:455-66.
112. Bhattacharya SK, Rockwood EJ, Smith SD, Bonilha VL, Crabb JS, Kuchtey RW, Robertson NG, Peachey NS, Morton CC, Crabb JW. Proteomics reveal Cochlin deposits associated with glaucomatous trabecular meshwork. J Biol Chem 2005; 280:6080-4.
113. Chinen A, Hamaoka T, Yamada Y, Kawamura S. Gene duplication and spectral diversification of cone visual pigments of zebrafish. Genetics 2003; 163:663-75.
114. Takechi M, Kawamura S. Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development. J Exp Biol 2005; 208:1337-45.
115. Mitton KP, Swain PK, Chen S, Xu S, Zack DJ, Swaroop A. The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation. J Biol Chem 2000; 275:29794-9.
116. Mears AJ, Kondo M, Swain PK, Takada Y, Bush RA, Saunders TL, Sieving PA, Swaroop A. Nrl is required for rod photoreceptor development. Nat Genet 2001; 29:447-52.
117. Liu Y, Shen Y, Rest JS, Raymond PA, Zack DJ. Isolation and characterization of a zebrafish homologue of the cone rod homeobox gene. Invest Ophthalmol Vis Sci 2001; 42:481-7.
118. Coolen M, Sii-Felice K, Bronchain O, Mazabraud A, Bourrat F, Retaux S, Felder-Schmittbuhl MP, Mazan S, Plouhinec JL. Phylogenomic analysis and expression patterns of large Maf genes in Xenopus tropicalis provide new insights into the functional evolution of the gene family in osteichthyans. Dev Genes Evol 2005; 215:327-39.
119. Hansen JN, Konkel DA, Leder P. The sequence of a mouse embryonic beta-globin gene. Evolution of the gene and its signal region. J Biol Chem 1982; 257:1048-52.
120. Nishioka Y, Leder P. The complete sequence of a chromosomal mouse alpha--globin gene reveals elements conserved throughout vertebrate evolution. Cell 1979; 18:875-82.
121. Saari JC. Biochemistry of visual pigment regeneration: the Friedenwald lecture. Invest Ophthalmol Vis Sci 2000; 41:337-48.
122. Fischmeister R, Hartzell HC. Volume sensitivity of the bestrophin family of chloride channels. J Physiol 2005; 562:477-91.
123. Caldwell GM, Kakuk LE, Griesinger IB, Simpson SA, Nowak NJ, Small KW, Maumenee IH, Rosenfeld PJ, Sieving PA, Shows TB, Ayyagari R. Bestrophin gene mutations in patients with Best vitelliform macular dystrophy. Genomics 1999; 58:98-101.
124. Bakall B, Marknell T, Ingvast S, Koisti MJ, Sandgren O, Li W, Bergen AA, Andreasson S, Rosenberg T, Petrukhin K, Wadelius C. The mutation spectrum of the bestrophin protein--functional implications. Hum Genet 1999; 104:383-9.
125. Bakall B, Marmorstein LY, Hoppe G, Peachey NS, Wadelius C, Marmorstein AD. Expression and localization of bestrophin during normal mouse development. Invest Ophthalmol Vis Sci 2003; 44:3622-8.
126. Kerscher S, Church RL, Boyd Y, Lyon MF. Mapping of four mouse genes encoding eye lens-specific structural, gap junction, and integral membrane proteins: Cryba1 (crystallin beta A3/A1), Crybb2 (crystallin beta B2), Gja8 (MP70), and Lim2 (MP19). Genomics 1995; 29:445-50.
127. Church RL, Wang JH. The human lens fiber-cell intrinsic membrane protein MP19 gene: isolation and sequence analysis. Curr Eye Res 1993; 12:1057-65.