Molecular Vision 2005; 11:1083-1100 <http://www.molvis.org/molvis/v11/a127/> Received 28 July 2005 | Accepted 31 August 2005 | Published 13 December 2005

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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: tvihteli@nd.edu

Abstract

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.

Introduction

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 [18]. 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 [23]. The ciliary region of the zebrafish eye is located within the posterior chamber between the base of the iris and the retinal margin [24]. 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 [27]. 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.

Methods

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.

Library construction

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 [48]. The cDNA synthesis and directional cloning into the Sal I-Not I sites of the pCMVSport6 vector (Invitrogen, Carlsbad, CA) were also previously described [48].

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 [52]. The insert sequences were identified by BLAST matches to the databases and grouped using GRIST [46]. 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 [53]. 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 [54]. 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 [55]. 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 [56]. 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 [57]. 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 [58]. 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 [70].

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.

Lens

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 [73]. 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 [48]. 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 [79].

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 [80], 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 [48] 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 [89]. The human LEP503 gene mapped to the same chromosomal region as a locus responsible for causing zonular pulverulent cataract [90].

Lens growth is dependent on the regulated proliferation and migration of the epithelial cells, and their differentiation into new fiber cells [91]. 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].

Anterior segment

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 [102]. 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 [103]. 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 [104]. 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 [106]. 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 [112]. 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 [112].

Retina

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 [113]. 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 [12], 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 [113]. The cone opsin genes are expressed in overlapping temporal and spatial patterns during development and in the adult retina [114].

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 [114]. MWS-1 and MWS-4 were the only green opsin subtypes we identified previously by hybridization screening of an adult whole eye cDNA library [20]. 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 [113].

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 [117] (personal communication, Pamela Raymond). Recently, a predicted gene for zebrafish nrl was identified in silico [118]. 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 [118], 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 [118]. 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.

Posterior segment

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 [21]. Recovery of the photosensitive rhodopsin conformation requires the formation of 11-cis-retinal from all-trans-retinal via the retinoid cycle [121]. 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 [121]. 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 [49]. 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 [49], 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 [125]. This suggests a specialized role for the bestrophin protein in the RPE of mammals that may not be shared by all vertebrate species.

Conclusions

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 [45]. 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 [73]. 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.

Acknowledgements

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.

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