Gene Expression Profile of the Rat Eye Iridocorneal Angle: NEIBank Expressed Sequence Tag Analysis

Abstract

purpose. To characterize gene expression pattern in the combined tissues of the rat iridocorneal angle by expressed sequence tag (EST) analysis, as part of the NEIBank project.

methods. RNA was extracted from dissected tissues of the rat iridocorneal angle (iris, ciliary body, trabecular meshwork, and Schlemm’s canal) and used to construct unamplified, non-normalized cDNA libraries in the pSPORT1 vector. Approximately 5000 clones were sequenced from the 5′-end. Clones were clustered and identified using the GRIST software, a procedure based on BLAST comparisons. Complete sequences of several novel cDNAs showing eye-preferred expression patterns were obtained. The expression patterns of several genes have been investigated by Northern blot and in situ hybridization, as well as by RT-PCR.

results. After analysis and removal of non-mRNA sequences, 2195 independent clusters, potentially representing individual eye angle-expressed clones were obtained. The expression profile of the combined rat eye angle tissues was more similar to that of the human iris than to human trabecular meshwork. Several cDNAs encoding transcription factors essential for normal eye development and function including Pax-6, Six3, c-Maf, Maf1, Sox-4, Foxc1, Rx, and Ldb2 were present among sequenced clones. A number of tested cDNAs showed eye-preferred expression patterns. Myocilin, which is abundant in human eye angle tissues, was not observed in the rat collection; however, transcripts for three other olfactomedin-domain proteins were seen. Latrotoxin receptor (CL1AA) and optimedin were shown to be expressed in the iris and ciliary body, as well as in the ganglion and inner nuclear cell layers of the retina, whereas the rat orthologue of the human HNOEL-iso gene was expressed in the iris and sclera and less actively in the trabecular meshwork, retina, and optic nerve.

conclusions. The iridocorneal libraries are a good source of novel uncharacterized genes and molecular markers for the tissues of the eye angle. Although myocilin is not abundantly expressed in rat eye angle, other olfactomedin-containing genes are expressed there and may play important roles in normal eye function and disease.

Glaucoma is a group of neurodegenerative disorders characterized by the death of retinal ganglion cells and by a specific deformation of the optic nerve head, known as glaucomatous cupping. Primary open-angle glaucoma, the most common form, is often associated with elevated intraocular pressure (IOP). Elevated IOP develops as a result of imbalance between aqueous humor production and outflow. In many glaucoma cases elevated intraocular pressure develops as a result of abnormally high resistance to the outflow of aqueous humor. The gene expression profiles of several human tissues affected in glaucoma, trabecular meshwork (TM), iris, ciliary body, and retina, have been examined by expressed sequence tag (EST) analysis of cDNA libraries. ^{1 2 3 4 5 6} Several SAGE libraries were also created from different areas of the human eye. ⁷ Because studies of the molecular mechanisms of glaucoma are difficult to conduct in humans, appropriate animal models are needed to give insight into the molecular events in the retina and the optic nerve. 

Several rat models of elevated pressure-induced optic nerve damage have been developed to study changes in the retina and the optic nerve. In these models, IOP elevation is achieved by injection of concentrated saline solution in the episcleral vein, ⁸ cauterization of episcleral vein, ⁹ trabecular laser photocoagulation after injection of india ink into the anterior chamber, ¹⁰ or a laser injury to the TM. ¹¹ Chronic IOP elevation is accompanied by death of the retinal ganglion cells, optic nerve degeneration, and optic nerve head remodeling similar to that observed in glaucoma in humans. ^{8 12 13 14}  

To study mechanisms of aqueous humor production and outflow, it is important to know the molecular composition of the tissues involved. Herein, we present characterization of more than 3400 cDNA clones from unamplified cDNA libraries derived from freshly isolated tissues of the rat iridocorneal angle (ICA) (iris, ciliary body, TM, and Schlemm’s canal). The expression patterns of genes in the rat ICA were compared to those in human eye tissues. Several novel genes were found in the library, and they were characterized in more detail. Some of these genes showed eye-preferred expression patterns. 

Materials and Methods

RNA Preparation and cDNA Libraries Construction

All experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wistar rats (Charles River Laboratory, Wilmington, MA) weighing 250 to 300 g were used for cDNA library construction and in all other experiments. Tissues of the ICA were dissected from 46 rat eyes under a dissection microscope. Total RNA was isolated from the indicated tissues (RNazol; TelTest, Friendswood, TX). 0.5 μg of total RNA was separated by electrophoresis on a 1.2% agarose-2.2 M formaldehyde gel to evaluate the quality of RNA samples. Total RNA (120 μg) were used for construction of the libraries. Poly(A)+RNA was prepared using an oligo(dT) cellulose affinity column. The cDNA was synthesized and directionally cloned in the pSPORT1 vector (Invitrogen, Carlsbad, CA) at Bioserve Biotechnology (Laurel MD). General details of the library construction for NEIBank cDNA libraries are described elsewhere. ^{2 3 15} In this case, as an additional measure to remove small contaminant fragments, the cDNA was run over a resin column (Sephacryl S-500 HR; Invitrogen-Gibco, Grand Island, NY) designed to fractionate cDNA more than 500 bp. The columns were run in TEN buffer (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 25 mM NaCl). Sublibraries (designated gw and gx) were made from the first two 35-μL fractions, containing cDNA. 

cDNA Sequencing and Bioinformatics

Methods for sequencing and bioinformatics analysis are described in detail elsewhere. ¹⁶ Briefly, randomly picked clones were sequenced at the NIH Intramural Sequencing Center (NISC). Clones were sequenced from the 5′ end. A specially developed software tool, GRIST (GRouping and Identification of Sequence Tags) was used to analyze the data and assemble the results in Web page format. ¹⁷ Clusters of sequences were also examined (SeqMan II; DNAstar, Madison, WI) to examine alternative transcripts. Sequences were searched through genome resources at http://www.ncbi.nlm.nih.gov/; National Center for Biotechnology [NCBI], Bethesda, MD; and http://genome.ucsc.edu/; University of Santa Cruz, CA). cDNA inserts of selected clones were sequenced with fluorescent dideoxynucleotides on an automated sequencer (Model 310; Applied Biosystems, Foster City, CA). The analysis of domain architecture in the deduced protein sequences was performed using the Simple Modular Architecture Research Tool (SMART) ^{18 19} (http://smart.embl.de/; provided in the public domain by the European Molecular Biology Laboratory, Heidelberg, Germany) and the Conserved Domain Architecture Retrieval Tool (CDART) ²⁰ (http://www.ncbi.nlm.nih.gov/; NCBI). 

Northern Blot and In Situ Hybridization

For Northern blot experiments, 2 μg of total RNA were separated on agarose gel as above, transferred to a membrane (Nitran; Schleicher & Schuell, Keene, NH) and hybridized with [³²P]-labeled cDNA probes for specific clones in hybridization solution (ExpressHyb; BD Biosciences-Clontech, Palo Alto, CA) at 68°C overnight. Membranes were washed in 2× SSC-0.1% SDS, and then in 1× SSC-0.1% SDS, and finally in 0.1× SSC-0.1% SDS solution at 65°. Filters were exposed for different amounts of time. Filters were stained with 0.02% methylene blue after autoradiography for normalization of the amount of loaded RNA. ²¹ In some cases, 1 μg ethidium bromide was added to RNA samples before separation, and RNA was visualized after electrophoresis under UV light. 

For in situ hybridization, rat eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH 7.4) overnight and processed for paraffin embedding. Serial sections (6 μm) were hybridized with specific [³³P]-labeled riboprobes. To prepare riboprobes, PCR fragments of the rat CL1AA (accession number AF081144, position 4253-5250), Cirl2 (accession number NM-134408, position 4367-5366), and Cirl3 (accession number NM-130822, position 4568-5279) cDNAs were cloned into a vector (pBluescriptII; Stratagene, La Jolla, CA). Plasmids were linearized by digestion with NotI, and antisense probes were transcribed from the T7 promoter. In situ hybridization, washes, and autoradiography were performed as previously described. ²²  

Semiquantitative RT-PCR

Total RNA (1 μg) was used for cDNA synthesis using reverse transcriptase (SuperScript II; Invitrogen, Carlsbad, CA) and oligo(dT)-primer. The amount of synthesized cDNA was evaluated by PCR using primers specific for cyclophilin and ribosomal protein Rpl19 depending on the source of RNA. PCR reactions were performed (PTC-200 Thermal Cycler; MJ Research, Watertown, MA) using polymerase (AmpliTaq; Applied Biosystems). Each PCR reaction was repeated at least twice. The thermal cycling parameters were as follows: 1 minute 30 seconds at 94° followed by 30 cycles of 30 seconds at 94°, 1 minute 30 seconds at 59°, 1 minute at 72° and final incubation for 5 minutes at 72°. PCR reaction products were analyzed by agarose gel-electrophoresis. After adjustment of cDNA concentration, relative abundances of mRNA for different genes were estimated. Primers for each gene were designed to cross introns when possible. Dilutions of cDNA samples were adjusted for each gene to give a linear range in PCR reactions. 

Results

Library Statistics

Rat ICA cDNAs were collected and cloned as two sublibraries designated gx and gw. For the gx and gw libraries, there were 1.3 × 10⁶ and 1.8 × 10⁶ primary cDNA recombinant clones with an average insert size of 1.73 and 1.38 kb, respectively. For each sublibrary, approximately 2500 clones were picked, and sequenced and data were combined for subsequent analysis. Both sublibraries contained 3% to 4% mitochondrial genome clones and approximately 9% rRNA inserts. After removal of non-mRNA clones and repetitive sequences and vector trimming there were 3420 high-quality sequence reads of average length 530 bp. Analysis of these clones through GRIST ¹⁷ resulted in 2195 clusters of sequences potentially representing individual genes expressed in the rat eye angle. Four hundred forty-eight clusters contained at least two clones, and 178 clusters contained at least 3 clones, representing mRNAs that may be highly or moderately abundant in the rat eye angle. Approximately 38% of the clones in the rat angle collection have no corresponding entry in GenBank (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by NCBI) at present and about half of these have no rat UniGene (http://www.ncbi.nlm.nih.gov/UniGene; provided in the public domain by NCBI) entry. Most of these clones have some sort of identified homologues in mouse or human, but even in these species, most cataloged genes have unknown biological functions. Information about all sequenced clones is available at http://neibank.nei.nih.gov/ (provided in the public domain by the National Eye Institute, Bethesda, MD). All clones were numbered according to their library designation and their position in 96-well plates (e.g., gx01d10). 

Gene Expression Profile of Rat Eye Angle Tissues

Table 1 provides a list of the 63 the most abundant clones in the rat eye ICA library, those represented by 5 or more clones. The removal of tissues from the rat eye angle is a delicate procedure. Rigorous measures were followed to minimize contamination of the dissected tissues by surrounding tissues. To evaluate possible contamination of the eye ICA dissected tissues by corneal cells, the expression profile of the rat eye ICA library was compared to that of corneal epithelial cells produced by SAGE analysis. ²³ Transketolase and ALDH3 mRNAs were the most abundant message in the mouse corneal SAGE library. Although there were five ALDH3-encoding clones in the rat ICA library, there was only one transketolase-encoding clone among the sequenced clones in this library. This indicated that the tissues of the rat ICA were not heavily contaminated by corneal tissue in the course of dissection. 

β-Actin cDNAs were the most abundant in the rat eye ICA library (95 clones) with γ-actin (4 clones) and α-actin (2 clones) being much less abundant. Although actins are ones of the most abundant proteins in many cell types, actin cDNA clones were not so abundant in the human TM ⁴ or iris cDNA libraries. ² Moreover, the relative distribution of actin isoforms was also different in these libraries. There were 11 α-actin and 2 β-actin cDNAs among 3459 sequenced clones in the human TM library, whereas there were 9 β-actin, 5 γ-actin and 2 α-actin cDNAs among approximately 2000 sequenced clones in the iris library. ^{2 4}  

Overall, the expression profile of the combined rat ICA tissues was strikingly different from human TM and more similar to that of the human iris. For example, 8 of the 10 most abundant clones in the human iris library were also represented in the rat eye ICA collection (opticin or oculoglycan and plasma protease [C1] inhibitor were two genes that were abundant in human iris but not observed in rat), whereas only 5 of the 10 most abundant clones in the human TM library were seen in rat. In particular, cDNAs for myocilin and matrix Gla proteins, which were very highly expressed in the human TM library, were missing from the rat ICA collection. Mutations in myocilin are associated with glaucoma in humans ²⁴ although the functional role of myocilin in TM is not well understood. Myocilin is an olfactomedin domain-containing protein, and most of the glaucoma-causing mutations in this gene affect this domain. ²⁵ Three other olfactomedin domain-containing proteins were represented by sequenced clones in the rat ICA collection. One of these cDNA clones encoded a novel protein named optimedin, which has been characterized in detail. ²⁶ The second transcript observed (one cDNA clone) encoded a divergent olfactomedin-containing protein similar to human HNOEL-iso protein (accession number NM-020190). The third member of this family detected (two cDNA clones) was latrotoxin receptor 1 (CL1AA; accession number AF081144) which contains the olfactomedin domain in the extracellular N-terminal portion of the protein. ²⁷  

Table 2 lists the transcription factors and proteins interacting with the transcriptional machinery identified among the sequenced clones. Several transcription factors essential for normal eye development and function, including Pax-6, Six3, c-maf, Maf1, Sox-4, Foxc1, Rx, and Ldb2, ²⁸ were present among sequenced clones. The list of the most abundant transcription factors identified in the rat eye ICA library was different from the list of such factors in the human TM and iris libraries. For example, v-fos, Pitx-2, cold shock domain A protein, and TSC-22 were abundant in the human TM library. ⁴ Of these, only TSC-22 was abundant in the rat eye ICA library but this transcription factor is abundant in many cDNA libraries. 

Expression Pattern of Selected Clones from the Eye Angle Library

Eight cDNA clones encoding novel or poorly characterized proteins were selected from the eye angle library and their expression patterns were analyzed by Northern blot hybridization. Figure 1 illustrates the results of these experiments for six cDNAs. Four cDNAs showed preferential expression in different eye structures. In particular, cDNAs gx01a07 and gx01a06 were highly expressed in the tissues of the ICA (Figs. 1A 1B) , whereas cDNA gw01d02 was highly expressed in the lens (Fig. 1C) . cDNA gx01d10 was highly expressed in the cornea and ICA (data not shown; Torrado M, Senatorov VV, Trivedi R, Fariss RN, and Tomarev SI, manuscript in preparation). cDNAs gw06g04 and gx01b08 were preferentially expressed in the retina and brain (Figs. 1E 1F) . cDNA gw01f11 was preferentially expressed in sclera and brain (data not shown), whereas cDNA gw01d04 was preferentially expressed in the tissues of the ICA, sclera, retina, brain, kidney, and lung (Fig. 1D) . 

Clone gw11g07 encoded optimedin and was expressed in the ICA tissues, retina, and brain, as described previously. ²⁶ Because olfactomedin domain-containing proteins may play important roles in normal eye development and disease, ^{24 26 29 30} expression of the CL1AA gene, ^{27 31} another member of the family that was present twice among the sequenced clones, was investigated by in situ hybridization. Two other genes, Cirl2 and Cirl3, which belong to the same family of latrotoxin receptors as the CL1AA gene, ^{32 33} were also included in this study, although Cirl2 and Cirl3 were not present among the sequenced clones. The results demonstrated (Fig. 2) that CL1AA and Cirl3 have very similar patterns of expression in the adult rat eye. They were expressed in the iris and ciliary body as well as in the ganglion and inner nuclear cell layers of the retina. The Cilr2 gene was expressed only in the inner nuclear layer of the retina, but not in the tissues of the eye angle. 

Clone gx21f10 is a rat orthologue of the human HNOEL-iso gene (accession number NM-020190). This gene encoded a divergent olfactomedin-containing protein. Its expression was studied by semiquantitative RT-PCR (Fig. 3) . The results demonstrated that this gene was more actively expressed in the iris and sclera than in the TM, retina, and optic nerve. Its expression was not detected in the lens, cornea, and brain in the conditions used in these experiments. 

On the basis of all these results, we concluded that the rat ICA library is a rich source of clones with interesting eye-preferred expression patterns. 

Sequencing of the Selected Clones

Some of these potentially eye-preferred clones were selected for full-length sequencing. Clone gx01a07 (accession number AY569010) contained an insert of 2882 bp, not counting the poly(A) tail, and encoded a novel conserved protein with a length of 541 amino acids (Fig. 4) . Analysis of the encoded sequence using the SMART package ^{18 19} suggested that it may represent a transmembrane protein with seven transmembrane helices (Fig. 4) . Recently a related cDNA clone has been identified in a rat prostate cDNA library (accession number BC062391). This clone and clone gx01a07 were different in the 3′-untranslated region (UTR) as a result of alternative splicing. However, these two clones encoded the same protein. The rat gene contains at least 12 exons. The rat protein has 98%, 96%, and 78% identity to mouse (AK044810), human (AK074984), and zebrafish (BC053246) proteins, respectively. It shows 45% identity to a Drosophila protein with unknown functions (NM_168450). The human orthologue maps to 1p31.2. In the human eye, this gene is expressed in the RPE-choroid, iris, and lens, as demonstrated by comparison of the rat sequence with the NEIBank. ^{2 15 34} Recently, the human gene has been shown to be involved in the activation of the NF-κB signaling pathway. ³⁵  

Clone gx01a06 (accession number AY569011) contained an insert with a length of 1790 bp and encoded a novel conserved protein with a length of 553 amino acids. Analysis of the protein sequence using the CDART package ²⁰ revealed that it contains an IMP-GMP–specific 5-nucleosidase domain (positions 50-551 in the rat protein sequence). The existence of this gene has been predicted recently on the basis of the computational analysis of the rat genome (accession number XM_214258). The encoded rat protein has 98%, 96%, and 74% identity to mouse (BC011230), human (AK022504), and Xenopus (BC046742) proteins, respectively. The rat gene consists of at least 14 exons, and its mRNA is alternatively spliced. The human orthologue maps to 3p21.31. In the human eye, this gene is expressed in the RPE/choroid and lens as judged by NEIBank EST analyses. ^{15 34}  

cDNA gw01d02 (accession number AY569012) had a length of 1347 bp without the poly(A) tail and encoded a protein with a length of 176 amino acids. This gene consists of only two exons. The encoded protein is related to mammary tumor receptor 2 isoform. ³⁶  

cDNA gw06g04 (accession number AY569014) had a length of 4391 bp and encoded a conserved protein with a length of 952 amino acids. This protein is a rat homologue of a recently identified postsynaptic membrane protein, calsynthenin-1. ³⁷ Calsyntenin-1 contains a cadherin repeat domain (positions 41-248 in the protein sequence) and a SCOP domain (positions 351-532 in the protein sequence), which is present in galectins, laminin G-like module, and calnexin. The cadherin repeat domain may mediate cell–cell contacts when bound to calcium. The rat calsyntenin-1 gene contains at least 17 exons, and its mRNA was alternatively spliced. In the human eye, calsyntenin-1 is expressed in the human lens, retina, and RPE-choroid. ^{3 15 34} In the mouse brain, the calsyntenin-1 gene is expressed in most neurons of central nervous system (CNS) with relatively little variation in its expression level. ³⁸  

cDNA gw01d04 (accession number AY569013) had a length of 2088 bp without the poly(A) tail and encoded a protein with a length of 475 amino acids. This protein was 99% identical with a recently described nonspecific mouse dipeptidase. ³⁹ Examination of sequences in GenBank showed that this gene has at least 12 exons and is alternatively spliced at the 5′-end as well as at internal positions. The human homologue maps to 18q22.3. 

cDNA gx01b08 was 96% and 98% identical with recently described human and mouse cDNAs, encoding stromal membrane-associated proteins. ⁴⁰ Although the gx01b08 cDNA insert was longer at the 5′-end than the corresponding human and mouse sequences, it was not complete at the 3′-end. The encoded protein contains a putative ArfGap domain, which is found in activators of small guanosine triphosphatases (GTPases) and may therefore be involved in the signal transduction. ^{41 42 43}  

Discussion

In the present study, we constructed cDNA libraries from the tissues of the rat ICA. We had two main goals in mind. The first was to characterize gene expression patterns in the tissues involved in aqueous humor production and outflow. We were also looking for novel genes that might be essential for normal function of eye tissues and might be involved in eye diseases such as glaucoma. 

The tissues of the ICA have various embryological origins and contain multiple cell types. The iris consists of the stroma and two layers of pigmented epithelium. Pigmented epithelium cells share a developmental origin with retinal pigmented epithelium cells, which are derived from the external walls of the optic vesicle and therefore have a neuroepithelial origin. Iris sphincter and dilator muscle also have neuroepithelial origin. The iris stroma is derived from cranial paraxial mesoderm and neural crest and contains small blood vessels, two types of pigmented cells, and myelinated and unmyelinated nerves, dendriform macrophages, and major histocompatibility complex (MHC) class II dendritic cells. The ciliary body consists of the epithelium, vascular stroma, and muscle. The layer of the ciliary body facing the vitreous is nonpigmented and is continuous with retina, whereas the external epithelium layer is pigmented and continuous with retinal pigmented epithelium and the iris pigmented epithelium. TM is derived from neural crest and consists of trabecular beams covered with endothelial cells. ⁴⁴  

β-Actin cDNAs were the most abundant in the rat ICA library. Although actins are among the most abundant proteins in many cell types, the level of expression seems to be higher in rat angle than in human TM or iris cDNA libraries. ^{2 4} Moreover, the relative distribution of actin isoforms was also different in these libraries. Although there is no necessary correlation between transcript and protein levels, these data may indicate high activity in synthesis or maintenance of the actin cytoskeleton in the tissues of the rat ICA. This idea is supported by the presence of cDNA clones for several actin-binding proteins involved in the regulation of actin microfilament (cofilin, actinins, coronin, Arp2/3, and profilin). ⁴⁵ Among actin isoforms, β-actin is expressed in all cell types, γ-actin is preferentially expressed in the neural tissues, whereas α-actin is considered to be cardiac or vessel specific. ⁴⁶ γ-Actin cDNAs were more abundant in the rat ICA and human iris libraries than α-actin cDNAs, ² whereas α-actin cDNAs were the most abundant in the human TM library and no γ-actin cDNAs were detected among sequenced clones in this library. ⁴  

The third most abundant group of clones in the rat ICA libraries corresponded to insulin-like growth factor protein 7 (IGFBP7), also known as mac25 and angiomodulin. IGFBP7 clones were also abundant in the human TM, iris, and RPE-choroid libraries. ^{2 4 15} IGFBP7 is also expressed in a many normal human tissues including heart, spleen, ovary, small intestine, and colon. ⁴⁷ It is a multifunctional secreted protein that exhibits growth-stimulatory activity in synergy with insulin or insulin-like growth factors and may interact with glycosaminoglycans and extracellular matrix proteins as well as immobilize chemokines. ⁴⁸ The high levels of IGFBP7 gene expression in the rat ICA and several human eye tissues indicate that the encoded protein may be essential for their function. 

Myocilin cDNAs formed the third most abundant group of clones in the human TM cDNA library. ⁴ Furthermore, 3 myocilin clones were also present among approximately 2000 sequenced clones in the human iris cDNA library. ² Although myocilin mRNA is expressed in the rat TM, ²⁶ we did not identify rat myocilin clones among sequenced clones in the rat ICA libraries. There are two possible explanations for the absence of myocilin clones. One is that TM tissue represents a relatively small fraction of the tissues used for library construction. The second explanation is that there are species-specific differences in gene expression patterns in the tissues of ICA and myocilin expression may be lower in rat than in human eye. Indeed, it is not only myocilin that is “missing” from rat ICA. Opticin/oculoglycan and plasma protease (C1) inhibitor clones, which are among the most abundant in the human iris cDNA library, are also absent from the rat collection. 

Although myocilin is missing, several other olfactomedin-domain proteins clones were represented among the sequenced clones in the eye angle library. One of them was optimedin which is expressed in the iris and ciliary body epithelial cells as judged by in situ hybridization. ²⁶ Another olfactomedin-related gene (accession number XM-227535) was preferentially expressed in the iris and sclera among eye tissues tested (Fig. 3) . This gene has been identified recently in a large-scale project designed to identify novel human secreted and transmembrane proteins. ⁴⁹ The calcium-independent latrophilin receptor genes (CL1AA, Cirl2, and Cirl3) encode membrane proteins containing the olfactomedin-related domain in the extracellular N-terminal part. ^{27 31 32 33 50} cDNAs encoding CL1AA were present twice among sequenced clones. In situ hybridization experiments demonstrated that the CL1AA and Cirl3 are expressed in the same rat eye tissues as the previously described optimedin. These genes were expressed in the epithelial cells of the iris and ciliary body as well as in the retinal ganglion and inner nuclear cell layers. Cirl2 was expressed only in the inner nuclear layer. CL1AA is a G-protein–coupled receptor that is able to bind α-latrotoxin. Natural ligands for this family of latrotoxin receptors have not been identified yet. It has been reported that CL1AA and Cirl3 are brain specific, whereas Cirl2 is ubiquitously expressed. ^{32 50} Our data demonstrate that these genes are expressed in the adult rat eyes where they may play an important role in G-protein–mediated signaling and in the formation of contacts between cells. 

Another important signaling mechanism in the eye is the Wnt-frizzled pathway that plays an important role in establishing cell polarity, cell proliferation, and specification of cell fate. ⁵¹ Wnt members can be divided into two functionally different classes: wnt1 and wnt5a. Wnt proteins belonging to the wnt1 class preferentially signal through β-catenin, which is considered to be the canonical pathway. Proteins of the Wnt5a class can stimulate intracellular Ca²⁺ release through interactions with the frizzled-2 receptor. Two cDNA clones corresponding to Wnt5a and two cDNA clones corresponding to frizzled-2 receptor are present in the rat ICA collection, whereas cDNAs encoding wnt4 and wnt10a are present once each. It is interesting to note that wnt4 is critical for kidney development ⁵² and may stimulate expression of C-type natriuretic peptide through the canonical signaling pathway. ⁵³ cDNA for atrial natriuretic peptide has been previously identified in the human ciliary body cDNA library ⁵⁴ and it has been suggested that natriuretic peptides may play a role in IOP regulation in human eyes. ⁵⁵ Three cDNA clones encoding secreted frizzled-related protein 1 were also present among sequenced clones. Secreted frizzled-related proteins are modulators of the Wnt-frizzled pathways. ⁵⁶  

Overall the EST analysis suggests several new avenues for further investigation. Sequencing of several cDNA inserts from the rat ICA library demonstrated that this library is a good source of novel or not well-characterized genes. Most of these cDNAs represented full coding sequences. Analysis of the promoter sequences of genes preferentially expressed in the tissues of the eye ICA may lead to the development of angle-specific or angle-preferred promoters. 

²  Present affiliations: the Department of Neuroscience, Georgetown University Medical Center, Washington, DC; the

³  Institute of Health Sciences, University of La Coruna, Campus de Oza, Edificio El Fortin, La Coruna, Spain; and

⁴  N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia.

 Supported by the National Eye Institute Intramural Program and by Russian Federation Basic Research Grant 02-04-48435 (RDZ).

 Submitted for publication March 16, 2004; revised April 13, 2004; accepted April 14, 2004.

 Disclosure: F. Ahmed, None; M. Torrado, None; R.D. Zinovieva, None; V.V. Senatorov, None; G. Wistow, None; S.I. Tomarev, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

The Most Abundant cDNA Clones in the Rat ICA cDNA Library

Rank	Gene Name	GenBank	UniGene	n	Chromosome Location of Human Homologue
1	Beta-actin	NM_031144	94978	92	7p15-p12
2	Ribosomal protein S2	X57432	2115	27	16p13.3
3	IGFBP7	XM_214014	1620	21	4q12
4	Basigin	NM_012783	2269	20	19p13.3
5	GNAS	AF107844	31	19	20q13.2-q13.3
6	Tyrosinase-related protein-2	XM_224517	26665	17	13q31-q32
7	Elongation factor 1a	NM_175838	965	14	6q14
8	Ferritin H subunit	NM_012848	54447	15	11q13
9	Apolipoprotein E	J00705	32351	14	19q13.2
10	TIMP-2	S82718	10161	13	17q25
11	Ornithine decarboxylase antizyme	D10706	266	13	19p13.3
12	Integral membrane protein 2B	XM_214228	107335	11	13q14.3
13	Aquaporin 1	NM_012778	1618	11	7p14
14	Cathepsin B	NM_022597	100909	11	8p22
15	CD63 antigen	NM_017125	11068	11	12q12-q13
16	Lamin A	X76297	44161	10	1q21.2-q21.3
17	Parathymosin	NM_031975	3609	10	17q12-q22
18	Vimentin	NM_031140	2710	10	10p13
19	Nuclease sensitive element binding protein 1	NM_031563	110976	10	1p34
20	Keratin 5	BC062086	6681	10	12q12-q13
21	Pmel17	XM_343146	38462	10	12q13-q14
22	Clusterin TRPM-2	M64723	1780	9	8p21-p12
23	Osteoactivin	AF184983	13778	9	7p15
24	Plasma glutathione peroxidase precursor	D00680	108074	8	5q32-q33.1
25	Protein kinase C delta-bindig protein	D85435	12281	8	11p15.4
26	Nbl1 neuroblastoma, suppression of tumorigenicity 1	NM_031609	4073	8	1p36.13-p36.11
27	Phosphatidylethanolamine-binding protein	X71873	29745	7	12q24.23
28	Ribophorin I (Rpn1)	NM_013067	4224	7	3q21.3-q25.2
29	Histone H3.3	XM_213961	9454	7	17q25
30	Transferrin	AF476964	91296	7	3q22.1
31	Mitochondrial ATP synthase alpha-subunit	NM_023093	40255	7	18q12-q21
32	Prothymosin-alpha	M60664	817	7	2q35-q36
33	GAPDH	XM_216453	91450	6	12p13
34	Calreticulin	NM_022399	974	6	19p13.3-p13.2
35	ATP synthase, H+ transporting, mitochondrial F1 complex, beta	XM_343136	92965	6	12p13-qter
36	Integral membrane protein 2C	XM_217467	8450	6	2q37
37	ATPase, Na+K+ transporting, alpha 1	M14511	2992	6	1p13
38	Prosaposin	BC061759	97173	6	10q21-q22
39	Calmodulin (pRCM1)	X13933	4166	6	14q24-q31
40	Connexin 43	NM_012567	10346	6	6q21-q23.2
41	Rap7a	NM_022526	1531	6	5p15.2
42	Mac-MARCKS protein (F52 gene)	AJ301677	2486	6	1p34.3
43	Eukaryotic translation initiation factor 5A	XM_213368	104607	6	17p13-p12
44	Ribosomal protein L13a	X68282	92211	6	10q24.1
45	NMDA receptor glutamate-binding chain	BC062074	5898	6	8q24.3
46	Fibulin 1	XM_243637	9375	6	22q13.3
47	Ribosomal protein L10	NM_031100	102157	5	Xq28
48	Ribosomal protein L8	X62145	9810	5	8q24.3
49	Calpain small subunit	U53859	3430	5	19q13.13
50	Myristoylated alanine-rich protein kinase C substrate	M59859	9560	5	6q22.2
51	Na+, K+-ATPase beta subunit	J02701	8925	5	1q24
52	Heterogeneous nuclear ribonucleoprotein L	XM_214878	33873	5	19q13.2
53	Calponin 3, acidic	NM_019359	57635	5	1p22-p21
54	B-cell translocation gene 1, (Btg1)	NM_017258	1000	5	12q22
55	Lysosomal-associated transmembrane protein 4A	NM_199384	108012	5	2p24.3
56	TSC-22	L25785	3545	5	13q14
57	Ribosomal protein L4 (Rp14)	NM_022510	1133	5	15q22
58	Enhancer-of-split and hairy-related protein 1	AF009329	10784	5	12p11.23-p12
59	CD81 antigen	NM_013087	1975	5	11p15.5
60	GABA-A receptor-associated protein	AF161588	8411	5	17p13.2
61	ALDH3	J03637	105627	5	17p11.2
62	Tumor protein, translationally controlled 1	NM_053867	36610	5	13q12-q14
63	ESTs (similar to CD99)	CK469078		5	(Xp22.32)

 This list is an edited detail from the output of the clustering procedure GRIST. ¹⁵ Where available, representative GenBank entries are shown, along with the current UniGene cluster, the number of ESTs identified in the rat ICA library, and location of human homologues.

Table 2.

 

Transcription Factors and Cofactors in the Rat Eye Angle Library

Rank	Gene Name	GenBank	UniGene	n
1	Nuclease sensitive element binding protein 1	NM_031563	110976	10
2	Nbl1 neuroblastoma, suppression of tumorigenicity 1	NM_031609	4073	8
3	TSC-22	L25785	3545	5
4	Enhancer-of-split and hairy-related protein 1	AF009329	10784	5
5	Cysteine rich protein 1 (Csrp1)	NM_017148	108075	4
6	Similar to ring protein 10	XM_213797	11656	4
7	Transcription factor MRG1	AF361476	31765	3
8	HMG17	AF329828	3517	3
9	Necdin	XM_218733	3096	3
10	Melanocyte-specific gene 1 protein (msg1)	AF104399	8163	3
11	Amino-terminal enhancer of split	L14462	11495	3
12	Chromobox homolog 6	XM_235484	101055	3
13	Hematopoietic PBX-interacting protein	XM_227420	21294	3
14	RYB-a	D28557	3306	2
15	RNA polymerase II transcription factor SIII p18 subunit	L42855	108140	2
16	Kruppel-like factor (Lklf)	XM_224722	92653	2
17	Nuclear LIM interactor-interacting factor 1	XM_217293	37030	2
18	Transcription factor EB	XM_236936	22098	2
19	Transcription factor BTF3	XM_215460	1858	2
20	General transcription factor III C 1	L28801	11288	2
21	Ovalbumin upstream promoter gamma nuclear receptor rCOUPg	AF003926	25840	2
22	SOX-4	XM_344594	15099	2
23	LPS-induced TNF-alpha factor	U53184	6940	2
24	Similar to general transcription factor III polypeptide 5	XM_342399	106946	2
25	Aryl-hydrocarbon receptor-interacting protein (Aip)	NM_172327	95160	2
26	Foxc1	(BC052011)	11816	2
27	Tripartite motif protein TRIM28	XM_344861	49293	2
28	Transcription factor HES-1	D13417	19727	2
29	Transcription factor MAZR	XM_223592	71473	2
30	LIM domain only 4	XM_342353	2517	1
31	Transcriptional enhancer factor 1	XM_341908	105443	1
32	LIM domain binding 2 (Ldb2)	XM_214054	94720	1
33	Hepatocarcinogenesis-related transcription factor	XM_214067	101044	1
34	Emx2	XM_217652	99323	1
35	General transcription factor II E	XM_224929	103659	1
36	RX	AF135839	87955	1
37	CREBBP/EP300 inhibitory protein 1	AY462245	108128	1
38	Kruppel-type zinc finger protein KROX-25	AF281635	105961	1
39	CCAAT binding transcription factor B subunit	M34238	10747	1
40	Pax transcription activation domain interacting protein	XM_231271	13768	1
41	Zinc finger protein 207	XM_221231	102608	1
42	Hmx1	XM_341238	24574	1
43	Zinc finger protein CRAZ2	XM_221977	38588	1
44	Modulator of estrogen induced transcription (Met)	XM_236381	49121	1
45	c-Maf	AY005471		1
46	Inhibitor of DNA binding 3	D10864	2760	1
47	RNA polymerase II polypeptide E	XM_216839	65038	1
48	Lhx9	AY273890	92463	1
49	Retinoic acid receptor gamma (RAR-gamma-B)	XM_217064	73054	1
50	Runt-related transcription factor binding protein	XM_225008	68832	1
51	General transcription factor II I	AF547390	27575	1
52	Idb4	NM_175582	22987	1
53	Tumor protein p53	NM_030989	54443	1
54	Winged helix/forkhead transcription factor HFH1	XM_214474	10101	1
55	Similar to zinc finger protein s11-6	(NM_133358)		1
56	Pax6	NM_013001	89724	1
57	Enhancer of rudimentary homolog	XM_216744	7881	1
58	Transcription factor USF2	AB047556	44637	1
59	Sequence-specific single-stranded DNA-binding protein	AF121893	2897	1
60	High mobility group protein A1a	AF507966	83614	1
61	Transcription factor UBF	XM_340913	22469	1
62	Zinc finger protein krox-20/egr-2	AB032420	89235	1
63	p53-related protein	AY093598	7990	1
64	Cas-associated zinc finger protein	AB019281	9637	1
65	Mitf	XM_232215	74693	1
66	Transcriptional regulating protein 132	XM_236953	46284	1
67	Ldb1a	XM_219948	102427	1
68	Fos-related antigen (Fra)	NM_030585	3228	1
69	Methyl-CpG binding protein MBD2	XM_214544	4085	1
70	Heat shock factor binding protein 1	AF522937	2578	1
71	Zic2 protein	XM_341381	64359	1
72	Kruppel-like factor 3	XM_223423	23326	1
73	v-Maf	NM_019316	10725	1
74	Similar to Adenovirus 5 E1A-binding protein (BS69 protein)	BC065308	38301	1
75	MYB binding protein 1a (Mybbp1a)	NM_031668	35696	1
76	Similar to zinc finger protein 175	XM_218548	79159	1
77	Hypoxia inducible factor 2 alpha (Hif-2a)	AJ277828	55138	1
78	Polymerase I-transcript release factor; PTRF	XM_220982	13589	1
79	Calmodulin binding transcription activator 2 (CAMTA2)	NM_015099	107362	1
80	Basic leucine zipper and W2 domains 1 (Bzw1)	BC061580	96215	1
81	Transducin-like enhancer 3 (Esp3)	AF186092	24106	1
82	Add1	L16995	95306	1
83	Nuclear factor I-A2 (NFIA)	AF112455	10550	1
84	Similar to Zinc finger protein 197	XM_236726	100996	1
85	Nuclear factor I/X	XM_213849	56776	1
86	Interferon regulatory factor 1 (IRF-1)	M34253	6396	1
87	Six3	AB030833	64582	1
88	Dopamine-inducible LIM-domain transcription factor DAT1	AF353304	25503	1
89	Tbx2	XM_220810	38282	1
90	Transcriptional activator protein Pur-alpha	XM_226016	41055	1
91	Zic	AF221839	48772	1
92	LIM domain-binding protein 1	XM_219948	102427	1
93	Amino-terminal enhancer of split	L14462	11495	1
94	Nuclear X box binding factor 1	XM_232897	55459	1
95	Transcription factor 19	BX883047	14867	1
96	Similar to ash1	XM_227409	72994	1
97	Zinc finger protein 162	XM_342001	1074	1
98	Hairy and enhancer of split 6	XM_237395	11980	1
99	Transcription factor BTEB4	XM_345792	105591	1
100	Elk-3	XM_343198	94638	1
101	Forkhead box F2	(NM_010225)		1
102	Similar to CCAAT/enhancer binding protein (C/EBP), beta	NM_024125	6479	1
103	Similar to retinoic acid receptor X-beta	XM_342095	49295	1
104	LIM homeodomain protein (LH-2)	L06804	81063	1

 There were no rat GenBank entries for some sequences. In such cases, mouse or human orthologues are shown in parenthesis.

Figure 1.

 

Northern blot analysis of selected genes expressed in rat tissues. The cDNA probes, used for these blots, are as follows: (A) gx01a07 (encoding a novel transmembrane protein); (B) gx01a06 (a novel protein); (C) gw01d02 (related to mammary tumor receptor 2 isoform); (D) gw01d04 (nonspecific dipeptidase); (E) gw06g04 (calsynthenin-1); (F) gx01b08 (stromal membrane-associated protein). Total RNA (2 μg) were loaded per lane. Loaded RNA was visualized by staining with ethidium bromide (not shown).

Figure 2.

 

Radioactive in situ hybridization of CL1AA (A, G), Cirl2 (C, I), and Cirl3 (E, K) antisense probes with rat eye sections. (A–F) correspond to the ICA area, whereas (G–L) show retina. The first and third rows show dark-field images. The second and fourth rows show the same regions in bright field after hematoxylin staining. c, cornea; cb, ciliary body; i, iris; inl, inner nuclear layer; onl, outer nucler layer; gcl, retinal ganglion cell layer; tm, trabecular meshwork.

Figure 3.

 

Semiquantitative RT-PCR analysis of the rat orthologue of the human HNOEL-iso gene expression in different rat eye tissues and brain. Primers 5′-CCGGAGAAATGAGAAATACGAT-3′ and 5′-CAGCCATGGTGAGGGTGAAG-3′ are located in different exons of the rat Noel-iso gene. Primers 5′-TCCTCCTTTCACAGAATTATTCC-3′ and 5′-AATTAGAGTTGTCCACAGTCGG-3′ were used to amplify cyclophilin cDNA for normalization of the amount of synthesized cDNA. Normalization with Rpl19 cDNA gave very similar results (data not shown).

Figure 4.

 

Encoded protein sequence (A) and exon–intron structure of the gene (B) corresponding to gx01a07 cDNA (accession number AY569010). (A) Yellow: potential signal peptide sequence; blue: transmembrane domains; black triangles: boundaries between different exons. (B) Diagram of gx01a07 gene structure drawn approximately to scale. The length of the last intron is not known.

The authors thank Jeff Touchman and Gerry Bouffard of NIH Intramural Sequencing Center and Don Smith, Patee Buchoff, and James Gao of the National Eye Institute for support in sequencing and organizing data; Irina Karavanova (National Institute of Child Health and Human Development) for help with in situ hybridization; and Patrick Chang for performing some of the RT-PCR experiments.