Molecular Vision 2007; 13:1828-1841 <http://www.molvis.org/molvis/v13/a204/> Received 3 December 2006 | Accepted 11 August 2007 | Published 2 October 2007

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Identification of genes specifically expressed by human Müller cells by use of subtractive hybridization

Caroline B. Lupien, Christian Salesse
 
 

Unité de Recherche en Ophtalmologie, Centre de recherche du CHUQ, Pavillon CHUL, and Département d'Ophtalmologie, Faculté de médecine, Université Laval, Ste-Foy, Québec, Canada

Correspondence to: Dr. Christian Salesse, Unité de Recherche en ophtalmologie, Salle S-5, Centre de recherche du CHUQ, Pavillon CHUL, 2705 Boul. Laurier, Ste-Foy, Québec, G1V 4G2, Canada; Phone: (418) 656-4141 ext. 47243; FAX: (418) 654-2131; email: Christian.Salesse@crchul.ulaval.ca

Abstract

Purpose: Müller cells are the predominant type of glial cells in the retina. They play a critical role in the retina. The purpose of this study was to generate a profile of the genes specifically expressed by human retinal Müller cells and to identify genes that may be responsible for retinal diseases.

Methods: Subtractive hybridization is a method process by which two populations of mRNA are compared in order to obtain clones of genes expressed in one population but not in the other. A cDNA subtraction library was constructed using RNA isolated from human Müller cells and human astrocytes. PCR-select differential screening was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced and analyzed using the NCBI BLASTN program to identify sequence homologies.

Results: We identified 194 clones specifically expressed in human Müller cells. Among these clones, 102 corresponded to known human genes. Of the remaining 94 clones, 75 corresponded to expressed sequence tags or genomic clones and 19 transcripts did not match with any sequence in databases, and are possibly novel genes.

Conclusions: The analysis of the subtraction library revealed genes that are specifically expressed by human Müller cells. Some of these genes are unidentified, novel genes that are specific to Müller cells as determined by RT-PCR and Northern blot analyses. These novel genes thus represent candidate genes for retinal diseases.

Introduction

The retina is divided into the retinal pigment epithelium and the neural retina. The neural retina consists of six neuron types that are in close contact with blood vessels and are supported by radial glial cells [1]. Four types of glial cells are found in the human retina [2], of which the Müller cells represent the main class. They extend throughout the whole thickness of the neural retina, forming the limits of the retina at the outer and inner limiting membranes (Insight). In addition to their function in structural support, Müller cells regulate the extracellular retinal environment by buffering the light-evoked variations of ions, in particular, the K⁺ concentration in the extracellular space [2]. Müller cells also remove glutamate from the extracellular space by active uptake, and they contain specific ion channels and transport systems [2]. The presence of cellular retinol and retinal-binding proteins in Müller cells suggests they may participate in the visual cycle of retinoid metabolism [3,4]. Müller cells are thought to perform many of the functions provided in the brain by oligodendroglia and astrocytes, which are absent or at least sparse in mammalian retinas. For instance, they synthesize and store glycogen, and they provide glucose to the retinal neurons [1]. Because Müller cells are the main support cells in the neural retina, it is not surprising that they play an active role in a number of metabolic processes that are vital to the normal retinal function.

The highly specialized function of the retina requires a large number of specifically expressed genes. The isolation of novel genes expressed exclusively or predominantly in the eye has contributed greatly to the understanding of retinal function and of the pathogenesis of retinal diseases. For example, several methods have been used to isolate retina and retinal pigment epithelium (RPE)-specific genes, including differential hybridization [5-7], expressed sequence tag (ESTs) mapping [8-11], microarray [12-16], SAGE [16-20], subtractive hybridization [21-27], and large-scale sequencing of retinal cDNA libraries [28-33]. Moreover, to our knowledge, only one proteomic study reported the protein expression profile of Müller cells [34].

We used suppressive subtractive hybridization (SSH) to identify genes that are specifically expressed by Müller cells. Subtractive hybridization has been previously used to compare induced versus noninduced states [35-37], disease versus nondisease [38-42], aging versus nonaging tissues [43,44], or various stages of differentiation and development [45-49] and to identify tissue-specific genes [25,50-53]. This method normalizes sequence abundance, achieves high enrichment of differentially expressed cDNAs by a single round of subtractive hybridization and allows el4imination of cDNAs that are common between two cell populations [54]. Because Müller cells and astrocytes share many similar functions in the retina, we chose to perform subtraction and differential screening of human retinal Müller cells with brain astrocytes. The sequence of 194 cDNAs specifically expressed by human retinal Müller cells was analyzed and compared with those from the GenBank, UCSC, and Ensembl databases. We found 136 of these cDNAs had previously been characterized, whereas others corresponded either to genomic clones or hypothetical proteins. Nineteen transcripts did not match with any sequence in databases, but mapped to a chromosomal location in the human genome, and are possibly novel genes, some of these genes being located in known loci regions of retinal pathologies.

Methods

Cell cultures and isolation of RNA

Isolation and culture of human Müller cells was performed as previously described [55]. This study was conducted in accordance with the Declaration of Helsinki and our institution's guidelines. Post-mortem human eyes from donors, whose ages ranged 2-40 years, were obtained from the Banque d'yeux Nationale Inc. (Québec, Canada). Human brain astrocytes (NHA6700) were purchased from Clonetics (Walkersville, MD) and cultured according to the manufacturer's protocol (ABM medium supplemented with 20 ng/ml human epidermal growth factor, 25 μg/ml insulin, 25 ng/ml progesterone, 50 μg/ml transferrin, 50 μg/ml gentamicin, 50 ng/ml amphotericin B, and 5% fetal bovine serum). Cells were cultured at 37 °C under 5% CO₂. When cultured cells reached confluency, total RNA from human retinal Müller cells and human astrocytes were isolated with the RNeasy Mini kit from Qiagen (Missisauga, Ontario, Canada) and poly (A)⁺ RNA of human astrocytes were subsequently obtained by using the Oligotex mRNA kit from Qiagen.

cDNA and suppressive subtractive hybridization

First strand cDNAs was synthesized using the SMART PCR cDNA synthesis kit from Clontech (Palo Alto, CA). First-strand synthesis was performed using 0.5 μg poly(A)⁺ RNA from human astrocytes and 2.5 μg total RNA from human Müller cells, according to the manufacturer's instructions. SSH was performed using the PCR-select cDNA subtraction kit according to the manufacturer's protocol (Clontech). The present study, focused on identifying genes that were specifically expressed by human Müller cells compared to human astrocytes. The cDNA synthesized from human Müller cells was therefore used as the tester cDNA whereas the cDNA generated from human astrocytes was used as the driver cDNA. Briefly, the tester and driver cDNAs were digested with a restriction enzyme, RsaI, to obtain shorter blunt-ended cDNA. The tester cDNA was then subdivided into two populations, and two different adaptors (adaptor 1 and adaptor 2) were then ligated to the 5' end of each strand of the tester cDNA. Two hybridizations were then performed with the tester and driver cDNAs: only the unhybridized sequences remained (representing cDNA expressed in the tester sample, i.e. Müller cells) and the hybridized sequences were eliminated. In the first round of hybridization, the concentration of high- and low-abundance sequences were equalized. During the second hybridization, only the remaining equalized and subtracted single-strand tester cDNAs bearing different end adaptors could reassociate and form double-strand tester molecules. The entire population of molecules was then subjected to PCR to amplify the desired differentially expressed sequences. Only molecules of the tester sample, which have two different adaptors, could be amplified exponentially. A second PCR amplification was performed using nested primers to further reduce any background PCR product and to enrich differentially expressed sequences, which were then directly inserted into the pGEM-T easy cloning vector system (Promega, Madison, WI).

Isolation and differential screening of cDNA clones

We used the PCR-Select Differential Screening kit (Clontech) for screening all clones obtained from the subtracted library. The first step was to amplify cDNA inserts by PCR and to array them on nylon membranes (Hybond+, Amersham Pharmacia Biotech, Baie d'Urfé, Québec, Canada). More than one thousand clones were randomly selected, and inserts were amplified with nested primer 1 (5'-TCG AGC GGC CGC CCG GGC AGG T-3') and the nested primer 2R (5'-AGC GTG GTC GCG GCC GAG GT-3'). These products were denaturated, neutralized, and applied to four nylon membranes. The second step was to hybridize each membrane with four different ³²P-labeled probes. The first probe was synthesized from the tester cDNA, the second one from the driver cDNA, the third one from the forward subtracted cDNA library, and the fourth one from the reverse subtracted cDNA library. The forward subtracted probe corresponds to the product of the subtracted library (Müller cells-astrocytes). To prepare the reverse subtracted probe, subtractive hybridization is performed where the original tester cDNA becomes the driver (Müller cells) whereas the driver cDNA becomes the tester (astrocytes). For more details about this protocol, see the user manual at Clontech. For the preparation of each of these probes, we used 50 μci of [α-³²P]-dCTP (3000Ci/mmol). Labeled cDNAs were purified with Chroma Spin-100 from Clontech. Nylon membranes were prehybridized at 72 °C and then hybridized with 10⁶cpm/ml cDNA probe at 72 °C overnight in the ExpressHyb Hybridization Solution from Clontech. Clones corresponding to differentially expressed mRNAs would hybridize only with the tester and forward probes, and not with the driver and reverse probes. The screening of the subtracted library was done following the manufacturer's instructions.

Sequencing and sequence analysis

After the PCR products were cloned in pGEM-T, the clones selected by differential screening were purified using the Qiaquick PCR purification kit from Qiagen and then sequenced using T7 and SP6 promoter primers with an ABI Prism 3100 automated sequencer (Applied Biosystems, Streetsville, Ontario, Canada). The nucleotide sequences were analyzed for similarities on the National Center for Biotechnology Information (NCBI) web site using the databases of the non-redundant nucleotide (nr), the EST (human and nonhuman) and the high throughput genomic sequences (htgs) using the Basic Local Alignment Search Tool program (BLASTN). We have also used the University of California at Santa Clara (UCSC) database as well as the Ensembl database (EMBL, Sanger, and HGMP). Poly (A), vector sequences, sequences with many ambiguities (N), and sequences with high homology to repetitive sequences were manually removed from the sequence data. Sequence identity was considered significant to a database entry when a match of p<1x10^-20 (smallest sum probability) and an aligned region >75% over the entire length were obtained. When a search revealed a significant match to more than one known gene, only the highest scoring hit was included in the data set.

Reverse transcription-polymerase chain reaction and Northern blot

For semiquantitative RT-PCR, total human RNA from brain, heart, kidney, liver, lung, muscle, pancreas, spleen, and uterus (Clontech) were used. Total RNA from human retina and Müller cells (prepared from human donor eyes; Banque d'Yeux Nationale Inc., Centre Hospitalier de l'Université Laval) was isolated using an RNA extraction kit (Qiagen) according to the manufacturer's instruction. Reverse transcription was performed using 5 μg total RNA and 0,2 μg oligo(dT) primer (Fermentas, Burlington, Ontario, Canada) following the manufacturer's protocol for synthesis of first strand cDNA. Gene-specific primers were designed to amplify cDNA fragments (Table 1). β-actin amplification was used to determine the integrity and the quality of the cDNA. Cycle parameters for β-actin (denaturation 94 °C, 1 min; annealing 60 °C, 1 min; extension 72 °C, 1 min) with a total number of 30 cycles. All other RT-PCR reactions were conducted for 35 cycles and 10 μl of the amplification products were separated by agarose gel electrophoresis and visualized under ultraviolet illumination.

The probes used for the Northern blot analyses have been prepared as following. A single band corresponding to the PCR product amplified with our selected primers was separated by agarose gel electrophoresis, extracted from the gel (DNA extraction kit from agarose gels, Millipore), purified (Qiaquick PCR purification kit, Qiagen), cloned in the pGEM-T vector, and then sequenced. These RT-PCR products of HMC-B33, HMC-C46, HMC-E8, HMC-E42, HMC-F2, HMC-Clone 65, HMC-Clone 92, HMC-Clone 106 and HMC-Clone 142 were labeled with α-³²P-dCTP using the random priming labelling kit of Promega (Prime-a-gene labelling system). PolyA+ RNA (1 μg/lane) from human retina was isolated with Oligotex Midi mRNA kit from Qiagen following the manufacturer's instructions. mRNA was separated by electrophoresis on a 1.2% fomaldehyde-agarose gel, and blotted to Nylon + membranes (Hybond N, Amersham) using 20X SSC as the transfer buffer. Filters were prehybridized for 2 h and hybridized overnight at 42 °C with ³²P-labeled probes (10⁸cpm/ml). The blot was then washed twice in 1X SSC, 0.1% SDS at room temperature for 20 min. and twice in 0.2X SSC, 0.1% SDS at 42 °C for 20 min. Blots were exposed on Kodak X-OMAT-AR at -80 °C for 24 h.

Results

Successful SSH decreases the concentration of ubiquitously expressed (housekeeping) genes, and increases the concentration of tissue-specific genes [54]. The effectiveness of the technique was tested by PCR with actin and cellular retinaldehyde binding protein (CRALBP) a gene known as a marker of Müller cells [56,57], before and after subtraction. Samples were analyzed after 18, 23, 28, and 33 cycles of PCR for actin. As expected, this housekeeping gene displayed a significant decrease in the amount of transcript after subtraction (23 cycles) compared to before subtraction (18 cycles; Figure 1A). These data indicate that the amount of β-actin transcript was reduced by over 20 fold on the basis of this five cycles difference as a result of normalization, which was consistent with the production of a successful subtraction library. In contrast and also as expected, the amount of transcript of CRALBP, increased after subtraction (Figure 1B). Altogether, these data demonstrate that subtraction was successful.

We obtained 1,342 clones following SSH. Inserts of each clone were amplified by PCR and then separated on 1% agarose gel. Clones containing no insert as well as multiple inserts were excluded such that only 777 clones were conserved for subsequent analysis. To identify cDNAs in the subtracted library that are preferentially or specifically expressed by human Müller cells, four different radiolabeled probes were used to screen four membranes (dot blots) containing the cDNAs of the subtracted libraries (the forward and the reverse subtraction libraries) as well as the cDNAs of human Müller cells and those of human astrocytes. Differential screening of these 777 clones resulted in the selection of 248 positive clones, which indicated that differential screening is essential for minimizing the false positive clones. All of these clones were sequenced in both directions. The length of the sequences of these clones ranged from 103 to 916 bases with an average of 483 bases. The nucleotide sequence of 54 out of these 248 positive clones was of insufficient quality to yield useful data for this study. Indeed, poly (A), vector sequences, sequences with many ambiguities (N) and sequences with high homology to repetitive sequences were manually removed from the sequence data. The remaining 194 sequences were analyzed by comparing them to NCBI, EMBL and UCSC databases as well as with human EST and nonhuman EST databases, using BLAST analysis. The results of this analysis showed that the majority (53%) of these clones corresponded to known human genes (Table 2). The analysis of the clone sequences showed that among these 194 sequences, 121 clones matched to human genes, 102 of them to known human genes and 19 to human hypothetical proteins (Table 2). Among the remaining 73 cDNAs, 34 clones matched to ESTs: 22 clones to human ESTs and 12 clones to non-human ESTs. Among the remaining 39 clones, 20 clones corresponded to human genomic sequences and 19 transcripts did not match any sequence in databases (Table 2).

A summary of the known human genes that are identical to at least one Müller cell cDNA is listed in Table 3. It can be seen that proteins found in the categories of translation/protein synthesis (22.3%), signaling molecules/growth factors/receptors (19.0%), protein processing/trafficking (9.9%), and cellular metabolism (7.4%) are the biological processes frequently represented in the subtracted library of human Müller cells. Several hypothetical proteins with an unknown function were also found and represented 15.7% of all cDNAs corresponding to known genes (Table 3). The human Müller cells subtracted cDNA library contained a certain number of redundant genes (Table 3). The 121 human Müller cell cDNA clones (known human genes and hypothetical proteins) corresponded to 72 different genes when taking redundancy into account (Table 2). Indeed, among these 121 positive cDNA clones, 57 were found only once, eight were found twice, three were found three times, and four were found six times or more, for a total of 72 different genes (Table 3). The gene most highly represented in the cDNA subtracted library (18 times) was from the translation/protein synthesis category, i.e., the eukaryotic translation elongation factor-1 alpha-1. Thus, when taking redundancy into account, we identified 72 known human genes (Table 3), 34 ESTs (Table 2), 20 human genomic clones (Table 2), and 19 unknown cDNA clones (hypothetical proteins; Table 2). In fact, redundancy has been found only for the category "cDNAs matching with known human genes" (Table 2). The 19 unknown cDNAs are new transcripts and they mapped to a chromosomal location in the human genome, and are thus possibly novel genes. These sequences were submitted to GenBank and their accession numbers (CK726457-CK726475) are provided in Table 4.

We compared the chromosomal location of human ESTs and novel Müller cells cDNAs identified in the subtracted library (Table 3 and Table 4) with the location of known retinal pathology loci on the RetNet website. RetNet provides a list of cloned and/or mapped genes that cause retinal diseases and, to date, 197 disease genes were reported. In total, 21 clones from the library were found to be located within known retinal pathology loci (Table 5). Thus, a large number of the uncharacterized Müller cells cDNAs were located in chromosomal regions of important retinal pathologies. Among these 21 cDNAs, 13 corresponded to hypothetical proteins, genomic clones or human ESTs. We chose the five human ESTs (HMC-E42, HMC-F2, HMC-Clone 92, HMC-Clone 106, and HMC-Clone-142) for further analyses because they corresponded to hypothetical proteins. Among the eight remaining cDNAs, four were chosen because they were novel and they were mainly expressed in Müller cells (HMC-B33, HMC-C46, HMC-E8 and HMC-Clone 65). These nine cDNAs were selected for further analyses by RT-PCR and Northern blot.

Semi-quantitative RT-PCR was used to investigate the specific or preferential expression of these nine cDNA sequences in human Müller cells. The RNA expression of these cDNAs was thus determined in 10 different tissues as well as in Müller cells. Northern blot analyses were performed to confirm that an amplicon was expressed for each of these clones and to determine the approximate length of the corresponding cDNA by using mRNA from human retina. The results obtained for the novel Müller cells cDNAs are presented in Figure 2 (HMC-B33 (Figure 2A,B), HMC-C46 (Figure 2C,D), HMC-E8 (Figure 2E,F), and HMC-Clone 65 (Figure 2G,H)). The RT-PCR data showed that HMC-B33 is expressed only in Müller cells, as a single transcript of 263 bp (Figure 2A). The HMC-Clone 65 was expressed both in Müller cells and retina, as a single transcript of 295 bp (Figure 2G). In contrast, clones HMC-C46 and HMC-E8 were expressed predominantly in Müller cells but also in other tissues (when normalized to actin expression in the same tissues, see Figure 2I). Indeed, HMC-C46 was expressed in heart, liver, spleen, kidney and uterus (Figure 2C), whereas HMC-E8 was expressed in Müller cells (Figure 2E) and a non specific PCR product, as determined by sequencing, of 500 bp could be seen in the brain, liver and retina (Figure 2E). Moreover, a 850 bp transcript can be seen for clone HMC-C46 together with an additional minor transcript at about 400 bp whereas clone HMC-E8 was expressed as a single transcript of 220 bp. The Northern blot analyses allowed identification of a single transcript of 0.5 Kb for HMC-B33 (Figure 2B), two different transcripts of 2.2 and 3.5 Kb for HMC-E8 (Figure 2D), three different transcripts of 2.2, 2.8 and 3.7 Kb for HMC-Clone 65 (Figure 2H) and five different transcripts of 0.6, 1.3, 2.3, 2.5, and 3.8 kb for HMC-C46 (Figure 2F). Figure 3 shows the results obtained for the five known ESTs (HMC-E42 (Figure 3A,B), HMC-F2 (Figure 3C,D), HMC-92 (Figure 3E,F), HMC-106 (Figure 3G,H), and HMC-Clone 142 (Figure 3I,J)). It can be seen that HMC-F2 (738 bp), HMC-Clone 106 (742 bp) and HMC-Clone 142 (293 bp) were only expressed in Müller cells whereas HMC-Clones E42 (205 bp) and 92 (300 bp) were expressed both in Müller cells and retina. In addition, HMC-E42 (Figure 3A) and HMC-Clone 92 (Figure 3E) were expressed as single transcripts. In contrast, more than one transcript could be seen for clones HMC-F2 (Figure 3C), HMC-Clone 106 (Figure 3G) and HMC-Clone 142 (Figure 3I). The Northern blot analyses allowed the identification of a single transcript of 1.5 Kb for HMC-E42 (Figure 3B), 1.4 Kb for HMC-Clone 92 (Figure 3F) and 1.1 Kb for HMC-Clone 142 (Figure 3J), two transcripts of 0.3Kb and 3.1Kb for HMC-Clone 106 (Figure 3H), and six transcripts for HMC-F2 (the three major transcripts corresponded to 2 Kb, 3.2 Kb and 4.9 Kb; Figure 3D).

We used Web tools for a more detailed analysis of each cDNA studied by RT-PCR and Northern blot (Figure 2 and Figure 3). The translation of a cDNA sequence using Translate allowed us to obtain one or more protein sequence with the different open reading frames. Among the different protein sequences obtained; we used the ORF Finder to determine the proper open reading frame. After choosing the most appropriate protein sequence, we looked for conserved domains with InterProScan, Conserved domain, and Pfscan servers. We have found a peptide signal for each translated protein sequence. We also found a transmembrane region for HMC-C46, and HMC-Clone 106. Two proteins that translated with the same open reading frame were found with the cDNA sequence of HMC-C46 which is consistent with the observation of several bands by Northern blot (Figure 2D). Moreover, 62% identity were obtained for HMC-E8 with a sequence of 47 amino acids from a full length sequence of 348 amino acids which corresponded to mitochondrial 39S ribosomal protein L3 (MRP-L3; NCBI accession number; P09001). The clone HMC-F2 was 56% identical with a sequence of 52 amino acids from a full length sequence of 148 amino acids, which corresponded to the single strand binding protein family (NCBI accession number; NP_003134). Finally, HMC-Clone 92 shared 82% identity with a sequence of 89 amino acids from a full length sequence of 1130 amino acids which encoded probable cation transporting ATPase3 (NCBI accession number; Q9H7F0).

Discussion

The present study describes the use of suppressive subtractive hybridization to screen for the first time clones that are differentially expressed by human retinal Müller cells. We chose this method because it was not dependent on the availability of previously cloned cDNA sets and it allowed cloning of unknown genes. This method also allowed for the normalization of frequent and rare cDNAs and for the subtraction of cDNAs that are common between two cell populations. The sequencing of 194 cDNAs from Müller cells resulted in the identification of several known genes as well as yet to be characterized novel cDNAs expressed by Müller cells.

The human Müller cells subtraction library was prepared by using Müller cells and cerebral astrocytes, which are both glial cells and thus share similar functions. Because retinal astrocytes have not yet been successfully isolated and cultured, cerebral astrocytes were used to prepare the subtractive hybridization library. Moreover, cerebral and retinal astrocytes have a common origin in the brain [58-60]. Indeed, nerve cells of the retina are derived from a germinal neuroepithelium in which individual progenitor cells can give rise to all classes of retinal neurons, as well as to Müller glial cells [60-63]. Retinal astrocytes are not derived from the retinal neuroepithelium but, rather, migrate into the retina from the optic stalk. All genes common to both Müller cells and astrocytes have been subtracted with the suppression subtractive hybridization method. Therefore, a number of genes known for metabolism such as aldehyde dehydrogenase, glutamate dehydrogenase, glutathione s-transferase and many others are not present in Table 3. Given that no differential hybridization, large-scale sequencing, microarray, SAGE or subtractive hybridization library of human Müller cells has been performed until now, the genes identified in Müller cells in the present study were compared with libraries using whole retinas [18,25,28,30,64,65]. However, such a comparison can not be extensive since the retina contains a large number of cells and that genes expressed specifically by Müller cells are likely to be expressed at low level and thus undetected in these retinal libraries. This could indeed be the case for six of the nine clones shown in Figure 2 and Figure 3 (HMC-B33, HMC-C46, HMC-E8, HMC-F2, HMC-clone 106, and HMC-clone 142). Distler and Dreher [66] estimated there are 8 to 8.6 millions Müller cells in the monkey retina. Moreover, there are approximately 127 million protoreceptors [1]. There are also amacrine, bipolar, ganglion, horizontal and interplexiform nerve cells in the mammalian retina. Ganglion cells account for approximately one third of these nerve cells. Given that there are approximately 1.2 millions ganglion cells in the monkey retina [67], it can be estimated that there are 3.6 millions nerve cells in the mammalian retina. Altogether, there are approximately 139 million cells in the mammalian retina. Müller cells thus account for approximately 6% of all retinal cells. Therefore, if the abundance of a given gene is low in Müller cells, it could be difficult to amplify this gene by RT-PCR using RNA extracted from the whole retina. In contrast, it should be possible to amplify a low abundance gene when using cDNA of Müller cells. However, the more sensitive Northern blot analyses (Figure 2 and Figure 3), which were performed with mRNA from whole retinal extracts clearly demonstrated that all clones are expressed by the whole retina (in addition to Müller cells).

One of the most redundant genes in the subtracted library (Table 3) is the eukaryotic translation elongation factor-1 alpha-1 within the translation/protein synthesis category. This protein is involved in the binding of aminoacyl-tRNAs to 80S ribosomes; there are more than 10 copies of this gene per haploid genome in humans [68]. It has also been found in two other studies presenting the profile of gene expression in the retina [18,30]. Other redundant genes like proteoglycan 1 secretory granule, serpine 2 and β amyloid protein have also been found in other studies on the retina [18,28,30] but not yet in Müller cells.

It is noteworthy that the following proteins found in the subtracted Müller cells library have also been identified in Müller cells by previous studies: protein tyrosine phosphatase (PTP), cadherin 11, transferrin, sulfatase, and dystrophin (Table 3). The first four proteins have been implicated in development and cell differentiation [69-73], and dystrophin is a protein involved in the homeostasis of Müller cells [74]. Protein tyrosine phosphorylation/dephosphorylation (PTP) is important in cell-cell interactions signaling that regulate growth and differentiation of multicellular organisms. In neural retina, high levels of protein phosphotyrosine are present in the plexiform layer and external limiting membrane, where cell-cell interactions are abundant [75]. As shown by Biscardi et al. [76], the phosphorylation process of protein tyrosine in the retina is preferentially associated with the plasmalemma of Müller cells at the site of glia-glia and glia-photoreceptor contact. Shock et al. (1995) demonstrated the expression of seven different PTPs in chick Müller cells. PTP identified in our subtracted Müller cells library (Table 3) is a potential signaling molecule for eye development [76]. Cadherins have been shown to be involved in cell adhesion, morphogenesis, cell growth and differentiation [77]. Faulkner-Jones et al. [70] identified 10 different cadherins in the postnatal mouse eye, cadherin 11 (CDH11) being the most frequent one. Marchong et al. [71] found that CDH11 mRNA levels decreased in the maturing murine retina, suggesting an important role during retinal development. CDH11 is highly expressed in a subset of cells of the inner nuclear layer of the retina [71]. These expression studies concur with the data reported by Honjo et al. [78], which showed CDH11 is localized in the retina and more specifically in Müller cells. CDH 11, which has been identified in our library (Table 3), could thus play an important role in the function of Müller cells. In mammalian, transferrins are responsible for the sequestration, transport and distribution of free iron [79] which is cytotoxic, promotes oxidation/lipid peroxidation, and is associated with age-related macular degeneration [80]. Moreover, several laboratories have suggested that transferrin plays an important role in neuronal differentiation (growth, neurotrophic, and differentiation-promoting factors) and in normal glial functions [72,81-84]. Some of these neuromodulator properties have been attributed to transferrin in retinal tissues [85]. Zeevalk and Hyndman [72] reported an immunoreactive pattern for transferrin in Müller cells. The transferrin protein identified in our library (Table 3), has also been previously detected in different studies of the gene expression profile of the human retina [13,18,30,65]. Recently, a subtractive hybridization study of the neuroepithelium known to contain glial progenitors identified sulfatase (Sulf1) [73], which has also been found in Müller cells subtracted library (Table 3). Sulf1 is a candidate gene involved in the process of glial cell specification. It could thus be involved in the developmental stage of Müller cells.

Dystrophin is a cytoskeletal membrane-associated protein [86]. In addition to the full-length dystrophin, four shorter isoforms have also been identified [87,88] (Dp260, Dp140, Dp116, and Dp71). Three of those isoforms are present in the retina [88-90] where Dp71 is localized exclusively in Müller cells [74]. In the rat retina, a protein complex including Dp71 [90,91] has been proposed to be responsible for the clustering of proteins that play a key role in K⁺ and water balance processes (AQP4 and Kir 4.1) in specific membrane areas of Müller cells [74]. In the absence of dystrophin Dp71, the retina is more vulnerable to vascular disorders, especially to ischemia [74] which is typical of diabetic retinopathy and glaucoma. Dystrophin Dp71 is the isoform identified in our subtracted Müller cells library (Table 3).

A large number of genes responsible for retinal diseases still remain unknown. We identified 19 new cDNA fragments, from which nine are located in chromosomal loci of retinal diseases, suggesting Müller cells could play a role in such diseases (Table 5). Among those, two cDNAs have similarities with known mitochondrial family proteins, HMC-E8 (mitochondrial ribosomal protein (MRP)) and HMC-F2 (single-strand binding protein). These are typical proteins involved in replication and repair. Mutations in mitochondrial DNA are known to be associated with a wide spectrum of human diseases with different clinical features, including neuromuscular disorders, diabetes, aplastic anemia, and deafness. Kenmochi et al. [92], found that certain MRP genes are present in candidate regions for retinitis pigmentosa and for disorders involving neural dysfunction such as Moebius syndrome and Hallervorden-Spatz syndrome. Because patients with mitochondrial disease frequently show features such as retinopathy, myopathy, and neuropathy, it is worthwhile to pursue the possibility that defects or mutations in the MRP genes may result in such pathological conditions.

The cDNA library shown in this study represents a significant resource for the gene expression profile of glial cells and more specifically of human Müller cells. A significant number of new clones (19 new cDNAs; hypothetical proteins) were found to be expressed by Müller cells, some of which have been shown by RT-PCR to be only expressed by these cells. The characterization of these possibly new genes is under way. This study presents the basis for more detailed expression analysis, characterization of several interesting genes, and future investigations into their putative roles in retinal diseases.

Acknowledgements

The authors thank la Banque d'Yeux Nationale Inc. for providing retinal tissues. This work was supported by the Natural Sciences and Engineering Research Council of Canada. C. Lupien is recipient of a fellowship from the Canadian Institutes of Health Research and the Vision Research Network from the "Fonds de la Recherche en Santé du Québec" (FRSQ). C. Salesse is a Chercheur boursier national from the FRSQ. The Banque d'Yeux Nationale is partly supported by the Réseau de Recherche en Santé de la Vision (FRSQ).

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