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Physiol. Genomics 25: 9-15, 2006. First published December 20, 2005; doi:10.1152/physiolgenomics.00121.2005
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Received 24 May 2005; accepted in final form 13 December 2005.
Physiological Genomics 25:9-15 (2006)
American Physiological Society © 2006 American Physiological Society

Call For Papers: Comparative Genomics

Genomic annotation of 15,809 ESTs identified from pooled early gestation human eyes

K. W. Choy1,2, C. C. Wang2,3, A. Ogura3, T. K. Lau2, M. S. Rogers2, K. Ikeo3, T. Gojobori3, D. S. C. Lam1 and C. P. Pang1

1 Departments of Ophthalmology and Visual Sciences, Chinese University of Hong Kong, Hong Kong
2 Departments of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong
3 Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Sokendai, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To complement cDNA libraries from the human eye at early gestation and to discover candidate genes associated with early ocular development, we used freshly dissected human eyeballs from week 9–14 of gestation to construct the early human fetal eye cDNA library. A total of 15,809 clones were isolated and sequenced from the unamplified and unnormalized library. We screened 11,246 good-quality ESTs, leading to the identification of 5,534 nonredundant clusters. Among them, 4,010 (72%) genes matched in the human protein database (Ensembl). The remaining 28% (1,524) corresponded to potentially novel or previously unidentified ESTs. We used BLASTX to compare our EST data with eight organisms and found common expression of a high portion of genes: Caenorhabditis briggsae (26%), Caenorhabditis elegans (27%), Anopheles gambiae (37%), Drosophila melanogaster (32%), Danio rerio (42%), Fugu rubripes (49%), Rattus norvegicusvalitus (52%), and Mus musculus (59%). Nevertheless, 48% (2,680 of 5,534) of the genes expressed in the early developing eye were not shared with current NEIBank human eye cDNA data. In addition, eight known retinal disease genes existed in our ESTs. Among them, six (COL11A1, BBS5, PDE6B, OAT, VMD2, and PGK1) were conserved among the genomes of other organisms, indicating that our annotated EST set provides not only a valuable resource for gene discovery and functional genomic analysis but also for phylogenetic analysis. Our foremost early gestation human eye cDNA library could provide detailed comparisons across species to identify physiological functions of genes and to elucidate evolutionary mechanisms.

expressed sequence tags; across species


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DEVELOPMENT OF THE HUMAN EYE involves complex interactive processes of molecular and cellular development of the neural and surface ectoderm, requiring multiple signaling pathways for proper cell specification and differentiation. Induction between cells is mediated by a complex signal transduction mechanism and involves a wide variety of signaling molecules and receptor proteins. Such intriguing biological processes are essentially governed by genes. To identify genes or cellular pathways that are selectively turned on or off in response to extrinsic factors or intrinsic genetic programs, it is necessary to deduce the catalogue of mRNAs expressed in a specific cell or tissue type at various stages of development. A systematic evaluation of transcripts and their expression levels at different stages of eye development should lead to better understanding of genes that contribute to cell patterning and differentiation as well as their underlying regulatory pathways.

During the last decade, a number of approaches have been utilized to identify cell- and tissue-specific genomes and transcriptomes for mouse, rat, and human tissues (3, 5, 11, 12a, 19a, 27, 30, 32, 33). Serial analysis of gene expression, subtractive hybridization, and analysis of expressed sequence tag (EST) databases have been used to identify differentially expressed tissue-specific genes (2, 5, 11, 27, 30, 32). On the basis of the occurrence of corresponding tags, the data obtained also provide important information for quantitative measurement. In particular, the data generated serve in the detection of functional alternatively spliced transcripts and computer-based methods for gene expression analysis (14, 15). Transcript databases have also been extensively excavated for extracting alternative splicing information within the same species. They also represent a potentially valuable resource for the discovery of alternative splice variants in other species (13). On the other hand, microarray-based global expression profiling of tissues in animal models with defects in a transcription factor gene can point to downstream regulatory targets and provide candidate genes for functional studies and cloning of disease loci. This approach has been utilized successfully in studies of the mouse retina (17, 20). A number of ESTs have been isolated from cDNA libraries that represent different stages of human or mouse eye tissues, including the mouse retina (3, 6), human adult retina and fovea (2, 4, 28), retinal pigmented epithelium (RPE) (9), cornea (22), and trabecular meshwork (10), and from the developing mouse retina (20).

Of the 110 candidate genes from the neural retina that have been identified, mutations in many of these have been associated with retinal and macular diseases (35). In addition, a large-scale transcription analysis of the embryonic retina in the mouse reveals the existence of thousands of expressed sequences with unknown functions (8, 20). Further characterization of novel ocular-specific ESTs therefore should lead to identification of transcripts with biological functions. However, the application of mouse, rat, and chick eye cDNA libraries is hindered by the lack of well-developed genetic and genomic resources. Furthermore, a majority of the transcribed sequences were mainly derived from cells in culture, from animal models, or from the adult, which have limited physiological and histological similarities to the human eye and are therefore not necessarily representative of the native human developing eye. A number of human fetal eye ESTs have been previously reported, but the actual timing (age) of the tissue used for obtaining these ESTs and library were either not recorded or at a much later developmental stages (33). In the present study, we attempted to establish a set of ESTs with deep coverage of expressed genes during early human eye development. We generated, annotated, and analyzed over 15,800 EST clones derived from a cDNA library constructed from developing human eyes in the first trimester of gestation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RNA isolation from fetal eyeballs.
Twenty-three eyes (at least 3 eyeballs from each stage) from fetuses between gestational weeks 9 and 14 were enucleated, with surrounding non-eye tissues including connective tissues and muscles removed, and placed immediately into a petri dish kept on ice containing diethylpyrocarbonate-treated PBS (without calcium chloride and magnesium chloride, pH 7.4). Institutional Review Board ethics approval and written consents were obtained before sample collection. Eyes were dissected in an RNase-free environment under a stereo zoom microscope. Only eyeballs with optic nerve heads and intact posterior and anterior segments were studied. mRNA was directly isolated using the QIAGEN RNeasy kit (Qiagen; Valencia, CA) following the manufacturer's protocol. mRNA preparations from gestational weeks 9-14 were pooled, and their integrity was determined by SYBR gold-stained agarose gels (Molecular Probes; Eugene, OR).

cDNA library construction.
Complete details of library construction are described elsewhere (19a, 23). Briefly, poly(A) RNA, isolated by oligo(dT) cellulose column chromatography (Qiagen), was used for the synthesis of cDNA. A NotI primer adaptor [5'-GAC TAG TTC TAG ATC GCG AGC GGC CGC CC(T)15–3'] and SuperScript II reverse transcriptase (Invitrogen; Carlsbad, CA) were used for the first-strand cDNA synthesis. After the second-strand cDNA synthesis by Escherichia coli DNA polymerase, cDNA fragments > 500 bp were fractionated on a Sephacryl S-500 column. Libraries were made from the first four fractions of 35 µl containing cDNA > 500 bp. SalI linkers were ligated onto the blunt ends of the cDNA. The NotI/SalI fragments were directionally cloned into the NotI/SalI sites of the pSPORT1 vector (Invitrogen). Plasmids were transferred into E. coli DH10B cells for amplification.

Sequence analysis and functional annotation.
High-throughput sequencing was performed on >15,809 individual clones isolated at the Center for Information Biology and DNA Data Bank of Japan. Data were analyzed using PHRED (8) to identify and trim quality reads. Vector, E. coli genome, and human mitochondrial sequences were trimmed or eliminated using Cross-match programs (23). EST sequences were assembled and clustered using PHRAP and Grouping and Identification of Sequence Tags (GRIST), a bioinformatics program that uses sequence match parameters derived from the BLAST program. BLASTN was used for making nonredundant data sets, assembling the sequences, and clustering the genes. BLASTX and the nonredundant protein database [National Center for Biotechnology Information (NCBI)] were used to find homologous genes among species, and BLASTN and the human genome (Goldenpath) were used to confirm genomic locations of ESTs. Functional annotation was conducted on the nonredundant data set of human eye ESTs based on the homologous genes obtained through the BLAST results. Gene ontology (FatiGO) was used for categorizing human eye ESTs with respect to gene function including molecular function, biological process, and cellular component (1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EST clustering of the cDNA library.
ESTs were obtained by sequencing from the 5'-end of individual clones. We picked and sequenced 15,809 clones in total. After removal of low-quality sequences and sequences representing mitochondrial and repetitive sequences, 11,246 high-quality (with good quality and containing no dust sequence) sequences were retained for further analysis (Table 1). Through the EST assembly system (or GRIST), a total of 11,246 high-quality ESTs were clustered and then assembled to 5,534 nonredundant clusters representing individual genes expressed in developing human fetal eye tissues. Homology search (BLASTX) using 5,534 clusters against the NCBI nonredundant database showed that ~28% (1,524 of 5,534) of the ESTs had no corresponding entry in GenBank (21). In our library, 3,658 clusters with a single occurrence, 1,582 clusters with two to four clones, and 295 clusters containing at least five clones represent mRNAs that may be moderately or highly abundant in the human fetal eye (Table 1).


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  		Table 1. Summary of the number of ESTs obtained in the human fetal eye cDNA library

 
Annotation and novelty of the human fetal eye EST database.
A number of sequence similarity searches were conducted to compare every EST to those in public or in our local databases. For ESTs with known gene matches in public databases, functional annotation was retrieved from NCBI UniGene (29) and LocusLink and analyzed by FatiGO (1). Of the 11,246 good-quality ESTs, 72% showed corresponding UniGene and LocusLink entries and 98% had matching human chromosomal locations (Table 1). Only 38 (0.7%) clusters contained >20 ESTs, and the majority (66%) consisted of a single EST (Table 1). Unknown and novel ESTs were clustered based on sequence similarity. In our library, 16 unknown ESTs occurred in at least 5 clones (https://cibexsv.genes.nig.ac.jp/embest). Eleven of them were novel sequences that did not match any database (see Supplemental Appendix SI).1 The redundancy of known ESTs in the libraries is elevated compared with that of unknown and novel ESTs. Although there are data sets of adult human eye ESTs in various databases, NEIBank contains the most up-to-date information of genes and proteins expressed in the eye and visual system. Compared with the NEIBank human whole eye unnormalized library (NbLib0079), our developmental stage ESTs have a similar proportion of genes involved in protein bindings (19% vs. 22%) but contained more genes related to cell communication (16% vs. 4%) and morphogenesis (7% vs. 2%). We also found that 48% (2,680 of 5,534) of the genes expressed in our early human fetal eye tissues were not shared with current NEIBank human eye cDNA data.

Table 2 summarizes the 30 most abundant genes of the library. In accordance with previous observations in NEIBank, translation factors [elongation factor-{alpha}1 (EEF1A)] and cell structure/cytoskeletal protein genes [tubulin and collagen, type III, {alpha}1 (COL3A1)] are among the most abundant and are essential for cell proliferation and the maintenance of cell and organ structure. Photoreceptor-specific genes, including rhodopsin and recoverin, were not detected, which is consistent with their late expression during development, reflecting the time at which photoreceptors mature in the human retina (3, 12).


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  		Table 2. Characteristics of the 30 most abundant clones in the human fetal eye library

 
Functional distribution of known ESTs.
As mentioned earlier, 1,524 of the 5,534 nonredundant clusters that represented expressed genes in the developing human fetal eye did not match with protein sequences in GenBank and 4,010 clusters did. These 4,010 clusters also had matched cDNA sequences of known genes (Table 1). Among them, 3,873 clusters with gene ontology at level 3 were data mined by FatiGO according to different ontology, including biological and molecular function. The two largest groups included proteins involved in nucleic acid binding (18%) and those involved in protein binding (18%). Following these were functional groups including ion binding (16%) and hydrolase activity (10%), and those involved in transferase and receptor activity were 8% and 4%, respectively (Fig. 1A). In general, at early developmental stages more genes (69%) appear to be involved in cell metabolism/physiological process compared with the mature retina, as reported in NEIBank (Fig. 1B).


Figure 1
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  		Fig. 1. Functional categorization using 3,873 expressed sequence tags (ESTs) with gene ontology at level 3. The number next to each category indicates the percentage of genes in that class. The most highly expressed genes were those involved in nucleic acid binding (A) and physiological processes (B).

 
In silico expression of candidate retinal disease genes.
We analyzed the chromosomal locations of all ESTs with known human gene matches. We first obtained the chromosomal location for each human ortholog, followed by the determination of the location of the corresponding human gene if available. Of the 5,534 nonredundant ESTs, we were able to obtain chromosomal locations for 5,420 clusters, 3,290 of which have mouse orthologs and mapping information based on the UniGene database (Table 1). We searched RetNet to determine whether any of those reported genes were expressed in our EST library (24). Of the 110 mapped and cloned retinal disease genes reported in RetNet, 8 genes were identified in our early fetal eye cDNA library (Table 3).


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  		Table 3. Candidate ocular disease genes identified in the human fetal eye cDNA library

 
We also performed real-time PCR analysis on total RNA extracted from different parts (cornea, lens, retina, etc.) of dissected human fetal eyes at week 11 to study the dopachrome tautomerase (DCT) and DnaJ [heat shock protein (Hsp)40] homolog, subfamily C, member 3 (DNAJC3) genes randomly selected from our EST library. The DCT transcript was differentially expressed in the RPE cell layer (Fig. 2A). The DNAJC3 gene was expressed in all eye tissues but at a lower level in the lens and retina compared with RPE cells (Fig. 2B).


Figure 2
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  		Fig. 2. Differential expression of dopachrome tautomerase (DCT; A) and DnaJ (heat shock protein 40) homolog, subfamily C, member 3 (DNAJC3; B) transcripts in gestational week 11 human fetal eye tissues. RPE, retinal pigmented epithelium; others, all other ocular tissues.

 
Conservation of human eye EST homologs across species.
Human-type camera eyes may have emerged since fishes in the vertebrate lineage (23). Hence, to identify human eye EST homologs conserved for eye organogenesis, we compared our EST data with other species by using BLASTX. To affirm the accuracy in identifying homologs, we used the proteins predicted or confirmed in the genomes of Caenorhabditis briggsae, Caenorhabditis elegans, Anopheles gambiae, Drosophila melanogaster, Danio rerio, Fugu rubripes, Rattus norvegicusvalitus, and Mus musculus, in which complete genomes have been sequenced so far. We found that 1,469 (26%) and 1,521 (27%) genes in our fetal eye library were conserved in C. briggsae and C. elegans. In insects, we found that 1,793 (32%) and 1,803 (32%) genes were conserved in A. gambiae and D. melanogaster. In fishes, 2,372 (42%) and 2,772 (49%) genes were conserved in D. rerio and F. rubripes. Finally, in mammals, we found that 2,874 (52%) and 3,293 (59%) genes were conserved in R. norvegicusvalitus and M. musculus. There was a small difference in the numbers of conserved genes in humans versus Fugu (fish) and humans versus Rattus (mammals). In contrast, 1,388 genes (25%) from our human fetal eye ESTs were not conserved in the other species we tested. Furthermore, we searched the conservation of the following retinal disease genes reported in RetNet across our ESTs and in the genomes of the above eight species: collagen, type 11, {alpha}1 [COL11A1 (BY797330)]; Bardet-Biedl syndrome gene [BBS5 (BY795410)]; ornithine aminotransferase [OAT (BY795807)]; phosphodiesterase 6, ß-subunit [PDE6B (BY796723)]; phosphoglycerate kinase-1 [PGK1 (BY796795)]; retinitis pigmentosa GTPase regulator [RPGR (BY797959)]; and vitelliform macular dystrophy gene [VMD2 (BY796537)]. Intriguingly, we found that six genes (COL11A1, BBS5, PDE6B, OAT, VMD2, and PGK1) were conserved among all species. This observation suggests that genetic retinal diseases are commonly conserved across species.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our cDNA library represents the first well-characterized ESTs from the early trimester human fetal eye. In correlation with the histological findings in the human eye (7), the physiological window we examined featured the occurrence of 1) retinal morphogenesis (the inner and outer neuroblastic layers differentiate into photoreceptor cells and ganglion cells); 2) morphological changes during lens formation (from spherical to ellipsoid shape and the development of secondary fibers at the equator of the lens); and 3) commencement of ciliary body/iris development with indentation of the outer pigmented layer of the neuroectoderm. Therefore, our library represents genes that might contribute to the configuration of the neural retina, maturation of the RPE, formation of a fibrous coated envelop (sclera), and development of the vitreous, hyaloid system, and aqueous outflow pathways and the formation of the anterior chamber. This is further evidenced by the fact that a detailed breakdown of functional groups by our cDNA library demonstrated that phototransduction-specific genes including rhodopsin (RHO) and rod cGMP-gated channel {alpha}-subunit (CNGA1) were absent in our cDNA library, which is in concordance with their lack of functional activity at this early stage of human eye development.

Of the 20 most abundant genes, 19 are known genes. However, only the crystallin family [{alpha}-crystallin A, {gamma}-crystallin S, ß-crystallin A4 (CRYBA4 gene), ß-crystallin A3, and ß-crystallin B1] has been known to have biological functions related to eye development, including an important role in lens structure and a protective effect against stress-induced protein aggregation in the retina. The rest of the 20 most abundant genes have been documented in many cDNA libraries. However, none of them have been known to play specific roles in eye function. In addition, eight of these genes {clone 6 (NADH dehydrogenase subunit 4, 5q31.1), clone 7 (putative senescence-associated protein, 12q24.32), clone 14 (parathyroid hormone-responsive osteosarcoma D1 protein, 17q11.2), clone 15 (novel, 5q14.1), clone 22 (decorin isoform a preprotein, 12q21.33), clone 24 (C2orf33 protein, 5q32), clone 25 (CRYBA4, 22q12.1), and clone 28 [heterogeneous nuclear ribonucleoprotein A1 (HNRPA1), 12q13.1]} have not been reported in other existing eye libraries according to NEIBank (Table 2), with the exception of CRYBA4 and HNRPA1, which were reported in Retinome (http://www.retinacentral.org/), an integrated platform for gene-related information on the adult mammalian retina (25). Our data therefore indicate that these highly expressed genes may play important roles in developmental and morphological processes of the human eye.

To explore the potential use of our library of uniquely expressed genes in early gestation for studying ocular diseases, we randomly selected two genes, DCT and DNAJC3, for examination of transcript expression (Fig. 2). In fact, DCT, which is expressed in melanoma, melanocytes, and the retina, was differentially expressed in the RPE cell layer (Fig. 2A). The DNAJC3 gene, a member of the Hsp40 family, was expressed in all eye tissues but at a lower level in the lens and retina compared with other ocular tissues and RPE cells (Fig. 2B). Interestingly, genetic association studies in microcoria confer 13q31–32 as a susceptibility locus. This is where DNAJC3 is located, implying that DNAJC3 might be a susceptibility gene for microcoria (26). This proportion is further supported by the fact that it is highly expressed in ocular structural tissues. DNAJC3 might be required for the proliferation and differentiation of ocular cells and maintenance of the shape of eye globe. Not all of the genes involved in the developmental process or biological function during human eye development were identified in our study. However, our library contains genes that may play important roles in developmental, structural, and morphological processes. For example, osteonectin (Table 2) is a matrix-associated protein that elicits changes in cell shape, inhibits cell cycle progression, and influences the synthesis of the extracellular matrix. EEF1A, which is highly expressed in chick and mouse retinas (3, 28), is involved in the binding of aminoacyl-tRNAs to 80S ribosomes. Its silencing by short interfering RNA was associated with DNA methylation of the targeted sequence (19). It is possible that some of the genes of unknown function that we found to be highly expressed during fetal eye development may emerge as regulators of this pathway. The presence of retinal disease genes [VMD2, COL11A1, BBS5, PDE6B, OAT, retinaldehyde binding protein gene (RLBP1), RPGR, and PGK1] in the early fetal developing eye indicates possible important roles in eye development. Structural or functional defects in these genes would lead to retinal disease.

All the ESTs reported in this study were available at our interactive database EMBEST (https://cibexsv.genes.nig.ac.jp/embest). All the 5,534 ESTs have been uploaded by the DNA Data Bank of Japan and accession numbers have been issued (Accession No. BY794942-BY800475). They are all available by retrieval tool getentry (http://getentry.ddbj.nig.ac.jp/). Compared with published human and mouse eye tissue EST libraries, the percentages of unique sequences in our study were not much lower (~50% vs. 67%) (13, 17, 35), although we have not normalized or subtracted our library. This indicated that the developing human eye expresses an exceedingly large and complex variety of genes. Only 2,854 of the ESTs that we found are present in NEIBank, which contains 8,810 genes expressed in the human eye. This indicates that many genes expressed in the mature human eye are not yet expressed between gestational weeks 9 and 14. For instance, 8 of our top 30 clusters were not reported in the current NEIBank database (Table 2).

Because a large fraction of retinal genes are mutated in retinal dystrophies, we anticipate that many of the still-unidentified retinal disease genes may be among the genes that we identify here. Indeed, by an in silico expression study, eight genes identified to cause retinal diseases were expressed in our library (Table 3). In addition, these 50 genes identified in our fetal eye cDNA library were placed within chromosomal intervals mapped to Mendelian retinal diseases loci (Table 3). Focusing on these genes might greatly reduce the number of genes that may be considered within the mapped interval. Our results should greatly speed up the identification process for ocular disease-associated genes. Our library may help in identification of the 48 additional Mendelian ocular disease genes that have been mapped but not yet identified (24).

From the known human eye EST homologs conserved for oculogenesis in other species, we found only a small difference in the numbers of conserved human eye genes between Fugu (fish) and Rattus (mammals). This indicates that there was only a small advance from fishes to mammals in terms of the evolution of the camera eye lineage. In contrast, 1,388 genes were not conserved in other species we tested, suggesting that these genes might have specific functions in human cells or tissues that might be appealing for further investigations. In conclusion, our study presents the first comprehensive human fetal eye cDNA library isolated from gestation weeks 9–14. Most significantly, we showed that a large number of clusters (2,680, 47%) in our fetal eye cDNA library are not shared with current NEIBank data (30), although 98% of the nonredundant sequences matched to human genomic sequences. Our investigation revealed a number of transcripts that are predominantly expressed in the human fetal eye, and many of these encode proteins of yet-undiscovered physiological functions. Our data has produced a rich resource for future eye developmental research. The genetic comparisons across species might facilitate sequencing, predicting the physiological function of genes, and elucidating evolutionary relationships in other closely related species.


   ACKNOWLEDGMENTS
 
We thank Dr. Winnie Li from the Department of Ophthalmology and Visual Sciences, Chinese University of Hong Kong, and Takezawa Umehara from National Institute of Genetics (Mishima, Japan) for technical support and experimental advice.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. P. Pang, Dept. of Ophthalmology and Visual Sciences, Chinese Univ. of Hong Kong, Hong Kong Eye Hospital, 147K Argyle St., Kowloon, Hong Kong (e-mail: cppang{at}cuhk.edu.hk).

1 The Supplemental Material for this article (Supplemental Appendix SI) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00121.2005/DC1. Back


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 RESULTS
 DISCUSSION
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