was performed on human eyes specifically looking at the fovea. ICC detects IRBP protein in some foveal cone precursor cells by Fwk 9. NRL-positive nuclei were first found in the fovea at Fwk 11 [38]. At Fwk 12, IRBP protein is still detected only in the fovea and now is seen in the apical region of the fovea cone precursors as a continuous band of immunoreactivity, indicating that many of the cells are expressing IRBP [39]. rtPCR of cDNA from whole eyes underestimates the time of onset of expression of IRBP but accurately recapitulates the order of expression. IRBP is expressed early in retinal development in cone precursors and is one of the earliest photoreceptor-specific proteins to be expressed.

VARIABILITY IN IRBP EXPRESSION

Conditions under which animals are raised and maintained also affect IRBP expression. Kutty et al. [40] demonstrated that ambient lighting conditions play a role in the regulation of IRBP but not S-antigen gene expression. Dark-reared mice showed a marked decrease in IRBP messenger RNA (mRNA) levels when compared to control animals. The dark-reared animals showed normal morphological development and normal distribution of IRBP protein. Light deprivation had no effect on IRBP protein levels, yet IRBP mRNA levels were decreased in these animals. The mechanism of light regulation on IRBP expression is unknown and could be at the level of gene transcription, RNA stability, or protein degradation.

Dietary intake of retinoids can also affect IRBP expression. Katz et al. [41] fed animals a diet containing vitamin A in the form of retinyl palmitate (+A) or retinoic acid (−A). It is known that the −A diet does not support visual function but can satisfy the vitamin A metabolic requirements of most other tissues. Retinal degenerative changes occurred in the animals fed the −A diet, and decreased levels of IRBP were also found in retinoiddeprived animals (animals on the −A diet). However, the localization of IRBP immunoreactivity was not affected in these animals. This work raises the possibility that the IRBP content of the retina is regulated by retinoids that are functioning in the visual cycle.

MOLECULAR BIOLOGY OF IRBP

The DNA sequence for IRBP reveals an internal quadruplication in the IRBP protein structure [42, 43]. Northern blot analyses using an IRBP cDNA clone as a probe have shown that the mRNA size varies depending on the species, approximately 8 kb (bovine [44]) or 4.4 kb (human [45]). IRBP mRNA has been detected in a number of animal orders, including primates, ungulates, rodents, lagomorphs [46], amphibians [47], chickens [48], and fish [49–51]. Northern blot analyses have also been performed to demonstrate IRBP tissue-specific expression. IRBP mRNA has been found in the retina and pineal gland but not in the lens or the liver [44, 52]. Surprisingly, there have been no reports on multiple-tissue Northern blots that were probed with IRBP to search for other tissues that might express this protein. In situ hybridization of selected organs has localized IRBP mRNA to the photoreceptor cells and pinealocytes [53]. IRBP is expressed in both rods and cones [54]. Recent searches of the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/ geo/) and related microarray resources suggest

that the bulk of expression is in the pineal and retina, with low-to-nonexistent levels elsewhere. The exception is some expression in the thymus of some mouse strains [55]. There are case reports of IRBP expression in cultured cell lines (e.g., 17440 Thy+ cells), the significance of which is unknown, and numerous accounts of IRBP expression in cultured retinoblastoma cell lines, as would be expected [56].

IRBP GENOMIC CLONING

We [43] were the first to describe the complete sequence of the IRBP gene. The bovine IRBP gene is compact considering the large size of its mRNA; it contains only four exons, two of which are large. The deduced amino acid sequence indicates that the protein contains a leader sequence, which would be expected in a secreted protein. We hypothesized that the unusual gene structure and fourfold repeat is the result of gene duplication and intron loss. Our group mapped the CAP site, the start of transcription, to denote where the promoter region of the gene and the gene itself begin. Other groups deduced the sequence for the human IRBP gene [45, 57]. Subsequently, we determined the sequence of the mouse gene [58]. More recently, the genomes of several vertebrates have been determined; as a result the IRBP gene structure is known in those species. These structures are discussed in more detail in the section Evolution of IRBP.

Comparison of the nucleotide sequences of the 5′ flanking region from mouse, bovine, and human reveals sequence similarities between all three species in two places: upstream from the CAP site for about 300 bp, the proximal promoter region, and another 260-bp stretch that is upstream a further 1.2 kb, the distal promoter region [59]. Although these IRBP orthologs do not contain a canonical TATA or CAAT box, the nucleotide sequence around the CAP site matches the consensus sequence for a transcriptional initiator, Py Py A+1 N T/A Py Py [60].

EVOLUTION OF IRBP

Because of IRBP’s location in the space between the photoreceptor outer segments and the RPE, IRBP might be necessary to maintain retinoid isomerization and chemical forms while retinoids cross back and forth [1, 61]. This hypothesis is consistent with the absence of IRBP in the invertebrates and the entirely different way that invertebrates reisomerize all-trans retinal back to 11-cis retinal while still bound to opsin in the photoreceptor cell. The mammalian IRBP gene includes a remarkable quadruplication of a 300-amino acid long repeat or module [58], while the zebrafish IRBP gene appeared to contain only two repeated modules [49].

These two different but related gene structures were puzzling. We recently expanded our search for variations in IRBP that might rectify those observed structures with prior prediction of a common ancestral gene, which was hypothesized to have appeared coincident with the vertebrate eye. We examined the IRBP gene ortholog from seven mammals: Xenopus tropicalis, the chicken, and four teleost fish. We also looked for the IRBP gene in urochordates, but we could not find an ortholog. The tetrapod IRBP gene retains a single common gene structure, with a large first exon encoding three full repeats and parts of the fourth and final repeat spread among the first through fourth exons (Fig. 1).

Fig. 2. The interphotoreceptor retinoid-binding protein (IRBP) gene locus in four teleost fish. The complete genome sequences have been obtained for four fish: zebrafish (A), tetraodon (B), fugu (C), and medaka (D). Each of these fish contains a single locus that contains all the IRBP gene paralogs in each whole genome. In one case, the locus contains only one gene (medaka), in a second species, there is one full copy of an IRBP gene and a small fragment of an additional gene (tetraodon), and in two species, there are two IRBP genes in the single locus (zebrafish and fugu). In the latter case, the two genes are oriented head to tail. Also, the two paralogous genes of the single locus are quite different in gene structure. The first gene contains only a single exon, which resembles exon 1 of the tetrapod gene (as though the remaining three exons were deleted). Gene 2 has four exons like the tetrapod gene, except the two internal repeats have been deleted from exon 1. (Reprinted from [119].)

The teleost fish IRBP gene locus exhibited marked differences from the tetrapods. The fish locus possesses a single IRBP gene in the Japanese rice fish (medaka; Orizias laptipes). Yet in the other three species (zebrafish, fugu, and tetraodon), there was clear evidence of an additional gene, or a remnant, upstream of the ortholog of the rice fish IRBP gene (Fig. 2). We were surprised to detect this upstream paralog, but its gene structure is illuminating. The upstream gene, in its full form, includes the first three repeats of the tetrapod IRBP gene, while the teleost fish second gene includes only the first and fourth repeats. Each teleost gene structure contains the pattern of exons that are predicted based on the content of the specified repeats from the tetrapod gene. This provides compelling evidence that the apparently unusual gene loci in the teleost fish all arose from a common ancestral gene that resembles the tetrapod gene in structure.

This is illustrated in Fig. 3 along with a model of the evolution of the IRBP gene. We suggest that the IRBP gene arose about the same time as the vertebrates. We expect that IRBP arose by exaptation of another superfamily member of the IRBP protein family, which includes numerous members, all of which contain a similar three-dimensional fold

Duplication, subfunctionalization

Exaptation recruits a protein family

member, which becomes the IRBP

gene after internal quadruplication

Ancestral vertebrate

No IRBP gene

Fig. 3. A model for the evolution of the interphotoreceptor retinoid-binding protein (IRBP) gene. The IRBP gene likely arose when the vertebrates diverged from the urochordates. This is the time period when nature “invented” the neural crest and the skull. The intent in this model is to highlight that the proposed early internal quadruplication of the IRBP gene occurred shortly after the vertebrate diverged from the urochordates (which lack the IRBP gene). This model is based on the work of Borst et al. [43] and Rajendran et al. [49]. The major revision is the addition of a two-gene IRBP locus in the teleosts. We propose that the teleost IRBP genes were formed as a result of the combination of an early whole-genome duplication and, following a short period of drift, compaction by unequal recombination to produce a two-gene locus in head-to-tail orientation [51]. A second possibility is a single-gene tandem duplication with subsequent drift of the paralogs [51]. (Reprinted from [119].)

with a substrate-binding site for hydrophobic substrates or ligands. We predict that this gene rapidly quadruplicated itself internally. Because the urochordates and the invertebrates all lack the IRBP gene, this pins the earliest appearance of IRBP to the vertebrates. This could be tested by further examination of sighted invertebrates, especially those having ciliate-type photoreceptors or intermediates having both ciliateand rhabdomeric-type photoreceptors. Scallops bearing numerous eyes might be an appropriate test case. This model predicts that shark rays and cartilaginous fish should possess a tetrapod-like fourrepeat, four-exon gene. The elephant shark whole-genome project is being completed and might reveal informative data in the near future. The genome and IRBP sequences from nonteleost bony fish such as gars and bowfin might be informative: We predict that they will possess a single IRBP gene much like the tetrapod structure.

In summary, the origins and evolution of the IRBP gene appear to coincide with the emergence of the vertebrates and the vertebrate style of eye. This coincidence may reflect fundamental changes needed with the inversion of the photoreceptor layer

between vertebrates and invertebrates and the development of the choroid and RPE as a source of nutrients for the outer layers of the retina. This inversion of layers may in part account for the Darwinian success of the vertebrates in terms of evolutionary advantage offered by a sensory system having heightened visual acuity and improved pattern and motion detection systems.

A final comment on the extraordinary teleost fish is needed: The teleost fish underwent a whole-genome duplication at about the time they diverged from other bony fish [62]. The resulting excess genetic material was freed from selective pressures, allowing three potential scenarios for each and every duplicate copy: deletion, neofunctionalization, or subfunction partitioning [63–65]. The term neofunctionalization simply represents the divergence of one of a pair of duplicate genes, one retaining the parent gene’s function while the other duplicate takes on a new and different function. Subfunction partitioning occurs when one of the daughter duplicates takes on one part of the parent’s gene functions, while the other daughter retains only complementary parts. Together the two genes complement each other and retain all the capabilities of the parent gene.

Neofunctionalization occurred in the zebrafish IRBP locus, where one of the genes is expressed in the photoreceptor cells and the pineal gland, as expected, while the other gene is newly expressed in the inner layers of the retina [51]. A guiding principle should be recognized: The teleost fish genomes represent a huge opportunity to investigate how multidomain proteins work. There are over 20,000 teleost fish, each with about a one third chance of bearing a neofunctionalized or subfunction-partitioned gene of interest. It is now possible to find unusual gene structures by database searching of the several teleost fish whole genomes that are publicly available. These new gene structures, having different functions or subfunctions, help to assign physiological roles to any human gene that previously was refractory to analysis.

IMPORTANCE OF THE STUDY OF THE CONTROL

OF GENE EXPRESSION

All cells with nuclei contain the same genetic information, yet cells are different from each other due to the expression of the different subsets of genes within each cell. The pattern of RNA abundance is characteristic of a particular cell type. The retina contains five major classes of neurons with specialized cell types within each class. The study of the control of gene expression begins the elucidation of what makes cells different from one and other. There are many different points at which gene expression can be controlled. The regulation of gene expression can occur at the level of transcription or translation. External signals can change the repertoire of genes expressed by a cell, as was seen by the dark rearing of animals and IRBP expression levels [40].

It was noticed in the late 1970s and early 1980s that the 5′ flanking region of mammalian genes contain conserved sequence motifs that are important in the control of transcription [66]. These DNA sequence features were initially used to build models of eukaryotic transcription based loosely on the classical works of molecular biology on bacterialpromoters, operators, and repressors, which usually are located immediately adjacent to the gene that they control [67, 68]. Elegant models of the process of eukaryotic RNA polymerase II complex formation and the initiation of RNA synthesis have since been built.

Within the niche of vision research, there is a notion that more or less the exact same transcriptional processes occur in the human eye, but with specialized sets of transcription factors and transcription factor binding sites (TFBSs) that ensure specificity and selectivity of transcription during the development of the eye and, following that, during the required maintenance of terminally differentiated cells of the eye.

Transcription factors are proteins that bind to sequence-specific regions of DNA and decipher (switch on) the genetic information that encodes the regulation of gene transcription (for review, see [66]). These proteins contain a DNA-binding module that is linked to one or more activation or repression modules. There is a variety of DNA-binding modules, including those containing helix-loop-helix (HLH), homeodomain, zinc finger, and leucine zipper motifs. Some transcription factors, such as those containing an HLH or homeodomain motif, can interact to form homoor heterodimers. Transcription factors are members of multigene families that have expanded during evolution [69].

As each gene that is expressed in the eye was sequenced, inevitably it was compared with known conserved TFBS motifs and patterns in the hunt for the elements that might control its promoter. Computer-mediated studies of this type can make numerous cross comparisons, among them cross-species comparisons of orthologs and cross comparisons of genes that share expression patterns. From these computer-mediated studies of DNA sequences, many candidate cis elements were predicted, and numerous studies sought to test whether the predicted motifs had any effect on the promoter activity of the gene in question. Great debates have arisen over the precise nucleotide sequence of a binding site or exactly which of a series of closely related trans factors is just the right one that stimulates cell-type- specific gene expression of someone’s favorite eye protein. The net result so far has been an incomplete story, but within the story certain promoters and transcription factors are well studied. A few relationships of TFBSs and the paired protein transcription factors show some promise for adequately describing the complex behavior, interrelatedness, and coregulation that generate proper levels of the myriad transcripts needed for visual function.

Initial work on the IRBP and other photoreceptor-specific promoters started in the middle-to-late 1980s and consisted of, for the most part, two joint strategies. Once sequence comparisons had been made that provided testable hypothesized cis elements, these joint experiments included first the manipulation of the 5′ flanking regions of the IRBP and other photoreceptor-specific protein genes (which included nested deletion series, point mutations, replacements, and combinations with other known cis elements) and the construction of reporter constructs. These constructs were introduced into retina cells or animals by transient transfection or creation of transgenic animals, usually mice or frogs (cf. [70]. Playing important parts in the transient transfection strategy was the implementation of permanently established cell lines such as Y79 and WERI from human retinoblastoma tumors [59, 71] and primary cultures of chick retina cells [72]. Many other variants on this approach have been used, including knockdown strategies and cotransfection of trans factor constructs for expression in cells normally devoid of any photoreceptor specific proteins. These promoter activity measurement studies have been best in the determination of the factors and sequences that stimulate or activate the promoter. They have been somewhat less successful in the measurement of circumstances that suppress or repress promoter activity.

The second component of the joint strategy was to study the physical interaction of the DNA (the putative TFBS) with a protein (the putative trans factor) by electrophoretic