Early expression of the gene for interphotoreceptor retinol-binding protein during photoreceptor differentiation suggests a critical role for the interphotoreceptor matrix in retinal development

Abstract

Interphotoreceptor retinol-binding protein (IRBP), the major protein component of the subretinal space, is in a strategic position to mediate cellular interactions between the retinal pigmented epithelium (RPE) and the neural retina. While IRBP appears to be involved in vitamin A transport during the visual cycle in the adult, the role of this protein during eye development has not been determined. As a first step to understanding the role of IRBP during retinal development, we have studied the expression of the mRNA for this glycolipoprotein during photoreceptor differentiation in the rat. A rat neural retina cDNA library was prepared from which an IRBP clone was isolated. The clone contains an open reading frame followed by a 3′ noncoding sequence ending in 10 adenosine residues. The coding region has an identity of 83.9 and 82.5% with the nucelotide sequence of human and bovine IRBP, respectively. Rats (Sprague-Dawley, Wistar, and Royal College of Surgeon pink-eyed controls) have a 6.4 and a 5.2-kb mRNA for IRBP which are present in a 1:4 ratio and thus are the only vertebrate known to definitely have more than one major form of the IRBP message. Genomic Southern blots are consistent with the hypothesis that there is only one allele of the IRBP gene, suggesting that the two forms are produced by alternative processing of the mRNA. To generate an antisense RNA probe for use in molecular titration assays and Northern blots, an Eco RI-Bam HI fragment from the coding region was subcloned in between flanking Sp6 and T7 promoters. Total RNA was prepared from undissected rat globes from postnatal days p0-p22. The expression of the mRNA for IRBP was studied by Northern blots and the level of the transcripts determined by solution hybridization assays. Approximately 105 IRBP mRNA transcripts/μg total eye RNA are present at birth. This increases to a final level of 3.1 × 106 transcripts/μg total RNA by p9. The one-half maximal level of the mRNA occurs at p4.2 which is 2 wk before the one-half maximal level of IRBP is reached in the subretinal space (Gonzalez-Fernandez, F., R. A. Landers, P. A. Glazebrook, S.-L. Fong, G. I. Liou, D. M. K. Lam, and C. D. B. Bridges. 1984. J. Cell Biol. 99:2092-2098). The expression of the mRNA for IRBP reflects the developmental emergence of the interphotoreceptor matrix as an important structure within the retina. The absolute level of IRBP is related to the volume of the subretinal space, which expands during development due to the elongation of the outer segments. The unexpected early expression of the mRNA for IRBP during retinal development suggests that the interphotoreceptor matrix is important not only to the adult during the visual cycle, but also plays a critical role during retinal development, perhaps facilitating the diffusion of hydrophobic nutrients and morphogens such as vitamin A from the RPE/choroid complex to the growing and differentiating neural retina.