Interphotoreceptor retinoid-binding protein (IRBP) is a retinal photoreceptor glycoprotein that has been suggested to serve as a retinoid transport vehicle in the rhodopsin cycle. Analysis of the timing of expression in developing mouse retinas has revealed that IRBP emerges prior to the last mitosis of the photoreceptor precursor cells whereas opsin emerges only in the postmitotic cells, suggesting that IRBP may be required for the initiation as well as the maintenance of differentiation. Our long term goal is to understand the process of photoreceptor differentiation. We propose to approach this goal through the study of IRBP regulation and function. The timing of demethylation of the IRBP and opsin genes during the terminal photoreceptor differentiation will be determined to indicate the timing for gene activation. The appearance of IRBP and opsin during development and the level at which their expression is regulated will be determined. The information obtained will be applied to early developing mouse models for retinal degeneration to determine whether IRBP is involved in the degenerative process. Analysis on transgenic mice has revealed that cis- acting elements regulating tissue-specific transcription of the IRBP gene reside within -156 of the gene. Gel shift and footprinting analyses in this upstream fragment have identified a retina-specific DNase I protected region. Based on these findings, cis- and trans-acting elements that regulate tissue-specific transcription of the IRBP gene and the ways in which these trans-acting elements are regulated will be determined. The information obtained should help understand the events occurring between the period of cell type commitment and initial photoreceptor differentiation. Transgenic mice that express antisense IRBP gene with or without a ribozyme designed to reduce IRBP expression will be used to study the function of IRBP. The expression of endogenous retinal genes and the transgenes will be measured and related to the retinal visual functions and retinal morphologies. A normal IRBP gene will be re-introduced into these mice and their phenotypes re-investigated. The information obtained from these studies will provide insights into the complex process of photoreceptor differentiation. The study of photoreceptor development at the molecular level should help form a basis for eventually tracing the genetic defects in human retina to their molecular source.
