Gene Profiling of Flow–Sorted Developing Mouse Rod Photoreceptors

Abstract

Abstract: : Purpose: Rod photoreceptors constitute over 75% of all cells in the mouse retina. Rods are born during an extended period of development; however, a delay of 5–12 days is observed before the expression of rhodopsin and other phototransduction genes. The goal of our studies is to elucidate the molecular pathways that lead to the differentiation of mature and functional rods from postmitotic rod precursors. Methods: The Nrl promoter was used to express green fluorescent protein (GFP) in transgenic mice. GFP–expressing cells were enriched by fluorescence–activated cell sorting (FACS). RNA from GFP+ cells was purified from developing retinas of Nrl–GFP mice and used for hybridization to Affymetrix GeneChips. Expression data were analyzed by statistical methods to identify differentially expressed genes. Co–expressed genes are being identified by clustering. Results: The Nrl promoter specifically directed expression of GFP to developing and mature rod photoreceptors and the pineal gland. Retinal GFP expression was detected as early as E12.5 within postmitotic cells, beginning 4–6 hours after completion of terminal S–phase. The expression increased postnatally and was closely correlated with rod birth. Gene profiles have been generated for GFP–expressing cells isolated from E16, P2, P6, P10 and adult retinas. Data analysis is in progress. Conclusions: Our data suggest that Nrl is one of the first molecular marker of newborn rod precursors. Analysis of the gene profiles from developing rods should yield novel insights into molecular pathways involved in rod differentiation.

Keywords: photoreceptors • retina • opsins