Gene expression changes in the developing ocular retardation eye

Abstract

Abstract: : Purpose: Chx10, a homeodomain transcription factor, plays a major role during retinal development. Initially expressed in dividing retinal neuroepithelial cells, Chx10 is down–regulated as differentiation proceeds; later, it is expressed selectively in bipolar cells. Ocular retardation (orJ) mutant mice carry a null allele of Chx10 (Burmeister et al., 1996) and exhibit an eye–specific phenotype that affects fundamental aspects of retinal development: (1) retinal neuroepithelial cell proliferation is reduced, leading to microphthalmia; (2) intraretinal retinal ganglion cell axon guidance is disrupted; and (3) bipolar cells fail to differentiate. Our goal was to define molecular changes in the developing orJ eye as a prelude to understanding the transcriptional regulatory function of Chx10. Methods: Mouse I–gene arrays, containing cDNAs of >6500 eye genes/ESTs printed in duplicate onto glass slides, were hybridized to target cDNAs generated from embryonic day 15.5 wild–type (+/+) and orJ/orJ eye mRNAs. At least four replicate hybridizations of independently prepared mRNA samples were performed, each hybridization being a comparison of one of these eye samples to a reference sample. Hybridized arrays were scanned and analyzed by DigitalGenome to generate intensity data. Data were normalized with locally weighted linear regression (loess) and print–tip–group methods using limma Package. Differential expression analysis was performed with multiple comparison correction (false–discovery rate) using BioConductor and SAM. Results: We identified more than 20 genes whose expression is significantly altered in the orJ/orJ eye. A majority of these genes were expressed at higher levels: these included H19, Igf2, Hsp86, and the regulatory subunit of the RNA binding protein (Dj1). We also identified a number of novel ESTs. We are currently using real–time PCR to verify and further quantify our findings, Affymetrix mouse GeneChips to expand the number of genes analyzed, and immunohistochemistry and in situ hybridization to examine spatiotemporal changes in expression patterns. Conclusions: In addition to gene products, identified previously using immunohistochemistry, whose expression is altered in the absence of Chx10 (e.g., Pax2 and islet1), these studies have identified several new candidate genes that may contribute to pathways regulated by Chx10.

Keywords: transcription factors • gene/expression • retinal development