ABSTRACT: The cornea continues to mature after birth to develop a fully functional, refractive and protective barrier tissue. Here we investigated the complex biological events underlying this process by profiling global genome-wide gene expression patterns of the immature postnatal day 10 and seven week-old adult mouse cornea. The lens and tendon were included in the study to increase the specificity of genes identified as up regulated in the corneal samples. Notable similarities in gene expression between the cornea and the tendon were in the mesenchymal extracellular matrix collagen (types I, III, V, VI) and proteoglycan (lumican, decorin and biglycan) genes. Expression similarities in the cornea and lens were limited to certain epithelial genes and the crystallins. Approximately 76 genes were over expressed in the cornea samples that showed basal expression levels in the lens and tendon. Thirty-two of these were novel with no known functions in the cornea. These include genes with a potential role in protection against oxidative stress (Dhcr24, Cdo1, Akr1b7, Prdx6), inflammation (Ltb4dh, Wdr1), ion-transport (Pdzk1ip1, Slc12a2, Slc25a17) and transcription (Zfp36l3, Pdzk1ip1). Direct comparison of the cornea of two ages showed selective up regulation of 50 and 12 genes in the P10 and adult cornea, respectively. Of the up regulated P10 genes several encode extracellular matrix collagens and proteoglycans that are stable components of the adult cornea and their high transcriptional activity at P10 indicate a period of active corneal growth and matrix deposition in the young cornea. Much less is known about the genes selectively over expressed in the adult cornea; some relate to immune response and innervations (Npy), and possibly to electron transport (Cyp24a1, Cyp2f2) and others of yet unknown functions in the cornea (Rgs10, Psmb8, Xlr4)). This study detected expression of genes with known functions in the cornea, providing additional validation of the microarray experiments. Importantly, it identified several novel genes whose functions have not been investigated in the cornea. Experiment Overall Design: Briefly, the corneas were dissected free of the limbus from postnatal day 10 (P10) and seven-week old (adult) mice. To enrich for stromal contribution, the epithelium was scraped off and the cornea placed in TRIzol (Invitrogen Life Technologies, Carlsbad, CA). The scraping off of the epithelium reduced, but did not abrogate epithelial contribution, as judged by the expression of known epithelial markers. The lens and the flexor digitorum longus (FDL) tendon were removed from seven-week old adult mice for isolation of total RNA. For the adult cornea three independent preparations of total RNA was generated. For all the other tissues two independent preparations of total RNA were used. The adult and the P10 microarrays were used as baselines in our previous study of corneal cells in culture to identify genes differentially expressed by cultured cells of the stroma (Chakravarti et al. 2004). Experiment Overall Design: The output fluorescence of the scanned images was analyzed first using the Affymetrix Microarray Suite 5.0 software to compute absolute expression, background calculation and detection call (present, absent or marginal). Background signal was between 42 to 55 arbitrary units, while the noise to signal ratio was between 1.42 to 1.78 arbitrary units. The expression data from all nine arrays were further analyzed as follows. The model-based DNA-Chip Analyzer (dChip) software (http://www.dchip.org/) was used to normalize the data from the image files for array-to-array comparison (Li and Wong 2001). This software uses an invariant-set algorithm as a basis for normalization and a model-based expression index to calculate the expression value for each transcript. To identify gene expression patterns that were statistically significantly different between any two given tissue types, we used the following criteria: (1) fold change ⥠2; (2) difference of mean expression index ⥠100 arbitrary units; (3) t-statistic, p < 0.05.
