Abstract
Purpose: :
Corneal disease-1 (Dstncorn1) mice are deficient for destrin, which functions to depolymerize filamentous actin (F-actin) into its monomeric form. Dstncorn1 mice exhibit an accumulation of F-actin in the corneal epithelial cells, and develop corneal epithelial cell hyperproliferation and stromal neovascularization. The purpose of this study was to determine the possible cellular mechanisms affected by changes in the actin cytoskeleton in vivo, and to identify genes that may be responsible for the development of Dstncorn1 phenotypes using microarray analysis. We also sought to compare the effects of Dstncorn1 and an allelic missense mutation, Dstncorn1-2J, which causes milder corneal phenotypes.
Methods: :
RNA samples extracted from corneas of P14 mutant mice, as well as wild-type mice, were used to generate cRNA probes for hybridization to Affymetrix mouse genome arrays. After pre-analysis processing of the data, a t-test was performed to identify differentially expressed genes. Hierarchial clustering was performed on differentially expressed probe sets. We used the DAVID Functional Annotation Tool to identify enriched gene ontology terms. Quantitative real-time PCR was used to confirm relative mRNA levels of differentially expressed genes. Immunohistochemical methods were used to examine the protein localization of differentially expressed genes.
Results: :
Our analysis showed that 1,226 and 202 genes are differentially expressed in the corneas of Dstncorn1 and Dstncorn1-2J mice, respectively. 81 genes were differentially expressed in both mutants, and a clustering analysis demonstrated that differential expression is more pronounced in Dstncorn1 mice. Functional annotation of the upregulated gene list demonstrated the GO term "cytoskeleton" to be the most enriched in both mutants. The upregulation of serum response factor (SRF) and its target genes, which is known to be affected by actin dynamics in vitro, was observed in Dstncorn1 mice.
Conclusions: :
This study demonstrated that aberrant actin dynamics lead to dramatic alterations of gene expression in the cornea. In particular, genes related to the structure of the cytoskeleton were most affected. We also have identified genes that may be responsible for the development of the phenotypes in destrin mutant mice, and provided strong evidence that the change in actin dynamics results in the activation of SRF-dependent transcription in vivo.
Keywords: cornea: basic science • gene microarray • cytoskeleton