Mutations in Destrin (Actin-Depolymerizing Factor) Lead to Corneal Epithelial Proliferation and Neovascularization in the Corn1 Mouse

Abstract

Abstract: : Purpose: Visual loss in many corneal diseases is due to changes in the corneal epithelium that is frequently associated with corneal neovascularization. Corneal disease-1 (corn1) is a spontaneous, autosomal recessive mutation that causes a dramatic increase in the rate of corneal epithelial proliferation and neovascularization. The purpose of this study was to elucidate the molecular basis for the corn1mutation. Methods: We used a positional cloning approach to identify the gene responsible for corn1. A high-resolution genetic map of the corn1 locus, on Chromosome 2, was generated using F2 intercrosses between A.BY/SnJ (A.BY)-corn1/corn1 and strains CAST/EiJ, DBA/2J and LP/J. A total of 1,862 F2 progeny were genotyped with flanking markers and phenotyped by slitlamp microscopy. A physical map of the critical area was assembled using bacterial artificial chromosome (BAC) clones. Candidate genes were tested for alterations by comparing coding sequences and expression levels between corn1 and wild-type mice. The expression pattern of destrin was studied by in situ hybridization and immunohistochemistry. Corneal whole-mounts were stained with FITC-phalloidin to visualize F-actin in the corneal epithelial cells. Results: The corn1 region was narrowed to 0.11 ± 0.05 cM flanked by markers D2Mit513 and D2Pjn420-9. Five transcripts were present in the annotated human BAC sequences that covered the corn1 minimal region. Northern analysis demonstrated absence of Dstn mRNA expression in homozygous corn1 mice. PCR analyses of genomic sequences established the presence of a ~35 kb deletion that encompasses the entire coding sequence of Dstn in corn1 mice. Expression analyses showed that destrin is highly expressed in the corneal epithelium, but not in the corneal stroma or endothelium. The corn1 corneal epithelial cells demonstrated a marked increase in the level of F-actin, a substrate for destrin, in a pattern similar to stress fibers. Conclusions: We identified the corn1 gene as destrin, which is an essential actin regulatory protein responsible for enhancing the turnover of actin in vivo. These results suggest a new pathway through which an aberrant actin cytoskeleton causes corneal epithelial proliferation.

Keywords: animal model • cornea: epithelium • positional cloning