Overexpression of Pitx3 in Lens Epithelial Cells Rescues the Phenotype of Aphakia Mice

Abstract

Abstract: : Purpose: The aphakia (ak) mutation in mice is a chromosome19 linked recessive eye defect, which causes congenital absence of the lens. The Pitx3 gene is in the vicinity of ak locus, and turn out to be deleted in the promoter and in the exon 1 region. Whole mount in situ hybridization indicates that Pitx3 is specifically expressed in the late-lens placode starting from E10.5 and subsequently expressed in lens epithelium. To test whether the Pitx3 mutation is the etiologic reason for aphakia and to investigate the possible role of Pitx3 in lens induction, we overexpressed Pitx3 gene in the ak/ak mouse lens. Methods: Pitx3 cDNA was linked to the FoxE3 promoter and DREAM promoter (a Delta-Enhancer-alpha-A-Minx chimeric promoter) to generate the ak/ak background transgenic mice by microinjection. The transgenic mice were characterized by histology, BrdU incorporation, immuno-staining and in situ hybridization analysis. Results: The FoxE3 transgenic mice develop lens in shape and in size. At embryonic day 15.5 (E15.5), expression of Pitx3 transgene was detected in the lens epithelial cells, and the BrdU pattern and the expression of lens markers such as alpha crystallin and beta crystallin in the transgenic lens are similar to the wild type lens, suggesting that the FoxE3-Pitx3 rescued the ak/ak lens degeneration. In contrast, the Dream-Pitx3 construct did not successfully rescue the ak/ak lens. Conclusions: Overexpression of Pitx3 in lens epithelium cells is sufficient to rescue the ak/ak phenotype. The DREAM-Pitx3 failed to rescued may have the possibilities: 1) either the Pitx3 gene is switched on too late, or this construct did not express normal Pitx3 protein; 2) The DREAM promoter is not active in the ak/ak background.

Keywords: transcription factors • transgenics/knock-outs • regeneration