European Association for Vision and Eye Research

Aim of the work is a functional analysis of the mouse mutant aphakia, which is characterized by missing eye lenses caused by 2 deletions in the Pitx3 promoter.

Expression profiles from aphakia and wild-type embryos during lens development (embryonic stages E 9.5 till E 13.5) were compared using the DNA Microarray Technology. The results were confirmed by Real Time PCR, and in cases of special interest by promoter assays.

23 genes showed significant differences in their expression patterns between aphakia and wild type, from which 15 are upregulated in the mutant. The most interesting results include the Clock gene which is upregulated in aphakia at E 11.5. It is involved in circadian regulation by acting as a transcription factor. We could corroborate the expression results with Real Time PCR.
Another promising gene is the transcription factor AP2. Its downregulation in the mutant at E 11.5 is supported by Real Time PCR results. As a binding site for AP2 is predicted in one of the deletions in the Pitx3 promoter in the mutant, we cotransfected the transcription factor with the Pitx3 promoter in the HEK 293 cell line, which is negative for AP2 expression. The transfection was successful as shown by RT-PCR, but no changes in promoter activity could be detected.

The loss of Pitx3 activity in the homozygous ak mutants lead to a stop of lens development at embryonic stage 10.5. We detected the first differences in gene expression in the microarrays at E 11.5. Further analysis by real-time PCR and in situ hybridization will reveal details of the correlations.