Expression analyses of Pitx3 transcripts and PITX3 protein in wild-type and miak mice.

A. Whole-mount in situ hybridization analysis in miak/+ and miak/miak in E11.5 embryos. The bottom panels show a magnified image of the eyes compared to the top panels. The arrows indicate the expression of Pitx3, detected as blue signals. Scale bar = 500 µm. B. The relative levels of Pitx3 mRNA in the eyes of wild-type (+/+) and miak/miak mice at E11.5, E12.5 and E14.5. Pitx3 mRNA expression was measured by real-time RT-PCR analysis using the Mm_Pitx3_1 primer set (Table S1). The values shown in the graph indicate the mean relative expression levels and the SDs of triplicate eye mRNAs. The expression levels in wild-type mice at E11.5 were assigned an arbitrary value of 1 for comparative purposes. ns, no significant differences; *P0.05; ***P0.001. C. Over-expression of PITX3 protein during lens development in miak mice. Confocal images show the lenses double-labeled with PITX3 antibody (red) and DAPI (blue) in wild-type and miak/miak mice at E10.5, E11.5 and E12.5. Strong PITX3 signals were observed in the miak/miak mice at E11.5 and E12.5 compared to the wild-type mice. Scale bar = 100 µm.