Figure 4

The Pax6^Aey18 paired domain mutation (deletion of exon 6) leads to reduced nuclear localisation of Pax6 in vivo and in vitro. A. Pancreatic endocrine cells of Pax6 wt embryos predominantly have a nuclear localization of Pax6 whereas it is found in the cytoplasm of many endocrine cells in Pax6^Aey18 paired domain mutants. In some cells, staining is predominantly cytoplasmatic (arrows) while others show equal cytoplasmatic and nuclear staining or largely exclusive nuclear staining similar to wt. B. To reconstruct the changes in the Pax6^Aey18 paired domain mutant mouse in vitro Exon 6 was deleted from an expression plasmid for canonical mouse Pax6. Wild type and mutated Pax6 where overexpressed in rat Ins1-E insulinoma cells. Cells where stained with anti-Pax6 and categorized into cells with pure nuclear, pure cytoplasmatic and mixed Pax6 localization. Only cells that clearly over-expressed the transcription factor over its endogenous level in Ins1-E cells where counted. After deletion of Exon 6 the percentage of cells with pure nuclear localization of Pax6 is substantially decreased (43% vs. 72% with wild type Pax6; *** p < 0,001). Representative confocal images of Ins1-E cells over-expressing Pax6 with either pure nuclear (wt) or pure cytoplasmatic localization (Pax6^Aey18) of the protein are shown in the right panels.