Purpose: The retina of some avian species contains two macular regions, making it an excellent model for retinal, and especially macular, development. Previous studies have provided evidence of the involvement of Pax6 in macular development. The purpose was to perform a comprehensive expression analysis of Pax6 isoforms in different regions of the pigeon retina.
Methods: The different mRNA transcripts were amplified by RT-PCR and characterized by sequencing analysis. Semiquantitative PCR and quantitative real-time PCR analyses were used to study the level of expression of each transcript. Western blot analysis was performed on both the cytosolic and nuclear cell fractions.
Results: An evolutionary analysis of all human-chicken retinal homologues revealed that Pax6 is one of the most conserved retinal genes. By alternative splicing and alternative initiation of transcription, Pax6 produces 41 different mRNA transcripts, encoding 17 protein isoforms in the pigeon retina, five of which are paired-less cytosolic proteins. Semiquantitative expression analysis revealed that the short, paired-less, transcripts have a relatively high level of expression. Quantitative real-time PCR analysis of the central macula, red area, and peripheral retina revealed a spatial and temporal expression profile indicating that many Pax6 transcripts take a part in macular development.
Conclusions: These data suggest that Pax6, a highly conserved gene, can maintain evolutionarily conserved variability at the protein level by alternative splicing and initiation mechanisms, allowing it to perform multiple functions. The variability in the length of the paired domain suggests that the different Pax6 isoforms activate different sets of genes.