Pax-6 regulates the development of the eye in animals ranging from jellyfish to humans. Knockout studies have shown that in addition to eyes Pax6 is also required for the normal differentiation of brain and pancreas. Heterozygous mutations in Pax6 cause aniridia in humans and small eye in rodents. Aniridia is a congenital bilateral disorder of the eye marked by complete or partial absence of the iris. Vision is progressively lost through cataracts, early onset of glaucoma, and corneal opacification. Pax6 functions as a transcription factor and has two DNA binding domains (a paired domain at the N terminus and a paired like homeodomain in the middle), a glycine-rich region that links the two DNA-binding domains, and a transactivation domain at the C terminus. There are two major alternatively spliced forms of Pax6 that differ by the presence or absence of 14 amino acids that are in the middle of the paired domain and are coded by an exon known as 5a. The +5a isoform is specific to and fully conserved among vertebrates. Mutation in the alternatively spliced region appear to cause a distinct eye abnormality. The inclusion of exon 5a in Pax 6 protein changes the specificity of the DNA-binding site, which indicates that the two forms may regulate different target genes. Our studies have indicated that the two isoforms behave differently even with the homeodomain DNA-binding sites. The focus of this proposal is (a) to determine the effects of missense mutations on Pax-6 function in vitro and in vivo to correlate the position of mutation with the type of functional abnormality and (b) to determine the function of the +5a isoform of Pax-6. The long term goal is to understand the role of Pax-6 in the cascade of eye development. A mouse model lacking exon 5a in the Pax-6 gene will be developed. The effect of loss of exon 5a will be determined by histological analysis of the tissues. The expression pattern of the +5a isoform will be determined in different tissues during development with +5a isoform specific antibodies. In vitro transactivation studies with combination of site-directed mutagenesis and DNA-binding assays will be performed to understand the DNA-binding of the Pax-6 and the +5a isoform. RT-PCR analysis will be used to identify other alternatively spliced forms of Pax-6. This work will provide insight into the role of the +5a isoform of Pax-66 in development and differentiation and define the effects of specific mutations found in aniridia patients.
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