MOLECULAR GENETIC ANALYSIS OF THE PAX6 GENE IN ANIRIDIA

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The long term objectives of this proposal are to study the function of a gene called PAX6, which is required for normal human eye development, and to identify genes which in turn are regulated by PAX6. PAX6 is a gene belonging to the paired box family and thus likely acts as a transcription factor. the mouse Pax-6 gene is mutated in small-eye, in which heterozygotes (Sey/+) have microphthalmia, absent anterior chambers, abnormally folded retinas, and small or absent lens. Homozygous Sey/Sey mice lack eyes and noses entirely. It has been proposed that a panocular disorder in man, called aniridia, is homologous to the mouse Small-eye mutation, because of similar, semi-dominant modes of inheritance, related phenotypes, and homologous chromosomal locations. to test the involvement of the human PAX6 gene in aniridia, and in eye development in general, we have cloned PAX6 and identified several independent point mutations in PAX6 in aniridia families, thus providing firm evidence that PAX6 is responsible for aniridia. The phenotype in aniridia and small-eye is potentially explained by an impairment in lens induction, since the development of anterior chamber structures, including the iris, is critically dependent on the influence of the lens. Moreover, this model is consistent with the intense expression of PAX6 in the optic vesicle, a known inducer of lens differentiation. The finding that a 50% reduction in PAX6 gene dosage results in a phenotype indicates that this gene product controls a critical, rate-limiting step in eye development. To gain further insight into the function of different parts of the PAX6 protein, we will analyze additional PAX6 mutations in aniridia. We will address the reliability of the SSCP analysis by sequencing multiple alleles of each exon from samples which are negative by SSCP. In addition, we will test the involvement of PAX6 in other potentially allelic disorders of eye development such as Peter's anomaly in man and the Coloboma mutation (Cm) in the mouse. the functional consequences of identifiable mutations in PAX6 will be tested by gel shift and footprint assay for their effect on DNA binding, and also for their effects on transcription. to this end, we will develop a cell culture system to dissect the role of the paired and homeo domains and the transcription regulating activity of the C-terminus. We propose to identify genes which are regulated by PAX6 during oculogenesis, by testing mouse and human homologs of Drosophila genes which are known targets for the Drosophila PAX6 homolog, paired. We will also employ alternative strategies including immuno-precipitation of genomic DNA or chromatin, and cDNA subtraction. Finally, we will test the sufficiency of PAX6 gene expression for lens induction by injecting a retroviral vector underneath surface ectoderm in the developing chick embryo. The availability of a cloned gene associated with ocular defects in both man and mouse provides a powerful system in which to study not only the basic embryology and genetics of ocular development, but also a disorder of decreased visual function in man.