ANALYSIS OF THE APHAKIA GENE REQUIRED FOR LENS FORMATION

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DESCRIPTION Congenital or acquired defects of the ocular lens such as cataract are a major cause of impaired vision and blindness. The long-term objective of this research proposal is an improved diagnosis, treatment or even prevention of lens disease by means of although understanding of the molecular mechanisms underlying development and maintenance of the lens. The proposed study is focused on isolation and analysis of a gene that is specifically required for lens development in the mouse. Mice homozygous for the autosomal recessive aphakia (ak) mutation are blind due to a absence of lens, iris and pupil with microphtalmia. During embryonic development, the earliest defects a partial randomization of spindle orientation in mitotic epithelial cells of the lens placode, as well as irregularities in the associated extracellular matrix (EM) A combined positional cloning and positional candidate gene approach will be used to identify the gene mutated in ak. The chromosomal interval containing the ak locus will be fine mapped by linkage analysis with polymorphic markers using homozygous offspring derived from an inter-specific cross. A high- resolution physical map of the locus will be constructed using existing YAC contigs as a starting point, further refined by construction of a BAC clone contig, and followed by exon trapping or cDNA selection to identify candidate genes. RNA isolated at appropriate developmental stages will be used to assess the expression of these genes in the normal and monkey eyes, and to generate cDNA to be used in mutation screening by SSCP and DNA sequence analysis. Furthermore,, to gain more precise information about the composition and role of the EM that intimately connects the optic cup with the lens placode, the expression, the expression of known EM constituents will be studied by immunolocalization and in situ hybridization techniques in normal and mutant eyes.