The general objectives of this application are to genetically define the aphakia (ak) locus as Pitx3 and to identify and characterize genes whose expression is regulated by ak in the developing eye. The spontaneous autosomal recessive mutation ak described in the mouse results in bilateral aphakia due to aberrant lens morphogenesis after the lens pit stage (Varnum and Stevens, 1968). The ak locus has been mapped to mouse chromosome 19 and high-resolution linkage mapping has positioned ak 0.2 +/- 0.2 cM from Pitx3, D19Mit8, and D19Mit9 (Semina et al., 2000). There is compelling evidence to suggest that the bicoid-related homeobox gene Pitx3 is a strong candidate for the ak locus. Mutational analysis of Pitx3 in ak mice revealed a 652bp deletion of the Pitx3 5' UTR (Semina et al., 2000). Mutations to Pitx3 in humans result in anterior segment mesenchymal dysgenesis and dominant cataracts (Semina et al., 1998). These observations suggest that, like ak, Pitx3 contributes to the development of the anterior segment and lens. The first objective of this proposal is to determine genetically if Pitx3 is ak using BAC transgenic complementation and targeted gene disruption methodologies. If Pixt3 is ak, then the long-term goal of this application is to (1) identify downstream target genes of Pitx 3 using Affymetrix GeneChip arrays and chromatin immunoprecipitation techniques and (2) characterize their expression in the developing mouse eye. Identification and characterization of Pitx3 target genes is a first step toward understanding the developmental pathways that regulate lens morphogenesis after the lens pit stage. However, if Pitx3 is not ak, then the goal of this application is to positionally clone ak. A BAC-based physical map of the critical region will be generated. Overlapping BACs will be tested for transgenic complementation and candidate ORFs in a BAC that rescues ocular development in ak embryos will be identified by cDNA selection. To identify the ak mutation, candidate gene expression will be characterized in ak mice and sequence data of genomic amplicons from ak mice will be analyzed.
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| Nunes, Irene; Tovmasian, Lucy T; Silva, Robert M et al. (2003) Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Natl Acad Sci U S A 100:4245-50 |
