Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice

Mouse aphakia (ak) is a recessive phenotype that spontaneously occurs in th e 129/Sv-SIJ strain and is characterized by small eyes that lack a lens. We have recently identified a homeobox-containing gene, Pitx3,and have shown that it is expressed in the developing lens and maps to chromosome 19 close to ak in mouse. Human PITX3 gene was found to underlie anterior segment dy sgenesis and cataracts. We have now obtained the entire sequence of the mou se Pitx3 gene including 10 kb of the 5' region and 5 kb of the 3' region. O f several microsatellite repeat regions identified within the Pitx3 sequenc e, one was informative for linkage analysis. No recombination was observed between ak and the Pitx3 marker, indicating that these two loci are closely linked (0.2 +/- 0.2 cM). Additionally, Pitx3 transcripts were not detected in the ak/ak mice either in the lens placode or at later developmental sta ges of the lens by in situ hybridization, Since no differences were previou sly found between ak/ak and wild-type sequences in the Pitx3 coding region, we hypothesized that an etiologic mutation is located in the promoter or o ther regulatory regions. To test this hypothesis we studied the 5' flanking region of the Pitx3 gene. This analysis revealed a deletion of 652 bp loca ted 2.5 kb upstream from the start point of the Pitx3 5' UTR sequence in ak /ak mice. The deletion cc-segregated with the ak mutation and was not detec ted in 16 samples from 10 different mouse strains including the founder str ains. Analysis of the 652 bp region identified sequences similar to consens us binding sites for transcription factors AP-2 and Maf that were shown to play a critical role in lens determination, These lines of evidence suggest that the abnormal ocular development in the aphakia mouse is due to the de letion upstream of the Pitx3 gene.