Pax6 encodes a sequence-specific transcription factor with a critical and evolutionarily conserved role in eye development. Pax6 expression commences at the earliest phases of eye development and its expression persists to adulthood in each eye tissue. Haploinsufficiency of Pax6 function results in aniridia in humans heterozygous for Pax6 mutation and in Small eye (Sey) in mice each characterized by severe iris hypoplasia. Homozygous mutation of mammalian Pax6 causes anopthalmia due to failure of the earliest event in eye development, formation of the lens placode. Pax6 function during early eye development of mice has been studied intensively, but the specific morphogenetic and biochemical lesions that underlie the iris abnormalities in aniridia have not been well detailed. Specifically it is not known if the severe iris hypoplasia that results from Pax6 haploinsufficiency is due to an essential role for Pax6 in the development of the iris itself or from an earlier defect in the patterning of the ciliary margin at the anterior tip of the optic cup. To circumvent early phenotypes resulting from Pax6 loss-of-function and determine role of Pax6 to development of the iris, we have developed a new conditional allele system. This technique exerts its regulation at the protein level with small molecules and is thereby both rapid and reversible - each property an advantage over other conditional allele strategies. We have created an allele of mouse Pax6, Pax6Frb*, in which the stability of Pax6 protein is controlled reversibly with rapamycin or analogs (called rapalogs). Pax6Frb*/Frb* embryos and Pax6Frb*/+ mice have the Sey phenotype in the absense of drug, but Pax6 stability and function can be rescued in the presence of rapamycin, which is specific for the product of the Pax6Frb* allele. We will regulate Pax6 function in Pax6Frb*/Frb* and Pax6Frb*/+ embryos at experimentally defined timepoints during development to determine sequentially the roles of Pax6 to iris development. In so doing we will delineate the mechanisms by which iris development fails in Pax6 heterozygous mice and by extrapolation in aniridia patients.
Mutation of the Pax6 gene leads to complete loss of the iris and other ocular abnormalities in humans and mice, but the specific functions for Pax6 during iris development are unclear. We have developed a technique and new mouse strain in which the function of Pax6 protein can be controlled with a drug, rapamycin. We will use conditionally regulate Pax6 during discrete points of mouse eye development to define the timepoints and mechanisms by which Pax6 controls iris development.
