Molecular Pathway in Retina Regeneration

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DESCRIPTION (provided by applicant): In the chick embryo, retinal pigment epithelial cells (rPEC) transdifferentiate to neural retina cells under the influence of Fibroblast Growth Factor-2 (FGF-2). The genes that are regulated by FGF-2 and cause the RPE to transdifferentiate and renew the lost retina are largely unknown. This study will determine the roles of microphthalmia (Mitf), a rPEC specific transciptional factor, Pax-6 a well known regulator of retina development, as well as the role of Sonic Hedgehog (Shh), known to be a key regulator of dorsal/ventml polarity during retinal development. Mitf is normally expressed in rPEC and its expression decreases when FGF-2 is present. The expression patterns of Mitf as well as its regulation suggest a role in the maintenance of rPEC cells and their ability to transdifferentiate to neural retina. Pax-6 has been shown to down regulate the expression of Mitf in in vitro assays and it seems to be essential for the process of transdifferentiation. Blocking the activity of Shh during optic cup formation induces the rPEC to transdifferentiate to neural retina. This is an indication that Shh may indirectly regulate Mift as well. This study will aid in elucidating the molecular pathway involved in retina regeneration and dissect the interaction between the aforementioned key molecules. Consequently, we can have at hand a model for inducing retina regeneration in higher vertebrates and possibly use such knowledge to treat retinal degenerative diseases. The studies in Aim 1 are designed to define the expression patterns of Mitf, Pax-6 and Shh genes and proteins during the process of chick retina regeneration via in situ hybridization, RTPCR, immunohistochemistry and Western Analysis. Aim 2 is designed to manipulate the process of retina regeneration by over-expressing Mitf, Pax-6 or Shh via retroviral constructs in the presence or absence of FGF-2, or by blocking the expression of Mitf, Pax-6, and Shh by the introduction of antisense oligonucleotides, and in the case of Shh also by blocking Shh pathway by specific antibodies, by expressing HIP or PTC (inhibitors of the Shh pathway) or by the use of cyclopamine. It is expected that upon transdifferentiation of the RPE into retinal cells Mift and Shh will be down- regulated and Pax-6 up- regulated. Manipulation of expression of these three genes should provide significant information for the induction of retina regeneration in other animals including humans.