Molecular Pathway in Retina Regeneration

描述（由申请人提供）：在鸡胚中，视网膜色素上皮细胞（rPEC）在成纤维细胞生长因子-2 （FGF-2）的影响下转分化为神经视网膜细胞。受FGF-2调控并导致RPE转分化和更新失去的视网膜的基因在很大程度上是未知的。本研究将确定小眼症（Mitf），一种rPEC特异性转录因子，Pax-6，一种众所周知的视网膜发育调节因子，以及Sonic Hedgehog (Shh)，一种在视网膜发育过程中被认为是背/鼻孔极性的关键调节因子的作用。Mitf通常在rPEC中表达，当FGF-2存在时，其表达减少。Mitf的表达模式及其调控提示在维持rPEC细胞及其向神经视网膜转分化的能力中起作用。在体外实验中，Pax-6已被证明可以下调Mitf的表达，它似乎对转分化过程至关重要。阻断视神经杯形成过程中Shh的活性可诱导rPEC向神经视网膜转分化。这表明Shh也可能间接调节mitt。本研究将有助于阐明参与视网膜再生的分子途径，并剖析上述关键分子之间的相互作用。因此，我们可以在手头有一个模型，以诱导视网膜再生的高等脊椎动物，并可能利用这些知识来治疗视网膜退行性疾病。Aim 1的研究旨在通过原位杂交、RTPCR、免疫组织化学和Western分析来确定鸡视网膜再生过程中Mitf、Pax-6和Shh基因和蛋白的表达模式。Aim 2旨在通过在存在或不存在FGF-2的情况下通过逆转录病毒构建物过度表达Mitf、Pax-6或Shh，或通过引入反义寡核苷酸阻断Mitf、Pax-6和Shh的表达，以及在Shh的情况下通过特异性抗体阻断Shh途径，通过表达HIP或PTC （Shh途径抑制剂）或使用环巴胺来操纵视网膜再生过程。预计在RPE转分化为视网膜细胞后，mir和Shh将下调，Pax-6上调。操纵这三个基因的表达将为诱导包括人类在内的其他动物的视网膜再生提供重要信息。

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.