Signaling pathways during chick retina regeneration

描述(申请人提供)：NEI的目标之一是支持研究人员寻求预防和治疗眼盲疾病，如老年性黄斑变性(AMD)、视网膜色素变性和糖尿病视网膜病变。我们已经建立了一个视网膜再生模型，在这个模型中我们可以很容易地操纵控制视网膜修复和再生的关键分子通路。胚胎雏鸡有能力在眼部睫状缘的视网膜干/祖细胞群中或在成纤维细胞生长因子(成纤维细胞生长因子)的辅助下，通过视网膜色素上皮(RPE)的转分化来再生其视网膜。我们已经确定，Hedgehog(HH)途径可以调节这些再生能力，与成纤维细胞生长因子途径相互作用，根据激活方式的不同，诱导或抑制再生。在这项提案中，我们想要描述参与这两种类型的再生的信号通路，并阐明调节这些过程的关键分子。Aim 1的研究将通过原位杂交、qRT-PCR、免疫组织化学和Western blotting来确定在胚胎鸡眼视网膜发育和再生过程中涉及成纤维细胞生长因子和HH信号通路的分子的表达模式，以进一步确定这两种再生方式所涉及的机制。目的2将确定成纤维细胞生长因子和HH通路如何调节细胞周期分子，以及HH如何刺激睫毛边缘再生。目的3将确定成纤维细胞生长因子如何在胚胎第4天(E4)诱导转分化，而不是在胚胎第5天(E5)，以及HH信号如何在E4负向调节这一过程。本课程还将讨论成纤维细胞生长因子和氢化氢对视网膜切除术后视网膜色素上皮细胞周期分子的调节。对于后两个目标，已经设计了体内和体外实验来测试分子/通路的抑制和过度激活，并将使用一系列诊断分析。AIM 4将通过微阵列分析提供参与视网膜再生的基因调控网络和途径的全球视角。在美国，视力受损和失明的主要原因主要是与年龄相关的眼病，包括与年龄相关的黄斑变性。目前还没有开发出有效的治疗方法来取代这些疾病中丢失的视网膜细胞。这项研究将确定参与视网膜细胞替换的分子，并可能为未来的治疗提供线索。

DESCRIPTION (provided by applicant): One of the goals of the NEI is to support researchers on the quest to prevent and treat eye blinding diseases such as Age-related Macular Degeneration (AMD), retinitis pigmentosa and diabetic retinopathy. We have established a retina regeneration model where we can easily manipulate key molecular pathways that could control retina repair and regeneration. The embryonic chick has the ability to regenerate its retina upon injury/removal from a population of retinal stem/progenitor cells located in the ciliary margin of the eye or via the transdifferentiation of retinal pigmented epithelium (RPE) with the aid of fibroblast growth factor (FGF). We have determined that the hedgehog (Hh) pathway can modulate these regenerative abilities interacting with the FGF pathway to either induce or inhibit regeneration depending on the mode of activation. In this proposal, we would like to delineate the signaling pathways involved in both types of regeneration and elucidate key molecules that regulate these processes. Studies in Aim 1 will determine the expression pattern of molecules involved in the FGF and Hh signaling pathways during retina development and regeneration in the embryonic chick eye via in situ hybridization, qRTPCR, immunohistochemistry and Western blotting to further define the mechanisms involved in both modes of regeneration. Aim 2 will determine how cell cycle molecules are regulated by the FGF and Hh pathways and how Hh stimulates regeneration from the ciliary margin. Aim 3 will determine how FGF induces transdifferentiation at embryonic day 4 (E4) but not at embryonic day 5 (E5) and how Hh signaling negatively regulates this process at E4. The regulation of cell cycle molecules by FGF and Hh in the RPE of retinectomized eyes will also be addressed. For these last two aims, in vivo and in vitro experiments have been designed to test for inhibition and over activation of molecules/pathways and a series of diagnostic assays will be used. Aim 4 will provide a global view of gene regulatory networks and pathways that are involved in retina regeneration via microarray analysis. The leading causes of vision impairment and blindness in the United States are primarily age related eye diseases including Age-related Macular Degeneration. No effective treatment has been developed to replace the lost retina cells in these diseases. This study will pinpoint molecules involved in the replacement of retina cells and could shed light into future treatments.