Molecular Mechanisms of Axenfeld-Rieger Syndrome

说明（由申请人提供）：说明申请的广泛、长期目标和具体目标，并提及项目与健康的相关性。简要描述研究设计和实现这些目标的方法。避免总结过去的成就和使用第一人称。本摘要旨在作为一个简洁和准确的描述拟议的工作时，从申请分开。如果申请获得资助，此描述将成为公共信息。因此，不包括专有/机密信息。PITX 2转录因子是脊椎动物正常眼睛发育所必需的。PITX 2的突变导致与发育性青光眼和其他系统性缺陷相关的Axenomy-Rieger综合征（ARS）。我们的研究证明了pitx 2在斑马鱼眼发育中的作用是保守的。利用斑马鱼模型，我们鉴定了在眼、脑和颅面发育过程中介导PITX 2/pitx 2表达的多个调控区域。这些研究发现了Axene-Rieger综合征的一种新机制：PITX 2上游远端调控元件的缺失。事实上，使用一个大型的ARS患者队列，我们发现几乎所有的经典ARS病例都可以通过PITX 2的核苷酸或拷贝数突变来解释，当检查编码区和调控区时。对鉴定的调控序列的进一步探索导致初步鉴定PITX 2/pitx 2的上游调控子，目前正在分析其在正常眼部发育和疾病中的作用。几个下游基因也已被确定和验证，在体外和体内试验。本申请将进一步评估我们鉴定的基因和一些其他已发表的因子在PITX 2/pitx 2通路中的作用。该提案的创新之一是，我们计划使用我们在前一个资助期内在眼周间充质（POM）细胞中表达GFP的pitx 2增强子转基因系和pitx 2敲低试剂方面产生的资源，以鉴定在不同发育阶段区分正常分化和pitx 2缺陷POM细胞的转录本。这将导致PITX 2/pitx 2的下游效应物的稳健的体内鉴定，以及有助于对正常眼睛发育具有高度意义的该迁移群体的一般表征。另一个主要创新是我们利用锌指核酸酶驱动的基因组编辑技术来产生在pitx 2编码区（无效等位基因）和CE 4调控元件中携带突变的pitx 2系，所述CE 4调控元件先前已显示参与pitx 2表达的不同方面，包括在POM细胞中的强存在。我们的主要假设是，PITX 2/pitx 2是一个保守的因素，是必不可少的诱导分化的眼周间充质细胞迁移到眼前段的发展眼睛。PITX 2表达的上游调节因子及其下游效应因子的发现将为脊椎动物胚胎眼发育的机制提供深入了解，并有可能确定人类青光眼表型的新原因，这些原因可以作为潜在的治疗靶点进一步探索。 PITX 2对眼睛的正常发育至关重要。该提案将通过鉴定调节PITX 2表达的其他基因和受PITX 2活性控制的基因来研究PITX 2如何参与眼睛发育。该项目的结果将更好地了解眼睛发育，并可能发现青光眼等人类眼部疾病的新原因，这可能会带来新的治疗机会。

DESCRIPTION (provided by applicant): State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This abstract is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. The PITX2 transcription factor is essential for normal eye development in vertebrates. Mutations in PITX2 result in Axenfeld-Rieger syndrome (ARS) associated with developmental glaucoma and other systemic defects. Our studies demonstrate conservation of the role of pitx2 in zebrafish ocular development. Using zebrafish model, we identified multiple regulatory regions mediating PITX2/pitx2 expression during eye, brain and craniofacial development. These studies led to the discovery of a novel mechanism of Axenfeld-Rieger syndrome: the deletion of the distant regulatory elements upstream of PITX2. In fact, using a large cohort of ARS patients we showed that virtually all classic ARS cases can be explained by nucleotide or copy number mutations in PITX2 when both coding and regulatory regions are examined. Further exploration of the identified regulatory sequences resulted in tentative identification of upstream regulators of PITX2/pitx2 that are currently being analyzed in terms of their role in normal ocular development and disease. Several downstream genes have also been identified and verified by in vitro and in vivo assays. The genes identified by us and some additional published factors will be further evaluated for their role in PITX2/pitx2 pathway in this application. One of the innovations of this proposal is that we plan to use the resources generated by us during the previous funding period in terms of pitx2 enhancer transgenic lines expressing GFP in periocular mesenchymal (POM) cells and pitx2 knockdown reagents to identify transcripts that distinguish normally differentiating and pitx2-deficient POM cells at different developing stages. This will result in robust in vivo identification of downstream effectors of PITX2/pitx2 as well as aid in general characterization of this migratory population with high significance to normal eye development. Another major innovation is our utilization of zinc finger nucleases- driven genome editing technology to generate pitx2 lines carrying mutations in pitx2 coding region (null allele) and CE4 regulatory element that has been previously shown to be involved in different aspects of pitx2 expression including strong presence in POM cells. Our main hypothesis is that PITX2/pitx2 is a conserved factor that is essential for inducing differentiation of periocular mesenchyme cells upon their migration into the anterior segment of the developing eye. Discovery of the upstream regulators of PITX2 expression and its downstream effectors will provide insight into the mechanisms of vertebrate embryonic eye development and are likely to identify new causes of human glaucoma phenotypes which can be further explored as potential therapeutic targets. PUBLIC HEALTH RELEVANCE: PITX2 is essential for normal eye development. This proposal will investigate how PITX2 is involved in eye development by identifying other genes that regulate PITX2 expression and genes that are controlled by PITX2 activities. The results of this project will provide better understanding of eye development and will likely identify new causes of human ocular conditions like glaucoma, which may lead to new treatment opportunities.