Molecular Mechanisms of Axenfeld-Rieger Syndrome

描述（由申请人提供）：说明申请的广泛、长期目标和具体目的，并参考项目与健康的关系。简要描述为实现这些目标的研究设计和方法。避免总结过去的成就和使用第一人称。当与应用程序分离时，此摘要旨在作为对所建议工作的简洁而准确的描述。如果该应用程序获得资助，则此描述将成为公共信息。因此，不要包含专有/机密信息。PITX2转录因子对脊椎动物的正常眼睛发育至关重要。PITX2突变导致Axenfeld-Rieger综合征（ARS），与发育性青光眼和其他系统性缺陷相关。我们的研究证明了pitx2在斑马鱼眼部发育中的保守作用。利用斑马鱼模型，我们确定了在眼睛、大脑和颅面发育过程中介导PITX2/ PITX2表达的多个调控区域。这些研究发现了Axenfeld-Rieger综合征的一种新机制：PITX2上游远端调控元件的缺失。事实上，通过对一大群ARS患者的研究，我们发现，当检查编码区和调控区时，几乎所有的经典ARS病例都可以用PITX2的核苷酸或拷贝数突变来解释。对已确定的调控序列的进一步探索，初步确定了PITX2/ PITX2的上游调控因子，目前正在分析其在正常眼部发育和疾病中的作用。一些下游基因也被体外和体内实验鉴定和验证。我们发现的基因和一些其他已发表的因子将在本应用中进一步评估其在PITX2/ PITX2通路中的作用。本提案的一个创新之处在于，我们计划利用我们在之前的资助期内在眼周间充质细胞（POM）中表达GFP的pitx2增强子转基因细胞系和pitx2敲低试剂方面产生的资源，鉴定在不同发育阶段区分正常分化和pitx2缺陷POM细胞的转录本。这将导致PITX2/ PITX2下游效应物在体内的强大鉴定，并有助于对这一对正常眼睛发育具有重要意义的迁移群体进行总体表征。另一项重大创新是我们利用锌指核酸酶驱动的基因组编辑技术生成pitx2系，该系携带pitx2编码区（零等位基因）和CE4调控元件的突变，这些突变先前已被证明与pitx2表达的不同方面有关，包括在POM细胞中强烈存在。我们的主要假设是PITX2/ PITX2是一个保守因子，在眼周间充质细胞迁移到发育中的眼睛前段时诱导其分化是必不可少的。发现PITX2表达的上游调控因子及其下游效应因子，将有助于深入了解脊椎动物胚胎眼发育的机制，并有可能发现人类青光眼表型的新原因，从而进一步探索潜在的治疗靶点。

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.