Molecular Mechanisms of Axenfeld-Rieger Syndrome

描述(由申请人提供)：说明申请的广泛、长期目标和具体目标，并参考项目与健康相关的内容。简明扼要地描述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。此摘要的目的是在脱离应用程序时，作为对拟议工作的简洁和准确的描述。如果申请得到资助，这一描述将成为公开信息。因此，不包括专有/机密信息。PITX2转录因子是脊椎动物眼睛正常发育所必需的。PITX2基因突变导致与发育性青光眼和其他系统性缺陷相关的阿森菲尔德-里格综合征(ARS)。我们的研究表明，在斑马鱼的眼睛发育中，Pitx2的作用是保守的。利用斑马鱼模型，我们确定了在眼、脑和颅面发育过程中调节PITX2/PITX2表达的多个调控区域。这些研究导致了阿森菲尔德-里格综合征的一种新机制的发现：PITX2上游的遥远调控元件的缺失。事实上，利用一大批ARS患者，我们表明，当同时检查编码区和调节区时，几乎所有经典的ARS病例都可以通过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导致初步确定了PITX2/PITX2的上游调控因子，目前正在分析它们在正常眼发育和疾病中的作用。几个下游基因也已经通过体外和体内试验得到了鉴定和验证。我们鉴定的基因和其他一些已发表的因子将在这一应用中进一步评估它们在PITX2/PITX2途径中的作用。这项建议的创新之一是，我们计划利用我们在上一个资助期产生的资源，在眼周间充质(POM)细胞中表达GFP的Pitx2增强子转基因株和Pitx2基因敲除试剂，以识别不同发育阶段正常分化和Pitx2缺陷的POM细胞的转录本。这将导致在体内可靠地识别PITX2/PITX2下游效应者，并有助于对这一对正常眼睛发育具有高度意义的迁徙群体的总体特征的描述。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来产生携带Pitx2编码区(空等位基因)和CE4调控元件突变的Pitx2系，此前已证明该突变元件参与Pitx2表达的不同方面，包括在POM细胞中的强烈存在。我们的主要假设是PITX2/PITX2是一种保守的因子，在眼周间充质细胞向发育中的眼前段迁移时，对于诱导其分化是必不可少的。PITX2表达的上游调控因子和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的认识，并有可能发现人类青光眼表型的新原因，可作为潜在的治疗靶点进行进一步探索。 与公众健康相关：PITX2对于正常的眼睛发育是必不可少的。这项提案将通过确定其他调控PITX2表达的基因和受PITX2活动控制的基因来研究PITX2如何参与眼睛发育。该项目的结果将提供对眼睛发育的更好了解，并可能确定导致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.