Oculomotor axon guidance in normal and abnormal development

这项建议描述了一个为期3年的学术生涯发展培训计划，重点是 了解斜视和眼球运动障碍的分子机制。我是一名儿科医生 波士顿儿童医院(BCH)和哈佛医学院(HMS)的眼科医生，目前正在接受 哈佛远景临床科学家发展计划(K12)。我希望继续我的研究和事业 发展，为独立的研究生涯做准备。我的长期目标包括了解 复杂性斜视的分子基础，阐明轴突引导的基本机制，并最终 设计治疗斜视和眼球运动障碍的新疗法，如异常再生。 拟议的研究计划侧重于识别对轴突引导重要的新分子途径。 动眼神经与先天性眼外肌纤维化的其他遗传因素 (CFEOM)。伊丽莎白·恩格尔博士将担任导师。她是一位世界上的专家，研究 动眼神经系统，并在生产力和指导方面有良好的记录。咨询委员会 包括大卫·亨特、丹尼尔·麦克阿瑟和拉里·本诺维茨将指导我的研究和职业发展。 该项目将在BCH丰富的研究环境中进行，可以获得HMS的资源， 马萨诸塞州眼耳医院和布罗德研究所。有大量的机会参加和 出席实验室会议、部门研讨会、全国会议和教学课程。 第一种实验方法使用了我开发的一种新的中脑-眼眶胚胎切片分析，其中 发育中的动眼神经在向眼眶生长的过程中是时间推移的图像。识别信号通路 在动眼神经生长和引导方面很重要，培养物用小分子细胞抑制物处理- 表面受体。候选受体的选择将基于运动神经元的转录图谱， E10.5和E11.5(未公布数据)。在薄片分析中干扰导向的分子的作用将是 通过检查轴突引导缺陷和异常神经支配的小鼠模型在体内定义。证明 使用CXCR4信号的抑制剂AMD3100已经建立了这一概念。体内初步数据 证实动眼神经轴突向背侧生长，而不是向腹侧生长，这一途径将被进一步研究。 第二种方法试图将这些发现与人类的眼球运动障碍联系起来，特别是 CFEOM。全基因组测序(WGS)正在对48个遗传未解决的CFEOM家系进行， 并将与布罗德研究所的丹尼尔·麦克阿瑟的团队合作进行分析。途径中的变体 切片分析中的扰动轴突引导将在分析中优先考虑。相反，在 WGS将在切片检测中进行测试，以帮助确定致病性。通过这些方法，我们将 确定在轴突引导中重要的新途径，开辟新的研究领域。该提案针对的是 对临床需求领域的研究不足，同时为我作为临床科学家的成功职业生涯做好准备。

This proposal describes a 3-year training program for the development of an academic career focused on understanding the molecular mechanisms of strabismus and ocular motility disorders. I am a pediatric ophthalmologist at Boston Children's Hospital (BCH) and Harvard Medical School (HMS), currently training in the Harvard Vision Clinical Scientist Development Program (K12). I wish to continue my research and career development to prepare for an independent research career. My long-term goals include understanding the molecular basis of complex strabismus, elucidating fundamental mechanisms of axon guidance, and ultimately devising new treatments for strabismus and ocular motility disorders, such as aberrant regeneration. The proposed research program focuses on identifying new molecular pathways important for axon guidance of the oculomotor nerve and additional genetic causes of Congenital Fibrosis of the Extraocular Muscles (CFEOM). Dr. Elizabeth Engle will serve as mentor. She is a world expert on the development of the oculomotor system and has a proven track record of productivity and mentorship. An advisory committee including David Hunter, Daniel MacArthur, and Larry Benowitz will guide my research and career development. The project will take place in the rich research environment of BCH, with access to the resources of HMS, Massachusetts Eye and Ear Infirmary, and the Broad Institute. There are extensive opportunities to attend and present at lab meetings, departmental seminars, national meetings, and didactic courses. The first experimental approach uses a novel midbrain-orbit embryonic slice assay I developed, in which the developing oculomotor nerve is time-lapse imaged as it grows towards the orbit. To identify signaling pathways important in oculomotor growth and guidance the cultures are treated with small molecule inhibitors of cell- surface receptors. Candidate receptors will be chosen based on transcriptional profiling of motor neurons at E10.5 and E11.5 (unpublished data). The role of molecules that perturb guidance in the slice assay will be defined in vivo by examining mouse models for axon guidance defects and aberrant innervation. Proof of concept has been established using an inhibitor of CXCR4 signaling, AMD3100. Preliminary data in vivo confirms oculomotor axon growth dorsally rather than ventrally, and this pathway will be further examined. The second approach seeks to relate these findings to human disorders of ocular motility, particularly CFEOM. Whole genome sequencing (WGS) is underway on 48 pedigrees with genetically unsolved CFEOM, and will be analyzed in collaboration with Daniel MacArthur's team at the Broad Institute. Variants in pathways that perturb axon guidance in the slice assay will be prioritized in the analysis. Conversely, variants identified in the WGS will be tested in the slice assay to help determine pathogenicity. Through these approaches, we will identify new pathways important in axon guidance, opening new areas of research. The proposal addresses an understudied area of clinical need while preparing me for a successful career as a clinician-scientist.