The Role of Cilia and Cerebrospinal Fluid Flow in Spine Development and Human Disease

项目总结/摘要 青少年特发性脊柱侧凸（AIS）是一种普遍的疾病，影响全球3%的儿童。有 目前对于AIS中出现的三维脊柱曲线没有已知的潜在基础，因此， 治疗仅限于侵入性手术干预或发病后的支具。当前的主要障碍 理解和治疗AIS是我们对疾病的潜在病因学的认识不足， 由于缺乏动物模型，情况更加复杂。这项建议的目的是 通过阐明AIS的原因，通过生成和表征 这种疾病的特殊斑马鱼模型，并通过了解疾病的机制基础。 在我的初步数据中，我证明了具有异常纤毛运动和CSF流动的斑马鱼突变体， 表现出晚发型脊柱曲线，模拟了人类AIS的定义特征。此外，我证明， 斑马鱼ptk7突变体（唯一存在的AIS模型）脊柱弯曲的原因可能源于 纤毛功能失调这使我对AIS的病因提出了一个新的假设： 部分由纤毛产生的脑脊液（CSF）流动导致AIS中的异常脊柱弯曲。为了验证这一 中心假设，我将通过定位和蛋白质组学研究来检查C21ORF59的功能， 已经发现通过连接纤毛运动性和纤毛极性对于纤毛介导的流动产生至关重要。以来 纤毛生物学的这两个方面之间的联系，都是生产流产生的关键， 我的新蛋白质组学方法将导致知识的重要增长。以及 通过研究纤毛产生水流的分子基础，我将产生并描述几种斑马鱼 AIS模型，以评估这些患者是否有CSF流动缺陷，并测试与 人AIS影响斑马鱼CSF流动产生。这将消除在这一领域取得进展的主要障碍。我 还将研究人类纤毛运动基因的变异是否会导致动物模型中的AIS。这些 实验将使我能够破译异常的纤毛运动和功能失调的脑脊液流动是如何与 人类AIS。最后，从指导阶段开始，延伸到独立阶段，我将执行 机制实验，以确定空间和时间的基础，AIS发病的斑马鱼，实验， 需要使用我们独特的温度敏感纤毛运动突变体这将允许我开始解释 脑脊液流动障碍如何导致脊柱弯曲我的建议将测试脊柱弯曲是否可以解决 通过恢复CSF流量和恢复的程度，可能提供一个原理证明， 脊柱弯曲可以进行非侵入性治疗，并为未来的治疗干预铺平了道路。 流行且知之甚少的疾病。因为我的研究强烈表明脑脊液流动对于维持 脊柱平直度，我结束了一系列有针对性的实验，旨在了解脑脊液流动是如何 在发育中的脊椎管中感觉到。

Project Summary/Abstract Adolescent Idiopathic Scoliosis (AIS) is a prevalent condition that impacts 3% of children worldwide. There is currently no known underlying basis for the three-dimensional spinal curves that occur in AIS and, as such, treatment is restricted to invasive surgical intervention or bracing post onset. A major obstacle to current understanding and treatment of AIS is our poor knowledge of the underlying etiology of the condition, a situation which is further compounded by our lack of animal models. The objective of this proposal is to address these barriers to progress by elucidating the cause of AIS, by generating and characterizing exceptional zebrafish models of the condition, and by understanding the mechanistic basis of the disease. In my preliminary data, I demonstrate that zebrafish mutants with abnormal cilia motility and CSF flow exhibit late-onset spinal curves that model the defining features of human AIS. Moreover, I demonstrate that the cause of spinal curves in zebrafish ptk7 mutants, the only existing AIS model, is likely to originate with dysfunctional cilia. This led me to propose a novel hypothesis for the cause of AIS: abnormal cerebrospinal fluid (CSF) flow, which is generated in part by cilia, causes the abnormal spinal curves in AIS. To test this central hypothesis, I will examine by localization and proteomic studies the function of C21ORF59, a protein I have found to be critical for cilia-mediated flow generation by linking cilia motility and cilia polarity. Since the links between these two facets of cilia biology, both critical for productive flow generation, are poorly understood, my novel proteomic approaches will lead to an important increase in knowledge. As well as investigating the molecular basis of flow generation by cilia, I will generate and characterize several zebrafish AIS models to assess whether these have defective CSF flow and to test whether mutations in genes linked to human AIS affect CSF flow generation in zebrafish. This will remove the major barrier to progress in this field. I will also investigate whether human variants in cilia motility genes cause AIS in animal models. These experiments will allow me to decipher how abnormal cilia motility and dysfunctional CSF flow are linked to human AIS. Lastly, beginning in the mentored phase but extending into the independent phase, I will perform mechanistic experiments to define the spatial and temporal basis for AIS onset in zebrafish, experiments that require the use of our unique temperature sensitive cilia motility mutant. This will allow me to begin to explain how dysfunctional CSF flow causes spinal curves. My proposal will test whether spinal curves can be resolved post-onset by restoring CSF flow and the extent of that recovery, potentially providing a proof-of-principle that spinal curves can be treated non-invasively and paving the way for future therapeutic interventions for this prevalent and poorly understood disease. Since my work strongly suggests that CSF flow is critical to maintain spine straightness, I end with a series of targeted experiments aimed at understanding how CSF flow is sensed in the developing spinal canal.