Uncovering the role of inppl1a in notochord vacuolation and the development of a straight body axis.

项目摘要 脊索是一种高度保守的发育组织，它沿着所有动物的前后轴延伸。 脊索动物，包括人类。它由内层空泡细胞和外层细胞组成。 分泌厚的细胞外基质的鞘细胞。限制性血管内空泡细胞的膨胀 鞘产生一个加压杆，支持运动的脊索动物，并最终模式的脊柱 脊椎动物因此，脊索和脊柱的发育是密切相关的， 脊索细胞的形成与脊柱侧凸和脊椎畸形有关。脊索是 在小鼠模型中难以研究的组织，因为它在出生时已经被脊柱取代。在 相比之下，斑马鱼的外部发育和光学透明度使其适合研究 涉及脊索发育和成熟的过程。该提案将使用定量图像 分析，斑马鱼遗传学和现代蛋白质组学方法来定义肌醇的作用 多聚磷酸酶样1a（inpp 1a）基因在脊索和脊柱发育中的作用。该基因的突变 导致斑马鱼的早期脊索缺陷和胸椎侧凸。在这个奖学金计划中，我将测试 假设inppl 1a调节脊索空泡膨胀，并最终调节 有三个具体目标的脊索。在目标1中，我将通过以下方法确定inpp 1a在脊索空泡化中的作用： 脊索细胞大小和空泡膨胀（1.1）以及内部空泡膜动力学的定量变化 （1.2）。我还将定义在脊索开发过程中inppl 1a的时间和空间需求， 药理学和分子遗传学方法（1.3）。在目标2中，我将评估 通过操纵机械应力（2.1）和椎骨矿化（2.2-2.3）的inpp 11 a突变脊索 在发展过程中。最后，在目标3中，我将定义脊索和脊柱中Inpp 11 a的蛋白质相互作用物 发展我将使用候选基因方法（3.1）和邻近依赖标记策略（3.2）来 鉴定Inpp 1a依赖性脊索液泡膨胀所需的其他蛋白质。为补充这些 方法，我还将使用现代蛋白质组学技术建立一个全面的脊索图谱 蛋白质相互作用网络（3.3）。在这样做的过程中，我将为未来的调查建立一个宝贵的资源， 参与脊索发育的蛋白质。总而言之，本提案中的工作将增加对以下方面的了解： 脊索细胞如何空泡化，最终将有助于我们了解人类骨骼健康， 疾病虽然脊索被认为是胚胎组织，但它与成人疾病有关， 包括椎间盘变性和脉络膜炎。此外，INPPL 1突变导致罕见的 软骨内骨疾病，视神经发育不良。因此，这项在斑马鱼中的工作将是重要的，因为它 将可能揭示inpp 11 a/INPPL 1在骨骼发育和疾病中的保守作用。

PROJECT SUMMARY The notochord is a highly conserved developmental tissue that extends along the anterior-posterior axis of all chordates, including humans. It is composed of inner vacuolated cells surrounded by an external layer of sheath cells that secrete a thick extracellular matrix. Inflation of the vacuolated cells within the restrictive sheath creates a pressurized rod that supports locomotion in chordates and ultimately patterns the spine of vertebrates. As such, the development of the notochord and spine are intimately linked, and defects in the formation of notochord cells have been linked to scoliosis and vertebral malformations. The notochord is a difficult tissue to study in mouse models since it is already replaced by the spine at the time of birth. In contrast, the external development and optical transparency of zebrafish make them suitable for investigating processes involved in notochord development and maturation. This proposal will use quantitative image analysis, zebrafish genetics, and modern proteomics approaches to define the role of the inositol polyphosphate phosphatase-like 1a (inppl1a) gene in notochord and spine development. Mutations in this gene cause early notochord defects and thoracic scoliosis in zebrafish. In this fellowship proposal, I will test the hypothesis that inppl1a regulates notochord vacuole inflation and, ultimately, the mechanical stability of the notochord with three Specific Aims. In Aim 1, I will determine the role of inppl1a in notochord vacuolation by quantifying changes in notochord cell size and vacuole inflation (1.1) and internal vacuole membrane dynamics (1.2). I will also define the temporal and spatial requirement of inppl1a during notochord development using pharmacological and molecular-genetic approaches (1.3). In Aim 2, I will evaluate the mechanical properties of inppl1a mutant notochords by manipulating mechanical stress (2.1) and vertebral bone mineralization (2.2-2.3) during development. Finally, in Aim 3, I will define the protein interactors of Inppl1a in notochord and spine development. I will use a candidate gene approach (3.1) and a proximity-dependent labeling strategy (3.2) to identify additional proteins required for Inppl1a-dependent notochord vacuole inflation. To supplement these approaches, I will also use modern proteomics techniques to build a comprehensive atlas of the notochord protein interaction network (3.3). In doing so, I will build an invaluable resource for future investigation of proteins involved in notochord development. Altogether, the work in this proposal will add to the knowledge of how notochord cells vacuolate and will ultimately benefit our understanding of human skeletal health and disease. Although the notochord is considered an embryonic tissue, it has been implicated in adult diseases, including intervertebral disc degeneration and chordoma. Additionally, mutations in INPPL1 cause the rare endochondral bone disorder, Opsismodysplasia. Therefore, this work in zebrafish will be significant because it will likely reveal a conserved role for inppl1a/INPPL1 in skeletal development and disease.