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

揭示 inppl1a 在脊索空泡化和直体轴发育中的作用。

基本信息

批准号：
10826125
负责人：
Brittney Voigt
金额：
$ 4.1万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-21 至 2026-09-20
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10826125
关键词：
Adult Anterior Atlases Basement membrane Biogenesis Biology Biotinylation Birth Bone Diseases Candidate Disease Gene Cell Size Cell Volumes Cell membrane Cell secretion Cells Chest Chordata Chordoma Clustered Regularly Interspaced Short Palindromic Repeats Cytoplasm Databases Defect Deposition Development Diameter Disease Embryo Epithelium Extracellular Matrix Fellowship Fertilization Fractionation Future GTPase-Activating Proteins Genes Genetic Genetic Screening Health Human INPPL1 gene Image Analysis Inositol Investigation Knowledge Label Length Lesion Link Locomotion Maps Mass Spectrum Analysis Mechanical Stress Mechanics Membrane Modernization Molecular Genetics Mutation Optics Pattern Phosphatidylinositols Phosphoric Monoester Hydrolases Physiologic calcification Plasma Cells Polyphosphates Predisposition Process Proteins Proteomics Resources Rod Role Severities Siblings Signal Pathway Skeletal Development Spinal Curvatures Structure Swelling Swimming Techniques Testing Thick Time Tissues Tretinoin Vacuole Vertebral Bone Vertebral column Vertebrates Vesicle Viscosity Work Zebrafish bone cartilaginous embryo tissue genetic approach in vivo intervertebral disk degeneration late endosome loss of function malformation marker transgenes mechanical properties mineralization mouse model mutant notochord notochord development opsismodysplasia pharmacologic premature pressure protein complex quantitative imaging scoliosis skeletal skeletal disorder spine bone structure trafficking

项目摘要

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.

项目概要脊索是一种高度保守的发育组织，沿着所有脊索的前后轴延伸脊索动物，包括人类。它由内层空泡细胞组成，外层是空泡细胞。分泌厚厚的细胞外基质的鞘细胞。限制性内空泡细胞的膨胀鞘形成一个加压杆，支持脊索动物的运动，并最终塑造脊索动物的脊柱脊椎动物。因此，脊索和脊柱的发育是密切相关的，脊索和脊柱的缺陷脊索细胞的形成与脊柱侧弯和椎骨畸形有关。脊索是一个在小鼠模型中研究很难的组织，因为它在出生时就已经被脊柱所取代。在相比之下，斑马鱼的外部发育和光学透明度使它们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磷酸磷酸酶样 1a (inppl1a) 基因在脊索和脊柱发育中的作用。该基因突变导致斑马鱼早期脊索缺陷和胸椎脊柱侧凸。在这个奖学金提案中，我将测试假设 inppl1a 调节脊索液泡膨胀，并最终调节脊索液泡的机械稳定性脊索具有三个具体目标。在目标 1 中，我将通过以下方式确定 inppl1a 在脊索空泡形成中的作用量化脊索细胞大小和液泡膨胀 (1.1) 以及内部液泡膜动力学的变化 (1.2)。我还将使用以下方法定义脊索发育过程中 inppl1a 的时间和空间要求药理学和分子遗传学方法（1.3）。在目标 2 中，我将评估机械性能 inppl1a 突变脊索通过操纵机械应力 (2.1) 和椎骨矿化 (2.2-2.3) 在开发过程中。最后，在目标 3 中，我将定义脊索和脊柱中 Inppl1a 的蛋白质相互作用因子发展。我将使用候选基因方法（3.1）和邻近依赖标记策略（3.2）来确定 Inppl1a 依赖性脊索液泡膨胀所需的其他蛋白质。为了补充这些方法，我还将使用现代蛋白质组学技术来构建脊索的综合图谱蛋白质相互作用网络（3.3）。通过这样做，我将为未来的调查建立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以下方面的知识：脊索细胞如何形成空泡，最终将有利于我们对人类骨骼健康的理解疾病。尽管脊索被认为是胚胎组织，但它与成人疾病有关，包括椎间盘退变和脊索瘤。此外，INPPL1 的突变会导致罕见的软骨内骨疾病，Opsismodysplasia。因此，这项针对斑马鱼的工作将具有重要意义，因为它可能会揭示 inppl1a/INPPL1 在骨骼发育和疾病中的保守作用。