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。

• 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
• 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

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.

项目摘要 脊索是一种高度保守的发育组织，沿着所有的前后轴延伸 弦，包括人类。它由内部吸尘单元组成，周围是 分泌厚细胞外基质的鞘细胞。限制性细胞的充气 鞘形成一个加压杆，该杆支持弦弦中的运动，并最终模式为 脊椎动物。因此，脊索和脊柱的发展与 脊索细胞的形成与脊柱侧弯和椎骨畸形有关。脊索是一个 在小鼠模型中很难研究的组织，因为它在出生时已经被脊柱取代。在 对比，斑马鱼的外部发展和光学透明度使它们适合研究 脊索发展和成熟涉及的过程。该建议将使用定量图像 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 脊索和脊柱发育中的多磷酸磷酸酶样1a（Inppl1a）基因。该基因的突变 引起斑马鱼的早期脊索缺陷和胸脊柱侧弯。在此奖学金建议中，我将测试 INPPL1A调节脊索液泡通胀的假设，最终是机械稳定性 具有三个特定目标的脊索。在AIM 1中，我将通过通过 量化脊索细胞大小和液泡膨胀（1.1）和内部液泡膜动力学的变化 （1.2）。我还将在努力训练期间定义inppl1a的时间和空间要求 药理和分子遗传学方法（1.3）。在AIM 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），Inppl1a突变体脊索 在开发过程中。最后，在AIM 3中，我将定义notochord和脊柱中Inppl1a的蛋白质相互作用者 发展。我将使用候选基因方法（3.1）和依赖性标签策略（3.2） 确定依赖Inppl1a依赖性的努力液膨胀所需的其他蛋白质。补充这些 方法，我还将使用现代蛋白质组学技术来构建Notochord的全面地图集 蛋白质相互作用网络（3.3）。这样，我将建立一个宝贵的资源 参与脊索发展的蛋白质。总之，该提案中的工作将增加 努力细胞如何空泡，最终将有益于我们对人类骨骼健康的理解和 疾病。尽管脊索被认为是胚胎组织，但它与成人疾病有关 包括椎间盘变性和脊索瘤。另外，Inppl1中的突变导致罕见 软骨内骨障碍，Opsismodyplasia。因此，斑马鱼中的这项工作将是重要的 可能会在骨骼发育和疾病中揭示INPPL1A/INPPL1的保守作用。