Investigating chemosensory cell heterogeneity in the upper airway epithelium with single cell RNA sequencing and high resolution light sheet microscopy

利用单细胞 RNA 测序和高分辨率光片显微镜研究上气道上皮化学感应细胞异质性

• 批准号: 10402936
• 负责人: Eric D. Larson
• 金额: $ 8.43万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-12 至 2023-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402936
• 关键词: 3-Dimensional; Acetylcholine; Address; Allergens; Anatomy; Anosmia; Anterior; Antibodies; Apical; Bacteria; Cells; Characteristics; Chemicals; Chemoreceptors; Chromium; Chronic; Complement Receptor; Data; Disease; Elements; Epithelial; Epithelial Attachment; Epithelial Cells; Etiology; Exposure to; Fingers; Free Nerve Ending; Functional disorder; Genes; Genomics; Goals; Head; Heterogeneity; Human; IgE; Image; Individual; Inflammation; Inflammatory Response; Inhalation; Irritants; Knowledge; Label; Lead; Libraries; Light; Location; Measures; Mediating; Methods; Microscopy; Molds; Molecular Profiling; Mucociliary Clearance; Mucous Membrane; Mucous body substance; Mus; Nasal cavity; Nerve Fibers; Nervous system structure; Nose; Optics; Pathway interactions; Pattern; Physiological; Play; Pollen; Population; Process; Property; Proteins; Quality of life; RNA Probes; Receptor Signaling; Research; Resolution; Respiratory System; Rhinitis; Rodent; Sampling; Scientist; Signal Pathway; Signal Transduction; Signaling Molecule; Smell Perception; Statistical Methods; Stimulus; Surface; TRP channel; TRPM5 gene; Taste Buds; Taste Perception; Techniques; Testing; Tight Junctions; Tissue Sample; Tissues; Transcript; Trigeminal System; Xenobiotics; airway epithelium; alpha-gustducin; base; cholinergic; chronic rhinosinusitis; experimental study; genetic manipulation; homoserine lactone; immunocytochemistry; innovation; innovative technologies; insight; lipophilicity; mouse model; nerve supply; particle; phospholipase C beta; prevent; receptor; receptor expression; reconstruction; respiratory; response; rhinosinusitis; single-cell RNA sequencing; transcriptome sequencing

Project Summary The nasal cavity is constantly exposed to airborne particles, some of which are potentially damaging to the respiratory tract. Noxious particles are trapped in the mucosal layer and are expelled through mucociliary clearance. Some particles trigger a local, protective inflammatory response through stimulation of solitary chemosensory cells that results in activation of peptidergic nerve fibers. Solitary chemosensory cells respond to numerous airborne irritants through receptors on their apical microvillar processes. The range of substances these cells are known to detect include bitter substances, homoserine lactones excreted by bacteria, and some odorous irritants. While these cells are generally protective, chronic stimulation could contribute to the etiology of airway disorders including chronic rhinosinusitis and conductive smell loss. The current proposal seeks to leverage innovative technologies to explore how this single population of cells is able to detect a wide variety of airborne irritants. First, single cell RNA-sequencing will be used to examine the heterogeneity of solitary chemosensory cells. The types of chemoreceptors on individual cells will be investigated and statistical methods will be used to cluster certain populations of cells based on the transcripts they contain. These results will help define specific signaling mechanisms by which certain stimuli could activate different classes of solitary chemosensory cells and signal to the nervous system or surrounding tissue. In the second part of this proposal, the distribution and innervation of solitary chemosensory cells in the entire nasal cavity will be explored using optical clearing methods and light sheet microscopy. The use of light sheet microscopy on optically cleared tissues prevents the need to physically section tissue samples for immunocytochemistry. By combining these methods, three dimensional reconstructions of large volumes of tissues can be generated to ascertain accurate spatial information. Specifically, subpopulations of solitary chemosensory cells and nerve fibers will be visualized using antibodies or RNA-probes. These experiments will address whether certain populations of solitary chemosensory cells are spatially segregated in the nasal cavity and whether certain populations are more or less likely to be innervated by peptidergic nerve fibers. The results from this study will be beneficial to chemosensory and respiratory system scientists. Currently, solitary chemosensory cells are usually referred to as a homogenous population of chemosensors in the nasal cavity that trigger inflammatory responses through a single mechanism; however, the results from this study will reshape current knowledge of solitary chemosensory cells by defining subtypes and examining their distribution patterns within the nose. These results will give insight to ways that specific stimuli could activate certain classes of solitary chemosensory cells and communicate this information to the nervous system or surrounding tissues.

项目摘要 鼻腔不断暴露于空中颗粒，其中一些可能会损害 呼吸道。有害的颗粒被困在粘膜层中，并通过粘膜纤毛排出 清除。一些颗粒通过刺激诱导局部保护性炎症反应 化学感应细胞会激活肽纤维纤维。孤立的化学感应细胞反应 通过受体在其顶端微绒毛过程中通过受体进行众多空中刺激物。物质范围 已知这些细胞可检测到包括苦味物质，被细菌排泄的均烯碱内酯，有些是 有刺激性的刺激性。尽管这些细胞通常具有保护性，但慢性刺激可能有助于病因 气道疾病，包括慢性鼻孔炎和导电气味丧失。当前的提议试图 利用创新技术来探讨这种单一的细胞如何能够检测到各种各样的细胞 空气刺激物。首先，将使用单细胞RNA测序来检查孤立的异质性 化学感应细胞。将研究单个细胞上的化学感受器的类型和统计 方法将根据其所包含的转录本来群集某些单元格的群体。这些结果 将有助于定义某些刺激可以激活不同类别的特定信号传导机制 孤立的化学感应细胞，并向神经系统或周围组织发出信号。在第二部分 提案，整个鼻腔中孤立化学感应细胞的分布和神经支配将是 使用光学清除方法和轻度显微镜进行了探索。轻微显微镜在 光学清除的组织可防止需要对免疫细胞化学的物理分割组织样品。经过 结合这些方法，可以生成大量组织的三维重建 确定准确的空间信息。具体而言，孤立的化学水敏细胞和神经的亚群 将使用抗体或RNA探针可视化纤维。这些实验将解决是否确定 鼻腔中孤立的化学感应细胞的群体在空间上被隔离，并且是否某些 种群或多或少地被肽能神经纤维支配。这项研究的结果将 对化学感应和呼吸系统科学家有益。目前，孤立的化学感应细胞是 通常被称为鼻腔中的化学传感器的同质种群，引发炎症 通过单一机制做出响应；但是，这项研究的结果将重塑当前的知识 通过定义亚型并检查其鼻子内的分布模式，孤立的化学感应细胞。 这些结果将深入了解特定刺激可以激活某些类别的单生 化学感应细胞并将这些信息传达给神经系统或周围组织。