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: 10046194
• 负责人: Eric D. Larson
• 金额: $ 15.55万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046194
• 关键词: 3-Dimensional; Acetylcholine; Address; Allergens; Anatomy; Anterior; Antibodies; Apical; Bacteria; Cells; Characteristics; Chemicals; Chemoreceptors; Chromium; Chronic; Complement Receptor; Data; Disease; Elements; Epithelial; Epithelial Attachment; Epithelial Cells; Epithelium; 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; Structure of respiratory epithelium; 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.

项目概要 鼻腔经常暴露在空气中的颗粒物中，其中一些颗粒物可能对鼻腔造成损害。 呼吸道。有毒颗粒被困在粘膜层并通过粘膜纤毛排出 清除。一些颗粒通过刺激孤立的炎症反应，引发局部保护性炎症反应。 导致肽能神经纤维激活的化学感应细胞。孤立的化学感应细胞做出反应 通过顶端微绒毛上的受体来对抗许多空气传播的刺激物。物质范围 已知这些细胞可以检测包括苦味物质、细菌分泌的高丝氨酸内酯以及一些 有气味的刺激物。虽然这些细胞通常具有保护性，但慢性刺激可能会导致病因学 气道疾病，包括慢性鼻窦炎和传导性嗅觉丧失。目前的提案旨在 利用创新技术探索单一细胞群如何能够检测多种 空气中的刺激物。首先，单细胞RNA测序将用于检查孤立的异质性 化学感应细胞。将研究并统计单个细胞上化学感受器的类型 方法将用于根据某些细胞群包含的转录本对它们进行聚类。这些结果 将有助于定义特定的信号机制，通过该机制某些刺激可以激活不同类别的 孤立的化学感应细胞并向神经系统或周围组织发出信号。在本篇的第二部分中 建议，整个鼻腔中孤立的化学感应细胞的分布和神经支配将是 使用光学透明方法和光片显微镜进行探索。光片显微镜的应用 光学透明的组织无需对组织样本进行物理切片以进行免疫细胞化学分析。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。具体来说，孤立化学感应细胞和神经细胞的亚群 纤维将使用抗体或 RNA 探针进行可视化。这些实验将解决某些 鼻腔中孤立的化学感应细胞群在空间上分离，并且是否某些 人群或多或少可能受到肽能神经纤维的支配。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 通常被称为鼻腔中引发炎症的同质化学传感器群 通过单一机制做出反应；然而，这项研究的结果将重塑当前的知识 通过定义亚型并检查它们在鼻子内的分布模式来识别孤立的化学感应细胞。 这些结果将让我们深入了解特定刺激可以激活某些类别的孤独的方式。 化学感应细胞并将此信息传递给神经系统或周围组织。