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

项目摘要 鼻腔经常暴露于空气中的颗粒物，其中一些颗粒物对鼻腔有潜在的损害。 呼吸道诺维汀颗粒被困在粘膜层，并通过粘膜纤毛排出 间隙一些颗粒通过刺激孤立性炎症反应而触发局部保护性炎症反应。 导致肽能神经纤维活化的化学感受细胞。孤立的化学感受细胞 通过其顶端微绒毛突起上的受体对许多空气中的刺激物产生刺激。物质的范围 已知这些细胞检测包括苦味物质、由细菌分泌的高丝氨酸内酯，以及一些 刺激性气味。虽然这些细胞通常具有保护作用，但慢性刺激可能导致病因学 包括慢性鼻窦炎和传导性嗅觉丧失。目前的建议旨在 利用创新技术探索这一单一细胞群如何能够检测各种各样的 空气传播的刺激物首先，将使用单细胞RNA测序来检查孤立的细胞的异质性。 化学感受细胞将对单个细胞上的化学感受器的类型进行研究和统计 方法将被用于基于它们所包含的转录物来聚类某些细胞群。这些结果 将有助于定义特定的信号传导机制，通过这些机制，某些刺激可以激活不同类型的 单独的化学感受细胞和信号到神经系统或周围组织。的第二部分 建议，整个鼻腔中孤立化学感受细胞的分布和神经支配将被 探索使用光学透明方法和光片显微镜。光片显微镜在 光学透明的组织避免了对用于免疫细胞化学的组织样品进行物理切片的需要。通过 结合这些方法，可以生成大体积组织的三维重建， 准确的空间信息。具体来说，孤立的化学感受细胞和神经细胞的亚群 纤维将使用抗体或RNA探针来可视化。这些实验将解决某些 单独的化学感受细胞群体在鼻腔中空间分离，并且某些化学感受细胞是否 群体或多或少可能受肽能神经纤维支配。这项研究的结果将 对化学感觉和呼吸系统科学家有益。目前，单独的化学感受细胞 通常被称为鼻腔中的化学传感器的同质群体， 然而，这项研究的结果将重塑目前对 通过定义亚型和检查它们在鼻子内的分布模式来观察孤立的化学感受细胞。 这些结果将使我们了解特定刺激可以激活某些类型的孤独 化学感受细胞并将此信息传递给神经系统或周围组织。

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.