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

项目摘要 鼻腔经常暴露在空气中的颗粒物中，其中一些可能会对鼻腔 呼吸道。有害颗粒被困在粘膜层，并通过粘液纤毛被排出。 通行证。一些颗粒通过刺激孤立体而触发局部的保护性炎症反应 能激活肽能神经纤维的化学感觉细胞。孤立的化学感觉细胞反应 通过其顶端微绒毛突起上的受体对许多空气传播的刺激物产生影响。物质的范围 已知这些细胞检测到的物质包括苦味物质、细菌排泄的高丝氨酸内酯以及一些 有气味的刺激物。虽然这些细胞通常是保护性的，但慢性刺激可能是病因之一。 呼吸道疾病，包括慢性鼻窦炎和传导性气味丧失。目前的提案旨在 利用创新技术探索这一单一细胞群体如何能够检测到各种各样的 空气中的刺激物。首先，将使用单细胞RNA测序来检测孤立子的异质性 化学感觉细胞。将对单个细胞上的化学感受器的类型进行调查和统计 方法将被用来根据细胞包含的转录本对某些细胞群体进行聚类。这些结果 将有助于定义特定的信号机制，通过这些机制，某些刺激可以激活不同类别的 孤立的化学感觉细胞，并向神经系统或周围组织发出信号。在本部分的第二部分 建议，孤立性化学感觉细胞在整个鼻腔的分布和神经支配 探索使用光学清晰法和光片显微镜。光片显微镜在医学上的应用 光学透明的组织避免了对组织样本进行物理切片进行免疫细胞化学的需要。通过 结合这些方法，可以生成大量组织的三维重建以 确定准确的空间信息。具体地说，孤立的化学感觉细胞和神经亚群 纤维将使用抗体或RNA探针进行可视化。这些实验将解决是否某些 鼻腔中孤立的化学感觉细胞群在空间上是分离的，以及某些 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 对化学感官和呼吸系统科学家有益。目前，孤立的化学感觉细胞是 通常指的是鼻腔中引发炎症的同质化学感受器群 然而，这项研究的结果将重塑目前对 通过定义亚型并检查它们在鼻子内的分布模式来确定孤立的化学感觉细胞。 这些结果将使我们深入了解特定刺激如何激活某些类别的孤立感 化学感觉细胞，并将这一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.