Profiling the transcriptome of globose basal cells of the olfactory epithelium at the single cell level

在单细胞水平上分析嗅上皮球状基底细胞的转录组

• 批准号: 9226320
• 负责人: JAMES E. SCHWOB
• 金额: $ 24.75万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226320
• 关键词: Acceleration; Activities of Daily Living; Address; Adult; Age; Aging; Antibodies; Area; Basal Cell; Bioinformatics; Biological; CRISPR/Cas technology; Categories; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Chimeric Proteins; Chronic; Data; Development; Disease; Doxycycline; Elderly; Environment; Epithelial; Epithelium; Exhibits; Exploratory/Developmental Grant; Expression Library; Functional disorder; Future; Gene Expression Profile; Genes; Goals; Grant; Hand; Harvest; Histones; Homeostasis; Immunohistochemistry; In Situ Hybridization; Individual; Injury; Kinetics; Knock-in; Knock-out; Knowledge; Label; Learning; Liquid substance; Mediating; Metaplasia; Mission; Mitotic; Molecular; Molecular Profiling; Mouse Strains; Multipotent Stem Cells; Mus; National Institute on Deafness and Other Communication Disorders; Natural regeneration; Nature; Neurons; Nose; Nutritional status; Odors; Olfactory Basal Cell; Olfactory Epithelium; Pathologic; Pathology; Population; Positioning Attribute; Proliferating; Quality of life; Recovery; Role; Safety; Sensory; Smell Perception; Stem cells; Structure of respiratory epithelium; TCF3 gene; Testing; Thymidine; Tissues; Transgenic Mice; Transgenic Organisms; adult stem cell; aged; analog; cell type; design; exhaustion; experience; homologous recombination; insight; molecular marker; neurogenesis; neuron loss; olfactory sensory neurons; progenitor; reconstitution; respiratory; single cell analysis; stem; stem cell biology; stemness; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

The capacity of the olfactory epithelium (OE) for replenishing the population of olfactory sensory neurons and for regenerating the epithelium after injury depends on the persistence and maintained function of stem cells within that adult epithelium. Decline in sensory function in the elderly is accompanied by pathological changes in the OE that emerge because the normally active olfactory stem and progenitor cells, namely globose basal cells (GBCs), become disordered and eventually depleted. At present, we can construct a flow diagram designed to encompass and sequence the various categories of GBCs, beginning with GBCs that function as multipotent progenitors, progressing through GBCs that act as transit amplifying progenitors, and reaching GBCs that give rise directly to neurons. We can also align those purported stages in the GBC hierarchy with the expression of various transcription factors. However, we lack a comprehensive understanding of this critical stem/progenitor cell population. Are these stages discrete or are they snapshots of a more fluid progression? How do we explain the plasticity in the progenitor capacity of some GBCs? How might aging disorder the progression? The current application proposes two specific aims designed to address the critical gaps in our knowledge of the active stem and progenitor cell population. Specific Aim 1 will generate transcriptomic profiles of the various functional categories of GBCs using transgenic mouse strains that express a fluorescent marker in conjunction with the transcription factor(s) gene(s) – Sox2, Ascl1, and Neurog1 – used to define the stages in the hierarchy. Specific Aim 2 will focus on and profile a specific kinetically- defined subset of GBCs that experience prolonged mitotic quiescence, as shown by the retention of the tagged histone fusion protein H2B/GFP, which is a feature common to many stem cell types. In both cases, cells labeled by expression of the fluorescent tags will be isolated by FACS, captured as single cells, and individually profiled. Analysis of the single cell expression libraries will be used for unbiased clustering of the cells and defining the genes whose expression differs across the clusters. The patterns of gene expression that differentiate the clusters will be validated by Q-PCR and either immunohistochemistry or in situ hybridization depending on antibody availability. At the conclusion of the analysis we will have achieved a comprehensive understanding of the GBC population and will clarify whether the existing flow diagram – which envisions distinct stages and abrupt transitions – is an accurate representation of the biology of this stem and progenitor cell-encompassing population. These advances, in turn, will inform our attempts to alleviate olfactory sensory dysfunction, particularly that which accompanies aging.

嗅觉上皮（OE）的能力补充嗅觉感觉神经元和 损伤后再生上皮取决于干细胞的持久性和维持功能 在那个成年上皮中。老年人的感觉功能下降是通过病理变化来实现的 在出现的OE中，因为正常活跃的嗅觉茎和祖细胞，即Globoso碱 细胞（GBC），变得无序并最终耗尽。目前，我们可以构建一个流程图 旨在涵盖和序列各种类别的GBC，首先是GBC的功能 多能祖细胞，通过GBC的发展，这些GBC充当过境的祖细胞，并达到 直接引起神经元的GBC。我们还可以使GBC层次结构中所谓的阶段与 各种转录因子的表达。但是，我们对此缺乏全面的理解 临界茎/祖细胞群。这些阶段是离散的还是更流畅的快照 进展？我们如何解释某些GBC的祖细胞能力的可塑性？老化会如何 混乱的进展？当前的申请提出了两个特定目标，旨在解决关键 我们对活性茎和祖细胞群的知识的差距。特定目标1将产生 使用转基因小鼠菌株对GBC的各种功能类别的转录组概况 与转录因子（S）基因 - SOX2，ASCL1和Neurog1一起表达荧光标记物 - 用于定义层次结构中的阶段。特定的目标2将重点关注并介绍特定的动力学 - 定义的GBC子集经历了延长有丝分裂静脉的经历，如标记的保留所示 Hisstone融合蛋白H2B/GFP，这是许多干细胞类型共有的特征。在这两种情况下，细胞 通过荧光标签的表达标记，将由FACS分离，以单个细胞捕获，并且 单独介绍。单细胞表达式文库的分析将用于无偏聚类 细胞并定义其表达在整个簇中的差异的基因。基因表达的模式 区分簇将通过Q-PCR和免疫组织化学或原位验证 杂交取决于抗体的可用性。分析结束时，我们将实现 对GBC人口的全面了解，并将阐明现有的流程图是否 - 设想不同的阶段和突然的过渡 - 是该茎生物学的准确表示 祖细胞包含的人群。这些进步反过来将为我们减轻我们的尝试 嗅觉感觉障碍，特别是涉及衰老的感觉障碍。