Interrogation of individual cells to identify lung progenitors and their niches

描述（由申请人提供）：识别和表征肺祖细胞及其信号龛对于理解健康的肺是如何建立和维持的，发育和维持途径的改变是如何导致或促成肺部疾病的，以及如何预防疾病和修复或替换受损的肺组织至关重要。由于肺部复杂的三维结构以及缺乏标记、追踪命运和操纵体内单个肺细胞基因表达的工具，对肺祖细胞及其生态位的识别和表征一直受到阻碍。在这里，我们建议开发这样的工具，通过适应用于小鼠肺的系统遗传方法和单细胞分辨率遗传工具（“克隆分析”），这些工具在过去十年中已被用于阐明模式生物果蝇的祖细胞和干细胞及其信号龛。我们将这种体内方法与高通量体外方法相结合，利用基因组学和微流体学的最新进展来表征个体肺祖细胞及其对体内实验中发现的信号的发育反应。这种联合方法是通用的，适用于整个肺和其他小鼠组织的祖细胞，尽管我们关注的是肺间质中特征不明显的祖细胞。具体目标是：

DESCRIPTION (provided by applicant): Identifying and characterizing lung progenitor cells and their signaling niches is crucial for understanding how a healthy lung is built and maintained, how alteration of development and maintenance pathways cause or contribute to lung disease, and how disease can be prevented and damaged lung tissue restored or replaced. Identifying and characterizing lung progenitor cells and their niches has been hampered by the complex three-dimensional structure of the lung and the lack of tools to mark, follow the fate, and manipulate gene expression in individual lung cells in vivo. Here we propose to develop such tools, by adapting for use in mouse lung the systematic genetic approaches and single cell resolution genetic tools ("clonal analysis") that have been used over the past decade to elucidate progenitor and stem cells and their signaling niches in the model organism Drosophila. We combine this in vivo approach with a high throughput in vitro approach that takes advantage of recent advances in genomics and microfluidics to characterize individual lung progenitor cells and their developmental responses to the signals identified in the in vivo experiments. This combined approach is general and applicable to progenitor cells throughout the lung and other mouse tissues, although we focus on the poorly characterized progenitor cells in lung mesenchyme. The specific aims are: To establish a general multi-color in vivo cell marking method to follow the fate of individual mouse lung cells, and to use this method to identify the number and types of progenitors cells in the mesenchyme of developing and mature mouse lung. To use high throughput in situ hybridization studies to localize all of the signaling and receiving centers in progenitor cell niches in the mouse lung; To determine the in vivo functions of the identified signaling and receiving centers in lung progenitor cell biology by constructing a library of transgenic mouse strains that can be used to create ectopic signaling centers and clonally inactivate each signaling pathway in the mouse lung; To establish a high throughput, microfluidic approach to isolate, propagate, profile, and assess the developmental potential of identified lung progenitor cells under defined culture conditions, and to use this to determine the number of molecularly and functionally distinct progenitor cell populations in lung mesenchyme; To establish methods for directing development of lung progenitor cells along specific lineages using the high throughput microfluidic approach to expose isolated, identified progenitor cells systematically to different concentrations, gradients, temporal patterns, and combinations of signaling molecules identified in the in vivo experiments.