Mechanical Forces and the Regulation of Airway Progenitor Cells

项目总结 肺部呼吸道的分支结构允许每年大约6升的空气输送。 在外部环境和肺泡之间的一分钟。呼吸道上皮树完成放气 交换、粘液产生和病原体清除，并阻止水、微粒和 微生物。为了完成这些不同的生物学功能，呼吸道上皮由几个部分组成 在胚胎发育过程中区别于普通祖细胞的不同细胞类型，其中第一种 是肺神经内分泌细胞。干扰特化上皮细胞类型的分化 对呼吸道形态发生有负面影响，肺内神经内分泌细胞数量异常多 发现于几种先天性和获得性肺部疾病中。随着它的分化，上皮细胞会分泌离子 和水穿过其根尖表面，导致液体以跨壁压力填充呼吸道的管腔。 高到足以使肺部充气。导致跨壁压力降低的缺陷与 既有肺发育不全，又有肺神经内分泌细胞增多，但其具体作用 压力和这个机械信号下游的分子信号是未知的。通过结合时间- 利用创新的微流控培养系统，我们发现跨壁压力 控制肺发育速度和神经内分泌细胞标志物的表达。使用NEXT- 世代序列分析，我们发现低跨壁压力降低了靶基因的表达 Noch，肺神经内分泌分化的主要调节者，以及YAP，一种已知的机械传感器。 在这里，我们假设跨壁压力协调不同细胞的生长和分化。 通过Notch和YAP信号传递上皮内的类型。我们将把微流控设备与 工程小鼠、高分辨率延时旋转圆盘共聚焦显微镜和下一代 测序分析确定压力、Notch和YAP在肺功能调节中的相对作用 神经内分泌祖细胞命运的决定。在具体目标1中，我们将使用微流控胸腔， 小鼠，时间推移成像和单细胞RNA测序，以确定物理上如何跨壁压力 调节肺发育中的肺神经内分泌群。在具体目标2中，我们将使用 微流控胸腔、报告小鼠和染色质免疫沉淀方法以确定 以及跨壁压力如何调节胚胎呼吸道上皮中的Notch信号。在具体目标3中， 我们将确定压力信号是否通过YAP影响肺神经内分泌分化 和Notch小路。这项工作将定义来自微环境的机械信号是如何 传递给发育中的呼吸道上皮细胞的第一个祖细胞命运决定。我们预计我们的结果是 将揭示对组织发育过程中祖细胞分化的机械控制的新见解 为肺部发育缺陷提出新的治疗靶点。

PROJECT SUMMARY The branched architecture of the airways of the lungs permit the transfer of approximately six liters of air per minute between the external surroundings and the alveoli. The airway epithelial tree accomplishes gas exchange, mucus production, and pathogen clearance and blocks the entry of water, particulates, and microbes. To accomplish these diverse biological functions, the airway epithelium is comprised of several distinct cell types that differentiate from common progenitors during embryonic development, the first of which is the pulmonary neuroendocrine cell. Disrupting the differentiation of the specialized epithelial cell types negatively affects airway morphogenesis, and abnormally high numbers of pulmonary neuroendocrine cells are found in several congenital and acquired diseases of the lung. As it differentiates, the epithelium secretes ions and water across its apical surface, causing fluid to fill the lumen of the airways with a transmural pressure high enough to inflate the lungs. Defects that cause a decrease in transmural pressure are associated with both underdeveloped lungs and an increase in pulmonary neuroendocrine cells, but the specific role of pressure and the molecular signaling downstream of this mechanical cue are unknown. By combining time- lapse confocal imaging with an innovative microfluidic culture system, we found that transmural pressure controls the rate of lung development and the expression of markers of neuroendocrine cells. Using next- generation sequencing analysis, we found that low transmural pressure decreases the expression of targets of Notch, the master regulator of pulmonary neuroendocrine differentiation, and YAP, a known mechanosensor. Here, we hypothesize that transmural pressure coordinates the growth and differentiation of the different cell types within the epithelium by signaling through Notch and YAP. We will combine microfluidic devices with engineered mice, high-resolution time-lapse spinning disk confocal microscopy, and next-generation sequencing analysis to define the relative roles of pressure, Notch, and YAP in the regulation of pulmonary neuroendocrine progenitor fate decisions. In Specific Aim 1, we will use microfluidic chest cavities, engineered mice, time-lapse imaging, and single cell RNA-sequencing to define physically how transmural pressure regulates the pulmonary neuroendocrine population in the developing lung. In Specific Aim 2, we will use microfluidic chest cavities, reporter mice, and chromatin immunoprecipitation approaches to define whether and how transmural pressure regulates Notch signaling in the embryonic airway epithelium. In Specific Aim 3, we will determine whether pressure signals through YAP to affect pulmonary neuroendocrine differentiation and the Notch pathway. This work will define how mechanical signals from the microenvironment are transmitted to the first progenitor fate decision in the developing airway epithelium. We expect that our results will reveal novel insights into mechanical control of progenitor differentiation during tissue development and suggest new therapeutic targets for defects in lung development.