Mechanical Forces and the Regulation of Airway Progenitor Cells

项目摘要 肺的气道的分支结构允许每个呼吸道输送大约六升空气。 在外部环境和肺泡之间的一分钟。气道上皮树完成气体 交换、粘液产生和病原体清除，并阻止水、颗粒和 微生物为了实现这些不同的生物学功能，气道上皮由几个细胞组成。 在胚胎发育过程中从共同的祖细胞分化出来的不同细胞类型，其中第一种是 是肺神经内分泌细胞破坏特化上皮细胞类型的分化 对气道形态发生有负面影响，肺神经内分泌细胞数量异常高， 在几种先天性和后天性肺部疾病中发现。在分化过程中，上皮细胞分泌离子 和水穿过其顶端表面，导致流体以跨壁压力填充气道的管腔 足以使肺部膨胀引起跨壁压降低的缺陷与以下因素有关： 肺发育不全和肺神经内分泌细胞增加，但 压力和这种机械信号下游的分子信号是未知的。结合时间- 通过创新的微流控培养系统的激光共聚焦成像，我们发现透壁压 控制肺发育的速率和神经内分泌细胞标志物的表达。使用next- 代测序分析，我们发现低跨壁压降低了靶基因的表达， Notch是肺神经内分泌分化的主要调节因子，雅普是一种已知的机械传感器。 在这里，我们假设跨壁压协调不同细胞的生长和分化， 通过Notch和雅普信号传导在上皮细胞内的类型。我们将把联合收割机微流体装置与 工程小鼠，高分辨率延时旋转圆盘共聚焦显微镜，以及下一代 测序分析，以确定压力、Notch和雅普在肺动脉高压调节中的相对作用。 神经内分泌祖细胞命运的决定。在具体目标1中，我们将使用微流体胸腔， 小鼠，延时成像和单细胞RNA测序，以确定物理上如何跨壁压 调节发育中的肺中的肺神经内分泌群。在具体目标2中，我们将使用 微流体胸腔，报告小鼠和染色质免疫沉淀方法，以确定是否 以及透壁压如何调节胚胎气道上皮中的Notch信号。在具体目标3中， 我们将确定压力信号是否通过雅普影响肺神经内分泌分化 和Notch途径这项工作将定义来自微环境的机械信号是如何 传递到发育中的气道上皮中的第一祖细胞命运决定。我们希望我们的结果 将揭示组织发育过程中祖细胞分化的机械控制的新见解， 为肺发育缺陷提供了新的治疗靶点。

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.