Mechanical Regulation of Mesenchyme and Mammalian Lung Development

描述(申请人提供)：呼吸道上皮树是通过分支形态发生在胚胎中塑造的，在这个过程中，新的子代分支从主茎侧面萌发(域分支)或从父分支的顶端分裂(平面或垂直分叉)。这些分支事件本质上是物理的，并且发生在动态的机械环境中，包括上皮本身的静态和相性的收缩 周围的呼吸道平滑肌收缩，以及因树管腔内液体的存在而引起的跨壁扩张压力。虽然胚胎肺的异常发育经常在胎儿身上观察到，这些缺陷导致这些组织室内的机械改变，但每个缺陷对驱动分支过程的物理贡献尚不清楚。WNTS下游的信号调节气道分支和平滑肌分化，并可能对发育中的肺的机械变化做出反应。在这里，我们假设，气道平滑肌的机械行为在驱动分支形态发生中起核心作用，并且上皮收缩和腔内液体压力都部分地通过改变控制平滑肌分化和收缩的信号通路来调节分支。我们将结合转基因小鼠与高分辨率实时旋转圆盘共聚焦显微镜、微流体设备、三维牵引力显微镜和计算模型来定义呼吸道上皮、平滑肌和腔液的机械行为如何协作来指导分支形态的发生。在具体目标1中，我们将使用转基因报告鼠来确定气道平滑肌的分化和收缩如何影响呼吸道上皮的区域分支和末端(平面和垂直)分支。在特定的目标2中，我们将使用微流控装置来控制胚胎肺外植体的跨壁压力，并确定跨壁压力在气道平滑肌分化、上皮分支和机械信号转导中的作用。在具体目标3中，我们将表征呼吸道上皮的收缩能力，量化上皮在分支过程中所施加的力，并确定上皮收缩如何引导周围间充质中的机械信号。这项工作将提供形态发生过程中组织间隔的完整机械肖像，并定义每个组件如何对塑造新分支所需的物理变化做出贡献。我们期望这项工作揭示的机械行为和信号通路将发现新的治疗选择，以治疗出现肺发育异常的胎儿和新生儿。

DESCRIPTION (provided by applicant): The airway epithelial tree is sculpted in the embryo via branching morphogenesis, a process in which new daughter branches sprout laterally off a main stem (domain branching) or split from the tip of a parent branch (planar or orthogonal bifurcations). These branching events are physical by nature and occur within a dynamic mechanical environment which includes the contractility of the epithelium itself, static and phasic contractions of the surrounding airway smooth muscle, and distending transmural pressures from the presence of fluid within the lumen of the tree. Although abnormal development of the embryonic lung is frequently observed in fetuses with defects that cause mechanical alterations in these tissue compartments, the physical contributions of each that are responsible for driving the branching process are unknown. Signaling downstream of Wnts regulates airway branching and smooth muscle differentiation, and is likely responsive to mechanical alterations in the developing lung. Here, we hypothesize that the mechanical behavior of airway smooth muscle plays a central role in driving branching morphogenesis, and that both epithelial contraction and luminal fluid pressure regulate branching in part by altering signaling pathways that control smooth muscle differentiation and contractility. We will combine transgenic reporter mice with high-resolution real-time spinning disk confocal microscopy, microfluidic devices, three-dimensional traction force microscopy, and computational modeling to define how the mechanical behaviors of the airway epithelium, smooth muscle, and luminal fluid collaborate to direct branching morphogenesis. In Specific Aim 1, we will use transgenic reporter mice to determine how airway smooth muscle differentiation and contraction affect domain branching and terminal (planar and orthogonal) bifurcations of the airway epithelium. In Specific Aim 2, we will use microfluidic devices to control the transmural pressure across embryonic lung explants and define the role of transmural pressure in airway smooth muscle differentiation, epithelial branching, and mechanical signaling. In Specific Aim 3, we will characterize the contractility of the airway epithelium, quantify the forces exerted by the epithelium during branching, and determine how epithelial contractility directs mechanical signaling in the surrounding mesenchyme. This work will provide a complete mechanical portrait of the tissue compartments during morphogenesis, and define how each component contributes to the physical changes required to sculpt a new branch. We expect that the mechanical behaviors and signaling pathways revealed by this work will uncover new therapeutic options to treat fetuses and neonates who present with abnormalities in lung development.