Mechanical Regulation of Mesenchyme and Mammalian Lung Development

描述（由申请人提供）：气道上皮树通过分支形态发生在胚胎中形成，这是一个新的子分支从主干侧向发芽（域分支）或从母分支尖端分裂（平面或正交分叉）的过程。这些分支事件本质上是物理性的，并且发生在动态机械环境中，所述动态机械环境包括上皮本身的收缩性、静态的和阶段性的 周围气道平滑肌的收缩，以及由于树的管腔内存在流体而引起的跨壁压力的膨胀。虽然在具有缺陷的胎儿中经常观察到胚胎肺的异常发育，这些缺陷导致这些组织隔室中的机械改变，但负责驱动分支过程的每个物理贡献是未知的。Wnts下游的信号传导调节气道分支和平滑肌分化，并且可能响应于发育中的肺中的机械改变。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着核心作用，并且上皮收缩和管腔流体压力部分地通过改变控制平滑肌分化和收缩性的信号通路来调节分支。我们将结合联合收割机转基因小鼠与高分辨率实时旋转圆盘共聚焦显微镜，微流控装置，三维牵引力显微镜，和计算建模，以确定如何机械行为的气道上皮细胞，平滑肌，管腔流体合作，以指导分支形态发生。在特定目标1中，我们将使用转基因报告小鼠来确定气道平滑肌分化和收缩如何影响气道上皮的结构域分支和末端（平面和正交）分叉。在具体目标2中，我们将使用微流体装置来控制跨胚胎肺外植体的跨壁压，并定义跨壁压在气道平滑肌分化、上皮分支和机械信号传导中的作用。在特定目标3中，我们将表征气道上皮的收缩性，量化上皮在分支期间施加的力，并确定上皮收缩性如何指导周围间充质中的机械信号传导。这项工作将提供一个完整的机械画像的组织隔间在形态发生，并定义如何每个组件有助于塑造一个新的分支所需的物理变化。我们期望这项工作揭示的机械行为和信号通路将揭示新的治疗选择，以治疗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.