Interplay between mechanical forces and retinoic acid in lung development

项目总结 出生后的呼吸需要呼吸道上皮及其周围的协调发展 间充质和间皮。在胎儿中，这些组织是对来自液体的外源力量的反应而形成的。 发育中的肺内和周围的压力通常在呼吸道的管腔内很高，因此 产生正的经肺压力。胎儿胸腔的机械环境是 被先天性横隔膜腹股沟(CDH)等疾病破坏，从而减少或逆转 对发育中的肺部施加压力，并导致肺发育不全，这是新生儿死亡的主要原因。 尽管包括维甲酸(RA)在内的几种生化信号已被认为与糖尿病的发病有关 CDH，调节肺的机械作用力和跨肺压下游的信号 发展是未知的。我们的初步和已发表的数据表明，经肺压力本身 调节RA生物合成途径、呼吸道上皮分支形态发生和气道顺畅 肌肉分化。在这里，我们建议利用我们创新的微流控平台，组织- 特定的基因敲除小鼠、荧光报告小鼠和CDH小鼠模型来揭示分子 在胚胎内连接压力、RA信号和形态发生和分化的机制 阿龙。我们将把这些方法与肺组织的延时成像、单细胞转录 分析，以及实时荧光力传感器。在具体目标1中，我们将检验压力的假设 以组织特异性的方式激活机械传感器YAP，以调节 RA生物合成途径相关基因的表达。在具体目标2中，我们将揭示 维甲酸的压力和组织特异性合成对呼吸道上皮细胞生长和形态发生及气道的影响 平滑肌分化。在具体目标3中，我们将测量压力和RA的相对影响 在上皮、间充质和间皮内传递张力、张力和流动性的信号。这项工作将， 第一次，确定了组织特异性机械力和分子信号的下游 调节肺早期形态发生的跨肺压力。我们预计我们的发现将 为治疗肺发育缺陷提出新的治疗靶点。

PROJECT SUMMARY Breathing after birth requires coordinated development of the airway epithelium and its surrounding mesenchyme and mesothelium. In the fetus, these tissues form in response to exogenous forces from fluid pressure within and around the developing lungs that is normally high in the lumen of the airways, thus generating a positive transpulmonary pressure. The mechanical environment of the fetal chest cavity is disrupted by conditions such as congenital diaphragmatic hernia (CDH), which reduces or reverses the pressure across the developing lungs and leads to pulmonary hypoplasia, a major cause of neonatal mortality. Although several biochemical signals, including retinoic acid (RA), have been implicated in the pathogenesis of CDH, the mechanical forces and signaling downstream of transpulmonary pressure that regulate lung development are unknown. Our preliminary and published data suggest that transpulmonary pressure itself regulates the RA-biosynthesis pathway, airway epithelial branching morphogenesis, and airway smooth muscle differentiation. Here, we propose to take advantage of our innovative microfluidic platforms, tissue- specific knockout mice, fluorescent reporter mice, and mouse models of CDH to uncover the molecular mechanisms that connect pressure, RA signaling, and morphogenesis and differentiation within the embryonic lung. We will combine these approaches with time-lapse imaging of lung explants, single-cell transcriptomic analysis, and real-time fluorescent force sensors. In Specific Aim 1, we will test the hypothesis that pressure activates the mechanosensor Yap in a tissue-specific manner to regulate the spatiotemporal pattern of expression of genes involved in the RA-biosynthesis pathway. In Specific Aim 2, we will uncover the effects of pressure and tissue-specific synthesis of RA on airway epithelial growth and morphogenesis and airway smooth muscle differentiation. In Specific Aim 3, we will measure the relative effects of pressure and RA signaling on tension, strain, and fluidity within the epithelium, mesenchyme, and mesothelium. This work will, for the first time, identify the tissue-specific mechanical forces and molecular signaling downstream of transpulmonary pressure that regulate early morphogenesis of the lung. We expect that our findings will suggest novel therapeutic targets for the treatment of defects in lung development.