Interplay between mechanical forces and retinoic acid in lung development

项目摘要 出生后的呼吸需要气道上皮及其周围的协调发展 间充质和间皮层。在胎儿中，这些组织的形成是对来自液体的外源性力量的反应。 发育中的肺内和周围的压力通常在气道腔中很高，因此 产生正的经肺压力。胎儿胸腔的力学环境是 被先天性腹股沟疝（CDH）等疾病破坏，这会减少或逆转 肺发育不良是新生儿死亡的主要原因。 尽管包括视黄酸（RA）在内的几种生化信号已经涉及到 CDH，调节肺的跨肺压的机械力和下游信号传导 发展未知。我们的初步和已发表的数据表明，穿髓压力本身 调节RA生物合成途径、气道上皮分支形态发生和气道平滑 肌肉分化在这里，我们建议利用我们创新的微流体平台，组织- 特异性敲除小鼠、荧光报告小鼠和CDH小鼠模型，以揭示CDH的分子机制。 连接压力、RA信号、胚胎内形态发生和分化的机制 肺。我们将联合收割机这些方法与肺移植物的延时成像，单细胞转录组学， 实时荧光力传感器。在具体目标1中，我们将检验压力 以组织特异性方式激活机械传感器雅普，以调节 参与RA生物合成途径的基因的表达。在具体目标2中，我们将揭示 压力和RA的组织特异性合成对气道上皮生长和形态发生以及气道 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、间充质和间皮内的张力、应变和流动性上的信号传导。这项工作将， 第一次，确定组织特异性机械力和下游的分子信号传导， 调节肺的早期形态发生的跨肺压。我们希望我们的发现 提出了治疗肺发育缺陷的新的治疗靶点。

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.