Development of a novel genetic approach to study lung mesenchymal cell signaling

描述（由申请人提供）：肺间充质细胞和上皮细胞之间的相互作用是正常肺发育和体内平衡所必需的。因此，肺间充质细胞生物学的改变将对胎儿肺形成和出生后肺损伤修复产生重大影响，从而导致许多肺部疾病的发病机制。尽管已经获得了几种复杂的小鼠遗传方法来研究肺上皮细胞生物学，但迄今为止缺乏肺间充质细胞特异性小鼠遗传方法已成为肺研究领域的关键障碍。我们假设Tbx4肺增强子驱动的rtTA（逆转录四环素依赖反激活因子）转基因小鼠系（Tbx4-rtTA）可用于构建新的肺间质特异性Tet-On遗传系统，以研究肺间质细胞信号在肺发育和损伤修复中的作用。因此，结合Tbx4-rtTA和TetO启动子驱动的显性阴性TGF-¿受体II （dnT2RII）转基因系，将为研究TGF-¿信号在肺间质中的特异性及其对肺发育的影响提供一个独特的工具。具体目标建立并表征肺间充质细胞特异性Tet-On遗传系统。一个5.5 kb的Tbx4基因肺增强子DNA元件将被用于构建Tbx4- rtta转基因小鼠。然后验证rtTA转基因表达在不同年龄的肺间质定位。此外，通过将Tbx4-rtTA和TetO-Cre小鼠与floxed-mT-mG荧光蛋白报告系统杂交，将生成肺间充质细胞特异性Tet-On遗传系统，并在小鼠中进行测试。具体目标2。利用已建立的肺间充质Tet-On遗传方法，解剖TGF-¿信号在体内肺间充质细胞发育中的调控作用。我们将利用新开发的肺间充质Tet-On系统表征肺间充质细胞TGF-¿信号，因为TGF-¿信号在调节肺发育、损伤修复和肺部疾病中发挥着极其重要的作用。通过生成Tbx4-rtTA/TetO-dnT2RII小鼠细胞系，可以通过dnT2RII诱导在体内不同水平和阶段可逆地抑制肺间质特异性TGF-¿信号。间充质TGF-¿信号在调节肺发育中的作用将被剖析。这些新的遗传方法的发展有可能大大扩展当前对肺间充质细胞生物学及其与肺部疾病的关系的认识。此外，通过将该系统应用于肺间质TGF-¿信号研究，我们将能够解剖TGF-¿信号在调节肺发育和成熟中的遗传剂量和时间窗口。成功建立这些独特的遗传系统将为确定间充质信号在肺发育和体内平衡以及肺部疾病病理生理中的作用提供有力的工具。此外，可能会发现新的治疗靶点。

DESCRIPTION (provided by applicant): Reciprocal interaction between lung mesenchymal and epithelial cells is essential for normal lung development and homeostasis. Therefore, alterations in lung mesenchymal cell biology will have a significant impact on both fetal lung formation and postnatal lung injury repair, resulting in the pathogenesis of many lung diseases. Although several sophisticated mouse genetic approaches have been obtained to study lung epithelial cell biology, the lack of lung mesenchymal cell-specific mouse genetic approaches to date has become a critical barrier to the field of lung research. We hypothesize that Tbx4 lung enhancer driven rtTA (reverse tetracycline- dependent transactivator) transgenic mouse line (Tbx4-rtTA) can be used to make a novel lung mesenchymal- specific Tet-On genetic system to study lung mesenchymal cell signaling in lung development and injury repair. Thus, combination of Tbx4-rtTA and TetO promoter driven dominant-negative TGF-¿ receptor II (dnT2RII) transgenic lines will provide a unique tool to study TGF-¿ signaling specifically in lung mesenchyme and its impact on lung development in vivo. Specific Aim 1. To develop and characterize a lung-mesenchymal cell-specific Tet-On genetic system. A 5.5 kb lung enhancer DNA element of Tbx4 gene will be used to make the Tbx4-rtTA transgenic mice. The lung mesenchymal localization of rtTA transgenic expression at different ages will then be verified. Furthermore, a lung mesenchymal cell-specific Tet-On genetic system will be generated and tested in mice by crossing Tbx4-rtTA and TetO-Cre mice with a floxed-mT-mG fluorescence protein reporter system. Specific Aim 2. To dissect the regulatory functions of TGF-¿ signaling in developing lung mesenchymal cells in vivo using the established lung mesenchymal Tet-On genetic approach. We will apply the newly developed lung mesenchymal Tet-On system to characterize TGF-¿ signaling in lung mesenchymal cells, as TGF- ¿ signaling plays extremely important roles in regulating lung development, injury repair, and lung diseases. By generating a Tbx4-rtTA/TetO-dnT2RII mouse line, lung mesenchymal-specific TGF-¿ signaling can be reversibly inhibited at various levels and stages in vivo through dnT2RII induction. The roles of mesenchymal TGF-¿ signaling in regulating lung development will then be dissected. Development of these novel genetic approaches has the potential to substantially extend current knowledge of lung mesenchymal cell biology and its relationship to lung diseases. Furthermore, by applying this system to lung mesenchymal TGF-¿ signaling research, we will be able to dissect the genetic dosages and time windows of TGF-¿ signaling in regulating lung development and maturation. Successful establishment of these unique genetic systems will provide powerful tools to define the role of mesenchymal signaling in lung development and homeostasis, and in the pathophysiology of lung diseases. Additionally, new therapeutic targets may be identified.