Molecular Mechanisms of Lung Branching Morphogenesis

描述（由申请人提供）：本提案验证以下假设：Alk3-Smad1信号可能通过一种新的wi -1/Wnt机制介导BMP4对小鼠肺发育的调节。因此，实验设计有三个具体目标：目标1。通过去除不同发育阶段肺上皮细胞中Alk3的功能，确定BMP I型受体Alk3在小鼠胚胎肺发育中的体内功能。在不同发育阶段，用多西环素诱导固定Alk3和SPC-rtTA/TetO-Cre转基因小鼠杂交的小鼠，实现肺上皮特异性Alk3条件性基因敲除。通过分析肺形态、细胞增殖分化和基因表达的动态变化，确定Alk3在肺发育中的具体调控功能。目标2。通过去除不同发育阶段肺上皮细胞中Smad1的功能，确定bmp特异性下游Smad1在小鼠胚胎肺发育中的体内功能。Smad1在不同发育阶段与SPC-rtTA/TetO-Cre转基因小鼠杂交后，Smad1在肺上皮中的功能被强力霉素诱导破坏。与Alk3研究一样，将对肺形态、细胞和分子生物学的动态变化进行表征，以确定Smad1在肺整体发育中的功能。目标3。探讨Alk3-Smad1诱导Wnt抑制因子-1调控肺远端上皮细胞分化的机制。我们的初步研究发现，在E18.5时，Alk3和Smad 1敲除小鼠肺中wi -1的表达均下调。因此，通过比较Alk3或Smad1敲除与妊娠后期对照小鼠肺的差异，可以确定WIF-1表达/Wnt经典通路激活与BMP调控的远端肺上皮细胞分化之间的关系。在培养的肺上皮细胞中，BMP-Smad激活对WIF-1的转录调控机制将进一步被解剖。此外，BMP-Smad信号通过wi -1介导的Wnt/ β -catenin通路下调调控肺上皮细胞变化的机制将在培养细胞中确定。与人类健康的相关性：阻断小鼠肺细胞内BMP信号可导致新生儿因空气呼吸衰竭而立即死亡。这些研究将提供肺发育和肺部疾病中BMP细胞内信号传导的基础知识，有助于我们了解先天性和新生儿疾病以及肺损伤修复。这些基本信息可能有助于未来设计新的治疗策略来预防和治疗发育性肺部疾病。

DESCRIPTION (provided by applicant): This proposal is testing the following hypothesis: Alk3-Smad1 signaling mediates BMP4 regulation of mouse lung development possibly through a novel WIF-1/Wnt mechanism. Experiments are therefore designed with three specific aims: Aim 1. To determine the in vivo function of BMP type I receptor Alk3 in mouse embryonic lung development by abrogation of Alk3 function in lung epithelial cells at different developmental stages. Lung epithelia specific Alk3 conditional gene knockout will be achieved by doxycycline-induced Cre expression in mice crossbred from floxed Alk3 and SPC-rtTA/TetO-Cre transgenic mice at different developmental stages. The specific regulatory function of Alk3 in lung development will then be determined by analyzing dynamic changes of lung morphology, cell proliferation and differentiation, and gene expression. Aim 2. To determine the in vivo function of BMP-specific downstream Smad1 in mouse embryonic lung development by abrogating Smad1 function in lung epithelial cells at different developmental stages. Smad1 function in lung epithelia will be abrogated in mice crossbred from floxed Smad1 and SPC-rtTA/TetO-Cre transgenic mice by doxycycline induction from different developmental stages. As in the Alk3 study, dynamic changes of lung morphology, cell and molecular biology will be characterized to determine the function of Smad1 in overall lung development. Aim 3. To determine the mechanism of Alk3-Smad1 induced Wnt inhibitory factor-1 in controlling distal lung epithelial cell differentiation. Our preliminary study found that WIF-1 expression was downregulated in both Alk3 and Smad 1 knockout mouse lungs at E18.5. Thus, the relationship between WIF-1 expression/Wnt canonical pathway activation and BMP regulated distal lung epithelial cell differentiation will be determined by comparison of Alk3 or Smad1 knockout and control mouse lungs at late gestation stages. Transcriptional regulatory mechanisms of WIF-1 by BMP-Smad activation will be further dissected in cultured lung epithelial cells. Furthermore, the mechanisms by which BMP-Smad signaling regulated lung epithelial cell changes through WIF-1-mediated downregulation of Wnt/beta-catenin pathway will be determined in cultured cells. Relevance to human health: Blocking intracellular BMP signaling in mouse lung results in immediate neonatal lethality due to failure of air breathing. These studies will provide fundamental knowledge about BMP intracellular signaling in lung development and pulmonary diseases, which will help us to understand congenital and neonatal disease as well as lung injury repair. This basic information may aid in the future design of novel therapeutic strategies to prevent and treat developmental pulmonary diseases.