Molecular Mechanisms of Lung Branching Morphogenesis

描述（由申请人提供）：本提案旨在验证以下假设：Alk 3-Smad 1信号传导可能通过新的WIF-1/Wnt机制介导BMP 4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。通过在不同发育阶段的肺上皮细胞中消除Alk 3功能来确定BMP I型受体Alk 3在小鼠胚胎肺发育中的体内功能。将在不同发育阶段的loxed Alk 3和SPC-rtTA/TetO-Cre转基因小鼠杂交的小鼠中通过强力霉素诱导Cre表达来实现肺上皮特异性Alk 3条件基因敲除。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Alk 3在肺发育中的特异性调节功能。目标二。通过在不同发育阶段的肺上皮细胞中消除Smad 1功能来确定BMP特异性下游Smad 1在小鼠胚胎肺发育中的体内功能。多西环素诱导不同发育阶段的SPC-rtTA/TetO-Cre转基因小鼠与floxed Smad 1杂交后，Smad 1在肺上皮细胞中的功能被消除。与Alk 3研究一样，将表征肺形态学、细胞和分子生物学的动态变化，以确定Smad 1在整个肺发育中的功能。目标3：探讨Alk 3-Smad 1诱导Wnt抑制因子1调控远端肺上皮细胞分化的机制。我们的初步研究发现，在E18.5，Alk 3和Smad 1基因敲除小鼠肺中WIF-1表达下调。因此，WIF-1表达/Wnt经典途径活化与BMP调节的远端肺上皮细胞分化之间的关系将通过比较Alk 3或Smad 1敲除小鼠和妊娠晚期对照小鼠肺来确定。WIF-1通过BMP-Smad激活的转录调节机制将在培养的肺上皮细胞中进一步剖析。此外，将在培养的细胞中确定BMP-Smad信号通过WIF-1介导的Wnt/β-连环蛋白途径的下调来调节肺上皮细胞变化的机制。与人类健康的相关性：阻断小鼠肺中的细胞内BMP信号传导会导致新生儿因空气呼吸失败而立即死亡。这些研究将为了解BMP细胞内信号在肺发育和肺部疾病中的作用提供基础知识，这将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.