Signaling pathways in lung stem cell differentiation

描述（由申请人提供）：肺修复和再生由沿着肺轴分布的不同干/祖细胞群进行，包括细支气管俱乐部（Clara）细胞、肺泡II型上皮细胞和细支气管肺泡干细胞（BASC）。这些上皮细胞类型也在日常基础上发挥作用，以促进气体交换和维持组织稳态。损伤后，信号指示干细胞产生受影响谱系的后代，但这些信号的分子特征仍然有限。我们已经开发了一种3D共培养试验，以在单细胞水平上询问BASC分化的调节。与BASC共培养的肺内皮细胞支持细支气管和肺泡细胞分化。最近，我们已经表明，调节共培养环境改变BASC分化结果。我们发现，BMP 4处理激活内皮细胞中的钙调神经磷酸酶依赖性转录因子NFATc 1，诱导血小板反应蛋白-1（Tsp 1）的表达，这是肺泡谱系特异性分化所必需和充分的。肺损伤的修复需要TSP 1的参与。因此，我们发现了一个新的信号传导轴，它在BASCs和内皮细胞之间起作用，以控制分化和损伤修复。我们现在寻求进一步表征TSP 1上游和下游的BMP-NFATc 1-TSP 1通路，并鉴定来自肺内皮细胞的调节BASC分化的其他信号。在目标1中，我们将定义BASCs中调节分化的分子。我们将检测候选TSP 1受体在3D共培养物和肺泡肺损伤样本中的表达。将通过来自体内Tsp 1操作的RNA-seq以无偏倚的方式鉴定TSP 1依赖性分化介质，并在3D共培养物中进行功能测试。在目标2中，我们将进一步描述由肺内皮细胞产生的影响肺泡分化的分泌因子。首先，我们将在BASC/内皮细胞共培养物中测试候选的BMP 4-NFATc 1靶点R-spondin 2，这是我们最近发现的一种Wnt途径配体，在BASC/内皮细胞共培养物中响应于BMP 4处理而被诱导。将使用谱系追踪方法在体内测试BASC中的R-spondin 2途径。我们还将进行RNA-Seq与NFATc 1-ChIP-Seq结合，以确定肺内皮细胞中的其他BMP 4靶点。我们的实验旨在确定多能肺干细胞如何被指示产生谱系特异性后代。这项工作将提供一个机会，发现机制的干细胞龛和治疗目标，增强肺损伤修复肺部疾病。

DESCRIPTION (provided by applicant): Lung repair and regeneration is carried out by different stem/progenitor cell populations distributed along the pulmonary axis, including the bronchiolar club (Clara) cells, the alveolar type II epithelial cells, and bronchioalveolar stem cels (BASCs). These epithelial cell types also function on a daily basis to facilitate gas exchange and maintain tissue homeostasis. After injury, signals instruct the stem cell to produce progeny of the affected lineage, but the molecular characterization of these signals remains limited. We have developed a 3D co-culture assay to interrogate the regulation of BASC differentiation at the single cell level. Lung endothelial cells co-cultured with BASCs support bronchiolar and alveolar cell differentiation. Recently, we have shown that modulating the co-culture environment alters BASC differentiation outcomes. We found that BMP4 treatment activates the calcineurin-dependent transcription factor NFATc1 in endothelial cells to induce expression of Thrombospondin-1 (Tsp1), which was necessary and sufficient for alveolar lineage-specific differentiation. TSP1 was required for alveolar lung injury repair in vivo. Thus, we have uncovered a new signaling axis that operates between BASCs and endothelial cells to control differentiation and injury repair. We now seek to further characterize the BMP-NFATc1-TSP1 pathway up and down stream of TSP1, and to identify additional signals from lung endothelial cells that regulate BASC differentiation. In Aim 1, we will define the molecules in BASCs that regulate differentiation. We will examine expression of candidate TSP1 receptors in 3D co-cultures and alveolar lung injury samples. TSP1-dependent mediators of differentiation will be identified in an unbiased fashion by RNA-seq from in vivo Tsp1-manipulation and functionally tested in 3D co-cultures. In Aim 2, we will further delineate the secreted factors produced by lung endothelial cells that influence alveolar differentiation. Initially we will test the candidat BMP4-NFATc1 target R-spondin2, a Wnt pathway ligand that we recently found was induced in response to BMP4 treatment, in BASC/endothelial cell co-cultures. A lineage tracing approach will be used to test the R-spondin2 pathway in BASCs in vivo. We will also perform RNA-Seq coupled with NFATc1-ChIP-Seq to identify additional BMP4 targets in lung endothelial cells. Our experiments aim to define how a multipotent lung stem cell is instructed to produce lineage-specific progeny. This work will provide an opportunity for the discovery of mechanisms operating in the stem cell niche and therapeutic targets for enhanced lung injury repair in lung disease.