Signaling pathways in lung stem cell differentiation

描述（由申请人提供）：肺修复和再生是由沿肺轴分布的不同干细胞/祖细胞群进行的，包括细支气管俱乐部细胞（Clara）、肺泡II型上皮细胞和细支气管肺泡干细胞（BASCs）。这些上皮细胞类型在日常生活中也起着促进气体交换和维持组织稳态的作用。损伤后，信号指示干细胞产生受影响谱系的后代，但这些信号的分子表征仍然有限。我们已经开发了一种3D共培养试验来询问单细胞水平上BASC分化的调节。肺内皮细胞与基底细胞共培养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会改变BASC分化结果。我们发现，BMP4治疗激活内皮细胞中钙调磷酸酶依赖的转录因子NFATc1，诱导血小板反应蛋白-1 （Tsp1）的表达，这对于肺泡谱系特异性分化是必要和充分的。体内肺泡肺损伤修复需要TSP1。因此，我们发现了一个新的信号轴，它在基底细胞和内皮细胞之间运作，控制分化和损伤修复。我们现在寻求进一步表征TSP1上下游的BMP-NFATc1-TSP1通路，并识别来自肺内皮细胞调节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TSP1受体在3D共培养和肺泡肺损伤样本中的表达。tsp1依赖的分化介质将通过体内tsp1操作的RNA-seq以无偏的方式进行鉴定，并在3D共培养中进行功能测试。在Aim 2中，我们将进一步描述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/内皮细胞共培养中测试候选BMP4- nfatc1靶点R-spondin2，这是我们最近发现的一种Wnt通路配体，可诱导响应BMP4治疗。一种谱系追踪方法将用于在体内测试BASCs中的R-spondin2通路。我们还将使用RNA-Seq结合NFATc1-ChIP-Seq来鉴定肺内皮细胞中的其他BMP4靶点。我们的实验旨在确定多能肺干细胞是如何被指示产生谱系特异性后代的。这项工作将为发现干细胞生态位的运作机制和治疗靶点提供机会，以增强肺部疾病的肺损伤修复。

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.