Signaling pathways in lung stem cell differentiation

描述（申请人提供）：肺修复和再生是由沿肺轴分布的不同干/祖细胞群进行的，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）。这些上皮细胞类型还每天发挥作用，促进气体交换并维持组织稳态。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。我们开发了一种 3D 共培养测定法来探讨单细胞水平上 BASC 分化的调节。肺内皮细胞与 BASC 共培养支持细支气管和肺泡细胞分化。最近，我们发现调节共培养环境可以改变 BASC 分化结果。我们发现BMP4处理激活内皮细胞中钙调神经磷酸酶依赖性转录因子NFATc1以诱导Thrombospondin-1 (Tsp1)的表达，这对于肺泡谱系特异性分化是必要且充分的。 TSP1 是体内肺泡肺损伤修复所必需的。因此，我们发现了一个在 BASC 和内皮细胞之间起作用以控制分化和损伤修复的新信号轴。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的调节 BASC 分化的其他信号。在目标 1 中，我们将定义 BASC 中调节分化的分子。我们将检查 3D 共培养物和肺泡肺损伤样本中候选 TSP1 受体的表达。 TSP1 依赖性分化介体将通过 RNA-seq 从体内 Tsp1 操作中以公正的方式进行鉴定，并在 3D 共培养物中进行功能测试。在目标 2 中，我们将进一步描述肺内皮细胞产生的影响肺泡分化的分泌因子。最初，我们将测试候选 BMP4-NFATc1 靶标 R-spondin2，这是我们最近发现的一种 Wnt 通路配体，在 BASC/内皮细胞共培养物中响应 BMP4 处理而被诱导。谱系追踪方法将用于测试体内 BASC 中的 R-spondin2 通路。我们还将进行 RNA-Seq 与 NFATc1-ChIP-Seq 结合，以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如何被指示产生谱系特异性后代。这项工作将为发现干细胞生态位中的运作机制和增强肺部疾病肺损伤修复的治疗靶点提供机会。

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.