Biology of Submucosal Gland Stem Cells in the Airway

项目摘要 已知气道粘膜下腺体（SMG）含有气道上皮的储备干细胞 （SAE）。在小鼠中，这个小生境只服务于气管;然而，在较大的哺乳动物中，如人类，猪和雪貂， SMG存在于整个叶内软骨气道中，并可用于更广泛的功能， 在疾病环境中维持近端传导气道上皮。在过去的六年中， 周期，该赠款已使用小鼠和雪貂遗传模型，以解决气道SMG生物学的多个方面， SMG干/祖细胞生物学和囊性纤维化（CF）肺发病机制。这项建议旨在确定 参与气道修复的腺肌上皮细胞（GMECs）亚群，以及Wnt- 控制它们受伤后行为的调节机制。根据我们的初步数据，我们假设 Lef-1和Sox 9转录因子差异控制Wnt反应性GMEC状态， GMECs在气道表面的定型、更新、迁移和增殖性扩张。目标1将定义 小鼠气管GMECs的生物学并利用一系列转基因系（交叉谱系追踪， 条件性敲除、Wnt-报告基因、Dox诱导型H2 B-GFP）来研究Lef-1和Sox 9在 调节控制GMEC承诺、更新、迁移和增殖的过程。目标2将使用 在雪貂中进行交叉谱系追踪，以确定GMEC亚型和腺导管细胞参与 在损伤后维持叶外和叶内SAE的稳态。我们假设腺体 导管是GMEC成熟为前基底细胞状态的场所，并且这种潜在的等级关系 在慢性气道疾病如CF中受到干扰。目标3将通过识别以下干扰来检验这一假设： 轻度和重度CF肺病背景下的GMEC和腺管小生境，使用VX-770-应答 CFTRG 551 D雪貂。这些研究的新方面包括第一次在一个物种（雪貂）中绘制非啮齿类动物的命运图。 密切模拟人类CF肺病和SMG生物学，并支持命运映射数据 证明雪貂GMECs（ACTA 2CreER）和腺导管细胞（KRT 7 CreER）参与SAE修复。这 研究还将揭示SMG干细胞隔室在叶外的行为差异， 叶内软骨气道，这在小鼠中无法解决，因为它们在叶内缺乏SMG 航空公司.该项目旨在提高我们对气道SMG中干细胞表型的理解， 调节它们参与SMG和表面气道修复的机制。鉴于GMECs可以 再生腺体和表面气道细胞类型，它们是CF中基因编辑的有吸引力的靶点， 从这项建议中获得的知识将加强这些努力。此外，这项工作将描绘 SMG干细胞小生境的疾病相关变化可能对CF气道的发病机制很重要 疾病和其他影响SMG的分泌过多疾病，如哮喘和慢性支气管炎。

Project Summary Airway submucosal glands (SMGs) are known to contain reserve stem cells for the surface airway epithelium (SAE). In mice, this niche serves only the trachea; however, in larger mammals such as humans, pigs and ferrets, SMGs are present throughout the intralobar cartilaginous airways and may serve the broader function of maintaining the proximal conducting airway epithelium in the setting of disease. During the previous six funding cycles, this grant has used mouse and ferret genetic models to address multiple aspects of airway SMG biology, SMG stem/progenitor cell biology, and cystic fibrosis (CF) lung pathogenesis. This proposal aims to identify the subpopulations of glandular myoepithelial cells (GMECs) that participate in airway repair, as well as the Wnt- regulated mechanisms that control their behavior following injury. Based on our preliminary data, we hypothesize that Lef-1 and Sox9 transcription factors differentially control Wnt-responsive GMEC states that orchestrate the commitment, renewal, migration, and proliferative expansion of GMECs on the airway surface. Aim 1 will define the biology of tracheal GMECs in mice and utilize an array of transgenic lines (intersectional lineage tracing, conditional knockout, Wnt-reporters, Dox-inducible H2B-GFP) to study the involvement of Lef-1 and Sox9 in regulating processes that control GMEC commitment, renewal, migration, and proliferation. Aim 2 will use intersectional lineage tracing in ferrets to define the participation of GMEC subtypes and gland ductal cells in maintaining the extralobar and intralobar SAE at homeostasis and following injury. We hypothesize that gland ducts are a site for GMEC maturation to a pre-basal cell state, and that this underlying hierarchical relationship is disturbed in chronic airway diseases such as CF. Aim 3 will test this hypothesis by identifying disturbances in GMEC and gland duct niches in the setting of mild and severe CF lung disease, using VX-770-responsive CFTRG551D ferrets. Novel aspects of these studies include the first non-rodent fate mapping in a species (ferret) that closely models CF lung disease and SMG biology of humans, and supporting fate mapping data demonstrating that ferret GMECs (ACTA2CreER) and gland ductal cells (KRT7CreER) participate in SAE repair. This research will also shed light on differences in the behavior of SMG stem cell compartments in the extralobar and intralobar cartilaginous airways, which cannot be addressed in mice because they lack SMGs in the intralobar airways. This project is designed to enhance our understanding of stem cell phenotypes in airway SMGs and the mechanisms that regulate their participation in SMG and surface airway repair. Given that GMECs can regenerate both glandular and surface airway cell types, they are an attractive target for gene editing in CF and such efforts will be enhanced by knowledge gained from this proposal. Furthermore, this work will delineate disease-associated changes to SMG stem cell niches that may be important for the pathogenesis of CF airway disease and other hypersecretory diseases that affect SMGs, such as asthma and chronic bronchitis.