Molecular Mechanisms of Mucociliary Epithelia and Airway Clearance

脊椎动物的传导气道由粘膜纤毛上皮排列，它清除气道，为抵抗病原体提供第一道防线。纤毛粘膜上皮由干细胞、分泌细胞和纤毛细胞组成，纤毛细胞形成运动突起（纤毛），突出到细胞外间隙。建立正确的纤毛细胞类型、组成和功能都是气道清除所必需的，任何一个过程的缺陷都会导致慢性肺部疾病并增加呼吸道感染的风险。肺部疾病是世界上最常见的死亡原因之一，但人们对导致肺部疾病的分子机制知之甚少。我提出的工作旨在通过研究通过Wnt信号通路的细胞间通讯如何调节黏毛上皮的基因表达和细胞类型组成，以及气道细胞中纤毛的形成和功能，来解决黏毛上皮的分子机制。因此，我将生成关于黏毛上皮基础生物学的关键数据，这将促进对导致慢性肺部疾病和呼吸道感染的分子机制的转化见解。为了研究这些分子机制，我将利用一套已建立的和新的粘膜纤毛研究模型系统，从快速发育和易于操作的青蛙胚胎，到遗传上可适应的小鼠，再到培养的人类气道干细胞。这将使我能够对Wnt信号通路在纤毛粘膜发育、再生和纤毛功能中的进化保守作用进行全面和比较的研究。通过操纵和分析不同细胞类型在发育、再生和纤毛形成关键阶段的Wnt信号活动，我将在基因组、细胞和组织水平上描述Wnt信号依赖过程。我将使用尖端的方法，包括转录组学，基因组学和crispr /Cas9的基因组编辑，结合生物信息学方法和功能研究来解决Wnt信号依赖性的生长调节网络，该网络协调粘液纤毛上皮的正常发育和细胞类型组成。此外，我将研究新发现的wnt调节基因如何促进气道清除。为了分析Wnt信号在纤毛形成和功能中的作用，我将研究最近发现的Cp110蛋白在Wnt依赖性纤毛协调行为中的新作用。我将采用蛋白质组学方法来确定cp110与哪些其他蛋白质合作，以及这个因子网络如何响应并可能介导纤毛细胞中的Wnt信号。在这些实验中，纤毛的形成和功能将使用遗传和生化方法以及先进的活细胞和超分辨率成像进行分析。该项目将解决气道生物学中的重要问题，从而获得对气道疾病机制的理解。

The vertebrate conducting airways are lined by a mucociliaryepithelium that clears the airways to confer a first line of defenseagainst pathogens. Mucociliary epithelia are composed of stem cells,secretory cells, and ciliated cells, the last of which form motileprotrusions (cilia) projecting into the extracellular space.Establishment of the correct cell type composition and function of ciliaare both required for airway clearance, and defects in either processcause chronic lung diseases and increase the risk for respiratoryinfections. Lung diseases are among the most common causes ofdeath worldwide, but little is known about the molecular mechanismsthat cause them. My proposed work aims to resolve the molecularmechanisms of mucociliary epithelia by investigating how cell-cellcommunication via the Wnt signaling pathway regulates geneexpression and cell type composition in mucociliary epithelia, as wellas cilia formation and function in airway cells. Thereby, I will generatecrucial data on the basic biology of mucociliary epithelia that willpromote translational insights into the molecular mechanisms leadingto chronic lung diseases and respiratory infections. To investigatethese molecular mechanisms, I will take advantage of a set ofestablished and new model systems for mucociliary research, rangingfrom quickly developing and easy to manipulate frog embryos, togenetically amenable mice, to human airway stem cells in culture.This will allow me to conduct comprehensive and comparative studieson the evolutionarily conserved roles of the Wnt signaling pathway inmucociliary development, regeneration and cilia function. Bymanipulating and analyzing Wnt signaling activity in different celltypes during key phases of development, regeneration and ciliaformation, I will characterize Wnt signaling-dependent processes atthe genomic, cell and tissue level. I will use cutting-edge methods,including transcriptomics, genomics, and genome editing byCRISPR/Cas9, in combination with a bioinformatics approach andfunctional studies to resolve the Wnt signaling-dependent generegulatory network that orchestrates normal development and celltype composition in mucociliary epithelia. Furthermore, I willinvestigate how newly discovered Wnt-regulated genes contribute toairway clearance. To analyze Wnt signaling in the context of ciliaformation and function, I will study recently discovered novel roles of the protein Cp110 in Wnt-dependent coordinated cilia behavior. I willemploy a proteomics approach to identify which other proteins Cp110cooperates with and how this network of factors responds to andpossibly mediates Wnt signaling in ciliated cells. Formation andfunction of cilia in these experiments will be analyzed using geneticand biochemical methods as well as advanced live-cell and superresolutionimaging. This project will address important questions inairway biology and, thereby, gain understanding of airway diseasemechanisms.