Molecular Mechanisms of Mucociliary Epithelia and Airway Clearance

脊椎动物的传导气道由粘液纤毛上皮覆盖，粘液纤毛上皮清除气道，以提供抵抗病原体的第一道防线。纤毛上皮由干细胞、分泌细胞和纤毛细胞组成，后者形成向细胞外空间突出的运动突起（纤毛）。纤毛的正确细胞类型组成和功能的建立都是气道清除所必需的，而这两个过程中的任何一个缺陷都会导致慢性肺部疾病并增加肺部感染的风险。肺部疾病是世界范围内最常见的死亡原因之一，但对导致这些疾病的分子机制知之甚少。我的工作旨在通过研究Wnt信号通路的细胞间通讯如何调节纤毛上皮的基因表达和细胞类型组成，以及纤毛在气道细胞中的形成和功能，来解决纤毛上皮的分子机制。因此，我将产生有关粘液纤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用于粘液纤毛研究的模型系统，从快速发育和易于操作的青蛙胚胎，到遗传学上适合的小鼠，再到培养的人类气道干细胞。这将使我能够对Wnt信号通路在粘液纤毛发育、再生和纤毛功能中的进化保守作用进行全面和比较研究。通过在发育、再生和纤毛形成的关键阶段操纵和分析不同细胞类型中的Wnt信号传导活性，我将在基因组、细胞和组织水平上表征Wnt信号传导依赖的过程。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信号依赖的基因调控网络，该网络协调粘膜纤毛上皮细胞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的背景下，我将研究最近发现的蛋白质Cp 110在Wnt依赖的协调纤毛行为中的新作用。我将采用蛋白质组学的方法来确定哪些其他蛋白质Cp 110合作，以及这个网络的因素如何响应，并可能介导Wnt信号在纤毛细胞。这些实验中纤毛的形成和功能将使用遗传学和生物化学方法以及先进的活细胞和超分辨率成像进行分析。本计画将探讨呼吸道生物学中的重要问题，进而了解呼吸道疾病的机制。

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.