Reverse Engineering the Alveolus: From cellular to microenvironment specification during development

 描述（由申请人提供）：肺泡介导气体交换，因此是肺的关键功能结构，也是许多重要和难治性肺疾病的发生部位。 疾病虽然其复杂的葡萄状结构由几种细胞类型组成（例如，上皮细胞、间充质细胞、内皮细胞和造血细胞），肺泡上皮细胞本身非常简单，仅包含两种肺泡细胞类型（AT）;形成肺泡上皮细胞的非常薄的AT 1细胞， 气体交换的位点和分泌防止肺泡塌陷的表面活性剂的立方形AT 2细胞。我们最近描述了这些细胞类型产生的细胞过程：双能祖细胞（BP）产生AT 1和AT 2的命运。我们最近还进行了单细胞基因表达谱的发展肺泡上皮细胞，并使用的数据来重建完整的基因表达程序的谱系层次从BP到AT 1和AT 2的命运。然而，控制这个程序的信号是未知的。我提出了发展肺泡上皮谱系层次结构的基因表达程序，并对周围肺泡细胞进行表达谱分析，以识别诱导肺泡上皮发育的关键受体及其同源配体联合收割机。了解在发育过程中如何确定AT命运的规范将提供重要的见解，以了解它如何在支气管肺发育不良（BPD），慢性阻塞性肺病（COPD），特发性肺纤维化（IPF）和肺腺癌等疾病中被破坏，并帮助设计再生策略以修复或替换患病组织。该计划的指导阶段将在斯坦福大学Mark Krasnow博士的实验室进行，该实验室是培养发育和干细胞生物学家的领先实验室。该建议的指导阶段侧重于确定控制肺泡发育的信号通路。为了实现这一目标，我将1）使用单一RNAseq表达谱来鉴定在发育中的AT 1和AT 2谱系中选择性表达的所有受体基因，并鉴定编码其同源配体的任何基因在周围肺泡间充质和内皮细胞中的表达。这将为控制肺泡上皮细胞的信号通路识别出杰出的候选者。 上皮细胞命运特化对于第二个目标，我将2）阐明一个候选信号传导途径的作用，即通过初始表达谱结果鉴定的Fgf 7-Fgfr 2途径，在肺泡发育中，通过确定该途径是否是肺泡上皮细胞命运特化所必需和充分的，以及该途径需要在哪些肺泡细胞中发挥其作用。初步数据表明，纯化的Fgf 7是一个强大的诱导剂肺泡细胞发育和肺泡形态发生在新建立的细胞培养试验，因此它提供了一个有前途的测试情况下，阐明的作用，确定候选信号通路在肺泡发育。如果成功，我将确定肺泡发育的第一个重要信号通路，以及识别和表征其他重要信号的框架。这将为肺发育提供关键信息，并为再生肺泡治疗提供合理的方法。从Krasnow博士的实验室，我将追求我的目标，找到一个终身职位，以完成独立阶段的建议。这一阶段的重点是表征肺泡微环境在发育过程中是如何构建的，肺泡发育的一个知之甚少的方面，对于理解肺泡形态发生和最终生物工程肺泡至关重要。为了实现这个目标，我将3）确定肺泡形成过程中肺泡微环境的组成和模式。将通过分析肺泡上皮细胞、间充质细胞和内皮细胞的细胞外基质（ECM）和ECM相关基因表达的单细胞转录谱，然后绘制肺泡发育期间ECM蛋白的空间分布图，以构建4-D图谱，描述它们彼此之间的关系和每种主要细胞类型以及在肺泡发育期间如何变化，来确定组成。我的长期目标是识别和表征发育中肺泡的细胞、控制信号和微环境，以指导健康肺泡的组织工程。我认为这项建议在三个方面是创新的。首先，它试图识别和表征诱导和引导肺泡发育的第一分子控制信号。其次，它开发了一种系统的体外和体内方法，以指导未来的研究，识别和表征其他重要的肺泡发育信号。最后，独立阶段是第一次尝试系统地描述肺泡中研究较少但同样重要的组成部分的形成;微环境。从这些肺泡发育图中，我希望有一天能够设计一个合成的肺泡微环境，然后用分离的祖细胞/干细胞接种它，并添加纯化的信号，为组织创造一个功能性肺泡。 替代疗法

 DESCRIPTION (provided by applicant): Alveoli mediate gas exchange and are thus the key functional structure of the lung, and they are also the site of many important and intractable lung diseases. While its complex grapelike structure is comprised of several cell types (e.g., epithelial, mesenchymal, endothelial, and hematopoietic), the alveolar epithelium itself is quite simple and contains only two alveolar cell types (AT); the exquisitely thin AT1 cells that form the site of gas exchange and the cuboidal AT2 cells which secrete the surfactant that prevents alveolar collapse. We recently described the cellular process by which these cell types arise: a bipotent progenitor (BP) gives rise to both AT1 and AT2 fates. We also recently conducted single cell gene expression profiling of the developing alveolar epithelium, and used the data to reconstruct the full gene expression program of the lineage hierarchy from BP to both AT1 and AT2 fate. However, the signals that control this program are unknown. I propose to gene expression program of the developing the alveolar epithelial lineage hierarchy with expression profiling of surrounding alveolar cells to identify a critical receptor and its cognate ligand combine the that induce alveolar epithelial development. Understanding how AT fate specification is determined during development will provide significant insight to how it is disrupted in diseases like bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung adenocarcinoma as well as help in devising regenerative strategies to repair or replace diseased tissue. The mentored phase of the proposal will be carried out at Stanford University in Dr. Mark Krasnow's laboratory, a leading lab for training developmental and stem cell biologists. The mentored phase of the proposal focuses on identifying a signaling pathway that controls alveolar development. Towards this goal, I will 1) Use single RNAseq expression profiling to identify all receptor genes selectively expressed in the developing AT1 and AT2 lineage and to identify expression in surrounding alveolar mesenchymal and endothelial cells of any genes that encode their cognate ligands. This will identify outstanding candidates for the signaling pathways that control alveolar epithelial fate specification. For the second aim, I will 2) Elucidate the role of one candidate signaling pathway, the Fgf7-Fgfr2 pathway identified by initial expression profiling results, in alveolar development, by determining if the pathway is necessary and sufficient for alveolar epithelial fate specification, and what alveolar cells the pathway is required in to exert its effet. Preliminary data show that purified Fgf7 is a powerful inducer of alveolar cell development and alveolar morphogenesis in a newly established cell culture assay, so it provides a promising test case for elucidating the role of an identified candidate signaling pathway in alveolar development. If successful, I will have identified the first important signaling pathway for alveolus development, and a framework for identifying and characterizing the other important signals. This will provide critical information on lung development and a rational approach to toward regenerative alveolar therapies. From Dr. Krasnow's laboratory, I will pursue my goal of finding a tenure track position to complete the the independent phase of the proposal. This phase focuses on characterizing how the alveolar microenvironment is constructed during development, a poorly understood aspect of alveolar development that is critical for understanding alveolar morphogenesis and ultimately bioengineering an alveolus. Toward this goal, I will 3) Determine the composition and pattern of the alveolar microenvironment during alveologenesis. Composition will be determined by analysis of single cell transcriptional profiles of alveolar epithelial, mesenchymal and endothelial cells for expression of extracellular matrix (ECM) and ECM-related genes, and then mapping the spatial distribution of the ECM proteins during alveolar development, to construct a 4-D map depicting their relationship to each other and each of the major cell types and how this changes during alveolar development. My long term goal is to identify and characterize the cells, control signals and microenvironment of the developing alveolus to guide tissue engineering of a healthy alveolus. I believe this proposal is innovative in three ways. First, it seeks to identify and characterize the first molecular control signals that induce and guide alveolar development. Second, it develops a systematic in vitro and in vivo approach to guide future studies identifying and characterizing other important alveolar development signals. Finally, the independent phase is the first attempt to systematically characterize the formation of the much less studied but equally important component of the alveolus; the microenvironment. From these maps of alveolar development I hope one day to be able to engineer a synthetic alveolar microenvironment and then seed it with isolated progenitors/stem cells and add purified signals to create a functional alveolus for tissue replacement therapies.