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

 描述(申请人提供)：肺泡是气体交换的媒介，因此是肺的主要功能结构，也是许多重要和顽固性肺的位置。 疾病。虽然其复杂的葡萄状结构由几种细胞类型(如上皮细胞、间充质细胞、内皮细胞和造血细胞)组成，但肺泡上皮本身非常简单，只包含两种类型的肺泡细胞(AT)；极薄的AT1细胞形成了 气体交换部位和立方的AT2细胞，后者分泌防止肺泡塌陷的表面活性物质。我们最近描述了这些细胞类型产生的细胞过程：一个双能祖细胞(BP)同时产生AT1和AT2命运。我们最近还对发育中的肺泡上皮细胞进行了单细胞基因表达谱分析，并利用这些数据重建了从BP到AT1和AT2命运的完整基因表达程序。然而，控制这个程序的信号是未知的。我建议结合周围肺泡细胞的表达谱建立肺泡上皮细胞系的基因表达程序，以确定一个关键的受体及其同源配体结合诱导肺泡上皮细胞的发育。了解AT命运规范在发育过程中是如何确定的，将有助于我们深入了解它在诸如支气管肺发育不良(BPD)、慢性阻塞性肺疾病(COPD)、特发性肺纤维化(IPF)和肺腺癌等疾病中是如何被破坏的，并有助于设计再生策略来修复或替换病变组织。该提案的指导阶段将在斯坦福大学马克·克拉斯诺博士的实验室进行，这是一个培训发育和干细胞生物学家的领先实验室。该提案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)使用单个RNAseq表达谱来确定在发育中的AT1和AT2谱系中选择性表达的所有受体基因，并确定任何编码其同源配体的基因在周围肺泡间充质细胞和内皮细胞中的表达。这将确定控制肺泡的信号通路的优秀候选者。 上皮性命运说明书。对于第二个目标，我将2)阐明一个候选信号通路，Fgf7-FGFR2通路在肺泡发育中的作用，通过确定该通路对肺泡上皮细胞命运的规定是否必要和充分，以及该通路需要在哪些肺泡细胞中发挥其作用。初步数据表明，在新建立的细胞培养实验中，纯化的Fgf7是一种强大的肺泡细胞发育和肺泡形态发生的诱导剂，因此它为阐明已确定的候选信号通路在肺泡发育中的作用提供了一个有前景的测试案例。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。这将提供有关肺发育的关键信息，并为再生性肺泡治疗提供合理的方法。在克拉斯诺博士的实验室里，我将继续追求我的目标，即找到一个终身教职跟踪职位，以完成该提案的独立阶段。这个阶段的重点是描述肺泡微环境是如何在发育过程中构建的，这是肺泡发育的一个鲜为人知的方面，对于理解肺泡的形态发生并最终对肺泡进行生物工程是至关重要的。为了实现这一目标，我将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单细胞转录谱来确定其组成，以寻找细胞外基质(ECM)和ECM相关基因的表达，然后绘制出这些ECM蛋白在肺泡发育过程中的空间分布，以构建一个4-D图来描述它们之间的关系以及每种主要细胞类型以及这种变化在肺泡发育过程中的变化。我的长期目标是鉴定和表征发育中的肺泡的细胞、控制信号和微环境，以指导健康的肺泡的组织工程。我认为这项提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个分子控制信号。其次，它开发了一种系统的体外和体内方法来指导未来的研究，识别和表征其他重要的肺泡发育信号。最后，独立阶段是首次尝试系统地描述肺泡的一个较少研究但同样重要的组成部分--微环境的形成。从这些肺泡发育图中，我希望有一天能够设计出一个合成的肺泡微环境，然后用分离的祖细胞/干细胞播种它，并加入纯化的信号，为组织创造一个有功能的肺泡 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.