LUNG ALVEOLAR TYPE 1 CELL MORPHOGENESIS

描述(申请人提供)：本提案的目的是确定肺泡I型细胞发育的分子机制，重点是I型细胞的主要特征之一，即它们广泛、薄、平的形状。妊娠晚期，前体肺远端上皮细胞形态由立方变为扁平，具有分化的I型细胞的结构和分子特征，形成较薄的肺泡气体交换面。我们假设，调控质膜生长和极化以及细胞骨架组织的基因在晚期胎儿I型细胞发育过程中起着关键的诱导和/或允许作用。我们将分析I型肺泡细胞的三个重要特征：形状、I型特异性基因表达和扁平化相关基因表达。我们将有选择地修改这些功能中的每一个，并研究其他两个功能中的更改。这一方法将提供关于启动和/或维持I型细胞形态发生的分子机制的重要信息。我们将在发育中的肺中研究在果蝇和线虫中发现的与上皮细胞扩张和扁平化相关的基因的作用，以及在T1a缺失突变小鼠中与细胞形状改变相关的基因的作用，其中I型细胞的形成受到损害。我们将确定这些基因在正常肺中表达的时间和地点。我们将利用从不同发育阶段的胎肺分离的表达绿色荧光蛋白的L前体细胞来评估它们在细胞扁平和扩散中的作用。我们将在体外增加或减少选定基因的表达，以确定对上皮细胞扁平和扩散的影响，以及对I型特异性基因表达的影响。我们将使用限制扩散的培养条件在体外调节上皮细胞的形状，并评估I型特异性和细胞扁平化相关基因的表达。最后，我们将在发育中的肺中评估这些基因在体内的作用，这些肺的类型I细胞分化受损。通过这些方法对I型细胞的分析将为调节胎肺中I型细胞的形成提供新的见解。这种调节可能对出生后肺生长中的I型细胞形态发生和成人肺损伤后的肺修复很重要。与公共卫生相关：当肺发育延迟或婴儿早产时，排列在肺泡内的细胞不成熟，无法有效地执行正常的气体交换过程。识别控制肺泡细胞形成的关键基因对于设计刺激新生儿肺成熟的新疗法非常重要。类似的机制也适用于感染或环境因素造成的损伤后肺泡细胞的愈合过程。因此，这些研究将提供对I型形成规律的新理解，这可能会改善成人急性和慢性肺部疾病的治疗。

DESCRIPTION (provided by applicant): The objective of this proposal is to define the molecular mechanisms of lung alveolar type I cell development, focusing on one of the principal features of type I cells, their extensive, thin, flat shape. In late gestation, precursor distal lung epithelial cells change their shape from cuboidal to flat, acquiring the structural and molecular features of differentiated type I cells to form the thin alveolar gas exchange surface. We hypothesize that genes that regulate plasma membrane growth and polarization, and cytoskeletal organization play a critical inductive and/or permissive role in the process of late fetal type I cell development. We will analyze three important features of alveolar type I cells: shape, type I specific gene expression, and flattening-related gene expression. We will selectively modify each of these features and study alterations in the other two. This approach will provide important information about the molecular mechanisms that initiate and/or sustain type I cell morphogenesis. We will study in the developing lung the roles of genes associated with epithelial cell expansion and flattening identified in Drosophila and C. elegans, and of genes associated with altered cell shape in T1a null mutant mouse, where type I cell formation is impaired. We will determine when and where these genes are expressed in normal lung. We will evaluate their role in cell flattening and spreading using type l-precursor cells isolated at different developmental stages from fetal lungs expressing GFP driven by the promoter of the type I cell gene T1a. We will increase or reduce expression of selected genes in vitro to determine effects on epithelial cell flattening and spreading and on type I specific gene expression. We will modulate the shape of epithelial cells in vitro using culture conditions that restrict spreading and evaluate type I specific and cell-flattening-related gene expression. Finally, we will evaluate the role of these genes in vivo using developing lungs with impaired type I cell differentiation. Analysis of type I cells by these approaches will provide new insights into the regulation of type I cell formation in the fetal lung. This regulation is likely important for type I cell morphogenesis in postnatal lung growth and in lung repair after injury in the adult lung. Relevance to Public Health: When lung development is delayed or babies are delivered prematurely the cells that line the lung alveoli are immature and cannot efficiently perform the normal process of gas exchange. Identifying the key genes that control alveolar cell formation is important to allow the design of new treatments to stimulate newborn lung maturation. Similar mechanisms could apply to the process of alveolar cell healing after injuries caused by infections or environmental factors. Therefore these studies will provide new understanding of the regulation of type I formation that likely will improve treatment of acute and chronic lung diseases in the adult.