α5β1 integrin mediates pulmonary epithelial cyst formation

Background: Formation of the epithelial cyst involves the establishment of apical–basolateral polarity through a series of cellular interactions that are in part mediated by the extracellular matrix (ECM). We report that in a three-dimensional multi-cellular self-assembly model of lung development, α5 integrin regulates epithelial cyst formation through organization of soluble fibronectin matrix into insoluble fibrils through a process called fibrillogenesis. Results: Dissociated murine embryonic lung cells self-assemble into three-dimensional pulmonary bodies that are dependent on α5β1 integrin mediated fibrillogenesis for cell–cell mediated self-assembly: compaction and epithelial cyst formation. Knockdown of α5 integrin resulted in a significant increase in another mediator of fibrillogenesis, αV integrin. Compensatory increased expression of another mediator of fibrillogenesis, αV integrin, was not sufficient to normalize epithelial cyst formation. Loss of α5 integrin-mediated fibrillogenesis perturbed the ability of clustered epithelial cells to establish clear polarity, loss of epithelial cell pyramidal shape, and disrupted apical F-actin–rich deposition. Lack of rich central epithelial localization of F-actin cytoskeleton and Podocalyxin suggests that loss of α5 integrin-mediated fibrillogenesis interferes with the normal cytoskeleton organization that facilitates epithelial cysts polarization. Conclusions: We conclude that lung epithelial cyst formation in development is mediated in part by α5β1 integrin dependent fibrillogenesis. Developmental Dynamics 246:475–484, 2016.