The trachea is a large tubular structure, which is part of the respiratory system, and the internal diameter of the trachea influences the efficiency of respiration. Malformations of the trachea, such as tracheostenosis, affect respiratory ability in the postnatal period; however, the developmental process of the trachea is still not well understood. Here, we analyzed tracheal development, focusing on single cell behaviors and the integration of single cells over long-term development.

We examined epithelial cell proliferation between E12.5 and P7. BrdU incorporation assay revealed that the ratio of proliferating epithelial cells at the late stage of tracheal development (E16.5) was 25%, markedly lower than the ratio of 50% at the middle stage (E12.5-E15.5). To define the relation between cell proliferation and trachea growth, we measured the length, diameter, and surface area of developing trachea by three-dimensional and quantitative method. These quantifications revealed that the trachea growth is accelerated through embryogenesis even though the epithelial cell proliferation was remarkably decreased at the late stage.

We hypothesized that the luminal surface of the trachea was enlarged through both proliferative and non-proliferative mechanisms, and that the non-proliferative mechanism plays a dominant role in the late stage. Histological analyses revealed that epithelial remodeling takes place during E14.5 to E18.5, in which the epithelial cells change its shape and alignment to create the typical pseudostratified columnar structure. We quantified individual epithelial cell shape by reconstructing three-dimensional cell morphology on PC from fluorescent images, which revealed sequential reorganization from un-uniformed to uniformed columnar cell shape and expansion of luminal surfaces during the late stage. These observations indicate that epithelial remodeling creates a pseudostratified epithelium and supports tracheal growth.