Time-lapse and cleared imaging of mouse embryonic lung explants to study three-dimensional cell morphology and topology dynamics.

Here, we present a protocol for collecting high-spatiotemporal-resolution datasets of undisturbed mouse embryonic epithelial rudiments using light-sheet fluorescence microscopy. We describe steps for rudiment dissection, clearing, and embedding for cleared and live imaging. We then detail procedures for light-sheet imaging followed by image processing and morphometric analysis. We provide protocol variations for imaging both growing and optically cleared lung explants to encourage the quantitative exploration of three-dimensional cell shapes, cell organization, and complex cell-cell dynamics. For complete details on the use and execution of this protocol, please refer to Gómez et al. (2021).1.

3D cell neighbour dynamics in growing pseudostratified epithelia.

During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organise their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organisation in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, that is, Euler's polyhedron formula, Lewis' law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimises the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organisation in growing epithelia emerges from simple physical principles.