Formation of 3D cell mounds at topological defects

In contrast with 2D epithelia, many tissues develop in 3D. These 3D architectures can result from the folding of a monolayer forming empty tubes, spheres or more complex shapes. They can also form bulk tridimensional tissues such as muscles or stratified epithelia, but also epithelial tumors in the context of EMT. Importantly, stacked muscle layers can be oriented perpendicularly in particular around large arteries or the intestine, thereby forming hydrostats. Mechanical cues have long been identified to be critical in the formation of these “crisscross” bilayers but the mechanism at play at the onset of their formation from a monolayer remains elusive. Here, we identify a mechanism by which a myoblast monolayer forms a crisscross bilayer in vitro. The cells first organize in a contractile active nematic monolayer in which most of the +1/2 topological defects self-propel. However, because of the production of extracellular matrix (ECM) by the cells, a subpopulation of these comet-like defects remain stationary even though the cells themselves are still motile. Crisscross bilayering occurs at these stationary defects by having cells of the head migrating on the cells of the tail. Since the cells keep the orientation they had in the initial defect prior to bilayering, the two stacked layers end up perpendicularly oriented. We then design mosaic substrates where microfabricated subcellular cues define domains presenting different orientations. Cells align with these microrails and form extended crisscross bilayers at the boundaries between domains. Finally, plating cells on substrates textured as integer “vortex” defects, we observe the formation of a large mounds at their core. These mounds result from long-range cell flows in the monolayer. We propose a permeation mechanism explaining the onset of the development of these multilayers. On a larger scale, we interpret the shape of mature mounds.