Crisscross multilayering of cell sheets

Abstract

Simple hydrostatic skeletons such as the Hydra's consist of two stacked layers of cells perpendicularly oriented. Although this crisscross architecture can be recapitulated in vitro, little is known on the formation of such multilayers starting from a monolayer. In the present article, we show that bilayering of myoblasts results from the organization and activity of the cells originally in the monolayer which can be described as a contractile active nematic. As expected, most of the +1/2 topological defects that are associated with this nematic order self-propel. However, a subpopulation of these defects remains immobile. Perpendicular bilayering occurs exclusively at these motionless defects. Indeed, cells located at the head of these defects converge toward the (immobile) core and accumulate there until they start migrating on top of the tail of the first layer while the tail cells migrate in the opposite direction under the head cells. Since the cells keep their initial orientations, the two stacked layers end up perpendicularly oriented. This concerted process leading to a bilayer is dependent on the apical secretion of Extra Cellular Matrix (ECM) by the cells. Indeed, we evidence the presence of ECM between the cell layers and at the apical surface of the topmost layer. ECM molecules are oriented in the direction of the cells that produce them, which may guide the migration of the subsequent cell layers on their apical side.

Spontaneous flow created by active topological defects

“…Experimental observations on nematic cell monolayers show that topological defects are preferential sites of cell extrusion [20,21] and multilayering [22]. Similar observations have been made in bacterial monolayers [23].…”

“…Similar observations have been made in bacterial monolayers [23]. In a recent work by T. Sarkar and colleagues [22] on the spontaneous organization of cell monolayers on a solid substrate, the authors measured the flow patterns around topological defects of charges ±1/2. Some of the +1/2 topological defects were found to be pinned at specific locations on the substrate, while others had a spontaneous motion as expected in active nematics.…”

“…The initial goal of this work was to explain the observation of Sarkar et al in ref [22] of motionless defects that are preferential sites for multilayer formation. We computed the self-advection velocity of motile defects and the force necessary to stall them.…”

“…This very general framework is based on symmetries and conservation laws [30] and has been shown to adequately describe monolayers of elongated cells that self-organize into a nematic phase [15,[31][32][33]. More specifically, we aim at describing the dynamics of nematic topological defects as observed experimentally in monolayers of myoblast cells [22]. Because we are interested in monolayers of cells at confluence, we consider the active nematic as a one-constituent dense phase, with a fixed cell density.…”

“…2.2 Stall force of a +1/2 defect Sarkar et al observed two classes of +1/2 topological defects in confluent monolayers of C2C12 mouse myoblasts [22]: motile topological defects with a flow characteristic of selfadvected defects, as presented in sec. 2.1, and stalled, nonmotile defects.…”