Ordering from the edge: Chiral multicellular pattern formation directed by actin self-organisation in cells

The mechanisms underlying both the establishment of mirror symmetry and deviations from it in development of bilateral multicellular organisms remain insufficiently understood. Actin cytoskeletons of individual cells exhibit intrinsic chirality, and a strong correlation exists between single-cell actin fibres chiral organisation and the collective alignment of cells confined to rectangular adhesive islands (2D-microtissues). Here, we demonstrate how multicellular chiral patterns can be inferred from the chiral behaviour of actin fibres in individual cells. By analysing chiral actin systems in cells with elliptical and semi-circular shapes, representing inner and boundary positions within 2D-microtissues, we defined the rules of chiral motile behaviour and formulated two models of cell alignment: (i) chiral rotation of inner cells and (ii) chiral tilting of boundary cells relative to island edges. In both models, neighbouring cells are also mutually aligned. Systematic variation of island area and aspect ratio, combined with dynamic observations, revealed the primary role of boundary cells. Chiral order first emerged at tissue boundaries and then propagated inward. This outside-in mechanism also explains why mirror-symmetric cell groups are difficult to achieve with uniformly chiral cells, but can be obtained either by reversing chirality in one subgroup or by enlarging the microtissue to minimise boundary influence.