Actin based cell chirality emerging at the boundary of 2D-microtissue directs chiral multicellular pattern formation.

The mechanisms underlying both the establishment of mirror (reflection) symmetry and deviations from it in the 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 semicircular 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 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 how intrinsically chiral cells can build mirror-symmetric tissues in bilateral organisms: either by reversing cell chirality in one half or by enlarging the tissue to minimise boundary influence.