Symmetry breaking in cultured mammalian cells

Dear Editor: The generation of natural forms, such as characteristic tissue patterns, involves a process of symmetry breaking in which structures with an initially uniform configuration spontaneously transform into structures with lower symmetry order (Ball, 1999). Symmetry breaking has been examined in biological systems, including formation of fruiting bodies in slime mold (Palsson and Cox, 1996), pattern formation in bacterial colonies (Budrene and Berg, 1991), and generation of animal coat patterns (Murray, 1993); however, the length scale of the characteristic patterns analyzed were orders of magnitude larger than the individual cell components that drive patterning. Here we describe a simplified in vitro model in which symmetry breaking and serf-organization can be visualized within small groups of mammalian cel! s migrating within a geometrically confined adhesive space on micropatterned culture substrates created with a microcontact printing technique (Chen et al., 1997). Development of mammalian tissues, such as capillary blood vessels, is mediated by coordinated migration of cells, which, in turn, is controlled by cell interactions with soluble morphogens, other cells, and insoluble extracellular matrix proteins, such as fibronectin (FN). To create a minimal in vitro model of migration-dependent pattern generation, capillary endothelial cells were cultured in medium containing motility factors (basic fibroblast growth factor + 10% serum) on small (-<50 X 50 ~m 2) FN-eoated adhesive islands that were surrounded by nonadhesive barrier regions. When two cells were cultured on a single island, the high symmetry order associated with the ordinarily random movement of cells on unconfined adhesive surfaces (Dunn and Brown, 1987) changed spontaneously. This transition resulted in directional cell migration and lower symmetry order, such that the entire muhicellular system synchronously rotated about its geometric center (Fig. 1A). Each cell in this dynamic pattern was led by a lamellipodium that wrapped around the trailing edge of the neighboring cell, thereby creating a sigmoid cell-cell interface reminiscent of the Yin-Yang (YY) symbol of Far Eastern religions (Capra, 1991) (Fig. 1B-D). The self-organization of stably rotating cellular ensembles was a robust property of the system and not a random transient phenomenon: YYs were always found to be present when the two-cell systems were analyzed and, once the pattern established itself, the cells rotated continuously over the entire period of observation (over 24 h). YYs were observed to rotate with similar angular velocity in groups of two to four cells cultured on islands of different size (30-50 ~m) and shape (circle versus square) (Fig. 1AD), with equal probability of clockwise or counter-clockwise migration. Emergence of this stable pattern in a group containing only two cells represents the simplest example of symmetry breaking in a mammalian cell population. A computer simulation based on an idealized minimal model in which two cells migrated on a circular track driven by random walk (Dunn and Brown, 1987) suggests that correlated migration similar to that we observed will spontaneously arise and be stable against perturbations if the normally random motility of these cells exhibits a certain minimal value for two basic parameters: persistence and dynamic coupling (Fig. 2). Persistence is the tendency of a cell to keep moving in the same direction; dynamic coupling indicates that one cell has an increased tendency to move in the same direction as its neighbor when the ceils touch (e.g., due to mechanical induction of polarized motion, Verkhovsky et al., 1999). Endothelial cells exhibit persistence values that are many-fold higher than those of other migrating cells, such as leukocytes (Stokes et al., 1991) and fibroblasts (Ware et al., 1998), and thus, the robustness of correlated migration we observed in endothelial cell populations is in agreement with the model. In fact, fibroblasts did not form YYs when cultured on the same FN-coated islands, and instead showed a straight cell-cell boundary (Fig 1E). The transition from random cell motility to coordinated directional migration of cellular ensembles in this simplified in vitro system represents a novel class of self-organization at the muhicellular level. Such a process generates patterns with characteristic features at the same length scale as the underlying components, namely, the individual cells, as is typical for tissue microarchitecture. This mechanism thus differs from the classical models based on reaction-diffusion (e.g., Turing model) which explain long-range (macroscopic) patterns (Murray, 1993) but averages out local inhomogeneities. Instead, our experimental model incorporates local adhesive and structural cues that are also known to play a key role in mammalian tissue morphogenesis (Huang and Ingber, 1999). Thus, it may provide a useful tool to identify how structural cues may lead to symmetry breaking and the emergence of complex tissue patterns through collective behavior among different cellular and molecular components.