The vertebrate body plan is overall symmetrical but left–right (LR) asymmetric in the shape and positioning of internal organs. Although several theories have been proposed, the biophysical mechanisms underlying LR asymmetry are still unclear, especially the role of cell chirality, the LR asymmetry at the cellular level, on organ asymmetry. Here with developing chicken embryos, we examine whether intrinsic cell chirality or handedness regulates cardiac C looping. Using a recently established biomaterial-based 3D culture platform, we demonstrate that chick cardiac cells before and during C looping are intrinsically chiral and exhibit dominant clockwise rotation in vitro. We further show that cells in the developing myocardium are chiral as evident by a rightward bias of cell alignment and a rightward polarization of the Golgi complex, correlating with the direction of cardiac tube rotation. In addition, there is an LR polarized distribution of N-cadherin and myosin II in the myocardium before the onset of cardiac looping. More interestingly, the reversal of cell chirality via activation of the protein kinase C signaling pathway reverses the directionality of cardiac looping, accompanied by a reversal in cellular biases on the cardiac tube. Our results suggest that myocardial cell chirality regulates cellular LR symmetry breaking in the heart tube and the resultant directionality of cardiac looping. Our study provides evidence of an intrinsic cellular chiral bias leading to LR symmetry breaking during directional tissue rotation in vertebrate development.

Our understanding of the left-right (LR) asymmetry of embryonic development, in particular the contribution of intrinsic handedness of the cell or cell chirality, is limited due to the confounding systematic and environmental factors during morphogenesis and a ack of physiologically relevant in vitro 3D platforms. Here we report an efficient two-layered biomaterial platform for determining the chirality of individual cells, cell aggregates, and self-organized hollow epithelial spheroids. This bioengineered niche provides a uniform defined axis allowing for cells to rotate spontaneously with a directional bias toward either clockwise or counterclockwise directions. Mechanistic studies reveal an actin-dependent, cell-intrinsic property of 3D chirality that can be mediated by actin cross-linking via α-actinin-1. Our findings suggest that the gradient of extracellular matrix is an important biophysicochemical cue influencing cell polarity and chirality. Engineered biomaterial systems can serve as an effective platform for studying developmental asymmetry and screening for environmental factors causing birth defects.cell chirality | left-right asymmetry | cell polarity | tissue morphogenesis | biomaterial A lmost all vertebrates have an asymmetric body plan, a deviation from which often leads to severe malformations (1, 2). In recent years, increasing evidence has suggested that embryonic and organ-specific left-right (LR) asymmetries, such as hindgut and genitalia rotation in Drosophila and symmetry breaking in pond snails (3-6), can arise from the LR bias at a cellular level, also termed cell chirality (7,8). In addition, this cellular asymmetry has been demonstrated in various models, including early asymmetry in Caenorhabditis elegans (9, 10), the chiral properties of Xenopus egg cortex (11, 12), asymmetric distribution of chirality related proteins at the early developmental stages of different animals (13), and migratory biases of cultured cells in vitro (12, 14-17). However, cell chirality is poorly understood in developing embryos, despite its scientific and clinical significance, due to complexities in imaging and molecular assays when dealing with animal models and confounding systematic and environmental factors that influence data explanation and hinder mechanistic findings. Therefore, it is of great importance to establish a biomimetic system for LR symmetry breaking that truly recapitulates 3D multicellular chiral morphogenesis.Cell chirality is a fundamental property of the cell, and the universality was not widely regarded until the recent use of microfabricated 2D in vitro systems (16,(18)(19)(20), including the 2D microcontact printing developed by us. In these systems, the cells were confined in a narrow area that allows the cells to exhibit their chiral biases in various formats, including cytoskeleton dynamics, cell migration, and multicellular biased alignment. With these new tools, cell chirality was found to be phenotype-dependent and related to the cross-linking of formin-associated actin bund...

Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left-right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Left-right symmetry breaking is a complex developmental process and an important part of embryonic axis development. As of yet, the biophysical mechanism behind LR asymmetry establishment remains elusive for the overall asymmetry of embryos as well as for the organ-specific asymmetry. Here, we demonstrate that inherent cellular chirality is observable in the cells of early embryonic stages using a 3D Matrigel bilayer system. Differentiation of human embryonic stem cells to three lineages corresponding to heart, intestine, and neural tissues demonstrates phenotype-specific inherent chiral biases, complementing the current knowledge regarding organ development. The existence of inherent cellular chirality early in development and its correlation with organ asymmetry implicate cell chirality as a possible regulator in LR symmetry breaking.