Cell generated contractility is a major driver of morphogenesis during processes such as epithelial bending and epithelial-to-mesenchymal transitions. Previous studies of contraction in embryos have relied on developmentally programmed cell shape changes such as those that accompany ventral furrow formation in Drosophila, bottle cell formation in Xenopus, ingression in amniote embryos, and neurulation in vertebrate embryos. We have identified three methods to reproducibly and acutely induce contraction in embryonic epithelial sheets: laser activation, electrical stimulation, and nano-perfusion with chemicals released by wounding. Contractions induced by all three methods occur over a similar time scale (1 to 2 min) and lead to reorganization of the F-actin cytoskeleton. By combining induced contractions with micro-aspiration we can simultaneously measure the stiffness of the tissue and the force and work done by contractions. Laser-activation allows real-time visualization of F-actin remodeling during contraction. Perfusion with cell-lysate suggests these three stimuli activate physiologically relevant pathways that maintain epithelial tension or trigger epithelial morphogenesis. Our methods provide the means to control and study cellular contractility and will allow dissection of molecular mechanisms and biomechanics of cellular contractility.

Dynamic mechanical culture systems are a widely studied approach for improving the functional mechanical properties of tissue engineering constructs intended for loading-bearing orthopedic applications such as tendon/ligament reconstruction. The design of effective mechanical stimulation regimes requires a fundamental understanding of the effects of cyclic strain parameters on the resulting construct properties. Toward this end, these studies employed a modular cyclic strain bioreactor system and fibroblast-seeded, porous polyurethane substrates to systematically investigate the effect of varying cyclic strain amplitude, rate, frequency, and daily cycle number on construct mechanical properties. Significant differences were observed in response to variation of all four loading parameters tested. In general, the highest values of elastic modulus within each experimental group were observed at low to intermediate values of the experimental variables tested, corresponding to the low to subphysiological range (2.5% strain amplitude, 25%/s strain rate, 0.1-0.5 Hz frequency, and 7,200-28,800 cycles/day). These studies demonstrate that fibroblasts are sensitive and responsive to multiple characteristics of their mechanical environment, and suggest that systematic optimization of dynamic culture conditions may be useful for the acceleration of construct maturation and mechanical function.

Multicellular organisms are generated by coordinated cell movements during morphogenesis. Convergent extension is a key tissue movement that organizes mesoderm, ectoderm, and endoderm in vertebrate embryos. The goals of researchers studying convergent extension, and morphogenesis in general, include understanding the molecular pathways that control cell identity, establish fields of cell types, and regulate cell behaviors. Cell identity, the size and boundaries of tissues, and the behaviors exhibited by those cells shape the developing embryo; however, there is a fundamental gap between understanding the molecular pathways that control processes within single cells and understanding how cells work together to assemble multi-cellular structures. Theoretical and experimental biomechanics of embryonic tissues are increasingly being used to bridge that gap. The efforts to map molecular pathways and the mechanical processes underlying morphogenesis are crucial to understanding: 1) the source of birth defects, 2) the formation of tumors and progression of cancer, and 3) basic principles of tissue engineering. In this paper, we first review the process of tissue convergent-extension of the vertebrate axis and then review models used to study the self-organizing movements from a mechanical perspective. We conclude by presenting a relatively simple "wedge-model" that exhibits key emergent properties of convergent extension such as the coupling between tissue stiffness, cell intercalation forces, and tissue elongation forces.

Spatiotemporal regulation of cell contractility coordinates cell shape change to construct tissue architecture and ultimately directs the morphology and function of the organism. Here we show that contractility responses to spatially and temporally controlled chemical stimuli depend much more strongly on intercellular mechanical connections than on biochemical cues in both stimulated tissues and adjacent cells. We investigate how the cell contractility is triggered within an embryonic epithelial sheet by local ligand stimulation and coordinates a long-range contraction response. Our custom microfluidic control system allows spatiotemporally controlled stimulation with extracellular ATP, which results in locally distinct contractility followed by mechanical strain pattern formation. The stimulationresponse circuit exposed here provides a better understanding of how morphogenetic processes integrate responses to stimulation and how intercellular responses are transmitted across multiple cells. These findings may enable one to create a biological actuator that actively drives morphogenesis.microfluidics | multicellular | mechanotransduction | signaling P hysiological control systems have evolved diverse strategies to sense the environment, transduce signals, and actuate contractile responses. One such strategy involves the actuation of nonmuscle cell contractility to drive a wide range of developmental processes as well as normal physiological and pathological disease states (1-5). The contractile behaviors of cells and their interactions in multicellular arrays are not only critical in shaping and guiding tissue formation (e.g., epithelial folding) for the successful outcome of development programs (6, 7), but also play a major role in the pathology of tumor growth, the invasionmetastasis cascade, wound healing, and tissue regeneration (8,9).From a signaling standpoint, embryonic development is a dynamic process where cells interact and coordinate force generation as their identities are patterned by spatiotemporally applied chemical stimuli. There has been considerable debate over the effectiveness of intra-versus intercellular signaling during development (10, 11); nevertheless, the cell-cell signaling and the coordination of a variety of multicellular responses are known to be mediated by gap-junction-dependent intercellular communication (12, 13). However, recent findings from studies of cell mechanics indicate that mechanical cues can be as potent as chemical factors in directing cell differentiation and behaviors and suggest a role in modulating signal transduction pathways (14, 15). Less clear is whether signal transduction can be modulated by mechanical connections during morphogenesis. Results and DiscussionTo investigate the complex integrated response of a multicellular system and investigate the mechanochemical response and actuation, we cultured an intact epithelial sheet adhered to a fibronectin extracellular matrix substrate within a custom microfluidic chamber and exposed a narrow band of 4-5 cells...

Embryonic development is guided by a complex and integrated set of stimuli that results in collective system-wide organization that is both time and space regulated. These regulatory interactions result in the emergence of highly functional units, which are correlated to frequency-modulated stimulation profiles. We have determined the dynamic response of vertebrate embryonic tissues to highly controlled, time-varying localized chemical stimulation using a microfluidic system with feedback control. Our approach has enabled localized spatiotemporal manipulation of the steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. Using this approach we investigated cell-scale responses to precisely controlled stimulation by tracking the redistribution of a GFP-tagged DEX-reporter constructed from the human glucocorticoid receptor (GR). We exposed defined regions of a single AC explant to different stimulation conditions—continuous stimulation, periodic stimulation, and no stimulation. We observed collective behavior of the GR transport into the nucleus was first-order. Furthermore, the dynamic response was well-modeled by a first-order differential equation with a single time derivative. The model predicted that responses to periodic stimulations closely matched the results of the frequency-based experiments. We find that stimulation with localized bursts versus continuous stimulation can result in highly distinct responses. This finding is critical as controlled space and time exposure to growth factors is a hallmark of complex processes in embryonic development. These complex responses to cellular signaling and transport machinery were similar to emergent behaviors in other complex systems, suggesting that even within a complex embryonic tissue, the overall system can converge toward a predictive first-order response.