Neural tube closure is a critical developmental event that relies on actomyosin contractility to facilitate specific processes such as apical constriction, tissue bending, and directional cell rearrangements. These complicated processes require the coordinated activities of Rho-Kinase (Rock), to regulate cytoskeletal dynamics and actomyosin contractility, and the Planar Cell Polarity (PCP) pathway, to direct the polarized cellular behaviors that drive convergent extension (CE) movements. Here we investigate the role of Shroom3 as a direct linker between PCP and actomyosin contractility during mouse neural tube morphogenesis. In embryos, simultaneous depletion of Shroom3 and the PCP components Vangl2 or Wnt5a results in an increased liability to NTDs and CE failure. We further show that these pathways intersect at Dishevelled, as Shroom3 and Dishevelled 2 co-distribute and form a physical complex in cells. We observed that multiple components of the Shroom3 pathway are planar polarized along mediolateral cell junctions in the neural plate of E8.5 embryos in a Shroom3 and PCPdependent manner. Finally, we demonstrate that Shroom3 mutant embryos exhibit defects in planar cell arrangement during neural tube closure, suggesting a role for Shroom3 activity in CE. These findings support a model in which the Shroom3 and PCP pathways interact to control CE and polarized bending of the neural plate and provide a clear illustration of the complex genetic basis of NTDs.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate. STEM CELLS

Neural tube defects arise from mechanical failures in the process of neurulation. At the most fundamental level, formation of the neural tube relies on coordinated, complex tissue movements that mechanically transform the flat neural epithelium into a lumenized epithelial tube (Davidson, 2012). The nature of this mechanical transformation has mystified embryologists, geneticists, and clinicians for more than 100 years. Early embryologists pondered the physical mechanisms that guide this transformation. Detailed observations of cell and tissue movements as well as experimental embryological manipulations allowed researchers to generate and test elementary hypotheses of the intrinsic and extrinsic forces acting on the neural tissue. Current research has turned toward understanding the molecular mechanisms underlying neurulation. Genetic and molecular perturbation have identified a multitude of subcellular components that correlate with cell behaviors and tissue movements during neural tube formation. In this review, we focus on methods and conceptual frameworks that have been applied to the study of amphibian neurulation that can be used to determine how molecular and physical mechanisms are integrated and responsible for neurulation. We will describe how qualitative descriptions and quantitative measurements of strain, force generation, and tissue material properties as well as simulations can be used to understand how embryos use morphogenetic programs to drive neurulation. Birth Defects Research 109:153-168, 2017. © 2016 Wiley Periodicals, Inc.

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...