Search

Directional migration during embryogenesis and tumor progression faces the challenge that numerous external signals need to converge to precisely control cell movement. The Rho guanine exchange factor (GEF) Trio is especially well suited to relay signals, as it features distinct catalytic domains to activate Rho GTPases. Here, we show that Trio is required for Xenopus cranial neural crest (NC) cell migration and cartilage formation. Trio cell-autonomously controls protrusion formation of NC cells and Trio morphant NC cells show a blebbing phenotype. Interestingly, the Trio GEF2 domain is sufficient to rescue protrusion formation and migration of Trio morphant NC cells. We show that this domain interacts with the DEP/C-terminus of Dishevelled (DVL). DVL - but not a deletion construct lacking the DEP domain - is able to rescue protrusion formation and migration of Trio morphant NC cells. This is likely mediated by activation of Rac1, as we find that DVL rescues Rac1 activity in Trio morphant embryos. Thus, our data provide evidence for a novel signaling pathway, whereby Trio controls protrusion formation of cranial NC cells by interacting with DVL to activate Rac1., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

During development cell-cell adhesion is not only crucial to maintain tissue morphogenesis and homeostasis, it also activates signalling pathways important for the regulation of different cellular processes including cell survival, gene expression, collective cell migration and differentiation. Importantly, gene mutations of adhesion receptors can cause developmental disorders and different diseases. Quantitative methods to measure cell adhesion are therefore necessary to understand how cells regulate cell-cell adhesion during development and how aberrations in cell-cell adhesion contribute to disease. Different in vitro adhesion assays have been developed in the past, but not all of them are suitable to study developmentally-related cell-cell adhesion processes, which usually requires working with low numbers of primary cells. In this review, we provide an overview of different in vitro techniques to study cell-cell adhesion during development, including a semi-quantitative cell flipping assay, and quantitative single-cell methods based on atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) or dual micropipette aspiration (DPA). Furthermore, we review applications of Förster resonance energy transfer (FRET)-based molecular tension sensors to visualize intracellular mechanical forces acting on cell adhesion sites. Finally, we describe a recently introduced method to quantitate cell-generated forces directly in living tissues based on the deformation of oil microdroplets functionalized with adhesion receptor ligands. Together, these techniques provide a comprehensive toolbox to characterize different cell-cell adhesion phenomena during development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Stem cells and developing tissues respond to long-range signaling molecules (morphogens), by starting different nuclear programs that decide about the cell fate. Cells sense the local morphogen concentration and the shape of the gradient. We developed a two-chambered microfluidic chip to reproduce the in vivo situation under shear stress free conditions. The gradient is generated in the lower part of our device and recognized by cells grown in the upper part in the microchamber. We tested our device by activating the Wnt/β-catenin signaling pathway in HeLa cells as proven by nuclear β-catenin accumulation in response to the Wnt pathway activator 6-bromoindirubin-3'-oxime (BIO). Applying the same readout system to a recombinant Wnt3a and Dkk-1 bipolar gradient we demonstrate that our microfluidic chip is suitable for morphogens as well as small molecules. More interestingly, our microfluidic device is highly flexible. While the generated gradients are stable for several hours and reproducible, we can change the kind and the shape of the gradient actively on demand. We also can switch from diffusion- to convection-based transport, thus applying the morphogen gradient either in a polarized or non-polarized manner.