Conserved physical mechanisms of cell and tissue elongation.

Living organisms have the ability to self-shape into complex structures appropriate for their function. The genetic and molecular mechanisms that enable cells to do this have been extensively studied in several model and non-model organisms. In contrast, the physical mechanisms that shape cells and tissues have only recently started to emerge, in part thanks to new quantitative in vivo measurements of the physical quantities guiding morphogenesis. These data, combined with indirect inferences of physical characteristics, are starting to reveal similarities in the physical mechanisms underlying morphogenesis across different organisms. Here, we review how physics contributes to shape cells and tissues in a simple, yet ubiquitous, morphogenetic transformation: elongation. Drawing from observed similarities across species, we propose the existence of conserved physical mechanisms of morphogenesis.

Inactivating negative regulators of cortical branched actin enhances persistence of single cell migration.

The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

Organizing collective cell migration through guidance by followers.

Morphogenesis, wound healing, and some cancer metastases rely on the collective migration of groups of cells. In these processes, guidance and coordination between cells and tissues are critical. While strongly adherent epithelial cells have to move collectively, loosely organized mesenchymal cells can migrate as individual cells. Nevertheless, many of them migrate collectively. This article summarizes how migratory reactions to cell-cell contacts, also called "contact regulation of locomotion" behaviors, organize mesenchymal collective cell migration. It focuses on one recently discovered mechanism called "guidance by followers", through which a cell is oriented by its immediate followers. In the gastrulating zebrafish embryo, during embryonic axis elongation, this phenomenon is responsible for the collective migration of the leading tissue, the polster, and its guidance by the following posterior axial mesoderm. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness. Along with other "contact regulation of locomotion" behaviors, this mechanism contributes to organizing collective migration of loose populations of cells.

La morphogénèse, la cicatrisation et certains types de métastases reposent sur la migration collective de groupes de cellules. Lors de ces processus, le guidage et la coordination entre cellules et entre tissus sont fondamentaux. Là où les tissus épithéliaux, très adhésifs, doivent se déplacer collectivement, les cellules mésenchymateuses, en ordre lâche, peuvent migrer individuellement. Cependant, de nombreuses cellules mésenchymateuses migrent de manière collective. Cet article résume comment les réactions migratoires aux contacts entre cellules, aussi appelées «  régulation de locomotion par contact  ¼ , organisent la migration collective des cellules mésenchymateuses. Il décrit en particulier un mécanisme récemment découvert, le «  guidage par les suiveuses  ¼ , par lequel une cellule est orientée par les suiveuses immédiatement en contact. Dans l'embryon de poisson-zèbre en gastrulation, lors de l'élongation du mésoderme axial, ce phénomène est responsable de la migration collective du tissu au front, le polster, et de son guidage par le tissu qui le suit, le mésoderme axial postérieur. Ce mécanisme de guidage par les suiveuses garantit la coordination des mouvements sur de longues distances ainsi que la robustesse du développement. Avec les autres processus de «  régulation de locomotion par contact  ¼ , ce mécanisme contribue à organiser la migration de groupe de cellules en ordre lâche.

Third harmonic imaging contrast from tubular structures in the presence of index discontinuity.

Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the presence of excitation field distortions resulting from sample heterogeneity. Numerical methods that account for these artifacts need to be established. In this work, we experimentally and numerically analyze the THG contrast obtained from stretched hollow glass pipettes embedded in different liquids. We also characterize the nonlinear optical properties of 2,2[Formula: see text]-thiodiethanol (TDE), a water-soluble index-matching medium. We find that index discontinuity not only changes the level and modulation amplitude of polarization-resolved THG signals, but can even change the polarization direction producing maximum THG near interfaces. We then show that a finite-difference time-domain (FDTD) modeling strategy can accurately account for contrast observed in optically heterogeneous samples, whereas reference Fourier-based numerical approaches are accurate only in the absence of index mismatch. This work opens perspectives for interpreting THG microscopy images of tubular objects and other geometries.

Guidance by followers ensures long-range coordination of cell migration through α-catenin mechanoperception.

Morphogenesis, wound healing, and some cancer metastases depend upon the migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, the extension of the embryonic axis is led by the mesendodermal polster that migrates toward the animal pole, followed by the axial mesoderm that undergoes convergence and extension. Here, we investigate how polster cells are guided toward the animal pole. Using a combination of precise laser ablations, advanced transplants, and functional as well as in silico approaches, we establish that each polster cell is oriented by its immediate follower cells. Each cell perceives the migration of followers, through E-cadherin/α-catenin mechanotransduction, and aligns with them. Therefore, directional information propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness.

Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads.

To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula.

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.

Analysis of In Vivo Cell Migration in Mosaic Zebrafish Embryos.

Being optically clear, the zebrafish embryo is a nice model system to analyze cell migration in vivo. This chapter describes a combination of injection and cell transplant procedures that allows creation of mosaic embryos, containing a few cells labeled differently from their neighbors. Rapid 5D confocal imaging of these embryos permits to simultaneously track and quantify the movement of large cell groups, as well as analyze the cellular or subcellular dynamics of transplanted cells during their migration. In addition, expression of a candidate gene can be modified in transplanted cells. Comparing behavior of these cells to control or neighboring cells allows determination of the role of the candidate gene in cell migration. We describe the procedure, focusing on one specific cell population during gastrulation, but it can easily be adapted to other cell populations and other migration events during early embryogenesis.

HMP-1/α-catenin promotes junctional mechanical integrity during morphogenesis.

Adherens junctions (AJs) are key structures regulating tissue integrity and maintaining adhesion between cells. During morphogenesis, junctional proteins cooperate closely with the actomyosin network to drive cell movement and shape changes. How the junctions integrate the mechanical forces in space and in time during an in vivo morphogenetic event is still largely unknown, due to a lack of quantitative data. To address this issue, we inserted a functional Fluorescence Resonance Energy Transfer (FRET)-based force biosensor within HMP-1/α-catenin of Caenorhabditis elegans. We find that the tension exerted on HMP-1 has a cell-specific distribution, is actomyosin-dependent, but is regulated differently from the tension on the actin cortex during embryonic elongation. By using time-lapse analysis of mutants and tissue-specific rescue experiments, we confirm the role of VAB-9/Claudin as an actin bundle anchor. Nevertheless, the tension exerted on HMP-1 did not increase in the absence of VAB-9/Claudin, suggesting that HMP-1 activity is not upregulated to compensate for loss of VAB-9. Our data indicate that HMP-1 does not modulate HMR-1/E-cadherin turnover, is required to recruit junctional actin but not stress fiber-like actin bundles. Altogether, our data suggest that HMP-1/α-catenin acts to promote the mechanical integrity of adherens junctions.