Shielding of actin by the endoplasmic reticulum impacts nuclear positioning.

Nuclear position is central to cell polarization, and its disruption is associated with various pathologies. The nucleus is moved away from the leading edge of migrating cells through its connection to moving dorsal actin cables, and the absence of connections to immobile ventral stress fibers. It is unclear how these asymmetric nucleo-cytoskeleton connections are established. Here, using an in vitro wound assay, we find that remodeling of endoplasmic reticulum (ER) impacts nuclear positioning through the formation of a barrier that shields immobile ventral stress fibers. The remodeling of ER and perinuclear ER accumulation is mediated by the ER shaping protein Climp-63. Furthermore, ectopic recruitment of the ER to stress fibers restores nuclear positioning in the absence of Climp-63. Our findings suggest that the ER mediates asymmetric nucleo-cytoskeleton connections to position the nucleus.

Blood Flow Forces in Shaping the Vascular System: A Focus on Endothelial Cell Behavior.

The endothelium is the cell monolayer that lines the interior of the blood vessels separating the vessel lumen where blood circulates, from the surrounding tissues. During embryonic development, endothelial cells (ECs) must ensure that a tight barrier function is maintained whilst dynamically adapting to the growing vascular tree that is being formed and remodeled. Blood circulation generates mechanical forces, such as shear stress and circumferential stretch that are directly acting on the endothelium. ECs actively respond to flow-derived mechanical cues by becoming polarized, migrating and changing neighbors, undergoing shape changes, proliferating or even leaving the tissue and changing identity. It is now accepted that coordinated changes at the single cell level drive fundamental processes governing vascular network morphogenesis such as angiogenic sprouting, network pruning, lumen formation, regulation of vessel caliber and stability or cell fate transitions. Here we summarize the cell biology and mechanics of ECs in response to flow-derived forces, discuss the latest advances made at the single cell level with particular emphasis on in vivo studies and highlight potential implications for vascular pathologies.

Blood Flow Limits Endothelial Cell Extrusion in the Zebrafish Dorsal Aorta.

Blood flow modulates endothelial cell (EC) response during angiogenesis. Shear stress is known to control gene expression related to the endothelial-mesenchymal transition and endothelial-hematopoietic transition. However, the impact of blood flow on the cellular processes associated with EC extrusion is less well understood. To address this question, we dynamically record EC movements and use 3D quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories in the zebrafish dorsal aorta. We find that ECs spread toward the cell extrusion area following the tissue deformation direction dictated by flow-derived mechanical forces. Cell extrusion increases when blood flow is impaired. Similarly, the mechanosensor polycystic kidney disease 2 (pkd2) limits cell extrusion, suggesting that ECs actively sense mechanical forces in the process. These findings identify pkd2 and flow as critical regulators of EC extrusion and suggest that mechanical forces coordinate this process by maintaining ECs within the endothelium.

Three-dimensional microscopy and image analysis methodology for mapping and quantification of nuclear positions in tissues with approximate cylindrical geometry.

Organogenesis involves extensive and dynamic changes of tissue shape during development. It is associated with complex morphogenetic events that require enormous tissue plasticity and generate a large variety of transient three-dimensional geometries that are achieved by global tissue responses. Nevertheless, such global responses are driven by tight spatio-temporal regulation of the behaviours of individual cells composing these tissues. Therefore, the development of image analysis tools that allow for extraction of quantitative data concerning individual cell behaviours is central to study tissue morphogenesis. There are many image analysis tools available that permit extraction of cell parameters. Unfortunately, the majority are developed for tissues with relatively simple geometries such as flat epithelia. Problems arise when the tissue of interest assumes a more complex three-dimensional geometry. Here, we use the endothelium of the developing zebrafish dorsal aorta as an example of a tissue with cylindrical geometry and describe the image analysis routines developed to extract quantitative data on individual cells in such tissues, as well as the image acquisition and sample preparation methodology.This article is part of the Theo Murphy meeting issue 'Mechanics of development'.

UV laser ablation to measure cell and tissue-generated forces in the zebrafish embryo in vivo and ex vivo.

Mechanically coupled cells can generate forces driving cell and tissue morphogenesis during development. Visualization and measuring of these forces is of major importance to better understand the complexity of the biomechanic processes that shape cells and tissues. Here, we describe how UV laser ablation can be utilized to quantitatively assess mechanical tension in different tissues of the developing zebrafish and in cultures of primary germ layer progenitor cells ex vivo.

Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly.

Epithelial spreading is a common and fundamental aspect of various developmental and disease-related processes such as epithelial closure and wound healing. A key challenge for epithelial tissues undergoing spreading is to increase their surface area without disrupting epithelial integrity. Here we show that orienting cell divisions by tension constitutes an efficient mechanism by which the enveloping cell layer (EVL) releases anisotropic tension while undergoing spreading during zebrafish epiboly. The control of EVL cell-division orientation by tension involves cell elongation and requires myosin II activity to align the mitotic spindle with the main tension axis. We also found that in the absence of tension-oriented cell divisions and in the presence of increased tissue tension, EVL cells undergo ectopic fusions, suggesting that the reduction of tension anisotropy by oriented cell divisions is required to prevent EVL cells from fusing. We conclude that cell-division orientation by tension constitutes a key mechanism for limiting tension anisotropy and thus promoting tissue spreading during EVL epiboly.

Forces driving epithelial spreading in zebrafish gastrulation.

Contractile actomyosin rings drive various fundamental morphogenetic processes ranging from cytokinesis to wound healing. Actomyosin rings are generally thought to function by circumferential contraction. Here, we show that the spreading of the enveloping cell layer (EVL) over the yolk cell during zebrafish gastrulation is driven by a contractile actomyosin ring. In contrast to previous suggestions, we find that this ring functions not only by circumferential contraction but also by a flow-friction mechanism. This generates a pulling force through resistance against retrograde actomyosin flow. EVL spreading proceeds normally in situations where circumferential contraction is unproductive, indicating that the flow-friction mechanism is sufficient. Thus, actomyosin rings can function in epithelial morphogenesis through a combination of cable-constriction and flow-friction mechanisms.