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Laser manipulation is widely used to study mechanics from the molecular to the tissue scale. We implemented optical tweezers to directly manipulate single cell-cell junctions in a developing tissue. We further extended the approach to two-point laser manipulation to enable extensive remodeling of cell-cell junctions. Here, we describe two-point laser manipulation and its implementation to probe the mechanics of cell junctions in the Drosophila embryo.

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II–arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined., Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and two small polar bodies, but the mechanisms underlying the required symmetry breaking and spindle rotation have remained elusive. This study shows that spindle rotation in activated mouse oocytes relies on spontaneous symmetry breaking resulting from an unstable configuration generated by cleavage furrow ingression and cortical chromosome attraction.

Morphogenesis relies on the active generation of forces, and the transmission of these forces to surrounding cells and tissues. Hence measuring forces directly in developing embryos is an essential task to study the mechanics of development. Among the experimental techniques that have emerged to measure forces in epithelial tissues, force inference is particularly appealing. Indeed it only requires a snapshot of the tissue, as it relies on the topology and geometry of cell contacts, assuming that forces are balanced at each vertex. However, establishing force inference as a reliable technique requires thorough validation in multiple conditions. Here we performed systematic comparisons of force inference with laser ablation experiments in four epithelial tissues from two animals, the fruit fly and the quail. We show that force inference accurately predicts single junction tension, tension patterns in stereotyped groups of cells, and tissue-scale stress patterns, in wild type and mutant conditions. We emphasize its ability to capture the distribution of forces at different scales from a single image, which gives it a critical advantage over perturbative techniques such as laser ablation. Overall, our results demonstrate that force inference is a reliable and efficient method to quantify the mechanical state of epithelia during morphogenesis, especially at larger scales when inferred tensions and pressures are binned into a coarse-grained stress tensor.

International audience; How individual cell behaviours lead to the emergence of global patterns is poorly understood. In the Xenopus embryonic epidermis, multiciliated cells (MCCs) are born in a random pattern within an inner mesenchymal layer, and subsequently intercalate at regular intervals into an outer epithelial layer. Using both experiments and mathematical modelling, we show that this transition from random to ordered distribution relies on mutual repulsion among motile immature MCCs, and affinity towards outer-layer intercellular junctions. Consistently, Arp2/3-mediated actin remodelling is required for MCC pattern emergence. Using multiple functional approaches, we show that the Kit tyrosine kinase receptor, expressed in MCCs, and its ligand Scf, expressed in outer-layer cells, are both required for regular MCC distribution. Membrane-associated Scf behaves as a potent adhesive cue for MCCs, while its soluble form promotes their mutual repulsion. On the other hand, Kit expression is sufficient to confer order to a disordered heterologous cell population. Our work reveals how a single signalling system can implement self-organised large-scale patterning.

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and two small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before the anaphase occurs. In metaphase II arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until the fertilization triggers the sister chromatids segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable the cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that the spindle rotation results from two antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling and second, an outward attraction exerted on both lots of chromatids by a RanGTP dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modelling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the lot of chromatids that eventually gets discarded are biologically predetermined.

SUMMARYHow individual cell behaviours lead to the emergence of global patterns is poorly understood. In theXenopusembryonic epidermis, multiciliated cells (MCCs) are born in a random pattern within an inner mesenchymal layer, and subsequently intercalate at regular intervals into an outer epithelial layer. Using both experiments and mathematical modelling, we show that this transition from chaotic to ordered distribution relies on mutual repulsion among motile immature MCCs, and affinity towards outer-layer intercellular junctions. Consistently, ARP2/3-mediated actin remodelling is required for MCC pattern emergence. Using multiple functional approaches, we show that the Kit tyrosine kinase receptor, expressed in MCCs, and its ligand Scf, expressed in outer-layer cells, are both required for regular MCC distribution. While Scf behaves as a potent adhesive cue for MCCs, Kit expression is sufficient to confer order to a disordered heterologous cell population. Our work reveals how a single signalling system can implement self-organised large-scale patterning.Highlights- Immature multiciliated cells transit from a disordered to an ordered pattern- The transition is a self-organising process based on repulsive and affinity movements- ARP2/3-dependent actin remodelling is required for pattern emergence- The SCF/KIT pathway promotes both repulsion and affinity movementseTOC blurbIn developingXenopusepidermis, immature multiciliated cells (MCCs), initially chaotically distributed within an inner layer, emerge in an orderly pattern among cells of the outer layer. This process involves MCC mutual repulsion and affinity towards outer-layer intercellular junctions. The SCF/KIT signalling pathway promotes both properties to allow regular MCC distribution.

Tissue remodeling during embryogenesis is driven by the apical contractility of the epithelial cell cortex. This behavior arises notably from Rho1/Rok induced transient accumulation of non-muscle myosin II (MyoII pulses) pulling on actin filaments (F-Actin) of the medio-apical cortex. While recent studies begin to highlight the mechanisms governing the emergence of Rho1/Rok/MyoII pulsatility in different organisms, little is known about how the F-Actin organization influences this process. Focusing onDrosophilaectodermal cells during germband extension and amnioserosa cells during dorsal closure, we show that the medio-apical actomyosin cortex consists of two entangled F-Actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1 dependent manner. The other forms a persistent and homogeneous network independent of Rho1. We identify the Frl/Fmnl formin as a critical nucleator of the persistent network since modulating its level, in mutants or by overexpression, decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue scale morphogenetic defects. Our work sheds new lights on how the F-Actin cortex offers multiple levels of regulation to affect epithelial cells dynamics.

Background - Understanding how molecular and physical cues orchestrate vascular morphogenesis is a challenge for developmental biology. Only little attention has been paid to the impact of mechanical stress caused by tissue growth on early blood distribution. Here we study the peripheral accumulation of blood in the chicken embryonic yolk sac, which precedes sinus vein formation. Results - We report that blood accumulation starts prior to heart-induced blood circulation. We hypothesized that the driving force for the primitive blood flow is a growth-induced gradient of tissue pressure in the yolk sac mesoderm. Therefore, we studied embryos in which heart development was arrested after two days of incubation, and found that yolk sac growth and blood peripheral accumulation still occurred. This suggests that tissue growth is sufficient to initiate the flow and the formation of the sinus vein, whereas heart contractions are not required. We designed a simple mathematical model which makes explicit the growth-induced pressure gradient and the subsequent blood accumulation, and show that growth can indeed account for the observed blood accumulation. Conclusions - This study shows that tissue growth pressure can drive early blood flow, and suggests that the mechanical environment, beyond hemodynamics, can contribute to vascular morphogenesis. This article is protected by copyright. All rights reserved.

International audience; It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. We previously showed that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation (Narbonne-Reveau et al., 2016). Using single-cell transcriptomics, clonal analysis and numerical modeling, we now identify a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin.

Morphogenesis relies on the active generation of forces, and the transmission of these forces to surrounding cells and tissues. Hence measuring forces directly in developing embryos is an essential task to study the mechanics of development. Among the experimental techniques that have emerged to measure forces in epithelial tissues, force inference is particularly appealing. Indeed it only requires a snapshot of the tissue, as it relies on the topology and geometry of cell contacts, assuming that forces are balanced at each vertex. However, establishing force inference as a reliable technique requires thorough validation in multiple conditions. Here we performed systematic comparisons of force inference with laser ablation experiments in three distinct Drosophila epithelia. We show that force inference accurately predicts single junction tensions, tension patterns in stereotyped groups of cells, and tissue-scale stress patterns, in wild type and mutant conditions. We emphasize its ability to capture the distribution of forces at different scales from a single image, which gives it a critical advantage over perturbative techniques such as laser ablation. Our results demonstrate that force inference is a reliable and efficient method to quantify the mechanics of epithelial tissues during morphogenesis.

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.

The mechanisms that govern the hierarchical organization of tumors are still poorly understood, especially in highly heterogeneous neural cancers. Previously, we had shown that aggressive neural tumors can be induced upon dedifferentiation of susceptible intermediate progenitors produced during early development (Narbonne-Reveau et al., 2016). Using clonal analysis, stochastic modelling and single-cell transcriptomics, we now find that such tumors rapidly become heterogeneous, containing progenitors with different proliferative potentials. We demonstrate that tumor heterogeneity emerges from the deregulated transition between two antagonistic RNA-binding proteins, Imp and Syncrip, that switch neural progenitors from a default self-renewing to a differentiation-prone state during development. Consequently, aberrant maintenance of Imp confers a cancer stem cell-like identity as Imp+ progenitors sustain tumor growth while being able to continuously generate Syncrip+ progenitors. The latter exhibit limited self-renewal likely due to Syncrip-mediated metabolic exhaustion. This study provides an example of how a subverted developmental transition establishes a hierarchical tumor.

Par quels mécanismes un organisme vivant, système d’une complexité physique étourdissante, émerge-t-il à partir d’un état initial très peu organisé ? Cette question a de tout temps constitué un défi conceptuel majeur pour les sciences naturelles.Vers 1900, Stéphane Leduc conçoit des expériences physiques dans lesquelles des formes jusque-là supposées spécifiques du vivant émergent spontanément. Il souligne les « facultés d’organisation » des forces physiques ordinaires qui, selon lui, président à la formation d’un organisme. Largement oubliées au 20esiècle durant l’ère génétique, ses idées préfigurent pourtant le concept moderne d’auto-organisation, et prennent tout leur sens aujourd’hui, alors que l’utilisation de concepts physiques explose en biologie du développement.

International audience; Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to our understanding of morphogenesis. Here, we use myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from myosin II pulses, we find that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which we use to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by myosin II pulses. We test this estimate by applying time-controlled forces on junctions with optical tweezers. Finally, we show that actin turnover participates in dissipation, as reducing it pharmacologically increases the reversibility of contractile events. Our results argue that active junctional deformation is stabilized by actin-dependent dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.

International audience; Adhesion molecules hold cells together but also couple cell membranes to a contractile actomyosin network, which limits the expansion of cell contacts. Despite their fundamental role in tissue morphogenesis and tissue homeostasis, how adhesion molecules control cell shapes and cell patterns in tissues remains unclear. Here we address this question in vivo using the Drosophila eye. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II, which leads to a highly contractile cell interface. Such differential regulation of contractility is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements. Our results establish a quantitative link between adhesion and contractility and reveal an unprecedented role of N-cadherin on cell shapes and cell arrangements.

Understanding how molecular and physical cues orchestrate vascular morphogenesis is a challenge for developmental biology. Only little attention has been paid to the impact of mechanical stress caused by tissue growth on early blood distribution. Here we study the peripheral accumulation of blood in the chicken embryonic yolk sac, which precedes sinus vein formation.We report that blood accumulation starts before heart-induced blood circulation. We hypothesized that the driving force for the primitive blood flow is a growth-induced gradient of tissue pressure in the yolk sac mesoderm. Therefore, we studied embryos in which heart development was arrested after 2 days of incubation, and found that yolk sac growth and blood peripheral accumulation still occurred. This suggests that tissue growth is sufficient to initiate the flow and the formation of the sinus vein, whereas heart contractions are not required. We designed a simple mathematical model which makes explicit the growth-induced pressure gradient and the subsequent blood accumulation, and show that growth can indeed account for the observed blood accumulation.This study shows that tissue growth pressure can drive early blood flow, and suggests that the mechanical environment, beyond hemodynamics, can contribute to vascular morphogenesis. Developmental Dynamics 246:573-584, 2017. © 2017 Wiley Periodicals, Inc.

Adhesion molecules hold cells together but also couple cell membranes to a contractile actomyosin network, which limits the expansion of cell contacts. Despite their fundamental role in tissue morphogenesis and tissue homeostasis, how adhesion molecules control cell shapes and cell patterns in tissues remains unclear. Here we address this question in vivo using the Drosophila eye. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II, which leads to a highly contractile cell interface. Such differential regulation of contractility is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements. Our results establish a quantitative link between adhesion and contractility and reveal an unprecedented role of N-cadherin on cell shapes and cell arrangements. DOI: http://dx.doi.org/10.7554/eLife.22796.001

International audience; Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell-cell and cell-ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell-cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

International audience; Branched structures are ubiquitous in nature, both in living and non-living systems. While the functional benefits of branching organogenesis are straightforward, the developmental mechanisms leading to the repeated branching of epithelia in surrounding mesoderm remain unclear. Both molecular and physical aspects of growth control seem to play a critical role in shape emergence and maintenance. On the molecular side, the existence of a gradient of growth-promoting ligand between epithelial tips and distal mesenchyme seems to be common to branched organs. On the physical side, the branching process seems to require a mechanism of real-time adaptation to local geometry, as suggested by the self-avoiding nature of branching events. In this paper, we investigate the outcomes of a general three-dimensional growth model, in which epithelial growth is implemented as a function of ligand income, while the mesenchyme is considered as a proliferating viscous medium. Our results suggest that the existence of a gradient of growth-promoting ligand between distal and proximal mesenchyme implies a growth instability of the epithelial sheet, resulting in spontaneous self-avoiding branching morphogenesis. While the general nature of the model prevents one from fitting the development of specific organs, it suggests that few ingredients are actually required to achieve branching organogenesis.

International audience; The arborescent architecture of mammalian conductive airways results from the repeated branching of lung endoderm into surrounding mesoderm. Subsequent lung's striking geometrical features have long raised the question of developmental mechanisms involved in morphogenesis. Many molecular actors have been identified, and several studies demonstrated the central role of Fgf10 and Shh in growth and branching. However, the actual branching mechanism and the way branching events are organized at the organ scale to achieve a self-avoiding tree remain to be understood through a model compatible with evidenced signaling. In this paper we show that the mere diffusion of FGF10 from distal mesenchyme involves differential epithelial proliferation that spontaneously leads to branching. Modeling FGF10 diffusion from sub-mesothelial mesenchyme where Fgf10 is known to be expressed and computing epithelial and mesenchymal growth in a coupled manner, we found that the resulting laplacian dynamics precisely accounts for the patterning of FGF10-induced genes, and that it spontaneously involves differential proliferation leading to a self-avoiding and space-filling tree, through mechanisms that we detail. The tree's fine morphological features depend on the epithelial growth response to FGF10, underlain by the lung's complex regulatory network. Notably, our results suggest that no branching information has to be encoded and that no master routine is required to organize branching events at the organ scale. Despite its simplicity, this model identifies key mechanisms of lung development, from branching to organ-scale organization, and could prove relevant to the development of other branched organs relying on similar pathways.