From heterogeneous morphogenetic fields to homogeneous regions as a step towards understanding complex tissue dynamics.

Within developing tissues, cell proliferation, cell motility and other cell behaviors vary spatially, and this variability gives a complexity to the morphogenesis. Recently, novel formalisms have been developed to quantify tissue deformation and underlying cellular processes. A major challenge for the study of morphogenesis now is to objectively define tissue sub-regions exhibiting different dynamics. Here, we propose a method to automatically divide a tissue into regions where the local deformation rate is homogeneous. This was achieved by several steps including image segmentation, clustering and region boundary smoothing. We illustrate the use of the pipeline using a large dataset obtained during the metamorphosis of the Drosophila pupal notum. We also adapt it to determine regions in which the time evolution of the local deformation rate is homogeneous. Finally, we generalize its use to find homogeneous regions for cellular processes such as cell division, cell rearrangement, or cell size and shape changes. We also illustrate it on wing blade morphogenesis. This pipeline will contribute substantially to the analysis of complex tissue shaping, and the biochemical and biomechanical regulations driving tissue morphogenesis.

Inferring cell junction tension and pressure from cell geometry.

Recognizing the crucial role of mechanical regulation and forces in tissue development and homeostasis has stirred a demand for in situ measurement of forces and stresses. Among emerging techniques, the use of cell geometry to infer cell junction tensions, cell pressures and tissue stress has gained popularity owing to the development of computational analyses. This approach is non-destructive and fast, and statistically validated based on comparisons with other techniques. However, its qualitative and quantitative limitations, in theory as well as in practice, should be examined with care. In this Primer, we summarize the underlying principles and assumptions behind stress inference, discuss its validity criteria and provide guidance to help beginners make the appropriate choice of its variants. We extend our discussion from two-dimensional stress inference to three dimensional, using the early mouse embryo as an example, and list a few possible extensions. We hope to make stress inference more accessible to the scientific community and trigger a broader interest in using this technique to study mechanics in development.

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis.

The orientation of cell division along the long axis of the interphase cell--the century-old Hertwig's rule--has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. Here we show that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and orienting cell division via the Dynein-associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJs emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues.

Modulation of junction tension by tumor suppressors and proto-oncogenes regulates cell-cell contacts.

Tumor suppressors and proto-oncogenes play crucial roles in tissue proliferation. Furthermore, de-regulation of their functions is deleterious to tissue architecture and can result in the sorting of somatic rounded clones minimizing their contact with surrounding wild-type (wt) cells. Defects in the shape of somatic clones correlate with defects in proliferation, cell affinity, cell-cell adhesion, oriented cell division and cortical contractility. Combining genetics, live-imaging, laser ablation and computer simulations, we aim to analyze whether distinct or similar mechanisms can account for the common role of tumor suppressors and proto-oncogenes in cell-cell contact regulation. In Drosophila epithelia, the tumor suppressors Fat (Ft) and Dachsous (Ds) regulate cell proliferation, tissue morphogenesis, planar cell polarity and junction tension. By analyzing the evolution over time of ft mutant cells and clones, we show that ft clones reduce their cell-cell contacts with the surrounding wt tissue in the absence of concomitant cell divisions and over-proliferation. This contact reduction depends on opposed changes of junction tensions in the clone bulk and its boundary with neighboring wt tissue. More generally, either clone bulk or boundary junction tension is modulated by the activation of Yorkie, Myc and Ras, yielding similar contact reductions with wt cells. Together, our data highlight mechanical roles for proto-oncogene and tumor suppressor pathways in cell-cell interactions.

Applying mechanical forces on Drosophila tissues in vivo using the StretchCo, a 3D-printable device.

Applying mechanical forces to tissues helps to understand morphogenesis and homeostasis. Additionally, recording the dynamics of living tissues under mechanical constraints is needed to explore tissue biomechanics. Here, we present a protocol to 3D-print a StretchCo device and use it to apply uniaxial mechanical stress on the Drosophila pupal dorsal thorax epithelium. We describe steps for 3D printing, polydimethylsiloxane (PDMS) strip cutting, and glue preparation. We detail procedures for PDMS strip mounting, tissue compaction, and live imaging upon force application. For additional details on the use and execution of this protocol, please refer to Cachoux et al. (2023)1 from which the StretchCo machine has been derived.

Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression.

The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA-negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaAneg daughter, which regains the size and behavior of its mother, and a neurogenic deltaApos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaAneg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations.

Epithelial apoptotic pattern emerges from global and local regulation by cell apical area.

Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, we know little about whether and how cell geometry controls apoptosis commitment in developing tissues. Here, we combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. We found that early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. We showed that these global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, we established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, our work provides important insights into the spatial regulation of cell death in tissues and improves our understanding of the mechanisms that control cell number and tissue size.

Apical stress fibers enable a scaling between cell mechanical response and area in epithelial tissue.

Biological systems tailor their properties and behavior to their size throughout development and in numerous aspects of physiology. However, such size scaling remains poorly understood as it applies to cell mechanics and mechanosensing. By examining how the Drosophila pupal dorsal thorax epithelium responds to morphogenetic forces, we found that the number of apical stress fibers (aSFs) anchored to adherens junctions scales with cell apical area to limit larger cell elongation under mechanical stress. aSFs cluster Hippo pathway components, thereby scaling Hippo signaling and proliferation with area. This scaling is promoted by tricellular junctions mediating an increase in aSF nucleation rate and lifetime in larger cells. Development, homeostasis, and repair entail epithelial cell size changes driven by mechanical forces; our work highlights how, in turn, mechanosensitivity scales with cell size.

Active cell migration is critical for steady-state epithelial turnover in the gut.

Steady-state turnover is a hallmark of epithelial tissues throughout adult life. Intestinal epithelial turnover is marked by continuous cell migration, which is assumed to be driven by mitotic pressure from the crypts. However, the balance of forces in renewal remains ill-defined. Combining biophysical modeling and quantitative three-dimensional tissue imaging with genetic and physical manipulations, we revealed the existence of an actin-related protein 2/3 complex-dependent active migratory force, which explains quantitatively the profiles of cell speed, density, and tissue tension along the villi. Cells migrate collectively with minimal rearrangements while displaying dual-apicobasal and front-back-polarity characterized by actin-rich basal protrusions oriented in the direction of migration. We propose that active migration is a critical component of gut epithelial turnover.

Biomechanics of cell rearrangements in Drosophila.

To acquire their adequate size and shape, living tissues grow and substantially deform as they develop. To do so, the cells making up the tissue can grow and deform as well, but they can also divide, intercalate and die. Among those cell behaviors, cell intercalation, also named cell rearrangement, is a major contributor to the morphogenesis of many cohesive tissues since it enables tissues to drastically deform as they develop while keeping their cohesiveness and avoiding extreme deformation of their cells. Here we review the mechanical principles and biological regulations at play during cell rearrangements in Drosophila tissues by first describing them in other cellular materials and by categorizing them. We then briefly discuss their quantifications and their interplay with other cell processes.

Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice.

Tissue repair is fundamental to our survival as tissues are challenged by recurrent damage. During mammalian skin repair, cells respond by migrating and proliferating to close the wound. However, the coordination of cellular repair behaviours and their effects on homeostatic functions in a live mammal remains unclear. Here we capture the spatiotemporal dynamics of individual epithelial behaviours by imaging wound re-epithelialization in live mice. Differentiated cells migrate while the rate of differentiation changes depending on local rate of migration and tissue architecture. Cells depart from a highly proliferative zone by directionally dividing towards the wound while collectively migrating. This regional coexistence of proliferation and migration leads to local expansion and elongation of the repairing epithelium. Finally, proliferation functions to pattern and restrict the recruitment of undamaged cells. This study elucidates the interplay of cellular repair behaviours and consequent changes in homeostatic behaviours that support tissue-scale organization of wound re-epithelialization.

Unified quantitative characterization of epithelial tissue development.

Understanding the mechanisms regulating development requires a quantitative characterization of cell divisions, rearrangements, cell size and shape changes, and apoptoses. We developed a multiscale formalism that relates the characterizations of each cell process to tissue growth and morphogenesis. Having validated the formalism on computer simulations, we quantified separately all morphogenetic events in the Drosophila dorsal thorax and wing pupal epithelia to obtain comprehensive statistical maps linking cell and tissue scale dynamics. While globally cell shape changes, rearrangements and divisions all significantly participate in tissue morphogenesis, locally, their relative participations display major variations in space and time. By blocking division we analyzed the impact of division on rearrangements, cell shape changes and tissue morphogenesis. Finally, by combining the formalism with mechanical stress measurement, we evidenced unexpected interplays between patterns of tissue elongation, cell division and stress. Our formalism provides a novel and rigorous approach to uncover mechanisms governing tissue development.

Mechanism of cytokinetic contractile ring constriction in fission yeast.

Cytokinesis involves constriction of a contractile actomyosin ring. The mechanisms generating ring tension and setting the constriction rate remain unknown because the organization of the ring is poorly characterized, its tension was rarely measured, and constriction is coupled to other processes. To isolate ring mechanisms, we studied fission yeast protoplasts, in which constriction occurs without the cell wall. Exploiting the absence of cell wall and actin cortex, we measured ring tension and imaged ring organization, which was dynamic and disordered. Computer simulations based on the amounts and biochemical properties of the key proteins showed that they spontaneously self-organize into a tension-generating bundle. Together with rapid component turnover, the self-organization mechanism continuously reassembles and remodels the constricting ring. Ring constriction depended on cell shape, revealing that the ring operates close to conditions of isometric tension. Thus, the fission yeast ring sets its own tension, but other processes set the constriction rate.

PTEN controls junction lengthening and stability during cell rearrangement in epithelial tissue.

Planar cell rearrangements control epithelial tissue morphogenesis and cellular pattern formation. They lead to the formation of new junctions whose length and stability determine the cellular pattern of tissues. Here, we show that during Drosophila wing development the loss of the tumor suppressor PTEN disrupts cell rearrangements by preventing the lengthening of newly formed junctions that become unstable and keep on rearranging. We demonstrate that the failure to lengthen and to stabilize is caused by the lack of a decrease of Myosin II and Rho-kinase concentration at the newly formed junctions. This defect results in a heterogeneous cortical contractility at cell junctions that disrupts regular hexagonal pattern formation. By identifying PTEN as a specific regulator of junction lengthening and stability, our results uncover how a homogenous distribution of cortical contractility along the cell cortex is restored during cell rearrangement to control the formation of epithelial cellular pattern.

Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.

During animal development, several planar cell polarity (PCP) pathways control tissue shape by coordinating collective cell behavior. Here, we characterize by means of multiscale imaging epithelium morphogenesis in the Drosophila dorsal thorax and show how the Fat/Dachsous/Four-jointed PCP pathway controls morphogenesis. We found that the proto-cadherin Dachsous is polarized within a domain of its tissue-wide expression gradient. Furthermore, Dachsous polarizes the myosin Dachs, which in turn promotes anisotropy of junction tension. By combining physical modeling with quantitative image analyses, we determined that this tension anisotropy defines the pattern of local tissue contraction that contributes to shaping the epithelium mainly via oriented cell rearrangements. Our results establish how tissue planar polarization coordinates the local changes of cell mechanical properties to control tissue morphogenesis.

Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia.

In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

Spontaneous creation of macroscopic flow and metachronal waves in an array of cilia.

Cells carrying cilia on their surface show many striking features: alignment of cilia in an array, two-phase asymmetric beating for each cilium, and existence of metachronal coordination with a constant phase difference between two adjacent cilia. We give simple theoretical arguments based on hydrodynamic coupling and an internal mechanism of the cilium derived from the behavior of a collection of molecular motors to account qualitatively for these cooperative features. Hydrodynamic interactions can lead to the alignment of an array of cilia. We study the effect of a transverse external flow and obtain a two-phase asymmetrical beating, faster along the flow and slower against the flow, proceeding around an average curved position. We show that an aligned array of cilia is able to spontaneously break the left-right symmetry and to create a global average flow. Metachronal coordination arises as a consequence of the internal mechanism of the cilia and their hydrodynamic couplings, with a wavelength comparable to that found in experiments. It allows the cilia to start beating at a lower adenosine-triphosphate threshold and at a higher frequency than for a single cilium. It also leads to a rather stationary flow, which might be its major advantage.