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.

Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila.

The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. Here, we found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, our data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.

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 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.

Systematic analysis of RhoGEF/GAP localizations uncovers regulators of mechanosensing and junction formation during epithelial cell division.

Cell proliferation is central to epithelial tissue development, repair, and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodeling to faithfully segregate the genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPase spatiotemporal regulation during epithelial cell division, we generated a transgenic fluorescently tagged library for the 48 Drosophila Rho guanine exchange factors (RhoGEFs) and GTPase-activating proteins (GAPs), and we systematically characterized their endogenous distributions by time-lapse microscopy. Therefore, we unveiled candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division. Building on these findings, we established that the conserved RhoGEF Cysts and RhoGEF4 play sequential and distinct roles to couple cytokinesis with de novo junction formation. During ring contraction, Cysts via Rho1 participates in the neighbor mechanosensing response, promoting daughter-daughter cell membrane juxtaposition in preparation to de novo junction formation. Subsequently and upon midbody formation, RhoGEF4 via Rac acts in the dividing cell to ensure the withdrawal of the neighboring cell membranes, thus controlling de novo junction length and cell-cell arrangements upon cytokinesis. Altogether, our findings delineate how the RhoGTPases Rho and Rac are locally and temporally activated during epithelial cytokinesis, highlighting the RhoGEF/GAP library as a key resource to understand the broad range of biological processes regulated by RhoGTPases.

Homeotic compartment curvature and tension control spatiotemporal folding dynamics.

Shape is a conspicuous and fundamental property of biological systems entailing the function of organs and tissues. While much emphasis has been put on how tissue tension and mechanical properties drive shape changes, whether and how a given tissue geometry influences subsequent morphogenesis remains poorly characterized. Here, we explored how curvature, a key descriptor of tissue geometry, impinges on the dynamics of epithelial tissue invagination. We found that the morphogenesis of the fold separating the adult Drosophila head and thorax segments is driven by the invagination of the Deformed (Dfd) homeotic compartment. Dfd controls invagination by modulating actomyosin organization and in-plane epithelial tension via the Tollo and Dystroglycan receptors. By experimentally introducing curvature heterogeneity within the homeotic compartment, we established that a curved tissue geometry converts the Dfd-dependent in-plane tension into an inward force driving folding. Accordingly, the interplay between in-plane tension and tissue curvature quantitatively explains the spatiotemporal folding dynamics. Collectively, our work highlights how genetic patterning and tissue geometry provide a simple design principle driving folding morphogenesis during development.

Multi-view confocal microscopy enables multiple organ and whole organism live-imaging.

Understanding how development is coordinated in multiple tissues and gives rise to fully functional organs or whole organisms necessitates microscopy tools. Over the last decade numerous advances have been made in live-imaging, enabling high resolution imaging of whole organisms at cellular resolution. Yet, these advances mainly rely on mounting the specimen in agarose or aqueous solutions, precluding imaging of organisms whose oxygen uptake depends on ventilation. Here, we implemented a multi-view multi-scale microscopy strategy based on confocal spinning disk microscopy, called Multi-View confocal microScopy (MuViScopy). MuViScopy enables live-imaging of multiple organs with cellular resolution using sample rotation and confocal imaging without the need of sample embedding. We illustrate the capacity of MuViScopy by live-imaging Drosophila melanogaster pupal development throughout metamorphosis, highlighting how internal organs are formed and multiple organ development is coordinated. We foresee that MuViScopy will open the path to better understand developmental processes at the whole organism scale in living systems that require gas exchange by ventilation.

Rapid and robust optogenetic control of gene expression in Drosophila.

Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. Here we describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. We show that it allows efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, we illustrate how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions.

Skin-resident immune cells actively coordinate their distribution with epidermal cells during homeostasis.

Organs consist of multiple cell types that ensure proper architecture and function. How different cell types coexist and interact to maintain their homeostasis in vivo remains elusive. The skin epidermis comprises mostly epithelial cells, but also harbours Langerhans cells (LCs) and dendritic epidermal T cells (DETCs). Whether and how distributions of LCs and DETCs are regulated during homeostasis is unclear. Here, by tracking individual cells in the skin of live adult mice over time, we show that LCs and DETCs actively maintain a non-random spatial distribution despite continuous turnover of neighbouring basal epithelial cells. Moreover, the density of epithelial cells regulates the composition of LCs and DETCs in the epidermis. Finally, LCs require the GTPase Rac1 to maintain their positional stability, density and tiling pattern reminiscent of neuronal self-avoidance. We propose that these cellular mechanisms provide the epidermis with an optimal response to environmental insults.

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.

Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands.

Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.

Mechanical induction and competence in epithelial morphogenesis.

Identifying the mechanisms that govern the precise sequence of tissue deformations and flows during development is a major topic in developmental biology. Recent studies have explored how the deformation or the flow of a tissue region can be induced by the activity of a neighboring region through mechanical coupling. Such a coupling process is akin to chemical induction, whereby differentiation in a region of competent cells is stimulated by a neighboring region through chemical induction: we therefore propose to name this phenomenon 'mechanical induction'. Focusing on examples of mechanically induced epithelial flow or planar deformation in vivo, this review aims at discussing the processes driving mechanical induction and the competence factors modulating the induced morphogenesis, in order to highlight the importance of integrating tissue and inter-tissue scales to understand morphogenesis.

Tricellular junctions.

Bosveld and Bellaïche discuss the composition and assembly of tricellular junctions, as well as their roles in cell packing, tissue mechanics and signalling.

Trpml controls actomyosin contractility and couples migration to phagocytosis in fly macrophages.

Phagocytes use their actomyosin cytoskeleton to migrate as well as to probe their environment by phagocytosis or macropinocytosis. Although migration and extracellular material uptake have been shown to be coupled in some immune cells, the mechanisms involved in such coupling are largely unknown. By combining time-lapse imaging with genetics, we here identify the lysosomal Ca2+ channel Trpml as an essential player in the coupling of cell locomotion and phagocytosis in hemocytes, the Drosophila macrophage-like immune cells. Trpml is needed for both hemocyte migration and phagocytic processing at distinct subcellular localizations: Trpml regulates hemocyte migration by controlling actomyosin contractility at the cell rear, whereas its role in phagocytic processing lies near the phagocytic cup in a myosin-independent fashion. We further highlight that Vamp7 also regulates phagocytic processing and locomotion but uses pathways distinct from those of Trpml. Our results suggest that multiple mechanisms may have emerged during evolution to couple phagocytic processing to cell migration and facilitate space exploration by immune cells.

Oriented cell divisions in epithelia: from force generation to force anisotropy by tension, shape and vertices.

Mitotic spindle orientation has been linked to asymmetric cell divisions, tissue morphogenesis and homeostasis. The canonical pathway to orient the mitotic spindle is composed of the cortical recruitment factor NuMA and the molecular motor dynein, which exerts pulling forces on astral microtubules to orient the spindle. Recent work has defined a novel role for NuMA as a direct contributor to force generation. In addition, the exploration of geometrical and physical cues combined with the study of classical polarity pathways has led to deeper insights into the upstream regulation of spindle orientation. Here, we focus on how cell shape, junctions and mechanical tension act to orient spindle pulling forces in epithelia, and discuss different roles for spindle orientation in epithelia.

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.

Mechanical Force-Driven Adherens Junction Remodeling and Epithelial Dynamics.

During epithelial tissue development, repair, and homeostasis, adherens junctions (AJs) ensure intercellular adhesion and tissue integrity while allowing for cell and tissue dynamics. Mechanical forces play critical roles in AJs' composition and dynamics. Recent findings highlight that beyond a well-established role in reinforcing cell-cell adhesion, AJ mechanosensitivity promotes junctional remodeling and polarization, thereby regulating critical processes such as cell intercalation, division, and collective migration. Here, we provide an integrated view of mechanosensing mechanisms that regulate cell-cell contact composition, geometry, and integrity under tension and highlight pivotal roles for mechanosensitive AJ remodeling in preserving epithelial integrity and sustaining tissue dynamics.

An in vitro method for studying subcellular rearrangements during cell polarization in Drosophila melanogaster hemocytes.

Thanks to the power of Drosophila genetics, this animal model has been a precious tool for scientists to uncover key processes associated to innate immunity. The fly immune system relies on a population of macrophage-like cells, also referred to as hemocytes, which are highly migratory and phagocytic, and can easily be followed in vivo. These cells have shown to play important roles in fly development, both at the embryonic and pupal stages. However, there is no robust assay for the study of hemocyte migration in vitro, which limits our understanding of the molecular mechanisms involved. Here, we contribute to fill this gap by showing that hemocytes adopt a polarized morphology upon ecdysone stimulation, allowing the study of the cytoskeleton rearrangements and organelle reorganization that take place during the first step of cell locomotion.

Actin Network Discussion during Mitotic Pseudo-Furrowing.

Two types of cortical actin networks act during mitotic pseudocleavage furrowing in the Drosophila syncytium, but how they interact has remained elusive. In this issue of Developmental Cell, Zhang et al. (2018) show how these networks shape each other and propose that furrowing is driven by actin polymerization-derived pushing forces.