A microfluidic mechano-chemostat for tissues and organisms reveals that confined growth is accompanied with increased macromolecular crowding.

Conventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellular assemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they often do not allow the control of compressive forces emerging when cells proliferate in a confined environment. Here, we present a generic microfluidic device to control both chemical and mechanical compressive forces. This device relies on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable closed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression.

DISSECT is a tool to segment and explore cell and tissue mechanics in highly deformed 3D epithelia.

Understanding morphogenesis strongly relies on the characterization of tissue topology and mechanical properties deduced from imaging data. The development of new imaging techniques offers the possibility to go beyond the analysis of mostly flat surfaces and image and analyze complex tissue organization in depth. An important bottleneck in this field is the need to analyze imaging datasets and extract quantifications not only of cell and tissue morphology but also of the cytoskeletal network's organization in an automatized way. Here, we describe a method, called DISSECT, for DisPerSE (Discrete Persistent Structure Extractor)-based Segmentation and Exploration of Cells and Tissues, that offers the opportunity to extract automatically, in strongly deformed epithelia, a precise characterization of the spatial organization of a given cytoskeletal network combined with morphological quantifications in highly remodeled three-dimensional (3D) epithelial tissues. We believe that this method, applied here to Drosophila tissues, will be of general interest in the expanding field of morphogenesis and tissue biomechanics.

Dissecting morphogenetic apoptosis through a genetic screen in Drosophila.

Apoptosis is an essential cellular process both in normal development and pathological contexts. Screens performed to date have focused on the cell autonomous aspect of the process, deciphering the apoptotic cascade leading to cell destruction through the activation of caspases. However, the nonautonomous aspect of the apoptotic pathway, including signals regulating the apoptotic pattern or those sent by the apoptotic cell to its surroundings, is still poorly understood. Here, we describe an unbiased RNAi-based genetic screen whose goal is to identify elements of the "morphogenetic apoptosis pathway" in an integrated model system, the Drosophila leg. We screened about 1,400 candidates, using adult joint morphology, morphogenetic fold formation, and apoptotic pattern as readouts for the identification of potential apoptosis-related genes. We identified 41 genes potentially involved in specific aspects of morphogenetic apoptosis: (1) regulation of the apoptotic process; (2) formation, extrusion, and elimination of apoptotic bodies; and (3) contribution to morphogenesis downstream of apoptosis.

Voicing the story behind the cover.

In this selection, we celebrate the art of science by highlighting some of the submitted cover images from the past year. In this collection, our authors share the stories behind their inspiration for how to portray their science to captivate a broader audience.

mBeRFP: a versatile fluorescent tool to enhance multichannel live imaging and its applications.

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins offers interesting new perspectives to bypass this limitation. Here, we provide a detailed characterization of mBeRFP for in vivo live imaging and its applications in Drosophila. Briefly, we show that a single illumination source is sufficient to stimulate mBeRFP and GFP simultaneously. We demonstrate that mBeRFP can be easily combined with classical green and red fluorescent proteins without any crosstalk. We also show that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications, such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano-biologists in their current research.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Functions of Arp2/3 Complex in the Dynamics of Epithelial Tissues.

Epithelia are sheets of cells that communicate and coordinate their behavior in order to ensure their barrier function. Among the plethora of proteins involved in epithelial dynamics, actin nucleators play an essential role. The branched actin nucleation complex Arp2/3 has numerous functions, such as the regulation of cell-cell adhesion, intracellular trafficking, the formation of protrusions, that have been well described at the level of individual cells. Here, we chose to focus on its role in epithelial tissue, which is rising attention in recent works. We discuss how the cellular activities of the Arp2/3 complex drive epithelial dynamics and/or tissue morphogenesis. In the first part, we examined how this complex influences cell-cell cooperation at local scale in processes such as cell-cell fusion or cell corpses engulfment. In the second part, we summarized recent papers dealing with the impact of the Arp2/3 complex at larger scale, focusing on different morphogenetic events, including cell intercalation, epithelial tissue closure and epithelial folding. Altogether, this review highlights the central role of Arp2/3 in a diversity of epithelial tissue reorganization.

Force-generating apoptotic cells orchestrate avian neural tube bending.

Apoptosis plays an important role in morphogenesis, and the notion that apoptotic cells can impact their surroundings came to light recently. However, how this applies to vertebrate morphogenesis remains unknown. Here, we use the formation of the neural tube to determine how apoptosis contributes to morphogenesis in vertebrates. Neural tube closure defects have been reported when apoptosis is impaired in vertebrates, although the cellular mechanisms involved are unknown. Using avian embryos, we found that apoptotic cells generate an apico-basal force before being extruded from the neuro-epithelium. This force, which relies on a contractile actomyosin cable that extends along the apico-basal axis of the cell, drives nuclear fragmentation and influences the neighboring tissue. Together with the morphological defects observed when apoptosis is prevented, these data strongly suggest that the neuroepithelium keeps track of the mechanical impact of apoptotic cells and that the apoptotic forces, cumulatively, contribute actively to neural tube bending.

Orchestration of Force Generation and Nuclear Collapse in Apoptotic Cells.

Apoptosis, or programmed cell death, is a form of cell suicide that is extremely important for ridding the body of cells that are no longer required, to protect the body against hazardous cells, such as cancerous ones, and to promote tissue morphogenesis during animal development. Upon reception of a death stimulus, the doomed cell activates biochemical pathways that eventually converge on the activation of dedicated enzymes, caspases. Numerous pieces of information on the biochemical control of the process have been gathered, from the successive events of caspase activation to the identification of their targets, such as lamins, which constitute the nuclear skeleton. Yet, evidence from multiple systems now shows that apoptosis is also a mechanical process, which may even ultimately impinge on the morphogenesis of the surrounding tissues. This mechanical role relies on dramatic actomyosin cytoskeleton remodelling, and on its coupling with the nucleus before nucleus fragmentation. Here, we provide an overview of apoptosis before describing how apoptotic forces could combine with selective caspase-dependent proteolysis to orchestrate nucleus destruction.

Getting started for migration: A focus on EMT cellular dynamics and mechanics in developmental models.

The conversion of epithelial cells into mesenchymal ones, through a process known as epithelial-mesenchymal transition (or EMT) is a reversible process involved in critical steps of animal development as early as gastrulation and throughout organogenesis. In pathological conditions such as aggressive cancers, EMT is often associated with increased drug resistance, motility and invasiveness. The characterisation of the upstream signals and main decision takers, such as the EMT-transcription factors, has led to the identification of a core molecular machinery controlling the specification towards EMT. However, the cellular execution steps of this fundamental shift are poorly described, especially in cancerous cells. Here we review our current knowledge regarding the stepwise nature of EMT in model organisms as diverse as sea urchin, Drosophila, zebrafish, mouse or chicken. We focus on the cellular dynamics and mechanics of the transitional stages by which epithelial cells progressively become mesenchymal and leave the epithelium. We gather the currently available pieces of the puzzle, including the overlooked property of EMT cells to produce mechanical forces along their apico-basal axis before detaching from their neighbours. We discuss the interplay between EMT and the surrounding tissue. Finally, we propose a conceptual framework of EMT cell dynamics from the very first hint of epithelial cell reorganisation to the successful exit from the epithelial sheet.

Arp2/3-dependent mechanical control of morphogenetic robustness in an inherently challenging environment.

Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, we show that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. We found that knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, our data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations.

Super-resolved live-cell imaging using random illumination microscopy.

Current super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.

Curling of epithelial monolayers reveals coupling between active bending and tissue tension.

Epithelial monolayers are two-dimensional cell sheets which compartmentalize the body and organs of multicellular organisms. Their morphogenesis during development or pathology results from patterned endogenous and exogenous forces and their interplay with tissue mechanical properties. In particular, bending of epithelia is thought to result from active torques generated by the polarization of myosin motors along their apicobasal axis. However, the contribution of these out-of-plane forces to morphogenesis remains challenging to evaluate because of the lack of direct mechanical measurement. Here we use epithelial curling to characterize the out-of-plane mechanics of epithelial monolayers. We find that curls of high curvature form spontaneously at the free edge of epithelial monolayers devoid of substrate in vivo and in vitro. Curling originates from an enrichment of myosin in the basal domain that generates an active spontaneous curvature. By measuring the force necessary to flatten curls, we can then estimate the active torques and the bending modulus of the tissue. Finally, we show that the extent of curling is controlled by the interplay between in-plane and out-of-plane stresses in the monolayer. Such mechanical coupling emphasizes a possible role for in-plane stresses in shaping epithelia during morphogenesis.

When the Community Silences Disruptive Elements: Multiscale Mechanical Coupling Buffers Morphogenetic Imprecisions.

How the high degree of shape reproducibility is conferred between individuals remains unclear. In this issue of Developmental Cell, Eritano et al. show that tissue-scale mechanical coupling corrects the intrinsic noise of gene expression pattern and tissue mechanics to ensure the high degree of precision of Drosophila cephalic furrow formation.

Mechanical impact of epithelial-mesenchymal transition on epithelial morphogenesis in Drosophila.

Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.

Mechanical Function of the Nucleus in Force Generation during Epithelial Morphogenesis.

Mechanical forces are critical regulators of cell shape changes and developmental morphogenetic processes. Forces generated along the epithelium apico-basal cell axis have recently emerged as essential for tissue remodeling in three dimensions. Yet the cellular machinery underlying those orthogonal forces remains poorly described. We found that during Drosophila leg folding cells eventually committed to die produce apico-basal forces through the formation of a dynamic actomyosin contractile tether connecting the apical surface to a basally relocalized nucleus. We show that the nucleus is anchored to basal adhesions by a basal F-actin network and constitutes an essential component of the force-producing machinery. Finally, we demonstrate force transmission to the apical surface and the basal nucleus by laser ablation. Thus, this work reveals that the nucleus, in addition to its role in genome protection, actively participates in mechanical force production and connects the contractile actomyosin cytoskeleton to basal adhesions.

Physical and functional cell-matrix uncoupling in a developing tissue under tension.

Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. Here, we have analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, we found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and is driven by myosin II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.This article has an associated 'The people behind the papers' interview.

A fluorescent toolkit for spatiotemporal tracking of apoptotic cells in living Drosophila tissues.

Far from being passive, apoptotic cells influence their environment. For example, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task that is currently challenging. In order to easily spot apoptotic cells in developing Drosophila tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP-, Cerulean- and Venus-derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and, notably, they can be combined with any kind of fluorophore.

Apoptotic forces in tissue morphogenesis.

It is now well established that apoptosis is induced in response to mechanical strain. Indeed, increasing compressive forces induces apoptosis in confined spheroids of tumour cells, whereas releasing stress reduces apoptosis in spheroids cultivated in free suspension (Cheng et al., 2009). Apoptosis can also be induced by applying a 100 to 250MPa pressure, as shown in different cultured cells (for review, see (Frey et al., 2008)). During epithelium development, the pressure caused by a fast-growing clone can trigger apoptosis at the vicinity of the clone, mediating mechanical cell competition (Levayer et al., 2016). While the effect of strain has long been known for its role in apoptosis induction, the reciprocal mechanism has only recently been highlighted. First demonstrated at the cellular level, the effect of an apoptotic cell on its direct neighbours has been analysed in different kinds of monolayer epithelium (Gu et al., 2011; Rosenblatt et al., 2001; Kuipers et al., 2014; Lubkov & Bar-Sagi, 2014). More recently, the concept of a broader impact of apoptotic cell behaviours on tissue mechanical strain has emerged from the characterisation of tissue remodelling during Drosophila development (Toyama et al., 2008; Monier et al., 2015). In the present review, we summarize our current knowledge on the mechanical impact of apoptosis during tissue remodelling.