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.

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.

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.

Apico-basal forces exerted by apoptotic cells drive epithelium folding.

Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.

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.

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.

Molecular and cellular mechanisms involved in leg joint morphogenesis.

In summary, the patterning of the presumptive leg depends on gradients of Dpp and Wg morphogens, which lead to the establishment of the proximo-distal axis marked by the expression of Hth, Dac and Dll in broad domains along the leg. Then, EGFR signaling specifies the tarsal region by regulating the expression of tarsal gap genes in different tarsal segments. This patterning is closely linked to the formation of rings of Notch activation in the distal part of each leg segment. These rings of Notch activation are further regulated by different mechanisms: (1) the maintenance of a sharp border of Dl expression, (2) the inhibition of N activation in cells located proximally to the ligands, thus restricting N activity specifically to the distal part of cells. This localised activation of Notch induces the expression of Dysfusion which controls the expression of both pro-apoptotic genes and RhoGTPase regulators. Finally, apoptotic cells appear within the pro-apoptotic domain, and while dying, generate a transient pulling force. This force constitutes a mechanical signal that propagates to the rest of the tissue and triggers cytoskeleton reorganisation specifically in the presumptive fold, where RhoGTPase regulators are expressed. Altogether, this complex array of patterning and signaling leads to precise cellular mapping of the developing leg to correctly position local cell shape modifications, inducing tissue folding.

The Hox gene Dfd controls organogenesis by shaping territorial border through regulation of basal DE-Cadherin distribution.

Hox genes are highly conserved selector genes controlling tissue identity and organogenesis. Recent work indicates that Hox genes also controls cell segregation and segmental boundary in various species, however the underlying cellular mechanisms involved in this function are poorly understood. In Drosophila melanogaster, the Hox gene Deformed (Dfd) is required for specification and organogenesis of the adult Maxillary (Mx) palp. Here, we demonstrate that differential Dfd expression control Mx morphogenesis through the formation of a physical boundary separating the Mx field and the Peripodial Epithelium (PE). We show that this boundary relies on DE-cadherin (DE-cad) basal accumulation in Mx cells controlled by differential Dfd expression. Indeed, Dfd controls boundary formation through cell autonomous basal redistribution of DE-cad which leads to subsequent fold at the Dfd expression border. Finally, the loss of Mx DE-cad basal accumulation and hence of Mx-PE folding is sufficient to prevent Mx organogenesis thus revealing the crucial role of boundaries in organ differentiation. Altogether, these results reveal that Hox coordination of tissue morphogenesis relies on boundary fold formation through the modulation of DE-cad positioning.

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments.

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.

DE-Cadherin regulates unconventional Myosin ID and Myosin IC in Drosophila left-right asymmetry establishment.

In bilateria, positioning and looping of visceral organs requires proper left-right (L/R) asymmetry establishment. Recent work in Drosophila has identified a novel situs inversus gene encoding the unconventional type ID myosin (MyoID). In myoID mutant flies, the L/R axis is inverted, causing reversed looping of organs, such as the gut, spermiduct and genitalia. We have previously shown that MyoID interacts physically with ß-Catenin, suggesting a role of the adherens junction in Drosophila L/R asymmetry. Here, we show that DE-Cadherin co-immunoprecipitates with MyoID and is required for MyoID L/R activity. We further demonstrate that MyoIC, a closely related unconventional type I myosin, can antagonize MyoID L/R activity by preventing its binding to adherens junction components, both in vitro and in vivo. Interestingly, DE-Cadherin inhibits MyoIC, providing a protective mechanism to MyoID function. Conditional genetic experiments indicate that DE-Cadherin, MyoIC and MyoID show temporal synchronicity for their function in L/R asymmetry. These data suggest that following MyoID recruitment by ß-Catenin at the adherens junction, DE-Cadherin has a twofold effect on Drosophila L/R asymmetry by promoting MyoID activity and repressing that of MyoIC. Interestingly, the product of the vertebrate situs inversus gene inversin also physically interacts with ß-Catenin, suggesting that the adherens junction might serve as a conserved platform for determinants to establish L/R asymmetry both in vertebrates and invertebrates.

Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis.

Morphogens are secreted signalling molecules that govern many developmental processes. In the Drosophila wing disc, the transforming growth factor beta (TGFbeta) homologue Decapentaplegic (Dpp) forms a smooth gradient and specifies cell fate by conferring a defined value of morphogen activity. Thus, neighbouring cells have similar amounts of Dpp protein, and if a sharp discontinuity in Dpp activity is generated between these cells, Jun kinase (JNK)-dependent apoptosis is triggered to restore graded positional information. To date, it has been assumed that this apoptotic process is only activated when normal signalling is distorted. However, we now show that a similar process occurs during normal development: rupture in Dpp activity occurs during normal segmentation of the distal legs of Drosophila. This sharp boundary of Dpp signalling, independently of the absolute level of Dpp activity, induces a JNK-reaper-dependent apoptosis required for the morphogenesis of a particular structure of the leg, the joint. Our results show that Dpp could induce a developmental programme not only in a concentration dependent manner, but also by the creation of a sharp boundary of Dpp activity. Furthermore, the same process could be used either to restore a normal pattern in response to artificial disturbance or to direct a morphogenetic process.

The Drosophila serine protease homologue Scarface regulates JNK signalling in a negative-feedback loop during epithelial morphogenesis.

In Drosophila melanogaster, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, we identified new JNK target genes during dorsal closure. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. Here, we show that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. We finally demonstrate that scaf acts as an antagonist by negatively regulating JNK activity. Overall, our results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis.

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.

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.

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.

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.

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.

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.

Strategies to establish left/right asymmetry in vertebrates and invertebrates.

Left/right (L/R) asymmetry is essential during embryonic development for organ positioning, looping and handed morphogenesis. A major goal in the field is to understand how embryos initially determine their left and right hand sides, a process known as symmetry breaking. A number of recent studies on several vertebrate and invertebrate model organisms have provided a more complex view on how L/R asymmetry is established, revealing an apparent partition between deuterostomes and protostomes. In deuterostomes, nodal cilia represent a conserved symmetry-breaking process; nevertheless, growing evidence shows the existence of pre-cilia L/R asymmetries involving active ion flows. In protostomes like snails and Drosophila, symmetry breaking relies on different mechanisms, involving, in particular, the actin cytoskeleton and associated molecular motors.