Toward a predictive understanding of epithelial cell death.

Epithelial cell death is highly prevalent during development and tissue homeostasis. While we have a rather good understanding of the molecular regulators of programmed cell death, especially for apoptosis, we still fail to predict when, where, how many and which specific cells will die in a tissue. This likely relies on the much more complex picture of apoptosis regulation in a tissular and epithelial context, which entails cell autonomous but also non-cell autonomous factors, diverse feedback and multiple layers of regulation of the commitment to apoptosis. In this review, we illustrate this complexity of epithelial apoptosis regulation by describing these different layers of control, all demonstrating that local cell death probability is a complex emerging feature. We first focus on non-cell autonomous factors that can locally modulate the rate of cell death, including cell competition, mechanical input and geometry as well as systemic effects. We then describe the multiple feedback mechanisms generated by cell death itself. We also outline the multiple layers of regulation of epithelial cell death, including the coordination of extrusion and regulation occurring downstream of effector caspases. Eventually, we propose a roadmap to reach a more predictive understanding of cell death regulation in an epithelial context.

DeXtrusion: automatic recognition of epithelial cell extrusion through machine learning in vivo.

Accurately counting and localising cellular events from movies is an important bottleneck of high-content tissue/embryo live imaging. Here, we propose a new methodology based on deep learning that allows automatic detection of cellular events and their precise xyt localisation on live fluorescent imaging movies without segmentation. We focused on the detection of cell extrusion, the expulsion of dying cells from the epithelial layer, and devised DeXtrusion: a pipeline based on recurrent neural networks for automatic detection of cell extrusion/cell death events in large movies of epithelia marked with cell contour. The pipeline, initially trained on movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion predictions in a large range of imaging conditions, and can also detect other cellular events, such as cell division or cell differentiation. It also performs well on other epithelial tissues with reasonable re-training. Our methodology could easily be applied for other cellular events detected by live fluorescent microscopy and could help to democratise the use of deep learning for automatic event detections in developing tissues.

Emerging roles and mechanisms of ERK pathway mechanosensing.

The coupling between mechanical forces and modulation of cell signalling pathways is essential for tissue plasticity and their adaptation to changing environments. Whilst the number of physiological and pathological relevant roles of mechanotransduction has been rapidly expanding over the last decade, studies have been mostly focussing on a limited number of mechanosensitive pathways, which include for instance Hippo/YAP/TAZ pathway, Wnt/ß-catenin or the stretch-activated channel Piezo. However, the recent development and spreading of new live sensors has provided new insights into the contribution of ERK pathway in mechanosensing in various systems, which emerges now as a fast and modular mechanosensitive pathway. In this review, we will document key in vivo and in vitro examples that have established a clear link between cell deformation, mechanical stress and modulation of ERK signalling, comparing the relevant timescale and mechanical stress. We will then discuss different molecular mechanisms that have been proposed so far, focussing on the epistatic link between mechanics and ERK and discussing the relevant cellular parameters affecting ERK signalling. We will finish by discussing the physiological and the pathological consequences of the link between ERK and mechanics, outlining how this interplay is instrumental for self-organisation and long-range cell-cell coordination.

LocalZProjector and DeProj: a toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images.

BACKGROUND: Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement. RESULTS: Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools. CONCLUSIONS: We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.

Solid stress, competition for space and cancer: The opposing roles of mechanical cell competition in tumour initiation and growth.

The regulation of cell growth, cell proliferation and cell death is at the basis of the homeostasis of tissues. While they can be regulated by intrinsic and genetic factors, their response to external signals emanating from the local environment is also essential for tissue homeostasis. Tumour initiation and progression is based on the misregulation of growth, proliferation and death mostly through the accumulation of genetic mutations. Yet, there is an increasing body of evidences showing that tumour microenvironment also has a strong impact on cancer initiation and progression. This includes the mechanical constrains and the compressive forces generated by the resistance of the surrounding tissue/matrix to tumour expansion. Recently, mechanical stress has been proposed to promote competitive interactions between cells through a process called mechanical cell competition. Cell population with a high proliferative rate can compact and eliminate the neighbouring cells which are more sensitive to compaction. While this emerging concept has been recently validated in vivo, the relevance of this process during tumour progression has never been discussed extensively. In this review, I will first describe the phenomenology of mechanical cell competition focusing on the main parameters and the pathways regulating cell elimination. I will then discuss the relevance of mechanical cell competition in tumour initiation and expansion while emphasizing its potential opposing contributions to tumourogenesis.

Cell mixing induced by myc is required for competitive tissue invasion and destruction.

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. Here we use the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Here we show, using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. We further show that elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. Our results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues.

Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction.

Cells and tissues are exposed to multiple mechanical stresses during development, tissue homoeostasis and diseases. While we start to have an extensive understanding of the influence of mechanics on cell differentiation and proliferation, how excessive mechanical stresses can also lead to cell death and may be associated with pathologies has been much less explored so far. Recently, the development of new perturbative approaches allowing modulation of pressure and deformation of tissues has demonstrated that compaction (the reduction of tissue size or volume) can lead to cell elimination. Here, we discuss the relevant type of stress and the parameters that could be causal to cell death from single cell to multicellular systems. We then compare the pathways and mechanisms that have been proposed to influence cell survival upon compaction. We eventually describe the relevance of compaction-induced death in vivo, and its functions in morphogenesis, tissue size regulation, tissue homoeostasis and cancer progression.

Staying away from the breaking point: Probing the limits of epithelial cell elimination.

Epithelial tissues are dramatically remodelled during embryogenesis and tissue homeostasis and yet need to maintain their sealing properties to sustain their barrier functions at any time. Part of these remodellings involve the elimination of a large proportion of cells through apoptosis. Cell extrusion, the remodelling steps leading to seamless dying cell expulsion, helps to maintain tissue cohesion. However, there is an intrinsic limit in the system that can only accommodate a certain proportion/rate of cell elimination as well as certain spatiotemporal distributions. What are then the critical conditions leading to epithelial rupture/tear/sealing defects upon cell elimination and which mechanisms ensure that such limits are never reached? In this short review, I document the conditions in which epithelial rupture has been observed, including in the contexts of epithelial cell death, and the mechanical parameters influencing tissue rupture, and review feedback mechanisms which help to keep the epithelia away from the breaking point.

Patterned apoptosis has an instructive role for local growth and tissue shape regulation in a fast-growing epithelium.

What regulates organ size and shape remains one fundamental mystery of modern biology. Research in this area has primarily focused on deciphering the regulation in time and space of growth and cell division, while the contribution of cell death has been overall neglected. This includes studies of the Drosophila wing, one of the best-characterized systems for the study of growth and patterning, undergoing massive growth during larval stage and important morphogenetic remodeling during pupal stage. So far, it has been assumed that cell death was relatively neglectable in this tissue both during larval stage and pupal stage, and as a result, the pattern of growth was usually attributed to the distribution of cell division. Here, using systematic mapping and registration combined with quantitative assessment of clone size and disappearance as well as live imaging, we outline a persistent pattern of cell death and clone elimination emerging in the larval wing disc and persisting during pupal wing morphogenesis. Local variation of cell death is associated with local variation of clone size, pointing to an impact of cell death on local growth that is not fully compensated by proliferation. Using morphometric analyses of adult wing shape and genetic perturbations, we provide evidence that patterned death locally and globally affects adult wing shape and size. This study describes a roadmap for precise assessment of the contribution of cell death to tissue shape and outlines an important instructive role of cell death in modulating quantitatively local growth and morphogenesis of a fast-growing tissue.

Cell polarity and extrusion: How to polarize extrusion and extrude misspolarized cells?

The barrier function of epithelia is one of the cornerstones of the body plan organization of metazoans. It relies on the polarity of epithelial cells which organizes along the apico-basal axis the mechanical properties, signaling as well as transport. This barrier function is however constantly challenged by the fast turnover of epithelia occurring during morphogenesis or adult tissue homeostasis. Yet, the sealing property of the tissue can be maintained thanks to cell extrusion: a series of remodeling steps involving the dying cell and its neighbors leading to seamless cell expulsion. Alternatively, the tissue architecture can also be challenged by local damages or the emergence of mutant cells that may alter its organization. This includes mutants of the polarity complexes which can generate neoplastic overgrowths or be eliminated by cell competition when surrounded by wild type cells. In this review, we will provide an overview of the regulation of cell extrusion in various tissues focusing on the relationship between cell polarity, cell organization and the direction of cell expulsion. We will then describe how local perturbations of polarity can also trigger cell elimination either by apoptosis or by cell exclusion, focusing specifically on how polarity defects can be directly causal to cell elimination. Overall, we propose a general framework connecting the influence of polarity on cell extrusion and its contribution to aberrant cell elimination.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Collective effects in epithelial cell death and cell extrusion.

Programmed cell death, notably apoptosis, is an essential guardian of tissue homeostasis and an active contributor of organ shaping. While the regulation of apoptosis has been mostly analysed in the framework of a cell autonomous process, recent works highlighted important collective effects which can tune cell elimination. This is particularly relevant for epithelial cell death, which requires fine coordination with the neighbours in order to maintain tissue sealing during cell expulsion. In this review, we will focus on the recent advances which outline the complex multicellular communications at play during epithelial cell death and cell extrusion. We will first focus on the new unanticipated functions of neighbouring cells during extrusion, discuss the contribution of distant neighbours, and finally highlight the complex feedbacks generated by cell elimination on neighbouring cell death.

Microtubule disassembly by caspases is an important rate-limiting step of cell extrusion.

The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, we showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

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.

Cell competition: Bridging the scales through cell-based modeling.

Cell competition is a context-dependent, cell-elimination process that has been proposed to rely on several overlapping mechanisms. A new study combining cell-based modeling and quantitative microscopy data helps to evaluate the main contributors of mutant cell elimination.

Robustness of epithelial sealing is an emerging property of local ERK feedback driven by cell elimination.

What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.

Cell Extrusion: Crowd Pushing and Sticky Neighbours.

Cell extrusion is a highly coordinated process allowing the removal of an epithelial cell from the tissue layer without disrupting its integrity. Two new studies shed new light on the complexity of cell-cell coordination at play during cell extrusion.

Multiple Influences of Mechanical Forces on Cell Competition.

Cell competition is a widespread process leading to the expansion of one cell population through the elimination and replacement of another. A large number of genetic alterations can lead to either competitive elimination of the mutated population or expansion of the mutated cells through the elimination of the neighbouring cells. Several processes have been proposed to participate in the preferential elimination of one cell population, including competition for limiting extracellular pro-survival factors, communication through direct cell-cell contact, or differential sensitivity to mechanical stress. Recent quantitative studies of cell competition have also demonstrated the strong impact of the shape of the interfaces between the two populations. Here, we discuss the direct and indirect contribution of mechanical cues to cell competition, where they act either as modulators of competitive interactions or as direct drivers of cell elimination. We first discuss how mechanics can regulate contact-dependent and diffusion-based competition by modulating the shape of the interface between the two populations. We then describe the direct contribution of mechanical stress to cell elimination and competition for space. Finally, we discuss how mechanical feedback also influences compensatory growth and triggers preferential expansion of one population.

Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation.

The plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighboring healthy cells. However, we know very little about the pathways that can trigger apoptosis upon tissue deformation, and the contribution of compaction-driven death to clone expansion has never been assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, we show first that tissue compaction induces cell elimination through the downregulation of epidermal growth factor receptor/extracellular signal regulated kinase (EGFR/ERK) pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Second, we assessed in vivo the contribution of compaction-driven death to pretumoral cell expansion. We found that the activation of the oncogene Ras in clones can downregulate ERK and activate apoptosis in the neighboring cells through their compaction, which eventually contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumor progression.

Cell Competition: How to Take Over the Space Left by Your Neighbours.

Fast-growing cells can expand in a tissue by eliminating and replacing the neighbouring wild-type cells. A new study provides an elegant explanation for how cell elimination contributes to the preferential expansion of the invading population.