Biology subjects

Levayer, R.

Publications and source records attributed to Levayer, R..

10 recordsLinked to original sources

bioRxiv2026

EpiCure (Epithelial Curation): a versatile and handy tool for curation of epithelial segmentation

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

developmental biology↗

bioRxiv2025

Integration of past caspase activity biases cell elimination in vivo

The fine tuning of apoptosis in epithelia is essential for regulating tissue size, shape, homeostasis and the maintenance of sealing properties. Regulation of cell death is mostly orchestrated by the activation of Caspases, proteases which were long thought to trigger an irreversible engagement in cell death. However, recent data in vivo and in vitro outline numerous non-apoptotic functions of caspases as well as quite ubiquitous sublethal activation of effector caspases during development. Yet, it remains unclear in many instances what drives the bifurcation between cell death engagement and cell survival upon caspase activation. The existence of a caspase activity threshold was generally considered to underpin this binary decision, but this was never assessed quantitatively in vivo especially at the single cell level. Using quantitative live imaging combined with machine learning and optogenetics in the Drosophila pupal notum (a single layer epithelium), we reveal for the first time the existence of a large heterogeneity of caspase sensitivity between cells, as well as the existence of distinct spatial domains with low or high sensitivity to caspases. Using correlative and perturbative experiments, we outline the central role of past exposure to sublethal caspase activity which sensitises cells for apoptosis for several hours. Integrating information about past caspase activation is sufficient to explain most of the global pattern of caspase sensitivity and predict at the single cell level which cells will engage in apoptosis. Finally, we demonstrate that past sublethal caspase activation in a subset of cells is sufficient to bias cell elimination at the clonal and single cell level, thus revealing an alternative mechanism of physiological cell competition. Altogether, this work reveals for the first time the existence of a new layer of apoptosis regulation in vivo downstream of effector caspases which can be developmentally regulated and bias clonal selection and the spatial pattern of cell death.

developmental biology↗

bioRxiv2025

Interfacial tension and growth both contribute to mechanical cell competition

Tissue plasticity and homeostasis rely on the mutual interplay between cell behaviour and mechanical inputs1. Yet, mechanical stress can also contribute to the evolution of some pathologies, notably by accelerating pretumoural cell expansion through the process of mechanical cell competition2-5. Mechanical cell competition is a conserved process in which one cell population is preferentially eliminated when mixed with another cell population due to its higher sensitivity to mechanical stress2,3,5-8. Most of the recent theoretical and experimental explorations of mechanical cell competition focused so far on the contribution of growth and pressure to cell elimination and were limited to few genetic contexts, including the activation of Ras in vivo2,8, and the mutation of the polarity gene scribble in mammalian cell culture3,4,9. However, it remains unclear whether other oncogenes can trigger similar mechanisms and whether growth is generally the only central regulator of cell compaction and cell elimination. Using the Drosophila pupal notum (a single layer epithelium), quantitative live imaging and vertex modelling, we revisited the mechanisms contributing to cell compaction and cell elimination during mechanical cell competition. Doing so, we outlined the co-existence of two modes of wild type (WT) cell compaction near oncogenic cells, namely the compaction driven by growth versus local compaction driven by increased tension at tumoural/WT cell interfaces in zones of high curvature (similar to "Laplace pressure"). We highlighted distinctive signatures in cell deformation and cell elimination distribution that can delineate these two modes of compaction, and we recapitulated them in silico and in vivo using genetic backgrounds affecting growth and/or interfacial tension independently. Altogether, this study reveals for the first time the contribution of interfacial tension-driven compaction to mechanical cell competition and outlines the co-existence of various modes of compaction during cell elimination and pretumoural clone expansion.

cell biology↗

bioRxiv2024

Developmental delay ensures global tissue size robustness upon local induction of apoptosis

The capacity of our tissues to cope with external and internal stress relies on the tight coupling between cell proliferation, cell growth and cell death. This coupling is assumed to be based on compensatory proliferation, where local mitogenic signals and mechanical inputs generated by dying cells promote neighbouring cell proliferation. However, compensatory proliferation was mostly studied in the context of massive death induction, irradiation, surgical tissue ablation or upon genetic perturbation of apoptosis execution. It remains thus unclear whether the same mechanism operates during physiological programmed cell death or upon mild induction of apoptosis, especially in vivo. Here, we use the Drosophila prospective wing (the larval wing disc), to study the impact of local induction of apoptosis on tissue size and proliferation pattern. We first confirmed that the wing could recover its final size and compensate for mild induction of apoptosis. However, using spatial statistics we found surprisingly that local induction of death is not associated with any local increase of proliferation, could it be upon clonal or compartment induction of apoptosis. Compensation is driven instead by a JNK dependant delay of growth and lengthening of the larval stage which is required to reach the final tissue target size. These results suggest that compensation is here driven by a global response rather that a local proliferation induction. Accordingly, while total tissue size is maintained despite local induction of apoptosis, this mechanism fails to correct the local reduction of cell number, hence modulating wing shape and proportion. Overall, this study opens novel perspectives on tissue size regulation and outlines the context-dependency of compensatory mechanisms.

developmental biology↗

bioRxiv2023

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

Epithelial cell death is highly prevalent during development and in adult tissues. It plays an essential role in the regulation of tissue size, shape, and turnover. Cell elimination relies on the concerted remodelling of cell junctions, so-called cell extrusion, which allows the seamless expulsion of dying cells. The dissection of the regulatory mechanism giving rise to a certain number and pattern of cell death was so far limited by our capacity to generate high-throughput quantitative data on cell death/extrusion number and distribution in various perturbed backgrounds. Indeed, quantitative studies of cell death rely so far on manual detection of cell extrusion events or through tedious systematic error-free segmentation and cell tracking. Recently, deep learning was used to automatically detect cell death and cell division in cell culture mostly using transmission light microscopy. However, so far, no method was developed for fluorescent images and confocal microscopy, which constitute most datasets in embryonic epithelia. Here, we devised DeXtrusion, a pipeline for automatic detection of cell extrusion/cell death events in larges movies of epithelia marked with cell contour and based on recurrent neural networks. The pipeline, initially trained on large movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion/cell death 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 markers of cell junctions with reasonable retraining.

developmental biology↗

bioRxiv2022

Patterned apoptosis modulates local growth and tissue shape in a fast-growing epithelium

What regulates organ size and shape remains one of the fundamental mysteries of modern biology. So far, 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 much more neglected. This includes studies of the Drosophila wing imaginal disc, the prospective fly wing which undergoes massive growth during larval stage, and represents one of the best characterised systems for the study of growth and patterning. So far, it has been assumed that cell death was relatively neglectable in this tissue 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, we show for the first time that cell death is not neglectable, and outline a persistent pattern of cell death and clone elimination in the disc. Local variation of cell death is associated with local variation of clone size, pointing to an impact of cell death on local growth which is not fully compensated by proliferation. Using morphometric analyses of adult wing shape and genetic perturbations, we provide evidence that patterned death affects locally and globally adult wing shape and size. This study describes a roadmap for accurate assessment of the contribution of cell death to tissue shape, and outlines for the first time an important instructive role of cell death in modulating quantitatively local growth and the morphogenesis of a fast-growing tissue.

developmental biology↗

bioRxiv2021

Microtubule disassembly by caspases is the rate-limiting step of cell extrusion

Epithelial cell death is essential for tissue homeostasis, robustness and morphogenesis. The expulsion of epithelial cells following caspase activation requires well-orchestrated remodeling steps leading to cell elimination without impairing tissue sealing. While numerous studies have provided insight about the process of cell extrusion, we still know very little about the relationship between caspase activation and the remodeling steps of cell extrusion. Moreover, most studies of cell extrusion focused on the regulation of actomyosin and steps leading to the formation of a supracellular contractile ring. However, the contribution of other cellular factors to cell extrusion has been poorly explored. Using the Drosophila pupal notum, a single layer epithelium where most extrusion events are caspase-dependent, we first showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration/dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. We confirmed that local and rapid increase/decrease of microtubules is sufficient to respectively expand/constrict cell apical area. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting steps of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

cell biology↗

bioRxiv2021

DProj: A toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images.

BO_SCPLOWACKGROUNDC_SCPLOWQuantitative imaging of epithelial tissues prompts for bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common pre-processing step consists in projecting the acquired 3D volume on a 2D plane mapping the tissue surface. Indeed, 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. RO_SCPLOWESULTSC_SCPLOWHere 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 user-friendly and 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. CO_SCPLOWONCLUSIONSC_SCPLOWWe find that LocalZProjector performs well even in situations where the volume to project also contains un-wanted 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.

bioinformatics↗

bioRxiv2020

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

While the pathways regulating apoptosis and cell extrusion are rather well described1,2, what regulates the precise spatio-temporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination, a widespread situation during embryogenesis3-6 and epithelial homeostasis7, where concomitant death of neighbours could impair the maintenance of epithelial sealing. However, the extent to which epithelial tissues can cope with concomitant cell death, and whether any mechanism regulates such occurrence have never been explored so far. Here, using the Drosophila pupal notum (a single layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first show that concomitant death of clusters of at least three cells is sufficient to transiently impair epithelial sealing. Such clustered extrusion was almost never observed in vivo, suggesting the existence of a mechanism preventing concomitant elimination of neighbours. Statistical analysis and simulations of cell death distribution in the notum highlighted a transient and local protective phase occurring near every dying cell. This protection is driven by a transient activation of ERK in the direct neighbours of extruding cells which reverts caspase activation and prevents elimination of cells in clusters. Altogether, this study demonstrates that the distribution of cell elimination in epithelia is an emerging property of transient and local feedbacks through ERK activation which is required to maintain epithelial sealing in conditions of high rate of cell elimination.

developmental biology↗

bioRxiv2018

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 neighbouring healthy cells. However we know very little about the pathways than can trigger apoptosis upon tissue deformation and the contribution of compaction driven death to clone expansion was never assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, we show that tissue compaction induces cell elimination through the downregulation of 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. Secondly, 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 also downregulate ERK and activate apoptosis in the neighbouring cells through their compaction, which contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumour progression.

developmental biology↗