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Epithelial cell death is highly prevalent during development and in adult tissues. It plays an essential role for the regulation of tissue size, shape and turnover. Cell elimination relies on the concerted remodeling of cell junctions, so called cell extrusion, which allow 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 relies so far on manual detection of cell extrusion event 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 theDrosophilapupal 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 marked cell junctions with reasonable retraining.

International audience; 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.

Additional file 1 Supplementary information for “LocalZProjector and DeProj: A toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images.”

Summary 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., Graphical abstract, Highlights • Simultaneous elimination of three neighboring cells is detrimental for epithelia • Biased cell-death distribution prevents the appearance of such clusters • This bias is driven by ERK pulses and caspase inhibition in the neighbors of dying cells • Clusters of elimination and transient sealing defects appear upon EGFR/ERK inhibition, How epithelia fine-tune the spatiotemporal distribution of cell death and cope with high rates of elimination remains unclear. Valon et al. shows that pulses of ERK induced near every dying cell prevent the simultaneous elimination of neighboring cells, hence maintaining epithelial sealing despite the high rates of cell elimination.

10 While the pathways regulating apoptosis and cell extrusion are rather well 11 described 1,2 , what regulates the precise spatio-temporal distribution of cell 12 elimination in tissues remains largely unknown. This is particularly relevant for 13 epithelia with high rates of cell elimination, a widespread situation during 14 embryogenesis 3-6 and epithelial homeostasis 7 , where concomitant death of 15 neighbours could impair the maintenance of epithelial sealing. However, the extent 16 to which epithelial tissues can cope with concomitant cell death, and whether any 17 mechanism regulates such occurrence have never been explored so far. Here, 18 using the Drosophila pupal notum (a single layer epithelium) and a new 19 optogenetic tool to trigger caspase activation and cell extrusion, we first show that 20 concomitant death of clusters of at least three cells is sufficient to transiently 21 impair epithelial sealing. Such clustered extrusion was almost never observed in 22 vivo, suggesting the existence of a mechanism preventing concomitant elimination 23 of neighbours. Statistical analysis and simulations of cell death distribution in the 24 notum highlighted a transient and local protective phase occurring near every 25 dying cell. This protection is driven by a transient activation of ERK in the direct 26 neighbours of extruding cells which reverts caspase activation and prevents 27 elimination of cells in clusters. Altogether, this study demonstrates that the 28 distribution of cell elimination in epithelia is an emerging property of transient and 29

International audience; 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.