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Many pathogens use M cells to access the underlying Peyer’s patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb), and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyer’s patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.

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.

Severe, often fatal, opportunistic fungal infections frequently arise following mucosal damage caused by trauma or cytotoxic chemotherapy. Interaction of fungal pathogens with epithelial cells that comprise mucosa is a key early event associated with invasion, and therefore, enhancing epithelial defense mechanisms may mitigate infection. Here we establish a model of mold and yeast infection mediated by inducible epithelial cell loss in larval zebrafish. Epithelial cell loss by extrusion promoted exposure of laminin that was associated with increased fungal attachment, invasion and larval lethality, while fungi defective in adherence or filamentation did not impact survival. Transcriptional profiling identified significant upregulation of the epidermal growth factor receptor ligand epigen (EPGN) upon mucosal damage. Treatment with recombinant human EPGN suppressed epithelial cell extrusion, leading to reduced fungal invasion and significantly enhanced survival. Together, these data support the concept of augmenting epithelial restorative capacity to attenuate pathogenic invasion of fungi associated with human disease.

International audience; The control of tissue growth, which is a key to maintain the protective barrier function of the epithelium, depends on the balance between cell division and cell extrusion rates [1, 2]. Cells within confluent epithelial layers undergo cell extrusion, which relies on cell-cell interactions [3] and actomyosin contractility [4, 5]. Although it has been reported that cell extrusion is also dependent on cell density [6, 7], the contribution of tissue mechanics, which is tightly regulated by cell density [8-12], to cell extrusion is still poorly understood. By measuring the multicellular dynamics and traction forces, we show that changes in epithelial packing density lead to the emergence of distinct modes of cell extrusion. In confluent epithelia with low cell density, cell extrusion is mainly driven by the lamellipodia-based crawling mechanism in the neighbor non-dying cells in connection with large-scale collective movements. As cell density increases, cell motion is shown to slow down, and the role of a supracellular actomyosin cable formation and its contraction in the neighboring cells becomes the preponderant mechanism to locally promote cell extrusion. We propose that these two distinct mechanisms complement each other to ensure proper cell extrusion depending on the cellular environment. Our study provides a quantitative and robust framework to explain how cell density can influence tissue mechanics and in turn regulate cell extrusion mechanisms.

Hematopoiesis leads to the formation of blood and immune cells. Hematopoietic stem cells emerge during development, from vascular components, via a process called the endothelial-to-hematopoietic transition (EHT). Here, we reveal essential biomechanical features of the EHT, using the zebrafish embryo imaged at unprecedented spatio-temporal resolution and an algorithm to unwrap the aorta into 2D-cartography. We show that the transition involves anisotropic contraction along the antero-posterior axis, with heterogenous organization of contractile circumferential actomyosin. The biomechanics of the contraction is oscillatory, with unusually long periods in comparison to other apical constriction mechanisms described so far in morphogenesis, and is supported by the anisotropic reinforcement of junctional contacts. Finally, we show that abrogation of blood flow impairs the actin cytoskeleton, the morphodynamics of EHT cells, and the orientation of the emergence. Overall, our results underline the peculiarities of the EHT biomechanics and the influence of the mechanical forces exerted by blood flow., eLife digest As humans, we have two major types of blood cell: our red blood cells transport oxygen around the body, while our white blood cells fight disease. Both types of cell come from the same stem cells, which first appear early in embryonic development. These stem cells emerge from the walls of major blood vessels, including the aorta – which carries blood from the heart. Stem cells have not yet decided which adult cell to become. Given the right signals, blood stem cells can form red blood cells or any of the different types of white blood cell. Understanding this process could allow scientists to recreate it in the laboratory, making blood stem cells that can give rise to all blood cells found in the body. But, this is not yet possible because we do not know all the conditions needed to make the cells and ensure they survive. One crucial gap in our understanding concerns the importance of blood flow. As the main blood vessel leaving the heart, the aorta experiences mechanical stress every time the heart beats. Lancino et al. wanted to find out whether this influences the development of the blood stem cells. Zebrafish embryos are transparent, making it easy to see their bodies developing under a microscope. Like humans, they also produce both red blood cells and white blood cells meaning Lancino et al. could watch the birth of blood stem cells in these embryos from a part of the aorta called the aortic floor. A new software tool unwrapped pictures of the tube-shaped blood vessel into flat, two-dimensional maps, making it possible to see how the aorta changed over time. This revealed that, as blood stem cells leave the aortic floor, they bend and contract with the direction of the blood flow. Rings of actin and myosin proteins that formed around the stem cells as they are born helped the process along, while stopping the heartbeat changed the way the blood cells emerged. Without any blood flow, the actin proteins did not assemble properly; the stem cells also emerged in the wrong direction and some of them even died. These findings show that physical forces play a role in the formation of blood stem cells. Understanding this process brings scientists a step closer to recreating the conditions for making different kinds of blood cells outside of the body.

The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer β-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.