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How tissues develop distinct structures remains poorly understood. We propose herein the Lego hypothesis of tissue morphogenesis, which states that during tissue morphogenesis, the topographical properties of cell surface adhesion molecules can be dynamically altered and polarised by regulating the spatiotemporal expression and localization of orientational cell adhesion (OCA) molecules cell-autonomously and non-cell-autonomously, thus modulating cells into unique Lego pieces for self-assembling into distinct cytoarchitectures. This concept can be exemplified by epithelial morphogenesis, in which cells are coalesced into a sheet by many types of adhesions. Among them, parallel OCAs (pOCAs) at the lateral cell membranes are essential for configuring cells in parallel. Major pOCAs include Na + /K + -ATPase-mediated adhesions, Crumbs-mediated adhesions, tight junctions, adherens junctions, and desmosomes. These pOCAs align in stereotypical orders along the apical-to-basal axis, and their absolute positioning is also regulated. Such spatial organization of pOCAs underlies proper epithelial morphogenesis. Thus, a key open question about tissue morphogenesis is how to regulate OCAs to make compatible adhesive cellular Lego pieces for tissue construction., (© 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

We propose a simple mathematical model to describe the mechanical relaxation of cells within a curved epithelial tissue layer represented by an arbitrary curve in two-dimensional space. This model generalises previous one-dimensional models of flat epithelia to investigate the influence of curvature for mechanical relaxation. We represent the mechanics of a cell body either by straight springs, or by curved springs that follow the curve's shape. To understand the collective dynamics of the cells, we devise an appropriate continuum limit in which the number of cells and the length of the substrate are constant but the number of springs tends to infinity. In this limit, cell density is governed by a diffusion equation in arc length coordinates, where diffusion may be linear or nonlinear depending on the choice of the spring restoring force law. Our results have important implications about modelling cells on curved geometries: (i) curved and straight springs can lead to different dynamics when there is a finite number of springs, but they both converge quadratically to the dynamics governed by the diffusion equation; (ii) in the continuum limit, the curvature of the tissue does not affect the mechanical relaxation of cells within the layer nor their tangential stress; (iii) a cell's normal stress depends on curvature due to surface tension induced by the tangential forces. Normal stress enables cells to sense substrate curvature at length scales much larger than their cell body, and could induce curvature dependences in experiments., Competing Interests: Declarations. Conflict of interest: Author Prof. Matthew J. Simpson is Editor-in-Chief of the Bulletin of Mathematical Biology. Ethics approval and consent to participate: Not applicable. Consent for publication: All authors gave final approval for publication and agreed to be held accountable for the work performed therein., (© 2025. The Author(s).)

Once multicellularity was thriving, a key development involved the emergence of epithelial layers that separated "inside" from "outside". Most epithelia then generate their own transepithelial electrical signals. So electrical forces were instrumental in the development of epithelial tissues, which themselves generate further electrical signals. Epithelia also developed extracellular basement membranes which act as spatially diverse scaffolds to organize multiple molecular interactions, dependent on electrical forces.Epithelia and basement membranes were constructed using electrical forces and their evolution had electrophysiological consequences., (© 2025. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Shape changes of epithelia during animal development, such as convergent extension, are achieved through the concerted mechanical activity of individual cells. While much is known about the corresponding large-scale tissue flow and its genetic drivers, fundamental questions regarding local control of contractile activity on the cellular scale and its embryo-scale coordination remain open. To address these questions, we develop a quantitative, model-based analysis framework to relate cell geometry to local tension in recently obtained time-lapse imaging data of gastrulating Drosophila embryos. This analysis systematically decomposes cell shape changes and T1 rearrangements into internally driven, active, and externally driven, passive, contributions. Our analysis provides evidence that germ band extension is driven by active T1 processes that self-organize through positive feedback acting on tensions. More generally, our findings suggest that epithelial convergent extension results from the controlled transformation of internal force balance geometry which combines the effects of bottom-up local self-organization with the top-down, embryo-scale regulation by gene expression., Competing Interests: FB, NC, ML, EW, BS No competing interests declared, (© 2024, Brauns, Claussen et al.)

Cell-based mechanical models, such as the Cell Vertex Model (CVM), have proven useful for studying the mechanical control of epithelial tissue dynamics. We recently developed a statistical method called image-based parameter inference for formulating CVM model functions and estimating their parameters from image data of epithelial tissues. In this study, we employed Bayesian statistics to improve the utility and flexibility of image-based parameter inference. Tests on synthetic data confirmed that both our non-hierarchical and hierarchical Bayesian models provide accurate estimates of model parameters. By applying this method to Drosophila wings, we demonstrated that the reliability of parameter estimation is closely linked to the mechanical anisotropies present in the tissue. Moreover, we revealed that the cortical elasticity term is dispensable for explaining force-shape correlations in vivo. We anticipate that the flexibility of the Bayesian statistical framework will facilitate the integration of various types of information, thereby contributing to the quantitative dissection of the mechanical control of tissue dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Regulation of salt and water balance occupies a dominant role in the physiology of many animals and often relies on the function of the renal system. In the mammalian kidney, epithelial ion and water transport requires high degree of coordination between the transcellular and paracellular pathways, the latter being defined by the intercellular tight junctions (TJs). TJs seal the paracellular pathway in a highly specialized manner, either by forming a barrier against the passage of solutes and/or water or by allowing the passage of ions and/or water through them. This functional TJ plasticity is now known to be provided by the members of the claudin family of tetraspan proteins. Unlike mammalian nephron, the renal structures of insects, the Malpighian tubules, lack TJs and instead have smooth septate junctions (sSJs) as paracellular barrier forming junctions. Many questions regarding the molecular and functional properties of sSJs remain open but research on model species have begun to inform our understanding. The goal of this commentary is to highlight key concepts and most recent findings that have emerged from the molecular and functional dissection of paracellular barriers in the mammalian and insect renal epithelia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No funding was received for this work., (Copyright © 2024. Published by Elsevier Inc.)

Despite the primary role of cell proliferation in tissue development and homeostatic maintenance, the interplay between cell density, cell mechanoresponse, and cell growth and division is not yet understood. In this article, we address this issue by reporting on an experimental investigation of cell proliferation on all time- and length-scales of the development of a model tissue, grown on collagen-coated glass or deformable substrates. Through extensive data analysis, we demonstrate the relation between mechanoresponse and probability for cell division, as a function of the local cell density. Motivated by these results, we construct a minimal model of cell division in tissue environment that can recover the data. By parameterizing the growth and the dividing phases of the cell cycle, and introducing such a proliferation model in dissipative particle dynamics simulations, we recover the mechanoresponsive, time-dependent density profiles in 2D tissues growing to macroscopic scales. The importance of separating the cell population into growing and dividing cells, each characterized by a particular time scale, is further emphasized by calculations of density profiles based on adapted Fisher-Kolmogorov equations. Together, these results show that the mechanoresponse on the level of a constitutive cell and its proliferation results in a matrix-sensitive active pressure. The latter evokes massive cooperative displacement of cells in the invading tissue and is a key factor for developing large-scale structures in the steady state., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cellular monolayers display various degrees of coordinated motion ranging from the small scale of just a few cells to large multi-cellular scales. This collective migration carries important physical cues for creating proper tissue morphology. Previous studies have demonstrated that the energetics of the epithelial monolayer show a linear variation with time in conjunction with an arrest in monolayer motion after confluency. However, little is known about how the energetics of monolayer development are affected by confined geometries. Here, we demonstrate that micropatterned epithelial monolayers display a non-linear change in energetic variables, which coincides with the large-scale coordination of migration. This non-linear scaling behavior was further seen to be associated with the biased alignment of cells and cell-cell adhesion. These findings provide a new understanding of how developing epithelia may be impacted by different conditions in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In vertebrates, many organs, such as the kidney and the mammary gland form ductal structures based on the folding of epithelial sheets. The development of these organs relies on coordinated sorting of different cell lineages in both time and space, through mechanisms that remain largely unclear. Tissues are composed of several cell types with distinct biomechanical properties, particularly at cell-cell and cell-substrate boundaries. One hypothesis is that adjacent epithelial layers work in a coordinated manner to shape the tissue. Using in vitro experiments on model epithelial cells, differential expression of atypical Protein Kinase C iota (aPKCi), a key junctional polarity protein, is shown to reinforce cell epithelialization and trigger sorting by tuning cell mechanical properties at the tissue level. In a broader perspective, it is shown that in a heterogeneous epithelial monolayer, in which cell sorting occurs, forces arising from epithelial cell growth under confinement by surrounding cells with different biomechanical properties are sufficient to promote collective cell extrusion and generate emerging 3D organization related to spheroids and buds. Overall, this research sheds light on the role of aPKCi and the biomechanical interplay between distinct epithelial cell lineages in shaping tissue organization, providing insights into the understanding of tissue and organ development., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The mechanics of epithelial tissues, which is governed by forces generated in various cell regions, is often investigated using two-dimensional models that account for the apically positioned actomyosin structures but neglect basolateral mechanics. We employ a more detailed three-dimensional model to study how lateral surface tensions affect the structure and rigidity of such tissues. We find that cells are apicobasally asymmetric, with one side appearing more ordered than the other depending on target cell apical perimeter. In contrast to the 2D model, which predicts a rigidity transition at large target perimeters, tissues in the 3D model remain solidlike across all parameter space.

Convergent extension of epithelial tissue is a key motif of animal morphogenesis. On a coarse scale, cell motion resembles laminar fluid flow; yet in contrast to a fluid, epithelial cells adhere to each other and maintain the tissue layer under actively generated internal tension. To resolve this apparent paradox, we formulate a model in which tissue flow in the tension-dominated regime occurs through adiabatic remodeling of force balance in the network of adherens junctions. We propose that the slow dynamics within the manifold of force-balanced configurations is driven by positive feedback on myosin-generated cytoskeletal tension. Shifting force balance within a tension network causes active cell rearrangements (T1 transitions) resulting in net tissue deformation oriented by initial tension anisotropy. Strikingly, we find that the total extent of tissue deformation depends on the initial cellular packing order. T1s degrade this order so that tissue flow is self-limiting. We explain these findings by showing that coordination of T1s depends on coherence in local tension configurations, quantified by a geometric order parameter in tension space. Our model reproduces the salient tissue- and cell-scale features of germ band elongation during Drosophila gastrulation, in particular the slowdown of tissue flow after approximately twofold elongation concomitant with a loss of order in tension configurations. This suggests local cell geometry contains morphogenetic information and yields experimentally testable predictions. Defining biologically controlled active tension dynamics on the manifold of force-balanced states may provide a general approach to the description of morphogenetic flow., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cell division is fundamental to all healthy tissue growth, as well as being rate-limiting in the tissue repair response to wounding and during cancer progression. However, the role that cell divisions play in tissue growth is a collective one, requiring the integration of many individual cell division events. It is particularly difficult to accurately detect and quantify multiple features of large numbers of cell divisions (including their spatio-temporal synchronicity and orientation) over extended periods of time. It would thus be advantageous to perform such analyses in an automated fashion, which can naturally be enabled using deep learning. Hence, we develop a pipeline of deep learning models that accurately identify dividing cells in time-lapse movies of epithelial tissues in vivo. Our pipeline also determines their axis of division orientation, as well as their shape changes before and after division. This strategy enables us to analyse the dynamic profile of cell divisions within the Drosophila pupal wing epithelium, both as it undergoes developmental morphogenesis and as it repairs following laser wounding. We show that the division axis is biased according to lines of tissue tension and that wounding triggers a synchronised (but not oriented) burst of cell divisions back from the leading edge., Competing Interests: JT, IC, PM, TL, HW No competing interests declared, (© 2023, Turley et al.)

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left-right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process.

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Local cells can actively create reverse bending (evagination) in invaginated epithelia, which plays a crucial role in the formation of elaborate organisms. However, the precise physical mechanism driving the evagination remains elusive. Here, we present a three-dimensional vertex model, incorporating the intrinsic cell polarity, to explore the complex morphogenesis induced by local mechanical modulations. We find that invaginated tissues can spontaneously generate local reverse bending due to the shift of the apicobasal polarity. Their exact shapes can be analytically determined by the local apicobasal differential tension and the internal stress. Our continuum theory exhibits three regions in a phase diagram controlled by these two parameters, showing curvature transitions from ordered to disordered states. Additionally, we delve into epithelial curvature transition induced by the nucleus repositioning, revealing its active contribution to the apicobasal force generation. The uncovered mechanical principles could potentially guide more studies on epithelial folding in diverse systems.

Epithelial tissues cover the surfaces and lumens of the internal organs of multicellular animals and crucially contribute to internal environment homeostasis by delineating distinct compartments within the body. This vital role is known as epithelial barrier function. Epithelial cells are arranged like cobblestones and intricately bind together to form an epithelial sheet that upholds this barrier function. Central to the restriction of solute and fluid diffusion through intercellular spaces are occluding junctions, tight junctions in vertebrates and septate junctions in invertebrates. As part of epithelial tissues, cells undergo constant renewal, with older cells being replaced by new ones. Simultaneously, the epithelial tissue undergoes relative rearrangement, elongating, and shifting directionally as a whole. The movement or shape changes within the epithelial sheet necessitate significant deformation and reconnection of occluding junctions. Recent advancements have shed light on the intricate mechanisms through which epithelial cells sustain their barrier function in dynamic environments. This review aims to introduce these noteworthy findings and discuss some of the questions that remain unanswered., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Abstract Background Diet-derived short-chain fatty acids (SCFAs) in the rumen have broad effects on the health and growth of ruminants. The microbe-G-protein-coupled receptor (GPR) and microbe-histone deacetylase (HDAC) axes might be the major pathway mediating these effects. Here, an integrated approach of transcriptome sequencing and 16S rRNA gene sequencing was applied to investigate the synergetic responses of rumen epithelium and rumen microbiota to the increased intake of dietary non-fiber carbohydrate (NFC) from 15 to 30% in the goat model. In addition to the analysis of the microbial composition and identification of the genes and signaling pathways related to the differentially expressed GPRs and HDACs, the combined data including the expression of HDACs and GPRs, the relative abundance of the bacteria, and the molar proportions of the individual SCFAs were used to identify the significant co-variation of the SCFAs, clades, and transcripts. Results The major bacterial clades promoted by the 30% NFC diet were related to lactate metabolism and cellulose degradation in the rumen. The predominant functions of the GPR and HDAC regulation network, under the 30% NFC diet, were related to the maintenance of epithelium integrity and the promotion of animal growth. In addition, the molar proportion of butyrate was inversely correlated with the expression of HDAC1, and the relative abundance of the bacteria belonging to Clostridum_IV was positively correlated with the expression of GPR1. Conclusions This study revealed that the effects of rumen microbiota-derived SCFA on epithelium growth and metabolism were mediated by the GPR and HDAC regulation network. An understanding of these mechanisms and their relationships to dietary components provides better insights into the modulation of ruminal fermentation and metabolism in the promotion of livestock production.

The rumen barriers, constituted by the microbial, physical and immune barrier, prevent the transmission of pathogens and toxins to the host tissue in the maintenance of host-microbe homeostasis. Ruminal short-chain fatty acids (SCFAs), which are the important signaling molecules derived from the rumen microbiota, regulate a variety of physiological functions of the rumen. So far, how the ruminal SCFAs regulate the function of rumen barriers is unclear. By the combined methods of transcriptome sequencing, 16S rRNA gene sequencing, and metagenome shotgun sequencing, we have investigated the regulatory effects of ruminal SCFAs on the functions of rumen barriers, by determining the composition and functions of epimural microbiota and on the structure and immunity of the rumen epithelium in goats receiving a 10% (LC group), 35% (MC group), or 65% concentrate diet (HC group). We found that, when the dietary concentrate shifted from 10 to 35%, the increase of total SCFA is associated with the diversification of epimural microbiota and the diversity of its gene pool. Within the microbial community, the relative abundance of genera Sphingobium, Acinetobacter, and Streptococcus increase mostly. Meantime, the signals on pathways concerning the mechanical connections and growth homeostasis in the rumen epithelium were upregulated. Under these conditions, the responses of immune components in the rumen epithelium decrease. However, when the dietary concentrate shifted from 35 to 65%, the increase of acetate and reduction of pH decrease the diversity of epimural microbiota and the diversity of its gene pool. Within the microbial community, the relative abundance of genera Sphingobium, Acinetobacter, and Streptococcus significantly decrease. Concomitantly, the signals on pathways concerning the cell growth and tight junction disruption were upregulated, while the signals on pathways concerning paracellular permeability were downregulated. Under these conditions, the signals on the pathways relating to the immune components increase. Our data thus indicates that diet-SCFA axis maintains the host-microbe homeostasis via promoting the diversification of epimural microbiota and maintaining the integrity of rumen epithelium in healthy animals, while via enhancing the activities of immune barrier in animal with lower rumen pH.

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications.

The formation of fluid- or gas-filled lumina surrounded by epithelial cells pervades development and disease. We review the balance between lumen pressure and mechanical forces from the surrounding cells that governs lumen formation. We illustrate the mechanical side of this balance in several examples of increasing complexity, and discuss how recent work is beginning to elucidate how nonlinear and active mechanics and anisotropic biomechanical structures must conspire to overcome the isotropy of pressure to form complex, non-spherical lumina., (© 2024 The Author(s).)

Thyroid epithelial cells organize as enclosed follicles containing thyroid hormone precursor, iodinated thyroglobulin, with lumina bordered by the cellular apices. Transepithelial transport determines composition of compartmental milieu essential for both prohormone formation and its downstream conversion to thyroxine. Hence, not only do follicular lumina function as storage vessels but also as physiological reaction chambers into which reactive components, together with the proper salts and water, are secreted. Polarized, two-dimensional cultures of pig thyroid epithelia, prepared using established protocols, provide a convenient system for assessing transport processes subserving hormone production. This chapter details established methods for growing and evaluating integrity of primary pig thyroid cultures for downstream analysis of transport and other key physiological functions., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In animals, epithelial tissues are barriers against the external environment, providing protection against biological, chemical, and physical damage. Depending on the organism's physiology and behavior, these tissues encounter different types of mechanical forces and need to provide a suitable adaptive response to ensure success. Therefore, understanding tissue mechanics in different contexts is an important research area. Here, we review recent tissue mechanics discoveries in three early divergent non-bilaterian systems-Trichoplax adhaerens, Hydra vulgaris, and Aurelia aurita. We highlight each animal's simple body plan and biology and unique, rapid tissue remodeling phenomena that play a crucial role in its physiology. We also discuss the emergent large-scale mechanics in these systems that arise from small-scale phenomena. Finally, we emphasize the potential of these non-bilaterian animals to be model systems in a bottom-up approach for further investigation in tissue mechanics., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, we know little about whether and how cell geometry controls apoptosis commitment in developing tissues. Here, we combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. We found that early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. We showed that these global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, we established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, our work provides important insights into the spatial regulation of cell death in tissues and improves our understanding of the mechanisms that control cell number and tissue size., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In order to understand morphogenesis, it is necessary to know the material properties or forces shaping the living tissue. In spite of this need, very few in vivo measurements are currently available. Here, using the early Drosophila embryo as a model, we describe a novel cantilever-based technique which allows for the simultaneous quantification of applied force and tissue displacement in a living embryo. By analyzing data from a series of experiments in which embryonic epithelium is subjected to developmentally relevant perturbations, we conclude that the response to applied force is adiabatic and is dominated by elastic forces and geometric constraints, or system size effects. Crucially, computational modeling of the experimental data indicated that the apical surface of the epithelium must be softer than the basal surface, a result which we confirmed experimentally. Further, we used the combination of experimental data and comprehensive computational model to estimate the elastic modulus of the apical surface and set a lower bound on the elastic modulus of the basal surface. More generally, our investigations revealed important general features that we believe should be more widely addressed when quantitatively modeling tissue mechanics in any system. Specifically, different compartments of the same cell can have very different mechanical properties; when they do, they can contribute differently to different mechanical stimuli and cannot be merely averaged together. Additionally, tissue geometry can play a substantial role in mechanical response, and cannot be neglected., Competing Interests: MC, JT, MO, KD, SB, CZ, KM, KD No competing interests declared

The vertex model is widely used to simulate the mechanical properties of confluent epithelia and other multicellular tissues. This inherently discrete framework allows a Cauchy stress to be attributed to each cell, and its symmetric component has been widely reported, at least for planar monolayers. Here, we consider the stress attributed to the neighbourhood of each tricellular junction, evaluating in particular its leading-order antisymmetric component and the associated couple stresses, which characterise the degree to which individual cells experience (and resist) in-plane bending deformations. We develop discrete potential theory for localised monolayers having disordered internal structure and use this to derive the analogues of Airy and Mindlin stress functions. These scalar potentials typically have broad-banded spectra, highlighting the contributions of small-scale defects and boundary layers to global stress patterns. An affine approximation attributes couple stresses to pressure differences between cells sharing a trijunction, but simulations indicate an additional role for non-affine deformations., (© 2022. The Author(s).)

Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis. Recently we observed the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 spliced (XBP1s) axis, enhancing glucose shunting to protein N glycosylation. To better understand the genomic targets of XBP1s, we identified its genomic targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1s in RSV infection. CUT&RUN identified 7086 binding sites in chromatin that were enriched in AP-1 motifs and GC-sequences. Of these binding sites, XBP1s peaks mapped to 4827 genes controlling Rho-GTPase signaling, N-linked glycosylation and ER-Golgi transport. Strikingly, XBP1s peaks were within 1 kb of transcription start sites of 2119 promoters. In addition to binding core mesenchymal transcription factors SNAI1 and ZEB1, we observed that hexosamine biosynthetic pathway (HBP) enzymes were induced and contained proximal XBP1s peaks. We demonstrate that IRE1α -XBP1s signaling is necessary and sufficient to activate core enzymes by recruiting elongation-competent phospho-Ser2 CTD modified RNA Pol II. We conclude that the IRE1α-XBP1s pathway coordinately regulates mesenchymal transcription factors and hexosamine biosynthesis in EMP by a mechanism involving recruitment of activated pSer2-Pol II to GC-rich promoters., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding our understanding of how mechanical stresses remodel cell-cell contacts. Here, we develop a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows us to produce junction extension and shrinkage through different push and pull manipulations at the edges of junctions. We use these observations to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, we show that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether our work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling.

Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right noses, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, i.e., cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping., Competing Interests: CR, SB, ME, AS, JH, IJ, BF, NJ No competing interests declared, (© 2023, Ringers et al.)

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.

Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1 Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context., (© 2022. The Author(s).)

Professor Hans H. Ussing (1911-2000) was one of the founding members of the field of epithelial cell biology. He is most famous for the electrophysiological technique that he developed to measure electrogenic ion flux across epithelial tissues. Ussing-style electrophysiology has been applied to multiple tissues and has informed fields as diverse as amphibian biology and medicine. In the latter, this technique has contributed to a basic understanding of maladies such as hypertension, polycystic kidney disease, cystic fibrosis, and diarrheal diseases to mention but a few. In addition to this valuable contribution to biological methods, Prof. Ussing also provided strong evidence for the concept of active transport several years before the elucidation of Na + K + ATPase. In addition, he provided cell biologists with the important concept of polarized epithelia with specific and different transporters found in the apical and basolateral membranes, thus providing these cells with the ability to conduct directional, active and passive transepithelial transport. My studies have used Ussing chamber electrophysiology to study the toad urinary bladder, an amphibian cell line, renal cell lines, and, most recently, choroid plexus cell lines. This technique has formed the basis of our in vitro mechanistic studies that are used in an iterative manner with animal models to better understand disease progress and treatment. I was honored to be invited to deliver the 2022 Hans Ussing Lecture sponsored by the Epithelial Transport Group of the American Physiological Society. This manuscript is a version of the material presented in that lecture.

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5-10%), re-differentiated monolayers with 11-27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Taste stem/progenitor cells from posterior mouse tongues have been used to generate taste bud organoids. However, the inaccessible location of taste receptor cells is observed in conventional organoids. In this study, we established a suspension-culture method to fine-tune taste bud organoids by apicobasal polarity alteration to form the accessible localization of taste receptor cells. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells enabled the direct application of calcium imaging to evaluate the taste response. Moreover, suspension-cultured organoids can be genetically altered. Suspension-cultured taste bud organoids harmoniously integrated with the recipient lingual epithelium, maintaining the taste receptor cells and gustatory innervation capacity. We propose that suspension-cultured organoids may provide an efficient model for taste research, including taste bud development, regeneration, and transplantation., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Keywords: epimural microbiota; rumen barrier; short-chain fatty acids; epithelium physiology; microbe-host interactions EN epimural microbiota rumen barrier short-chain fatty acids epithelium physiology microbe-host interactions 1 1 1 09/29/21 20210923 NES 210923 There is an error in the Funding statement. Rumen barrier, epimural microbiota, short-chain fatty acids, epithelium physiology, microbe-host interactions. [Extracted from the article]

Interactions of developing T cells with Aire + medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEC hi ) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire + mTEC hi . However, it remains unknown whether thymocytes control the precursors of Aire + mTEC hi that are contained in mTEC lo cells or other mTEC lo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4 + thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4 + thymocytes induce key transcriptional regulators in mTEC lo and control the composition of mTEC lo subsets, including Aire + mTEC hi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEC lo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4 + thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4 + thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity., Competing Interests: NL, NB, JS, NP, JC, PF, MG, MI No competing interests declared, (© 2022, Lopes et al.)

Introduction: The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes., Material and Methods: We included healthy pre- (n = 17) and postmenopausal (n = 8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n = 4], late follicular [n = 3], early luteal [n = 5], late luteal [n = 5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-β) and canonical wingless-type MMTV integration site (Wnt) pathways., Results: The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p = 0.016 and p = 0.032, respectively) and low PI3K activity compared with the late follicular phase (p = 0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p = 0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-β pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube., Conclusions: We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle., (© 2021 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

The orthotopic transplantation assay has provided important insights into mammary development, stem cell function, and tumorigenesis. Technically, it consists in grafting mammary tissue fragments, organoids, mammospheres, or isolated cells into the fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed, thereby allowing growth and differentiation of mammary epithelial cells in their physiological environment. Here, we describe how is conducted transplantation of epithelial fragments and cells isolated from mouse mammary glands, report the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells, and highlight the strengths and limitations of this in vivo grafting assay., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Epithelial tissues form folded structures during embryonic development and organogenesis. Whereas substantial efforts have been devoted to identifying mechanical and biochemical mechanisms that induce folding, whether and how their interplay synergistically shapes epithelial folds remains poorly understood. Here we propose a mechano-biochemical model for dorsal fold formation in the early Drosophila embryo, an epithelial folding event induced by shifts of cell polarity. Based on experimentally observed apical domain homeostasis, we couple cell mechanics to polarity and find that mechanical changes following the initial polarity shifts alter cell geometry, which in turn influences the reaction-diffusion of polarity proteins, thus forming a feedback loop between cell mechanics and polarity. This model can induce spontaneous fold formation in silico, recapitulate polarity and shape changes observed in vivo, and confer robustness to tissue shape change against small fluctuations in mechanics and polarity. These findings reveal emergent properties of a developing epithelium under control of intracellular mechano-polarity coupling., Competing Interests: The authors have declared that no competing interests exist.

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by diverse genetic variants for the CF transmembrane conductance regulator (CFTR) protein. Among these, p.Phe508del is the most prevalent variant. The effects of this variant on the physiology of each tissue remains unknown. This study is aimed at predicting cell signaling pathways present in different tissues of fibrocystic patients, homozygous for p.Phe508del. The study involved analysis of two microarray datasets, E-GEOD-15568 and E-MTAB-360 corresponding to the rectal and bronchial epithelium, respectively, obtained from the ArrayExpress repository. Particularly, differentially expressed genes (DEGs) were predicted, protein-protein interaction (PPI) networks were designed, and centrality and functional interaction networks were analyzed. The study reported that p.Phe508del-mutated CFTR-allele in homozygous state influenced the whole gene expression in each tissue differently. Interestingly, gene ontology (GO) term enrichment analysis revealed that only "neutrophil activation" was shared between both tissues; however, nonshared DEGs were grouped into the same GO term. For further verification, functional interaction networks were generated, wherein no shared nodes were reported between these tissues. These results suggested that the p.Phe508del-mutated CFTR-allele in homozygous state promoted tissue-specific pathways in fibrocystic patients. The generated data might further assist in prediction diagnosis to define biomarkers or devising therapeutic strategies., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Joice de Faria Poloni et al.)

Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1β (IRE1β), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1β arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues., (© 2021. The Author(s).)

The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel formation, using immunohistochemical techniques. Decalcified mandibular sections (10 µm) including incisors were used from Wistar rats ages 10-12 weeks. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and the ion transporters (Na + /H + exchanger (NHE1) and Na + /Ca 2+ exchanger (NCX)) for 24 h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were examined by fluorescence microscopy. Haematoxylin-eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within enamel organ cells and connective tissue covering suggesting complex cellular interactions and indicating a new functional concept and possible complementary role during enamel maturation. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis is suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation which functions to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca 2+ towards enamel., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The collective polarization of cellular structures and behaviors across a tissue plane is a near universal feature of epithelia known as planar cell polarity (PCP). This property is controlled by the core PCP pathway, which consists of highly conserved membrane-associated protein complexes that localize asymmetrically at cell junctions. Here, we introduce three new mouse models for investigating the localization and dynamics of transmembrane PCP proteins: Celsr1, Fz6 and Vangl2. Using the skin epidermis as a model, we characterize and verify the expression, localization and function of endogenously tagged Celsr1-3xGFP, Fz6-3xGFP and tdTomato-Vangl2 fusion proteins. Live imaging of Fz6-3xGFP in basal epidermal progenitors reveals that the polarity of the tissue is not fixed through time. Rather, asymmetry dynamically shifts during cell rearrangements and divisions, while global, average polarity of the tissue is preserved. We show using super-resolution STED imaging that Fz6-3xGFP and tdTomato-Vangl2 can be resolved, enabling us to observe their complex localization along junctions. We further explore PCP fusion protein localization in the trachea and neural tube, and discover new patterns of PCP expression and localization throughout the mouse embryo., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

We studied electrophysiological changes in rabbit esophageal epithelium following acute (AS) and chronic stress (CS). Esophageal tissue was placed in Ussing chamber and the potential difference U between the luminal and abluminal sides, the short-circuit current I sc , as well as the tissue resistance R were measured. The initial values of these parameters for each sample were determined after the samples were stabilized in Ringer solution. Then, the tissues were exposed for 1 h to normal Ringer solution or Ringer solution with pH 4.0 and pH 1.7 with or without pepsin (0.25 mg/ml). Fluorescein was added to the luminal side of the sample to measure its permeability. In the AS group, U at Ringer solution (pH 1.7)+pepsin was significantly decreased in comparison with the baseline and control values (by 46 and 22%, respectively, p<0.05). R decreased by 74% in comparison with baseline, which little differed from the decrease in control samples exposed to Ringer solution (pH 1.7)+pepsin (by 62%). CS did not change U relative to baseline values, while changes in R were similar to those in the AS group. In the AS group, the permeability of the esophageal tissue perfused with Ringer solution (pH 1.7)+pepsin was significantly higher than in both the control and CS groups. AS, but not CS, made the esophageal epithelium more sensitive to the effects of noxious agents, disrupted barrier properties, and increased permeability. The effects of stress on gastroesophageal reflux disease symptoms can be related to severe exposure to acid and/or pepsin; however, the mechanisms other than epithelial defense should be evaluated., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications. [ABSTRACT FROM AUTHOR]

We recently established an in vitro culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a desirable distance (∼100 μm). For complete details on the use and execution of this protocol, please refer to Lu et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. Here, we show that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, we show that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. We present experimental and computational evidence that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)