Bayesian parameter inference for epithelial mechanics.

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.

Vertex remodeling during epithelial morphogenesis.

Epithelial cells adhere to each other via intercellular junctions that can be classified into bicellular junctions and tricellular contacts (vertices). Epithelial morphogenesis involves cell rearrangement and requires remodeling of bicellular junctions and vertices. Although our understanding of how bicellular junction mechanics drive epithelial morphogenesis has advanced, the mechanisms underlying vertex remodeling during this process have only received attention recently. In this review, we outline recent progress in our understanding of how cells reorganize cell adhesion and the cytoskeleton to trigger the displacement and resolution of cell vertices. We will also discuss how cells achieve the optimal balance between the structural flexibility and stability of their vertices. Finally, we introduce new modeling frameworks designed to analyze mechanics at cell vertices. Integration of live imaging and modeling techniques is providing new insights into the active roles of cell vertices during epithelial morphogenesis.

Mechanical convergence in mixed populations of mammalian epithelial cells.

Tissues consist of cells with different molecular and/or mechanical properties. Measuring the forces and stresses in mixed-cell populations is essential for understanding the mechanisms by which tissue development, homeostasis, and disease emerge from the cooperation of distinct cell types. However, many previous studies have primarily focused their mechanical measurements on dissociated cells or aggregates of a single-cell type, leaving the mechanics of mixed-cell populations largely unexplored. In the present study, we aimed to elucidate the influence of interactions between different cell types on cell mechanics by conducting in situ mechanical measurements on a monolayer of mammalian epithelial cells. Our findings revealed that while individual cell types displayed varying magnitudes of traction and intercellular stress before mixing, these mechanical values shifted in the mixed monolayer, becoming nearly indistinguishable between the cell types. Moreover, by analyzing a mixed-phase model of active tissues, we identified physical conditions under which such mechanical convergence is induced. Overall, the present study underscores the importance of in situ mechanical measurements in mixed-cell populations to deepen our understanding of the mechanics of multicellular systems.

Coronin-1 promotes directional cell rearrangement in Drosophila wing epithelium.

Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. In this study, we investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. We observed that Coronin-1 localizes at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1) and cofilin, actin disassemblers and known regulators of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1 and cofilin, highlighting the role of ABPs in the complex process of morphogenesis.Key words: morphogenesis, cell rearrangement, actin binding proteins (ABPs).

Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium.

Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, we found that M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, we developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.

Hiatal Hernia with Prolapse of the Pancreas Causing Bile Duct Stricture and Liver Function Disorders: A Case Report and Literature Review.

Hiatal hernia is a common condition in elderly patients, but the additional presence of prolapse of the pancreas is extremely rare. We herein report an 89-year-old woman who presented with liver function disorders and abdominal pain. Her laboratory tests revealed cholestasis, and imaging examinations showed stenosis of the common bile duct pulled toward the hernia sac. She was diagnosed with a common bile duct stricture due to pancreatic herniation and underwent laparoscopic surgery. Our review of the literature identified three types of pancreatic herniations: asymptomatic, bile duct complication, and acute pancreatitis. Pancreatic head herniation tends to induce bile duct complications.

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition.

Image-based parameter inference for epithelial mechanics.

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.

Bowel Obstruction due to Shiitake Mushrooms: Diagnostic Features on Computed Tomography.

Shiitake mushrooms are edible mushrooms popular in East Asian cuisine. We herein report a 69-year-old man with abdominal distension and vomiting after ingesting several pieces of sautéed Shiitake mushrooms. Abdominal computed tomography (CT) revealed ring-shaped and crescent-shaped low-density objects (-100 to -300 Hounsfield units) in the ileum. Based on the specific shapes and CT numbers of the foreign bodies, he was diagnosed with small bowel obstruction due to Shiitake mushrooms. After conservative treatment, he passed four pieces of Shiitake mushrooms. Despite the rarity, the condition can be diagnosed before exploratory surgery by careful and detailed interpretation of CT findings.

Discovery of an F-actin-binding small molecule serving as a fluorescent probe and a scaffold for functional probes.

Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. There is a growing demand for practical and accessible tools for dissecting the role of the actin cytoskeleton in cellular function, and the discovery of a new actin-binding small molecule is an important advance in the field, offering the opportunity to design and synthesize of new class of functional molecules. Here, we found an F-actin­binding small molecule and introduced two powerful tools based on a new class of actin-binding small molecule: One enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green light­controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

AIP1 and cofilin ensure a resistance to tissue tension and promote directional cell rearrangement.

In order to understand how tissue mechanics shapes animal body, it is critical to clarify how cells respond to and resist tissue stress when undergoing morphogenetic processes, such as cell rearrangement. Here, we address the question in the Drosophila wing epithelium, where anisotropic tissue tension orients cell rearrangements. We found that anisotropic tissue tension localizes actin interacting protein 1 (AIP1), a cofactor of cofilin, on the remodeling junction via cooperative binding of cofilin to F-actin. AIP1 and cofilin promote actin turnover and locally regulate the Canoe-mediated linkage between actomyosin and the junction. This mechanism is essential for cells to resist the mechanical load imposed on the remodeling junction perpendicular to the direction of tissue stretching. Thus, the present study delineates how AIP1 and cofilin achieve an optimal balance between resistance to tissue tension and morphogenesis.

From cells to tissue: A continuum model of epithelial mechanics.

A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

Difference in Dachsous Levels between Migrating Cells Coordinates the Direction of Collective Cell Migration.

In contrast to extracellular chemotactic gradients, how cell-adhesion molecules contribute to directing cell migration remains more elusive. Here we studied the collective migration of Drosophila larval epidermal cells (LECs) along the anterior-posterior axis and propose a migrating cell group-autonomous mechanism in which an atypical cadherin Dachsous (Ds) plays a pivotal role. In each abdominal segment, the amount of Ds in each LEC varied along the axis of migration (Ds imbalance), which polarized Ds localization at cell boundaries. This Ds polarity was necessary for coordinating the migratory direction. Another atypical cadherin, Fat (Ft), and an unconventional myosin Dachs, both of which bind to Ds, also showed biased cell-boundary localizations, and both were required for the migration. Altogether, we propose that the Ds imbalance within the migrating tissue provides the directional cue and that this is decoded by Ds-Ft-mediated cell-cell contacts, which restricts lamellipodia formation to the posterior end of the cell.

A mechanical model for diversified insect wing margin shapes.

The wings in different insect species are morphologically distinct with regards to their size, outer contour (margin) shape, venation, and pigmentation. The basis of the diversity of wing margin shapes remains unknown, despite the fact that gene networks governing the Drosophila wing development have been well characterised. Among the different types of wing margin shapes, smoothly curved contour is the most frequently found and implies the existence of a highly organised, multicellular mechanical structure. Here, we developed a mechanical model for diversified insect wing margin shapes, in which non-uniform bending stiffness of the wing margin is considered. We showed that a variety of spatial distribution of the bending stiffness could reproduce diverse wing margin shapes. Moreover, the inference of the distribution of the bending stiffness from experimental images indicates a common spatial profile among insects tested. We further studied the effect of the intrinsic tension of the wing blade on the margin shape and on the inferred bending stiffness. Finally, we implemented the bending stiffness of the wing margin in the cell vertex model of the wing blade, and confirmed that the hybrid model retains the essential feature of the margin model. We propose that in addition to morphogenetic processes in the wing blade, the spatial profile of the bending stiffness in the wing margin can play a pivotal role in shaping insect wings.

Measuring forces and stresses in situ in living tissues.

Development, homeostasis and regeneration of tissues result from a complex combination of genetics and mechanics, and progresses in the former have been quicker than in the latter. Measurements of in situ forces and stresses appear to be increasingly important to delineate the role of mechanics in development. We review here several emerging techniques: contact manipulation, manipulation using light, visual sensors, and non-mechanical observation techniques. We compare their fields of applications, their advantages and limitations, and their validations. These techniques complement measurements of deformations and of mechanical properties. We argue that such approaches could have a significant impact on our understanding of the development of living tissues in the near future.

Unified quantitative characterization of epithelial tissue development.

Understanding the mechanisms regulating development requires a quantitative characterization of cell divisions, rearrangements, cell size and shape changes, and apoptoses. We developed a multiscale formalism that relates the characterizations of each cell process to tissue growth and morphogenesis. Having validated the formalism on computer simulations, we quantified separately all morphogenetic events in the Drosophila dorsal thorax and wing pupal epithelia to obtain comprehensive statistical maps linking cell and tissue scale dynamics. While globally cell shape changes, rearrangements and divisions all significantly participate in tissue morphogenesis, locally, their relative participations display major variations in space and time. By blocking division we analyzed the impact of division on rearrangements, cell shape changes and tissue morphogenesis. Finally, by combining the formalism with mechanical stress measurement, we evidenced unexpected interplays between patterns of tissue elongation, cell division and stress. Our formalism provides a novel and rigorous approach to uncover mechanisms governing tissue development.

Filamin acts as a key regulator in epithelial defence against transformed cells.

Recent studies have shown that certain types of transformed cells are extruded from an epithelial monolayer. However, it is not known whether and how neighbouring normal cells play an active role in this process. In this study, we demonstrate that filamin A and vimentin accumulate in normal cells specifically at the interface with Src- or RasV12-transformed cells. Knockdown of filamin A or vimentin in normal cells profoundly suppresses apical extrusion of the neighbouring transformed cells. In addition, we show in zebrafish embryos that filamin plays a positive role in the elimination of the transformed cells. Furthermore, the Rho/Rho kinase pathway regulates filamin accumulation and filamin acts upstream of vimentin in the apical extrusion. This is the first report demonstrating that normal epithelial cells recognize and actively eliminate neighbouring transformed cells and that filamin is a key mediator in the interaction between normal and transformed epithelial cells.

Robustness of force and stress inference in an epithelial tissue.

During morphogenesis, the shape of a tissue emerges from collective cellular behaviors, which are in part regulated by mechanical and biochemical interactions between cells. Quantification of force and stress is therefore necessary to analyze the mechanisms controlling tissue morphogenesis. Recently, a mechanical measurement method based on force inference from cell shapes and connectivity has been developed. It is non-invasive, and can provide space-time maps of force and stress within an epithelial tissue, up to prefactors. We previously performed a comparative study of three force-inference methods, which differ in their approach of treating indefiniteness in an inverse problem between cell shapes and forces. In the present study, to further validate and compare the three force inference methods, we tested their robustness by measuring temporal fluctuation of estimated forces. Quantitative data of cell-level dynamics in a developing tissue suggests that variation of forces and stress will remain small within a short period of time (~minutes). Here, we showed that cell-junction tensions and global stress inferred by the Bayesian force inference method varied less with time than those inferred by the method that estimates only tension. In contrast, the amplitude of temporal fluctuations of estimated cell pressures differs less between different methods. Altogether, the present study strengthens the validity and robustness of the Bayesian force-inference method.

The mechanical anisotropy in a tissue promotes ordering in hexagonal cell packing.

Many epithelial tissues pack cells into a honeycomb pattern to support their structural and functional integrity. Developmental changes in cell packing geometry have been shown to be regulated by both mechanical and biochemical interactions between cells; however, it is largely unknown how molecular and cellular dynamics and tissue mechanics are orchestrated to realize the correct and robust development of hexagonal cell packing. Here, by combining mechanical and genetic perturbations along with live imaging and Bayesian force inference, we investigate how mechanical forces regulate cellular dynamics to attain a hexagonal cell configuration in the Drosophila pupal wing. We show that tissue stress is oriented towards the proximal-distal axis by extrinsic forces acting on the wing. Cells respond to tissue stretching and orient cell contact surfaces with the stretching direction of the tissue, thereby stabilizing the balance between the intrinsic cell junction tension and the extrinsic force at the cell-population level. Consequently, under topological constraints of the two-dimensional epithelial sheet, mismatches in the orientation of hexagonal arrays are suppressed, allowing more rapid relaxation to the hexagonal cell pattern. Thus, our results identify the mechanism through which the mechanical anisotropy in a tissue promotes ordering in cell packing geometry.

Bayesian inference of force dynamics during morphogenesis.

During morphogenesis, cells push and pull each other to trigger precise deformations of a tissue to shape the body. Therefore, to understand the development of animal forms, it is essential to analyze how mechanical forces coordinate behaviors of individual cells that underlie tissue deformations. However, the lack of a direct and non-invasive force-measurement method has hampered our ability to identify the underlying physical principles required to regulate morphogenesis. In this study, by employing Bayesian statistics, we develop a novel inverse problem framework to estimate the pressure of each cell and the tension of each contact surface from the observed geometry of the cells. We confirmed that the true and estimated values of forces fit well in artificially generated data sets. Moreover, estimates of forces in Drosophila epithelial tissues are consistent with other readouts of forces obtained by indirect or invasive methods such as laser-induced destruction of cortical actin cables. Using the method, we clarify the developmental changes in the patterns of tensile force in the Drosophila dorsal thorax. In summary, the batch and noninvasive nature of the described force-estimation method will enable us to analyze the mechanical control of morphogenesis at an unprecedented quantitative level.