Project description:Epithelial monolayer formation depends on the architecture and composition of the microtubule cytoskeleton. Microtubules control bidirectional trafficking and determine the positioning of structural cellular proteins. We studied the role of tubulin tyrosination in epithelial cell shape and motility. Tubulin tyrosine ligase (TTL), the enzyme that adds tyrosine to the carboxy terminus of detyrosinated α-tubulin, was depleted or overexpressed in 2D epithelial monolayers as well as in 3D intestinal organoids. We demonstrate qualitatively and quantitatively that in the absence of TTL the cells comprise high levels of detyrosinated tubulin, change their shape into an initial flat morphology and retardedly acquire a differentiated columnar epithelial cell shape. Enhanced adhesion and accelerated migration patterns of TTL-knockout cells combined with reverse effects in TTL-overexpressing cells indicate that the loss of TTL affects the organization of cell adhesion foci. Precipitation of detyrosinated tubulin with focal adhesion scaffold components coincides with increased quantities and persistence of focal adhesion plaques. Our results indicate that the equilibrium between microtubules enriched in detyrosinated or tyrosinated tubulin modulates epithelial tissue formation, cell morphology, and adhesion.
Project description:The mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching, however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.
Project description:Internal organs such as the heart demonstrate apparent left-right (LR) asymmetric morphology and positioning. Cellular chirality and associated LR biased mechanical behavior such as cell migration have been attributed to LR symmetry breaking during embryonic development. Mathematical models have shown that chiral directional migration can be driven by cellular intrinsic torque. Tissue jamming state (i.e., solid-like vs fluid-like state) strongly regulates collective migratory behavior, but how it might affect chiral morphogenesis is still unknown. Here, we develop a cell vertex model to study the role of tissue rigidity or jamming state on chiral morphogenesis of the cells on a patterned ring-shaped tissue, simulating a previously reported experimental setup for measuring cell chirality. We simulate chirality as torsional forces acting on cell vertices. As expected, the cells undergo bidirectional migration at the opposing (inner and outer) boundaries of the ring-shaped tissue. We discover that more fluid-like tissues (unjammed) demonstrate a stronger chiral cell alignment and elongation than more solid-like (jammed) tissues and maintain a bigger difference in migration velocity between opposing tissue boundaries. Finally, we find that fluid-like tissues undergo more cell-neighbor exchange events. This study reveals that chiral torque is sufficient to achieve a biased cellular alignment as seen in vitro. It further sheds light on the mechanical regulation of chiral morphogenesis of tissues and reveals a role of cell density-independent tissue rigidity in this process.
Project description:Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodeling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behavior under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviors, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodeling and continuous strain relaxation. First, junctions must overcome a critical strain threshold to trigger tension remodeling, resulting in irreversible junction length changes. Second, there is a continuous relaxation of junctional strain that removes mechanical memory from the system. This enables pulsatile contractions to further remodel cell shape via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodeling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.
Project description:Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.
Project description:Epithelial folding is a universal biological phenomenon in morphogenesis, typical examples being brain gyri, villi of the intestinal tract, and imaginal discs in invertebrates. During epithelial morphogenesis, the physical constraints imposed by the surrounding microenvironment on epithelial tissue play critical roles in folding morphology. In this study, we focused on the asymmetry of the environmental constraints sandwiching the epithelial sheet and introduced the degree of asymmetry, which indicates whether the basal or apical side of the epithelium is closer to the constraint wall. Then, we investigated the relationship between the degree of asymmetry and epithelial folding morphology using three-dimensional vertex simulations. The results show that the folding patterns of the epithelial sheets change from spot patterns to labyrinth patterns and then to hole patterns as the degree of asymmetry changes. Furthermore, we examined the pattern formation in terms of the equation of out-of-plane displacement of the sheet derived from the mechanical energy functional.
Project description:The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.
Project description:Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.
Project description:BACKGROUND:Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. RESULTS:We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting ?6+?1 integrins and the nonmuscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. CONCLUSIONS:Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.
Project description:During embryonic development, many organs form by extensive branching of epithelia through the formation of clefts and buds. In cleft formation, buds are delineated by the conversion of epithelial cell-cell adhesions to cell-matrix adhesions, but the mechanisms of cleft formation are not clear. We have identified Btbd7 as a dynamic regulator of branching morphogenesis. Btbd7 provides a mechanistic link between the extracellular matrix and cleft propagation through its highly focal expression leading to local regulation of Snail2 (Slug), E-cadherin, and epithelial cell motility. Inhibition experiments show that Btbd7 is required for branching of embryonic mammalian salivary glands and lungs. Hence, Btbd7 is a regulatory gene that promotes epithelial tissue remodeling and formation of branched organs.