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The periodic circumferential cytoskeleton supports various tubular tissues1-5. Radial expansion of the tube lumen causes anisotropic tensile stress, which can be exploited as a geometric cue6. However, the molecular machinery linking anisotropy to robust circumferential patterning is poorly understood. Here, we aimed to reveal the emergent process of circumferential actin cable formation in a Drosophila tracheal tube7. During luminal expansion, sporadic actin nanoclusters emerge and exhibit circumferentially biased motion and fusion. RNAi screening revealed the formin family protein, DAAM, as an essential component of tensile stress sensing, and non-muscle myosin II as a component required for nanocluster fusion. A coarse-grained molecular dynamics simulation demonstrated that crosslinkers play a crucial role in nanocluster formation and cluster-to-cable transition occurs in response to mechanical anisotropy. Altogether, we propose that an actin nanocluster is an organizational unit that senses stress in the cortical membrane and builds a higher-order cable structure.

In development and homeostasis, multi-cellular systems exhibit spatial and temporal heterogeneity in their biochemical and mechanical properties. Nevertheless, it remains unclear how spatiotemporally heterogeneous forces affect the dynamical and mechanical properties of confluent tissue. To address this question, we study the dynamical behavior of the two-dimensional cellular vertex model for epithelial monolayers in the presence of fluctuating cell-cell interfacial tensions, which is a biologically relevant source of mechanical spatiotemporal heterogeneity. In particular, we investigate the effects of the amplitude and persistence time of fluctuating tension on the tissue dynamics. We unexpectedly find that the long-time diffusion constant describing cell rearrangements depends non-monotonically on the persistence time, while it increases monotonically as the amplitude increases. Our analysis indicates that at low and intermediate persistence times tension fluctuations drive motion of vertices and promote cell rearrangements, while at the highest persistence times the tension in the network evolves so slowly that rearrangements become rare., Comment: 16 pages, 14 figures

Migration of cell populations is a fundamental process in morphogenesis and disease. The mechanisms of collective cell migration of epithelial cell populations have been well studied. It remains unclear, however, how the highly motile mesenchymal cells, which migrate extensively throughout the embryo, are connected with each other and coordinated as a collective. During gastrulation in chick embryos, the mesoderm cells, that are formed by an epithelial-to-mesenchymal transition (EMT), migrate in the 3D space between ectoderm and endoderm of the embryo. Using live imaging and quantitative analysis, such as topological data analysis (TDA), we found that the mesoderm cells undergo a novel form of collective migration, in which they form a meshwork structure while moving away from the primitive streak. This meshwork is supported by N-cadherin-mediated cell-cell adhesion, which undergoes rapid reorganization. Overexpressing a mutant form of N-cadherin decreases the speed of tissue progression and the directionality of the collective cell movement, whereas the speed of individual cells remains unchanged. To investigate how this meshwork arises and how it contributes to the cell movement, we utilized an agent-based theoretical model, showing that cell elongation, cell-cell adhesion, and cell density are the key parameters for the meshwork formation. These data provide novel insights into how a supracellular structure of migrating mesenchymal cells forms and how it facilitates efficient migration during early mesoderm formation.

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the ‘telescope model’, a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments. Live imaging and single-cell transcriptomics of mouse hair follicles reveal their development from 2D concentric zones in the placode to 3D longitudinal compartments, one of which is a stem cell compartment.

Pattern formation and the mechanics of a mixture of actin filaments and myosin motors that is confined by a rigid membrane is investigated. By using a coarse-grained molecular dynamics model, we demonstrate that the competition between the depletion force and the active force of the motors gives rise to actin accumulation in the membrane vicinity. The resulting actomyosin structure exerts pressure on the membrane, that, due to nematic alignment of the filaments, converges to a constant for large motor active force. The results are independent of filament length and membrane curvature, indicating the universality of this phenomenon. Thus, this study proposes a novel mechanism by which the compounds of the cytoskeleton can self-organize into a higher-order structure., 13 pages, 7 figures

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a ‘transition zone’. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium–basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

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, how they interact 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 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.

Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell–cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia form and found that they sporadically grew from around E-cadherin–based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also support c-lamellipodia formation by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.

Coordination between cell differentiation and proliferation during development requires the balance between asymmetric and symmetric modes of cell division. However, the cellular intrinsic cue underlying the binary choice between these two division modes remains elusive. Here we show evidence inCaenorhabditis elegansthat the invariable lineage of the division modes is programmed by the balance between antagonizing complexes of partitioning-defective (PAR) proteins. By uncoupling unequal inheritance of PAR proteins from that of fate determinants during zygote division, we demonstrated that changes in the balance between PAR-2 and PAR-6 are sufficient to re-program the division modes from symmetric to asymmetric andvice versain two-cell stage embryos. The division mode adopted occurs independently of asymmetry in cytoplasmic fate determinants, cell-size asymmetry, and cell-cycle asynchrony between the sister cells. We propose that the balance between antagonizing PAR proteins represents an intrinsic self-organizing cue for binary specification of the division modes during development.

Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell-cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia are generated, and found that they sporadically grew from Ecadherin-based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also generate c-lamellipodia by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events

A synthetic mammalian reaction-diffusion pattern has yet to be created, and Nodal-Lefty signaling has been proposed to meet conditions for pattern formation: Nodal is a short-range activator whereas Lefty is a long-range inhibitor. However, this pattern forming possibility has never been directly tested, and the underlying mechanisms of differential diffusivity of Nodal and Lefty remain unclear. Here, through a combination of synthetic and theoretical approaches, we show that a reconstituted Nodal-Lefty network in mammalian cells spontaneously gives rise to a pattern. Surprisingly, extracellular Nodal is confined underneath the cells, resulting in a narrow distribution compared with Lefty. The short-range distribution requires the finger 1 domain of Nodal, and transplantation of the finger 1 domain into Lefty shortens the distribution of Lefty, successfully preventing pattern formation. These results indicate that the differences in localization and domain structures between Nodal and Lefty, combined with the activator-inhibitor topology, are sufficient for reaction-diffusion patterning., Nodal-Lefty signaling combines a short-range activator and a long-range inhibitor for axis formation and left-right patterning. Here the authors reconstitute a synthetic biology activator-inhibitor circuit of Nodal-Lefty to drive pattern formation in mammalian cells.

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.

Coordination between cell differentiation and proliferation during development requires the balance between asymmetric and symmetric modes of cell division. However, the cellular intrinsic cue underlying the choice between these two division modes remains elusive. Here, we show evidence in Caenorhabditis elegans that the invariable lineage of the division modes is specified by the balance between antagonizing complexes of partitioning-defective (PAR) proteins. By uncoupling unequal inheritance of PAR proteins from that of fate determinants during cell division, we demonstrate that changes in the balance between PAR-2 and PAR-6 can be sufficient to re-program the division modes from symmetric to asymmetric and vice versa in two daughter cells. The division mode adopted occurs independently of asymmetry in cytoplasmic fate determinants, cell-size asymmetry, and cell-cycle asynchrony between sister cells. We propose that the balance between PAR proteins represents an intrinsic self-organizing cue for the specification of the two division modes during development.

During embryonic development, epithelial sheets fold into complex structures required for tissue and organ functions. Although substantial efforts have been devoted to identifying molecular mechanisms underlying epithelial folding, far less is understood about how forces deform individual cells to sculpt the overall sheet morphology. Here we describe a simple and general theoretical model for the autonomous folding of monolayered epithelial sheets. We show that active modulation of intracellular mechanics along the basal-lateral as well as the apical surfaces is capable of inducing fold formation in the absence of buckling instability. Apical modulation sculpts epithelia into shallow and V-shaped folds, whereas basal-lateral modulation generates deep and U-shaped folds. These characteristic tissue shapes remain unchanged when subject to mechanical perturbations from the surroundings, illustrating that the autonomous folding is robust against environmental variabilities. At the cellular scale, how cells change shape depends on their initial aspect ratios and the modulation mechanisms. Such cell deformation characteristics are verified via experimental measurements for a canonical folding process driven by apical modulation, indicating that our theory could be used to infer the underlying folding mechanisms based on experimental data. The mechanical principles revealed in our model could potentially guide future studies on epithelial folding in diverse systems.

Biophysical mechanisms underlying collective cell migration of eukaryotic cells have been studied extensively in recent years. One mechanism that induces cells to correlate their motions is contact inhibition of locomotion, by which cells migrating away from the contact site. Here, we report that tail-following behavior at the contact site, termed contact following locomotion (CFL), can induce a non-trivial collective behavior in migrating cells. We show the emergence of a traveling band showing polar order in a mutant Dictyostelium cell that lacks chemotactic activity. We find that CFL is the cell–cell interaction underlying this phenomenon, enabling a theoretical description of how this traveling band forms. We further show that the polar order phase consists of subpopulations that exhibit characteristic transversal motions with respect to the direction of band propagation. These findings describe a novel mechanism of collective cell migration involving cell–cell interactions capable of inducing traveling band with polar order., eLife digest The cells of animals and many other living things are able to migrate together in groups. This collective cell migration plays crucial roles in many processes in animals such as forming organs and limbs, and healing wounds. A soil-dwelling amoeba called Dictyostelium discoideum – or just Dicty for short – is commonly used as a model to study how groups of cells migrate collectively. Individual Dicty cells may live alone but sometimes many cells come together to form a larger mobile structure called a “slug”. Chemical signals coordinate how the cells collectively migrate to form the multicellular slug. Mutant Dicty cells that lack these chemical signal processes can still move together as a band that travels across a surface. This movement resembles a type of collective motion that has previously been observed in physics experiments using self-propelled particles. However, it remains unclear how this collective behavior works. Hayakawa et al. have now combined genetics, cell biology and computational approaches to study how groups of the mutant Dicty cells migrate together. The experiments showed that the traveling band is dynamically maintained by cells joining or leaving, and that this turnover is caused by simple interactions between the cells known as “contact following locomotion”. Contact following locomotion has been also reported in mammalian cells so the findings of Hayakawa et al. may aid research into how animals develop and how errors in cell migration may lead to diseases. Further studies are required to find out whether other cells showing contact following locomotion also travel in a band.

The pluripotent epiblast gives rise to all tissues and organs in an adult body. Its differentiation starts at gastrulation when the epiblast generates mesoderm and endoderm germ layers through a process called epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast’s cellular morphology and its pluripotency is not well understood. In this work, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit; whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and its developmental function is to mediate planar symmetry-breaking within an epithelialized epiblast, taking place after epiblast MET but before gastrulation EMT.

Morphological constancy is universal in developing systems. It is unclear whether precise morphogenesis stems from faithful mechanical interpretation of gene expression patterns. We investigate the formation of the cephalic furrow, an epithelial fold that is precisely positioned with a linear morphology. Fold initiation is specified by a precise genetic code with single-cell row resolution. This positional code activates and spatially confines lateral myosin contractility to induce folding. However, 20% of initiating cells are mis-specified because of fluctuating myosin intensities at the cellular level. Nevertheless, the furrow remains linearly aligned. We find that lateral myosin is planar polarized, integrating contractile membrane interfaces into supracellular "ribbons." Local reduction of mechanical coupling at the "ribbons" using optogenetics decreases furrow linearity. Furthermore, 3D vertex modeling indicates that polarized, interconnected contractility confers morphological robustness against noise. Thus, tissue-scale mechanical coupling functions as a denoising mechanism to ensure morphogenetic precision despite noisy decoding of positional information.

Epiblast is composed of pluripotent cells which will give rise to all cell lineages in a human body. It forms a single-cell layered epithelium conserved among all amniotic vertebrates (birds, reptiles and mammals) and undergoes complex morphogenesis both before and during gastrulation. Our knowledge of the amniote epiblast is based on data acquired through cellular and molecular analyses of early chick and mouse embryos in vivo and mammalian pluripotent stem cells (PSCs) in vitro. Very few studies have been published on biomechanical characteristics of the amniote epiblast, largely due to lack of experimental tools for measuring and perturbing biomechanical properties. Also missing is a conceptual framework that can integrate both biomechanical and molecular parameters of the epiblast. This review is aimed at providing a background based on which epiblast morphogenesis, including its transition between the epithelial and mesenchymal states, can be understood from a biomechanical perspective. This simple developmental biology system is suitable for testing a multitude of theoretical models in biomechanics, leading to a better understanding of biomechanical logics and constraints governing multicellular organization.

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity., 細胞が対称性を破る仕組みを解明 --極性を持たない細胞に非対称性を与える--. 京都大学プレスリリース. 2019-07-12.

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the dynamic processes that occur during polarization are not well understood. Here, we artificially reconstructed Par-dependent polarity using non-polarizedDrosophilaS2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Par-complex patches resembling Par-islands exist inDrosophilamitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.

Morphological constancy is universal in developing systems. It is unclear whether precise morphogenesis stems from faithful mechanical interpretation of gene expression patterns. We investigate the formation of the cephalic furrow, an epithelial fold that is precisely positioned with a linear morphology. Fold initiation is specified by a precise genetic code with single-cell row resolution. This positional code activates and spatially confines lateral myosin contractility to induce folding. However, 20% of initiating cells are mis-specified due to fluctuating myosin intensities at the cellular level. Nevertheless the furrow remains linearly aligned. We find that lateral myosin is planar polarized, integrating contractile membrane interfaces into supracellular “ribbons.” Local reduction of mechanical coupling at the “ribbons” using optogenetics decreases furrow linearity. Furthermore, 3D vertex modelling indicates that polarized, interconnected contractility confers morphological robustness against noise. Thus, tissue-scale mechanical coupling functions as a denoising mechanism to ensure morphogenetic precision despite noisy decoding of positional information.