Scaling of dorsal-ventral patterning by embryo size-dependent degradation of Spemann's organizer signals.

Spemann's organizer plays a key role in dorsal-ventral (DV) patterning in the amphibian embryo by secreting diffusible proteins such as Chordin, an antagonist to ventralizing bone morphogenetic proteins (BMPs). The DV patterning is so robust that an amphibian embryo with its ventral half surgically removed can develop into a smaller but proportionally patterned larva. Here, we show that this robust patterning depends on facilitated Chordin degradation and requires the expression of the Chordin-proteinase inhibitor Sizzled on the opposite side. Sizzled, which is stable and diffuses widely along the DV axis, stabilizes Chordin and expands its distribution in the ventral direction. This expanded Chordin distribution, in turn, limits BMP-dependent Sizzled production, forming an axis-wide feedback loop for shaping Chordin's activity. Using bisection assays, we demonstrate that Chordin degradation is dynamically controlled by embryo-size-coupled Sizzled accumulation. We propose a scaling model that enables the DV pattern to adjust proportionally to embryonic axis size.

Tracing the origin of hair follicle stem cells.

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.

Structure formation induced by non-reciprocal cell-cell interactions in a multicellular system.

Collective cellular behavior plays a crucial role in various biological processes, ranging from developmental morphogenesis to pathological processes such as cancer metastasis. Our previous research has revealed that a mutant cell of Dictyostelium discoideum exhibits collective cell migration, including chain migration and traveling band formation, driven by a unique tail-following behavior at contact sites, which we term "contact following locomotion" (CFL). Here, we uncover an imbalance of forces between the front and rear cells within cell chains, leading to an additional propulsion force in the rear cells. Drawing inspiration from this observation, we introduce a theoretical model that incorporates non-reciprocal cell-cell interactions. Our findings highlight that the non-reciprocal interaction, in conjunction with self-alignment interactions, significantly contributes to the emergence of the observed collective cell migrations. Furthermore, we present a comprehensive phase diagram, showing distinct phases at both low and intermediate cell densities. This phase diagram elucidates a specific regime that corresponds to the experimental system.

Mesenchymal-epithelial transition regulates initiation of pluripotency exit before gastrulation.

The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through 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 cellular morphology and its pluripotency is not well understood. Here, 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 that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.

Autonomous epithelial folding induced by an intracellular mechano-polarity feedback loop.

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.

Non-monotonic fluidization generated by fluctuating edge tensions in confluent tissues.

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.

Three-Dimensional Cell Geometry Controls Excitable Membrane Signaling in Dictyostelium Cells.

Phosphatidylinositol (3-5)-trisphosphate (PtdInsP3) is known to propagate as waves on the plasma membrane and is related to the membrane-protrusive activities in Dictyostelium and mammalian cells. Although there have been a few attempts to study the three-dimensional (3D) dynamics of these processes, most studies have focused on the dynamics extracted from single focal planes. However, the relation between the dynamics and 3D cell shape remains elusive because of the lack of signaling information about the unobserved part of the membrane. Here, we show that PtdInsP3 wave dynamics are directly regulated by the 3D geometry (i.e., size and shape) of the plasma membrane. By introducing an analysis method that extracts the 3D spatiotemporal activities on the entire cell membrane, we show that PtdInsP3 waves self-regulate their dynamics within the confined membrane area. This leads to changes in speed, orientation, and pattern evolution, following the underlying excitability of the signal transduction system. Our findings emphasize the role of the plasma membrane topology in reaction-diffusion-driven biological systems and indicate its importance in other mammalian systems.

Biomechanical regulation of EMT and epithelial morphogenesis in amniote epiblast.

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.

Propagation of regulatory fluctuations induces coordinated switching of flagellar motors in chemotaxis signaling pathway of single bacteria.

The random motion of E. coli is driven by multiple flagella motors. When all motors rotate in the counter clockwise direction, the bacteria swims smoothly. A recent experimental report by Terasawa et al. [Biophys J,100,2193,(2011)] demonstrated that a coordination of the motors can occur through signaling pathways, and perturbation of a regulatory molecule disrupted the coordination. Here, we develop a mathematical model to show that a large temporal fluctuation in the regulator concentration can induce a correlated switching of the multiple motors. Such a large fluctuation is generated by a chemotaxis receptor cluster in unilateral cell pole, which then exhibits a spatial propagation through the cytoplasm from the receptor position to the motor around cell periphery. Our numerical simulation successfully reproduces synchronized switching and the lag time in the motions of two distant motors, which has been observed experimentally. We further show that the large fluctuation in the regulator concentration at the motor positions can expand the dynamic range that the motor can respond, which confers robustness to the signaling system.

Epithelial Folding Driven by Apical or Basal-Lateral Modulation: Geometric Features, Mechanical Inference, and Boundary Effects.

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.

Visualization of Ca2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells.

The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca(2+) signaling, serving as both a source and sink of Ca(2+), and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca(2+) dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca(2+) dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca(2+) indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca(2+) concentration ([Ca(2+)]ER) in dendrites and spines. In this experimental system, the recovery of [Ca(2+)]ER takes several seconds, and recovery half-time depends on the extent of ER Ca(2+) depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca(2+), rather than Ca(2+) reuptake, is the dominant mechanism for the replenishment of the local [Ca(2+)]ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca(2+) diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca(2+) spikes in PCs, cumulatively increased [Ca(2+)]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca(2+) within the neurons, and as a leaky integrator of Ca(2+) spike-inducing synaptic inputs.

Cell Chirality Induces Collective Cell Migration in Epithelial Sheets.

During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

Self-organization and advective transport in the cell polarity formation for asymmetric cell division.

Anterior-Posterior (AP) polarity formation of cell membrane proteins plays a crucial role in determining cell asymmetry, which depends not only on the several genetic process but also biochemical and biophysical interactions. The mechanism of AP formation of Caenorhabditis elegans embryo is characterized into the three processes: (i) membrane association and dissociation of posterior and anterior proteins, (ii) diffusion into the membrane and cytosol, and (iii) active cortical and cytoplasmic flows induced by the contraction of the acto-myosin cortex. We explored the mechanism of symmetry breaking and AP polarity formation using self-recruitment model of posterior proteins. We found that the AP polarity pattern is established over wide range in the total mass of polarity proteins and the diffusion ratio in the cytosol to the membrane. We also showed that the advective transport in both membrane and cytosol during the establishment phase affects optimal time interval of establishment and positioning of the posterior domain, and plays a role to increase the robustness in the AP polarity formation by reducing the number of posterior domains for the sensitivity of initial conditions. We also demonstrated that a proper ratio of the total mass to cell size robustly regulate the length scale of the posterior domain.

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos.

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.

Excitable signal transduction induces both spontaneous and directional cell asymmetries in the phosphatidylinositol lipid signaling system for eukaryotic chemotaxis.

Intracellular asymmetry in the signaling network works as a compass to navigate eukaryotic chemotaxis in response to guidance cues. Although the compass variable can be derived from a self-organization dynamics, such as excitability, the responsible mechanism remains to be clarified. Here, we analyzed the spatiotemporal dynamics of the phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) pathway, which is crucial for chemotaxis. We show that spontaneous activation of PtdInsP3-enriched domains is generated by an intrinsic excitable system. Formation of the same signal domain could be triggered by various perturbations, such as short impulse perturbations that triggered the activation of intrinsic dynamics to form signal domains. We also observed the refractory behavior exhibited in typical excitable systems. We show that the chemotactic response of PtdInsP3 involves biasing the spontaneous excitation to orient the activation site toward the chemoattractant. Thus, this biased excitability embodies the compass variable that is responsible for both random cell migration and biased random walk. Our finding may explain how cells achieve high sensitivity to and robust coordination of the downstream activation that allows chemotactic behavior in the noisy environment outside and inside the cells.

Modeling the self-organized phosphatidylinositol lipid signaling system in chemotactic cells using quantitative image analysis.

A key signaling event that is responsible for gradient sensing in eukaryotic cell chemotaxis is a phosphatidylinositol (PtdIns) lipid reaction system. The self-organization activity of this PtdIns lipid system induces an inherent polarity, even in the absence of an external chemoattractant gradient, by producing a localized PtdIns (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)]-enriched domain on the membrane. Experimentally, we found that such a domain could exhibit two types of behavior: (1) it could be persistent and travel on the membrane, or (2) be stochastic and transient. Taking advantage of the simultaneous visualization of PtdIns(3,4,5)P(3) and the enzyme phosphatase and tensin homolog (PTEN), for which PtdIns(3,4,5)P(3) is a substrate, we statistically demonstrated the inter-dependence of their spatiotemporal dynamics. On the basis of this statistical analysis, we developed a theoretical model for the self-organization of PtdIns lipid signaling that can accurately reproduce both persistent and transient domain formation; these types of formations can be explained by the oscillatory and excitability properties of the system, respectively.

Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis.

Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold.

Asymmetric PTEN distribution regulated by spatial heterogeneity in membrane-binding state transitions.

The molecular mechanisms that underlie asymmetric PTEN distribution at the posterior of polarized motile cells and regulate anterior pseudopod formation were addressed by novel single-molecule tracking analysis. Heterogeneity in the lateral mobility of PTEN on a membrane indicated the existence of three membrane-binding states with different diffusion coefficients and membrane-binding lifetimes. The stochastic state transition kinetics of PTEN among these three states were suggested to be regulated spatially along the cell polarity such that only the stable binding state is selectively suppressed at the anterior membrane to cause local PTEN depletion. By incorporating experimentally observed kinetic parameters into a simple mathematical model, the asymmetric PTEN distribution can be explained quantitatively to illustrate the regulatory mechanisms for cellular asymmetry based on an essential causal link between individual stochastic reactions and stable localizations of the ensemble.

Emergence of periodic circumferential actin cables from the anisotropic fusion of actin nanoclusters during tubulogenesis.

The periodic circumferential cytoskeleton supports various tubular tissues. Radial expansion of the tube lumen causes anisotropic tensile stress, which can be exploited as a geometric cue. However, the molecular machinery linking anisotropy to robust circumferential patterning is poorly understood. Here, we aim to reveal the emergent process of circumferential actin cable formation in a Drosophila tracheal tube. During luminal expansion, sporadic actin nanoclusters emerge and exhibit circumferentially biased motion and fusion. RNAi screening reveals the formin family protein, DAAM, as an essential component responding to tissue anisotropy, and non-muscle myosin II as a component required for nanocluster fusion. An agent-based model simulation suggests 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 responds to stress in the cortical membrane and builds a higher-order cable structure.

Intracellular encoding of spatiotemporal guidance cues in a self-organizing signaling system for chemotaxis in Dictyostelium cells.

Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to move directionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases. It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activates the motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network have been reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues. Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision of polarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for the sensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-time waves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.