Hedgehog signaling is a principal inducer of Myosin-II-driven cell ingression in Drosophila epithelia

Cell constriction promotes epithelial sheet invagination during embryogenesis across phyla. However, how this cell response is linked to global patterning information during organogenesis remains unclear. To address this issue, we have used the Drosophila eye and studied the formation of the morphogenetic furrow (MF), which is characterized by cells undergoing a synchronous apical constriction and apicobasal contraction. We show that this cell response relies on microtubules and F-actin enrichment within the apical domain of the constricting cell as well as on the activation of nonmuscle myosin. In the MF, Hedgehog (Hh) signaling is required to promote cell constriction downstream of cubitus interruptus (ci), and, in this context, Ci155 functions redundantly with mad, the main effector of dpp/BMP signaling. Furthermore, ectopically activating Hh signaling in fly epithelia reveals a direct relationship between the duration of exposure to this signaling pathway, the accumulation of activated Myosin II, and the degree of tissue invagination.     

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.     

Mechanics of head fold formation: investigating tissue-level forces during early development

During its earliest stages, the avian embryo is approximately planar. Through a complex series of folds, this flat geometry is transformed into the intricate three-dimensional structure of the developing organism. Formation of the head fold (HF) is the first step in this cascading sequence of out-of-plane tissue folds. The HF establishes the anterior extent of the embryo and initiates heart, foregut and brain development. Here, we use a combination of computational modeling and experiments to determine the physical forces that drive HF formation. Using chick embryos cultured ex ovo, we measured: (1) changes in tissue morphology in living embryos using optical coherence tomography (OCT); (2) morphogenetic strains (deformations) through the tracking of tissue labels; and (3) regional tissue stresses using changes in the geometry of circular wounds punched through the blastoderm. To determine the physical mechanisms that generate the HF, we created a three-dimensional computational model of the early embryo, consisting of pseudoelastic plates representing the blastoderm and vitelline membrane. Based on previous experimental findings, we simulated the following morphogenetic mechanisms: (1) convergent extension in the neural plate (NP); (2) cell wedging along the anterior NP border; and (3) autonomous in-plane deformations outside the NP. Our numerical predictions agree relatively well with the observed morphology, as well as with our measured stress and strain distributions. The model also predicts the abnormal tissue geometries produced when development is mechanically perturbed. Taken together, the results suggest that the proposed morphogenetic mechanisms provide the main tissue-level forces that drive HF formation.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.     

Active reinforcement of externally imposed folding in amphibians embryonic tissues

Although the folding of epithelial layers is one of the most common morphogenetic events, the underlying mechanisms of this process are still poorly understood. We aimed to determine whether an artificial bending of an embryonic cell sheet, which normally remains flat, is reinforced and stabilized by intrinsic cell transformations. We observed both reinforcement and stabilization in double explants of blastocoel roof tissue from Xenopus early gastrula embryos. The reinforcement of artificial bending occurred over the course of a few hours and was driven by the gradual apical constriction and radial elongation of previously compressed cells situated at the bending arch of the concave layer of explant. Apical constriction was associated with actomyosin contraction and endocytosis-mediated engulfing of the apical cell membranes. Cooperative apical constrictions of the concave layer of cells produced a tensile force that extended over the entire surface of the explant and correlated with apical contraction of the concave side cells. In the explants taken from the anterior regions of the embryo, this reinforcement was more stable and the bending better expressed than in those taken from suprablastoporal areas. The morphogenetic role of cell responses to the bending force is discussed.     

α-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium

Apical actomyosin activity in animal epithelial cells influences tissue morphology and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the nonmetazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from nonmetazoans to vertebrates and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in?a simple model organism.     

How the Sequence of a Gene Specifies Structural Symmetry in Proteins

Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules.     

Mechanical roles of apical constriction,cell elongation,and cell migration during neural tube formation in Xenopus

Neural tube closure is an important and necessary process during the development of the central nervous system. The formation of the neural tube structure from a flat sheet of neural epithelium requires several cell morphogenetic events and tissue dynamics to account for the mechanics of tissue deformation. Cell elongation changes cuboidal cells into columnar cells, and apical constriction then causes them to adopt apically narrow, wedge-like shapes. In addition, the neural plate in Xenopus is stratified, and the non-neural cells in the deep layer (deep cells) pull the overlying superficial cells, eventually bringing the two layers of cells to the midline. Thus, neural tube closure appears to be a complex event in which these three physical events are considered to play key mechanical roles. To test whether these three physical events are mechanically sufficient to drive neural tube formation, we employed a three-dimensional vertex model and used it to simulate the process of neural tube closure. The results suggest that apical constriction cued the bending of the neural plate by pursing the circumference of the apical surface of the neural cells. Neural cell elongation in concert with apical constriction further narrowed the apical surface of the cells and drove the rapid folding of the neural plate, but was insufficient for complete neural tube closure. Migration of the deep cells provided the additional tissue deformation necessary for closure. To validate the model, apical constriction and cell elongation were inhibited in Xenopus laevis embryos. The resulting cell and tissue shapes resembled the corresponding simulation results.

     

Actomyosin contractility and microtubules drive apical constriction in Xenopus bottle cells

Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation, and organogenesis. However, the cell biology driving cell shape changes is poorly understood, especially in vertebrates. The beginning of Xenopus laevis gastrulation is marked by the apical constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a crevice at the blastopore lip. We found that bottle cells contribute significantly to gastrulation, as their shape change can generate the force required for initial blastopore formation. As actin and myosin are often implicated in contraction, we examined their localization and function in bottle cells. F-actin and activated myosin accumulate apically in bottle cells, and actin and myosin inhibitors either prevent or severely perturb bottle cell formation, showing that actomyosin contractility is required for apical constriction. Microtubules were localized in apicobasally directed arrays in bottle cells, emanating from the apical surface. Surprisingly, apical constriction was inhibited in the presence of nocodazole but not taxol, suggesting that intact, but not dynamic, microtubules are required for apical constriction. Our results indicate that actomyosin contractility is required for bottle cell morphogenesis and further suggest a novel and unpredicted role for microtubules during apical constriction.     

Movement of an Epithelial Layer Isolated from Early Embryos of the Newt, Cynops pyrrhogaster II. Formation of a Blastoporal Groove and Archenteron in a Superficial Epithelial Layer Isolated from Initial Gastrula

The roles of folding movement of epithelial layer in amphibian gastrulation were investigated. A superficial epithelial layer was isolated from the vegetal hemisphere of the initial gastrula (stage 11) of the newt, Cynops pyrrhogaster . The isolated epithelial layers were cultured and morphogenetic movements of the epithelial layers were analysed. Two types of folding movement, folding toward the apical side in the blastopore-forming region and folding toward the basal side in the dorsal marginal zone, arose autonomously in the cultured epithelial layers. These movements caused morphogenesis similar to the formation of the blastoporal groove and archenteron in the control embryo. Treatment with chemical reagents that affect the morphogenetic movement of cells and electron microscopy of the submembranous microfilaments layer (SML) suggested that contraction of actin filaments in the SML was involved in both types of folding movement but that they are controlled, respectively, by different mechanisms in terms of involvement of Ca²⁺ ions. The present results suggest that two types of folding movement arise in the superficial epithelial layer of the embryo and play important roles in the formation of the blastoporal groove and archenteron during early steps of amphibian gastrulation.     

Apical constriction: A cell shape change that can drive morphogenesis

Biologists have long recognized that dramatic bending of a cell sheet may be driven by even modest shrinking of the apical sides of cells. Cell shape changes and tissue movements like these are at the core of many of the morphogenetic movements that shape animal form during development, driving processes such as gastrulation, tube formation, and neurulation. The mechanisms of such cell shape changes must integrate developmental patterning information in order to spatially and temporally control force production—issues that touch on fundamental aspects of both cell and developmental biology and on birth defects research. How does developmental patterning regulate force-producing mechanisms, and what roles do such mechanisms play in development? Work on apical constriction from multiple systems including Drosophila, Caenorhabditis elegans, sea urchin, Xenopus, chick, and mouse has begun to illuminate these issues. Here, we review this effort to explore the diversity of mechanisms of apical constriction, the diversity of roles that apical constriction plays in development, and the common themes that emerge from comparing systems.     

Neural fold formation at newly created boundaries between neural plate and epidermis in the axolotl

According to a recent model, the cortical tractor model, neural fold and neural crest formation occurs at the boundary between neural plate and epidermis because random cell movements become organized at this site. If this is correct, then a fold should form at any boundary between epidermis and neural plate. To test that proposition, we created new boundaries in axolotl embryos by juxtaposing pieces of neural plate and epidermis that would not normally participate in fold formation. These boundaries were examined superficially and histologically for the presence of folds, permitting the following observations. Folds form at each newly created boundary, and as many folds form as there are boundaries. When two folds meet they fuse into a hollow "tube" of neural tissue covered by epidermis. Sections reveal that these ectopic folds and "tubes" are morphologically similar to their natural counterparts. Transplanting neural plate into epidermis produces nodules of neural tissue with central lumens and peripheral nerve fibers, and transplanting epidermis into neural plate causes the neural tube and the dorsal fin to bifurcate in the region of the graft. Tissue transplanted homotypically as a control integrates into the host tissue without forming folds. When tissue from a pigmented embryo is transplanted into an albino host, the presence of pigment allows the donor cells to be distinguished from those of the host. Mesenchymal cells and melanocytes originating from neural plate transplants indicate that neural crest cells form at these new boundaries. Thus, any boundary between neural plate and epidermis denotes the site of a neural fold, and the behavior of cells at this boundary appears to help fold the epithelium. Since folds can form in ectopic locations on an embryo, local interactions rather than classical neural induction appear to be responsible for the formation of neural folds and neural crest.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.     

Invagination of Ectodermal Placodes Is Driven by Cell Intercalation-Mediated Contraction of the Suprabasal Tissue Canopy

Ectodermal organs such as teeth, hair follicles, and mammary glands begin their development as placodes. These are local epithelial thickenings that invaginate into mesenchymal space. There is currently little mechanistic understanding of the cellular processes driving the early morphogenesis of these organs and of why they lead to invagination rather than simple tissue thickening. Here, we show that placode invagination depends on horizontal contraction of superficial layers of cells that form a shrinking and thickening canopy over underlying epithelial cells. This contraction occurs by cell intercalation and is mechanically coupled to the basal layer by peripheral basal cells that extend apically and centripetally while remaining attached to the basal lamina. This process is topologically analogous to well-studied apical constriction mechanisms, but very different from them both in scale and molecular mechanism. Mechanical cell–cell coupling is propagated through the tissue via E-cadherin junctions, which in turn depend on tissue-wide tension. We further present evidence that this mechanism is conserved among different ectodermal organs and is, therefore, a novel and fundamental morphogenetic motif widespread in embryonic development.     

Relaxation-expansion model for self-driven retinal morphogenesis: a hypothesis from the perspective of biosystems dynamics at the multi-cellular level

The generation of complex organ structures such as the eye requires the intricate orchestration of multiple cellular interactions. In this paper, early retinal development is discussed with respect to the structure formation of the optic cup. Although recent studies have elucidated molecular mechanisms of retinal differentiation, little is known about how the unique shape of the optic cup is determined. A recent report has demonstrated that optic-cup morphogenesis spontaneously occurs in three-dimensional stem-cell culture without external forces, indicating a latent intrinsic order to generate the structure. Based on this self-organizing phenomenon, we introduce the "relaxation-expansion" model to mechanically interpret the tissue dynamics that enable the spontaneous invagination of the neural retina. This model involves three consecutive local rules (relaxation, apical constriction, and expansion), and its computer simulation recapitulates the optic-cup morphogenesis in silico.     

Modelling apical constriction in epithelia using elastic shell theory

Apical constriction is one of the fundamental mechanisms by which embryonic tissue is deformed, giving rise to the shape and form of the fully-developed organism. The mechanism involves a contraction of fibres embedded in the apical side of epithelial tissues, leading to an invagination or folding of the cell sheet. In this article the phenomenon is modelled mechanically by describing the epithelial sheet as an elastic shell, which contains a surface representing the continuous mesh formed from the embedded fibres. Allowing this mesh to contract, an enhanced shell theory is developed in which the stiffness and bending tensors of the shell are modified to include the fibres’ stiffness, and in which the active effects of the contraction appear as body forces in the shell equilibrium equations. Numerical examples are presented at the end, including the bending of a plate and a cylindrical shell (modelling neurulation) and the invagination of a spherical shell (modelling simple gastrulation).     

Molecular responses of rat tracheal epithelial cells to transmembrane pressure

Smooth muscle constriction in asthma causes the airway to buckle into a rosette pattern, folding the epithelium into deep crevasses. The epithelial cells in these folds are pushed up against each other and thereby experience compressive stresses. To study the epithelial cell response to compressive stress, we subjected primary cultures of rat tracheal epithelial cells to constant elevated pressures on their apical surface (i.e., a transmembrane pressure) and examined changes in the expression of genes that are important for extracellular matrix production and maintenance of smooth muscle activation. Northern blot analysis of RNA extracted from cells subjected to transmembrane pressure showed induction of early growth response-1 (Egr-1), endothelin-1, and transforming growth factor-beta1 in a pressure-dependent and time-dependent manner. Increases in Egr-1 protein were detected by immunohistochemistry. Our results demonstrate that airway epithelial cells respond rapidly to compressive stresses. Potential transduction mechanisms of transmembrane pressure were also investigated.