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The Birth of Morphomechanics
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The morphogenic process of convergent thickening (CT) was originally described as the mediolateral convergence and radial thickening of the explanted ventral involuting marginal zone (IMZ) of Xenopus gastrulae (Keller and Danilchik 1988). Here we show that CT is expressed in all sectors of the pre-involution IMZ, which transitions to expressing convergent extension (CE) after involution. CT occurs without CE and drives symmetric blastopore closure in ventralized embryos. Assays of tissue affinity and tissue surface tension measurements suggest CT is driven by increased interfacial tension between the deep IMZ and the overlying epithelium. The resulting minimization of deep IMZ surface area drives a tendency to shorten the mediolateral (circumblastoporal) aspect of the IMZ, thereby generating tensile force contributing to blastopore closure (Shook et al. 2018). These results establish CT as an independent force-generating process of evolutionary significance and provide the first clear example of an oriented, tensile force generated by an isotropic, Holtfreterian/Steinbergian tissue affinity change.
Cleavage is the earliest developmental stage. During this stage, the fertilized oocyte gives rise to a cluster of smaller cells (blastomeres) with a particular spatial pattern (a cleavage pattern). Different metazoan species have different cleavage patterns, but most of them fit into a small set of basic types. The relationship between the phylogeny of a given species and its cleavage pattern is far from direct, but most taxa seem to use the same basic cell processes (such as directed cell division or cell adhesion) to build their cleavage patterns. We assess which are those mechanisms in the first section of this chapter. In a second section, we explore how the combined action of these mechanisms can account for the emergence of particular cleavage patterns in different metazoan taxa and the evolutionary transitions between them.
Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells’ fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies.
Living cells are constantly exposed to mechanical stimuli arising from the surrounding extracellular matrix (ECM) or from neighboring cells. The intracellular molecular processes through which such physical cues are transformed into a biological response are collectively dubbed as mechanotransduction and are of fundamental importance to help the cell timely adapt to the continuous dynamic modifications of the microenvironment. Local changes in ECM composition and mechanics are driven by a feed forward interplay between the cell and the matrix itself, with the first depositing ECM proteins that in turn will impact on the surrounding cells. As such, these changes occur regularly during tissue development and are a hallmark of the pathologies of aging. Only lately, though, the importance of mechanical cues in controlling cell function (e.g., proliferation, differentiation, migration) has been acknowledged. Here we provide a critical review of the recent insights into the molecular basis of cellular mechanotransduction, by analyzing how mechanical stimuli get transformed into a given biological response through the activation of a peculiar genetic program. Specifically, by recapitulating the processes involved in the interpretation of ECM remodeling by Focal Adhesions at cell-matrix interphase, we revise the role of cytoskeleton tension as the second messenger of the mechanotransduction process and the action of mechano-responsive shuttling proteins converging on stage and cell-specific transcription factors. Finally, we give few paradigmatic examples highlighting the emerging role of malfunctions in cell mechanosensing apparatus in the onset and progression of pathologies.
Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 mN during gastrulation and over 4 mN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 mN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms.
The ability of cells to respond to mechanical forces is critical for numerous biological processes. Emerging evidence indicates that external mechanical forces trigger changes in nuclear envelope structure and composition, chromatin organization and gene expression. However, it remains unclear if these processes originate in the nucleus or are downstream of cytoplasmic signals. Here we discuss recent findings that support a direct role of the nucleus in cellular mechanosensing and highlight novel tools to study nuclear mechanotransduction.
Cell adhesion and cell–cell contacts are pre-requisites for proper metabolism, protein synthesis, cell survival, and cancer metastasis. Major transmembrane receptors are the integrins, which are responsible for cell matrix adhesions, and the cadherins, which are important for cell-cell adhesions. Adherent cells are anchored via focal adhesions (FAs) to the extracellular matrix, while cell-cell contacts are connected via focal adherens junctions (FAJs). Force transmission over considerable distances and stress focusing at these adhesion sites make them prime candidates for mechanosensors. Exactly which protein(s) within FAs and FAJs or which membrane component of ion channels sense, transmit, and respond to mechano-chemical signaling is currently strongly debated and numerous candidates have been proposed.
Arrays of microposts of different heights generate substrates with different flexibility, on which cells can be grown.
Recently, the term tensotaxis was proposed to describe the phenomenon that tensile stress or strain affects cell migration. Even so, less attention has been paid to the effects of compressive stress on cell behavior. In this study, by using an injection-molded method combined with photoelastic technology, we developed residual stress gradient-controlled poly-L-lactide discs. After culturing NIH-3T3 fibroblasts on the stress gradient substrate, the cell distributions for high- and low-stress regions were measured and compared. Our results showed that there were significantly more cells in the low-compressive stress region relative to their high-stress analogs (p < 0.05). In addition, NIH-3T3 fibroblasts in the low-compressive stress region expressed more abundant extensive filopodia. These findings provide greater insight into the interaction between cells and substrates, and could serve as a useful reference for connective tissue development and repair.
Research in cellular mechanotransduction often focuses on how extracellular physical forces are converted into chemical signals at the cell surface. However, mechanical forces that are exerted on surface-adhesion receptors, such as integrins and cadherins, are also channelled along cytoskeletal filaments and concentrated at distant sites in the cytoplasm and nucleus. Here, we explore the molecular mechanisms by which forces might act at a distance to induce mechanochemical conversion in the nucleus and alter gene activities.
In Xenopus laevis embryos at the early gastrula stage, circumferential tensions of embryonic ectoderm were relaxed by making sagittal or transversal slits in the ventral parts of embryos and inserting into surgical cuts the sectors of homologous tissue from same-stage embryos. Changes in tensile patterns were controlled by measuring cell surface angles. Immediate decreases in surface cell wall tension as related to transversal wall tension were registered. Within minutes of the operation, the lobopodial activity of the inner ectodermal surface increased. The subsequent gastrulation movements were disturbed, germ layers partially mixed and archenteron reduced. The areas of extensive cell columnarization in the ectoderm of operated embryos were less regularly arranged and were extended much more ventrally than in intact embryos. Ventro-dorsal migration and latero-medial intercalation of mesodermal cells also were suppressed. As the operated embryos developed, we observed increases in the total amount of neural tissue, associated sometimes with duplication and even triplication of neural tubes, duplication of otic vesicles, partial fusion of axial rudiments, suppression of mesodermal segmentation and branching or bending of notochord. In the gravest cases the antero-posterior embryo polarity was disturbed. In some cases we observed the formation of axial rudiments in ventral implants. The role of tensions in determining the patterns of morphogenetic cell movements and in establishing the morphological order of normal development is discussed.
It is well known that in embryonic tissues at the key stages of morphogenesis there arise stable, stage--specific tension fields. These fields occur due to particular pattern of morphologically polarized cells. Some basic properties have been understood previously. 1. Morphologically polarized and isotropic shapes correspond to the alternative stable states of embryonic cells. 2. Polarization can be transmitted between the adjacent cells via intercellular contacts. 3. The tension fields at particular stage of development determine the pattern of morphogenetic movements. In this paper the physical model is suggested which interprets the selforganization of tension fields in embryonic tissues. The polarization in some region of tissue is assumed to generate the elastic tension in the surrounding cells thus restricting the propagation of cell polarization. It is shown that the properties underlined are sufficient to provide spontaneous subdivision of the cellular layer into the domains of polarized and unpolarized stretched cells. The proportion of polarized and unpolarized areas is determined and size--invariant.
Reactions of embryonic tissues to a distributed and concentrated stretching are described and compared with the mechanics of the normal gastrulation movements. A role of mechanically stressed dynamic cell structures in the gastrulation, demarcation of notochord borders and in providing proportionality of the axial rudiments is demonstrated. A morphomechanical scheme of amphibian gastrulation is presented.
The first explanations of the mechanisms of development of living organisms were proposed in antiquity. At that time two competing ideas existed, about the strict determination of embryonic structures (we call it the “Hippocrates line”) and about the possible formation of structures from the unstructured condition (“Aristotle line”). We can trace the opposition between the “Hippocrates line” and “Aristotle line” from antiquity till the present time. At the end of the XIX century, experimental investigation of the mechanisms of integrity of development had started. In the XX century, the “Aristotle line” finds its expression in the Morphogenetic Field Theory of A.G. Gurwitsch, according to which cells of the organism are integrated in an organic whole. Since the 1970s, mechanical forces and tensions have been considered as integral factors of ontogenesis. One of the most productive scientific teams which worked in this area was the laboratory of Professor L.V. Beloussov from the Lomonossov Moscow State University, Russia. In the 1970s, Lev Beloussov and his colleagues discovered the presence of “passive” and “active” (i.e. metabolically-dependent) mechanical stresses in the tissues of developing organisms, their organization and stage-specific patterns. In 1980–1990 s, a lot of experimental data about the role of the patterns of mechanical stresses in morphogenesis and cell differentiation was accumulated. Based on the experimental data, Professor Beloussov and his colleagues developed a theory of the regulation of the development of living organisms on the basis of the interaction of passive and active mechanical stresses (Belousov-Mittenthal Theory), which forms the basis of a new science – morphomechanics.
We have revised a mathematical model of epithelial morphogenesis by Belintsev et al. (1987) (BB model) taking into account the oscillatory nature of morphogenesis and stability analysis (Cherdantsev, 2014). Following the BB model in considering the feedback control of cell shape changes by mechanical forces, we modify it to represent epithelial surface movements observed in different types of Metazoan gastrulation. Basing on these observations, we argue that the epithelial surface movement is that of an incompressible fluid supplemented by a positive feedback between the movement and spreading of the surface flow. Dipole interactions between sources and sinks of surface energy provide a single mechanism both of short-ranged and long-ranged regulation of collective cell and surface movements whose basic variables are the space averaged epithelial surface curvature and lateral pressure within the epithelial surface flux negatively related to its velocity. The short-ranged activation means a movement of the surface up to the lateral pressure gradient under non-linear feedback control of the surface flexure. The break of this feedback with equalization of the surface curvature is sufficient for a self-restriction of the movement spreading. Owing to bistable interdependence between the lateral pressure and epithelial surface curvature, we get a generic oscillatory contour in which the same region oscillates being alternately a sink and source of the surface flow. The opposite phase oscillations of the lateral pressure and curvature allow for both directional propulsion of the surface through the same region and spatial differentiation based on parametric differences between the large-scaled regions that correspond to sources and sinks of the surface.
In 1935 Ervin Bauer formulated a basic principle of organization of living matter defined as the stable non-equilibrium state. The homeostatic stable non-equilibrium is realized internally by selecting the trajectories supporting stable configurations from those disturbing it and supported by the dynamical structure of metabolism consisting of metabolic cycles, feedback loops and feedforward constraints. In the developing systems, according to Bauer, the principle of stable non-equilibrium is transformed into the principle of increasing external work which is grounded in the hyper-restorative non-equilibrium. The basis of this principle, as formulated by Lev Beloussov, is a hyper-restorative process during conformational relaxation of biomacromolecules which adds extra energy in the system and governs its complexification in which a new optimal coordinate pattern is searched. At the quantum level, this complexification is determined by the parametric refinement process in the field of possibilities. The complexification process takes place at the level of the cytoskeleton which represents a macroscopic enzymatic system and can generate differentiation waves that spread between cells. This may be associated with the function of cytoskeleton of transmitting signals and generating impulses. Different types of waves during morphogenesis correspond to different ranges of wavelengths of emission, from biophoton radiation to the hypersound waves in transmission of neural impulses. It is concluded that biological morphogenesis is based on the hyper-restorative non-equilibrium supported by the functional structure of the cytoskeleton.
Mechanical forces and interactions participate in ontogenesis at all scale levels: intracellular, cellular, and supra-cellular, the latter including tissue level. This concept, now almost trivial, was finding its way with difficulties, and the works of L.V. Beloussov have played a decisive role in its establishment. The continuum approach presented in this study makes it possible to take at the tissue level into account both relative motion of cells and forces that control this motion. The characteristics which allow us to take into account general active properties of the cell medium are described, possible mechanisms represented by these characteristics are discussed, and a concise review of our results obtained to date is presented. In the strain rate tensor, two separate components are distinguished, one of them being related to deformation of individual cells and the other to cell rearrangement. A separate phase (submedium) that corresponds to active subcellular elements associated with rearrangement-controlling active stresses is also introduced. Within this general approach two specific models are considered. The first made it possible to establish general mechanisms whose account enabled us to satisfactorily describe the experimental results of L.V. Beloussov and collaborators, concerning mechano-dependent reactions of embryonic epithelium explants. On the assumptions that the active stress responds to cell shape deviations and the rearrangement strain rate component depends on the active stresses developed by pseudopodia, the cell shape and tissue stress evolution observed experimentally in stretched explants, as well as their post-release deformation, are reproduced. The second particular model considers self-organization in a conglomerate of loosely connected cells in the presence of a fluid phase. In this case, the active stress was assumed to nonlocally depend on the density of cells and the rearrangement strain rate on the active and passive stresses. Due to loss of stability of the spatially homogeneous state, various structures similar to those observed in embryogenesis develop. In particular, within the conglomerate, a cavity can be formed, a certain level of the fluid pressure being necessary for this.
This reference work provides an comprehensive and easily accessible source of information on numerous aspects of Evolutionary Developmental Biology. The work provides an extended overview on the current state of the art of this interdisciplinary and dynamic scientific field. The work is organized in thematic sections, referring to the specific requirements and interests in each section in far detail. “Evolutionary Developmental Biology – A Reference Guide” is intended to provide a resource of knowledge for researchers engaged in evolutionary biology, developmental biology, theoretical biology, philosophy of sciences and history of biology.
https://link.springer.com/referencework/10.1007/978-3-319-33038-9
There is still no consensus on the mechanisms regulating the formation and maintenance of morphological structures in the individual development of living organisms. The hypothesis that the mechanical forces are important for biological morphogenesis was put forward more than 100 years ago. In recent decades, studies indicating the regulatory role of mechanical stresses at different levels of organization of life have appeared. The signaling mechanisms that are responsible for the reception of mechanical influences and reprogramming of the properties of cells and tissues are studied. Since the mid-1970s, the principles of selfstressed structures or the tensegrity (tensional integrity) theory have been applied to understand the structure and functions of living structures in statics and dynamics. According to this standpoint, the cell can be represented as a self-stressed structure in which microtubules function as rigid rods and microfilaments serve as elastic threads. Such a system is anchored to extracellular matrix through cellular contacts, since it is adjusted to the external patterns of mechanical stresses. The notion of living systems as self-stressed structures provides a fresh look at the mechanotransduction, developing organism integrity, and biological morphogenesis. Although the application of the ideas of tensegrity to biological systems has not yet received broad support among biologists, the influence of these ideas on the formation of modern mechanobiology and understanding the crucial role of cytoskeletal structures in cellular processes should be mentioned.
It is well established that cells sense chemical signals from their local microenvironment and transduce them to the nucleus to regulate gene expression programmes. Although a number of experiments have shown that mechanical cues can also modulate gene expression, the underlying mechanisms are far from clear. Nevertheless, we are now beginning to understand how mechanical cues are transduced to the nucleus and how they influence nuclear mechanics, genome organization and transcription. In particular, recent progress in super-resolution imaging, in genome-wide application of RNA sequencing, chromatin immunoprecipitation and chromosome conformation capture and in theoretical modelling of 3D genome organization enables the exploration of the relationship between cell mechanics, 3D chromatin configurations and transcription, thereby shedding new light on how mechanical forces regulate gene expression.
The seminal observation that mechanical signals can elicit changes in biochemical signalling within cells, a process commonly termed mechanosensation and mechanotransduction, has revolutionized our understanding of the role of cell mechanics in various fundamental biological processes, such as cell motility, adhesion, proliferation and differentiation. In this Review, we will discuss how the interplay and feedback between mechanical and biochemical signals control tissue morphogenesis and cell fate specification in embryonic development. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Cell movements during embryogenesis produce mechanical tensions that shape the embryo and can also regulate gene expression, thereby affecting cell differentiation. Increasing evidence indicates that mechano-sensitive regulation of gene expression plays important roles during embryogenesis by coupling the processes of morphogenesis and differentiation. However, the molecular mechanisms of this phenomenon remain poorly understood. This review focuses on the molecular mechanisms that "translate" mechanical stimuli into gene expression. This article is protected by copyright. All rights reserved.
The transcriptional regulators YAP and TAZ are the focus of intense interest given their remarkable biological properties in development, tissue homeostasis and cancer. YAP and TAZ activity is key for the growth of whole organs, for amplification of tissue-specific progenitor cells during tissue renewal and regeneration, and for cell proliferation. In tumors, YAP/TAZ can reprogram cancer cells into cancer stem cells and incite tumor initiation, progression and metastasis. As such, YAP/TAZ are appealing therapeutic targets in cancer and regenerative medicine. Just like the function of YAP/TAZ offers a molecular entry point into the mysteries of tissue biology, their regulation by upstream cues is equally captivating. YAP/TAZ are well known for being the effectors of the Hippo signaling cascade, and mouse mutants in Hippo pathway components display remarkable phenotypes of organ overgrowth, enhanced stem cell content and reduced cellular differentiation. YAP/TAZ are primary sensors of the cell's physical nature, as defined by cell structure, shape and polarity. YAP/TAZ activation also reflects the cell "social" behavior, including cell adhesion and the mechanical signals that the cell receives from tissue architecture and surrounding extracellular matrix (ECM). At the same time, YAP/TAZ entertain relationships with morphogenetic signals, such as Wnt growth factors, and are also regulated by Rho, GPCRs and mevalonate metabolism. YAP/TAZ thus appear at the centerpiece of a signaling nexus by which cells take control of their behavior according to their own shape, spatial location and growth factor context.
Morphogenesis is the remarkable process by which cells self-assemble into complex tissues and organs that exhibit specialized form and function during embryological development. Many of the genes and chemical cues that mediate tissue and organ formation have been identified; however, these signals alone are not sufficient to explain how tissues and organs are constructed that exhibit their unique material properties and three-dimensional forms. Here, we review work that has revealed the central role that physical forces and extracellular matrix mechanics play in the control of cell fate switching, pattern formation, and tissue development in the embryo and how these same mechanical signals contribute to tissue homeostasis and developmental control throughout adult life.
Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.
The movements of blastomere surfaces marked with carbon particles during cytokinesis of the Ist–IVth cleavage divisions in the eggs of the gastropodsLymnaea stagnalis, L. palustris, Physa acuta and Ph. fontinalis have been studied by time-lapse cinematographic methods. The vitelline membrane was removed with trypsin. At 2- and 4-cell stages shifts of nuclei have also been studied.Symmetrical as well as asymmetrical surface movements (in respect to the furrow plane) have been revealed.
Symmetrical surface movements at the beginning of cytokinesis consist mainly in contraction of the furrow zone and in expansion of the more peripheral regions; between these there is a stationary zone. After the end of cytokinesis the furrow region expands.Considerableasymmetrical surface movements have also been observed in all four divisions. From anaphase until the end of cytokinesis each of the two sister blastomeres rotates with respect to the other in such a way, that if viewed along the spindle axis, the blastomere nearest to the observer rotates dexiotropically in a dextral species and laeotropically in a sinistral species (primary rotations). After the completion of cytokinesis the blastomeres may rotate in a reverse direction. The latter rotations are less pronounced in the IInd and IIIrd divisions and most pronounced in the IVth division. Blastomeres with the vitelline membrane intact retain a slight capacity for primary rotations. In normal conditions nuclei of the first two blastomeres shift mainly laeotropically in dextral species, but dexiotropically in sinistral species, being carried along by the reverse surface rotations.The invariable primary asymmetrical rotations of blastomeres seem to be the basis of enantiomorphism in molluscan cleavage. They are assumed to be determined by an asymmetrical structure of the contractile ring carrying out the cytokinesis.
Time-lapse videomicrographic and SEM analyses of normal and microsurgically altered gastrulation show that the morphogenetic movements of the dorsal marginal zone (DMZ)—extension, convergence, and involution—all result from behavior that occurs after the marginal zone has involuted. Before its involution, the DMZ shows no detectable capacity for autonomous convergence or extension. If its involution is prevented, the DMZ will show convergence and extension but only at developmental stages at or beyond the stage at which it normally would have involuted. Thus autonomous convergence and extension, which have been ascribed to the DMZ are, in fact, properties of the dorsal mesodermal mantle (DMM) and the archenteron roof. SEM analysis of cell shape and packing patterns, suggest that cells of the DMM merge (interdigitate) mediolaterally, between one another, beginning just beyond the point of involution. This behavior is thought to reduce the width and increase the length (postinvolution convergence and extension) of the DMM. The decrease in circumference (width) at the vegetal-most part of the newly involuted DMM forms a constriction ring just inside the blastopore. Constriction and concurrent elongation of the DMM act in concert to move the blastoporal lip vegetally. The DMZ is passively pulled vegetally and over the blastoporal lip as deep cells are recruited for participation in mediolateral interdigitation at the vegetal end of the DMM.
Dynamic mechanical processes shape the embryo and organs during development. Little is understood about the basic physics of these processes, what forces are generated, or how tissues resist or guide those forces during morphogenesis. This review offers an outline of some of the basic principles of biomechanics, provides working examples of biomechanical analyses of developing embryos, and reviews the role of structural proteins in establishing and maintaining the mechanical properties of embryonic tissues. Drawing on examples we highlight the importance of investigating mechanics at multiple scales from milliseconds to hours and from individual molecules to whole embryos. Lastly, we pose a series of questions that will need to be addressed if we are to understand the larger integration of molecular and physical mechanical processes during morphogenesis and organogenesis.
Mechanical deformations associated with embryonic morphogenetic movements have been suggested to actively participate in the signaling cascades regulating developmental gene expression. Here we develop an appropriate experimental approach to ascertain the existence and the physiological relevance of this phenomenon. By combining the use of magnetic tweezers with in vivo laser ablation, we locally control physiologically relevant deformations in wild-type Drosophila embryonic tissues. We demonstrate that the deformations caused by germ band extension upregulate Twist expression in the stomodeal primordium. We find that stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/beta-catenin, which is required for Twist mechanical induction in the stomodeum. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, resulting in larval lethality. These experiments show that mechanically induced Twist upregulation in stomodeal cells is necessary for subsequent midgut differentiation.
Submicroscopic rearrangements are described. They serve as the bases of rapid (up to 20 min) changes in the form of the common frog neurula explants: formation of filamentous layer under "naked" surface, appearance of lobopodia on "naked" surface, their "flow", cell polarization and submersion. In all these processes an active part appears to be played by microtubules and microfilaments the bundles of which are always oriented along the long axes of active cells or the directions of passive mechanical tensions. In the cells which are not yet polarized the microtubules form under the surface adjacent to the already polarized cell. This may be considered as one of the chains of cooperative cell polarization.
The movements of cells in the oral field of anuran embryos were followed by means of orto- and heterotopic transplantations of the ectoderm fragments. To mark individual ectoderm regions, the embryos were used which differed from each other by colour. The cells polarize along the apical-basal layer axis and the polarization spreads along the layer as a waver embracing consecutively new and new cells. The waves of morphogenetic rearrangements from the spatial deploiments of morphogenetic movement, i. e. the transition between the local morphologies of the layer which replace one another in time is swept continuously in space. The presence of spatial sweep is the necessary condition for the structural stability of morphogenetic movements.
The lines of mechanical tension (cross-lines) in axial rudiments of the amphibian embryo represent bands of polarized cells. They form in the inner layers of the rudiments as separate bundles of polarized cell which, then, merge, attain the external surface and gather in lengthy planes (cross-planes) and, later, degrade. The primary inductor induces the formation of cross-lines in the ventral ectoderm of the early gastrula. The growth of cross-lines in considered as one of the types of contact cells polarization. The morphogenetic role of contact polarization is discussed. The connection between the subsequent tension patterns is based on the fact that the lines of exit of the cross-planes on the surface of the embryo coincide with the direction of the previously established tensions.
The concept of morphogenesis is determined and the mathematical image of the developing system is considered. A certain amount of stable and unstable states and slow changes of the potential relief (parameters) are inherent in the latter. The developing systems are intermediate between the deterministic and statistic ones. They are distinctly multiple-levelled. The microprocesses of morphogenesis and the laws of macromorphogenesis are described, the instabilities and stable periods of morphogenesis are considered. All of them in the multiple-leveled system tend to the formation of through hierarchies, or cascades, where the upper levels parametrize the lower ones. This tendency increases as the evolutionary progress proceeds. The genetic regulation is considered also as a parametric regulation in the domains of instabilities. The approach contemplated may be considered both as the generalization of the available data and as the programme of investigation, more adequate to the biological reality than the causal-analytical methodology.
The still unsegemented axial mesoderm was cross cut at different distances form the last somite under the normal physiological conditions, at the low temperature and under the effect of cytochalasin B. Under the normal conditions the inner cells of the axial mesoderm formed a lens-shaped cleft immediately after the operation; this movement was suppressed at the low temperature and absent under the effect of cytochalasin B. This suggests the active contractility of the mesodermal syncytium. The wound margin near the last somite acquired the epithelial structure already within several minutes after the cut, whereas in the more caudal regions it preserved the connective tissue character for a long time. The importance of the axial mesoderm contractility and its ability of epithelization for the somitogenesis is discussed.
The immediate relaxation deformations have been studied in the embryonic frog tissues from the late blastula stage till the early tail bud stage. As a result, the maps of mechanical stresses were constructed which were characterized by the existence of distinct tension-lines dissecting the embryonic tissues (cross-lines). A few discrete moments of development were established when new cross-lines formed or the previous ones disappeared; they are separated by the topologically invariant periods of development. The cross-lines play an important role in morphogenesis in that they determine the ways of active cell migration and trace the boundaries between different anlages. The orientation of the cross-lines coincides, as a rule, with the direction of predominant tension of tissues during the preceding topologically invariant period of development.
Changes in the shape and cell architecture of pieces of epithelial and neural ectoderm, mesoderm, neural tube, and combined ectomesodermal fragments from embryos of Rana temporaria 0-60 min after isolation were studied. The fragments were capable of changing their shape quickly (actually during separation) or after a latent period of several minutes. Rapid deformations were not prevented by cooling or by moderate doses of cyanide; as a rule they were connected with contraction of the surface area of the cells of the fragment and they can be regarded as relaxation to forms with lower mechanical energy. The direction of the deformation usually coincides with the subsequent normal morphogenesis of the particular anlage. Deformations with a latent period are suppressed by cooling and by the addition of cyanide, which lead to an increase in the surface area of individual cells, but they reduce the total surface area of the fragment. The shape of the fragments becomes more complex: they become irregularly twisted, they form folds, and they separate into spherical regions with stretched surfaces ("drops"). These processes are connected with the performance of positive mechanical work by the intracellular contractile systems. The reasons why the fragments become more complex in shape are discussed.
1. Embryos of Rana temporaria have been dissected and shape alterations of different parts of the embryo, taking place within 1 h of separation, have been studied. Two categories of deformation have been revealed.
2. The first category comprises those deformations which take place immediately after separation. They are insensitive to cooling, cyanide and Cytochalasin B treatment. These deformations, which consist of a shortening of initially elongated cells, are considered to be the passive relaxations of previously established elastic tensile stresses.
3. Deformations of the second category proceed more slowly. They are inhibited by cooling, cyanide and Cytochalasin B treatment, are accompanied by elongation and migration of cells and occasionally lead to rather complex morphodifferentiations of isolated fragments. These processes are considered to be the result of the active work of intracellular contractile systems, either pre-existing or induced de novo.
4. By analysing the arrangement of the passive deformations we have constructed maps of mechanical stresses in embryos from late blastula up to the early tail-bud stage. At several embryonic stages drastic transformations of the stress pattern occur, these transformations being separated by periods during which the pattern of stress distribution remains topologically constant.
5. A correlation between the arrangement of stress lines and the presumptive morphological pattern of the embryo is pointed out.
6. Some possible relations between tensile tissue stresses and active mechanochemical processes are discussed.
One of the most universal events in morphogenesis is the formation of domains of morphologically polarized cells in the initially homogeneous epithelial sheets. We investigate the possibility of considering this process as a phenomenon of self-organization which is based upon the following experimentally proven mechanochemical cell properties: (1) a capacity of individual cells for morphological polarization considered as a bistable "all-or-none" transition of a cell from a non-polarized to a polarized state; (2) transmission of this capacity from one cell to another on their contacts; (3) feedback relations between co-operative cell polarization and tangential elastic tensions in a cell sheet: cell polarization increases tangential tensions whereas the latter inhibit further cell polarization. We have constructed a phenomenological model which formally expresses the above properties. Its mathematical description includes but few macroscopic parameters available to experimental investigation and controlled changes. The analysis of the collective dynamic regimes of cell polarization demonstrates that variations of some non-specific parameters leads to spontaneous transition in the morphology of cell layers accompanied by symmetry breaking (Turing's instability). Under these conditions either long-range ordered patterns of cell polarization (including hexagonal cell nets) or non-regular spotted structures can emerge. In the particular case of a sheet having fixed complete dimensions and lacking any external elastic bonds a stable macrostate is created; it corresponds to the sheet's binary subdivision into polarized and non-polarized cell domains of size-invariant proportions. The model conclusions are compared with the morphogenetical processes in sea-urchin development, the morphogenesis of skin derivates and artificially induced budding in hydrozoa.
The locomotory behavior of tissue cells cultured on various artificial substrata was studied by time-lapse cinemicrography. Cells were able to spread more completely on certain more wettable substrata and to accumulate preferentially on these substrata according to a consistent hierarchy of cell-substratum affinity, which was the same for all cell types. Cell responses to variations in substrata suggest that substratum adhesiveness is the determining factor, but that cells accumulate on more adhesive substrata as the result of unequal competition between several actively locomotory ruffled lamellae around their margin. The increased overlapping between cells cultured on less adhesive substrata was found to be attributable to factors other than a decrease of contact inhibition of locomotion.
In the explants of lateral mesoderm, together or without the overlaying ectoderm, taken from the neurula-early tail bud of amphibian embryos (Xenopus laevis, Rana temporaria) within 5-30 min, several types of cytological transformations have been observed, some of these being: formation of a microfilamentous layer under the naked explant surface, morphological polarization, and unilateral spreading of cells. These processes are described at the light and electron microscopy levels. It is suggested that these may be of relay character, that is, each transformed cell induces the same transformation in the adjacent one. The cytological transformations are correlated with metabolic ones described elsewhere.
We have studied the generation of spatial patterns created by mechanical (rather than chemical) instabilities. When dissociated fibroblasts are suspended in a gel of reprecipitated collagen, and the contraction of the gel as a whole is physically restrained by attachment of its margin to a glass fibre meshwork, then the effect of the fibroblasts' traction is to break up the cell-matrix mixture into a series of clumps or aggregations of cells and compressed matrix. These aggregations are interconnected by linear tracts of collagen fibres aligned under the tensile stress exerted by fibroblast traction. The patterns generated by this mechanical instability vary depending upon cell population density and other factors. Over a certain range of cell concentrations, this mechanical instability yields geometric patterns which resemble but are usually much less regular than the patterns which develop normally in the dermis of developing bird skin. We propose that an equivalent mechanical instability, occurring during the embryonic development of this skin, could be the cause not only of the clumping of dermal fibroblasts to form the feather papillae, but also of the alignment of collagen fibres into the characteristic polygonal network of fibre bundles - which interconnect these papillae and which presage the subsequent pattern of the dermal muscles serving to control feather movements. More generally, we suggest that this type of mechanical instability can serve the morphogenetic functions for which Turing's chemical instability and other reaction-diffusion systems have been proposed. Mechanical instabilities can create physical structures directly, in one step, in contrast to the two or more steps which would be required if positional information first had to be specified by chemical gradients and then only secondarily implemented in physical form. In addition, physical forces can act more quickly and at much longer range than can diffusing chemicals and can generate a greater range of possible geometries than is possible using gradients of scalar properties. In cases (such as chondrogenesis) where cell differentiation is influenced by the local population density of cells and extracellular matrix, the physical patterns of force and distortion within this extracellular matrix should even be able to accomplish the spatial control of differentiation, usually attributed to diffusible 'morphogens'.
When tissue cells are cultured on very thin sheets of cross-linked silicone fluid, the traction forces the cells exert are
made visible as elastic distortion and wrinkling of this substratum. Around explants this pattern of wrinkling closely resembles
the "center effects" long observed in plasma clots and traditionally attributed to dehydration shrinkage.