Gastrulation in amphibian embryos, regarded as a succession of biomechanical feedback events

Gastrulation in amphibian embryos is a composition of several differently located morphogenetic movements which are perfectly coordinated with each other both in space and time. We hypothesize that this coordination is mediated by biomechanical interactions between different parts of a gastrulating embryo based upon the tendency of each part to hyper-restore the value of its mechanical stress. The entire process of gastrulation in amphibian embryos is considered as a chain of these mutually coupled reactions, which are largely dependent upon the geometry of a given embryo part. We divide gastrulation into several partly overlapped steps, give a theoretical interpretation for each of them, formulate the experiments for testing our interpretation and describe the experimental results which confirm our point of view. Among the predicted experimental results are: inhibition of radial cell intercalation by relaxation of tensile stresses at the blastula stage; inversion of convergent intercalation movements by relaxation of circumferential stresses at the early gastrula stage; stress-promoted reorientation of axial rudiments, and others. We also show that gastrulation is going on under a more or less constant average value of tensile stresses which may play a role as rate-limiting factors. A macro-morphological biomechanical approach developed in this paper is regarded as complementary to exploring the molecular machinery of gastrulation.     

On the work of the developmental biophysics laboratory of the embryology department of Moscow State University

The laboratory is engaged in morphomechanics—the study of self-organization of mechanical forces that create the shape and structure of the embryonic primordia. As part of its work, the laboratory described pulsating modes of mechanical stresses in hydroids, identified and mapped mechanical stresses in the tissues of amphibian embryos, and studied morphogenetic reorganization caused by the relaxation and reorientation of tensions. The role of mechanical stresses in maintaining the orderly architectonics of the embryo is shown. Mechano-dependent genes are detected. Microstrains of embryonic tissues and stress gradients associated with them are described. A model of hyper-recovery of mechanical stresses as a possible driving force of morphogenesis is proposed.     

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.     

Mechanical feedback in morphogenesis and cell differentiation

Studies of mechanical stresses and mechanical feedback at the cell level are reviewed. It is shown that cells and embryonic tissues respond to external mechanical stresses and can generate such stresses themselves. Regular feedback loops between external (passive) and internal (active) mechanical stresses have been established. They are essential for cell survival, determination of the direction of their differentiation, and selforganization of morphogenetic processes. Relevant experimental data are presented, and models of mechanical feedback loops are discussed.     

Mechanical stress inference for two dimensional cell arrays

Many morphogenetic processes involve mechanical rearrangements of epithelial tissues that are driven by precisely regulated cytoskeletal forces and cell adhesion. The mechanical state of the cell and intercellular adhesion are not only the targets of regulation, but are themselves the likely signals that coordinate developmental process. Yet, because it is difficult to directly measure mechanical stress in vivo on sub-cellular scale, little is understood about the role of mechanics in development. Here we present an alternative approach which takes advantage of the recent progress in live imaging of morphogenetic processes and uses computational analysis of high resolution images of epithelial tissues to infer relative magnitude of forces acting within and between cells. We model intracellular stress in terms of bulk pressure and interfacial tension, allowing these parameters to vary from cell to cell and from interface to interface. Assuming that epithelial cell layers are close to mechanical equilibrium, we use the observed geometry of the two dimensional cell array to infer interfacial tensions and intracellular pressures. Here we present the mathematical formulation of the proposed Mechanical Inverse method and apply it to the analysis of epithelial cell layers observed at the onset of ventral furrow formation in the Drosophila embryo and in the process of hair-cell determination in the avian cochlea. The analysis reveals mechanical anisotropy in the former process and mechanical heterogeneity, correlated with cell differentiation, in the latter process. The proposed method opens a way for quantitative and detailed experimental tests of models of cell and tissue mechanics.     

The Effect of Temperature Gradients on Stress Development During Cryopreservation via Vitrification

This study addresses the problem of thermal stress development in bulky specimens during cryopreservation via vitrification (vitreous means glassy in Latin). While this study is a part of an ongoing effort to associate the developing mechanical stress with the relevant physical properties of the cryopreserved media and to its the thermal history, the current paper focuses exclusively on the role of temperature gradients. Temperature gradients arise due to the high cooling rates necessary to facilitate vitrification; the resulting non-uniform temperature distribution leads to differential thermal strain, possibly resulting in cracking. The cooling rate is assumed constant on the outer surface in this study, and the material properties are assumed constant. It is demonstrated that under these assumptions, mechanical stress develops only when the temperature distribution in the specimen approaches thermal equilibrium at a cryogenic storage temperature. It is shown that the maximum possible stresses for a given cooling rate can be computed with a simple thermo-elastic analysis; these stresses are associated with cooling to sufficiently low temperatures and are independent of the variation of viscosity with temperature. Analytic estimates for these stresses are obtained for several idealized shapes, while finite element analysis is used to determine stresses for geometries used in cryopreservation practice. Stresses that develop under a wider range of storage temperatures are also studied with finite element analysis, and the results are summarized with suitable normalizations. It is found that no stresses arise if cooling ceases above the set-temperature, which defines the transition from viscous-dominated to elastic-dominated behavior; the set-temperature is determined principally by the dependency of viscosity upon temperature. Strategies for rapidly reaching low temperatures and avoiding high stresses are inferred from the results.     

A simple model for estimating the active reactions of embryonic tissues to a deforming mechanical force

Active reactions of embryonic tissues to mechanical forces play an important role in morphogenesis. To study these reactions, experimental models that enable to evaluate the applied forces and the deformations of the tissues are required. A model based upon the active intrusion of a living early gastrula Xenopus embryo into a tube half the embryo in diameter is described. The intrusion is initially triggered by a suction force of several dozen Pa but then continues in the absence of external driving force, stopping immediately after the entire embryo has penetrated into the tube. The process can be stopped by cytoskeletal drugs or by the damage of the part of the embryo still non-aspirated and is associated with the transversal contraction and meridional elongation of the non-aspirated part of the embryo surface and quasi-periodic longitudinal contractions/extensions of the cells within the part already aspirated. We suggest that this reaction is an active response to the embryo deformation and discuss its morphogenetic role. The problem of estimating the elastic modules of embryonic tissues is also discussed.     

An anisotropic-viscoplastic model of plant cell morphogenesis by tip growth

Plant cell morphogenesis depends critically on two processes: the deposition of new wall material at the cell surface and the mechanical deformation of this material by the stresses resulting from the cell's turgor pressure. We developed a model of plant cell morphogenesis that is a first attempt at integrating these two processes. The model is based on the theories of thin shells and anisotropic viscoplasticity. It includes three sets of equations that give the connection between wall stresses, wall strains and cell geometry. We present an algorithm to solve these equations numerically. Application of this simulation approach to the morphogenesis of tip-growing cells illustrates how the viscoplastic properties of the cell wall affect the shape of the cell at steady state. The same simulation approach was also used to reproduce morphogenetic transients such as the initiation of tip growth and other non-steady changes in cell shape. Finally, we show that the mechanical anisotropy built into the model is required to account for observed patterns of wall expansion in plant cells.     

Cell surface galactosyltransferase as a recognition molecule during development

Recent results from our laboratory suggest that a variety of cellular interactions during development are mediated, in part, by the binding of a cell surface enzyme, galactosyltransferase (GalTase), to its specific lactosaminoglycan (LAG) substrate on adjacent cell surfaces and in the extracellular matrix. Our present interest in surface GalTase developed from earlier biochemical studies of a series of morphogenetic mutations in the mouse which map to the T/t-complex. These studies identified a specific defect in the regulation of surface GalTase activity on morphogenetically abnormal cells, while eight other enzymes showed normal activity. This led us to consider the unique function of surface GalTase in those cell interactions that are influenced by mutations of the T/t-complex. By using a multidisciplinary approach, which included genetic, biochemical and immunological probes, we have found that GalTase functions as a surface receptor during fertilization, early embryonic cell adhesions, and embryonic cell migration on basal lamina matrices. Recently, we have examined the expression of surface GalTase during spermatogenesis, as well as the fate of sperm GalTase following the acrosome reaction. This paper summarizes the results of these studies, as well as others, which suggest that GalTase functions as a surface receptor during those cell interactions regulated by the T/t-complex alleles.     

Morphomechanics: goals, basic experiments and models

Morphomechanics is a branch of developmental biology, studying the generation, space-time patterns and morphogenetic role of mechanical stresses (MS) which reside in embryonic tissues. All the morphogenetically active embryonic tissues studied in this respect have been shown to bear substantial mechanical stresses of tension or pressure. MS are indispensable for organized cell movements, expression of a number of developmentally important genes and the very viability of cells. Even a temporary relaxation of MS leads to an increase in the morphological variability and asymmetry of embryonic rudiments. Moreover, MS may be among the decisive links of morphogenetic feedback required for driving forth embryonic development and providing its regular space-time patterns. We hypothesize that one such feedback is based upon the tendency of cells and tissues to hyperrestore (restore with an overshoot) their MS values after any deviations, either artificial or produced by neighboring morphogenetically active tissues. This idea is supported by a number of observations and experiments performed on the tissue and individual cell levels. We describe also the models demonstrating that a number of biologically realistic stationary shapes and propagating waves can be generated by varying the parameters of the hyperrestoration feedback loop. Morphomechanics is an important and rapidly developing branch of developmental and cell biology, being complementary to other approaches.     

Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of <Emphasis Type="Italic">Ocotea catharinensis</Emphasis>

In this study we examined the effect of polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) on growth, morphology evolution, endogenous PAs levels and nitric oxide (NO) release in Ocotea catharinensis somatic embryo cultures. We observed that Spd and Spm reduced culture growth, permitted embryo morphogenetic evolution from the earliest to last embryo development stages, increased endogenous PAs levels, and induced NO release in O. catharinensis somatic embryos. On the other hand, Put had little effect on these parameters. Spd and Spm could successfully be used to promote somatic embryo maturation in O. catharinensis. The results suggest that Spd and Spm have an important role during the growth, development and morphogenetic evolution of somatic embryos, through alterations in the endogenous nitric oxide and PAs metabolism in this species.     

ERECTA family genes regulate development of cotyledons during embryogenesis

Receptor-like kinases are important regulators of plant growth. Often a single receptor is involved in regulation of multiple developmental processes in a variety of tissues. ERECTA family (ERf) receptors have previously been linked with stomata development, above-ground organ elongation, shoot apical meristem function, flower differentiation and biotic/abiotic stresses. Here we explore the role of these genes during embryogenesis. ERfs are expressed in the developing embryo, where their expression is progressively limited to the upper half of the embryo. During embryogenesis ERfs redundantly stimulate the growth of cotyledons by promoting cell proliferation and inhibiting premature stomata differentiation.     

Cell division events are essential for embryo patterning and morphogenesis: studies on dominant-negative cdc2aAt mutants of arabidopsis

During plant development, cell division events are coordinately regulated, leading to specific growth patterns. Experimental evidence indicates that the morphogenetic controls that act at the vegetative plant growth stage are flexible and tolerate distortions in patterns and frequencies of cell division. To address questions concerning the relationship between cell division and embryo formation, a novel experimental approach was used. The frequencies of cell division were reduced exclusively during embryo development of Arabidopsis by the expression of a dominant cdc2a mutant. The five independent transgenic lines with the highest levels of the mutant cdc2a affected embryo formation. In the C13 line, seeds failed to germinate. The C1, C5 and C12 lines displayed a range of distortions on the apical-basal embryo pattern. In the C3 line, the shoot apical meristem of the seedlings produced leaves defective in growth and with an incorrect phyllotactic pattern. The results demonstrate that rates of cell division do not dictate cellular differentiation of embryos. Nevertheless, whereas cell divisions are uncoupled from vegetative development, they are instrumental in elaborating embryo structures and modulating embryo and seedling morphogenesis.     

Relocations of cell convergence sites and formation of pharyngula-like shapes in mechanically relaxed <Emphasis Type="Italic">Xenopus</Emphasis> embryos

Influence of the relaxation of mechanical tensions upon collective cell movements, shape formation, and expression patterns of tissue-specific genes has been studied in Xenopus laevis embryos. We show that the local relaxation of tensile stresses within the suprablastoporal area (SBA) performed at the early-midgastrula stage leads to a complete arrest of normal convergent cell intercalation towards the dorsal midline. As a result, SBA either remains nondeformed or protrudes a strip of cells migrating ventralwards along one of the lateral lips of the opened blastopore. Already, few minutes later, the tissues in the ventral lip vicinity undergo abnormal transversal contraction/longitudinal extension resulting in the abnormal cell convergence toward ventral (rather than dorsal) embryo midline. Within a day, the dorsally relaxed embryos acquire pharyngula-like shapes and often possess tail-like protrusions. Their antero-posterior and dorso-ventral polarity, as well as expression patterns of pan-neural (Sox3), muscular cardiac actin, and forebrain (Otx2) genes substantially deviate from the normal ones. We suggest that normal gastrulation is permanently controlled by mechanical stresses within the blastopore circumference. The role of tissue tensions in regulating collective cell movements and creating pharyngula-like shapes are discussed.     

Identification of emergent motion compartments in the amniote embryo

Abstract

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ～400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis

The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing embryo.     

Cell adhesion receptors in mechanotransduction

Integrins and cadherins are tri-functional: they bind ligands on other cells or in the extracellular matrix, connect to the cytoskeleton inside the cell, and regulate intracellular signaling pathways. These adhesion receptors therefore transmit mechanical stresses and are well positioned to mediate mechanotransduction. Studies of cultured cells have shown that both integrin- and cadherin-mediated adhesion are intrinsically mechanosensitive. Strengthening of adhesions in response to mechanical stimulation may be a central mechanism for mechanotransduction. Studies of developing organisms suggest that these mechanisms contribute to tissue level responses to tension and compression, thereby linking morphogenetic movements to cell fate decisions.     

Is mechano‐sensitive expression of twist involved In mesoderm formation?

Mesoderm invagination, the first morphogenetic movement of gastrulation in the early Drososphila embryo, is controlled by the expression of the twist and snail genes. Our knowledge concerning epistatic relationships between these genes implies the existence of a poorly understood biochemical maintenance of twist expression during mesoderm invagination by the snail gene. In the light of a review detailing the role of these genes in the cell shape changes leading to invagination, and of recent findings showing the expression of twist as mechanically sensitive, we suggest that the expression of twist in the mesoderm could alternatively be maintained by mechanical strains developed during mesoderm invagination.