Stress-generating tissue deformations in Xenopus embryos: Long-range gradients and local cell displacements.

BACKGROUND: Although the role of endogenous mechanical stresses in regulating morphogenetic movements and cell differentiation is now well established, many aspects of mechanical stress generation and transmission in developing embryos remain unclear and require quantitative studies. RESULTS: By measuring stress-bearing linear deformations (caused by differences in cell movement rates) in the outer cell layer of blastula - early tail-bud Xenopus embryos, we revealed a set of long-term tension-generating gradients of cell movement rates, modulated by short-term cell-cell displacements much increasing the rates of local deformations. Experimental relaxation of tensions distorted the gradients but preserved and even enhanced local cell-cell displacements. During development, an incoherent mode of cell behavior, characterized by extensive cell-cell displacements and poorly correlated cell trajectories, was exchanged for a more coherent regime with the opposite characteristics. In particular, cell shifts became more synchronous and acquired a periodicity of several dozen minutes. CONCLUSIONS: Morphogenetic movements in Xenopus embryos are mediated by mechanically stressed dynamic structures of two different levels: extended gradients and short-term cell-cell displacements. As development proceeds, the latter component decreases and cell trajectories become more correlated. In particular, they acquire common periodicities, making morphogenesis more coherent.

[On the Work of the Developmental Biophysics Laboratory of the Embryology Department of Moscow State University].

The laboratory is engaged in morphomechanics­the 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.

Morphogenesis can be driven by properly parametrised mechanical feedback.

A fundamental problem of morphogenesis is whether it presents itself as a succession of links that are each driven by its own specific cause-effect relationship, or whether all of the links can be embraced by a common law that is possible to formulate in physical terms. We suggest that a common biophysical background for most, if not all, morphogenetic processes is based upon feedback between mechanical stresses (MS) that are imposed to a given part of a developing embryo by its other parts and MS that are actively generated within that part. The latter are directed toward hyper-restoration (restoration with an overshoot) of the initial MS values. We show that under mechanical constraints imposed by other parts, these tendencies drive forth development. To provide specificity for morphogenetic reactions, this feedback should be modulated by long-term parameters and/or initial conditions that are set up by genetic factors. The experimental and model data related to this concept are reviewed.

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.

Neuro-mesodermal patterns in artificially deformed embryonic explants: a role for mechano-geometry in tissue differentiation.

The mutual arrangement of neural and mesodermal rudiments in artificially bent double explants of Xenopus laevis suprablastoporal areas was compared with that of intact explants. While some of the bent explants straightened or became spherical, most retained and actively reinforced the imposed curvature, creating folds on their concave sides and expanding convex surfaces. In the intact explants, the arrangement of neural and mesodermal rudiments exhibited a distinct antero-posterior polarity, with some variability. In the bent explants, this polarity was lost: the neural rudiments were shifted towards concave while the mesodermal tissues moved towards the convex side, embracing the neural rudiments in a horseshoe-shaped manner. We associate these drastic changes in neuro-mesodermal patterning with the active extension and contraction of the convex and concave sides, respectively, triggered by the imposed deformations. We speculate that similar events are responsible for the establishment of neuro-mesodermal patterns during normal development.

A common biomechanical model for the formation of stationary cell domains and propagating waves in the developing organisms.

Many important morphogenetic processes that take place in the development of an animal start from the segregation of a homogeneous layer of cells into a different number of the domains of columnar and flattened cells. In many cases, waves of cell shape transformation travel throughout embryonic tissues. A biomechanical model is presented which embraces both kinds of event. The model is based on the idea of interplay between short- and long-range factors. While the former promote the spreading of a given cell state along a cell row in the recalculation direction, long-range factors are associated with self-generated tensions which, after exceeding a certain threshold, induce active cell extension and hence the rise of tangential pressure. Different kinds of biologically realistic stationary structures, as well as various kinds of the running waves, can be modelled under different parameter values. Moreover, the current model can be coupled with the previous one (Beloussov and Grabovsky, Comput. Methods Biomech. Biomed. Eng., 6: 53-63 (2003)) permitting a common causal chain to be created, moving from the state of an initial homogeneous cell layer towards the complicated shapes of embryonic rudiments.

A Geometro-mechanical model for pulsatile morphogenesis.

A model is proposed which imitates the morphogenesis of several species of the lower invertebrate animals, the hydroid polyps and permits the derivation of the geometry (surface curvature) of each developmental stage from that of the preceding stage. The model is based upon two experimentally verified assumptions. First, neighbouring cells are assumed to compress each other laterally in a regular and species-specific pulsatile manner. It is this pressure, and/or an active cell reaction to it, which changes the curvature of a cell layer. Secondly, cell layers are assumed to have quasi-elastic properties tending to smooth out their curvature. With our model, the different pulsatile patterns of cell-cell pressure are reproduced and the elasticity parameters are modulated. As a result, within a large zone of parameter values (a so-called "morphogenetic zone", MZ) realistic shapes of the rudiments are reproduced. The main principles of the model can also be used for interpreting the morphogenesis of other groups of animals. A suggested model emphasizes the self-organizing properties of a "stressed geometry" of embryonic rudiments.

Biophotonic patterns of optical interactions between fish eggs and embryos.

The optical (non-substantial) interactions between various biological samples have been evident in a number of cases mainly by the effects on their functional activity and developmental patterns. However, the mechanisms of these interactions have remained obscure. Effect of optical interaction has been observed on the intensity and Fourier patterns of biophoton emission of fish embryos. We demonstrate that: (1) the short-term optical interactions are accompanied by a gradual decrease of a total emission intensity of the interacting batches; (2) this effect is spread laterally to that part of a batch which does not have any direct optical contacts with its partner; and (3) the long-term optical contacts lead to a mutual exchange of spectral characteristics of interacting batches in which the total spectral density values are reversed (often with an overshoot). The reversal rate depends upon the developmental distance between the optical partners and the initial differences of their spectral characteristics. The results are discussed in terms of a sub-radiance and Le Chatelier principle.

Morphogenetic fields: outlining the alternatives and enlarging the context.

Several alternative properties which we define as deterministic or field ones are formulated and analyzed in their relations to the realms of morphogenesis and biophoton emission. In spite of all the differences between these two groups of events both of them share the properties of non-additivity, delocalization, self-focusing and several others which we relate to the field phenomena. To a large extent, the field properties of the biological systems are associated with a set of oscillations of different time periods. We suggest that even such deterministic events as, for example, a ligand-receptor coupling are acting, within an activated cell, as the switches and/or modulators of its field properties.

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos.

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant's layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant's cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell's trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar "embryoid" shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events.

Studies in developmental cytomechanic.

One of the most promising trends in modern developmental and cell biology, recently defined as <>, or <>, is directed towards revealing the role of mechanical stresses, chemomechanical transduction and active stress responses of cells antissues of developing embryos. We review here the results obtained in this field by our research group and compare them with those from other labs. Our studies relate to the buds of hydroid polypes and to amphibian embryos. We describe the space-temporal patterns of mechanical stresses in these species, analyze their morphogenetical role and the tissue responses to the experimental modulations of stress patterns. In hydroid polypes we explore also the molecular events involved in mechanochemical coupling. A model, linking the passive mechanical stresses with the active stress-responses of embryonic tissues is suggested. We consider these investigations as a first approach to a developing embryo as to an <>.

Biomechanical feedback in morphogenesis, as exemplified by stretch responses of amphibian embryonic tissues.

We explore the idea that morphogenetical processes may be self-regulated by the biomechanical feedback established between the active stress-generating devices and the passive stresses of stretching and (or) compression, these feedback directed towards hyperrestoration (restoration with overlapping) of the initial stress values. As an example, a stretch-induced behaviour of the pieces of ventral ectoderm of Xenopus laevis early gastrulae is considered. By stretching the explants in 1.3-1.7 times, we induced several active poststretching cell responses, including further autonomous elongation of an explant in the stretch direction and contraction in the perpendicular direction, as well as more complicated shape changes. At the cellular level, these responses were associated with the return of the stretched cells to isodiametrical shapes and with the production of extensive cell protrusions along the stretch direction. As shown by dissections, the stretch-induced tissue tensions were considerably diminished in the poststretching period. The results obtained are discussed within the framework of the hyperrestoration hypothesis.

X-ray microanalysis of ion contents in vacuoles and cytoplasm of the growing tips of a hydroid polyp as related to osmotic changes and growth pulsations.

Growth pulsations (GP) in hydroid polyps are associated with changes in vacuolar patterns which can be imitated by altering external osmolarity. With the use of X-ray spectroscopy we measured the elemental contents in the vacuoles and cytoplasm of the growing tips of a hydroid polyp, Podocoryne carnea, under various tonicity conditions. Under hypertonic condition which arrested the samples at the retraction phase of normal GP, the elemental content within the vacuolar compartment appeared to be similar to that of the external medium, confirming our previous conclusion about the dehermetization of the vacuolar compartment under these conditions. Under hypotonical condition which arrested samples at the extension GP phase (vacuoles isolated) element ratio data displayed an obvious bimodality. At least one of the data groups could be characterized by a significant increase in the concentrations of sodium and potassium, as related to Cl, Ca and Mg, and in comparison to the same ratios in hypotonical samples and those in the external medium. We suggest that under hypotonical conditions the isolated vacuolar compartment is formed by influx of sodium and potassium ions. These cations are accompanied by anions other than chloride. Potassium appears to be transferred into the vacuoles from the cytoplasm while the sodium derives from the external environment.

Generative rules for the morphogenesis of epithelial tubes.

A finite elements model imitating the morphogenesis of smoothly curved tubular epithelial rudiments is suggested. It is based upon the experimentally proved assumption of the lateral (tangential) pressure between adjacent epithelial cells. The main idea of the model is that under a non-zero local curvature the lateral cell-cell pressure acquires the radial components which are absent under zero curvature. In the framework of the model we investigate the roles of initial geometry, the different coefficients relating the local curvatures and radial cell shifts, and of visco-elastical cell-cell linkages in the shaping process. We also employ the different temporal regimes (both periodical and constant) of the lateral pressure exerted and the different overall durations of the modelling. As a result, we get a set of biologically realistical shapes, almost all of them belonging to the same basical "trefoiled" archetype. Among the variables explored, shaping was most affected by the changes in visco-elastical coefficients, in the temporal regimes and in the overall duration of the modelling. The model shows that rather complicated and realistical shapes of epithelial rudiments can be obtained without assuming any initial regional differences inside cell layers. The model may be useful for understanding the principles underlying both genetical and epigenetical regulation of the morphogenesis.

Effects of relaxation of mechanical tensions upon the early morphogenesis of Xenopus laevis embryos.

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.

Mechanics of animal development.

Morphogenetic movements are active processes created by forces located within the moving cells themselves. As a rule, these forces are generated by cytoskeleton structures (contraction of actin microfilaments organized as subcortical bundles or actine gel) and by the membranes and vacuole mediated processes of osmotic water transport. The intercellular forces lead to contraction and thus give rise to long-range mechanical stresses, mostly tensile ones. Tensile stresses create the regularly space/time arranged fields which remain topologically invariable within certain developmental periods, which then change drastically. The model of epithelial morphogenesis is discussed which postulates the segregation of an initially homogeneous cell sheet to the proportional domains of polarized and tangentially stretched cells as a result of self-organization. Some other models which tend to explain the different kinds of fold formation are also suggested. One of the models implies a simple "curvature increasing rule" which derives a common trefoiled archetype as a fundamental trend of development of an epithelial rudiment.

The role of external tensions in differentiation of Xenopus laevis embryonic tissues.

Explants extirpated from Xenopus laevis embryos at the early gastrula stage were placed on pieces of hydrophilized latex film which were then either stretched or remained intact. In explants cultivated on the intact films most cells emigrated out of the explants and remained undifferentiated, whereas the explants on the films stretched for 10 min or more developed a normal set of rudiments. In the explants of suprablastoporal zone stretched perpendicularly to the cranio-caudal direction, the axial organs were oriented in the direction of stretching. In the stretched explants, unlike the intact ones, a system of microfilament-associated intercellular contacts was formed within a few minutes.