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Although the folding of epithelial layers is one of the most common morphogenetic events, the underlying mechanisms of this process are still poorly understood. We aimed to determine whether an artificial bending of an embryonic cell sheet, which normally remains flat, is reinforced and stabilized by intrinsic cell transformations. We observed both...
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... (early gastrula) as described by Nieuwkoop and Faber (1956). Pieces of blastocoel roofs that were excised from homologous regions of a pair of the same stage embryos were fused by their inner surfaces, extensively bent along their mid-lines and inserted vertically into a groove dig with a knife into an agarose substrate to fix the imposed bending (Fig. 1A). The bending plane was oriented either parallel or perpendic- ular to the anteroposterior axis of the embryo. Because the bending orientation did not affect the obtained results, we de- scribe these results together. We examined the bending re- sponse of sandwiches prepared from three different zones of the blastocoel roof arranged in ...
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... We measured the apical indices (AI) of epithelial cells situated at the concave or convex sides of the bending arch from pho- tographs of microscopic sections of Epon embedded samples. AI is the ratio L/W between the maximal cell length (L) in the direction perpendicular to its apical surface and cell width (W), as observed on transverse sections (Fig. 1B). Each mea- surement was performed on at least five normal (non-treated) samples and six samples treated with any of the cytoskeletal drugs. In each case, the measurements were made on 50 cells per ...
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... the cytoskeletal drugs used significantly inhibited the AI increase of concave side cells without noticeably affecting the convex side cells. Inhibition was mostly due to the sup- pression of apical constriction rather than the suppression of radial elongation (Fig. 10, cf A-C). The most pronounced ef- fects were generated by ML-7 or blebbistatin treatment, both of which are known to decouple acto-myosin interactions. Less pronounced defects were observed after treatment with cytochalasin D or with inhibitors of microtubules assembly (nocodazole and ...
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... samples prepared from all regions of the blasto- coel roof demonstrated an AI increase in the concave side cells situated at the bending arch, the amount of this increase gradually diminished in the anteroposterior direction (Fig. 11B). This diminishment was due to a reduction in radial cell lengths, as the amount of apical constriction remained constant ( Fig. 11C and D). The sandwiches prepared from pos- terior regions showed a greater tendency to unbend by crawl- ing out of the slit. After 2 h of incubation, approximately one half of the bent sandwiches prepared ...
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... from all regions of the blasto- coel roof demonstrated an AI increase in the concave side cells situated at the bending arch, the amount of this increase gradually diminished in the anteroposterior direction (Fig. 11B). This diminishment was due to a reduction in radial cell lengths, as the amount of apical constriction remained constant ( Fig. 11C and D). The sandwiches prepared from pos- terior regions showed a greater tendency to unbend by crawl- ing out of the slit. After 2 h of incubation, approximately one half of the bent sandwiches prepared from the SBA (suprabl- astoporal area) moved out of the slit and straightened; in the remaining explants, the imposed curvature was ...
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... ( Kornikova et al., 2009). Therefore, the forces involved in active bending are within the range of those involved in normal morphogenesis. A peculiar and unexpected result of our experiments was that the effect of bending on tissues located in the blastopore vicinity was less pronounced and highly reversible compared to ventrally located tissues (Fig. 11). In subsequent develop- mental stages, the suprablastoporal area (SBA) generates the neural fold; therefore, the bending response could be ex- pected to be mechanically induced much easier within the SBA than in tissues that do not typically participate in these deformations. The observed result indicates the opposite and suggests that ...
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Citations
... 12 In the cooperative process, groups of compressed cells acquire a bottle-like shape to reduce the compression. 50 As a result, folds and grooves cover the surface of the embryo. 12 In the gastrulae of D. pumila, similar compressed cells with a bottle-like shape can be observed, but they are less common ( Figure 5C). ...
Background
In almost all metazoans examined to this respect, the axial patterning system based on canonical Wnt (cWnt) signaling operates throughout the course of development. In most metazoans, gastrulation is polar, and embryos develop morphological landmarks of axial polarity, such as blastopore under control/regulation from cWnt signaling. However, in many cnidarian species, gastrulation is morphologically apolar. The question remains whether сWnt signaling providing the establishment of a body axis controls morphogenetic processes involved in apolar gastrulation.
Results
In this study, we focused on the embryonic development of Dynamena pumila, a cnidarian species with apolar gastrulation. We thoroughly described cell behavior, proliferation, and ultrastructure and examined axial patterning in the embryos of this species. We revealed that the first signs of morphological polarity appear only after the end of gastrulation, while molecular prepatterning of the embryo does exist during gastrulation. We have shown experimentally that in D. pumila, the direction of the oral‐aboral axis is highly robust against perturbations in cWnt activity.
Conclusions
Our results suggest that morphogenetic processes are uncoupled from molecular axial patterning during gastrulation in D. pumila. Investigation of D. pumila might significantly expand our understanding of the ways in which morphological polarization and axial molecular patterning are linked in Metazoa.
... Mounting evidence points to a direct relation between membrane reservoir and trafficking pathways with tension in the regulation of cell shape changes and movements in morphogenetic processes (Dai and Sheetz, 1995;Sheetz and Dai, 1996;Dai et al., 1998;Apodaca, 2002;Gauthier et al., 2011Gauthier et al., , 2012Kremnyov et al., 2012;Diz-Munoz et al., 2013). During morphogenesis, as tissues change their shapes and sizes, cell membranes dynamically change their area, composition and links to the cortex. ...
Morphogenesis in early embryos demands the coordinated distribution of cells and tissues to their final destination in a spatio-temporal controlled way. Spatial and scalar differences in adhesion and contractility are essential for these morphogenetic movements, while the role that membrane remodeling may play remains less clear. To evaluate how membrane turnover modulates tissue arrangements we studied the role of endocytosis in zebrafish epiboly. Experimental analyses and modeling have shown that the expansion of the blastoderm relies on an asymmetry of mechanical tension in the yolk cell generated as a result of actomyosin-dependent contraction and membrane removal. Here we show that the GTPase Rab5ab is essential for the endocytosis and the removal of the external yolk cell syncytial layer (E-YSL) membrane. Interfering in its expression exclusively in the yolk resulted in the reduction of yolk cell actomyosin contractility, the disruption of cortical and internal flows, a disequilibrium in force balance and epiboly impairment. We conclude that regulated membrane remodeling is crucial for directing cell and tissue mechanics, preserving embryo geometry and coordinating morphogenetic movements during epiboly.
... Biological relevance of the proposed feedback as well as underlying cellular responces can be demonstrated in experiments with artificially bent explants of embryonic tissues of X. laevis (Kremnyov et al., 2012).Cells at the concave side of bent double explants of blastocoel roof demonstrated two morphologically distinct phases of radial lengthening (i.e. increase of length-to-width ratio of individual cells). Both phases were accompanied by extensive endocytosys, spatially restricted to the concave groove, in slight disagreement with the initial hypothesis. ...
... Another disagreement between predictions based on the curvature increasing feedback, and experimental results (Kremnyov et al., 2012) was the lack of supposed active extension at the convex side (Fig. 5E). In experiment, cells at the fold arch at the convex side did not demonstrate significant changes in length-to-width ratio, contrary to intact explants with their length-to-width ratio decrease (cell flattening) to the end of the observation (Fig. 5F). ...
... In experiment, cells at the fold arch at the convex side did not demonstrate significant changes in length-to-width ratio, contrary to intact explants with their length-to-width ratio decrease (cell flattening) to the end of the observation (Fig. 5F). This difference might be caused by unaccounted cell rearrangements in depth of the explants and lack of quantification of stress values within cell layers in (Kremnyov et al., 2012). ...
The article is devoted to physical views on embryo development as a combination of structurally stable dynamics and symmetry-breaking events in the general process of self-organization. The first corresponds to the deterministic aspect of embryo development. The second type of processes is associated with sudden increase of variability in the periods of symmetry-breaking, which manifests unstable dynamics. The biological basis under these considerations includes chemokinetics (a system of inductors, repressors, and interaction with their next surrounding) and morphomechanics (i.e. mechanotransduction, mechanosensing, and related feedback loops). Although the latter research area is evolving rapidly, up to this time the role of mechanical properties of embryonic tissues and mechano-dependent processes in them are integrated in the general picture of embryo development to a lesser extent than biochemical signaling. For this reason, the present article is mostly devoted to experimental data on morphomechanics in the process of embryo development, also including analysis of its limitations and possible contradictions. The general system of feedback-loops and system dynamics delineated in this review is in large part a repetition of the views of Lev Beloussov, who was one of the founders of the whole areas of morphomechanics and morphodynamics, and to whose memory this article is dedicated.
... Another way to redistribute tensions was to bend forcibly tissue pieces, stretching thus their convex surfaces and compressing the concave ones. After stopping deformations, progressive deepening of just slightly outlined invaginations could be observed on the concave sides ( Fig. 4 cf a-d; see for details [57,59]). Common to all the above described reactions was the tendency not only to restore stress values perturbed during experimental interventions but to do it with certain overshoots. ...
... Common to all the above described reactions was the tendency not only to restore stress values perturbed during experimental interventions but to do it with certain overshoots. Indeed, as shown by morphometric measurements, the relaxation of tensions or slight compression triggered cells tangential contractions and endocytotic engulfment of cell membrane to the amount enough for hyper-restoring the initial tensions [56,57,59,11]. Similarly, as shown in Fig. 3, the external stretching, by inducing cells convergence-extension, hyper-restores the initial state of a moderate tension by producing internal pressure, and thus reversing the sign of stresses. ...
Morphogenesis in living tissues is the paramount example of a time- and space-dependent orchestration of living matter where shape and order emerge from undifferentiated initial conditions. The genes encode the protein expression that eventually drives the emergence of the phenotype, while energy supply and cell-to-cell communication mechanisms are necessary to such a process. The overall control of the system likely exploits the laws of chemistry and physics through robust and universal processes. Even if the identification of the communication mechanisms is a question of fundamental nature, a long-standing investigation settled in the realm of chemical factors only (also known as morphogens) faces a number of apparently unsolvable questions. In this paper, we investigate at what extent mechanical forces, alone or through their biological feedbacks, can direct some basic aspects of morphogenesis in development biology. In this branch of mechano-biology, we discuss the typical rheological regimes of soft living matter and the related forces, providing a survey on how local mechanical feedbacks can control global size or even gene expression. We finally highlight the pivotal role of nonlinear mechanics to explain the emergence of complex shapes in living matter.
... Pronounced overshoots can be traced in experimentally bent double explants (sandwiches) prepared either from a suprablastoporal area (SBA) of early gastrula Xenopus embryos (Kornikova et al. 2010) or from the ventral ectoderm of the same stage embryos (Kremnyov et al. 2012). So far as under the action of external force, the concave side of a bent sandwich should be shrunk and the convex side 116 4 Morphomechanical Feedbacks extended, the overshoots should be manifested by active contraction of the concave surfaces and active extension of the convex ones. ...
... b 1 -b 2 Cross sections of similar samples few minutes and 3 h after bending. c A gradual narrowing of artificially imposed fold in the sandwiches prepared from Xenopus early gastrula ventral ectoderm (from Kremnyov et al. 2012) 4.2 Evidences for Hyper-restoration of Mechanical Stresses in antiphase, promoting thus each other elongation (for details, see . If the lateral rudiment is removed, the adjacent wall of the central one ( Fig. 4.2a-c, left walls) will retard its longitudinal growth and hence become understretched (relatively relaxed) as compared with the opposite wall of the same rudiment which retained normal contacts with its lateral neighbor (Fig. 4.2a-c, right walls). ...
We start from reviewing several ubiquitous approaches to morphogenesis and argue that for a more adequate presentation of morphogenesis, they should be replaced by explanatory constructions based upon the self-organization theory (SOT). The first step on this way will be in describing morphogenetic events in terms of the symmetry theory, to distinguish the processes driven either toward increase or toward decrease of the symmetry order and to use Curie principle as a clue. We will show that the only way to combine this principle with experimental data is to conclude that morphogenesis passes via a number of instabilities. The latter, in their turn, point to the domination of nonlinear regimes. Accordingly, we come to the realm of SOT and give a survey of the dynamic modes which it provides. By discussing the physical basis of embryonic self-organization, we focus ourselves on the role of mechanical stresses. We suggest that many (although no all) morphogenetic events can be regarded as retarded relaxations of previously accumulated elastic stresses toward a restricted number of metastable energy wells.
... If preparing a sandwich from BRA tissue and try to fasten it in the bending state (by pulling into a groove) in the first dozens of seconds it resists bending as an elastic body, attempting to straighten back again. Within few minutes however it not only takes an imposed shape but tries to reinforce it actively by increasing the bending curvature due to contraction of the apical cell surfaces [20] (Figure 3). Such a passage from passive to active bending seems to play an important role in making embryonic shapes and may be hence recommended for bioengineering purposes. ...
... Red converged arrows in (C) display active contractile forces while blue lines depict stretched surfaces which later on (E) create numerous undulations (see below for comments). IC: inflated cavities in the inner parts of tissue sandwiches[20]. ...
In this paper we review experimental approaches used in our research group for deforming embryonic tissues in amphibian embryos by relaxing pre-existed tensions, stretching samples in given directions or bending them. In these experiments , owing to the active tissue reactions to changes in mechanical stresses, they change their shapes in predictable ways. In some cases the changes in geometry dictate reconstruction of cell differentiation patterns. We suggest that these results may orient bioengineers in elaborating new technologies permitting to endow artificial organs by required shapes.
... Pronounced overshoots can be traced in experimentally bent double explants (sandwiches) prepared either from a suprablastoporal area (SBA) of early gastrula Xenopus embryos (Kornikova et al. 2010) or from the ventral ectoderm of the same stage embryos (Kremnyov et al. 2012). So far as under the action of external force, the concave side of a bent sandwich should be shrunk and the convex side 116 4 Morphomechanical Feedbacks extended, the overshoots should be manifested by active contraction of the concave surfaces and active extension of the convex ones. ...
... b 1 -b 2 Cross sections of similar samples few minutes and 3 h after bending. c A gradual narrowing of artificially imposed fold in the sandwiches prepared from Xenopus early gastrula ventral ectoderm (from Kremnyov et al. 2012) 4.2 Evidences for Hyper-restoration of Mechanical Stresses in antiphase, promoting thus each other elongation (for details, see . If the lateral rudiment is removed, the adjacent wall of the central one ( Fig. 4.2a-c, left walls) will retard its longitudinal growth and hence become understretched (relatively relaxed) as compared with the opposite wall of the same rudiment which retained normal contacts with its lateral neighbor (Fig. 4.2a-c, right walls). ...
Regular patterns of mechanical stresses are perfectly expressed on the macromorphological level in the embryos of all taxonomic groups studied in this respect. Stress patterns are characterized by the topological invariability retained during prolonged time periods and drastically changing in between. After explanting small pieces of embryonic tissues, they are restored within several dozens minutes. Disturbance of stress patterns in developing embryos irreversibly breaks the long-range order of subsequent development. Morphogenetically important stress patterns are established by three geometrically different modes of cell alignment: parallel, perpendicular, and oblique. The first of them creates prolonged files of actively elongated cells. The second is responsible for segregation of an epithelial layer to the domains of columnar and flattened cells. The model of this process, demonstrating its scaling capacities, is described. The third mode which follows the previous one is responsible for making the curvatures. It is associated with formation of “cell fans,” the universal devices for shapes formation due to slow relaxation of the stored elastic energy.
... Pronounced overshoots can be traced in experimentally bent double explants (sandwiches) prepared either from a suprablastoporal area (SBA) of early gastrula Xenopus embryos (Kornikova et al. 2010) or from the ventral ectoderm of the same stage embryos (Kremnyov et al. 2012). So far as under the action of external force, the concave side of a bent sandwich should be shrunk and the convex side 116 4 Morphomechanical Feedbacks extended, the overshoots should be manifested by active contraction of the concave surfaces and active extension of the convex ones. ...
... b 1 -b 2 Cross sections of similar samples few minutes and 3 h after bending. c A gradual narrowing of artificially imposed fold in the sandwiches prepared from Xenopus early gastrula ventral ectoderm (from Kremnyov et al. 2012) 4.2 Evidences for Hyper-restoration of Mechanical Stresses in antiphase, promoting thus each other elongation (for details, see . If the lateral rudiment is removed, the adjacent wall of the central one ( Fig. 4.2a-c, left walls) will retard its longitudinal growth and hence become understretched (relatively relaxed) as compared with the opposite wall of the same rudiment which retained normal contacts with its lateral neighbor (Fig. 4.2a-c, right walls). ...
This book outlines a unified theory of embryonic development, assuming morphogenesis to be a multi-level process including self-organizing steps while also obeying general laws. It is shown how molecular mechanisms generate mechanical forces, which in the long run lead to morphological changes. Questions such as how stress-mediated feedback acts at the cellular and supra-cellular levels and how executive and regulatory mechanisms are mutually dependent are addressed, while aspects of collective cell behavior and the morphogenesis of plants are also discussed. The morphomechanical approach employed in the book is based on the general principles of self-organization theory.
... Pronounced overshoots can be traced in experimentally bent double explants (sandwiches) prepared either from a suprablastoporal area (SBA) of early gastrula Xenopus embryos (Kornikova et al. 2010) or from the ventral ectoderm of the same stage embryos (Kremnyov et al. 2012). So far as under the action of external force, the concave side of a bent sandwich should be shrunk and the convex side 116 4 Morphomechanical Feedbacks extended, the overshoots should be manifested by active contraction of the concave surfaces and active extension of the convex ones. ...
... b 1 -b 2 Cross sections of similar samples few minutes and 3 h after bending. c A gradual narrowing of artificially imposed fold in the sandwiches prepared from Xenopus early gastrula ventral ectoderm (from Kremnyov et al. 2012) 4.2 Evidences for Hyper-restoration of Mechanical Stresses in antiphase, promoting thus each other elongation (for details, see . If the lateral rudiment is removed, the adjacent wall of the central one ( Fig. 4.2a-c, left walls) will retard its longitudinal growth and hence become understretched (relatively relaxed) as compared with the opposite wall of the same rudiment which retained normal contacts with its lateral neighbor (Fig. 4.2a-c, right walls). ...
An attempt is made to reconstruct the natural successions of the developmental events on the basis of a common mechanically based trend. It is formulated in terms of a hyper-restoration (HR) hypothesis claiming that embryonic tissue responds to any external deforming force by generating its own one, directed toward the restoration of the initial stress value, but as a rule overshooting it in the opposite side. We give a mathematical formulation of this model, present a number of supporting evidences, and describe several HR-driven feedbacks which may drive forth morphogenesis. We use this approach for reconstructing in greater detail the gastrulation of the embryos from different taxonomic groups. Also, we discuss the application of this model to cytotomy, ooplasmic segregation, and shape complication of tubular rudiments (taking hydroid polyps as examples). In addition, we review the perspectives for applying morphomechanical approach to the problem of cell differentiation.
... For example, as a tissue changes shape, an individual cell may encounter varying levels of chemical signals that influence its properties. Mechanical feedback is also likely to be important during epithelial morphogenesis [56], and in several systems, myosin appears to be recruited to cellular regions under tension [57]. Work has already been done to incorporate diffusible chemical signals, juxtacrine signaling, and/or mechanical feedback into 2D vertex models [37,[58][59][60]. ...
The folding of epithelial sheets, accompanied by cell shape changes and rearrangements, gives rise to three-dimensional structures during development. Recently, some aspects of epithelial morphogenesis have been modeled using vertex models, in which each cell is approximated by a polygon; however, these models have been largely confined to two dimensions. Here, we describe an adaptation of these models in which the classical two-dimensional vertex model is embedded in three dimensions. This modification allows for the construction of complex three-dimensional shapes from simple sheets of cells. We describe algorithmic, computational, and biophysical aspects of our model, with the view that it may be useful for formulating and testing hypotheses regarding the mechanical forces underlying a wide range of morphogenetic processes.
