Biomechanics and the Thermotolerance of Development

Successful completion of development requires coordination of patterning events with morphogenetic movements. Environmental variability challenges this coordination. For example, developing organisms encounter varying environmental temperatures that can strongly influence developmental rates. We hypothesized that the mechanics of morphogenesis would have to be finely adjusted to allow for normal morphogenesis across a wide range of developmental rates. We formulated our hypothesis as a simple model incorporating time-dependent application of force to a viscoelastic tissue. This model suggested that the capacity to maintain normal morphogenesis across a range of temperatures would depend on how both tissue viscoelasticity and the forces that drive deformation vary with temperature. To test this model we investigated how the mechanical behavior of embryonic tissue (Xenopus laevis) changed with temperature; we used a combination of micropipette aspiration to measure viscoelasticity, electrically induced contractions to measure cellular force generation, and confocal microscopy to measure endogenous contractility. Contrary to expectations, the viscoelasticity of the tissues and peak contractile tension proved invariant with temperature even as rates of force generation and gastrulation movements varied three-fold. Furthermore, the relative rates of different gastrulation movements varied with temperature: the speed of blastopore closure increased more slowly with temperature than the speed of the dorsal-to-ventral progression of involution. The changes in the relative rates of different tissue movements can be explained by the viscoelastic deformation model given observed viscoelastic properties, but only if morphogenetic forces increase slowly rather than all at once.     

Epithelial machines that shape the embryo

Embryonic form and the shape of many organs are the product of forces acting within and on epithelial sheets. Analysis of these processes requires both consideration of the mechanical operation of these multicellular machines and an understanding of how epithelial sheets are integrated with surrounding tissues. From the diverse array of epithelial morphogenetic movements seen during embryogenesis we review examples of epithelial sheet bending, Drosophila ventral furrow formation and ascidian gastrulation, and direct measurements of epithelial mechanics from Xenopus laevis. We present these examples as works-in-progress and highlight opportunities for future studies into both the direct consequence of force production and embryonic tissue mechanics and potential roles of signaling from biomechanical processes.     

The Fog signaling pathway: Insights into signaling in morphogenesis

Epithelia form the building blocks of many tissue and organ types. Epithelial cells often form a contiguous 2-dimensional sheet that is held together by strong adhesions. The mechanical properties conferred by these adhesions allow the cells to undergo dramatic three-dimensional morphogenetic movements while maintaining cell–cell contacts during embryogenesis and post-embryonic development. The Drosophila Folded gastrulation pathway triggers epithelial cell shape changes that drive gastrulation and tissue folding and is one of the most extensively studied examples of epithelial morphogenesis. This pathway has yielded key insights into the signaling mechanisms and cellular machinery involved in epithelial remodeling. In this review, we discuss principles of morphogenesis and signaling that have been discovered through genetic and cell biological examination of this pathway. We also consider various regulatory mechanisms and the system?s relevance to mammalian development. We propose future directions that will continue to broaden our knowledge of morphogenesis across taxa.     

Emergent material properties of developing epithelial tissues

Background

Force generation and the material properties of cells and tissues are central to morphogenesis but remain difficult to measure in vivo. Insight is often limited to the ratios of mechanical properties obtained through disruptive manipulation, and the appropriate models relating stress and strain are unknown. The Drosophila amnioserosa epithelium progressively contracts over 3 hours of dorsal closure, during which cell apices exhibit area fluctuations driven by medial myosin pulses with periods of 1.5–6 min. Linking these two timescales and understanding how pulsatile contractions drive morphogenetic movements is an urgent challenge.

Results

We present a novel framework to measure in a continuous manner the mechanical properties of epithelial cells in the natural context of a tissue undergoing morphogenesis. We show that the relationship between apicomedial myosin fluorescence intensity and strain during fluctuations is consistent with a linear behaviour, although with a lag. We thus used myosin fluorescence intensity as a proxy for active force generation and treated cells as natural experiments of mechanical response under cyclic loading, revealing unambiguous mechanical properties from the hysteresis loop relating stress to strain. Amnioserosa cells can be described as a contractile viscoelastic fluid. We show that their emergent mechanical behaviour can be described by a linear viscoelastic rheology at timescales relevant for tissue morphogenesis. For the first time, we establish relative changes in separate effective mechanical properties in vivo. Over the course of dorsal closure, the tissue solidifies and effective stiffness doubles as net contraction of the tissue commences. Combining our findings with those from previous laser ablation experiments, we show that both apicomedial and junctional stress also increase over time, with the relative increase in apicomedial stress approximately twice that of other obtained measures.

Conclusions

Our results show that in an epithelial tissue undergoing net contraction, stiffness and stress are coupled. Dorsal closure cell apical contraction is driven by the medial region where the relative increase in stress is greater than that of stiffness. At junctions, by contrast, the relative increase in the mechanical properties is the same, so the junctional contribution to tissue deformation is constant over time. An increase in myosin activity is likely to underlie, at least in part, the change in medioapical properties and we suggest that its greater effect on stress relative to stiffness is fundamental to actomyosin systems and confers on tissues the ability to regulate contraction rates in response to changes in external mechanics.

     

Convergence and extension movements mediate the specification and fate maintenance of zebrafish slow muscle precursors

During vertebrate gastrulation, concurrent inductive events and cell movements fashion the body plan. Convergence and extension (C&E) gastrulation movements narrow the vertebrate embryonic body mediolaterally while elongating it rostrocaudally. Segmented somites are shaped and positioned by C&E alongside the notochord and differentiate into skeleton, fast, and slow muscles during somitogenesis. In zebrafish, simultaneous inactivation of non-canonical Wnt signaling components Knypek and Trilobite strongly impairs C&E gastrulation movements. Here we show that knypek;trilobite double mutants exhibit a severe deficit in slow muscles and their precursor, adaxial cells, revealing essential roles of C&E movements in adaxial cell development. Adaxial cells become distinguishable in the presomitic mesoderm during late gastrulation by their expression of myogenic factors and axial-adjacent position. Using cell tracing analyses and genetic manipulations, we demonstrate that C&E movements regulate the number of prospective adaxial cells specified during gastrulation by determining the size of the interface between the inductive axial and target presomitic tissues. During segmentation, when the range of Hedgehog signaling from the axial tissue declines, tight apposition of prospective adaxial cells to the notochord, which is achieved by convergence movements, is necessary for their continuous Hedgehog reception and fate maintenance. We provide direct evidence to show that the deficiency of adaxial cells in knypek;trilobite double mutants is due to impaired C&E movements, rather than an alteration in Hedgehog signal and its reception, or a cell-autonomous requirement for Knypek and Trilobite in adaxial cell development. Our results underscore the significance of precise coordination between cell movements and inductive tissue interactions during cell fate specification.     

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation.     

Morphogenetic origin of natural variation

We studied individual pathways of gastrulation in two related amphibian species making an emphasis on the developmental dynamics of normal variation in the geometry of gastrulation movements. Analyzing the variation dynamics, we show that the linear succession of developmental stages is a secondary phenomenon disguising self-oscillations that lie at the heart of the dorsal blastopore lip morphogenesis. Characteristic features of the equations derived to describe the oscillations are, first, their dependence only on the movement geometry and, second, including of the dynamics of spatial variance directly into the movement equations, making it clear that the reasons for variability of morphogenesis are the same that for morphogenesis itself. The equations describing morphogenetic oscillations are mathematically similar to those describing natural selection in that the system tends to minimize its variance, individual or within-individual one, but the spatially uniform state turns to be unstable. Comparing of the dynamics of natural developmental variation in gastrulation in two frog species shows that, depending on the mechanics and geometry mass cell movements, different types of gastrulation movements have different proportions of the between- to within-individual differences, which strongly influences the choice of characters subject to evolution. Instead of being a source of constraints imposed on externally guided evolutionary trends, morphogenesis becomes a driving force of the adaptively silent, but directional evolution of the developing systems, which seems to be the only possible way of originating of the evolutionary novelties, both in evolution and ontogeny of the biological structures.     

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.     

Controlling the mechanical properties of three-dimensional matrices via non-enzymatic collagen glycation

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.     

Surprisingly simple mechanical behavior of a complex embryonic tissue

Background

Previous studies suggest that mechanical feedback could coordinate morphogenetic events in embryos. Furthermore, embryonic tissues have complex structure and composition and undergo large deformations during morphogenesis. Hence we expect highly non-linear and loading-rate dependent tissue mechanical properties in embryos.

Methodology/Principal Findings

We used micro-aspiration to test whether a simple linear viscoelastic model was sufficient to describe the mechanical behavior of gastrula stage Xenopus laevis embryonic tissue in vivo. We tested whether these embryonic tissues change their mechanical properties in response to mechanical stimuli but found no evidence of changes in the viscoelastic properties of the tissue in response to stress or stress application rate. We used this model to test hypotheses about the pattern of force generation during electrically induced tissue contractions. The dependence of contractions on suction pressure was most consistent with apical tension, and was inconsistent with isotropic contraction. Finally, stiffer clutches generated stronger contractions, suggesting that force generation and stiffness may be coupled in the embryo.

Conclusions/Significance

The mechanical behavior of a complex, active embryonic tissue can be surprisingly well described by a simple linear viscoelastic model with power law creep compliance, even at high deformations. We found no evidence of mechanical feedback in this system. Together these results show that very simple mechanical models can be useful in describing embryo mechanics.     

Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. Here, we present high-resolution 3D digital datasets of frog gastrulae, and morphometrics that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, we combined these tools with time-lapse analysis of gastrulation, and examined both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling. Remarkably, although inhibition of Xdsh signaling disrupted both convergent extension and blastopore closure, mesendoderm internalization proceeded very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, we found a surprising degree of dissociability between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

Cell movements during the initial phase of gastrulation in the sea urchin embryo.

The morphogenetic processes responsible for the initial phase of gastrulation in sea urchin embryos are not known. Here we report observations of the size and position of clones of cells derived from horseradish peroxidase (HRP)-injected mesomeres and macromeres. The displacement of these clones during the initial phase of gastrulation suggests that involution is a mechanism involved in primary invagination. Experiments with embryos marked with vital dyes indicate that movements occur only during a brief phase coincident with the invagination of the vegetal plate. Counts of cells derived from HRP-injected mesomeres and macromeres suggest it unlikely that localized growth in the vegetal plate is involved in gastrulation. An analysis of changes in cell shape during the initial phase of gastrulation indicates that there is a stage-dependent shift from cells being columnar to having their apices skewed toward the vegetal plate and an increase in the proportion of cells having basal processes during gastrulation. When embryos are grown in the presence of monoclonal antibodies to the apical lamina or monovalent fragments of these antibodies, the initial phase of gastrulation is delayed and they form partial exogastrulae. Analysis of embryos marked with HRP indicate that the antibody treatments interfere with the cellular movements observed in untreated embryos. We conclude that directed movements of cells within the blastoderm, probably employing tractoring on components of the hyaline layer, cause the buckling of the vegetal plate and displacement of presumptive endoderm cells seen during the initial phase of gastrulation.     

Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

Mathematical modelling of the cellular mechanics of plants

The complex mechanical behaviour of plant tissues reflects the complexity of their structure and material properties. Modelling has been widely used in studies of how cell walls, single cells and tissue respond to loading, both externally applied loading and loads on the cell wall resulting from changes in the pressure within fluid-filled cells. This paper reviews what approaches have been taken to modelling and simulation of cell wall, cell and tissue mechanics, and to what extent models have been successful in predicting mechanical behaviour. Advances in understanding of cell wall ultrastructure and the control of cell growth present opportunities for modelling to clarify how growth-related mechanical properties arise from wall polymeric structure and biochemistry.     

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker

Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.     

Tensile properties of vascular smooth muscle cells: bridging vascular and cellular biomechanics

Vascular walls change their dimensions and mechanical properties adaptively in response to blood pressure. Because these responses are driven by the smooth muscle cells (SMCs) in the media, a detailed understanding of the mechanical environment of the SMCs should reveal the mechanism of the adaptation. As the mechanical properties of the media are highly heterogeneous at the microscopic level, the mechanical properties of the cells should be measured directly. The tensile properties of SMCs are, thus, important to reveal the microscopic mechanical environment in vascular tissues; their tensile properties have a close correlation with the distribution and arrangement of elements of the cytoskeletal networks, such as stress fibers and microtubules. In this review, we first introduce the experimental techniques used for tensile testing and discuss the various factors affecting the tensile properties of vascular SMCs. Cytoskeletal networks are particularly important for the mechanical properties of a cell and its mechanism of mechanotransduction; thus, the mechanical properties of cytoskeletal filaments and their effects on whole-cell mechanical properties are discussed with special attention to the balance of intracellular forces among the intracellular components that determines the force applied to each element of the cytoskeletal filaments, which is the key to revealing the mechanotransduction events regulating mechanical adaptation. Lastly, we suggest future directions to connect tissue and cell mechanics and to elucidate the mechanism of mechanical adaptation, one of the key issues of cardiovascular solid biomechanics.