Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90° rotation, (5) Π-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in Xenopus laevis embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90 degrees rotation, (5) pi-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

Artificially Applied Tensions Normalize Development of Relaxed Xenopus laevisEmbryos

Relaxation of tensions of the surface of Xenopus laevisembryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by the incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

Effects of mechanical stretching of embryonic tissues on axial structure formation in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Effect of mechanical stretch on the differentiation of axial anlages and Chordin gene expression was studied in sandwich explants prepared from embryonic tissues of Xenopus laevis at the early gastrula stage in two variants: with dissected or intact dorso-medial region. In the first case, convergent cell movements were suppressed and properly organized axial organs (notochord and somites) were almost completely absent. However, they developed if the explants of such type were artificially stretched in the ventro-dorsal direction. In this case, axial organs elongated in the line of stretching, that is in the direction vertical to their normal orientation. Segmented mesoderm was always in contact with the chord anlage. In situ hybridization revealed that the area of Chordin gene expression was also extended in the direction of stretching. PCR showed that Chordin gene expression in stretched explants with disrupted dorso-medial region was statistically at the same level as in the explants with intact dorso-medial region. At the same time, the corresponding gene expression in unstretched explants with disrupted dorso-medial region was statistically higher. The obtained data indicate that mechanical stretch and associated cell movements are a necessary and sufficient condition for the formation of proper histological structure of axial organs and regulation of Chordin gene expression.     

Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

Tensotaxis--a collective movement of embryonic cells up along the gradients of mechanical tensions]

We have examined the active collective movement of ectodermal cells from early gastrula of Xenopus laevis towards the point source of stretching, using techniques of videomicroscopy and scanning electron microscopy. We define this mode of cell movement as tensotaxis. This movement begins near the source of tension 5-10 min after the beginning of stretching and is spread in a relay fashion to more distant cells. As a result, a considerable fraction of observed cells more towards the source of stretching over a considerable territory at a rate of 0.6-3 mu/min. Subsequently, these movements are replaced by cell intercalation roughly oriented in the direction transverse to that of tissue stretching. It is proposed that tensotaxis is initiated by asymmetric deformation of the embryonic tissue due to the concentration (focusing) of a stretching force and contains both passive and active components. Data are presented supporting the view that, during normal development, tensotaxis may determine the movement of embryonic cells towards the blastopore and can also participate in other morphogenetic processes.     

Neural plate morphogenesis and axial stretching in “notochord-defective” Xenopus laevis embryos

The morphogenetic movements of neural ectoderm cells associated with neural plate development and neural fold fusion were examined in notochord-defective embryos. Those movements were apparently normal in embryos which displayed a notochord reduced in size or which completely lacked a notochord. Likewise, axial stretching in the anterior-posterior direction was also normal in “notochord-defective” embryos. A role for the anuran notochord in directing neural fold fusion and axial stretching can, therefore, be ruled out.     

On the origin of tiger bush

We propose a model which describes the dynamics of vast classes of terrestrial plant communities growing in arid or semi-arid regions throughout the world. On the basis of this model, we show that the vegetation stripes (tiger bush) formed by these communities result from an interplay between short-range cooperative interactions controlling plant reproduction and long-range self-inhibitory interactions originating from plant competition for environmental resources. Isotropic as well as anisotropic environmental conditions are discussed. We find that vegetation stripes tend to orient themselves in the direction parallel or perpendicular with respect to a direction of anisotropy depending on whether this anisotropy influences the interactions favouring or inhibiting plant reproduction; furthermore, we show that ground curvature is not a necessary condition for the appearance of arcuate vegetation patterns. In agreement within situ observations, we find that the width of vegetated bands increases when environmental conditions get more arid and that patterns formed of stripes oriented parallel to the direction of a slope are static, while patterns which are perpendicular to this direction exhibit an upslope motion.     

Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyl

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.     

Effect of 5-bromodeoxyuridine on appearance of cell-surface saccharides in organ cultures of embryonic pancreas

Rat pancreatic rudiments from Day 15 embryos cultured for 11 days with 20 μM 5-bromodeoxyuridine (BrdU) show selective suppression of acinar cell cytodifferentiation with production of fluid-filled vacuoles lined by undifferentiated cells. Using a battery of lectin-ferritin conjugates (Con A, RCA I, WGA, SBA, and Ulex lectin) we have shown that the majority of these cells express cell-surface glycoconjugate patterns reminiscent of both protodifferentiated cells from Day 15 rudiments and of adult centroacinar (i.e., duct-like) cells. A smaller population of the undifferentiated cells which contain abundant elements of the rough-surfaced endoplasmic reticulum expresses surface saccharide patterns equivalent to those of acinar cells in Day 19 rudiments. These cells, however, lack zymogen granules characteristic of acinar cells in Day 19 rudiments and are similar morphologically to presumptive acinar cells in Day 17 rudiments. Culture of Day 14 pancreatic rudiments with BrdU leads to growth only of undifferentiated cells with duct-like cell-surface saccharide patterns. We interpret these results to indicate that (1) the differentiation program for the acinar cell plasmalemma is established earlier than that for its intracellular organelles; (2) these two developmental programs are independently regulated; and (3) the progenitor of the acinar cell in the protodifferentiated rudiment may be related to the centroacinar or duct-like cell.     

Fibroblast responses to cyclic mechanical stretching depend on cell orientation to the stretching direction

Fibroblasts in intact tendons align with stretching direction, but they tend to orient randomly in healing tendons. Therefore, a question arises: Do fibroblast responses to mechanical stretching depend on their orientation? To address this question, human patellar tendon fibroblasts were grown in custom-made silicone dishes that possess microgrooved culture surfaces. The direction of the microgrooves was either parallel or normal to the direction of cyclic uniaxial stretching. Fibroblasts grown in these microgrooves had a polar morphology and oriented along the direction of the microgrooves regardless of the stretching conditions. Tendon fibroblasts expressed higher levels of alpha-smooth muscle actin when they were oriented parallel to the stretching direction than when they were oriented normal to the stretching direction. Also, cyclic stretching of the fibroblasts perpendicular to their orientation induced a higher activity level of secretory phospholipase A(2) compared with stretching of the cells parallel to their orientation. Thus, these results show that fibroblast responses to mechanical stretching depend on cell orientation to the stretching direction.     

Mechanodependent cell movements in the axial rudiments of <Emphasis Type="Italic">Xenopus</Emphasis> gastrulae

Sandwich explants of the suprablastoporal area of Xenopus early-mid gastrula and same stages of entire embryos were stretched with two needles perpendicular to the direction of natural elongation of the axial rudiments. The changes in the embryonic shape and histological structure were monitored as well as the arrangement of descendants of one of dorsal blastomers labeled with fluorescein-dextran at the 16-cell stage. A substantial fraction of stretched explants reoriented along the applied stretch direction. The arrangement dynamics of fluorescein-dextran-labeled cells and explant shape demonstrate that this is an active response based on convergent intercalation of cells induced by stretching. Stretched gastrulae demonstrated arrested gastrulation, dorsoventral extension of the blastopore, and ventral flow of labeled cells towards the lateral lips of the blastopore, which was also mediated by convergent intercalation and tensotaxis. The obtained data are discussed in terms of the hypothesis of mechanical stress hyper-restoration.     

Effect of Partial H2O-D2O Replacement on the Anisotropy of Transverse Proton Spin Relaxation in Bovine Articular Cartilage

Anisotropy of transverse proton spin relaxation in collagen-rich tissues like cartilage and tendon is a well-known phenomenon that manifests itself as the “magic-angle” effect in magnetic resonance images of these tissues. It is usually attributed to the non-zero averaging of intra-molecular dipolar interactions in water molecules bound to oriented collagen fibers. One way to manipulate the contributions of these interactions to spin relaxation is by partially replacing the water in the cartilage sample with deuterium oxide. It is known that dipolar interactions in deuterated solutions are weaker, resulting in a decrease in proton relaxation rates. In this work, we investigate the effects of deuteration on the longitudinal and the isotropic and anisotropic contributions to transverse relaxation of water protons in bovine articular cartilage. We demonstrate that the anisotropy of transverse proton spin relaxation in articular cartilage is independent of the degree of deuteration, bringing into question some of the assumptions currently held over the origins of relaxation anisotropy in oriented tissues.     

Relationship between streaming potential and compressive stress in bovine intervertebral tissue

The intervertebral disc is formed by the nucleus pulposus (NP) and annulus fibrosus (AF), and intervertebral tissue contains a large amount of negatively charged proteoglycan. When this tissue becomes deformed, a streaming potential is induced by liquid flow with positive ions. The anisotropic property of the AF tissue is caused by the structural anisotropy of the solid phase and the liquid phase flowing into the tissue with the streaming potential. This study investigated the relationship between the streaming potential and applied stress in bovine intervertebral tissue while focusing on the anisotropy and loading location. Column-shaped specimens, 5.5 mm in diameter and 3 mm thick, were prepared from the tissue of the AF, NP and the annulus–nucleus transition region (AN). The loading direction of each specimen was oriented in the spinal axial direction, as well as in the circumferential and radial directions of the spine considering the anisotropic properties of the AF tissue. The streaming potential changed linearly with stress in all specimens. The linear coefficients k_e of the relationship between stress and streaming potential depended on the extracted positions. These coefficients were not affected by the anisotropy of the AF tissue. In addition, these coefficients were lower in AF than in NP specimens. Except in the NP specimen, the k_e values were higher under faster compression rate conditions. In cyclic compression loading the streaming potential changed linearly with compressive stress, regardless of differences in the tissue and load frequency.     

A three-dimensional constitutive model for the stress relaxation of articular ligaments

A new nonlinear constitutive model for the three-dimensional stress relaxation of articular ligaments is proposed. The model accounts for finite strains, anisotropy, and strain-dependent stress relaxation behavior exhibited by these ligaments. The model parameters are identified using published uniaxial stress–stretch and stress relaxation data on human medial collateral ligaments (MCLs) subjected to tensile tests in the fiber and transverse to the fiber directions (Quapp and Weiss in J Biomech Eng Trans ASME 120:757–763, 1998; Bonifasi-Lista et al. in J Orthop Res 23(1):67–76, 2005). The constitutive equation is then used to predict the nonlinear elastic and stress relaxation response of ligaments subjected to shear deformations in the fiber direction and transverse to the fiber direction, and an equibiaxial extension. A direct comparison with stress relaxation data collected by subjecting human MCLs to shear deformation in the fiber direction is presented in order to demonstrate the predictive capabilities of the model.     

Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering

Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1+/-0.4MPa, compared to highly anisotropic scaffolds whose modulus was 11.6+/-3.1MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.     

Surface microdeformations and regulation of cell movements in <Emphasis Type="Italic">Xenopus</Emphasis> development

The velocities and directions of movements of individual outer ectodermal cells of Xenopus embryos in the course of normal development from the blastula to the early tail-bud stage, as well as after mechanical relaxation in the early gastrula, were measured. An alternation of the periods of directed movements of large cell masses and local cell wanderings was detected. In both cases, the trajectories of individual cells consisted primarily of orthogonal segments. Cell movements were measured on two scales. At a smallscale consideration (time intervals of the order of several hours and distances of the order of tens of microns), fairly slight linear stretching and compressive deformations were detected, which looked like gentle smooth gradients along which the upward morphogenetic movements of cells were directed. At a large-scale consideration (time intervals of the order of tens of minutes and distances of the order of microns), quasi-periodic fluctuations of velocities of individual cells partly correlated in time were found. The differences between these velocities generated microdeformations, which reached several tens of percent and developed within time intervals not more than 10 min. Measurements of relative magnitudes of mechanical forces influencing the cell walls suggests that microdeformations generate local stretching and compressive deformations modulating smoother tension gradients.     

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.     

Dichroism of Photosensitive Pigment in Rhabdoms of the Crayfish Orconectes

Microspectrophotometric measurements of isolated crayfish rhabdoms illuminated transversely show that their photosensitive absorption exhibits a dichroic ratio of 2 in situ. The major absorption axis matches the axial direction of the closely parallel microvilli comprising the receptor organelle. Since these microvilli are regularly oriented transversely in about 24 layers, with the axes of the microvilli at 90° in alternate layers, transverse illumination of a properly oriented rhabdom displays alternate dichroic and isotropic bands. Because all the microvilli from any one cell share the same orientation, the layers of microvilli constitute two sets of orthogonal polarization analyzers when illuminated along the normal visual axis. Furthermore, since the dichroic ratio is 2 and transverse absorption in isotropic bands is the same as that in the minor absorbing axis of dichroic bands, the simplest explanation of the analyzer action is that the absorbing dipoles of the chromophores, as in rod and cone outer segments, lie parallel to the membrane surface but are otherwise randomly oriented. The rhabdom's functional dichroism thus arises from its specific fine structural geometry.