[Surface Microdeformations and Regulation of Cell Movements in Xenopus 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 small-scale 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.

[Mechano-geometric generative rules of morphogenesis].

The ideographical approach aimed at detecting specific causative relationships within the process of development prevails in modern embryology. The present work considers the possibilities of using the nomothetic approach aimed at putting forward nonspecific general laws based on the general scientific theory of self-organization and can be formulated in morphomechanical terms based on feedback links between passive and active mechanical stress. The perspectives of this approach and the involvement of genetic factors in the regulation of feedback links are discussed.

[Statistical study of rapid mechanodependent cell movements in deformed explants of African clawed frog Xenopus laevis embryonic tissues].

Computer analysis of artificially deformed (stretched or compressed) double explants (sandwiches) of the blastocoel roof (BRs) and suprablastoporal region (SBRs) of African clawed frog Xenopus laevis early gastrula has been performed using frames of time-lapse microfilming. During the first 14 min after cutting off, the velocities and displacement angles of several hundreds of cells relative to one another, as well as to fixed points and the extension axis, were measured in the control and deformed samples. It has been found that the deformation of samples leads to a rapid reorientation of large cell masses and increase in the velocities of movements along the extension axes or perpendicularly to the compression axes. In addition, an increase in the velocities of mutual cell displacements in the stretched BRs and cell convergence to the extension axes have been observed. Comparison of different angular sectors demonstrates a statistically significant positive correlation between the mean velocities of cell movements and the number of cells moving within an individual sector. This suggests cooperativity of mechanodependent cell movements. In general, these results demonstrate an important role of mechanical factors in regulation of collective cell movements.

[Passive and active reactions of embryonic tissues to the action of dosed mechanical forces].

With the help of a suction manometric device, the relation between the deformation of Xenonus laevis embryo at the gastrula and neurula stages and the value of the applied force has been studied. Stiffness modules of embryonic tissues were in the order of several dozens of Pascal and they were inversely proportional during deformation from 40 to 20%. At the gastrula stage, a uniform or an increasing rate of expansion of the embryo body in the suction capillary with the diameter of approximately half that of the embryo was observed for 30 min after the action of the suction forces. The length of the stretched portion of the embryo correlates with the value of its deformation at the first minute. As a result of the expansion, the total body surface area of the deformed embryo increases more than twice compared to intact embryos. After expelling the embryo from the capillary, its surface reduced and the deformation became smoothened within 5 min, which indicates the existence of tensional force in the expanded embryo. These data confirm that, at the embryo gastrula stage, external mechanical forces do not only passively deform the embryo but also initiate the active expansion of the embryo which takes place at zero external force and overcomes the tensional resistance of tissues. The mechanism of active expansion and its link with the processes of normal morphogenesis are discussed.

[Parametric models of ontogenetic diversities].

Modeling of morphogenesis demonstrates that they form rather wide regions of structural stability and narrow zones of instability in parametric space. Within instability zones, small parameter shifts lead to drastic changes in the morphology of buds. These particular zones are the sources of ontogenetic diversities and represent the reserve for evolutionary variation. A topical problem is to construct models based on universal schemes of negative feedbacks between dynamic components of ontogenesis; moreover, the specificity of ontogenesis should be determined by the values of genetic and epigenetic parameters.

[Changes in topology and geometry of the embryonic epithelium of Xenopus during relaxation of mechanical tension].

The paper presents the results of statistical evaluation of the changes of cellular apex connections, apical angles, and apical indices of ventral cells of the epiectodermal gastrula of Xenopus during the first HC-four hours after the relaxation of mechanical tension. In the unrelaxed epithelium, an overwhelming majority of cells have three apical connections, apical angles close to 120 degrees, and apical indices around one (isodiametric cells); after relaxation, the number of cells with more than three connections, the number of apical angles deviating substantially from 120 degrees, and the percentage of columnar cells with high apical index increase. Apices with more than three connections tend to gather in enclosed groups, forming a smooth line of cell walls. The length and curvature of cell walls with four apical connections significantly exceeds those same indicators for cells with three apical connections. The observed changes in topology and geometry of cells correspond to reconstructions observed during normal morphogenesis. They are considered in terms of the hyper-restoration model of mechanical tension in relaxed epithelial layers.

[Mechanodependent cell movements in the axial rudiments of Xenopus 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.

["Our standpoint different from common ..." (scientific heritage of Alexander Gurwitsch)].

The work of prominent Russian biologist Alexander Gavrilovich Gurwitsch (1874-1954) on the theory of organism development are reviewed. Alexander Gurwitsch introduced the concept of embryonic (morphogenetic, biological, and cellular) field and proposed several revisions of it from 1912 to 1944. Although neither of them can be considered as a final theory of development, his the persistent search for the invariant law that allows the shape (spatial structure) to be proposed for each next developmental stage from the previous shape is of imperishable methodological interest. Alexander Gurwitsch anticipated many ideas of the future theory of self-organization. His theoretical constructions are explicit and experiment-oriented but absolutely not esoteric. They represent a highly important and original contribution to theoretical biology and are an essential step to further development of the ontogenetic theory.

[Mechanical feedback in morphogenesis and cell differentiation].

The review is concerned with studies of mechanical stresses and mechanical feedbacks on the cellular level. The dependence of responses of cells and embryonic tissues on mechanical stresses and their ability to generate these stresses by themselves have been shown. Regular feedbacks between external (passive) and internal (active) mechanical stresses have been established that are required for the viability of cells, determine the direction of their differentiation, and provide the self-organization of morphogenetic processes. Relevant experimental data, are presented and the models of mechanical feedbacks are discussed.

[Ultraweak emissions of the developing Xenopus laevis eggs and embryos].

We measured ultraweak emissions of the Xenopus laevis eggs and embryos during normal development and under the influence of stress factors in a spectral range of 250 to 800 nm using a photomultiplier. The registered emissions were analyzed by several basic characteristics: mean intensity, histograms, kurtosis, linear trends, and Fourier spectra. We followed relationships between these parameters and developmental stage, as well as the number of individuals in optic contact with each other. The ultraweak emissions did not differ from the background at all developmental stages according to the mean intensity. But Fourier analysis revealed the reliable presence of a number of spectral lines of ultraweak emission, predominantly in the ranges of 10-20 and 30-40 Hz, in the embryos at developmental stages 2 to 11. The intensity of ultraweak emissions reliably decreased within the first 10 min after egg activation and fertilization, as well as in the case of optic interaction between groups of embryos. Sharp cooling, increase in osmotic medium pressure, and transfer in a Ca(2+)- and Mg(2+)-free medium induced a short term (approximately 1-5 min) increase in the mean intensity of ultraweak emission. We studied specific features of ultraweak emissions from different parts of the embryo. The intensity of emission from the animal part of early blastula exceeded those from the vegetal area and entire embryo. Separated fragments of the lateral ectoderm at the neurula stage had higher mean intensities of ultraweak emission than intact embryos at the same developmental stages.

[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.

[A morphomechanical aspect of epigenesis].

Epigenesis in classical embryology is regarded as self-complication of spatial organization of the embryo during its development. The reality of the phenomenon of self-complication at the cellular and supracellular levels has been demonstrated by classical experimental embryology. Today, in light of studies of cell differentiation mechanisms, this problem acquired a molecular aspect. However, the attempt to solve it within the limits of molecular level leads to the paradox of "unreducible complexity". The discovery of a physical factor that concurrently would influence the processes of supracellular and molecular levels would be the best way to solve the problem of self-complication. The mechanical tension in cells and tissues of a developing organism may play the role of such factor. The paper considers facts on the role of mechanical stresses in morphogenesis and gene expression.

[Kinetics of the reaction of yolk cell surface in the loach to puncture and mechanic deformation].

Circumferential and radial components of the yolk cell surface movements were measured in the loach embryos at the late blastula stage within 40-50 min after puncture or indentation by an obliquely directed glass rod. The yolk cell surface was preliminarily marked by coal particles. It was shown that even closely located regions of the surface differed markedly in the rate and direction of their movements. In the vicinity of puncture, the yolk cell surface at first contracted in both circumferential and radial directions and then widened, but did not reach the initial values. In more remote areas, this surface continued to contract in the circumferential direction, but was extended in the radial direction. The degree of its contraction along different radii was unequal. The reaction to oblique indentation was anisotropic: the closest area of the yolk cell surface, located along the direction of indentation, contracted in both circumferential and radial directions and formed a fold "leaking" onto the rod, while the opposite area contracted in the circumferential direction, but extended in the radial direction. A conclusion was drawn that the yolk cell surface is a multivariant mechanosensitive system. Its active responses to mechanical influences obey the same patterns as multicellular embryonic tissues.

[Structurization of cortical layer of loach yolk cell after wounding as a "minimal" model of morphogenesis]. / Strukturizatsiia kortikal'nogo sloia zheltochnoi kletki v'iuna posle naneseniia rany kak "minimal'naia" model' morfogeneza.

Structural rearrangements of the yolk cell surface were studied in loach embryos using SEM and TEM, which take place within 30 min after a point-like puncture at the late blastula stage. The effects of sucking off or addition of a part of yolk, lowered temperature, and absence of Ca2+ on structurization were studied. Around the area of puncture, the yolk granules were submerged, the number of vesicles increased, and numerous membrane folds were formed. The folds were aggregated to form two sharply distinct types of structures: a group of rounded evaginations around the site of puncture and a system of radial folds in the periphery. Small radial folds are aggregated in radial strands, several dozens folds in each. Sucking off a part of yolk accelerated the above processes, while addition of yolk, cooling, and absence of Ca2+ in the incubation medium slowed down or suppressed these processes. The observed structurization can be considered as self-organization at the level of the yolk cell cortical level, largely similar to that during normal morphogenesis at the level of multicellular sheets. Hence, the membrane dynamics in the yolk cell wall after its damage can be considered as one of simplified ("minimal") models of morphogenesis. A study of this model makes it possible to narrow down the circle of factors essential for self-organization of morphogenetic processes.

[A quantitative study of regional and stage specific reaction of African clawed frog embryonic tissues on mechanical stress]. / Kolichestvennoe issledovanie regional'nykh i stadiespetsifichnykh reaktsii émbrional'nykh tkanei shportsevoi liagushki na mekhanicheskuiu razgruzku.

Residual deformation of fragments of the embryonic tissues preserved after relaxation of the stretching force serve as a criterion of active redistribution of their cells caused by this stretching. We measured residual deformations of the Xenopus laevis ventral and dorsal ectoderm at the early gastrula and lateral ectoderm at the late gastrula-early neurula after stretching of varying time and force. While the samples responded to moderate (up to 40%) short-term stretching as elastic bodies (residual deformations were absent), residual deformation appeared in the early gastrula tissues after 30-60-min stretching, which were more pronounced in the ventral tissues than in the dorsal ones. On the contrary, a contractile reaction developed in the late gastrula-early neurula tissues in response to 60-min stretching, which almost relaxed residual deformation within 20 min after unloading. A conclusion was drawn that gastrulation and neurulation proceed under the conditions of relaxing and nonrelaxing mechanical tensions, respectively. Mechanical bases and morphogenetic role of the described reactions is discussed.

[Statistical and frequency-amplitude characteristics of ultra-weak emissions of loach eggs and embryos under normal conditions and on their optic interactions. II. Changes in characteristics of ultra-weak emissions upon optic interaction of groups of embryos of different ages. ]. / Statisticheskie i chastotno-amplitudnye kharakteristiki sverkhslabykh izluchennii iaitsekletok i zarodyshei vyiuna v norme i pri ikh opticheskikh vzaumodeistviakh. II. Izmenenie kharakteristik sverkhslabykh izluchenii pri opticheskom vzaimodeistvii raznovozrastnykh grupp zarodyshei.

We compared the characteristics of ultraweak emissions from groups of loach embryos of different ages in the presence or absence of optic interaction. The percentage of zero values of emission gradually increased during the first hour of optic interaction. The number and height of rare big pulses estimated by the value of kurtosis increased in parallel. In addition, the correlation between the Fourier spectra of optically interacting samples decreased at a higher rate than in the absence of optical contact. Just after the 1-hour optic interaction was terminated, the number of high pulses decreased in a younger interacting group and increased in the older one and the farther away the partner groups were in developmental stages, the more pronounced these differences were. Measurements of the Fourier spectra after long-term (12-22-hour) optic interactions have shown that an "exchange" of autocorrelation characteristics of the spectra took place among the samples: the sums of autocorrelation coefficients were inverted in the vast majority of cases, often with an "overshoot" or, at least, were smoothed over with reference to the control samples. We conclude that the previously described effects of optic interactions between groups of loach embryos of different ages could be due to changes in the frequency spectra of their ultraweak emissions.

[Statistical and frequency-amplitude characteristics of ultra weak emissions of the loach eggs and embryos under the normal conditions and during their optic interactions. I. Characteristics of ultra weak emission in normal development and the optic role of egg envelope]. / Statisticheskie i chastotno-amplitudnye kharakteristiki sverkhslabykh izluchenii iaitsekletok i zarodyshei v'iuna v norme i pri ikh opticheskikh vzaimodeistviiakh. I. Kharakteristiki sverkhslabykh izluchenii v normal'nom razvitii i opticheskaia rol' iaitsevykh obolochek.

Ultraweak emissions of groups comprising several dozens of unfertilized and fertilized loach eggs, embryos, larvae, and their egg envelopes were measured on a photomultiplier tube. The envelopes absorbed the light from external sources but readily gave it back in the absence of embryos. We carried out statistical and frequency-amplitude analyses of ultraweak emissions and studied the autocorrelation structure of their frequency spectra. The frequencies of signals with different intensity underwent regular changes during development. Cascades of short-term (< or = 1 ms) flashes timed (during cleavage) to furrowing were a characteristic element of ultraweak emission. The Fourier spectra of developing embryos had pronounced frequency-amplitude peaks and higher, than in unfertilized eggs and inanimate samples, mutual correlation during successive time intervals. Stage-specific translational symmetry of the frequency spectra of ultraweak emissions was demonstrated, which suggests the presence in groups of embryos of a coordinated system of harmonic oscillators. The latter underwent regular changes during development. The measurement of ultraweak emissions represents a unique non-invasive method of analysis of these oscillators.

[Correlation of fine structures of distributions of amplitudes of a photomultiplier dark current fluctuations with the Earth rotations about its axis]. / Korreliatsiia tonkoi struktury raspredeleniia amplitud fluktuatsii temnovogo toka fotoumnozhitelei s vrashcheniem Zemli vokrug svoei osi.

The fine structures of distributions of photomultiplier dark current fluctuations measured in two laboratories 2000 km distant from other: in the international Institute of Biophysics (Neuss, Germany) and in the Moscow State University (Moscow, Russia) were compared. It is shown that similar forms of appropriate histograms are apparently more often realized at both locations at the same local time. This confirms the previous conclusion that the fine structure of distributions correlates with rotation of the Earth about its axis.

[Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos]. / Iskusstvenno nalozhennye natiazheniia normalizuiut razvitie relaksirovannykh zarodyshei shportsevoi liagyshki.

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.

[Work of morphogenesis in the Department of Embryology of Moscow State University: toward understanding the dynamic architecture of development]. / Raboty po morfogenezu na kafedre émbriologii MGU: k poznaniiu dinamicheskoi arkhitektury razvitiia.

This is a review of studies on morphogenesis carried out at the Department of Embryology, Moscow State University, over the past 30 years. The main direction of studies has been to reveal and describe the properties of self-organizing fields of mechanical stresses in developing embryos.