Statistical and Frequency-Amplitude Characteristics of Ultraweak Emissions of the Loach Eggs and Embryos under the Normal Conditions and during Their Optic Interactions. 1. Characteristics of Ultraweak Emission in Normal Development and the Optic Role of Egg Envelopes

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 (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.     

Statistical and Frequency-Amplitude Characteristics of Ultraweak Emissions of the Loach Eggs and Embryos under the Normal Conditions and upon Their Optic Interactions. 2. Changes in Characteristics of Ultraweak Emissions upon Optic Interaction of Groups of Embryos of Different Ages

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.     

Overexpression of camello, a member of a novel protein family, reduces blastomere adhesion and inhibits gastrulation in Xenopus laevis

Vertebrate gastrulation involves complex coordinated movements of cells and cell layers to establish the axial structures and the general body plan. Adhesion molecules and the components of extracellular matrix were shown to be involved in this process. However, other participating molecules and detailed mechanisms of the control of gastrulation movements remain largely unknown. Here, we describe a novel Xenopus gene camello (Xcml) which is expressed in the suprablastoporal zone of gastrulating embryos. Injection of Xcml RNA into dorsovegetal blastomeres retards or inhibits gastrulation movements. Database searches revealed a family of mammalian mRNAs encoding polypeptides highly similar to Xcml protein. Characteristic features of the camello family include the presence of the central hydrophobic domain and the N-acetyltransferase consensus motifs in the C-terminal part, as well as functional similarity to Xcml revealed by overexpression studies in Xenopus embryos. Xcml expression results in the decrease of cell adhesion as demonstrated by the microscopic analysis and the blastomere aggregation assay. Cell fractionation and confocal microscopy data suggest that Xcml protein is localized in the secretory pathway. We propose that Xcml may fine tune the gastrulation movements by modifying the cell surface and possibly extracellular matrix proteins passing through the secretory pathway.     

Gastrulation in amphibian embryos, regarded as a succession of biomechanical feedback events

Gastrulation in amphibian embryos is a composition of several differently located morphogenetic movements which are perfectly coordinated with each other both in space and time. We hypothesize that this coordination is mediated by biomechanical interactions between different parts of a gastrulating embryo based upon the tendency of each part to hyper-restore the value of its mechanical stress. The entire process of gastrulation in amphibian embryos is considered as a chain of these mutually coupled reactions, which are largely dependent upon the geometry of a given embryo part. We divide gastrulation into several partly overlapped steps, give a theoretical interpretation for each of them, formulate the experiments for testing our interpretation and describe the experimental results which confirm our point of view. Among the predicted experimental results are: inhibition of radial cell intercalation by relaxation of tensile stresses at the blastula stage; inversion of convergent intercalation movements by relaxation of circumferential stresses at the early gastrula stage; stress-promoted reorientation of axial rudiments, and others. We also show that gastrulation is going on under a more or less constant average value of tensile stresses which may play a role as rate-limiting factors. A macro-morphological biomechanical approach developed in this paper is regarded as complementary to exploring the molecular machinery of gastrulation.     

A Quantitative Study of Regional and Stage Specific Reaction of Xenopus laevisEmbryonic Tissues on Mechanical Load

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.     

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.     

The protein encoded by the germ plasm RNA Germes associates with dynein light chains and functions in Xenopus germline development

Germ plasm plays a prominent role in germline formation in a large number of animal taxons. We previously identified a novel maternal RNA named Germes associated with Xenopus germ plasm. In the present work, we addressed possible involvement of Germes protein in germ plasm function. Expression in oocytes followed by confocal microscopy revealed that the EGFP fused to Germes, in contrast to the free EGFP, co-localized with the germ plasm. Overexpression of intact Germes and Germes lacking both leucine zipper motifs (GermesDeltaLZs) resulted in a statistically significant reduction of the number of primordial germ cells (PGCs). Furthermore, the GermesDeltaLZs mutant inhibited PGC migration and produced abnormalities in germ plasm intra-cellular distribution at tailbud stages. To begin unraveling biochemical interactions of Germes during embryogenesis, we searched for Germes partners using yeast two-hybrid (YTH) system. Two closely related sequences were identified, encoding Xenopus dynein light chains dlc8a and dlc8b. Tagged versions of Germes and dlc8s co-localize in VERO cells upon transient expression and can be co-immunoprecipitated after injection of the corresponding RNAs in Xenopus embryos, indicating that their interactions occur in vivo. We conclude that Germes is involved in organization and functioning of germ plasm in Xenopus, probably through interaction with motor complexes.     

Involvement of the guanine nucleotide exchange factor xLARG in the epiboly of <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryo animal pole cells

The development of multicellular organisms is a complicated coordinated process of the movement of groups of embryonic cells, which is controlled by many regulatory systems. At present little is known about the regulation of the earliest manifestations of movement in embryogenesis: epiboly and radial intercalation. The coordinators of these processes may be small GTPases of the Rho family and their activators, guanine nucleotide exchange factors. It has been shown in this work that overexpression of a guanine nucleotide exchange factor xLARG in Xenopus laevis embryos leads to an increase in the amount of the active form of xLARG. In addition, an increase in the expression of xLARG disturbs the process of radial intercalation. The data obtained suggest that xLARG is involved in maintaining the xLARG activation level necessary for the occurrence of epiboly.