Experimental approaches to the study of the formation of axial structures during early development of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

The effect of mechanical extension on the differentiation of axial mesoderm in double explants (sandwiches) of Xenopus laevis embryonic tissues isolated during the early gastrula–late neurula developmen-tal period is studied. In explants at the early gastrula stage, artificial extension orients and stimulates isolated differentiation of the notochord and somites as well as their joint formation. Moreover, extension facilitated the formation of the normal anatomical structure of the notochord and affected expression of Chordin gene. At the late gastrula stage, the effect of artificial extension on joint somite–notochord differentiation was weaker. At the stage of late neurula, somites were sometimes formed in explants lacking a notochord anlage. Thus, at earlier stages, the formation of somites was stimulated by contacts with the notochord and joint development of both structures was mechanical dependent, while at the later stages, somites developed inde-pendently of the notochord. Thus, the role of tissue extension is primarily the establishment of normal mor-phology and expression of Chordin was located in the direction of extension.     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

Electrophoretic spectra of nuclear proteins from embryos ofXenopus laevis

Summary The changes in saline-soluble, 0.35 M NaCl-soluble and the residual fraction of nuclear proteins during early development ofXenopus were studied by analytical electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The fractions were obtained by consecutive extraction of nuclei from the blastula, neurula and tail-bud stage of development. No qualitative and only limited quantitative differences were found when the proteins of any of the three fractions isolated from the neurula stage were compared with the proteins of the corresponding fraction isolated from the tail-bud stage. But the electrophoretic pattern of each of the three fractions of the nuclear proteins from the blastula stage differs significantly from the electrophoretic pattern of the same fraction isolated from the neurula or tail-bud stage. Compared with the blastula stage, in the two later stages the relative amounts of chromosomal proteins with apparent molecular weights below 30,000 are decreased. Proteins which migrate in electrophoresis in the positions of the very lysine-rich histones and of the proteins of the nuclear ribonucleo-protein particles are indicated among the chromosomal proteins of the blastula stage, and are visible as strong bands in the electrophorogram of 0.35 M NaCl-soluble proteins extracted from neurula or tail-bud stage nuclei.     

Alterations in lateral lipid mobility in the plasma membrane of urodelean ectodermal cells during gastrulation

The mobility characteristics of lipids were studied in the plasmalemma of dissociated presumptive ectodermal cells from embryos of Pleurodeles Waltl at different stages of development, from early blastula to early neurula, using a Fluorescence Recovery After Photobleaching technique (FRAP), after incorporation of the lipophilic fluorescent probe 5N-(hexadecanoyl)-aminofluoresceine (HEDAF) into the cell plasma membrane. At all stages of development, fluorescence recovery was found to extrapolate to 100%, which suggested that the lipid phase in these plasma membranes can be regarded as dynamically homogeneous (no immobilized fraction). It appears as a continuum over a wide cell surface area, in which lipids are free to move laterally. The lateral diffusion coefficient of the probe, obtained from statistical analysis of the fluorescence recovery data, was found to decrease significantly from blastula to gastrula, slightly increasing at the neurula stage. These changes in the dynamic properties of the lipid probe HEDAF during gastrulation suggest that the lipid phase of the plasma membrane of these ectodermal cells undergo structural changes. The results lend support to the idea that the plasma membrane of these cells is actively involved in the morphogenetic movements which characterize the development of the embryo.     

Quantity of functionally changed cells as an identificator of the moment of the organizmus transfer to the next period of development

As "the threshold gears are peculiar to all processes which are flowing past in the world", the problem of looking up of quantitative criterion determining transition of a biological system from one condition to another, is one of the main problems of natural sciences. For analysis of the given problem we investigated some stages of forwardness of marine aster Asterias rubens (mean and late blastula, early and late gastrula, early larva), dis- tinguishing among themselves by morphological characters. Change of structure of cell populations at each stage of development A. rubens analyzed with the help of method of quantitative assessment of functional condition of cells genome. The method is based on cytophotometryc analysis of cell populations coloured by Feulgen (quantifying of DNA in cell) and Naftol yellow S (quantifying of histones in a cell). As the criterion of estimation of functional condition of the cell was used the parameter CFAGEN, coefficient of functional activity of cell genome deduced from histone/DNA ratio after estimation of the absorbency of the nucleus coloured by two chromophores. The data obtained showed that at the moment of organism transition from one stage of development (late blastula) to another (early gastrula) the difference in CFAGEN values between these stages was statistically authentic. Thus comparison of cell distribution histograms has show that cell population conforming to the early gastrula contains 33 % cells with the level of genome functional activity that differs from this one in the cell population conforming to the late blastula. Just in the same moment, the transition of an organism from the stage of late gastrula to the stage of larva is accompanied by statistically authentic distinction in CFAGEN between these stages and availability of more than 1/3 cells (34 %) of the population at the larva stage showing genome functional activity different from that at the late gastrula stage. Thus, it is established, that the biological system passes to qualitatively new condition, if quantity of members of this system changes (increaser or decreases comparatively to a standard point of reference: the norm, previous system status and etc.) not less than on 1/3.     

ULTRASTRUCTURE OF THE 'GERMINAL PLASM' IN XENOPUS EMBRYOS AFTER CLEAVAGE

The endodermal location of 'germinal plasm'-bearing cells (GPBCs) and the ultrastructure of the 'germinal plasm' were studied in Xenopus laevis embryos at gastrula, neurula, tailbud and younger tadpole stages. Primordial germ cells (PGCs) of feeding tadpoles were also observed ultrastructurally.
GPBCs were found in the inner endoderm and in the yolk plug region at the late gastrula stage, in the middle and in the dorsal part of the endoderm cell mass at the late neurula and late tailbud stages, respectively. At the younger tadpole stage they were observed in the uppermost dorsal part of the endoderm. Germinal granules were always present in GPBCs at all stages examined but were not found in PGCs of feeding tadpoles. Irregularly shaped-stringlike bodies (ISBs) which seemed to have changed from germinal granules were first noticed in GPBCs at the late neurula stage, and were still present in PGCs of tadpoles, while 'granular materials' were not seen in GPBCs until the feeding tadpole stages. These facts and ultrastructural similarities shared by these organelles lead us to conclude that the change of the germinal granule through ISB, to the 'granular material' takes place during the differentiation of GPBCs into PGCs.     

Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl

Summary The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.     

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.     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

Alteration of cell adhesion system in amphibian ectoderm cells during primary embryonic induction: changes in reaggregation pattern of induced neurectoderm cells and ultrastructural features of the reaggregate

Summary Cell adhesion was studied during primary embryonic induction. The disaggregation rate and reaggregation patterns were analysed in the ectoderm cells of various developing Cynopus gastrulae and neurulae. The neurectoderm cells disaggregated more slowly with gastrulation, and the neural plate cells of early neurula showed a lesser capacity for disaggregation. Although no differences in reaggregation were found between dorsal and ventral ectoderm at the early gastrula stage, there were significant differences between the induced neurectoderm and the non-induced ventral epidermal cells at the late gastrula stage. Neural plate cells of the early neurula stage were seen to form a chain-like reaggregate, but the ventral epidermal cells of the same embryo formed a cluster-like spherical reaggregate. Scanning electron microscope observations of reaggregates also showed significant differences in adhesive properties between induced neurectoderm and non-induced epidermal cells. The adhesion field of the induced neurectoderm cells was smooth, differing from the distinct ridges of the non-induced epidermal cells. These results suggest that changes in the cell adhesion system, resulting in the formation of a columnar cell shape, may occur immediately after a neural-inducing action.     

Curie-point pyrolysis, gas-liquid chromatography of xylan

Levels of nucleotides and sugar nucleotides in embryos of Bufo arenarum at the stages of morula, gastrula, and neurula have been measured. The total amounts of purine nucleoside diphosphates decreased from morula to gastrula, but increased sharply from gastrula to neurula. The levels of ADP followed this pattern, but those of GDP did not change significantly through the three stages. Purine nucleoside triphosphate levels, which had increased immediately after fertilization, remained almost constant through morula, gastrula, and neurula. As with the purine nucleoside diphosphates, the adenine nucleotide decreased from morula to gastrula, and increased from gastrula to neurula. In contrast, the level of GTP showed a sharp maximum at gastrula. The total pyrimidine nucleoside triphosphate did not change significantly from morula through neurula. As in previous stages of development, only uridine sugar nucleotides were detected. A sharp increase of the galactosyl ester of nucleotides was found at gastrula.     

鳙鱼ARISTICHTHYS NOBILIS早期发育的一些生物化学上的变化

本实验第一部分对鳙鱼卵及胚胎在不同的发育时期蛋白质合成的速度进行了研究,实验采用微量克氏定氮法,对鳙卵从未受精卵到受精卵、尾芽期和出膜期,共测定了十三个不同的发育时期。实验结果表明了在鱼的不同胚胎发育时期蛋白质合成速度有着明显的差异。 实验的第二部分对鳙鱼胚胎发育过程氨基酸组成和氨基酸含量进行了测定,其结果显示出鳙鱼卵和胚胎的各发育时期氨基酸的组分十分相似,但氨基酸的含量在各个时期是有差异的。     

An inducible system for the study of FGF signalling in early amphibian development

The use of a novel inducible FGF signalling system in the frog Xenopus laevis is reported. We show that the lipophilic, synthetic, dimerizing agent AP20187 is able to rapidly activate signalling through an ectopically expressed mutant form of FGFR1 (iFGFR1) in Xenopus embryos. iFGFR1 lacks an extracellular ligand binding domain and contains an AP20187 binding domain fused to the intracellular domain of mouse FGFR1. Induction of signalling by AP20187 is possible until at least early neurula stages, and we demonstrate that ectopically expressed iFGFR1 protein persists until late neurula stages. We show that activation of signalling through iFGFR1 can mimic a number of previously reported FGF activities, including mesoderm induction, repression of anterior development, and neural posteriorization. We show that competence to morphological posteriorization of the anteroposterior axis by FGF signalling only extends until about stage 10.5. We demonstrate that the competence of neural tissue to express the posterior markers Hoxa7 and Xcad3, in response to FGF signalling, is lost by the end of gastrula stages. We also show that activation of FGF signalling stimulates morphogenetic movements in neural tissue until at least the end of the gastrula stage.     

Lithium induces changes in the plasma membrane protein pattern of early amphibian embryos

Summary— The plasma membrane protein pattern of Rana ridibunda embryos subjected to lithium (Li) treatment at various stages of development was examined by two-dimensional gel electrophoresis. Differences were observed at the neurula stage not only as compared to controls but among lithium-treated embryos as well. Of particular interest was the presence of proteins, specific for the gastrula stage, in lithium-treated embryos. The results are discussed in relation to the well-known effect of lithium on amphibian morphogenesis.