Changing complexity of endogenous lectin activities during juvenile development of Xenopus laevis

Galactoside-binding lectin has been isolated from whole Xenopus laevis embryos and tadpoles at four development stages: st. 24–26, 32, 41 and 47. The main lectin activity at st. 24–26 is -galactoside specific, producing a 34/35.5K doublet on SDS-PAGE. Later in development, lectin activities specific for a wide range of other sugars appear concommitant with the detection of a number of new protein bands on SDS-PAGE gels. The greatest variety of new lectin activities exists at st. 32 when lectins specific for all of the main sugar families found in nature are detected. After this stage and up to st. 47 (the beginning of metamorphosis), fewer different lectin activities are again detected. The results suggest that a complex, developmentally regulated battery of different lectins are present during early Xenopus development, perhaps with stage-specific roles to play in the control of tissue morphogenesis.     

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.     

Possible mechanism for the inhibition of lectin-erythrocyte interaction in presence of endogenous lectin receptor

The presence of hydrophobic sites in the lectin-I molecule was indicated by hydrophobic probes like 1-anilinonapthalene-8-sulfonic acid (ANS), 2-p-toluidinyl napthalene-6-sulfonic acid (TNS), N-phenyl-1-napthylamine (NA) and rose bengal (RB). This was further confirmed by amino acid modifications in the hydrophobic region of the lectin-I molecule. The binding of ANS, TNS, NA and RB to lectin-I was affected in the presence of NaCl. The involvement of hydrophobic interactions in rice-bean lectin-I-endogenous lectin receptor (ELR) complex were indicated by alterations in the circular dichroism and fluorescence emission spectra. The percentage of -conformation (55–63%) of lectin-I was decreased by addition of ELR. ELR on reacting with lectin-I reduced the fluorescence emissions of the hydrophobic probes while fluorescence emission of ANS, TNS, NA and RB were greatly enhanced in presence of lectin-I alone. N-aceyl-galactosamine did not change the fluorescence emissions of any of the hydrophobic probes in presence or in absence of lectin-I. This demonstrates that carbohydrate and hydrophobic sites may be different and non-interacting. It is proposed that the ELR in reacting with lectin-I, induced conformational changes in the lectin-I molecule and thereby affected its erythroagglutinating activity with human blood group A erythrocytes.     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos

Summary Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.     

The fate of oocyte nuclear proteins during early development ofXenopus laevis

Summary The localization and movements of four nuclear proteins, originally contained in the germinal vesicle ofXenopus oocytes, were followed through early development from cleavage to late neurula. The study made use of monoclonal antibodies directed against germinal vesicle proteins. Biochemical methods showed that all proteins persist in the embryo without a change in molecular size or gross concentration. At early stages the proteins are localized preferentially in the cytoplasm of the animal hemisphere. They shift from the cytoplasm to the nucleus at stages specific for the individual proteins. During mitosis the proteins are released from the nucleus into the cytoplasm.     

The lens proteins in adult and embryos of the periodic albino mutant ofXenopus laevis

Summary The crystallins of normal and a^p mutants ofX. laevis have been studied using biochemical (electrophoresis in agar and polyacrylamide gels, isoelectric focusing) and immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoabsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The immunochemical analysis was carried out with rabbit antisera prepared against electrophoretic fractions of the mutant lens.Crystallins of adultX. laevis (a^p/a^p; ++/++) are heterogenous as judged by electrophoretic mobility, isoelectric point, antigenic and species specificity.No qualitative nor quantitative differences were found between crystallins of normal and mutant animals at the level of the protein subunits. These conclusions, however, are valid only for those crystallins, which are solubilized at pH 9.0.Immunofluorescence studies showed that crystallins appear in the normal and mutant embryos at practically the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found.Some of the gamma and, perhaps, beta-crystallins appear first; alpha-crystallins appear later. It has been shown for the first time that some gamma-crystallins are formed at advanced developmental stages.The periodic albino mutation does not affect the function of genes coding for crystallins either in embryos or in the adultX. laevis.     

Quantitative aspects of RNA synthesis in normal and lithium-treated embryos ofXenopus laevis

Summary The treatment ofXenopus early embryos with lithium chloride produces exogastruale — embryos which fail to gastrulate normally and in which the rates of cell division are reduced. In the present study estimations of incorporations of (5-³H) uridine and the specific activities of the 5-ribonucleotide precursor pools showed that exogastrulae have higher rates of RNA synthesis per cell than control neurulae. Sub-cellular fractionations showed that a greater proportion of labelled RNA was retained in the nuclei of exogastrulae than of neurulae, while neurulae showed a greater incorporation into polysomes.     

Endogenous lectin secretion into the extracellular matrix of early embryos of Xenopus laevis

An endogenous galactoside-binding lectin with subunit molecular weight of 43,000-45,000, previously detected in unfertilized eggs of Xenopus laevis, persists at high levels in embryos through gastrulation. During embryonic development the lectin is found in cytoplasmic vesicles, and then is secreted into extracellular matrix which is prominent around the blastopore and on the roof of the blastocoel. The lectin is also found in the extracellular material in the developing neural fold. The presence of lectin at sites of active morphogenetic movements raises the possibility that it participates in the formation of an extracellular matrix that influences these processes.     

EMA,an epithelial membrane-associated antigen during early development and morphogenesis ofXenopus laevis

Summary We raised monoclonal antibodies against a membrane fraction ofXenopus neurulae in order to detect tissue-specific cell-surface markers. Here we describe a monoclonal antibody that recognizes an epithelial membrane-associated antigen (EMA) in immunohistological stainings. The tissue-specific and membrane-associated antigen detected in immunohistological stainings could serve as useful marker in epithelium differentiation and membrane organization of the early embryo. In tadpoles and adults EMA was found in specific epithelial tissues derived from different germ layers such as kidney, skin, gut, pancreas, epiphysis and choroid plexus. In the cleaving embryo this antibody stained newly formed membranes between blastomeres from the two-cell stage onwards. Cytoplasmic staining in large oocytes and early embryos was also observed. The possibility that the cytoplasmic signal represents a maternal store of membrane material is discussed.     

Immuno-electron-microscopic study of fibronectin in gastrulating amphibian embryos

Summary The ultrastructural organization of fibronectin (FN) in early amphibian embryos (Ambystoma mexicanum, Pleurodeles waltlii) was studied with the use of antibodies directed against amphibian plasmatic FN. Scanning and transmission electron microscopy combined with immunogold labeling of FN revealed that the extracellular matrix that covers the inner surface of the ectodermal layer consists of FN-containing fibrils. During gastrulation, the mesodermal cells appear to be devoid of FN. These cells extend filopodia adhering to the FN-containing fibrils and are spreading along them. These findings suggest that FN may be involved in contact formation between mesodermal cells and the extracellular matrix that serves as a substratum for migration.     

Localization of cortical granule lectin ligand in Xenopus laevis egg jelly

The block to polyspermy in Xenopus laevis involves an interaction between a cortical granule lectin, released at fertilization, and a ligand located in the egg extracellular matrix. The egg extracellular matrix in X. laevis consists of a vitelline envelope and three distinct jelly layers, designated J1, J2 and J3. To localize cortical granule lectin ligand in the egg extracellular matrix, we used enzyme-linked lectin assays that showed that cortical granule lectin ligands were absent in J2, J3 and the vitelline envelope. Cortical granule lectin bound to a ligand(s) in J1 in a galactose-dependent fashion. In addition, we separated egg jelly macromolecules electrophoretically and, in conjunction with western blotting, have shown that J1 contains two major, high molecular weight ligands for cortical granule ligand. Finally, using confocal microscopy, we demonstrated that the ligand(s) for cortical granule lectin occupies a 20–30 μm thick band in a region of J1 just proximal to the vitelline envelope.     

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant’s layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant’s cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell’s trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar ”embryoid” shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events. Received: 26 April 1999 / Accepted: 30 August 1999     

Cleavage rate of diandric triploid mouse embryos during the preimplantation period.

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used 1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, 2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and 3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning microscopy of disaggregated and aggregated preimplantation mouse embryos

Summary Blastomeres isolated from 8-and 16-cell embryos (that is 1/8 and 1/16) show a smooth surface at their point of contact with other blastomeres and a microvillous free surface. Microvilli reappear completely on the smooth surface of 52% of 1/8 embryos and partially on 88% of 1/16 embryos if cultured in vitro for 6 h. When 2-to 8-cell embryos are aggregated to 8-cell embryos and forced apart after 1–3 h, the contact surface of the 8-cell embryos has become smooth. Fixed 8-cell embryos are also able to induce complete disappearance of microvilli on the contact surface of a living 8-cell embryo. Embryos having more than 8 cells do not induce complete disappearance of microvilli on the contact surface of 8-cell embryos. Aggregates of late morulae do not show complete disappearance of microvilli at their contact surfaces but rather a loosening of their peripheral blastomeres.Our results show that isolated 1/8 and 1/16 embryos tend to recover from regionalization, that the process of aggregation of embryos having 8 cells or less is similar to compaction and that embryos having more than 8 cells seem to aggregate by cell sorting. The processes of compaction, adhesion and reassortment are briefly discussed. We submit that blastomere regionalization, which depends on cell to cell contact, may be the spatial basis of embryonic regulation and of the inside-outside normal differentiation of early mouse embryos.     

Development of xenopus laevis embryos in a static magnetic field

Xenopus laevis embryos were exposed to a DC magnetic field (2.5 kG) for periods up to 1 week. The previously reported stabilization of cell membranes by stationary magnetic fields could not be demonstrated.     

Immunolocalization and functional role of Sclerotium rolfsii lectin in development of fungus by interaction with its endogenous receptor

Many fungi are known to secrete lectins, but their functional roles are not clearly understood. Sclerotium rolfsii, a soilborne plant pathogenic fungus capable of forming fruiting bodies called sclerotial bodies, secrete a cell wall-associated Thomsen-Friedenreich antigen-specific lectin. To understand the functional role of this lectin, we examined its occurrence and expression during development of the fungus. Furthermore, putative endogenous receptors of the lectin were examined to substantiate the functional role of the lectin. Immunolocalization studies using FITC-labeled lectin antibodies revealed discrete distribution of lectin sites at the branching points of the developing mycelia and uniformly occurring lectin sites on the mature sclerotial bodies. During development of the fungus the lectin is expressed in small amounts on the vegetative mycelia and reaching very high levels in mature sclerotial bodies with a sudden spurt in secretion at the maturation stage. Capping of the lectin sites on the sclerotial bodies by lectin antibodies or haptens inhibit strongly the germination of these bodies, indicating functional significance of the lectin. At the maturation stage the lectin interacts with the cell wall-associated putative endogenous receptor leading to the aggregation of mycelium to form sclerotial bodies. The lectin-receptor complex probably acts as signaling molecule in the germination process of sclerotial bodies. Using biotinylated lectin, the receptors were identified by determining the specific lectin binding to lipid components, extracted from sclerotial bodies, and separated on thin-layer chromatograms. Preliminary characterization studies indicated that the receptors are glycosphingolipids and resemble inositolphosphoceramides. These findings together demonstrate the importance of lectin-receptor interactions to explain hitherto speculated functional role of the lectins and also the glycosphingolipids of fungi.