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.     

The effects of Tunicamycin and 2-deoxy-D-glucose on the development ofXenopus laevis embryos

Summary Tunicamycin and 2-deoxy-D-glucose were applied toXenopus laevis embryos in the first cleavage furrow, blastula and early gastrula stages. No effect was observed with 2-deoxy-D-glucose up to the concentration 0.1 M. The effect of Tunicamycin is dose- and stage-dependent. At the concentration of 5 g/ml cleaving embryos are arrested at the onset of gastrulation and their cells exhibit decreased intercellular adhesivity, while embryos treated in the blastula and early gastrula stages may develop up to the late neurula and tail-bud stage, respectively. Higher concentrations (up to 20 g/ml) drastically affect cleavage. Concentrations of 4 to 1 g/ml allow embryos to develop up to more advanced stages; however, developmental defects are the rule. Concentrations of less than 1 g/ml do not affect development.     

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.     

The metamorphic switch in hemoglobin phenotype ofXenopus laevis involves erythroid cell replacement

Summary To elucidate the cellular basis of hemoglobin transition inXenopus laevis the distribution of larval and adult hemoglobins was analyzed by indirect immunofluorescence in the circulating erythrocytes during metamorphosis. In addition, the morphological characteristics as well as the capacity for synthesis of DNA and hemoglobin in the erythrocytes were followed during the same developmental period. Our quantitative analysis on the distribution of larval and adult hemoglobins suggests that they are localized in different cells. Hemoglobin transition, therefore, most likely reflects replacement of the larval erythrocyte population by new cells which are committed to adult globin synthesis. Since hemoglobin transition is not accompanied by an increase in the abundance of immature erythroid cells with active DNA synthesis, we assume that the presumptive adult erythroid cells are released into circulation at a relatively advanced stage of maturation. The decline in the synthesis of DNA and larval hemoglobin further indicates that cessation of cell renewal in the larval erythrocyte population may represent a decisive step in hemoglobin transition.     

Fission yeast tmsl protein abrogates normal development in Xenopus laevis embryos

Recently we cloned tms1 (a putative dehydrogenase) by complementation of a human tumour-derived mutant p53 induced growth arrest in fission yeast. Microinjection of purified tmsl protein into Xenopus laevis embryos abrogated normal embryo development by causing cleavage retardation or cleavage arrest of injected blastomeres in a concentration dependant manner, whereas injection of specific affinity purified tms1 antiserum showed no significant morphological defects. Microinjection of tms1 protein together with affinity purified tms1 antibody resulted in a significantly reduced number of cleavage arrested embryos.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

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.     

Two recessive mutations with maternal effect upon colour and cleavage ofXenopus l. laevis eggs

Summary Pale eggs and partial cleavage are two mutations with a maternal effect that are found in the same family ofXenopus l. laevis. The pale eggs have animal hemispheres of a yellow to beige colour and give rise to normally pigmented tadpoles and frogs. The cells of pale embryos contain fewer melanosomes than those of controls. The partial cleavage eggs are characterized by an abnormality of cleavage visible from the eight-cell stage onwards, by abnormal yolk platelet distribution and abnormal cytological features. Cleaved, syncytial and uncleaved areas are observed in these eggs, which are lethal at the blastula stage.     

Calcium signalling during neural induction in Xenopus laevis embryos

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

Surface changes during development and involution of the cement gland of Xenopus laevis

Summary The cement gland was studied from stage 17, when the anlage is established, to stage 49, shortly before its disappearance. At early stages, the apical membrane is covered by small microvilli that are more abundant than in the surrounding epiblast cells. Vesicular protrusions along the cell boundaries are also more numerous in the gland cells.When the gland reaches maturity, the apical membranes of gland cells differentiate into two regions. In the cranial, kidney-shaped region, the membranes are very narrow and protrude above the level of cell boundaries. Long and slender villi raise from the surface adjacent to cell boundaries. Apical surfaces in the caudal portion are larger and flattened. Cell boundaries are lined with shorter and thicker surface projections. At these stages, the bordering cells are covered with secretion vesicles.During involution the number of cells is progressively reduced. The area of the caudal portion increases relative to the area of the cranial portion. Apical surfaces become more flattened. Surface projections become much shorter and invade the whole of the apical surface. Bordering cells lose their secretion vesicles and their apical surface becomes ruffled with numerous short wrinkles. The significance of the apical structures and their evolution is discussed.     

Spatially and temporally regulated alpha6 integrin cleavage during Xenopus laevis development

The α6 integrin is essential for early nervous system development in Xenopus laevis. We have previously reported a uPA cleaved form of integrin α6 (α6p), in invasive human prostate cancer tissue, whose presence correlates with increased migration and invasive capacity. We now report that α6 is cleaved during the normal development of Xenopus in a spatially and temporally controlled manner. In addition, unlike normal mammalian tissues, which lack α6p, the major form of the α6 integrin present in adult Xenopus is α6p. The protease responsible for the cleavage in mammals, uPA, is not involved in the cleavage of Xenopus α6. Finally, overexpression of a mammalian α6 mutant which cannot be cleaved leads to developmental abnormalities suggesting a potential role for the cleavage in development.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.     

Neural-inducing activity of nuclei and nuclear fractions from Xenopus laevis embryos

Summary From embryos (Xenopus laevis) of different developmental stages nuclei were isolated which exert neural inducing activity in the biological test. The active material could partly be extracted from the nuclei. Experiments for the isolation of nuclear ribonucleoprotein (RNP) particles have shown that the activity is localized at least in part in these particles. On the other hand, some neural inducer is not detached from chromatin and the nuclear matrix even with ionic detergents. Inducing activity was found in germinal vesicles and to a higher degree in the cytoplasm of oocytes, but in a masked, biologically inactive state.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Exogenous sphingosine enters Xenopus laevis embryos grown in petri dishes and it is metabolized

Xenopus embryos of different developmental stages were exposed to 0.1 M [1-³H]sphingosine. Labeled sphingosine was quickly absorbed by Xenopus embryos. The amount of radioactivity absorbed increased with embryo age and appeared to be linearly correlated (R=0.97) to the embryo surface area. About 45% of the total radioactivity associated to the embryos was found in the skin, 22% in the intestine, 15% in the heart, 12% in the liver and 6% in the brain.A portion of [1-³H]sphingosine entered very rapidly the biosynthetic pathway of sphingolipids; after 30 min of incubation, in fact, only a small amount of free radioactive sphingosine could be detected. Sphingomyelin was the main radioactive sphingolipid synthesized; radioactive ceramide, galactosylceramide and lactosylceramide could also be recognized and quantified. On the contrary, the amount of radioactive gangliosides was hardly detectable.A portion of [1-³H]sphinogosine absorbed by Xenopus embryos (30 to 60% depending on the developmental stage) entered the catabolic pathway producing radioactive phosphoethanolamine that was recycled for the biosynthesis of radioactive phosphatidylethanolamine. This phospholipid was produced mainly in the intestine and in the skin, while sphingomyelin was the main radioactive lipid in the heart, liver and brain.     

Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.