Tadpole Xenopus laevis hemoglobin. Correlation between structure and functional properties

Perutz & Brunori (1982) proposed that the COOH-terminal His and Ser F9 of the beta-chains of fish and amphibian hemoglobins are responsible for their Root effect and part of their alkaline Bohr effect. Analysis of the kinetics of carbon monoxide binding by hemoglobin from the tadpole of Xenopus laevis supports that model and suggests an explanation for the absence of an alkaline Bohr effect in many aquatic Anura and Urodela.     

Induction and patterning of the telencephalon in Xenopus laevis

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.     

Obtaining of Agr2 Specific Antibodies and Determination of the Agr2 Protein Distribution Pattern during Early Embryonic Development and Tadpole Regeneration in Xenopus laevis

Russian Journal of Developmental Biology - The Agr (anterior gradient) group proteins belong to the family of proteins with a noncanonical thioredoxin motif and are involved in the regulation of...     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

Induction of Vitellogenin Synthesis in Xenopus laevis Tadpoles

Oestradiol induces vitellogenin synthesis in vitro in liver taken from Xenopus laevis tadpoles that are in late metamorphosis. Inducibility first appears at the end of prometamorphosis, and the response to oestradiol increases during the completion of metamorphosis. Oestradiol continuously present during development does not influence the stage at which tadpole liver becomes inducible. It seems that the acquisition of inducibility is part of the normal development of the liver, and independent of both the supply of oestrogen and the sex of the tadpole.     

Pattern regulation in defect embryos of Xenopus laevis

Defect embryos of 24 series were prepared by removing increasing numbers of blastomeres from an 8-cell embryo of Xenopus laevis. They were cultured and their development was examined macroscopically when controls reached a tailbud stage or later. Results show that most of defect embryos of 12 series develop normally, and some of them become normal frogs. Each of these defect embryos contain at least two animal blastomeres, one dorsal, and one ventral blastomere of the vegetal hemisphere. This suggests that a set of these four blastomeres of the three types is essential for complete pattern regulation.     

Cell intercalation during notochord development in Xenopus laevis

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high-resolution time-lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamelliform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid- to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well-defined geometric and mechanical environment.     

Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Induction of meiosis of Xenopus laevis oocytes by mianserine

Maturation of Xenopus laevis oocytes can be induced by mianserine, a tricyclic antidepressant. K⁺-free medium facilitates this maturation process. Mianserine must be kept in contact with the oocytes during the whole process of maturation for maximal efficiency. It is inactive after injection into the oocytes. Mianserine induces the formation of maturation-promoting factor (MPF) in the treated oocytes. Mianserine-induced maturation is strongly inhibited by theophylline, even in K⁺-free medium. Progesterone displays synergistic effects with mianserine for the induction of maturation. Likewise, oestradiol shows cooperative maturing effects with progesterone as well as with mianserine. It is suggested that mianserine exerts its primary effects on oocyte maturation by inhibiting a membrane adenylate cyclase.     

Induction of glucose-regulated proteins in Xenopus laevis A6 cells

We have characterized the induction of glucose-regulated proteins (GRPs) in Xenopus laevis A6 cells, a kidney epithelial cell line. Exposure of A6 cells to medium in which 2-deoxyglucose replaced galactose resulted in enhanced synthesis of two proteins at 78 and 98 kd. The 78 kd protein was determined by two-dimensional PAGE to consist of two isoelectric variants with pls of 5.3 and 5.2 whereas the 98 kd protein resolved into a single spot with a pl of 5.1. The 78 kd protein cross-reacted with antiserum against chicken GRP78 (glucose-regulated protein), suggesting that the Xenopus protein shares homology with a previously characterized GRP. This was supported by the finding that a rat GRP78 probe hybridized with a 2-deoxyglucose-inducible mRNA. Synthesis of the two proteins was also induced by tunicamycin, 2-deoxygalactose, and dithiothreitol. However, the GRPs were not induced by glucosamine or calcium ionophore A23187 at concentrations and exposure periods that have previously been shown to elicit a GRP response in mammalian and avian cells. Enhanced synthesis of the two GRPs by 2-deoxyglucose was transient, reaching maximal levels by 12-24 h and decreasing to near control levels by 48 h. Removal of the stress at the point of peak synthesis resulted in decreased synthesis of both proteins within 6 h and a return to control levels within 24 h of recovery. These data suggest that Xenopus cells have a GRP response that is similar, but not identical, to that found in mammalian cells.     

Histochemical and Enzyme Digestion Studies on Neural Induction in Xenopus laevis

The purpose of this work was to obtain information on the chemical constitution and functional significance of the extracellular materials (granules and fibrils) observed, in previous investigations, at the ecto-mesodermal junction during neural induction.
The results indicate that the granules are composed mainly of RNA and the fibrils of glycosaminoglycans, and that neither of these morphological features are essential mediators of the inductive stimuli required for the formation of the nervous system.
It is therefore suggested that the mechanism of neural induction depends either on the passage of diffusible substances between the two tissues or on direct contacts between the membranes of their constituent cells.     

Development of behaviour in the hindlimb of Xenopus laevis

The paper is one of a series of studies of the ontogeny of the innervation of the vertebrate limb in which the histogenesis of the nerves is correlated with the development of the pattern of behaviour in the limb. Here, the motility of the developing limb in tadpoles of Xenopus laevis is described, both in the normal larva and those in which the spinal cord is isolated from the brain. In spinal tadpoles the responses of the limb to electrical stimulation are correlated with its normal behaviour.     

Cell migration in the formation of the pronephric duct in Xenopus laevis

To determine if cell migration is involved in the formation of the pronephric duct in Xenopus, we used morphometry, ablation, and videomicroscopy of vitally stained cells to study duct formation. In St 23-24 (Nieuwkoop and Faber, 1956) embryos, a ridge of cells forms caudal to the pronephric rudiment. The ridge lengthens at approximately the same rate as the embryonic trunk from St 23 to St 31. Ablation experiments demonstrated that the ridge constitutes the pronephric duct rudiment (PDR); when the ridge was ablated at St 23-24, little or no duct formation occurred, whereas a duct formed when the pronephric rudiment was ablated and the ridge left intact. Vital dye injections showed that the PDR forms from the intermediate mesoderm ventral to myotomes IV-VIII. From St 29/30 to St 33/34, the PDR actively elongates along the ventral edge of the myotomes as far as myotome XIV, where it joins the cloaca as the pronephric duct. Videomicroscopy of vitally stained cells showed that the PDR elongates throughout its length and does not incorporate additional cells from the mesoderm over which it elongates. The results strengthen the case for a common mode of pronephric duct formation among amphibian species.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.