Early Steroid Metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris Embryos

Embryos of Xenopus laevis , Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos.
In Xenopus incubations progesterone was converted to 5α-pregnane-3,20-dione, 17α-hydroxy-4pregnen-3-one, 4-androstene-3,17-dione and 17α,20α:-dihydroxy-4-pregnen-3-one, indicating 5α-reductase, 17α-hydroxylase, 19–20-desmolase and 20α-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19–20-desmolase was found. In Rana oocytes were also not evidence of 17α-hydroxylase activity. All identified activities except 20α-hydroxylase were common to embryos of all three species.
It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthetized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis .
Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells.
These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.     

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to facilitate cross-presentation of chaperoned antigens and elicit innate and adaptive T cell responses. Xenopus skin graft rejection provides an excellent platform to study the ability of gp96 to elicit classical MHC class Ia (class Ia) restricted T cell responses. Additionally, the Xenopus model system also provides an attractive alternative to mice for exploring the ability of gp96 to generate responses against tumors that have down-regulated their class Ia molecules thereby escaping immune surveillance. Recently, we have developed an adoptive cell transfer assay in Xenopus clones using peritoneal leukocytes as antigen presenting cells (APCs), and shown that gp96 can prime CD8 T cell responses in vivo against minor histocompatibility skin antigens as well as against the Xenopus thymic tumor 15/0 that does not express class Ia molecules. We describe here the methodology involved to perform these assays including the elicitation, pulsing and adoptive transfer of peritoneal leukocytes, as well as the skin graft and tumor transplantation assays. Additionally we are also describing the harvesting and separation of peripheral blood leukocytes used for flow cytometry and proliferation assays which allow for further characterization of the effector populations involved in skin rejection and anti-tumor responses.Download video file.^{(128M, mp4)}     

Dissection,Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation^1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates ^12,14-18.While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells ^7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues ^{8,19-22,24-33}. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level ^{5,8,21,24,27-30,33-39}. Xenopus laevis, a classic model system for the study of early neural development ^{19,27,29,31-32,40-42}, serves as a particularly suitable system for retinal primary cell culture ^10,38,43-45.Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction ^25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products ^10,24,44-45.However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).     

Specification and Establishment of Dorsal-Ventral Polarity in Eggs and Embryos of Xenopus laevis

Dorsal-ventral (D-V) polarization in Xenopus eggs and embryos is achieved by passing through a series of complicated phenomena such as initial specification of the polarity before first cleavage, establishment of polarity during cleavage stages resulting in an acquisition of a unique developmental capacity by each blastomere, regional differentiation of mesoderm, and finally neural induction by Spemann's organizer. In order to gain an insight into basic mechanisms which govern D-V polarization, experimental modifications or perturbations of the body axis of embryos, including physical or chemical treatments of eggs, altered orientation of eggs under the normal gravity, centrifugation, manipulation of blastomeres, cytoplasmic withdrawal, and bisection or partial ligation of fertilized eggs are reviewed: all data are consistent with the concept that a cytoplasmic activity which becomes localized in the dorsal side of the egg is responsible or indispensable for the establishment of the D-V axis. The cytoplasmic activity is tentatively called "anterodorsal structure-forming activity." A model which explains the specification, establishment, and realization of D-V polarity in Xenopus laevis is proposed.     

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.     

Formation of the Neural Plate and the Mesoderm in Normally Developing Embryos of Xenopus laevis

Normally developing embryos of Xenopus were fixed at various stages between the blastula and early tail bud stage, and their serial sections were examined. The marginal belt of the blastula was characterized by abundance of cells with RNA-rich peripheral cytoplasm called mesoplasm. At the early gastrula stage, the marginal belt was folded into two layers giving rise to mesodermal material and marginal ectoderm. During gastrulation, the mesodermal material, which consisted of RNA-rich cells, spread to enclose the blastocoel and the endoderm, and a large part of it was shifted to the dorsal side of the embryo. It gradually established the mesodermal layer. The notochord was formed on the dorsal lip of the blastopore by involution, separately from preformed mesodermal material. The RNA-rich cells in the marginal ectoderm became columnar, forming a broad belt in the marginal zone. This belt was deformed and shifted to the dorsal side during gastrulation, eventually establishing the neural plate showing quantitative differentiation along the head-tail axis. Possible mechanisms involved in the formation of the neural plate and mesoderm were discussed with reference to the organizer and the mesoplasm.     

Cyclin A1/Cdk2 is Sufficient but not Required for the Induction of Apoptosis in Early Xenopus laevis Embryos

The role of cyclin-dependent kinases in cell proliferation is well characterized, whereas their somewhat paradoxical role in catalyzing apoptosis is less understood. One Cdk complex implicated in both cell proliferation and cell death is cyclin A/Cdk2. During early embryonic development of Xenopus laevis, distinct isoforms of cyclin A are expressed at different times. From fertilization through gastrulation, cyclin A1 is the predominant isoform. Cyclin A1 dimerizes with Cdk2 but not Cdk1. In contrast, cyclin A2 is expressed at a low level until gastrulation, when it becomes the major A-type cyclin and associates with both Cdk1 and Cdk2. When Xenopus embryos are treated with ionizing radiation (IR) prior to the midblastula transition (MBT), cyclin A1 protein persists beyond the MBT and forms an active complex with Cdk2. During this window of cyclin A1/Cdk2 activity, the embryo undergoes apoptosis. To test the hypothesis that cyclin A1-associated activity is a mediator of apoptosis, cyclin A1 protein level and associated kinase activity were measured in embryos treated with aphidicolin to induce apoptosis. Both cyclin A1 content and associated kinase activity were sustained after the MBT as embryos underwent apoptosis. To determine whether cyclin A1/Cdk2 was sufficient to induce apoptosis, recombinant cyclin A1/Cdk2 complex was injected into single-celled embryos, which induced apoptosis after the MBT. However, morpholinos targeting translation of cyclins A1 and A2 did not block apoptosis in embryos treated with X-rays or aphidicolin. These data indicate that cyclin A1/Cdk2 is sufficient, but not required for apoptosis during early development.     

Xenopus laevis lectin is localized at several sites in Xenopus oocytes, eggs, and embryos

The endogenous lectin of Xenopus laevis oocytes, unfertilized eggs, and blastula-stage embryos was immunohistochemically localized using a highly specific antiserum. Each tissue was examined with several techniques, including paraformaldehyde or glutaraldehyde fixation, frozen or plastic sections, and immunofluorescence or immunoperoxidase staining. In oocytes and unfertilized eggs, lectin was detected in association with yolk platelets, cortical granules, and the vitelline envelope. In embryos, cortical granules had disappeared and lectin was found in the cleavage furrows between the embryonic cells. The distribution of the lectin suggests that it plays more than one role in this developing system.     

Development of Microtubule-Dependence of the Chromosome Cycle at the Midblastula Transition in Xenopus laevis Embryos

Animal-cap cells isolated from Xenopus laevis morulae and blastulae are cultured for 2 to 6 hr in medium containing nocodazole, Colcemid or taxol, at concentrations completely inhibiting cell division. At 20°C, cells from each control embryo undergo synchronous cell cycles up to the 12th, with a period of 32 min, of which 60% represents the chromosome condensation (mitotic or M-) phase, and the average mitotic index remains near 50%. Cells treated with nocodazole, Colcemid or taxol before 12th cleavage undergo chromosome cycles with a similar period as controls, albeit without chromosome segregation, and the average mitotic index remains near 50%. From the 12th to 15th cycles, control cycles become asynchronous, their period gradually increases 2 to 3 times, and the mitotic index declines to 10%. In cells treated after 12th cleavage with taxol, the mitotic index declines, similarly to control cells. However, in nocodazole-treated cells, it increases steadily, and exceeds 70% at 2 hr of treatment, but gradually declines to 40% at 6 hr. Therefore, while inhibition of microtubule activities does not significantly alter the timing of chromosome condensation cycles during synchronous cleavage, inhibition of microtubule assembly can prolong M-phase during asynchronous cleavage after the midblastula transition.     

Circadian Locomotor Activity Rhythms in the African Clawed Frog, Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.     

Circadian Locomotor Activity Rhythms in the African Clawed Frog,Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.     

XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos

Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.     

A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.     

The Cytoskeletal Protein Zyxin Modulates the Expression of the Target Genes of the Shh Signaling Cascade in the Cells of the Neural Plate of Embryos of the Spur-Toed Frog Xenopus laevis

Russian Journal of Bioorganic Chemistry - It has been shown earlier in a study of the role of the cytoskeletal protein zyxin in cell differentiation in the primordium of the central nervous system...     

The expression of creatine kinase isozymes in Xenopus tropicalis,Xenopus laevis laevis,and their viable hybrid

Starch gel electrophoresis of creatine kinase (CK) isozymes of Xenopus tropicalis shows that at least two different genes code for CK in this diploid (2n=20) species. These genes seem to be orthologous to the CK-A and CK-C genes of extant crossopterygian fish. Additional isozymes may be interpreted either as products of duplicate genes or, more probably, as epigenetically modified forms of the homodimers A^tA^t and C^tC^t, respectively. The originally tetraploid species X. laevis laevis (2n=36), which may have arisen by hybridization of diploid ancestors some 30–40 million years ago, has retained expression of all duplicate CK-A and CK-C genes. Differential expression during ontogenesis (CK-A genes) and in different adult tissues (CK-C genes) indicates that divergence occurred not only with respect to the primary sequence of these duplicate genes, but also with respect to the regulation of their expression. In the interspecific hybrid X. 1. laevis × X. tropicalis, all parental CK genes appear to be expressed simultaneously in the heart. However, several subunit combinations cannot be detected on the zymograms.This work was supported by Swiss National Foundation for Scientific Research Grant 3.775.0.80.     

Preservation of Xenopus laevis rDNA-containing plasmid, pXlr101A, injected into the fertilized egg of Xenopus laevis

A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pXlr101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pXlr101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.