South African Clawed Toad Research Articles

<b>Microvascularization of the Pancreas in Larval and Adult </b><i><b>Xenopus laevis</b></i><b> – Histomorphology and Scanning Electron Microscopy of Vascular Corrosion </b><b>Casts </b>

The microvascularization of the pancreas of larval and adult South African Clawed Toads Xenopus laevis, was studied by scanning electron microscopy of vascular corrosion casts and light microscopy of paraplast embedded Goldner-stained serial tissue sections. We showed that branches of left and right gastric artery, hepatic artery and anterior intestinal artery, namely anterior pancreatic, anterior middle pancreatic, posterior middle pancreatic and caudal pancreatic arteries supply and pancreatic veins drain the adult pancreas into hepatic portal vein, anterior and middle gastric vein, gastroduodenal vein, and anterior duodenal vein. In premetamorphosis the pancreas showed a dense but immature vascular bed with signs of ongoing sprouting and non-sprouting angiogenesis (=intussusceptive microvascular growth; IMG). During metamorphic climax the pancreas shrinked dramatically paralleled by vascular regression.The larval pancreas had an underdeveloped ductal system which in the course of pancreas remodeling during metamorphic climax developed into a complex ductal system. In adult Xenopus laevis the pancreas showed intralobular islets of Langerhans only and an insuloacinar portal vessel system as described sofar in reptiles, birds and mammals. Islets in Xenopus located superficially and within deeper regions and emitted both insulo-acinar portal vessels and insulo-venous efferent vessels. Intrainsular microvascular patterns found suggest that in Xenopus islets blood flows first to β-cells and subsequently to the other endocrine cells present in the endocrine pancreas. Key words: Xenopus, pancreas, microvascularization, vascular casts, histomorphology

Mesodermal origin of median fin mesenchyme and tail muscle in amphibian larvae.

Mesenchyme is an embryonic precursor tissue that generates a range of structures in vertebrates including cartilage, bone, muscle, kidney, and the erythropoietic system. Mesenchyme originates from both mesoderm and the neural crest, an ectodermal cell population, via an epithelial to mesenchymal transition (EMT). Because ectodermal and mesodermal mesenchyme can form in close proximity and give rise to similar derivatives, the embryonic origin of many mesenchyme-derived tissues is still unclear. Recent work using genetic lineage tracing methods have upended classical ideas about the contributions of mesodermal mesenchyme and neural crest to particular structures. Using similar strategies in the Mexican axolotl (Ambystoma mexicanum), and the South African clawed toad (Xenopus laevis), we traced the origins of fin mesenchyme and tail muscle in amphibians. Here we present evidence that fin mesenchyme and striated tail muscle in both animals are derived solely from mesoderm and not from neural crest. In the context of recent work in zebrafish, our experiments suggest that trunk neural crest cells in the last common ancestor of tetrapods and ray-finned fish lacked the ability to form ectomesenchyme and its derivatives.

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

Using Transgenic Animal Models in Neuroendocrine Research

Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.

Intracellular signal transduction by the extracellular calcium-sensing receptor of Xenopus melanotrope cells

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist l-phenylalanine ( l-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G s/PKA nor G q/PKC pathways are involved. However, pertussis toxin (G i/o protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaR-evoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of l-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.

Possible antimicrobial defense by free sugars on the epidermal surface of aquatic vertebrates

Biochemical, lectin histochemical, and densitometrical methods demonstrated contents and spectra of free sugars in the skin of aquatic vertebrates (fishes: cod, marine trout, freshwater trout, perch; amphibians: Koa-Tao-Island caecilian, smooth newt, edible frog, South-African clawed toad; mammals: northern fur seal, common seal, otter, Capybara). Total free sugar contents, particu- larly sialic acids, were relatively higher in freshwater species (perch, Capybara) than in marine species. In amphibians, the caecilian and the newt showed higher amounts of free sugars than the anurans; strong reactions indicated a specific involvement of α-L-fucose in mucus functions. Lectin histochemistry demonstrated positive reactions in the stratum superficiale, the outer stratum spinosum, and specific cells of the fish epidermis. The integumental free sugar spectrum of the amphibians was narrow, with generally lower reaction intensities, except for mucus glands. In the mammals, the epidermal cells exhibited rather weak lectin histochemical staining, except for the northern fur seal and the Capybara, where the upper vital epidermis and corneal cells reacted strongly. The mammalian apocrine skin glands, particularly, exhibited positive reaction staining in their secretory endpiece. The free sugar spectrum was similar in all species: α-D-mannose, β-D-N-acetylglucosamine, α-D-N-acetylgalactosamine, α-D-galactose, β-D-galactose, α-L-fucose, sialic acids (mainly in fish epidermis and skin glands). High concentrations of free sugars on the skin surface possibly impede attacks of commensal skin micro-inhabitants (bacteria, fungi) against the integrity of the epidermis. This features a basic or general biological mechanism that operates before the specific immune system is activated.

Endocrine disrupting chemicals (EDC) with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: II. Effects on gonad histomorphology

A number of man-made chemicals has been shown to mimic endogenous hormones and to induce alterations of reproductive physiology in wild populations. Of particular importance are compounds that mimic estrogens and androgens (and their antagonists), because of their central role in reproductive function. In this study, male and female adult South African clawed toads ( Xenopus laevis) were exposed to ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT) and flutamide (FLU) as (anti)estrogenic and (anti)androgenic model compounds, respectively, at a concentration of 10 − 8 M, and to water from the river Lambro (LAM), a contaminated watercourse from Northern Italy. Potential disrupting effects on reproduction were studied by histological analyses of gonads. The strongest adverse effects were observed in EE2 and LAM exposed males, e.g. tubule mean diameter reduction, spermatogenic nest breakdown and interlobular wall thickening. In both groups, the occurrence of small oocytes within the seminiferous tubules was observed. In TAM and MDHT exposed females slight oocyte atresia and occurrence of spermatogenic nests were observed. In contrast to previous studies addressing the alteration of molecular biomarkers in the same experimental setup, histological analyses of gonads were very sensitive and indicated an adverse effect of water from Lambro River on reproductive physiology of X. laevis.

Cancer Resistance in Amphibians

While spontaneous tumours may occasionally develop in inbred and isogenic strains of Xenopus laevis, the South African clawed toad, they are extremely rare in natural and laboratory populations. Only two amphibian neoplasms, the renal adenocarcinoma of Rana pipiens and the lymphosarcoma of Xenopus laevis, have been extensively explored. Amphibians are resistant to the development of neoplasia, even following exposure to "direct-acting" chemical carcinogens such as N-methyl-N-nitrosourea, that are highly lymphotoxic, thus diminishing immune reactivity. Regenerative capacity in adults, and a dramatic metamorphosis which remodels much of the larval body to produce the adult form, are unique to amphibian vertebrates, and the control mechanisms involved may protect against cancer. For example, naturally rising corticosteroid titres during metamorphosis will impair some T-cell functions, and the removal of T-regulatory (suppressor) functions inhibits the induction of altered-self tolerance. Altered-self tolerance is not as effectively induced in adult Xenopus laevis as in mammals, so cancer cells with new antigenicity are more likely be rejected in amphibians. Amphibian immunocytes tend to undergo apoptosis readily in vitro, and, unlike mammalian immunocytes, undergo apoptosis without entering the cell cycle. Cells not in the cell cycle that die from nuclear damage (apoptosis), will have no opportunity to express genetic instability leading to cell transformation. We suggest that all these factors, rather than any one of them, may reduce susceptibility to cancer in amphibians.

Bleomycin‐induced DNA damage and repair in Xenopus laevis and Xenopus tropicalis

Microgel cell electrophoresis has been used with various species to measure breakage of DNA and DNA repair following exposure to the radiomimetic antibiotic, bleomycin. With humans, a high degree of DNA damage is considered to be predictive of cancer susceptibility. Non-isogeneic Xenopus laevis, the South African clawed toad, rarely develop spontaneous or induced cancers. Here, we investigate bleomycin-induced DNA damage and repair in splenic lymphocytes of this species to test consistency with cancer predictability. As X. laevis is pseudotetraploid in nature, while Xenopus tropicalis is diploid, we additionally explore the effect of polyploidy on DNA damage and repair in these vertebrates. The results show that higher doses of bleomycin are required to induce comparable levels of DNA damage in both Xenopus species, than in humans. X. tropicalis, the diploid, is more bleomycin-sensitive than is X. laevis. Additionally, repair rates of damaged DNA of X. laevis lymphocytes are more rapid than those of X. tropicalis, although both are hours slower than human leukocytes. While no data exist on cancer susceptibility in X. tropicalis, the results suggest greater susceptibility to cancer than X. laevis, but less than in humans. Thus, polyploidy serves as a protection against DNA damage and allows more rapid repair.

Evidence that urocortin I acts as a neurohormone to stimulate αMSH release in the toad Xenopus laevis

We have raised the hypothesis that in the South African clawed toad Xenopus laevis, urocortin 1 (UCN1), a member of the corticotropin-releasing factor (CRF) peptide family, functions not only within the brain as a neurotransmitter/neuromodulator but also as a neurohormone, promoting the release of α-melanophore-stimulating hormone (αMSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland. This hypothesis has been investigated by (1) assessing the distribution of UCN1 and CRF by light immunocytochemistry, (2) determining the subcellular presence of UCN1 in the neural lobe of the pituitary gland by immuno-electron microscopy applying high-pressure freezing and cryosubstitution, and (3) testing the effect of UCN1 on MSH release from toad melanotrope cells using in vitro superfusion. In the X. laevis brain, the main site of UCN1-positive somata was found to be the Edinger–Westphal nucleus. UCN1 immunoreactivity (ir) also occurs in the nucleus posteroventralis tegmenti, central gray, nucleus reticularis medius, nucleus motorius nervi facialis, and nucleus motorius nervi vagi. UCN1 occurs together with CRF in the nucleus motorius nervi trigemini, and in the magnocellular nucleus, which send a UCN1- and CRF-containing fiber tract to the median eminence. Strong UCN1-ir and CRF-ir were found in the external zone of the median eminence. From the internal zone of the median eminence, UCN1-ir fibers, but few CRF-ir fibers, were found to project to the pituitary neural lobe, where they form numerous neurohemal axon terminals. Ultrastructurally, two types of terminal containing UCN1-ir secretory granules were distinguished: type A contains large, moderately electron-dense, round secretory granules and type B is filled with smaller, strongly electron-dense, ellipsoid secretory granules. In vitro superfusion studies showed that UCN1 stimulated the release of αMSH from melanotrope cells in a dose-dependent manner. Our results support the hypothesis that in X. laevis, UCN1 released from neurohemal axon terminals in the pituitary neural lobe functions as a stimulatory neurohormone for αMSH release from melanotrope cells of the pituitary intermediate lobe.

Expression and characterization of the extracellular Ca(2+)-sensing receptor in melanotrope cells of Xenopus laevis.

The extracellular Ca(2+)-sensing receptor (CaR) is expressed in many different organs in various species, ranging from mammals to fish. In some of these organs, this G protein-coupled receptor is involved in the control of systemic Ca(2+) homeostasis, whereas in other organs its role is unclear (e.g. in the pituitary gland). We have characterized the CaR in the neuroendocrine melanotrope cell of the intermediate pituitary lobe of the South African clawed toad Xenopus laevis. First, the presence of CaR mRNA was demonstrated by RT-PCR and in situ hybridization. Then it was shown that activation of the CaR by an elevated extracellular Ca(2+) concentration and different CaR-activators, including L-phenylalanine and spermine, stimulates both Ca(2+) oscillations and secretion from the melanotrope. Furthermore, it was revealed that activation of the receptor stimulates Ca(2+) oscillations through opening of voltage-operated Ca(2+) channels in the plasma membrane of the melanotropes. Finally, it was shown that the CaR activator L-phenylalanine could induce the biosynthesis of proopiomelanocortin in the intermediate lobe. Thus, in this study it is demonstrated that the CaR is present and functional in a defined cell type of the pituitary gland, the amphibian melanotrope cell.

Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni 2+

We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na + channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance ( C m) measurements and noise analysis to evaluate the effects of cAMP and Ni 2+ on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker–channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current ( I ami) and conductance ( G ami). Furthermore, C m was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni 2+ (2.5 mM) inhibited about 60% of the rENaC current and conductance while C m remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni 2+ on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.

Control of chromosomal DNA replication in the early Xenopus embryo.

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis.

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.

Intussusceptive microvascular growth in the lung of larval Xenopus laevis Daudin: a light microscope, transmission electron microscope and SEM study of microvascular corrosion casts.

The remodeling of the uniform wide, plexus-like capillary bed of the lung of metamorphosing tadpoles of the South African clawed toad Xenopus laevis (Daudin) is studied from developmental stages 54 to 65 by scanning electron microscopy (SEM) of microvascular corrosion casts (VCCs), light microscopy (LM) and transmission electron microscopy (TEM). VCCs reveal that the remodeling of the existing uniform, plexus-like lung capillary bed into well-defined alveolar capillary meshworks starts in the caudal lung and then gradually proceeds cranially. Vascular remodeling is entirely by intussusceptive microvascular growth through insertion and enlargement of new and fusion of pre-existing capillary meshes. Analyses of lung tissue serial sections at the LM and TEM level confirm the presence of intracapillary cushions and tissue posts and correlate these structures in respect of size and location to the round to slit-like imprints and tiny "holes" found in VCCs. Additionally, SEM of VCCs give clear evidence that intussusceptive microvascular growth is also involved in the remodeling and maturation of alveolar arterioles and venules.

Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition

Inflammation is avoided in apoptosis by early removal of dying cells by macrophages (MØs). In mammalian cells, an early aspect of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet of the cell membrane to the surface. PS recognition can serve as a signal for triggering removal of dying cells. PS expression on splenocytes and thymocytes of Xenopus laevis was quantified using FITC-Annexin and flow cytometry following exposure in vitro to several known apoptogens for this species. All apoptogens used induced PS expression. Dose dependency and the kinetics of PS expression following exposure to the calcium ionophore, A23187, were also examined. Peritoneal exudate cells (PEC’s) were cultured with A23187-treated thymocytes to test MØ capacity for recognition of PS. MØ binding to apoptotic thymocytes was reduced following exposure of PEC’s to a water soluble analogue of PS, phospho- L-serine. The presence of a phagocytic PS-dependent recognition system in amphibia is supportive of the evolutionary conservation of this function in mammals that is crucial in limiting inflammation induced by dying cells.

Stretch-independent activation of the mechanosensitive cation channel in oocytes of Xenopus laevis

Oocytes of the South African clawed toad Xenopus laevis possess in their plasma membrane a so-called stretch-activated cation channel (SAC) which is activated by gently applying positive or negative pressure (stretch) to the membrane patch containing the channels. We show here that this mechanosensitive channel acted as a spontaneously opening, stretch-independent non-selective cation channel (NSCC) in more than half of the oocytes that we investigated. In 55% of cell-attached patches (total number of patches, 58) on 30 oocytes from several different donors, we found NSCC opening events. These currents were increased by elevating the membrane voltage or raising the temperature. NSCC and SAC currents shared some properties regarding the relative conductances of Na +>Li +>Ca 2+, gating behaviour and amiloride sensitivity. Stretch-independent currents could be clearly distinguished from stretch induced SAC currents by their voltage and temperature dependence. Open events of NSCC increased strongly when temperature was raised from 21 to 27°C. NSCC currents could be partly inhibited by high concentrations of extracellular Gd 3+ and amiloride (100 and 500 μM, respectively). We further show exemplarily that NSCC can seriously hamper investigations when oocytes are used for the expression of foreign ion channels. In particular, NSCC complicated investigations on cation channels with small conductance as we demonstrate for a 4 pS epithelial Na + channel (ENaC) from guinea pig distal colon. Our studies on NSCCs suggest the involvement of these channels in oocyte temperature response and ion transport regulation. From our results we suggest that NSCC and SAC currents are carried by one protein operating in different modes.

Expression of a Fas-like proapoptotic molecule on the lymphocytes of Xenopus laevis

Ligation of the externally expressed Fas (APO 1/CD95) molecule will initiate programmed cell death (apoptosis), in many mammalian developing and adult cells. Fas-induced apoptosis has not been demonstrated with the cells of any non-mammalian vertebrate. We immunostained suspensions of splenocytes from adult Xenopus laevis, the South African clawed toad, with a polyclonal rabbit anti-human Fas antibody raised against the amino acid residues 321–335 of human Fas. The binding was specific, as it was dramatically reduced by preincubation of the antibody with the Fas peptide used to make it, but not with a Fas-ligand (FasL) peptide. The binding was enhanced after in vitro exposure of the splenocytes to phytahemagglutinin (PHA), a T cell mitogen and apoptogen in this species. Sections of developing Xenopus larval tissue were also immunostained with the polyclonal rabbit anti-human Fas antibody. Consistant binding of thymocytes and splenocytes was not observed until early metamorphosis in these immunological sites. A monoclonal mouse anti-human Fas antibody, previously used to stimulate apoptosis in mammalian cells, induced significant levels of apoptosis in adult Xenopus splenocytes and additionally, bound specifically to a splenocyte extract, as assayed by ELISA. Thus, a molecule on Xenopus splenocytes shares both structural and functional homologies with human Fas, indicating the evolutionary conservation within vertebrates of this means of initiating apoptosis.

In situ lymphocyte apoptosis in larval xenopus laevis, the South African clawed toad

During Anuran metamorphosis larval structures regress, adult structures form anew and impaired T cell immune functions are noted, as are alterations in endogenous glucocorticoid titers. In situ histological data, after staining for DNA fragmentation, reveal patterns of lymphocyte suicide in the thymus and spleen of non-antigenically challenged, laboratory bred, developing larvae, that do not correlate with either impaired immune functions or plasma glucocorticoid titers. Apoptotic levels in the thymus are high in premetamorphic stages, low during prometamorphosis and high again, after metamorphic climax, reflecting a periodic removal of thymocytes. Lymphocytic apoptosis in the spleen is low during premetamorphosis, rises in prometamorphic stages, principally within the red pulp, reaching a peak at climax, before declining as metamorphosis is completed.