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Sample records for amphibian xenopus laevis

  1. Xenopus laevis and Emerging Amphibian Pathogens in Chile.

    PubMed

    Soto-Azat, Claudio; Peñafiel-Ricaurte, Alexandra; Price, Stephen J; Sallaberry-Pincheira, Nicole; García, María Pía; Alvarado-Rybak, Mario; Cunningham, Andrew A

    2016-12-01

    Amphibians face an extinction crisis with no precedence. Two emerging infectious diseases, ranaviral disease caused by viruses within the genus Ranavirus and chytridiomycosis due to Batrachochytrium dendrobatidis (Bd), have been linked with amphibian mass mortalities and population declines in many regions of the globe. The African clawed frog (Xenopus laevis) has been indicated as a vector for the spread of these pathogens. Since the 1970s, this species has been invasive in central Chile. We collected X. laevis and dead native amphibians in Chile between 2011 and 2013. We conducted post-mortem examinations and molecular tests for Ranavirus and Bd. Eight of 187 individuals (4.3 %) tested positive for Ranavirus: seven X. laevis and a giant Chilean frog (Calyptocephallela gayi). All positive cases were from the original area of X. laevis invasion. Bd was found to be more prevalent (14.4 %) and widespread than Ranavirus, and all X. laevis Bd-positive animals presented low to moderate levels of infection. Sequencing of a partial Ranavirus gene revealed 100 % sequence identity with Frog Virus 3. This is the first report of Ranavirus in Chile, and these preliminary results are consistent with a role for X. laevis as an infection reservoir for both Ranavirus and Bd.

  2. Lethal and sublethal effects of three insecticides on two developmental stages of Xenopus laevis and comparison with other amphibians.

    PubMed

    Yu, Shuangying; Wages, Mike R; Cai, Qingsong; Maul, Jonathan D; Cobb, George P

    2013-09-01

    It has been suggested that Xenopus laevis is less sensitive than other amphibians to some chemicals, and therefore, that the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) may have limited use in risk assessments for other amphibians. However, comparisons are based mostly on results of FETAX, which emphasizes embryos. Larval X. laevis may be more sensitive to chemicals than embryos and may serve as a better life stage in risk assessments. The present study was conducted to determine the lethal and sublethal effects of 3 insecticides (malathion, endosulfan, and α-cypermethrin) on X. laevis embryos and larvae and to compare toxicity of X. laevis with that of other amphibians. All 3 insecticides have different modes of action, and they caused mortality, malformations, and growth inhibition in both developmental stages. Compared with embryos, larvae were more sensitive to endosulfan and α-cypermethrin but not to malathion. Xenopus laevis larvae had low sensitivity to endosulfan, median sensitivity to malathion, and high sensitivity to α-cypermethrin/cypermethrin relative to other larval amphibians. Our results suggest that X. laevis larvae may generate more protective toxicity estimates in risk assessments than embryos. Xenopus laevis may have limited use in evaluating risk of organochlorine insecticides to other amphibians but may provide useful toxicity thresholds for pyrethroid and perhaps organophosphorus insecticides.

  3. Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis.

    PubMed

    Guselnikov, S V; Grayfer, L; De Jesús Andino, F; Rogozin, I B; Robert, J; Taranin, A V

    2015-11-01

    The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

  4. Molecular cloning and developmental expression of the caveolin gene family in the amphibian Xenopus laevis.

    PubMed

    Razani, Babak; Park, David S; Miyanaga, Yuko; Ghatpande, Ashwini; Cohen, Justin; Wang, Xiao Bo; Scherer, Philipp E; Evans, Todd; Lisanti, Michael P

    2002-06-25

    Caveolae are approximately 50-100 nm invaginations of the plasma membrane thought to form as a result of a local accumulation of cholesterol, sphingolipids, and a unique family of three proteins known as the caveolins: Cav-1, -2, and -3. Here, we report the identification, sequence, and developmental expression of the three caveolin genes in the amphibian Xenopus laevis. Sequence comparisons show that Xenopus Cav-1, -2, and -3 are approximately 80, 64, and 45% identical, respectively, to their counterparts in humans. Furthermore, Northern blotting experiments demonstrate that the Xenopus caveolins have tissue-specific expression profiles consistent with those previously reported in adult mammals. In the adult frog, Xenopus Cav-1 and Cav-2 are most abundantly expressed in the fat body and the lungs, while Xenopus Cav-3 is primarily expressed in muscle tissue types (heart and skeletal muscle). However, our temporal and spatial analyses of these expression patterns during embryogenesis reveal several novel features, with possible relevance to developmental signaling. Transcripts encoding Xenopus Cav-1 and -2 first appear in the notochord of neurula stage embryos, which represents a key signaling tissue. In contrast, Xenopus Cav-3 shows a highly specific punctate expression pattern in the embryonic epidermis, similar to previous patterns implicated in Notch signaling. These findings are in striking contrast to their steady-state expression patterns in the adult frog. Taken together, our results show that the Xenopus caveolin gene family is present and differentially expressed in both embryonic and adult tissues. This report is the first detailed study of caveolin gene expression in a developing embryo.

  5. Mechanisms of amphibian macrophage development: characterization of the Xenopus laevis colony-stimulating factor-1 receptor.

    PubMed

    Grayfer, Leon; Edholm, Eva-Stina; Robert, Jacques

    2014-01-01

    Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1R tissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidney CSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liver CSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrow CSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrow CSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpole macrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.

  6. Perturbation of Organogenesis by the Herbicide Atrazine in the Amphibian Xenopus laevis

    PubMed Central

    Lenkowski, Jenny R.; Reed, J. Michael; Deininger, Lisa; McLaughlin, Kelly A.

    2008-01-01

    Background Exposure to anthropogenic chemicals during development can disrupt the morphogenesis of organ systems. Use of the herbicide atrazine has been debated in recent years because of its implicated, but poorly characterized, effects on vertebrates. Previous studies primarily examined the effects of atrazine exposure during metamorphosis or early developmental stages of amphibians. Objectives We sought to identify and characterize the susceptibility during the often-overlooked developmental stage of organ morphogenesis. Methods We used a static renewal experimental treatment to investigate the effects of 10, 25, and 35 mg/L atrazine from early organ morphogenesis through the onset of tadpole feeding in the aquatic amphibian model system, Xenopus laevis. We quantified malformations of the body axis, heart, and intestine, as well as apoptosis in the midbrain and pronephric kidney. Results We found a significant dose-dependent increase in the percentage of atrazine-exposed tadpoles with malformations of multiple tissues including the main body axis, circulatory system, kidney, and digestive system. Incidence of apoptotic cells also increased in the both midbrain and kidney of atrazine-exposed tadpoles. Conclusions Our results demonstrate that acute atrazine exposure (10–35 mg/L for ≤ 48 hr) during early organ morphogenesis disrupts proper organ development in an amphibian model system. The concurrent atrazine-induced apoptosis in the pronephric kidney and midbrain begins to elucidate a mechanism by which atrazine may disrupt developmental processes in nontarget organisms. PMID:18288322

  7. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with relying on tail resorption as a measu...

  8. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with relying on tail resorption as a measu...

  9. Chytrid fungus infections in laboratory and introduced Xenopus laevis populations: assessing the risks for U.K. native amphibians

    PubMed Central

    Tinsley, Richard C.; Coxhead, Peter G.; Stott, Lucy C.; Tinsley, Matthew C.; Piccinni, Maya Z.; Guille, Matthew J.

    2015-01-01

    The chytrid fungus Batrachochytrium dendrobatidis (Bd) is notorious amongst current conservation biology challenges, responsible for mass mortality and extinction of amphibian species. World trade in amphibians is implicated in global dissemination. Exports of South African Xenopus laevis have led to establishment of this invasive species on four continents. Bd naturally infects this host in Africa and now occurs in several introduced populations. However, no previous studies have investigated transfer of infection into co-occurring native amphibian faunas. A survey of 27 U.K. institutions maintaining X. laevis for research showed that most laboratories have low-level infection, a risk for native species if animals are released into the wild. RT-PCR assays showed Bd in two introduced U.K. populations of X. laevis, in Wales and Lincolnshire. Laboratory and field studies demonstrated that infection levels increase with stress, especially low temperature. In the U.K., native amphibians may be exposed to intense transmission in spring when they enter ponds to spawn alongside X. laevis that have cold-elevated Bd infections. Exposure to cross-infection has probably been recurrent since the introduction of X. laevis, >20 years in Lincolnshire and 50 years in Wales. These sites provide an important test for assessing the impact of X. laevis on Bd spread. However, RT-PCR assays on 174 native amphibians (Bufo, Rana, Lissotriton and Triturus spp.), sympatric with the Bd-infected introduced populations, showed no foci of self-sustaining Bd transmission associated with X. laevis. The abundance of these native amphibians suggested no significant negative population-level effect after the decades of co-occurrence. PMID:25843959

  10. The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like receptors.

    PubMed

    Guselnikov, Sergey V; Reshetnikova, Evdokiya S; Najakshin, Alexander M; Mechetina, Ludmila V; Robert, Jacques; Taranin, Alexander V

    2010-03-01

    In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3-4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRgamma subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

  11. Caging, but not air deprivation, slows tadpole growth and development in the amphibian Xenopus laevis.

    PubMed

    Rose, Christopher S

    2014-08-01

    Xenopus laevis tadpoles raised in submerged cages in normoxic water develop more slowly than tadpoles raised with access to air. This study distinguishes between the effects of being caged and being deprived access to air on development and growth. Tadpoles were raised in high and low density control tanks and in cages in the same tank that were either completely submerged or with the top exposed to air. Experiments were repeated with the cages in different positions relative to the air stones and with and without the water flow from air stones supplemented with a pump. Whereas caging tadpoles has a large effect on their development and growth, additionally depriving them of air has a small effect and this effect can be removed by optimizing water flow through the cage. The effect of caging, though significant in this study, is small compared to the variation in growth and developmental rates that is commonly encountered within and among controls in lab studies. Caging effects can also be diminished by optimizing rearing conditions and/or having exceptionally vigorous tadpoles. The effects of air deprivation and caging thus pose less of a problem for experimenting on air-deprived (AD) and air-restored Xenopus tadpoles than their inherent variability in growth and developmental rates and their susceptibility to growth and developmental arrest. Further, the effect of air deprivation in this air-breathing amphibian does not pose a conflict with evolutionary hypotheses for lung loss involving lengthening of the larval period and delay in the onset of air breathing.

  12. Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis.

    PubMed

    Ramsey, Jeremy P; Reinert, Laura K; Harper, Laura K; Woodhams, Douglas C; Rollins-Smith, Louise A

    2010-09-01

    Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.

  13. Characterization of gamma-aminobutyric acid receptors in the neurointermediate lobe of the amphibian Xenopus laevis.

    PubMed

    Verburg-van Kemenade, B M; Jenks, B G; Lenssen, F J; Vaudry, H

    1987-02-01

    The neurotransmitter gamma-aminobutyric acid (GABA) is involved in the regulation of secretion of MSH from the intermediate lobe of Xenopus laevis. The purpose of this study was to identify the GABA receptor(s) involved by determination of the effect of specific receptor agonists and antagonists on the release of immunoreactive MSH from superfused neurointermediate lobes of Xenopus. Exogenous GABA induces a rapid inhibition of MSH secretion. There was no evidence for a transitory stimulatory effect of GABA as reported for the rat melanotropes. Both the GABA agonists (GABAa) homotaurine and isoguvacine and the GABA agonist (GABAb) baclofen inhibited MSH release in a dose-dependent manner. In vivo, homotaurine and baclofen caused aggregation of pigment in dermal melanophores. The MSH release-inhibiting effect of homotaurine and isoguvacine could be antagonized by the specific GABAa receptor antagonist bicuculline. However, bicuculline and picrotoxin failed to block the effect of exogenous GABA. We conclude that in the neurointermediate lobe of Xenopus laevis both GABAa and GABAb receptors are present, suggesting a dual inhibitory regulation.

  14. Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.

    PubMed

    Wendel, Emily S; Yaparla, Amulya; Koubourli, Daphne V; Grayfer, Leon

    2017-03-01

    Infections of amphibians by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to the amphibian declines, yet much remains unknown regarding amphibian antiviral immunity. Notably, amphibians represent an important step in the evolution of antiviral interferon (IFN) cytokines as they are amongst the first vertebrates to possess both type I and type III IFNs. Accordingly, we examined the roles of type I and III IFNs in the skin of FV3-challenged amphibian Xenopus laevis) tadpoles and adult frogs. Interestingly, FV3-infected tadpoles mounted type III IFN responses, whereas adult frogs relied on type I IFN immunity. Subcutaneous administration of type I or type III IFNs offered short-term protection of tadpoles against FV3 and these type I and type III IFNs induced the expression of distinct antiviral genes in the tadpole skin. Moreover, subcutaneous injection of tadpoles with type III IFN significantly extended their survival and reduced FV3 dissemination. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2014-01-01

    Macrophages are integral to amphibian immunity against RVs, as well as to the infection strategies of these pathogens. Although CSF-1 was considered to be the principal mediator of macrophage development, the IL-34 cytokine, which shares no sequence identity with CSF-1, is now believed to contribute to vertebrate monopoiesis. However, the respective roles of CSF-1- and IL-34-derived macrophages are still poorly understood. To delineate the contribution of these macrophage populations to amphibian immunity against the RV FV3, we identified the Xenopus laevis IL-34 and transcriptionally and functionally compared this cytokine with the previously identified X. laevis CSF-1. The X. laevis CSF-1 and IL-34 displayed strikingly nonoverlapping developmental and tissue-specific gene-expression patterns. Furthermore, only CSF-1 but not IL-34 was up-regulated in the kidneys of FV3-challenged tadpoles. Intriguingly, recombinant forms of these cytokines (rXlCSF-1, rXlIL-34) elicited morphologically distinct tadpole macrophages, and whereas rXlCSF-1 pretreatment decreased the survival of FV3-infected tadpoles, rXlIL-34 administration significantly prolonged FV3-challenged animal survival. Compared with rXlIL-34-elicited macrophages, macrophages derived by rXlCSF-1 were more phagocytic but also significantly more susceptible to in vitro FV3 infections. By contrast, rXlIL-34-derived macrophages exhibited significantly greater in vitro antiranaviral activity and displayed substantially more robust gene expression of the NADPH oxidase components (p67phox, gp91phox) and type I IFN. Moreover, FV3-challenged, rXlIL-34-derived macrophages exhibited several orders of magnitude greater up-regulation of the type I IFN gene expression. This marks the first report of the disparate roles of CSF-1 and IL-34 in vertebrate antiviral immunity. PMID:25190077

  16. Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.

    PubMed

    Grayfer, Leon; Robert, Jacques

    2014-12-01

    Macrophages are integral to amphibian immunity against RVs, as well as to the infection strategies of these pathogens. Although CSF-1 was considered to be the principal mediator of macrophage development, the IL-34 cytokine, which shares no sequence identity with CSF-1, is now believed to contribute to vertebrate monopoiesis. However, the respective roles of CSF-1- and IL-34-derived macrophages are still poorly understood. To delineate the contribution of these macrophage populations to amphibian immunity against the RV FV3, we identified the Xenopus laevis IL-34 and transcriptionally and functionally compared this cytokine with the previously identified X. laevis CSF-1. The X. laevis CSF-1 and IL-34 displayed strikingly nonoverlapping developmental and tissue-specific gene-expression patterns. Furthermore, only CSF-1 but not IL-34 was up-regulated in the kidneys of FV3-challenged tadpoles. Intriguingly, recombinant forms of these cytokines (rXlCSF-1, rXlIL-34) elicited morphologically distinct tadpole macrophages, and whereas rXlCSF-1 pretreatment decreased the survival of FV3-infected tadpoles, rXlIL-34 administration significantly prolonged FV3-challenged animal survival. Compared with rXlIL-34-elicited macrophages, macrophages derived by rXlCSF-1 were more phagocytic but also significantly more susceptible to in vitro FV3 infections. By contrast, rXlIL-34-derived macrophages exhibited significantly greater in vitro antiranaviral activity and displayed substantially more robust gene expression of the NADPH oxidase components (p67(phox), gp91(phox)) and type I IFN. Moreover, FV3-challenged, rXlIL-34-derived macrophages exhibited several orders of magnitude greater up-regulation of the type I IFN gene expression. This marks the first report of the disparate roles of CSF-1 and IL-34 in vertebrate antiviral immunity.

  17. Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.

    PubMed

    Kurth, Thomas; Berger, Jürgen; Wilsch-Bräuninger, Michaela; Kretschmar, Susanne; Cerny, Robert; Schwarz, Heinz; Löfberg, Jan; Piendl, Thomas; Epperlein, Hans H

    2010-01-01

    In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY FOR THYROID DISRUPTION

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with reliance on tail resorption as a meas...

  19. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY FOR THYROID DISRUPTION

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with reliance on tail resorption as a meas...

  20. Agglutination of Jelly Coat and Cortical Granule Components and the Block to Polyspermy in the Amphibian Xenopus laevis

    PubMed Central

    Wyrick, Ron E.; Nishihara, Tatsuro; Hedrick, Jerry L.

    1974-01-01

    A block to polyspermy in amphibians is established at fertilization by the conversion of the vitelline envelope to the fertilization envelope. In Xenopus laevis a major ultrastructural change in the envelope at fertilization is the appearance of an electron-dense layer, termed the F layer, between the envelope and the inner-most jelly coat layer, J1. The F layer is derived, at least in part, from materials released from the cortical granules. Further definition of the origin and chemical nature of the F layer was sought by using isolated cortical granule (CG) exudate and jelly coat layer J1. In double diffusion experiments, the isolated components interacted in an agglutination reaction producing a band of precipitation. The agglutination involved α-galactoside residues and metal ions (Ca++). Employing chemically modified jelly, we demonstrated that sulfhydryl-disulfide interchanges were not involved in the agglutination and, with 35S-labeled jelly, that the agglutinating J1 component possessed sulfate esters. Both the CG exudate and the J1 components contained carbohydrate, as evidenced by their lectin reactivity. A number of ionic polymers, both natural and synthetic, were tested as chemical analogs of CG exudate and J1; none gave an agglutination band. Dissolved jelly coat material from eggs of two different species of frogs agglutinated with CG exudate, while jelly from sea urchin eggs and hyaluronic acid from mammalian eggs did not. Thus, the agglutination reaction was chemically and phylogenetically specific. An electron-dense layer, similar to the F layer, formed on the outer of the vitelline envelope when jellied unfertilized eggs were immersed in CG exudate; such eggs were not fertilizable. We suggest that in Xenopus laevis, and perhaps other organisms as well, an agglutination type of reaction between cortical granule components and egg integuments is a participant in the structural and molecular events establishing a block to polyspermy. Images PMID

  1. A developmental analysis of periodic albinism in the amphibian Xenopus laevis.

    PubMed

    Eagleson, Gerald W; van der Heijden, Roel A; Roubos, Eric W; Jenks, Bruce G

    2010-09-01

    The periodic albino of Xenopus laevis displays a transitory presence of black melanin pigment in the embryo but looses this during tadpole development. This mutation, involving a recessive allele, affects melanogenesis in dermal melanophore pigment cells. It has been suggested that the mutation is intrinsic to the melanophore cell itself or, alternatively, reflects malfunction in the neuroendocrine system that regulates melanophore cell function. This latter system, involving pituitary melanotrope cells which produces alpha-melanophore stimulating hormone (alpha-MSH), is responsible for stimulating the production and dispersion of melanin pigment in dermal melanophores. The purpose of the present study was to determine to which degree the albinism is intrinsic to the melanophore or involves neuroendocrine malfunction. Experiments involved transplantation of presumptive melanophores from wild-type to albino embryos, and vice versa, immunocytochemical analysis of the albino neuroendocrine system and the creation of wild-type/albino parabiotic animals to determine if the neuroendocrine system of the albino can support melanotrope cell function. We show that the albino has a functional neuroendocrine system and conclude that the defect in the albino primarily affects the melanophore cell, possibly rendering it incapable of responding to alpha-MSH. It is also apparent from our results that in later stages of development the cellular environment of the melanotrope cell does become important to its development, but the nature of the critical cellular factors involved remains to be determined.

  2. Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test.

    PubMed

    Mouchet, F; Gauthier, L; Mailhes, C; Ferrier, V; Devaux, A

    2006-06-01

    Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan. Copyright 2006 Wiley Periodicals, Inc.

  3. Comparative study of the comet assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene, ethyl methanesulfonate, and methyl methanesulfonate: establishment of a positive control in the amphibian comet assay.

    PubMed

    Mouchet, F; Gauthier, L; Mailhes, C; Ferrier, V; Devaux, A

    2005-02-01

    The present investigation explored the potential use of the comet assay (CA) as a genotoxicity test in the amphibian Xenopus laevis and compared it with the French standard micronucleus test (MNT). Benzo[a]pyrene (B[a]P), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS) were used as model compounds for assessing DNA damage. Damage levels were measured as DNA strand breaks after alkaline electrophoresis of nuclei isolated from larval amphibian erythrocytes using the CA in order to establish a positive control for further ecotoxicological investigations. The results led to the selection of MMS as a positive control on the basis of the higher sensitivity of Xenopus laevis to this compound. The CA and MNT were compared for their ability to detect DNA damage with the doses of chemical agents and exposure times applied. EMS and MMS were shown to increase micronucleus and DNA strand break formation in larval erythrocytes concurrently. However, B[a]P increased micronucleus formation but not that of DNA strand breaks. Time-dose experiments over 12 days of exposure suggest that the CA provides an earlier significant response to genotoxicants than does the MNT. In Xenopus the CA appears to be a sensitive and suitable method for detecting genotoxicity like that caused by EMS and MMS. It can be considered a genotoxicity-screening tool. The results for B[a]P show that both tests should be used in a complementary manner on Xenopus.

  4. Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

    PubMed

    Haselman, Jonathan T; Sakurai, Maki; Watanabe, Naoko; Goto, Yasushi; Onishi, Yuta; Ito, Yuki; Onoda, Yu; Kosian, Patricia A; Korte, Joseph J; Johnson, Rodney D; Iguchi, Taisen; Degitz, Sigmund J

    2016-12-01

    The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l(-1) BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l(-1) treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l(-1) ) and 100% of the genetic males in the 3.0 and 6.0 mg l(-1) treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina.

    PubMed

    Yoshii, Chika; Ueda, Yoko; Okamoto, Mitumasa; Araki, Masasuke

    2007-03-01

    In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.

  6. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    PubMed Central

    Martini, Federica; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  7. PHYLOGENETIC AND DEVELOPMENTAL STUDY OF CD4, CD8 α AND β T CELL CO-RECEPTOR HOMOLOGS IN TWO AMPHIBIAN SPECIES, XENOPUS TROPICALIS AND XENOPUS LAEVIS

    PubMed Central

    Chida, Asiya Seema; Goyos, Ana; Robert, Jacques

    2010-01-01

    CD4 and CD8 co-receptors play critical roles in T cell development and activation by interacting both with T cell receptors and MHC molecules. Although homologs of these genes have been identified in many jawed vertebrates, there are still unresolved gaps concerning their evolution and specialization in MHC interaction and T cell function. Using experimental and computational procedures we identified CD4, CD8α and CD8β gene homologs both in Xenopus tropicalis, whose full genome has been sequenced, and its sister species X. laevis. Multiple alignments of deduced amino acid sequences reveal a poor conservation of the residues involved in binding of CD4 to MHC class II, and CD8α to class I in non-mammalian species, presumably related to the coevolutionary pressure of MHC I and II genes. Phylogenetic study suggests that Xenopodinae co-receptor genes are more closely related to their homologs in other tetrapods than those of bony fish. Furthermore, the developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus as early as four days post-fertilization (dpf) at the onset of thymic organogenesis. CD8β expression is specific to adult surface CD8+ T cells and thymocytes, and is first detected in the thymus at five dpf in parallel with productive TCRγ transrcipts, whereas productive TCRβ and α rearrangements are not detected before 7–9 dpf. PMID:21075137

  8. Endocrine disruption by environmental gestagens in amphibians - A short review supported by new in vitro data using gonads of Xenopus laevis.

    PubMed

    Ziková, Andrea; Lorenz, Claudia; Hoffmann, Frauke; Kleiner, Wibke; Lutz, Ilka; Stöck, Matthias; Kloas, Werner

    2017-08-01

    Endocrine disruption caused by various anthropogenic compounds is of persisting concern, especially for aquatic wildlife, because surface waters are the main sink of these so-called endocrine disruptors (ED). In the past, research focused on (anti)estrogenic, (anti)androgenic, and (anti)thyroidal substances, affecting primarily reproduction and development in vertebrates; however, other endocrine systems might be also targeted by ED. Environmental gestagens, including natural progestogens (e.g. progesterone (P4)) and synthetic progestins used for contraception, are supposed to affect vertebrate reproduction via progesterone receptors. In the present paper, we review the current knowledge about gestagenic effects in amphibians, focussing on reproduction and the thyroid system. In addition, we support the literature data with results of recent in vitro experiments, demonstrating direct impacts of the gestagens levonorgestrel (LNG) and P4 on sexually differentiated gonads of larval Xenopus laevis. The results showed a higher susceptibility of female over male gonads to gestagenic ED. Only in female gonads LNG, but not P4, had direct inhibitory effects on gene expression of steroidogenic acute regulatory protein and P450 side chain cleavage enzyme, whereas aromatase expression decreased in reaction to both gestagens. Surprisingly, beyond the expected ED effects of gestagens on reproductive physiology in amphibians, LNG drastically disrupted the thyroid system, which resembles direct effects on thyroid glands and pituitary along the pituitary-thyroid axis disturbing metamorphic development. In amphibians, environmental gestagens not only affect the reproductive system but at least LNG can impact also development by disruption of the thyroid system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro.

    PubMed Central

    Pickford, D B; Morris, I D

    1999-01-01

    There is currently little evidence of pollution-induced endocrine dysfunction in amphibia, in spite of widespread concern over global declines in this ecologically diverse group. Data regarding the potential effects of endocrine-disrupting contaminants (EDCs) on reproductive function in amphibia are particularly lacking. We hypothesized that estrogenic EDCs may disrupt progesterone-induced oocyte maturation in the adult amphibian ovary, and tested this with an in vitro germinal vesicle breakdown assay using defolliculated oocytes from the African clawed frog, Xenopus laevis. While a variety of natural and synthetic estrogens and xenoestrogens were inactive in this system, the proestrogenic pesticide methoxychlor was a surprisingly potent inhibitor of progesterone-induced oocyte maturation (median inhibitive concentration, 72 nM). This inhibitory activity was specific to methoxychlor, rather than to its estrogenic contaminants or metabolites, and was not antagonized by the estrogen receptor antagonist ICI 182,780, suggesting that this activity is not estrogenic per se. The inhibitory activity of methoxychlor was dose dependent, reversible, and early acting. However, washout was unable to reverse the effect of short methoxychlor exposure, and methoxychlor did not competitively displace [3H]progesterone from a specific binding site in the oocyte plasma membrane. Therefore, methoxychlor may exert its action not directly at the site of progesterone action, but downstream on early events in maturational signaling, although the precise mechanism of action is unclear. The activity of methoxychlor in this system indicates that xenobiotics may exert endocrine-disrupting effects through interference with progestin-regulated processes and through mechanisms other than receptor antagonism. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:10090707

  10. Probing the Xenopus laevis inner ear transcriptome for biological function

    PubMed Central

    2012-01-01

    Background The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Results Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. Conclusions We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the

  11. Xenopus laevis in Developmental and Molecular Biology.

    ERIC Educational Resources Information Center

    Dawid, Igor B.; Sargent, Thomas D.

    1988-01-01

    Discusses the advantages of Xenopus laevis as an experimental animal in the study of embryogenesis in vertebrates. Summarizes the contributions of this system to the analysis of ribosomal and 5S RNA genes, and the diverse and highly productive applications of the oocyte injection technology. (RT)

  12. Xenopus laevis in Developmental and Molecular Biology.

    ERIC Educational Resources Information Center

    Dawid, Igor B.; Sargent, Thomas D.

    1988-01-01

    Discusses the advantages of Xenopus laevis as an experimental animal in the study of embryogenesis in vertebrates. Summarizes the contributions of this system to the analysis of ribosomal and 5S RNA genes, and the diverse and highly productive applications of the oocyte injection technology. (RT)

  13. XENOPUS LAEVIS: A CULTURING AND REARING PROTOCOL

    EPA Science Inventory

    Xenopus laevis are used extensively here at MED-Duluth as a model for assessing development toxicity to xenobiotics. As a result, a culturing system has been developed that provides eggs and tadpoles of consistent high quality for use by researchers at the facility. The methods ...

  14. XENOPUS LAEVIS: A CULTURING AND REARING PROTOCOL

    EPA Science Inventory

    Xenopus laevis are used extensively here at MED-Duluth as a model for assessing development toxicity to xenobiotics. As a result, a culturing system has been developed that provides eggs and tadpoles of consistent high quality for use by researchers at the facility. The methods ...

  15. Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

    SciTech Connect

    Muccioli, G.; Guardabassi, A.; Pattono, P. )

    1990-03-01

    The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrence of prolactin specific binding sites in Xenopus laevis choroid plexus cells.

  16. Amphibian (Xenopus sp.) iodothyronine deiodinase ...

    EPA Pesticide Factsheets

    The U.S. EPA-MED amphibian thyroid group is currently screening chemicals for inhibition of human iodothyronine deiodinase activity as components of the thyroid system important in human development. Amphibians are a bellwether taxonomic group to gauge toxicity of chemicals in the environment. Amphibian thyroid function is not only important in development but also metamorphosis. Xenopus sp. have been used extensively as model organisms and are well characterized genetically. We propose to screen a list of chemicals (selected from the human DIO screening results) to test for inhibition of Xenopus deiodinases. Large quantities of the enzymes will be produced using an adenovirus system. Our preliminary results show that there may be catalytic differences between human and Xenopus deiodinases. The Twin Ports Early Career Scientists is a new group formed within the Duluth-Superior scientific community. This presentation will provide a basic introduction to my research and our mission at EPA, and help to establish networking and collaboration relationships across disciplines and institutions.

  17. Amphibian (Xenopus sp.) iodothyronine deiodinase ...

    EPA Pesticide Factsheets

    The U.S. EPA-MED amphibian thyroid group is currently screening chemicals for inhibition of human iodothyronine deiodinase activity as components of the thyroid system important in human development. Amphibians are a bellwether taxonomic group to gauge toxicity of chemicals in the environment. Amphibian thyroid function is not only important in development but also metamorphosis. Xenopus sp. have been used extensively as model organisms and are well characterized genetically. We propose to screen a list of chemicals (selected from the human DIO screening results) to test for inhibition of Xenopus deiodinases. Large quantities of the enzymes will be produced using an adenovirus system. Our preliminary results show that there may be catalytic differences between human and Xenopus deiodinases. The Twin Ports Early Career Scientists is a new group formed within the Duluth-Superior scientific community. This presentation will provide a basic introduction to my research and our mission at EPA, and help to establish networking and collaboration relationships across disciplines and institutions.

  18. Xendorphin B1, a novel opioid-like peptide determined from a Xenopus laevis brain cDNA library, produces opioid antinociception after spinal administration in amphibians

    PubMed Central

    Stevens, Craig W.; Tóth, Géza; Borsodi, Anna; Benyhe, Sándor

    2007-01-01

    Prodynorphins (PDYNs) from the African clawed frog (Xenopus leavis), originally described as ‘proxendorphins’, are novel members of the family of opioid-like precursor polypeptides and were recently discovered based on polymerase chain reaction (PCR) isolates from a Xenopus brain cDNA library. This amphibian prodynorphin was found in two isoforms, XenPDYN-A and XenPDYN-B, consisting of 247 and 279 amino acids, respectively. Each prepropeptide contains five potential opioid-like peptides, collectively named xendorphins. One of these, xendorphin B1 (XenPDYN-B sequence 96–111: YGGFIRKPDKYKFLNA), is a hexadecapeptide that displaced [3H]naloxone and the radiolabelled kappa opioid, [3H]dynorphin A (1–17), with nanomolar affinity from rat brain membranes. Using the acetic acid pain test, the present study examined the antinociceptive effects of spinally administered xendorphin B1 in amphibians. Xendorphin B1 produced a long-lasting and dose-dependent antinociceptive effect in the Northern grass frog (Rana pipiens) with an ED50 value of 44.5 nmol/frog. The antinociceptive effects of xendorphin B1 were significantly blocked by pretreatment with the non-selective opioid antagonist, naltrexone. This is the first report of the in vivo characterization of a non-mammalian prodynorphin-derived peptide and suggests that xendorphin peptides may play a role in the modulation of noxious information in vertebrates. PMID:17292806

  19. Maternal transfer of antibodies to eggs in Xenopus laevis.

    PubMed

    Poorten, Thomas J; Kuhn, Raymond E

    2009-02-01

    The immune system of the African clawed frog, Xenopus laevis, includes nearly the full repertoire of lymphoid organs and immune cell types found in mammals. In contrast to the mammalian immune system, the development of the amphibian immune system occurs in the open environment. Oviparity necessitates a rapid ontogeny of the immune system. X. laevis larvae become immunocompetent about 2 weeks after fertilization of the egg. During this 2-week window, larvae cannot mount an adaptive immune response to potential pathogens and presumably must depend on innate responses. In the present study, the possibility of maternal transfer of antibodies to eggs was examined. Adult female X. laevis were injected three times at weekly intervals with the hapten-carrier complex, trinitrophenylated bovine serum albumin (TNP-BSA). The sera of immunized frogs demonstrated antibody activity to BSA, TNP-BSA, and, importantly, trinitrophenylated ovalbumin (TNP-OVA) when examined by enzyme-linked immunosorbent assay (ELISA). Reactivity to TNP-OVA confirmed that antibodies were produced against TNP. The adult female frogs were induced to lay eggs by injection of human chorionic gonadotropin. Next, membrane-free extracts of the eggs were treated with protease inhibitors in order to prevent proteolysis of proteins found in the eggs. On analysis by ELISA, it was found that TNP-specific antibodies were present in the egg extracts. This demonstrated the transfer of antigen-specific antibodies from adult females to eggs in X. laevis.

  20. Regulative development of Xenopus laevis in microgravity

    NASA Astrophysics Data System (ADS)

    Black, S.; Larkin, K.; Jacqmotte, N.; Wassersug, R.; Pronych, S.; Souza, K.

    To test whether gravity is required for normal amphibian development, Xenopus leavis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.

  1. Regulative development of Xenopus laevis in microgravity

    NASA Technical Reports Server (NTRS)

    Black, S.; Larkin, K.; Jacqmotte, N.; Wassersug, R.; Pronych, S.; Souza, K.

    1996-01-01

    To test whether gravity is required for normal amphibian development, Xenopus leavis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.

  2. Bisphenol A induces feminization in Xenopus laevis tadpoles.

    PubMed

    Levy, Gregor; Lutz, Ilka; Krüger, Angela; Kloas, Werner

    2004-01-01

    To evaluate possible estrogenic effects of bisphenol A (BPA) in an amphibian model, Xenopus laevis tadpoles were exposed to BPA and 17beta-estradiol (E2) during larval development. After metamorphosis, the gonadal phenotype was determined by gross morphology, and testes were further examined histologically to validate the results. BPA treatment altered the normal sex ratio toward females depending on the BPA concentrations added. Chemical analysis showed a time-dependent decline of BPA during semistatic exposure, indicating that BPA is taken up and metabolized to some extent by tadpoles. In addition, tadpoles were exposed to BPA and E2 for 2 weeks during sensitive stages of sexual differentiation. Afterward, the expression of an estrogenic biomarker, estrogen receptor (ER) mRNA, was assessed by semiquantitative RT-PCR. Both BPA and E2 up-regulated ER mRNA significantly. In conclusion, these results show clear evidence that BPA induces feminization in X. laevis tadpoles, revealing an estrogenic potency of BPA that influences sexual development in amphibians.

  3. Vascular regression during amphibian metamorphosis--a scanning electron microscope study of vascular corrosion casts of the ventral velum in tadpoles of Xenopus laevis Daudin.

    PubMed

    Aichhorn, H; Lametschwandtner, A

    1996-09-01

    We used scanning electron microscopy and vascular casting to study gross arterial supply, venous drainage, and microvascular patterns of the fully developed ventral velum of tadpoles of Xenopus laevis Daudin and analyzed changes of the velar vascular bed from prometamorphosis to metamorphic climax in a qualitative and quantitative manner. The multilayered, highly secretory ventral velum is supplied bilaterally by an anterior and a posterior velar artery, branches of the external carotid artery. Velar arterioles branch mainly dichotomously and form a flat two-dimensional capillary meshwork overlying the tops of filterplates I-IV. Thymopharyngeal veins, dorsal branches of the filter plates veins, and the internal jugular veins drain the velum toward the venous sinus of the heart. Location, architecture, and the drainage of the velar microvascular bed into the venous sinus make a significant contribution of the velar capillaries to gas exchange unlikely. Instead, velar capillaries rather serve the nutrition of the secretory epithelium. The overall morphology of velar vessels from prometamorphosis to metamorphic climax--deduced from vascular corrosion casts--points to atonic vessels with increased leakage indicated by adhering globular extravasations, and to obstructed or blind ending vessels evidenced by the tapered and/or rounded blind ending cast vessels. The significant decrease in the size of the ventral velum during the metamorphic cycle was paralleled by a miniaturization of the velar vascular bed. We hypothetize that this miniaturization occurs by a shortening and fusion of capillary mesh elements. Our findings in corrosion casts, particularly the miniaturization of the velar microvascular bed and the morphology of the regressing capillaries, point to profound morphologic and ultrastructural changes in velar vessels; a study on the fine structure of the microvascular bed of the ventral velum in metamorphic tadpoles is in progress.

  4. Vocal competition in male Xenopus laevis frogs

    PubMed Central

    Tobias, Martha L.; Corke, Anna; Korsh, Jeremy; Yin, David; Kelley, Darcy B.

    2011-01-01

    Male Xenopus laevis frogs produce underwater advertisement calls that attract gravid females and suppress calling by male competitors. Here we explore whether groups of males establish vocal ranks and whether auditory cues alone suffice for vocal suppression. Tests of male–male pairs within assigned groups reveal linear vocal dominance relations, in which each male has a defined rank. Both the duration over which males interact, as well as the number of competitive opportunities, affect linearity. Linear dominance across the group is stable for about 2 weeks; rank is dynamic. Males engage in physical interactions (clasping) while paired but clasping and vocal rank are not correlated. Playbacks of advertisement calls suppress calling and calls from high- and low-ranking males are equally effective. Thus, auditory cues alone suffice to suppress vocal behavior. Playback intensities equivalent to a nearby male advertising effectively suppress calling while low-intensity playbacks are either ineffective or stimulate vocal behavior. X. laevis advertisement calls are biphasic, composed of alternating fast and slow click trills. Approximately half the males tested are more vocally suppressed by all slow than by all fast trills; thus, these males can distinguish between the two phases. The fully aquatic family Pipidae diverged from terrestrial ancestors approximately 170 mya. Vocal suppression in the X. laevis mating system may represent the translation of an ancient anuran social strategy to underwater life. PMID:21442049

  5. Vocal competition in male Xenopus laevis frogs.

    PubMed

    Tobias, Martha L; Corke, Anna; Korsh, Jeremy; Yin, David; Kelley, Darcy B

    2010-11-01

    Male Xenopus laevis frogs produce underwater advertisement calls that attract gravid females and suppress calling by male competitors. Here we explore whether groups of males establish vocal ranks and whether auditory cues alone suffice for vocal suppression. Tests of male-male pairs within assigned groups reveal linear vocal dominance relations, in which each male has a defined rank. Both the duration over which males interact, as well as the number of competitive opportunities, affect linearity. Linear dominance across the group is stable for about 2 weeks; rank is dynamic. Males engage in physical interactions (clasping) while paired but clasping and vocal rank are not correlated. Playbacks of advertisement calls suppress calling and calls from high- and low-ranking males are equally effective. Thus, auditory cues alone suffice to suppress vocal behavior. Playback intensities equivalent to a nearby male advertising effectively suppress calling while low-intensity playbacks are either ineffective or stimulate vocal behavior. X. laevis advertisement calls are biphasic, composed of alternating fast and slow click trills. Approximately half the males tested are more vocally suppressed by all slow than by all fast trills; thus, these males can distinguish between the two phases. The fully aquatic family Pipidae diverged from terrestrial ancestors approximately 170 mya. Vocal suppression in the X. laevis mating system may represent the translation of an ancient anuran social strategy to underwater life.

  6. Genes for Xenopus laevis U3 small nuclear RNA.

    PubMed Central

    Savino, R; Hitti, Y; Gerbi, S A

    1992-01-01

    Genomic Southern blots showed there are only 14 to 20 copies of U3 snRNA genes per somatic genome in Xenopus laevis, unlike the highly repetitive, tandem arrangement of other snRNA genes in this organism. Sequencing of two U3 snRNA genes from lambda clones of a genomic library revealed striking similarity upstream, but much more divergence downstream. Consensus motifs common to other U snRNA genes were also found: a distal sequence element (DSE, octamer motif at -222 to -215), a proximal sequence element (PSE, at -62 to -52) and a 3' Box (15 or 16 bp downstream of the U3 genes). The DSE of mammals also has an inverted CCAAT motif specific for U3 snRNA genes, and we find this is conserved in the amphibian U3 snRNA genes. The Xenopus inverted CCAAT motif is exactly one helical turn further upstream of the octamer motif than its mammalian counterpart, suggesting interaction of putative transcription factors bound to these motifs. Mutation of the inverted CCAAT motif and part of an adjacent Sp1 site greatly depresses transcription of the mutant U3 snRNA gene in Xenopus oocytes, implying a role in transcriptional efficiency. Electrophoretic mobility shift assays implicate transcription factor binding to this region. Images PMID:1437561

  7. An age-dependent sensitivity of the roll-induced vestibuloocular reflex to hypergravity exposure of several days in an amphibian (Xenopus laevis).

    PubMed

    Sebastian, C E; Pfau, K; Horn, E R

    1998-01-01

    In tadpoles of the Southern Clawed Toad (Xenopus laevis), the effects of an exposure to hypergravity of several days duration on the development of the roll-induced static vestibuloocular reflex (rVOR) were investigated. Special attention was given to the onset of the 9 or 12 days lasting 3G-period during early life. First recordings of the rVOR characteristics for complete 360 degrees rolls of the tadpoles were performed 24 hrs after the end of the 3G-period. The rVOR peak-to-peak amplitudes as well as the VOR-gain for a roll angle of 15 degrees from 3G-and 1G-samples recorded at the 2nd and 3rd day after 3G-termination agreed for the youngest group, but were reduced by approx. 30% in the older tadpoles. Long-term observations lasting up to 8 weeks after termination of the 3G-period, demonstrated (i) an early retardation of the development, and (ii) a developmental acceleration in all groups so that after 2 weeks in the stage 6/9- and 33/36-samples and after 8 weeks in the stage 45-tadpoles, the rVOR-amplitude as well as the rVOR-gain for a 15 degrees roll were at the same level in both the 3G- and the 1G-samples. The results support the existence of a sensitive period for the rVOR development, and additionally demonstrate the importance of the period of the first appearance of the rVOR for the development of adaptive properties of the underlying neuronal network. They also demonstrate the dominant efficiency of genetic programs in the functional development of the vestibular system. Methodological approaches are discussed which will be useful in the further description of the critical period. They include studies on the neuronogenesis and synaptic maturation within the vestibular pathways as well as on the fundamentals of buoyancy control during swimming. A modular but closed mini-system for experimental use is described which allows survival periods lasting many weeks and multiple types of treatments of developing aquatic animals in orbit, controlled

  8. An age-dependent sensitivity of the roll-induced vestibuloocular reflex to hypergravity exposure of several days in an amphibian ( Xenopus laevis)

    NASA Astrophysics Data System (ADS)

    Sebastian, C. E.; Pfau, K.; Horn, E. R.

    In tadpoles of the Southern Clawed Toad ( Xenopus laevis), the effects of an exposure to hypergravity of several days duration on the development of the roll-induced static vestibuloocular reflex (rVOR) were investigated. Special attention was given to the onset of the 9 or 12 days lasting 3G-period during early life. First recordings of the rVOR characteristics for complete 360 ° rolls of the tadpoles were performed 24 hrs after the end of the 3G-period. The rVOR peak-to-peak amplitudes as well as the VOR-gain for a roll angle of 15 ° from 3G-and 1G-samples recorded at the 2nd and 3rd day after 3G-termination agreed for the youngest group, but were reduced by approx. 30% in the older tadpoles. Long-term observations lasting up to 8 weeks after termination of the 3G-period, demonstrated (i) an early retardation of the development, and (ii) a developmental acceleration in all groups so that after 2 weeks in the stage 6/9- and 33/36-samples and after 8 weeks in the stage 45-tadpoles, the rVOR-amplitude as well as the rVOR-gain for a 15 ° roll were at the same level in both the 3G- and the 1G-samples. The results support the existence of a sensitive period for the rVOR development, and additionally demonstrate the importance of the period of the first appearance of the rVOR for the development of adaptive properties of the underlying neuronal network. They also demonstrate the dominant efficiency of genetic programs in the functional development of the vestibular system. Methodological approaches are discussed which will be useful in the further description of the critical period. They include studies on the neuronogenesis and synaptic maturation within the vestibular pathways as well as on the fundamentals of buoyancy control during swimming. A modular but closed mini-system for experimental use is described which allows survival periods lasting many weeks and multiple types of treatments of developing aquatic animals in orbit, controlled automatically.

  9. Structure of four acidic oligosaccharides from the jelly coat surrounding the eggs of Xenopus laevis.

    PubMed

    Plancke, Y; Wieruszeski, J M; Alonso, C; Boilly, B; Strecker, G

    1995-07-15

    Novel acidic oligosaccharides were released by reductive beta-elimination from the jelly coat eggs of the Anuran Xenopus laevis. According to the structural analysis of these oligosaccharide-alditols, the following structures are proposed: [sequence: see text] where Kdn, 3-deoxy-D-glycero-D-galactononulosonic acid. These results confirm the species specificity of the glycanic structures present in the secretion of amphibian oviducts, and may form the basis of a specific egg-sperm recognition process.

  10. Evidence that far-infrared radiation promotes growth of xenopus laevis

    NASA Astrophysics Data System (ADS)

    Shiurba, Robert; Hirabayashi, Tatsuo; Kiyokawa, Shin; Fukui, Akimasa; Miyanaga, Yuko; Kojima, Issey; Asashima, Makoto

    1999-01-01

    In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment.

  11. PROGRESS TOWARDS DEVELOPMENT OF AN AMPHIBIAN-BASED THYROID SCREENING ASSAY USING XENOPUS LAEVIS: ORGANISMAL AND THYROIDAL RESPONSES TO THE MODEL COMPOUNDS 6-PROPYLTHIOURACIL, METHIMAZOLE, AND THYROXINE.

    EPA Science Inventory

    The data presented in this manuscript specifically addresses the development and standardization needs associated with an amphibian thyroid axis screening assay. A protocol for an amphibian growth and reproduction test has been requested by the Office of Science Council and Polic...

  12. PROGRESS TOWARDS DEVELOPMENT OF AN AMPHIBIAN-BASED THYROID SCREENING ASSAY USING XENOPUS LAEVIS: ORGANISMAL AND THYROIDAL RESPONSES TO THE MODEL COMPOUNDS 6-PROPYLTHIOURACIL, METHIMAZOLE, AND THYROXINE.

    EPA Science Inventory

    The data presented in this manuscript specifically addresses the development and standardization needs associated with an amphibian thyroid axis screening assay. A protocol for an amphibian growth and reproduction test has been requested by the Office of Science Council and Polic...

  13. Polystyrene nanoparticles affect Xenopus laevis development

    NASA Astrophysics Data System (ADS)

    Tussellino, Margherita; Ronca, Raffaele; Formiggini, Fabio; Marco, Nadia De; Fusco, Sabato; Netti, Paolo Antonio; Carotenuto, Rosa

    2015-02-01

    Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay- Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona" effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.

  14. Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4-tert-octylphenol or 17β-trenbolone exposure in Xenopus laevis from embryo to juvenile.

    PubMed

    Haselman, Jonathan T; Kosian, Patricia A; Korte, Joseph J; Olmstead, Allen W; Iguchi, Taisen; Johnson, Rodney D; Degitz, Sigmund J

    2016-12-01

    The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17β-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l(-1) ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l(-1) ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l(-1) ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l(-1) treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is

  15. Aeromonas hydrophila infection in Xenopus laevis.

    PubMed

    Hubbard, G B

    1981-06-01

    Aeromonas hydrophila caused severe disease in a group of Xenopus laevis within 3 weeks of receipt. The primary clinical signs were marked pallor of the skin, petechiation, lethargy, anorexia, and edema. The duration of the outbreak was approximately 45 days during which time 21 frogs became sick and 18 died, despite tetracycline therapy. Pale skin, subcutaneous edema and hemorrhages, ascites, and pale livers were seen at necropsy. Aeromonas hydrophila was isolated from skin, and the same organism was isolated in pure culture from skeletal muscle. Tetracycline treatment via stomach tube was effective if given early in the course of the disease. The outbreak was controlled by removing sick frogs, feeding twice per week, changing the water several hours after feeding, and maintaining the frogs where the ambient temperature was 22 degrees C or lower. The pallor of the skin and general malaise were produced experimentally by crowding normal frogs, changing the water infrequently, and increasing ambient temperatures. Mild disease was reproduced experimentally by subcutaneous injection of Aeromonas hydrophila into apparently healthy frogs.

  16. Development of the larval amphibian growth and development assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile

    EPA Science Inventory

    The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan’s Ministry of Environment to support risk assessment. The assay is employed as a ...

  17. Development of the larval amphibian growth and development assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile

    EPA Science Inventory

    The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan’s Ministry of Environment to support risk assessment. The assay is employed as a ...

  18. Xenopus laevis - A success story in biological research in Space

    NASA Astrophysics Data System (ADS)

    Horn, E.

    A feature of sensory, neuronal and motor systems is the existence of a critical period during their development. Environmental modifications, in particular stimulus depri-vation, during this period of life affects development in a long-term manner. For gravity sensory systems, space flights offer the only opportunity for deprivation conditions. Studies in the amphibian Xenopus laevis presented the most complete picture. The presentation demonstrates the importance of Xenopus laevis as an ex-perimental model animal in the past and even for future research in Space. Studies are presented which range from fertilization in Space and anatomical studies during early development under weightlessness up to post-flight studies on the anatomy of the peripheral sense organ, the spinal motor activity and behavior. Gravity depriva-tion induces anatomical as well as behavioral and neurophysiological modifications, which are normalized either during flight (thickening of the blastocoel roof) or after reentry in 1g-conditions (swimming and reflex behavior, spinal motor activity). The physiological changes can be explained by mechanisms of physiological adaptation. However, the studies also revealed stages which were insensitive to gravity depriva-tion; they point to the existence of a critical period. Observations on morphological mal-formations are described which are reversible after termination of microgravity and which are linked to a depression of vestibular reflex behavior. They might be caused by a competition between dorsalization and ventralization inducing growth factors. This observation offers the possibility for a genetic approach in finding ba-sics for microgravity effects on the development of Xenopus, and in a general frame, on the development of vertebrates including men. At the present stage of research, it remains open whether adaptive processes during exposure to altered gravity or the existence of a critical period in vestibular development are responsible for

  19. Effective RNAi-mediated β2-microglobulin loss of function by transgenesis in Xenopus laevis

    PubMed Central

    Nedelkovska, Hristina; Edholm, Eva-Stina; Haynes, Nikesha; Robert, Jacques

    2013-01-01

    Summary To impair MHC class I (class I) function in vivo in the amphibian Xenopus, we developed an effective reverse genetic loss of function approach by combining I-SceI meganuclease-mediated transgenesis with RNAi technology. We generated transgenic outbred X. laevis and isogenetic laevis/gilli cloned lines with stably silenced expression of β2-microglobulin (b2m) critical for class I function. Transgenic F1 frogs exhibited decreased surface class I expression on erythrocytes and lymphocytes, decreased frequency of peripheral CD8 T cells and impaired CD8 T cell-mediated skin allograft rejection. Additionally, b2m knockdown increased susceptibility to viral infection of F0 transgenic larvae. This loss of function strategy offers new avenues for studying ontogeny of immunity and other developmental processes in Xenopus. PMID:23519478

  20. Embryonic development of Xenopus laevis under static magnetic fields up to 6.34 T

    NASA Astrophysics Data System (ADS)

    Ueno, S.; Shiokawa, K.; Iwamoto, M.

    1990-05-01

    A possible influence of intense magnetic fields on the embryonic development of frogs was studied in reference to a potential hazardous problem in magnetic resonance imaging technology. Fertilized eggs of African clawed toads, Xenopus laevis, were cultured in Amphibian Ringer solution under static magnetic fields up to 6.34 T for varying lengths of time, and their cleavage and early embryonic development were followed to examine the possibility of teratogenic effects. Fertilized eggs cultured under the static magnetic field for 6 h followed normal course of cleavage and normally developed into feeding tadpoles. Results were unchanged even when fertilized eggs were cultured for 18 h from the cleavage stage to the neurula stage under a magnetic field of 4.5 T. We conclude that static magnetic fields up to 6.34 T do not affect appreciably the rapid cleavage and the following cell multiplication and differentiation in Xenopus laevis.

  1. Wnt antagonism initiates cardiogenesis in Xenopus laevis

    PubMed Central

    Schneider, Valerie A.; Mercola, Mark

    2001-01-01

    Heart induction in Xenopus occurs in paired regions of the dorsoanterior mesoderm in response to signals from the Spemann organizer and underlying dorsoanterior endoderm. These tissues together are sufficient to induce heart formation in noncardiogenic ventral marginal zone mesoderm. Similarly, in avians the underlying definitive endoderm induces cardiogenesis in precardiac mesoderm. Heart-inducing factors in amphibians are not known, and although certain BMPs and FGFs can mimic aspects of cardiogenesis in avians, neither can induce the full range of activities elicited by the inducing tissues. Here we report that the Wnt antagonists Dkk-1 and Crescent can induce heart formation in explants of ventral marginal zone mesoderm. Other Wnt antagonists, including the frizzled domain-containing proteins Frzb and Szl, lacked this activity. Unlike Wnt antagonism, inhibition of BMP signaling did not promote cardiogenesis. Ectopic expression of GSK3β, which inhibits β-catenin-mediated Wnt signaling, also induced cardiogenesis in ventral mesoderm. Analysis of Wnt proteins expressed during gastrulation revealed that Wnt3A and Wnt8, but not Wnt5A or Wnt11, inhibited endogenous heart induction. These results indicate that diffusion of Dkk-1 and Crescent from the organizer initiate cardiogenesis in adjacent mesoderm by establishing a zone of low Wnt3A and Wnt8 activity. PMID:11159911

  2. Overexpression of Xenopus laevis growth hormone stimulates growth of tadpoles and frogs

    PubMed Central

    Huang, Haochu; Brown, Donald D.

    2000-01-01

    The role of growth hormone (GH) in amphibian metamorphosis is ambiguous based on experiments in which mammalian GH was administered to tadpoles and frogs. We have reexamined the effects of GH by producing transgenic Xenopus laevis that overexpress the cDNA encoding X. laevis GH. These transgenic tadpoles take the same length of time to reach metamorphosis as control tadpoles, but the transgenic tadpoles are twice as large. After metamorphosis, the transgenic frogs grow at a greatly accelerated rate and develop skeletal abnormalities reminiscent of acromegaly. The transgenic frogs are larger than mature frogs in a few months and die in about 1 year. At as early as 10 months of age, the males have mature sperm. We conclude that the growth-promoting effects of GH in this amphibian closely resemble those described for mammals. Although excess GH increases the size of the tadpole, it does not alter the developmental programs involved in metamorphosis. PMID:10618393

  3. Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs.

    PubMed

    Nakade, Shota; Sakuma, Tetsushi; Sakane, Yuto; Hara, Yoshihiro; Kurabayashi, Atsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Obara, Masanobu

    2015-10-01

    Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences.

  4. Cytoplasmic effect on gene function in Xenopus laevis.

    PubMed

    Yu, H J; Shi, C P; Niu, M C

    1987-05-01

    The pigmentation gene of Xenopus laevis is dominant and that of albino aP mutant recessive. Heterologous haploid hybrids are produced by UV inactivation of the egg nuclei during second polar body formation in the mutant sperm-fertilized Xenopus eggs. During development of these hybrids, melanin appeared in the eye and melanophores in the skin at stages comparable to those of the wild type, but much earlier than in the albino mutant. The number and intensity of pigment cells are intermediate between the black Xenopus and albino mutant. While a number of pigment cells remain in the hybrids, those in the albino eventually degenerate. Therefore, the development and maintenance of pigmentation in heterologous hybrids are contributed by Xenopus cytoplasm. Tadpole tail-tips were squashed and stained for chromosome counting. The results show that Xenopus and mutants are diploid (36 chromosomes) and heterologous haploid hybrids have 18 chromosomes.

  5. Trichodina xenopodus, a Ciliated Protozoan, in a Laboratory-Maintained Xenopus laevis

    PubMed Central

    Collymore, Chereen; White, Julie R; Lieggi, Christine

    2013-01-01

    A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding. PMID:24209965

  6. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    PubMed Central

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-01-01

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved. Images PMID:6296782

  7. pdzrn3 is required for pronephros morphogenesis in Xenopus laevis.

    PubMed

    Marracci, Silvia; Vangelisti, Alberto; Raffa, Vittoria; Andreazzoli, Massimiliano; Dente, Luciana

    2016-01-01

    Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.

  8. Twin Xenopus laevis embryos appearing from flattened eggs.

    PubMed

    Sato, Eiji

    2014-01-01

    Remarkable progress has recently been made in molecular biology of double axis formation in Xenopus laevis. Leaving aside, for the time being, the problem of the gene expressions regulating Xenopus laevis development, here I show that pulse treatment could induce formation of a secondary axis in a fertilized Xenopus laevis egg. At 3 min after insemination, metal oxides were added to Xenopus fertilized eggs, and then twin embryos appeared. Zirconium oxide (ZrO2) was the most effective metal oxide for producing twin embryos. ZrO2 was added to the fertilized eggs, and 30 sec later, the eggs were dejellied with cysteine solution and washed within 7 min after insemination. The fertilized eggs began flattening at around 15 min after insemination. When the degree of flattening (the vertical length of the egg divided by the horizontal length) of the eggs at the 16- and 32-cell stages became less than 0.4 degrees, production of twin embryos occurred. Many flattened eggs at less than 0.4 degrees formed twin embryos. The third cleavage of eggs treated with metal oxides was meridional, while the normal third cleavage was horizontal.

  9. Toxicity of selenium to developing Xenopus laevis embryos.

    PubMed

    Browne, C L; Dumont, J N

    1979-07-01

    Se in the form of sodium selenite is toxic to Xenopus laevis embryos and tadpoles continuously exposed to concentrations above 1 ppm. Concentrations of 2 ppm and above result in severe developmental abnormalities and increased mortality. Uptake and loss of radioactive Se from water are rapid, but depuration is not complete indicating that some Se can remain bound by the organism. The facts that Se is toxic at low levels to Xenopus embryos and tadpoles, can cause developmental abnormalities, and accumulates in tissues suggest that increased release of Se compounds into the environment poses a potential threat to aquatic organisms.

  10. A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis.

    PubMed

    Furlow, J David; Neff, Eric S

    2006-03-01

    Thyroid hormone induces the complete metamorphosis of anuran tadpoles into juvenile frogs. Arguably, anuran metamorphosis is the most dramatic effect of a hormone in any vertebrate. Recent advances in pharmacology and molecular biology have made the study of this remarkable process in the frog Xenopus laevis attractive to developmental biologists and endocrinologists alike. In particular, the availability of a straightforward transgenesis assay and the near completion of the Xenopus tropicalis genome are enabling significant advances to be made in our understanding of the major remaining problems of metamorphosis: the extraordinary tissue specificity of responses, the precise timing of morphological changes, the degree of cell autonomy of hormone responses and developmental competence. We argue that X. laevis metamorphosis presents an exciting opportunity for understanding the role of thyroid hormone in vertebrate development.

  11. Xenopus laevis a success story of biological research in space

    NASA Astrophysics Data System (ADS)

    Horn, Eberhard R.

    2006-01-01

    The clawed toad Xenopus laevis is a common experimental animal used in many disciplines of life sciences, such as integrative, developmental and molecular biology or experimental medicine. Since 30 years, Xenopus is used in biological research in space. Important milestones were the years 1975, when Xenopus embryos flew for the first time on the Russian space station Salut-4 and 1994, when Xenopus eggs were successfully fertilized for the first time in space during the Japanese Spacelab mission STS-47 and developed in microgravity to vital tadpoles. Most Xenopus studies were related to embryogenesis and development. Observations during and after altered gravity revealed changes such as the thickening of the blastocoel roof, the dorsalization of the tail, and modifications of vestibular reflexes, fictive and freely swimming. Many changes were reversible even during microgravity exposure. Studies about the vestibuloocular reflex or synapse formation revealed an age-related sensitivity to altered gravity. Xenopus offers useful tools for studies about microgravity effects on living systems. Its oocyte is a suitable model to study ion channel function in space; the dorsalization model can be used to analyse growth factor sensibilities. Hardware for life support of adults, tadpoles and embryos (cf. SUPPLY unit in combination with miniaquaria) as well as for controlled experiments in space are prerequisites for an extension of research with Xenopus. The application aspect is based on the fact that fundamental research per se brings benefit to man.

  12. Waterborne infectivity of the ranavirus Frog-Virus 3 in Xenopus laevis

    PubMed Central

    Robert, Jacques; George, Erica; De Jesús Andino, Francisco; Chen, Guangchun

    2011-01-01

    Ranaviruses like Frog Virus 3 (FV3) are responsible of emerging infectious diseases spreading worldwide to fish, amphibian and reptilian species. We have developed, in Xenopus laevis, an experimental model to investigate viral transmission. We show that FV3 released in water by immunocompromised infected adults can infect adult and larval stages of Xenopus within 3 hours of exposure. Time course of virus load and viral transcription in different tissues suggests that early waterborne FV3 infection through the digestive tract leads to dissemination in the kidney. Finally, a fraction of adult macrophages becomes infected following exposure to waterborne FV3 as visualized by fluorescence microscopy using macrophage- and FV3-specific antibodies. Little cytopathicity and apoptosis were detected in infected macrophages, which is consistent with our proposition that macrophages are permissive to FV3. These data highlight the efficiency of FV3 infectivity by the water route and the ability of FV3 to adapt to its hosts. PMID:21783222

  13. 'Immobile' (im), a recessive lethal mutation of Xenopus laevis tadpoles.

    PubMed

    Droin, A; Beauchemin, M L

    1975-10-01

    'Immobile' (im) is a recessive lethal mutation discovered in the F3 of a Xenopus (Xenopus laevis laevis) originating from a mesodermal nucleus of a neurula transplanted into an enucleated egg. The im embryos do not contract after mechanical stimulation nor do they present any spontaneous contraction from the neurula stage onwards. Development proceeds normally during the first days after which deformation of the lower jaw and tail are observed. The im tadpoles die when normal controls are at the feeding stage. Nevous and muscular tissues are histologically normal in the mutant tadpoles; at advanced stages, however, an irregularity in the path of the myofibrils is observed which is especially conspicuous in the electron microscope. Cholinesterases and ATPase are present in the mutant muscles. Parabiosis and chimerae experiments have shown that parabionts and grafts behave according to their own genotype. Cultures of presumptive axial systems with or without ectoderm lead to the conclusion that, first of all, the abnormality is situated in the mesodermal cells and secondly that the first muscular contractions in normal Xenopus laevis are of myogenic origin. The banding pattern of the myofibrils is normal as was shown by obtaining contractions of glycerol extracted in myoblasts with ATP. It seems therefore that in this mutation, the abnormality is situated in the membraneous system of the muscular cell, sarcoplasmic reticulum and/or tubular system as is probably the case in the mdg mutation of the mouse.

  14. In vivo Assessment and Potential Diagnosis of Xenobiotics that Perturb the Thyroid Pathway: Proteomic Analysis of Xenopus laevis Brain Tissue following Exposure to Model T4 Inhibitors

    EPA Science Inventory

    As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression profiles in the brain of an amphibian model (Xenopus laevis) following in vivo exposur...

  15. In vivo Assessment and Potential Diagnosis of Xenobiotics that Perturb the Thyroid Pathway: Proteomic Analysis of Xenopus laevis Brain Tissue following Exposure to Model T4 Inhibitors

    EPA Science Inventory

    As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression profiles in the brain of an amphibian model (Xenopus laevis) following in vivo exposur...

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

    PubMed Central

    Nedelkovska, Hristina; Cruz-Luna, Tanya; McPherson, Pamela; Robert, Jacques

    2010-01-01

    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. PMID:20972386

  17. Primary structure of 12 neutral oligosaccharide-alditols released from the jelly coats of the anuran Xenopus laevis by reductive beta-elimination.

    PubMed

    Strecker, G; Wieruszeski, J M; Plancke, Y; Boilly, B

    1995-02-01

    The O-linked oligosaccharides of the jelly coat surrounding the eggs of Xenopus laevis were analysed by 1H-NMR spectroscopy. Among the 12 neutral oligosaccharide-alditols which have been characterized, three of them possess the following unusual structures: [sequence: see text] As previously observed for six other amphibian species, the carbohydrate chains of the jelly coat of Xenopus eggs display a high species specificity which could support a biological role during the fertilization processes.

  18. Serum clinical biochemical and hematologic reference ranges of laboratory-reared and wild-caught Xenopus laevis.

    PubMed

    Wilson, Sabrina; Felt, Stephen; Torreilles, Stéphanie; Howard, Antwain; Behan, Colleen; Moorhead, Roberta; Green, Sherril

    2011-09-01

    The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis).

  19. Early development of the thymus in Xenopus laevis

    PubMed Central

    Lee, Young-Hoon; Williams, Allison; Hong, Chang-Soo; You, Youngjae; Senoo, Makoto; Saint-Jeannet, Jean-Pierre

    2012-01-01

    Background Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3 and foxn1, and evaluated the neural crest contribution to thymus development. Results In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T cell differentiation in frogs. Conclusions These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms. PMID:23172757

  20. Protocadherin-9 involvement in retinal development in Xenopus laevis.

    PubMed

    Izuta, Yusuke; Taira, Tetsuro; Asayama, Ayako; Machigashira, Mika; Kinoshita, Tsutomu; Fujiwara, Miwako; Suzuki, Shintaro T

    2015-04-01

    Biological roles of most protocadherins (Pcdhs) are a largely unsolved problem. Therefore, we cloned cDNA for Xenopus laevis protocadherin-9 and characterized its properties to elucidate the role. The deduced amino acid sequence was highly homologous to those of mammalian protocadherin-9 s. X. laevis protocadherin-9 expressed from the cDNA in L cells showed basic properties similar to those of mammalian Pcdhs. Expression of X. laevis protocadherin-9 was first detected in stage-31 embryos and increased as the development proceeded. In the later stage embryos and the adults, the retina strongly expressed protocadherin-9, which was mainly localized at the plexiform layers. Injection of morpholino anti-sense oligonucleotide against protocadherin-9 into the fertilized eggs inhibited eye development; and eye growth and formation of the retinal laminar structure were hindered. Moreover, affected retina showed abnormal extension of neurites into the ganglion cell layer. Co-injection of protocadherin-9 mRNA with the morpholino anti-sense oligonucleotide rescued the embryos from the defects. These results suggest that X. laevis protocadherin-9 was involved in the development of retina structure possibly through survival of neurons, formation of the lamina structure and neurite localization.

  1. Xenopus laevis oocyte maturation is affected by metal chlorides.

    PubMed

    Marin, Matthieu; Slaby, Sylvain; Marchand, Guillaume; Demuynck, Sylvain; Friscourt, Noémie; Gelaude, Armance; Lemière, Sébastien; Bodart, Jean-François

    2015-08-01

    Few studies have been conducted using Xenopus laevis germ cells as oocytes, though these cells offer many advantages allowing both electrophysiological studies and morphological examination. Our aim was to investigate the effects of metal (cadmium, lead, cobalt and zinc) exposures using cell biology approaches. First, cell survival was evaluated with both phenotypical and electrophysiological approaches. Secondly, the effect of metals on oocyte maturation was assessed with morphological observations and electrophysiological recordings. From survival experiments, our results showed that metal chlorides did not affect cell morphology but strongly depolarized X. laevis oocyte resting potential. In addition, cadmium chloride was able to inhibit progesterone-induced oocyte maturation. By contrast, zinc, but also to a lesser extent cadmium, cobalt and lead, were able to enhance spontaneous oocyte maturation in the absence of progesterone stimulation. Finally, electrophysiological recordings revealed that some metal chlorides (lead, cadmium) exposures could disturb calcium signaling in X. laevis oocyte by modifying calcium-activated chloride currents. Our results demonstrated the high sensitivity of X. laevis oocytes toward exogenous metals such as lead and cadmium. In addition, the cellular events recorded might have a predictive value of effects occurring later on the ability of oocytes to be fertilized. Together, these results suggest a potential use of this cellular lab model as a tool for ecotoxicological assessment of contaminated fresh waters. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Preparation of Xenopus laevis retinal cryosections for electron microscopy.

    PubMed

    Tam, Beatrice M; Yang, Lee Ling; Bogėa, Tami H; Ross, Bradford; Martens, Garnet; Moritz, Orson L

    2015-07-01

    Transmission electron microscopy is the gold standard for examination of photoreceptor outer segment morphology and photoreceptor outer segment abnormalities in transgenic animal models of retinal disease. Small vertebrates such as zebrafish and Xenopus laevis tadpoles have been used to generate retinal disease models and to study outer segment processes such as protein trafficking, and their breeding capabilities facilitate experiments involving large numbers of animals and conditions. However, electron microscopy processing and analysis of these very small eyes can be challenging. Here we present a methodology that facilitates processing of X. laevis tadpole eyes for electron microscopy by introducing an intermediate cryosectioning step. This method reproducibly provides a well-oriented tissue block that can be sectioned with minimal effort by a non-expert, and also allows retroactive analysis of samples collected on slides for light microscopy.

  3. Genome evolution in the allotetraploid frog Xenopus laevis.

    PubMed

    Session, Adam M; Uno, Yoshinobu; Kwon, Taejoon; Chapman, Jarrod A; Toyoda, Atsushi; Takahashi, Shuji; Fukui, Akimasa; Hikosaka, Akira; Suzuki, Atsushi; Kondo, Mariko; van Heeringen, Simon J; Quigley, Ian; Heinz, Sven; Ogino, Hajime; Ochi, Haruki; Hellsten, Uffe; Lyons, Jessica B; Simakov, Oleg; Putnam, Nicholas; Stites, Jonathan; Kuroki, Yoko; Tanaka, Toshiaki; Michiue, Tatsuo; Watanabe, Minoru; Bogdanovic, Ozren; Lister, Ryan; Georgiou, Georgios; Paranjpe, Sarita S; van Kruijsbergen, Ila; Shu, Shengquiang; Carlson, Joseph; Kinoshita, Tsutomu; Ohta, Yuko; Mawaribuchi, Shuuji; Jenkins, Jerry; Grimwood, Jane; Schmutz, Jeremy; Mitros, Therese; Mozaffari, Sahar V; Suzuki, Yutaka; Haramoto, Yoshikazu; Yamamoto, Takamasa S; Takagi, Chiyo; Heald, Rebecca; Miller, Kelly; Haudenschild, Christian; Kitzman, Jacob; Nakayama, Takuya; Izutsu, Yumi; Robert, Jacques; Fortriede, Joshua; Burns, Kevin; Lotay, Vaneet; Karimi, Kamran; Yasuoka, Yuuri; Dichmann, Darwin S; Flajnik, Martin F; Houston, Douglas W; Shendure, Jay; DuPasquier, Louis; Vize, Peter D; Zorn, Aaron M; Ito, Michihiko; Marcotte, Edward M; Wallingford, John B; Ito, Yuzuru; Asashima, Makoto; Ueno, Naoto; Matsuda, Yoichi; Veenstra, Gert Jan C; Fujiyama, Asao; Harland, Richard M; Taira, Masanori; Rokhsar, Daniel S

    2016-10-20

    To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

  4. Genome evolution in the allotetraploid frog Xenopus laevis

    PubMed Central

    Session, Adam M.; Uno, Yoshinobu; Kwon, Taejoon; Chapman, Jarrod A.; Toyoda, Atsushi; Takahashi, Shuji; Fukui, Akimasa; Hikosaka, Akira; Suzuki, Atsushi; Kondo, Mariko; van Heeringen, Simon J.; Quigley, Ian; Heinz, Sven; Ogino, Hajime; Ochi, Haruki; Hellsten, Uffe; Lyons, Jessica B; Simakov, Oleg; Putnam, Nicholas; Stites, Jonathan; Kuroki, Yoko; Tanaka, Toshiaki; Michiue, Tatsuo; Watanabe, Minoru; Bogdanovic, Ozren; Lister, Ryan; Georgiou, Georgios; Paranjpe, Sarita S.; van Kruijsbergen, Ila; Shu, Shengquiang; Carlson, Joseph; Kinoshita, Tsutomu; Ohta, Yuko; Mawaribuchi, Shuuji; Jenkins, Jerry; Grimwood, Jane; Schmutz, Jeremy; Mitros, Therese; Mozaffari, Sahar; Suzuki, Yutaka; Haramoto, Yoshikazu; Yamamoto, Takamasa S.; Takagi, Chiyo; Heald, Rebecca; Miller, Kelly; Haudenschild, Christian; Kitzman, Jacob; Nakayama, Takuya; Izutsu, Yumi; Robert, Jacques; Fortriede, Joshua; Burns, Kevin; Lotay, Vaneet; Karimi, Kamran; Yasuoka, Yuuri; Dichmann, Darwin S.; Flajnik, Martin F.; Houston, Douglas W; Shendure, Jay; DuPasquier, Louis; Vize, Peter D.; Zorn, Aaron M.; Ito, Michihiko; Marcotte, Ed; Wallingford, John B.; Ito, Yuzuru; Asashima, Makoto; Ueno, Naoto; Matsuda, Yoichi; Veenstra, Gert Jan C.; Fujiyama, Asao

    2017-01-01

    To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression. PMID:27762356

  5. A dissociation factor from embryos of Xenopus laevis.

    PubMed

    Decroly, M; Goldfinger, M

    1975-04-16

    A dissociating factor has been extracted from the ribosomal KCl wash and from the cytosol of developing embryos of Xenopus laevis. No dissociating activity could be detected in the KCl wash of ribosomes from full grown oocytes and unfertilized eggs. As in bacteria, the acitivity of the dissociation factor seems to be correlated with the rate of protein synthesis suggesting a physiological role of the dissociation factor. The possibility that the dissociation factor might be one of the components which limits the rate of protein synthesis in the oocytes is discussed.

  6. Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis.

    PubMed

    Oka, Tomohiro; Tooi, Osamu; Mitsui, Naoko; Miyahara, Maki; Ohnishi, Yuta; Takase, Minoru; Kashiwagi, Akihiko; Shinkai, Tadashi; Santo, Noriaki; Iguchi, Taisen

    2008-05-30

    There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

  7. Skin wound healing in different aged Xenopus laevis.

    PubMed

    Bertolotti, Evelina; Malagoli, Davide; Franchini, Antonella

    2013-08-01

    Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8- and 15-month-old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re-epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti-inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti-tumor necrosis factor (TNF)-α, iNOS, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti-α-SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar-like pattern. The quantitative PCR analysis demonstrated an up-regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar-like tissue formation. Copyright © 2013 Wiley Periodicals, Inc.

  8. E2F and its developmental regulation in Xenopus laevis.

    PubMed Central

    Philpott, A; Friend, S H

    1994-01-01

    The transcription factor E2F has been implicated in cell cycle control by virtue of its association with cyclins, cyclin-dependent kinases, and pRb-related tumor suppressor gene products. Eggs and embryos from the frog Xenopus laevis have been used to investigate the characteristics of E2F-like molecules in the Xenopus cell cycle and throughout early development. We find multiple E2F species in Xenopus eggs, at least one of which is modified by phosphorylation. The vast majority of E2F remains in the free form throughout the very early embryonic cell cycle, and it also remains predominantly free until some time after the mid-blastula transition, the onset of zygotic transcription. At this time, E2F complexes significantly to pRb but not to cdk2, although cdk2 binding is found in tissue culture cells from a very advanced stage in embryogenesis. This suggests that the complexing of E2F to cyclins, cyclin-dependent kinases, and tumor suppressor gene products may be controlled separately in early Xenopus development. Thus, the association of E2F with other molecules may not result solely from processes affecting cell cycle progression but may also reflect developmental and differentiation cues. Images PMID:8007993

  9. Thyroid endocrine disruption of azocyclotin to Xenopus laevis during metamorphosis.

    PubMed

    Li, Meng; Cao, Chuyan; Li, Shuying; Gui, Wenjun; Zhu, Guonian

    2016-04-01

    Organotin compounds are ubiquitous contaminants that are frequently detected in the environment and in biota, which raises concern about their risk to wildlife and human health. In the present study, Nieuwkoop & Faber stage 51 Xenopus laevis tadpoles were exposed to different concentrations of azocyclotin (0, 0.02, 0.1 and 0.5μg/L) for 21 days, during which time the tadpoles underwent morphological development. Exposure to azocyclotin caused an inhibitory effect on the pre-metamorphic development of X. laevis (e.g., a shortened hind limb length). Azocyclotin induced an alteration of the triiodothyronine (T3) content, which indicated thyroid endocrine disruption. Real-time PCR was performed to examine the expression levels of the genes involved in the thyroid hormone (TH) signaling pathway. Significant down-regulation of the type 2 deiodinase gene was observed, which may be partially responsible for the decreased T3 concentrations. Furthermore, the expression of T3 responsive genes, including thyroid hormone receptor, basic transcription element binding protein, 2tromelysins-3 and matrix metalloproteinase 2, were down-regulated in tadpoles, suggesting that azocyclotin induced a decrease in the T3 contents and, in turn, affected the mRNA expression of downstream genes involved in multiple physiological responses. Chemical analysis showed that azocyclotin could accumulate in X. laevis after 21 days of exposure. In conclusion, the results of the present study showed that azocyclotin could alter the mRNA expression of genes involved in TH signaling as well as the thyroid hormone concentrations in X. laevis tadpoles, leading to endocrine disruption of thyroid system, and that azocyclotin had obvious inhibitory effects on X. laevis metamorphosis.

  10. Swimming kinematics and respiratory behaviour of Xenopus laevis larvae raised in altered gravity

    NASA Technical Reports Server (NTRS)

    Fejtek, M.; Souza, K.; Neff, A.; Wassersug, R.

    1998-01-01

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  11. Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui.

    PubMed

    Nath, Kimberly; Boorech, Jamie L; Beckham, Yvonne M; Burns, Mary M; Elinson, Richard P

    2005-01-15

    Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.

  12. Swimming kinematics and respiratory behaviour of Xenopus laevis larvae raised in altered gravity.

    PubMed

    Fejtek, M; Souza, K; Neff, A; Wassersug, R

    1998-06-01

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  13. Swimming kinematics and respiratory behaviour of xenopus laevis larvae raised in altered gravity

    PubMed

    Fejtek; Souza; Neff; Wassersug

    1998-05-21

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  14. Corticosterone promotes emergence of fictive air breathing in Xenopus laevis Daudin tadpole brainstems.

    PubMed

    Fournier, Stéphanie; Dubé, Pierre-Luc; Kinkead, Richard

    2012-04-01

    The emergence of air breathing during amphibian metamorphosis requires significant changes to the brainstem circuits that generate and regulate breathing. However, the mechanisms controlling this developmental process are unknown. Because corticosterone plays an important role in the neuroendocrine regulation of amphibian metamorphosis, we tested the hypothesis that corticosterone augments fictive air breathing frequency in Xenopus laevis. To do so, we compared the fictive air breathing frequency produced by in vitro brainstem preparations from pre-metamorphic tadpoles and adult frogs before and after 1 h application of corticosterone (100 nmol l(-1)). Fictive breathing measurements related to gill and lung ventilation were recorded extracellularly from cranial nerve rootlets V and X. Corticosterone application had no immediate effect on respiratory-related motor output produced by brainstems from either developmental stage. One hour after corticosterone wash out, fictive lung ventilation frequency was increased whereas gill burst frequency was decreased. This effect was stage specific as it was significant only in preparations from tadpoles. GABA application (0.001-0.5 mmol l(-1)) augmented fictive air breathing in tadpole preparations. However, this effect of GABA was no longer observed following corticosterone treatment. An increase in circulating corticosterone is one of the endocrine processes that orchestrate central nervous system remodelling during metamorphosis. The age-specific effects of corticosterone application indicate that this hormone can act as an important regulator of respiratory control development in Xenopus tadpoles. Concurrent changes in GABAergic neurotransmission probably contribute to this maturation process, leading to the emergence of air breathing in this species.

  15. Susceptibility of early life stages of Xenopus laevis to cadmium

    SciTech Connect

    Herkovits, J.; Perez-Coll, C.S.; Cardellini, P.; Pavanati, C.

    1997-02-01

    The susceptibility of Xenopus laevis to cadmium during different stages of development was evaluated by exposing embryos to cadmium concentrations ranging from 0.1 to 10 mg Cd{sup 2+}/L for 24, 48, and 72 h and assessing lethality and malformations. Susceptibility increased from the two blastomeres stage (stage 2) to stage 40, in which the 24-h LC100 was 1.13 mg Cd{sup 2+}/L, and resistance increased from this stage onward. Malformations occurred at all developmental stages evaluated, the most common being reduced size, incurvated axis, underdeveloped or abnormally developed fin, microcephaly, and microphtalmy. Scanning electron microscopy revealed changes in the ectodermal surface ranging from slightly vaulted cells to a severe reduction in the number of ciliated cells as the concentration of cadmium increased. The intraspecific variation evaluated in embryos (from four sets of parents) at seven developmental stages, expressed as the coefficient of variation of the LC100, ranged from 10 to 112% and reflects the capacity of Xenopus laevis to adapt to changing environmental conditions at different embryonic stages.

  16. Mapping neurogenesis onset in the optic tectum of Xenopus laevis

    PubMed Central

    Herrgen, Leah; Akerman, Colin J.

    2016-01-01

    Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior-posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increased restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. PMID:27358457

  17. Cutaneous acariasis in the African clawed frog (Xenopus laevis).

    PubMed

    Ford, Timothy R; Dillehay, Dirck L; Mook, Deborah M

    2004-12-01

    Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.

  18. Mechanisms driving neural crest induction and migration in the zebrafish and Xenopus laevis

    PubMed Central

    Klymkowsky, Michael W; Rossi, Christy Cortez

    2010-01-01

    The neural crest is an evolutionary adaptation, with roots in the formation of mesoderm. Modification of neural crest behavior has been critical for the evolutionary diversification of the vertebrates and defects in neural crest underlie a range of human birth defects. There has been a tremendous increase in our knowledge of the molecular, cellular and inductive interactions that converge on defining the neural crest and determining its behavior. While there is a temptation to look for simple models to explain neural crest behavior, the reality is that the system is complex in its circuitry. In this review, our goal is to identify the broad features of neural crest origins (developmentally) and migration (cellularly) using data from the zebrafish (teleost) and Xenopus laevis (tetrapod amphibian) in order to illuminate where general mechanisms appear to be in play and, equally importantly, where disparities in experimental results suggest areas of profitable study. PMID:20962584

  19. Sex-specific response to hypoxia in a reduced brainstem preparation from Xenopus laevis.

    PubMed

    Rousseau, Jean-Philippe; Fournier, Stéphanie; Kinkead, Richard

    2016-04-01

    Respiratory reflexes and tolerance to hypoxia show significant sexual dimorphism. However, the data supporting this notion originates exclusively from mammals. To determine whether this concept is limited to this group of vertebrates, we examined the sex-specific response to acute hypoxia in an adult reduced brainstem preparation from Xenopus laevis. Within the first 5min of exposure to hypoxic aCSF (98% N2/2% CO2), recordings of respiratory-related activity show a stronger increase in fictive breathing frequency in males than females. This initial response was followed by a decrease in respiratory-related activity; this depression occurred 6min sooner in males than females. These results represent new evidences of sexual dimorphism in respiratory control in amphibians and provide potential insight in understanding the homology with other groups of vertebrates, including mammals.

  20. Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

    PubMed

    Rakshit, Tatini; Senapati, Subhadip; Sinha, Satyabrata; Whited, A M; Park, Paul S-H

    2015-01-01

    Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

  1. Eugenol for anesthesia of African clawed frogs (Xenopus laevis).

    PubMed

    Guénette, Sarah A; Hélie, Pierre; Beaudry, Francis; Vachon, Pascal

    2007-05-01

    To determine the level of anesthesia attained in Xenopus laevis frogs with eugenol at different doses and by different routes of administration. Prospective experimental trial. Sixty X. laevis nonbreeding female frogs weighing between 90 and 140 g. Three different routes of administration were tested - subcutaneous injections into the dorsal lymph sacs, topical administration using a gauze patch, and immersion in a bath containing eugenol. Following the determination of the best route of administration, the acetic acid test, the withdrawal reflex, righting reflex, heart rate, and respiratory frequency were used to evaluate central nervous system depression following eugenol bath administration. In an additional group, the response to a surgical incision of the abdominal wall was evaluated. The pharmacokinetics of eugenol were determined following bath immersion administration, and pharmacokinetic parameters were calculated following blood concentration determination by tandem liquid chromatography/mass spectrometry analyses. It was not possible to induce anethesia with subcutaneous and patch administration, independent of the eugenol dose administered. The immersion bath was the only efficacious route for anesthesia inducing surgical anesthesia for at least 30 minutes with postoperative analgesia. Histopathology of selected tissues (heart, lung, liver, kidneys, eyes) showed no evidence of lesions 24 hours following bath immersion. The elimination half-life (T(1/2)) was 4 hours. When administered as a single-bath immersion (dose 350 mg L(-1)) for 15 minutes, eugenol may serve as an effective anesthetic in X. laevis frogs for short surgical procedures.

  2. Metamorphic remodeling of the olfactory organ of the African clawed frog, Xenopus laevis.

    PubMed

    Dittrich, Katarina; Kuttler, Josua; Hassenklöver, Thomas; Manzini, Ivan

    2016-04-01

    The amphibian olfactory system undergoes massive remodeling during metamorphosis. The transition from aquatic olfaction in larvae to semiaquatic or airborne olfaction in adults requires anatomical, cellular, and molecular modifications. These changes are particularly pronounced in Pipidae, whose adults have secondarily adapted to an aquatic life style. In the fully aquatic larvae of Xenopus laevis, the main olfactory epithelium specialized for sensing water-borne odorous substances lines the principal olfactory cavity (PC), whereas a separate olfactory epithelium lies in the vomeronasal organ (VNO). During metamorphosis, the epithelium of the PC is rearranged into the adult "air nose," whereas a new olfactory epithelium, the adult "water nose," forms in the emerging middle cavity (MC). Here we performed a stage-by-stage investigation of the anatomical changes of the Xenopus olfactory organ during metamorphosis. We quantified cell death in all olfactory epithelia and found massive cell death in the PC and the VNO, suggesting that the majority of larval sensory neurons is replaced during metamorphosis in both sensory epithelia. The moderate cell death in the MC shows that during the formation of this epithelium some cells are sorted out. Our results show that during MC formation some supporting cells, but not sensory neurons, are relocated from the PC to the MC and that they are eventually eliminated during metamorphosis. Together our findings illustrate the structural and cellular changes of the Xenopus olfactory organ during metamorphosis.

  3. Inner Ear Formation during the Early Larval Development of Xenopus Laevis

    PubMed Central

    Quick, Quincy A.; Serrano, Elba E.

    2010-01-01

    The formation of the eight independent endorgan compartments (sacculus, utricle, horizontal canal, anterior canal, posterior canal, lagena, amphibian papilla, and basilar papilla) of the Xenopus laevis inner ear is illlustrated as the otic vesicle develops into a complex labyrinthine structure. The morphology of transverse sections and whole mounts of the inner ear was assessed in seven developmental stages (28, 31, 37, 42, 45, 47, 50) using brightfield and laser scanning confocal microscopy. The presence of mechanosensory hair cells in the sensory epithelia was determined by identification of stereociliary bundles in cryosectioned tissue and whole mounts of the inner ear labeled with the fluorescent F-actin probe, Alexa-488 phalloidin. Between stages 28 and 45 the otic vesicle grows in size, stereociliary bundles appear and increase in number, and the pars inferior and pars superior become visible. The initial formation of vestibular compartments with their nascent stereociliary bundles is seen by larval stage 47, and all eight vestibular and auditory compartments with their characteristic sensory fields are present by larval stage 50. Thus in Xenopus, inner ear compartments are established between stages 45 and 50, a two week period during which the ear quadruples in length in the anteroposterior dimension. The anatomical images presented here demonstrate the morphological changes that occur as the otic vesicle forms the auditory and vestibular endorgans of the inner ear. These images provide a resource for investigations of gene expression patterns in Xenopus during inner ear compartmentalization and morphogenesis. PMID:16217737

  4. Negative Effects of Low Dose Atrazine Exposure on the Development of Effective Immunity to FV3 in Xenopus laevis

    PubMed Central

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B. Paige; Robert, Jacques

    2014-01-01

    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxiciology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses. PMID:24984115

  5. Negative effects of low dose atrazine exposure on the development of effective immunity to FV3 in Xenopus laevis.

    PubMed

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B Paige; Robert, Jacques

    2014-11-01

    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxicology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses.

  6. Trpc2 is expressed in two olfactory subsystems, the main and the vomeronasal system of larval Xenopus laevis.

    PubMed

    Sansone, Alfredo; Syed, Adnan S; Tantalaki, Evangelia; Korsching, Sigrun I; Manzini, Ivan

    2014-07-01

    Complete segregation of the main olfactory epithelium (MOE) and the vomeronasal epithelium is first observed in amphibians. In contrast, teleost fishes possess a single olfactory surface, in which genetic components of the main and vomeronasal olfactory systems are intermingled. The transient receptor potential channel TRPC2, a marker of vomeronasal neurons, is present in the single fish sensory surface, but is already restricted to the vomeronasal epithelium in a terrestrial amphibian, the red-legged salamander (Plethodon shermani). Here we examined the localization of TRPC2 in an aquatic amphibian and cloned the Xenopus laevis trpc2 gene. We show that it is expressed in both the MOE and the vomeronasal epithelium. This is the first description of a broad trpc2 expression in the MOE of a tetrapod. The expression pattern of trpc2 in the MOE is virtually undistinguishable from that of MOE-specific v2rs, indicating that they are co-expressed in the same neuronal subpopulation.

  7. Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.

    PubMed

    Berger, Jens; Hardt, Martin; Clauss, Wolfgang G; Fronius, Martin

    2010-07-01

    A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

  8. Posttranslational modification and secretion of vitellogenin in Xenopus laevis

    SciTech Connect

    Gottlieb, T.A.

    1981-08-01

    The thesis represents an attempt to elucidate the intracellular pathway linking the synthesis and secretion of the yolk precursor protein vitellogenin. More specifically, how the hepatocyte attaches both phosphate and carbohydrate to the peptide backbone prior to secretion is considered. A model which summarizes the accumulated data depicting various aspects of these processes is also presented. Pulse-chase experiments demonstrate the subcellular sites of vitellogenin phosphorylation. The results of these experiments indicate that approximately 70% of the phsosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of the total incorporated phosphate. The presence of a divalent-cation requiring protein kinase (E.C. 2.7.1,37, ATP:protein phosphotransferase) is demonstrated in the microsomes derived from the liver of estrogenized female Xenopus laevis. We present data showing that this protein kinase is responsible for phosphorylating hepatic precursors to serum vitellogenin in vivo. Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. The results indicate that the oligosaccharide component of vitellogenin in Xenopus is a complex type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.

  9. Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation.

    PubMed

    Shimizu-Nishikawa, Keiko; Nishimatsu, Shin-ichiro; Nishikawa, Akio

    2012-05-01

    Fates of digits in amniotes, i.e., free or webbed digits, are determined by the size of programmed interdigital cell death (ICD) area. However, no (or very few) cell death has thus far been observed in developing limb buds of non-amniotic terrestrial vertebrates including other anuran or urodela amphibians. We speculate that the undetectable situation of amphibian ICD is the result of their less frequency due to slow developmental speed characteristic to most amphibian species. Here, we present three strategies for detecting difficult-to-find ICD in the frog, Xenopus laevis. (1) Addition of triiodo-L-thyronine (T(3)) accelerated two to three times the limb development and increased two to four times the appearance frequency of vital dye-stainable cells in limb buds of the accelerated tadpoles (stage 54 to 55). (2) Application of human bone morphogenetic protein-4 to the autopods of tadpoles at stage 53 to 54 enhanced digital cartilage formation and induced vital dye-stainable cells around the enhanced digital cartilages within 2 d. (3) In cell culture, T(3) increased the chondrogenic and cell death activities of limb mesenchymal cells. The augmentation of both activities by T(3) was stronger in the forelimb cells than in the hindlimb cells. This situation is well coincided with the limb fates of non-webbed forelimbs and webbed hindlimbs in X. laevis adulthood. Collectively, all three approaches showed that it become possible to detect X. laevis ICD with appropriate strategies.

  10. Prepatterning and patterning of the thalamus along embryonic development of Xenopus laevis

    PubMed Central

    Bandín, Sandra; Morona, Ruth; González, Agustín

    2015-01-01

    Previous developmental studies of the thalamus (alar part of the diencephalic prosomere p2) have defined the molecular basis for the acquisition of the thalamic competence (preparttening), the subsequent formation of the secondary organizer in the zona limitans intrathalamica, and the early specification of two anteroposterior domains (rostral and caudal progenitor domains) in response to inducing activities and that are shared in birds and mammals. In the present study we have analyzed the embryonic development of the thalamus in the anuran Xenopus laevis to determine conserved or specific features in the amphibian diencephalon. From early embryonic stages to the beginning of the larval period, the expression patterns of 22 markers were analyzed by means of combined In situ hybridization (ISH) and immunohistochemical techniques. The early genoarchitecture observed in the diencephalon allowed us to discern the boundaries of the thalamus with the prethalamus, pretectum, and epithalamus. Common molecular features were observed in the thalamic prepatterning among vertebrates in which Wnt3a, Fez, Pax6 and Xiro1 expression were of particular importance in Xenopus. The formation of the zona limitans intrathalamica was observed, as in other vertebrates, by the progressive expression of Shh. The largely conserved expressions of Nkx2.2 in the rostral thalamic domain vs. Gbx2 and Ngn2 (among others) in the caudal domain strongly suggest the role of Shh as morphogen in the amphibian thalamus. All these data showed that the molecular characteristics observed during preparttening and patterning in the thalamus of the anuran Xenopus (anamniote) share many features with those described during thalamic development in amniotes (common patterns in tetrapods) but also with zebrafish, strengthening the idea of a basic organization of this diencephalic region across vertebrates. PMID:26321920

  11. Spiral Calcium Wave Propagation and Annihilation in Xenopus laevis Oocytes

    NASA Astrophysics Data System (ADS)

    Lechleiter, James; Girard, Steven; Peralta, Ernest; Clapham, David

    1991-04-01

    Intracellular calcium (Ca2+) is a ubiquitous second messenger. Information is encoded in the magnitude, frequency, and spatial organization of changes in the concentration of cytosolic free Ca2+. Regenerative spiral waves of release of free Ca2+ were observed by confocal microscopy in Xenopus laevis oocytes expressing muscarinic acetylcholine receptor subtypes. This pattern of Ca2+ activity is characteristic of an intracellular milieu that behaves as a regenerative excitable medium. The minimal critical radius for propagation of focal Ca2+ waves (10.4 micrometers) and the effective diffusion constant for the excitation signal (2.3 x 10-6 square centimeters per second) were estimated from measurements of velocity and curvature of circular wavefronts expanding from foci. By modeling Ca2+ release with cellular automata, the absolute refractory period for Ca2+ stores (4.7 seconds) was determined. Other phenomena expected of an excitable medium, such as wave propagation of undiminished amplitude and annihilation of colliding wavefronts, were observed.

  12. Sexually differentiated central pattern generators in Xenopus laevis.

    PubMed

    Zornik, Erik; Yamaguchi, Ayako

    2008-06-01

    Understanding the neural mechanisms that underlie the function of central pattern generators (CPGs) presents a formidable challenge requiring sophisticated tools and well-chosen model systems. In this article, we describe recent work on vocalizations of the African clawed frog Xenopus laevis. These behaviors are driven by sexually differentiated CPGs and are exceptionally well suited to this objective. In particular, a simplified mechanism of vocal production (independent of respiratory musculature) allows straightforward interpretations of nerve activity with respect to behavior. Furthermore, the development of a fictively vocalizing isolated brain, together with the finding of rapid androgen-induced masculinization of female vocalizations, provides an invaluable tool for determining how new behaviors arise from existing circuits.

  13. Sexually differentiated central pattern generators in Xenopus laevis

    PubMed Central

    Zornik, Erik; Yamaguchi, Ayako

    2008-01-01

    Understanding the neural mechanisms that underlie the function of central pattern generators (CPGs) presents a formidable challenge requiring sophisticated tools and well-chosen model systems. In this article, we describe recent work on vocalizations of the African clawed frog Xenopus laevis. These behaviors are driven by sexually differentiated CPGs and are exceptionally well suited to this objective. In particular, a simplified mechanism of vocal production (independent of respiratory musculature) allows straightforward interpretations of nerve activity with respect to behavior. Furthermore, the development of a fictively vocalizing isolated brain, together with the finding of rapid androgen-induced masculinization of female vocalizations, provides an invaluable tool for determining how new behaviors arise from existing circuits. PMID:18471902

  14. PHENOBARBITAL AFFECTS THYROID HISTOLOGY AND LARVAL DEVELOPMENT IN THE AFRICAN CLAWED FROG XENOPUS LAEVIS

    EPA Science Inventory

    The abstract highlights our recent study to explore endocrine disrupting effects of phenobarbital in the African clawed frog, Xenopus laevis. In mammals, this chemical is known to induce the biotransforming enzyme UDP-glucuronosyltransferase (UDPGT) resulting in increased thyroid...

  15. Long term effects of carbaryl exposure on antiviral immune responses in Xenopus laevis.

    PubMed

    De Jesús Andino, Francisco; Lawrence, B Paige; Robert, Jacques

    2017-03-01

    Water pollutants associated with agriculture may contribute to the increased prevalence of infectious diseases caused by ranaviruses. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable experimental platform for evaluating the effects of common waterborne pollutants, such as the insecticide carbaryl. Following 3 weeks of exposure to 10 ppb carbaryl, X. laevis tadpoles exhibited a marked increase in mortality and accelerated development. Exposure at lower concentrations (0.1 and 1.0 ppb) was not toxic, but it impaired tadpole innate antiviral immune responses, as evidenced by significantly decreased TNF-α, IL-1β, IFN-I, and IFN-III gene expression. The defect in IFN-I and IL-1β gene expression levels persisted after metamorphosis in froglets, whereas only IFN-I gene expression in response to FV3 was attenuated when carbaryl exposure was performed at the adult stage. These findings suggest that the agriculture-associated carbaryl exposure at low but ecologically-relevant concentrations has the potential to induce long term alterations in host-pathogen interactions and antiviral immunity.

  16. Biochemical response to exposure to six textile dyes in early developmental stages of Xenopus laevis.

    PubMed

    Güngördü, Abbas; Birhanli, Ayse; Ozmen, Murat

    2013-01-01

    The present study was undertaken to determine the toxic effect of a lethal concentration of six different commercially used textile dyes on the 46th stage of Xenopus laevis tadpoles. The tadpoles were exposed to Astrazon Red FBL, Astrazon Blue FGRL, Remazol Red RR, Remazol Turquoise Blue G-A, Cibacron Red FN-3G, and Cibacron Blue FN-R for 168 h in static test conditions, and thus, 168-h median lethal concentrations (LC(50)s) of each dye were determined to be 0.35, 0.13, 112, 7, 359, and 15.8 mg/L, respectively. Also, to evaluate the sublethal effects of each dye, tadpoles were exposed to different concentrations of dyes (with respect to 168-h LC(50)s) for 24 h. The alteration of selected enzyme activities was tested. For this aim, glutathione S-transferase (GST), carboxylesterase, and lactate dehydrogenase (LDH) were assayed. After dye exposure, the GST induction or inhibition and LDH induction indicated some possible mechanisms of oxidative stress and deterioration in aerobic respiration processes induced by the tested dyes. Findings of the study suggest that selected biomarker enzymes are useful in understanding the toxic mechanisms of these dyes in X. laevis tadpoles as early warning indicators. Therefore, these selected biomarkers may evaluate the effect of environmental factors, such as textile dye effluents and other industrial pollutants, on amphibians in biomonitoring studies.

  17. Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

    PubMed

    Tedeschi, Gabriella; Pagliato, Lara; Negroni, Manuela; Montorfano, Gigliola; Corsetto, Paola; Nonnis, Simona; Negri, Armando; Rizzo, Angela Maria

    2011-03-01

    Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

  18. Teratogenic effects of five anticancer drugs on Xenopus laevis embryos.

    PubMed

    Isidori, Marina; Piscitelli, Concetta; Russo, Chiara; Smutná, Marie; Bláha, Luděk

    2016-11-01

    In recent years, the environmental presence of pharmaceuticals - including anticancer drugs - is an emerging issue. Because of the lack of appropriate critical studies about anticancer drug effects in frogs, the aim of the present study was to investigate lethal and teratogenic effects of five anticancer drugs widely used in large quantities, i.e. 5-flourouracil, capecitabine, cisplatin, etoposide, and imatinib, in the embryos of the South African clawed frog, Xenopus laevis, using FETAX - Frog Embryo Teratogenesis Assay in Xenopus. None of the studied anticancer drugs induced statistically significant mortality within the concentrations tested (0.01-50mg/L, depending on the studied compound), and no growth inhibition of embryos after a 96-h exposure was observed. Except for cisplatin, the other pharmaceuticals induced an increase of developmental malformations such as abdominal edema, axial flexure, head, eyes, gut and heart malformations with statistically significant effects observed at the highest concentrations tested (50mg/L for 5-flourouracil; 30mg/L for etoposide and 20mg/L for capecitabine and imatinib). The results indicate that anticancer drugs can affect embryogenesis mechanisms.

  19. Arrested development in Xenopus laevis tadpoles: how size constrains metamorphosis.

    PubMed

    Rot-Nikcevic, Irena; Wassersug, Richard J

    2004-05-01

    Xenopus laevis tadpoles that arrest development and remain as larvae for several years sometimes occur spontaneously in laboratory populations. These tadpoles cease development at an early hindlimb stage, but continue to grow and develop into grossly deformed giants. Giant tadpoles lack thyroid glands, and differ in morphology and behaviour from normal larvae. They are negatively buoyant, typically with small and partially solidified lungs, and have greatly enlarged fat bodies. Giant tadpoles have mature gonads with eggs and sperm, whereas normal tadpoles of the same stage have undifferentiated gonads. Larval reproduction has never been reported in anurans, but gonadal development decoupled from metamorphosis brings these giants the closest of any anurans to being truly neotenic. We discuss behavioural and morphological factors that may hinder both reproduction in giant Xenopus larvae and the evolution of neoteny in anurans in general. Experimental treatment with exogenous thyroid hormone induces some, but not complete, metamorphic changes in these giants. The limbs and head progress through metamorphosis; however, all tadpoles die at the stage when the tail would normally be resorbed. The disproportionate growth of tissues and organs in giant tadpoles may preclude complete metamorphosis, even under exogenous thyroid hormone induction.

  20. A quantitative adverse outcome pathway model for thyroid axis disruption in Xenopus laevis tadpoles

    EPA Science Inventory

    The development of Xenopus laevis tadpoles is tightly controlled by the thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). Toxicity testing efforts have shown that several compounds interfere with development in X. laevis tadpoles by disrupting the thyroid axis a...

  1. A quantitative adverse outcome pathway model for thyroid axis disruption in Xenopus laevis tadpoles

    EPA Science Inventory

    The development of Xenopus laevis tadpoles is tightly controlled by the thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). Toxicity testing efforts have shown that several compounds interfere with development in X. laevis tadpoles by disrupting the thyroid axis a...

  2. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    PubMed

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  3. Molecular cloning and characterization of novel ficolins from Xenopus laevis.

    PubMed

    Kakinuma, Yuji; Endo, Yuichi; Takahashi, Minoru; Nakata, Munehiro; Matsushita, Misao; Takenoshita, Seiichi; Fujita, Teizo

    2003-04-01

    Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins and are thought to play crucial roles in host defense through opsonization and complement activation. To elucidate the evolution of ficolins and the primordial complement lectin pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its lectin activity.

  4. Recovery capabilities of Xenopus laevis after exposure to Cadmium and Zinc.

    PubMed

    Mouchet, F; Teaniniuraitemoana, V; Baudrimont, M; Daffe, G; Gauthier, L; Gonzalez, P

    2015-11-01

    The present investigation evaluates the recovery capabilities of Xenopus laevis following 12days of exposure to 30μg CdL(-1) and 1000μg ZnL(-1) alone or mixed, followed by a depuration phase in laboratory conditions. Focused endpoints, which were investigated at different times of depuration, are bioaccumulation of Cd and Zn, micronucleus induction, quantification of metallothioneins (MTs), and expression of genes involved in metal toxicity mechanisms. The results show that at the end of the contamination phase, there was higher metal bioaccumulation capability and MT synthesis in remaining tissues than in the liver. An increased expression of genes involved in detoxification and oxidative stress mechanisms was observed, suggesting an additive effect of both metals and a higher Zn regulation in the liver. During the depuration phase, the results show the recovery capability of Xenopus from 7days of depuration related to metamorphosis processes, which were observed at the end of the experiment. The results confirm the relevance of the amphibian model and the complementarities between a marker of genotoxicity, MT production, bioaccumulation and transcriptional analysis in the evaluation of the ecotoxicological impact. The results also highlight the reversible effects of Cd and Zn toxicity.

  5. Molecular and cellular characterization of urinary bladder-type aquaporin in Xenopus laevis.

    PubMed

    Shibata, Yuki; Katayama, Izumi; Nakakura, Takashi; Ogushi, Yuji; Okada, Reiko; Tanaka, Shigeyasu; Suzuki, Masakazu

    2015-10-01

    In contrast to many anuran amphibians, water is not reabsorbed from the urinary bladder in aquatic Xenopus, thereby helping to prevent excessive water influx. However, little is known about the molecular mechanisms for this process. In the present study, we have identified urinary bladder-type aquaporin, AQP-x2, in Xenopus laevis by cDNA cloning. The predicted amino acid sequence contained six putative transmembrane domains and the two conserved Asn-Pro-Ala motifs, characteristic of AQPs. The sequence also contained a putative N-glycosylation site and phosphorylation motifs for protein kinase A and protein kinase C. The oocyte swelling assay showed that AQP-x2 facilitated water permeability. Reverse transcription-PCR analysis indicated that AQP-x2 mRNA was expressed in the urinary bladder and lung, and faintly in the kidney. Immunomicroscopical study further localized AQP-x2 protein to the cytoplasm of granular cells in the luminal epithelium of the urinary bladder whilst AQP3 was observed along the basolateral side of these cells. In vitro stimulation of the urinary bladder with 10(-8)M vasotocin (AVT), 10(-8)M hydrin 1, or 10(-8)M hydrin 2 had no clear effect on the subcellular distribution of AQP-x2. When the AVT concentration was increased to 10(-6)M, however, AQP-x2 was partially transferred to the apical plasma membrane. The treatment with hydrin 1 or hydrin 2 at the same concentration failed to induce the translocation to the apical membrane. On the other hand, AQP3 remained along the basolateral side even after the treatment with vasotocin or hydrins. The results suggest that the poor responsiveness of AQP-x2 to neurohypophyseal peptides may be a main cause for the little water permeability of the urinary bladder of X. laevis.

  6. Environmentally relevant concentrations of ammonium perchlorate inhibit development and metamorphosis in Xenopus laevis.

    PubMed

    Goleman, Wanda L; Urquidi, Lina J; Anderson, Todd A; Smith, Ernest E; Kendall, Ronald J; Carr, James A

    2002-02-01

    We determined whether environmentally relevant concentrations of ammonium perchlorate alter development and metamorphosis in Xenopus laevis. Eggs and larvae were exposed to varying concentrations of ammonium perchlorate or control medium for 70 d. Most treatment-related mortality was observed within 5 d after exposure and was due in large part to reduced hatching success. The 5- and 70-d median lethal concentrations (LC50s) were 510 +/- 36 mg ammonium perchlorate/L and 223 +/- 13 mg ammonium perchlorate/L, respectively. Ammonium perchlorate did not cause any concentration-related developmental abnormalities at concentrations below the 70-d LC50. Ammonium perchlorate inhibited metamorphosis in a concentration-dependent manner as evident from effects on forelimb emergence, tail resorption, and hindlimb growth. These effects were observed after exposure to ammonium perchlorate concentrations in the parts-per-billion range, at or below concentrations reported in surface waters contaminated with ammonium perchlorate. Ammonium perchlorate significantly inhibited tail resorption after a 14-d exposure in the U.S. Environmental Protection Agency (U.S. EPA) Endocrine Disruptor Screening and Testing Committee (EDSTAC) Tier I frog metamorphosis assay for thyroid disruption in amphibians. We believe that ammonium perchlorate may pose a threat to normal development and growth in natural amphibian populations.

  7. Developmental Toxicity of Drinking Water Disinfection By-Products to Embryos of the African Clawed Frog (Xenopus laevis)

    DTIC Science & Technology

    2005-06-10

    developmental toxicity tests with embryos of the South African clawed frog Xenopus laevis used to evaluate four individual DWDB; bromodichloromethane...SUBJECT TERMS Developmental toxicity; FETAX; water disinfection by-products; frogs ; Xenopus laevis; embryo malformations; embryo mortality...Disinfection By-Products to Embryos of the African Clawed Frog (Xenopus laevis) L. M. Brennan,1 M. W. Toussaint,1 D. M. Kumsher,1 W. E. Dennis,’ A. B

  8. Dose-Dependent Early Life Stage Toxicities in Xenopus laevis Exposed In Ovo to Selenium.

    PubMed

    Massé, Anita J; Muscatello, Jorgelina R; Janz, David M

    2015-11-17

    Selenium (Se) is a developmental toxicant in oviparous vertebrates. The adverse reproductive effects of Se toxicity have been predominantly investigated in fishes and birds with only a few studies focusing on amphibians. The objective of this study was to determine tissue-based toxicity thresholds for early life stage Se toxicities in Xenopus laevis as a consequence of in ovo exposure through maternal transfer of dietary Se. Following a 68-day dietary exposure to food augmented with l-selenomethionine (SeMet) at measured concentrations of 0.7 (control), 10.9, 30.4, or 94.2 μg Se/g dry mass (d.m.), adult female X. laevis were bred with untreated males, and resulting embryos were incubated until 5 days postfertilization (dpf). The measured Se concentrations in eggs were 1.6, 10.8, 28.1, and 81.7 μg Se/g d.m., respectively. No biologically significant effects were observed on fertilization success, hatchability, or mortality in offspring. Frequency and severity of morphological abnormalities were significantly greater in 5 dpf tadpoles from the highest exposure group when compared to the control, with eye lens abnormalities being the most prominent of all abnormalities. The estimated EC10 value for frequency of total early life stage abnormalities was 44.9 μg Se/g egg d.m., which suggests that this amphibian species is less sensitive to in ovo Se exposure than most of the fish species studied to date.

  9. The Xenopus alcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes

    PubMed Central

    2014-01-01

    Background The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family. Results Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not

  10. The Xenopus alcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes.

    PubMed

    Borràs, Emma; Albalat, Ricard; Duester, Gregg; Parés, Xavier; Farrés, Jaume

    2014-03-20

    The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family. Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not present in amphibians

  11. Effects of perfluorooctanesulfonate and perfluorobutanesulfonate on the growth and sexual development of Xenopus laevis.

    PubMed

    Lou, Qin-Qin; Zhang, Yin-Feng; Zhou, Zhen; Shi, Ya-Li; Ge, Ya-Nan; Ren, Dong-Kai; Xu, Hai-Ming; Zhao, Ya-Xian; Wei, Wu-Ji; Qin, Zhan-Fen

    2013-09-01

    Perfluorobutanesulfonate (PFBS), as a substitute for perfluorooctanesulfonate (PFOS), is widespread in the environment and biotic samples as well as PFOS. To investigate effects of PFOS and PFBS on the growth and sexual development of amphibians, we exposed Xenopus laevis tadpoles at a series of concentrations of PFOS and PFBS (0.1; 1; 100; 1,000 μg/l) as well as 17-beta-estradiol (E2, 100 ng/l) and 5 alpha-androstan-17-beta-ol-3-one (DHT, 100 ng/l) from stage 46/47 to 2 months postmetamorphosis. We found that neither PFOS nor PFBS had a significant effect on the survival and growth. However, they caused hepatohistological impairment at higher concentrations (100; 1,000 μg/l). Unlike E2, PFOS at all concentrations did not alter the sex ratio and induce intersex, but caused degeneration of spermatogonia in testes except for the lowest concentration. PFBS had no effect on the sex ratio and gonadal histology. PFOS and PFBS promoted expression of estrogen receptor (ER) and androgen receptor (AR), but not affected aromatase expression in the brain. The increase in expression of ER and AR suggests an increase in the responsiveness to the corresponding sex hormone and potential effects on sexual development. Our results show that PFBS as well as PFOS have adverse effects on hepato-histology and sexual development on X. laevis. Also, PFOS- and PFBS-induced increase in ER and AR expression highlights the need to further study effects of PFOS and PFBS on subsequently gonadal development, sexual dimorphism, and secondary sex characteristics in X. laevis. It is debatable that PFBS is widely used as a substitute of PFOS.

  12. Endocrine effects of 2,2{prime},4,4{prime}-tetrachlorobiphenyl in the African clawed frog (Xenopus laevis)

    SciTech Connect

    Diana, S.; Hansen, L.; Foley, G.; Beasley, V.

    1995-12-31

    Ortho-substituted polychlorinated biphenyls are known to exhibit estrogenic activity and, in some cases, to enhance excretion of tetraiodothyronine (T4), resulting in hypothyroxinemia in mammals. Since thyroxine activity is essential for amphibian metamorphosis, and amphibian sex determination can be altered or reversed by exposure to exogenous estrogens or androgens, the effects of exposure of larvae of the African clawed frog (Xenopus laevis) to 2,2{prime},4,4{prime}-tetrachlorobiphenyl (CB 47) were investigated. Eggs and larvae of X. laevis were exposed to nominal concentrations of CB 47 of 0.05 or 0.25 ppm (1 ppm was found to result in 100% mortality) throughout the period of larval development, and effects on rates of metamorphosis and body growth and on gonad morphology were determined. Stage of metamorphosis, body length and body weight did not differ between treatment and control groups, following exposure to these sub-lethal concentrations, at any time during larval development. Effects of exposure on gonad morphology will be discussed. The failure of CB 47 to delay or prevent metamorphosis under these conditions may be due to poor responsiveness of hepatic UDP-glucuronyl transferases to induction, or novel systems of thyroxine and/or PCB transport, metabolism and excretion in larval amphibians.

  13. Characterization of X-OCRL, a Xenopus laevis homologue of OCRL-1, the Lowe oculocerebrorenal syndrome candidate gene

    SciTech Connect

    Reilly, D.S.; Nussbaum, R.L.

    1994-09-01

    The Lowe oculocerebrorenal syndrome (OCRL) is an X-linked disease characterized by congenital cataract, mental retardation, and renal tubular dysfunction. A candidate cDNA, OCRL-1, was identified by positional cloning and mutations in OCRL-1 have been detected in patients with Lowe syndrome. The OCRL-1 nucleotide sequence encodes a predicted protein of 968 amino acids and shares 51% amino acid identity with a human inositol polyphosphate-5-phosphatase. This suggests that the underlying defect in OCRL may be due to a defect in inositol phosphate metabolism. The isolation of OCRL-1 provides the opportunity to investigate its function through the use of animal model systems. We have isolated a partial cDNA clone encoding an OCRL-1 homologue, X-OCRL, from the South African clawed frog, Xenopus laevis. We used a portion of the human cDNA to screen a Xenopus laevis embryo cDNA library and isolated four positive clones. One clone, 42-5A, is a 650 bp insert with over 75% amino acid identity to the corresponding region of the human OCRL-1 sequence. 42-5A detects messenger RNA in adult Xenopus brain, stomach, small intestine, skin, muscle, lung, blood, and oviduct. X-OCRL messenger RNA is first detected during late gastrula and continues to be expressed throughout Xenopus development. In situ hybridization studies are underway to identify the cellular localization of X-OCRL expression in Xenopus embryos and adult tissues. We are especially interested in characterizing X-OCRL expression during formation of the amphibian lens since congenital cataracts are a constant feature of the human disease.

  14. Transient effects of microgravity on early embryos of Xenopus laevis

    NASA Astrophysics Data System (ADS)

    de Mazière, A.; Gonzalez-Jurado, J.; Reijnen, M.; Narraway, J.; Ubbels, G. A.

    In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity (mug) during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and mug. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in mug and on earth. Analysis of the video tapes revealed that the cortical contraction in mug starts earlier than at 1 g on earth. After recovery of the eggs fertilized in mug and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the mug embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

  15. Valproate-induced neurodevelopmental deficits in Xenopus laevis tadpoles.

    PubMed

    James, Eric J; Gu, Jenny; Ramirez-Vizcarrondo, Carolina M; Hasan, Mashfiq; Truszkowski, Torrey L S; Tan, Yuqi; Oupravanh, Phouangmaly M; Khakhalin, Arseny S; Aizenman, Carlos D

    2015-02-18

    Autism spectrum disorder (ASD) is increasingly thought to result from low-level deficits in synaptic development and neural circuit formation that cascade into more complex cognitive symptoms. However, the link between synaptic dysfunction and behavior is not well understood. By comparing the effects of abnormal circuit formation and behavioral outcomes across different species, it should be possible to pinpoint the conserved fundamental processes that result in disease. Here we use a novel model for neurodevelopmental disorders in which we expose Xenopus laevis tadpoles to valproic acid (VPA) during a critical time point in brain development at which neurogenesis and neural circuit formation required for sensory processing are occurring. VPA is a commonly prescribed antiepileptic drug with known teratogenic effects. In utero exposure to VPA in humans or rodents results in a higher incidence of ASD or ASD-like behavior later in life. We find that tadpoles exposed to VPA have abnormal sensorimotor and schooling behavior that is accompanied by hyperconnected neural networks in the optic tectum, increased excitatory and inhibitory synaptic drive, elevated levels of spontaneous synaptic activity, and decreased neuronal intrinsic excitability. Consistent with these findings, VPA-treated tadpoles also have increased seizure susceptibility and decreased acoustic startle habituation. These findings indicate that the effects of VPA are remarkably conserved across vertebrate species and that changes in neural circuitry resulting from abnormal developmental pruning can cascade into higher-level behavioral deficits.

  16. The thymus and tail regenerative capacity in Xenopus laevis tadpoles.

    PubMed

    Franchini, Antonella; Bertolotti, Evelina

    2012-07-01

    A morphofunctional analysis of the thymus from differently aged Xenopus laevis tadpoles during regeneration of the tail is reported. In stage 50 larvae, competent to regenerate, the appendage cut provoked thymic structural modifications that affected the medullary microenvironment cells and changes in TNF-α immunoreactivity. Mucocyte-like cells, multicellular epithelial cysts, myoid cells and cells immunoreactive to TNF-α increased in number. Increased numbers of lymphocytes were also found in regenerating areas and, at the end of regeneration, thymic structural and immunocytochemical patterns were restored to control levels. The observed cellular responses and the induction of molecules critical for thymus constitutive processes suggest a stimulation of thymic function after tail amputation. In older larvae, whose capacity to form a new complete and correctly patterned tail was reduced, thymic morphological changes were more severe and may persist throughout the regeneration process with a significant reduction in organ size. In these larvae the histological patterns and the marked thymic decrease may be related to the events occurring during regeneration, i.e. the higher inflammatory response and the reduced tail regenerative potential.

  17. Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis

    PubMed Central

    Pai, Vaibhav P.; Aw, Sherry; Shomrat, Tal; Lemire, Joan M.; Levin, Michael

    2012-01-01

    Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (Vmem) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating Vmem of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca2+ channel-dependent pathway transduces the Vmem signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that Vmem is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases. PMID:22159581

  18. Transient effects of microgravity on early embryos of Xenopus laevis.

    PubMed

    De Mazière, A; Gonzalez-Jurado, J; Reijnen, M; Narraway, J; Ubbels, G A

    1996-01-01

    In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and microgravity. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in microgravity and on earth. Analysis of the video tapes revealed that the cortical contraction in microgravity starts earlier than at 1 g on earth. After recovery of the eggs fertilized in microgravity and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the microgravity embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

  19. Circadian rhythm of interrenal activity in Xenopus laevis.

    PubMed

    Thurmond, W; Kloas, W; Hanke, W

    1986-02-01

    Young specimens of Xenopus laevis were kept under constant environmental conditions (artifical light from 600 to 1800 hr, feeding between 800 and 900 hr) and the concentrations of aldosterone and corticosterone in the serum were measured every 3 hr. Furthermore, the kidneys containing the interrenals were removed and their corticosteroid release under stimulation by the pars distalis and mammalian ACTH was determined. Under these conditions, the corticosteroid levels in the serum were maximal from 900 to 1200 hr (corticosterone, 7.7 +/- 0.47 ng/ml; aldosterone, 2.8 +/- 0.27 ng/ml) and minimal during the night (corticosterone, 5.2 +/- 0.43 ng/ml; aldosterone, 1.7 +/- 0.18 ng/ml). The basal secretion rate of the interrenals in vitro showed the opposite course (corticosterone, 24 +/- 3 to 72 +/- 8 pg/min/tissue; aldosterone, 46 +/- 5 to 68 +/- 9 pg/min/tissue). Stimulation by the pars distalis and mammalian ACTH clearly increased the secretion rate. After both types of stimulation the original rhythm was lost for aldosterone but still present for corticosterone. The ratio of the amounts of corticosterone/aldosterone was greater than 1.0 in the serum but less than 1.0 in the incubation fluid. It decreased significantly after stimulation in vitro by pars distalis or ACTH.

  20. A restriction map of Xenopus laevis mitochondrial DNA.

    PubMed

    Cordonnier, A M; Vannier, P A; Brun, G M

    1982-08-01

    The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.

  1. Budgett’s frog (Lepidobatrachus laevis): a new amphibian embryo for developmental biology

    PubMed Central

    Amin, Nirav M.; Womble, Mandy; Ledon-Rettig, Cris; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette

    2015-01-01

    The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett’s frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett’s frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett’s tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett’s frog model provides inimitable advantages for developmental studies—and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution. PMID:26169245

  2. Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology.

    PubMed

    Amin, Nirav M; Womble, Mandy; Ledon-Rettig, Cristina; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette

    2015-09-15

    The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies-and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution.

  3. Identification and Bioinformatics Analyses of the Basic Helix-loop-helix Transcription Factors in Xenopus laevis.

    PubMed

    Liu, Wuyi; Li, Fengmei

    2015-04-01

    Xenopus laevis is a long established model organism for developmental, behavioral and neurological studies. Herein, an updated genome-wide survey was conducted using the ongoing genome project of Xenopus laevis and 106 non-redundant Basic Helix-Loop-Helix (bHLH) genes were identified in the Xenopus laevis genome databases. Gene Ontology (GO) enrichment statistics showed 51 significant GO annotations of biological processes and molecular functions and 5 significant KEGG pathways and a number of Xenopus laevis bHLH genes play significant role in specific development or special physiology processes like the development processes of muscle and eye and other organs. Furthermore, each sub-group of the bHLH family has its special gene functions except for the common GO term categories. Molecular phylogenetic analyses revealed that among these identified bHLH proteins, 105 sequences could classified into 39 families with 46, 25, 10, 5, 16 and 3 members in the corresponding high-order groups A, B, C, D, E and F, respectively with an addition bHLH member categorized as an orphan. The present study provides much useful information for further researches on Xenopus laevis.

  4. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    SciTech Connect

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  5. A comparison of host-defense peptides in skin secretions of female Xenopus laevis × Xenopus borealis and X. borealis × X. laevis F1 hybrids.

    PubMed

    Mechkarska, Milena; Prajeep, Manju; Leprince, Jérôme; Vaudry, Hubert; Meetani, Mohammed A; Evans, Ben J; Conlon, J Michael

    2013-07-01

    Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (XLB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (XBL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1 was present in low concentration. No peptides were identified in secretions of either XLB or XBL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis×X. muelleri F1 hybrids and no novel peptide expression. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Development of the larval amphibian growth and development assay: Effects of chronic 4-tert-octylphenol or 17ß-trenbolone exposure in Xenopus laevis from embryo to juvenile

    EPA Science Inventory

    The Larval Amphibian Growth and Development Assay (LAGDA) is a Tier II test guideline being developed by the US Environmental Protection Agency under the Endocrine Disruptor Screening Program. The LAGDA was designed to evaluate effects of chronic chemical exposure on growth, thy...

  7. Development of the larval amphibian growth and development assay: Effects of chronic 4-tert-octylphenol or 17ß-trenbolone exposure in Xenopus laevis from embryo to juvenile

    EPA Science Inventory

    The Larval Amphibian Growth and Development Assay (LAGDA) is a Tier II test guideline being developed by the US Environmental Protection Agency under the Endocrine Disruptor Screening Program. The LAGDA was designed to evaluate effects of chronic chemical exposure on growth, thy...

  8. Morphological, biochemical, transcriptional and epigenetic responses to fasting and refeeding in intestine of Xenopus laevis.

    PubMed

    Tamaoki, Keiji; Okada, Reiko; Ishihara, Akinori; Shiojiri, Nobuyoshi; Mochizuki, Kazuki; Goda, Toshinao; Yamauchi, Kiyoshi

    2016-01-01

    Amphibians are able to survive for several months without food. However, it is unclear what molecular mechanisms underlie their survival. To characterize the intestinal responses to fasting and refeeding, we investigated morphological, biochemical, transcriptional and epigenetic changes in the intestine from adult male Xenopus laevis. Frogs were fed for 22 days, fasted for 22 days, or fasted for 21 days and refed for 1 day. Fasting reduced, and refeeding recovered partially or fully, morphological parameters (wet weight of the intestine, circumference of the epithelial layer and number of troughs in a villus-trough unit), activities of digestive enzymes and plasma biochemical parameters (glucose, triglycerides, cholesterol and free fatty acids). Reverse transcription-quantitative polymerase chain reaction analysis revealed overall suppression of the transcript levels by fasting, with various recovery rates on refeeding. Chromatin immunoprecipitation assays on the selected genes whose transcript levels declined with fasting and recovered quickly with refeeding, showed several euchromatin marks in histone (acetylation and methylation) and RNA polymerase II modifications (phosphorylation) with fasting, and returned to the feeding levels by refeeding. The mRNA levels of these genes responded to fasting and refeeding to greater extents than did the pre-mRNA levels, suggesting the involvement of post-transcriptional regulation. Our results demonstrate that the X. laevis intestine may undergo overall metabolic suppression at least at the transcriptional level to save energy during fasting and quickly recovered to moderate nutritional deficiency by refeeding, and suggest that these dietary responses of the intestine are epigenetically and post-transcriptionally regulated.

  9. Extinction of an introduced warm-climate alien species, Xenopus laevis, by extreme weather events.

    PubMed

    Tinsley, Richard C; Stott, Lucy C; Viney, Mark E; Mable, Barbara K; Tinsley, Matthew C

    Invasive, non-native species represent a major threat to biodiversity worldwide. The African amphibian Xenopus laevis is widely regarded as an invasive species and a threat to local faunas. Populations originating at the Western Cape, South Africa, have been introduced on four continents, mostly in areas with a similar Mediterranean climate. Some introduced populations are also established in cooler environments where persistence for many decades suggests a capacity for long-term adaptation. In these cases, recent climate warming might enhance invasion ability, favouring range expansion, population growth and negative effects on native faunas. In the cool temperate UK, populations have been established for about 50 years in Wales and for an unknown period, probably >20 years, in England (Lincolnshire). Our field studies over 30 and 10 years, respectively, show that in favourable conditions there may be good recruitment, fast individual growth rates and large body size; maximum longevity exceeds 23 years. Nevertheless, areas of distribution remained limited, with numbers <500 in each population. In 2010, only a single individual was captured at each locality and further searching failed to record any others in repeated sampling up to 2014. We conclude that both populations are now extinct. The winters of 2009-2010 and 2010-2011 experienced extreme cold and drought (December 2010 was the coldest in 120 years and the third driest in 100 years). The extinction of X. laevis in these areas indicates that even relatively long-established alien species remain vulnerable to rare extreme weather conditions.

  10. Cutaneous nitrogen excretion in the African clawed frog Xenopus laevis: effects of high environmental ammonia (HEA).

    PubMed

    Cruz, Melissa J; Sourial, Mary M; Treberg, Jason R; Fehsenfeld, Sandra; Adlimoghaddam, Aida; Weihrauch, Dirk

    2013-07-15

    Ammonia is a highly toxic molecule and often introduced in considerable amounts into aquatic environments due to anthropogenic activities. Many aquatic and semi-aquatic amphibians utilize, in addition to their kidneys, the skin for osmoregulation and nitrogen excretion. In the present study the effects of prolonged (7-21 days) exposure to high environmental ammonia (HEA, 1 mmol l(-1) NH4Cl) on cutaneous nitrogen excretion and gene expression of key-transporters involved in nitrogen excretion and acid-base regulation were investigated in the fully aquatic African clawed frog, Xenopus laevis. The study revealed that X. laevis excretes predominately ammonia of which approximately 50% is excreted via the skin. Both the ventral and dorsal skin were capable to generate a net ammonia efflux, which was significantly activated by 10 mmol l(-1) of the phosphodiesterase blocker theophylline. The obtained data further suggest that the ammonia efflux was promoted by an acidification of the unstirred boundary layer, likely generated by an apical localized V-ATPase, with NH3 being transported via cutaneous expressed ammonia transporters, Rhbg and Rhcg. Prolonged HEA exposure did significantly reduce the net-flux rates over the ventral skin with Vmax changing from 256 nmol cm(-2) h(-1) in control frogs to 196 nmol cm(-2) h(-1) in HEA exposed animals. Further, prolonged HEA exposure caused a decrease in mRNA expression levels of the ammonia transporter Rhbg, Na(+)/K(+)-ATPase (α-subunit) and V-ATPase (subunit H) in the ventral and dorsal skin and the kidney. In contrast, Rhcg expression levels were unaffected by HEA in skin tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Transcriptional changes in African clawed frogs (Xenopus laevis) exposed to 17α-ethynylestradiol during early development.

    PubMed

    Tompsett, Amber R; Higley, Eric; Pryce, Sara; Giesy, John P; Hecker, Markus; Wiseman, Steve

    2015-03-01

    Although the past two decades have witnessed a significant increase in the number of studies investigating effects of estrogenic chemicals on amphibians, to date little is known about specific molecular interactions of estrogens with the hypothalamus-pituitary-gonadal-hepatic axis in developing amphibians. Here, tissue-specific functional sets of genes, derived previously from studies of fishes exposed to endocrine active chemicals, were evaluated in Xenopus laevis exposed to 17α-ethynylestradiol (EE2) throughout their early development. Specifically, transcriptional responses of X. laevis exposed to 0.09, 0.84, or 8.81 µg EE2/L were characterized during sexual differentiation [31 day post hatch (dph)] and after completion of metamorphosis during the juvenile stage (89 dph). While at 31 dph there were no consistent effects of EE2 on abundances of transcripts,at 89 dph X. laevis exhibited significant alterations in expression of genes involved in steroid signaling and metabolism, synthesis of cholesterol, and vitellogenesis. Specifically, expression of androgen receptor, farnesyl diphosphate synthase, estrogen receptor α, and vitellogenin A2 was significantly greater (>2-fold) than in controls while expression of farnesoid x-activated receptors α and β was significantly less (>2-fold reduction) than in controls. These results support the hypothesis that sets of genes derived from studies in teleost fish can be extrapolated for use in amphibians during the juvenile stage but not in sexually undifferentiated individuals. Furthermore, changes in abundances of transcripts of the here utilized sets of genes in animals sampled post sexual differentiation were in accordance with developmental effects and alterations of gonadal histology reported in a parallel study. This set of genes might be useful for predicting potential adverse outcomes at later life-stages.

  12. Tissue-specific selenium accumulation and toxicity in adult female Xenopus laevis chronically exposed to elevated dietary selenomethionine.

    PubMed

    Massé, Anita J; Muscatello, Jorgelina R; Hogan, Natacha S; Janz, David M

    2017-04-01

    Selenium (Se) is a developmental toxicant that is also capable of altering the bioenergetic and endocrine status of adult fish. To date, aquatic ecotoxicological research has predominantly focused on the toxic effects of Se in fish, and minimal information has been published related to amphibians. The objective of the present study was to investigate the potential toxicity associated with chronically elevated dietary Se consumption in adult female amphibians utilizing the model species Xenopus laevis. Adult X. laevis females were fed a diet augmented with L-selenomethionine at measured concentrations of 0.7 µg Se/g (control), 10.9 µg Se/g, 30.4 µg Se/g, or 94.2 µg Se/g dry mass for 68 d, after which they were bred with untreated males. Ovary, egg, liver, muscle, and blood samples were collected from female frogs after completion of the exposure period and subsequent breeding to ascertain Se tissue distribution, muscle and liver triglyceride and glycogen levels, and plasma cortisol concentrations. The concentrations of Se measured in female tissues excluding the liver were significantly increased in proportion with dietary intake. No significant differences were observed among treatment groups with respect to biometric indices, energy stores, or stress response of adult female X. laevis after Se exposure, which suggests that this amphibian species is capable of accumulating substantial quantities of this element in their tissues with no adverse effects on fitness. Environ Toxicol Chem 2017;36:1047-1055. © 2016 SETAC. © 2016 SETAC.

  13. Quantifying calcium fluxes underlying calcium puffs in Xenopus laevis oocytes

    PubMed Central

    Bruno, Luciana; Solovey, Guillermo; Ventura, Alejandra C.; Dargan, Sheila; Dawson, Silvina Ponce

    2010-01-01

    Summary We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al., 2005 [1]. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca2+ release comes from a region of width ~ 450 nm, that the release duration is peaked around 18ms and that the underlying Ca2+ currents range between 0.12 and 0.95pA. All these parameters are independent of IP3 concentration. We explore what distributions of channels that open during a puff, Np, and what relations between current and number of open channels, I(Np), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al, 2006 [2]. To this end, we use simple “mean field” models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I(Np) ~Np for small Np and I(Np)~Np1/α (α>1) for large Np, provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of ~400 puffs obtained using the dye Oregon green. PMID:20097419

  14. Composite transposable elements in the Xenopus laevis genome.

    PubMed Central

    Garrett, J E; Knutzon, D S; Carroll, D

    1989-01-01

    Members of two related families of transposable elements, Tx1 and Tx2, were isolated from the genome of Xenopus laevis and characterized. In both families, two versions of the elements were found. The smaller version in each family (Tx1d and Tx2d) consisted largely of two types of 400-base-pair tandem internal repeats. These elements had discrete ends and short inverted terminal repeats characteristic of mobile DNAs that are presumed to move via DNA intermediates, e.g., Drosophila P and maize Ac elements. The longer versions (Tx1c and Tx2c) differed from Tx1d and Tx2d by the presence of a 6.9-kilobase-pair internal segment that included two long open reading frames (ORFs). ORF1 had one cysteine-plus-histidine-rich sequence of the type found in retroviral gag proteins. ORF2 showed more substantial homology to retroviral pol genes and particularly to the analogs of pol found in a subclass of mobile DNAs that are supposed retrotransposons, such as mammalian long interspersed repetitive sequences, Drosophila I factors, silkworm R1 elements, and trypanosome Ingi elements. Thus, the Tx1 elements present a paradox by exhibiting features of two classes of mobile DNAs that are thought to have very different modes of transposition. Two possible resolutions are considered: (i) the composite versions are actually made up of two independent elements, one of the retrotransposon class, which has a high degree of specificity for insertion into a target within the other, P-like element; and (ii) the composite elements are intact, autonomous mobile DNAs, in which the pol-like gene product collaborates with the terminal inverted repeats to cause transposition of the entire unit. Images PMID:2550791

  15. Histochemical identification of sialylated glycans in Xenopus laevis testis

    PubMed Central

    Valbuena, Galder; Alonso, Edurne; Ubago, María Martínez; Madrid, Juan Francisco; Díaz-Flores, Lucio; Sáez, Francisco José

    2012-01-01

    Carbohydrate chains of glycoprotein and glycosphingolipids are highly diverse molecules involved in many cell functions, including cell recognition, adhesion and signalling. Sialylated glycans are of special interest because the terminal position of sialic acid (NeuAc) in glycans linked by different ways to subterminal monosaccharides has been shown to be involved in several biological processes, as occurs with gangliosides, which have been reported as being essential in spermatogenesis in mammals. Some glycan-binding proteins, the lectins, which specifically recognize glycan sequences, have been extensively used to characterize tissue and cell carbohydrates by means of cytochemical techniques. The aim of the present work was to determine the presence of NeuAc by means of histochemical techniques in the testis of Xenopus laevis, an animal model widely used in cell and molecular biology research. However, considering that some NeuAc-binding lectins are capable of binding to N-acetylglucosamine (GlcNAc), other GlcNAc-binding lectins were also assayed. The results showed that NeuAc is mainly expressed in the interstitium, and only a weak labelling in the male germ cells was observed. Most NeuAc was located in O-linked oligosaccharides, but some masked NeuAc in N-glycans were identified in primary and secondary spermatogonia and spermatocytes. By contrast, GlcNAc was widely expressed in all germ cell types. Deglycosylative pre-treatments suggest that both N- and O-glycans and/or glycolipids could be responsible for this labelling. In addition, GlcNAc in O-linked oligosaccharides has been identified in spermatogonial cells. The acrosome of spermatids was always negative. Variations of glycan expression have been found in different cell types, suggesting that glycosylation is modified during spermatogenetic development. PMID:22881213

  16. Atrazine and malathion shorten the maturation process of Xenopus laevis oocytes and have an adverse effect on early embryo development.

    PubMed

    Ji, Qichao; Lee, Jessica; Lin, Yu-Huey; Jing, Guihua; Tsai, L Jillianne; Chen, Andrew; Hetrick, Lindsay; Jocoy, Dylan; Liu, Junjun

    2016-04-01

    The use of pesticides has a negative impact on the environment. Amphibians have long been regarded as indicator species to pollutants due to their permeable skin and sensitivity to the environment. Studies have shown that population declines of some amphibians are directly linked with exposure to agricultural contaminants. In the past, much of the studies have focused on the toxic effect of contaminants on larvae (tadpoles), juvenile and adult frogs. However, due to the nature of their life cycle, amphibian eggs and early embryos are especially susceptible to the contaminants, and any alteration during the early reproductive stages may have a profound effect on the health and population of amphibians. In this study, we analyzed the effect of atrazine and malathion, two commonly used pesticides, on Xenopus laevis oocyte maturation and early embryogenesis. We found that both atrazine and malathion shortened the frog oocyte maturation process and resulted in reduced Emi2 levels at cytostatic factor-mediated metaphase arrest, and a high level of Emi2 is critically important for oocyte maturation. Furthermore, frog embryos fertilized under the influence of atrazine and/or malathion displayed a higher rate of abnormal division that eventually led to embryo death during early embryogenesis.

  17. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis

    PubMed Central

    Peña-Villalobos, Isaac; Narváez, Cristóbal

    2016-01-01

    ABSTRACT Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR) and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level) in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group) and hyper-osmotic (340 mOsm NaCl; HYP group) environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis. PMID:27334694

  18. A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis

    PubMed Central

    Golding, Anne; Levin, Michael; Kaplan, David L.

    2016-01-01

    In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis) model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device’s observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies. PMID:27257960

  19. Oral immunization of the African clawed frog (Xenopus laevis) upregulates the mucosal immunoglobulin IgX

    PubMed Central

    Du, Christina C.; Mashoof, Sara M.; Criscitiello, Michael F.

    2011-01-01

    The frog Xenopus laevis is a model species for developmental biology but is also of significant interest to comparative immunologists. Amphibians are the oldest group of organisms in which both the B lymphocytes of some species undergo immunoglobulin (Ig) class switch recombination and also have a dedicated mucosal Ig isotype. The purpose of this study was to test the hypothesis that frog IgX would be produced in response to oral immunization. In order to facilitate studies of humoral, and especially mucosal immunity, in this model species, we developed a gavage technique for oral immunization. The result of this oral administration of antigen to frogs was assayed by the induction of the mucosal antibody isotype, IgX, in plasma by enzyme linked immunosorbant assay (ELISA), and a significant IgX upregulation was detected compared to frogs receiving systemic immunization into the coelom. These data are consistent with the view that IgX is the functional analog of mammalian IgA and mandate further studies of the relationship between IgX and IgA. Additionally, the gavage technique should be adaptable for functional studies of gut-associated immunology in other small aquatic vertebrates. PMID:22100190

  20. Nuclear Receptor Corepressor Recruitment by Unliganded Thyroid Hormone Receptor in Gene Repression during Xenopus laevis Development

    PubMed Central

    Sachs, Laurent M.; Jones, Peter L.; Havis, Emmanuelle; Rouse, Nicole; Demeneix, Barbara A.; Shi, Yun-Bo

    2002-01-01

    Thyroid hormone receptors (TR) act as activators of transcription in the presence of the thyroid hormone (T3) and as repressors in its absence. While many in vitro approaches have been used to study the molecular mechanisms of TR action, their physiological relevance has not been addressed. Here we investigate how TR regulates gene expression during vertebrate postembryonic development by using T3-dependent amphibian metamorphosis as a model. Earlier studies suggest that TR acts as a repressor during premetamorphosis when T3 is absent. We hypothesize that corepressor complexes containing the nuclear receptor corepressor (N-CoR) are key factors in this TR-dependent gene repression, which is important for premetamorphic tadpole growth. To test this hypothesis, we isolated Xenopus laevis N-CoR (xN-CoR) and showed that it was present in pre- and metamorphic tadpoles. Using a chromatin immunoprecipitation assay, we demonstrated that xN-CoR was recruited to the promoters of T3 response genes during premetamorphosis and released upon T3 treatment, accompanied by a local increase in histone acetylation. Furthermore, overexpression of a dominant-negative N-CoR in tadpole tail muscle led to increased transcription from a T3-dependent promoter. Our data indicate that N-CoR is recruited by unliganded TR to repress target gene expression during premetamorphic animal growth, an important process that prepares the tadpole for metamorphosis. PMID:12446772

  1. Calcium channel kinetics of melanotrope cells in Xenopus laevis depend on environmental stimulation.

    PubMed

    Zhang, Hongyan; Langeslag, Michiel; Breukels, Vincent; Jenks, Bruce G; Roubos, Eric W; Scheenen, Wim J J M

    2008-03-01

    We have tested the hypothesis that the type and kinetics of voltage-activated Ca(2+) channels in a neuroendocrine cell depend on the cell's long-term external input. For this purpose, the presence and kinetics of both low (LVA) and high-voltage-activated (HVA) L-type Ca(2+) channels have been assessed in melanotrope pituitary cells of the amphibian Xenopus laevis. The secretory activity of this cell type can readily be manipulated in vivo by changing the animal's environmental light condition, from a black to a white background. We here show that, compared to white background-adapted Xenopus, melanotropes from black background-adapted frogs have (1) a much larger size, as revealed by their 2.5 times larger membrane capacitance (P<0.001), (2) a 2 times higher HVA current density (P<0.05), (3) a clearly smaller Ca(2+)-dependent inactivation (10%; P<0.05), (4) L-type channels with 5 times slower activation and inactivation kinetics (P<0.05), and (5) slower kinetics of L-type channels that become faster and more similar to those in white-background adapted cells when the intracellular Ca(2+)-buffering capacity is reduced. Furthermore, white-adapted melanotropes possess LVA-type Ca(2+) channels, which are lacking from cells from black-adapted animals. The melanotrope calmodulin mRNA level does not differ between the two adaptation states. These results indicate that HVA L-type channel kinetics differ in relation to environmentally induced changes in cellular secretory state, probably mediated via intracellular Ca(2+)-buffering, whereas the occurrence of LVA Ca(2+) channels may depend on environmentally controlled channel gene expression.

  2. Innate immune responses and permissiveness to ranavirus infection of peritoneal leukocytes in the frog Xenopus laevis.

    PubMed

    Morales, Heidi D; Abramowitz, Lara; Gertz, Jacqueline; Sowa, Jessica; Vogel, Ashley; Robert, Jacques

    2010-05-01

    Ranaviruses such as frog virus 3 ([FV3] family Iridoviridae) are increasingly prevalent pathogens that infect reptiles, amphibians, and fish worldwide. Whereas studies in the frog Xenopus laevis have revealed the critical involvement of CD8 T-cell and antibody responses in host resistance to FV3, little is known about the role played by innate immunity to infection with this virus. We have investigated the occurrence, composition, activation status, and permissiveness to infection of peritoneal leukocytes (PLs) in Xenopus adults during FV3 infection by microscopy, flow cytometry, and reverse transcription-PCR. The total number of PLs and the relative fraction of activated mononucleated macrophage-like cells significantly increase as early as 1 day postinfection (dpi), followed by NK cells at 3 dpi, before the peak of the T-cell response at 6 dpi. FV3 infection also induces a rapid upregulation of proinflammatory genes including arginase 1, interleukin-1beta, and tumor necrosis factor alpha. Although PLs are susceptible to FV3 infection, as evidenced by apoptotic cells, active FV3 transcription, and the detection of viral particles by electron microscopy, the infection is weaker (fewer infectious particles), more transitory, and involves a smaller fraction (less than 1%) of PLs than the kidney, the main site of infection. However, viral DNA remains detectable in PLs for at least 3 weeks postinfection, past the point of viral clearance observed in the kidneys. This suggests that although PLs are actively involved in anti-FV3 immune responses, some of these cells can be permissive and harbor quiescent, asymptomatic FV3.

  3. Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis

    PubMed Central

    Moreno, Nerea; González, Agustín

    2017-01-01

    The complexity of the pallium during evolution has increased dramatically in many different respects. The highest level of complexity is found in mammals, where most of the pallium (cortex) shows a layered organization and neurons are generated during development following an inside-out order, a sequence not observed in other amniotes (birds and reptiles). Species-differences may be related to major neurogenetic events, from the neural progenitors that divide and produce all pallial cells. In mammals, two main types of precursors have been described, primary precursor cells in the ventricular zone (vz; also called radial glial cells or apical progenitors) and secondary precursor cells (called basal or intermediate progenitors) separated from the ventricle surface. Previous studies suggested that pallial neurogenetic cells, and especially the intermediate progenitors, evolved independently in mammalian and sauropsid lineages. In the present study, we examined pallial neurogenesis in the amphibian Xenopus laevis, a representative species of the only group of tetrapods that are anamniotes. The pattern of pallial proliferation during embryonic and larval development was studied, together with a multiple immunohistochemical analysis of putative progenitor cells. We found that there are two phases of progenitor divisions in the developing pallium that, following the radial unit concept from the ventricle to the mantle, finally result in an outside-in order of mature neurons, what seems to be the primitive condition of vertebrates. Gene expressions of key transcription factors that characterize radial glial cells in the vz were demonstrated in Xenopus. In addition, although mitotic cells were corroborated outside the vz, the expression pattern of markers for intermediate progenitors differed from mammals. PMID:28396626

  4. Immune Defenses against Batrachochytrium dendrobatidis, a Fungus Linked to Global Amphibian Declines, in the South African Clawed Frog, Xenopus laevis▿

    PubMed Central

    Ramsey, Jeremy P.; Reinert, Laura K.; Harper, Laura K.; Woodhams, Douglas C.; Rollins-Smith, Louise A.

    2010-01-01

    Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections. PMID:20584973

  5. The colloidal thyroxine (T4) ring as a novel biomarker of perchlorate exposure in the African clawed frog Xenopus laevis

    USGS Publications Warehouse

    Hu, F.; Sharma, Bibek; Mukhi, S.; Patino, R.; Carr, J.A.

    2006-01-01

    The purpose of this study was to determine if changes in colloidal thyroxine (T4) immunoreactivity can be used as a biomarker of perchlorate exposure in amphibian thyroid tissue. Larval African clawed frogs (Xenopus laevis) were exposed to 0, 1, 8, 93, and 1131 ??g perchlorate/l for 38 and 69 days to cover the normal period of larval development and metamorphosis. The results of this study confirmed the presence of an immunoreactive colloidal T4 ring in thyroid follicles of X. laevis and demonstrated that the intensity of this ring is reduced in a concentration-dependent manner by perchlorate exposure. The smallest effective concentration of perchlorate capable of significantly reducing colloidal T4 ring intensity was 8 ??g perchlorate/l. The intensity of the immunoreactive colloidal T4 ring is a more sensitive biomarker of perchlorate exposure than changes in hind limb length, forelimb emergence, tail resorption, thyrocyte hypertrophy, or colloid depletion. We conclude that the colloidal T4 ring can be used as a sensitive biomarker of perchlorate-induced thyroid disruption in amphibians. ?? Copyright 2006 Oxford University Press.

  6. Effects of low dose endosulfan exposure on brain neurotransmitter levels in the African clawed frog Xenopus laevis.

    PubMed

    Preud'homme, Valérie; Milla, Sylvain; Gillardin, Virginie; De Pauw, Edwin; Denoël, Mathieu; Kestemont, Patrick

    2015-02-01

    Understanding the impact of pesticides in amphibians is of growing concern to assess the causes of their decline. Among pesticides, endosulfan belongs to one of the potential sources of danger because of its wide use and known effects, particularly neurotoxic, on a variety of organisms. However, the effect of endosulfan was not yet evaluated on amphibians at levels encompassing simultaneously brain neurotransmitters and behavioural endpoints. In this context, tadpoles of the African clawed frog Xenopus laevis were submitted to four treatments during 27 d: one control, one ethanol control, and two low environmental concentrations of endosulfan (0.1 and 1 μg L(-1)). Endosulfan induced a significant increase of brain serotonin level at both concentrations and a significant increase of brain dopamine and GABA levels at the lower exposure but acetylcholinesterase activity was not modified by the treatment. The gene coding for the GABA transporter 1 was up-regulated in endosulfan contaminated tadpoles while the expression of other genes coding for the neurotransmitter receptors or for the enzymes involved in their metabolic pathways was not significantly modified by endosulfan exposure. Endosulfan also affected foraging, and locomotion in links with the results of the physiological assays, but no effects were seen on growth. These results show that low environmental concentrations of endosulfan can induce adverse responses in X. laevis tadpoles. At a broader perspective, this suggests that more research using and linking multiple markers should be used to understand the complex mode of action of pollutants.

  7. Effects of 17α-ethynylestradiol on sexual differentiation and development of the African clawed frog (Xenopus laevis).

    PubMed

    Tompsett, Amber R; Wiseman, Steve; Higley, Eric; Pryce, Sara; Chang, Hong; Giesy, John P; Hecker, Markus

    2012-11-01

    Several studies have shown that exposure of amphibians, including the African clawed frog (Xenopus laevis), to potent estrogens at critical times during development results in feminization and/or demasculinization. However, genotyping of X. laevis has only recently become possible, so studies performed in the past were rarely able to make explicit linkages between genetic and phenotypic sex. Therefore, to further characterize this relationship, X. laevis tadpoles were exposed during development to 0.09, 0.84, or 8.81 μg/L 17α-ethynylestradiol (EE2), which is the estrogen analog commonly used in oral contraceptives. Exposure to all concentrations of EE2 tested resulted in significant delays in time to metamorphosis. Genotyping showed that genetic sex ratios were similar among treatments. However, morphological evaluation revealed that a significant number of individuals with a male genotype displayed mixed sex and abnormal phenotypes. Additionally, both genetic males and females exposed to EE2 exhibited greater presence of vitellogenin protein relative to the respective controls. Since estrogens function downstream of the initial molecular signals of sexual differentiation, it is likely that genetic male animals received mixed endogenous male and exogenous female signals that caused disordered sexual development. The production of vitellogenin was probably temporally separated and independent from primary effects on sexual differentiation, and might have contributed to delays in metamorphosis observed in individuals exposed to EE2. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis.

    PubMed

    Matsuda, Yoichi; Uno, Yoshinobu; Kondo, Mariko; Gilchrist, Michael J; Zorn, Aaron M; Rokhsar, Daniel S; Schmid, Michael; Taira, Masanori

    2015-01-01

    Xenopus laevis (XLA) is an allotetraploid species which appears to have undergone whole-genome duplication after the interspecific hybridization of 2 diploid species closely related to Silurana/Xenopus tropicalis (XTR). Previous cDNA fluorescence in situ hybridization (FISH) experiments have identified 9 sets of homoeologous chromosomes in X. laevis, in which 8 sets correspond to chromosomes 1-8 of X. tropicalis (XTR1-XTR8), and the last set corresponds to a fusion of XTR9 and XTR10. In addition, recent X. laevis genome sequencing and BAC-FISH experiments support this physiological relationship and show no gross chromosome translocation in the X. laevis karyotype. Therefore, for the benefit of both comparative cytogenetics and genome research, we here propose a new chromosome nomenclature for X. laevis based on the phylogenetic relationship and chromosome length, i.e. XLA1L, XLA1S, XLA2L, XLA2S, and so on, in which the numbering of XLA chromosomes corresponds to that in X. tropicalis and the postfixes 'L' and 'S' stand for 'long' and 'short' chromosomes in the homoeologous pairs, which can be distinguished cytologically by their relative size. The last chromosome set is named XLA9L and XLA9S, in which XLA9 corresponds to both XTR9 and XTR10, and hence, to emphasize the phylogenetic relationship to X. tropicalis, XLA9_10L and XLA9_10S are also used as synonyms.

  9. Fertilization and development of eggs of the South African clawed toad, Xenopus laevis, on sounding rockets in space.

    PubMed

    Ubbels, G A; Berendsen, W; Kerkvliet, S; Narraway, J

    1992-01-01

    Egg rotation and centrifugation experiments strongly suggest a role for gravity in the determination of the spatial structure of amphibian embryos. Decisive experiments can only be made in Space. Eggs of Xenopus laevis, the South African clawed toad, were the first vertebrate eggs which were successfully fertilized on Sounding Rockets in Space. Unfixed, newly fertilized eggs survived reentry, and a reasonable number showed a seemingly normal gastrulation but died between gastrulation and neurulation. Only a few reached the larval stage, but these developed abnormally. In the future, we intend to test whether this abnormal morphogenesis is due to reentry perturbations, or due to a real microgravity effect, through perturbation of the reinitiation of meiosis and other processes, or started by later sperm penetration.

  10. Fertilization and development of eggs of the South African clawed toad, Xenopus laevis, on sounding rockets in space

    NASA Astrophysics Data System (ADS)

    Ubbels, Geertje A.; Berendsen, Willem; Kerkvliet, Sonja; Narraway, Jenny

    Egg rotation and centrifugation experiments strongly suggest a role for gravity in the determination of the spatial structure of amphibian embryos. Decisive experiments can only be made in Space. Eggs of Xenopus laevis, the South African clawed toad, were the first vertebrate eggs which were successfully fertilized on Sounding Rockets in Space. Unfixed, newly fertilized eggs survived reentry, and a reasonable number showed a seemingly normal gastrulation but died between gastrulation and neurulation. Only a few reached the larval stage, but these developed abnormally. In the future, we inted to test whether this abnormal morphogenesis is due to reentry perturbations, or due to a real microgravity effect, through perturbation of the reinitiation of meiosis and other processes, or started by later sperm penetration.

  11. A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin.

    PubMed

    Hoffmann, W; Richter, K; Kreil, G

    1983-01-01

    A variety of peptides closely related to mammalian hormones and neurotransmitters are secreted from amphibian skin. Using cDNA clones of mRNA isolated from skin of Xenopus laevis, we have been searching for precursors of some of these constituents. Here we present the sequences of parts of cloned mRNAs which code for precursors of a novel peptide. In the predicted polypeptides, pairs of basic residues flank a sequence of 25 amino acids terminating with glycine, the signal for the formation of a terminal amide. The predicted final product liberated from these precursors would be a peptide comprised of 24 amino acids starting with tyrosine and ending with leucine amide, which has therefore been designated PYLa. This peptide can form an amphipathic helix similar to that found in peptides with cytotoxic, bacteriostatic and/or lytic properties.

  12. Neural transduction in Xenopus laevis lateral line system.

    PubMed

    Strelioff, D; Honrubia, V

    1978-03-01

    1. The process of neural excitation in hair cell systems was studied in an in vitro preparation of the Xenopus laevis (African clawed toad) lateral line organ. A specially designed stimulus chamber was used to apply accurately controlled pressure, water movement, or electrical stimuli, and to record the neural responses of the two afferent fibers innervating each organ or stitch. The objective of the study was to determine the characteristics of the neural responses to these stimuli, and thus gain insight into the transduction process. 2. A sustained deflection of the hair cell cilia due to a constant flow of water past the capula resulted in a maintained change in the mean firing rate (MFR) of the afferent fibers. The data also demonstrated that the neural response was proportional to the velocity of the water flow and indicated that both deflection and movement of the cilia were the effective physiological stimuli for this hair cell system. 3. The preparations responded to sinusoidal water movements (past the capula) over the entire frequency range of the stimulus chamber, 0.1-130 Hz, and were most sensitive between 10 and 40 Hz. The variation of the MFR and the percent modulation indicated that the average dynamic range of each organ was 23.5 dB. 4. The thresholds, if any, for sustained pressure changes and for sinusoidal pressure variations in the absence of water movements were very high. Due to the limitations of the stimulus chamber it was not possible to generate pressure stimuli of sufficient magnitude to elicit a neural response without also generating suprathreshold water-movement stimuli. Sustained pressures had no detectable effect on the neural response to water-movement stimuli. 5. The preparations were very sensitive to electrical potentials applied across the toad skin on which the hair cells were located. Potentials which made the ciliated surfaces of the hair cells positive with respect to their bases increased the MFR of the fibers, whereas

  13. Regulation of cyclin E stability in Xenopus laevis embryos

    NASA Astrophysics Data System (ADS)

    Brandt-(Webb), Yekaterina

    Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus

  14. Endocrine effects of environmental pollution on Xenopus laevis and Rana temporaria.

    PubMed

    Bögi, C; Schwaiger, J; Ferling, H; Mallow, U; Steineck, C; Sinowatz, F; Kalbfus, W; Negele, R D; Lutz, I; Kloas, W

    2003-10-01

    To determine the capacity of sewage treatment work effluents to disrupt the endocrine system under semifield conditions, two amphibian species, Xenopus laevis and Rana temporaria, were exposed to the effluent of a regional sewage treatment plant in South Bavaria during larval development until completion of metamorphosis. Exposure was carried out in river water (Würm) as a reference, and a 1:12-mixture sewage effluent representing the real situation on the spot, and in a higher concentration of sewage using a 1:2 mixture. An accidental impact of industrial wastewater into the reference and dilution medium, Würm, which was caused by a spate in the respective area during the sensitive period of sex differentiation of amphibian larvae, is assumed to be responsible for the relatively high percentage of females observed by histological analysis in all treatment groups. All of these values were higher than those determined in controls exposed to artificial tap water in laboratory experiments conducted in a comparable study design. Sex ratios between species, revealed by the semifield study with decreasing portions of females from control to 1:12 to 1:2, were strongly correlated. Determination of biomarker-mRNA-levels in Xenopus liver using semiquantitative RT-PCR at the end of the experimental phase, when exposure regime has turned into the initially expected situation with the highest load of potential estrogens in the effluent, followed by 1:2 and 1:12 mixture, resulted in a significant increase of Vitellogenin-mRNA in female juveniles exposed to the highest portion of sewage, whereas expression of both androgen and estrogen receptor-mRNA showed no clear differences. The results concerning the induction of estrogenic biomarkers are in accordance with our findings for estrogen receptor binding of sample extracts from the Würm and sewage taken in parallel at the end of the experiment, when sewage extracts possessed a much higher ability to displace [3H]estradiol from

  15. Effects of the biocide methylisothiazolinone on Xenopus laevis wound healing and tail regeneration.

    PubMed

    Delos Santos, Nicole; Azmat, Summer; Cuenca, Yesenia; Drenth, Jessica; Lauper, Julia; Tseng, Ai-Sun

    2016-12-01

    The South African clawed frog, Xenopus laevis, has a strong history as a suitable model for environmental studies. Its embryos and transparent tadpoles are highly sensitive to the environment and their developmental processes are well described. It is also amenable for molecular studies. These characteristics enable its use for rapid identification and understanding of exposure-induced defects. To investigate the consequences of chemical exposure on aquatic animals, Xenopus laevis embryos and tadpoles were exposed to the biocide, methylisothiazolinone (MIT). Frog tadpoles exposed to MIT following tail amputation lost their natural regenerative ability. This inhibition of regeneration led to a failure to regrow tissues including the spinal cord, muscle, and notochord. This MIT-dependent regenerative defect is due to a failure to close the amputation wound. A wound healing assay revealed that while untreated embryos close their wounds within one day after injury, MIT-treated animals maintained open wounds that did not reduce in size and caused lethality. Concomitant exposure of MIT with chemicals containing thiol groups such as glutathione and N-acetyl cysteine restored normal wound healing and regeneration responses in tadpoles. Together these results indicate that exposure to MIT impairs developmental wound repair and tissue regeneration in Xenopus laevis. Thus, this study reveals new aspects of MIT activity and demonstrates that Xenopus laevis is a well-suited model for facilitating future research into chemical exposure effects on injury responses. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. cnrip1 is a regulator of eye and neural development in Xenopus laevis.

    PubMed

    Zheng, Xiaona; Suzuki, Toshiyasu; Takahashi, Chika; Nishida, Eisuke; Kusakabe, Morioh

    2015-04-01

    Cannabinoid receptor interacting protein 1 (CNRIP1), which has been originally identified as the binding partner of cannabinoid receptor 1 (CNR1), is evolutionarily conserved throughout vertebrates, but its physiological function has been unknown. Here, we identify a developmental role of CNRIP1 using Xenopus laevis embryos. During early embryogenesis, expression of Xenopus laevis cnrip1 is highly restricted to the animal region of gastrulae where neural and eye induction occur, and afterward it is seen in neural and other tissues with a temporally and spatially regulated pattern. Morpholino-mediated knockdown experiments indicate that cnrip1 has an essential role in early eye and neural development by regulating the onset of expression of key transcription factor genes, sox2, otx2, pax6 and rax. Also, over-expression experiments suggest that cnrip1 has a potential to expand sox2, otx2, pax6 and rax expression. These results suggest an instructive role of Xenopus laevis cnrip1 in early eye and neural development. Furthermore, Xenopus laevis cnr1 knockdown leads to eye defects, which are partly similar to, but milder than, those caused by cnrip1 knockdown, suggesting a possible functional similarity between CNRIP1 and CNR1. This study is the first characterization of an in vivo role of CNRIP1 in the context of whole organisms.

  17. METAMORPHIC INHIBITION OF XENOPUS LAEVIS BY SODIUM PERCHLORATE: EFFECTS ON DEVELOPMENT AND THYROID HISTOLOGY

    EPA Science Inventory

    The perchlorate anion inhibits thyroid hormone (TH) synthesis via inhibition of the sodium-iodide symporter. It is, therefore, a good model chemical to aid in the development of a bioassay to screen chemicals for effects on thyroid function. Xenopus laevis larvae were exposed to ...

  18. CONCENTRATION DEPENDENT ACCUMULATION OF [3H]-DELTAMETHRIN IN XENOPUS LAEVIS OOCYTES.

    EPA Science Inventory

    Pyrethroid insecticides such as deltamethrin have been demonstrated to target and disrupt voltage-sensitive sodium channels (VSSCs). VSSCs were expressed in Xenopus laevis oocytes and used to study the effects of deltamethrin on VSSCs. This study evaluated the amount of deltameth...

  19. Chronic sublethal exposure to silver nanoparticles disrupts thyroid hormone signaling during Xenopus laevis metamorphosis.

    PubMed

    Carew, Amanda C; Hoque, M Ehsanul; Metcalfe, Chris D; Peyrot, Caroline; Wilkinson, Kevin J; Helbing, Caren C

    2015-02-01

    Nanoparticles (NPs) are engineered in the nanoscale (<100 nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver particles (AgNPs) are the most prevalent NPs in consumer products due to their strong antimicrobial action. While AgNP toxicity at high concentrations has been thoroughly investigated, the sublethal effects at or below regulatory guidelines are relatively unknown. Amphibian metamorphosis is mediated by thyroid hormone (TH), and initial studies with bullfrogs (Rana catesbeiana) indicate that low concentrations of AgNPs disrupt TH-dependent responses in premetamorphic tadpole tailfin tissue. The present study examined the effects of low, non-lethal, environmentally-relevant AgNP concentrations (0.018, 0.18 or 1.8 μg/L Ag; ∼10 nm particle size) on naturally metamorphosing Xenopus laevis tadpoles in two-28 day chronic exposures beginning with either pre- or prometamorphic developmental stages. Asymmetric flow field flow fractionation with online inductively coupled plasma mass spectrometry and nanoparticle tracking analysis indicated a mixture of single AgNPs with homo-agglomerates in the exposure water with a significant portion (∼30-40%) found as dissolved Ag. Tadpoles bioaccumulated AgNPs and displayed transient alterations in snout/vent and hindlimb length with AgNP exposure. Using MAGEX microarray and quantitative real time polymerase chain reaction transcript analyses, AgNP-induced disruption of five TH-responsive targets was observed. The increased mRNA abundance of two peroxidase genes by AgNP exposure suggests the presence of reactive oxygen species even at low, environmentally-relevant concentrations. Furthermore, differential responsiveness to AgNPs was observed at each developmental stage. Therefore, low concentrations of AgNPs had developmental stage-specific endocrine disrupting effects during TH-dependent metamorphosis.

  20. Induction of cardiac muscle differentiation in isolated animal pole explants of Xenopus laevis embryos.

    PubMed

    Logan, M; Mohun, T

    1993-07-01

    We have isolated a cDNA fragment encoding a portion of the myosin heavy chain alpha-isoform (XMHC alpha) in the amphibian, Xenopus laevis. The XMHC alpha transcript is highly enriched in adult heart RNA and is expressed exclusively in embryonic heart tissue. It therefore provides a tissue-specific marker for cardiac muscle differentiation during early embryogenesis. Using an RNAase protection assay, we can detect the onset of cardiac muscle differentiation in an anterior, ventral region of tailbud embryos, many hours before the appearance of a beating heart. Whole-mount in situ RNA hybridisation indicates that expression of the XMHC alpha gene is restricted to the developing heart primordium. XMHC alpha gene expression can also be induced in isolated animal pole explants of blastulae by treatment with the growth factor, activin A. Induction is dose-dependent, requiring high doses of the growth factor compared with that required for myotomal (skeletal) muscle differentiation. In contrast, no XMHC alpha transcripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differentiation. Activin-induced explants show a similar temporal pattern of XMHC alpha gene expression to that found in normal embryogenesis. Furthermore, cells expressing this gene appear clustered in one or two foci within fused explant aggregates, which often show regular, spontaneous contractions after several days in culture. These results show that terminal differentiation of cardiac muscle can occur in growth factor-induced explants and may be distinguished from skeletal muscle differentiation by the dose and nature of the inducing factor.

  1. CONCENTRATION DEPENDENT ACCUMULATION OF [3H]-DELTAMETHRIN IN SODIUM CHANNEL N AV1.2 EXPRESSING XENOPUS LAEVIS OOCYTES.

    EPA Science Inventory

    Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown....

  2. CONCENTRATION DEPENDENT ACCUMULATION OF [3H]-DELTAMETHRIN IN SODIUM CHANNEL N AV1.2 EXPRESSING XENOPUS LAEVIS OOCYTES.

    EPA Science Inventory

    Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown....

  3. Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis.

    PubMed

    Lutz, Ilka; Jie, Zhang; Opitz, Robert; Kloas, Werner; Ying, Xu; Menzel, Ralph; Steinberg, Christian E W

    2005-12-01

    According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS1500 was applied. The in vivo results showed that HS1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSHbeta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals.

  4. Environmentally relevant concentrations of ammonium perchlorate inhibit thyroid function and alter sex ratios in developing Xenopus laevis.

    PubMed

    Goleman, Wanda L; Carr, James A; Anderson, Todd A

    2002-03-01

    Embryos and larvae of the South African frog Xenopus laevis were exposed to ammonium perchlorate (AP) or control medium for 70 d. The dosage levels (59 ppb, 14,140 ppb) bracketed a range of perchlorate concentrations measured in surface waters at the Longhorn Army Ammunition Plant (LHAAP) in Karnack, Texas, USA. The experiment also included a 28-d nontreatment recovery period to assess the reversibility of AP effects. There were no significant effects of AP on mortality or hatching success. There were no effects of AP on developmental abnormalities such as bent/asymmetric tails or edema. Ammonium perchlorate inhibited forelimb emergence, the percentage of animals completing tail resorption, and hindlimb development during the 70-d exposure period. Only the upper AP concentration reduced whole-body thyroxine content, whereas both concentrations caused significant hypertrophy of the thyroid follicular epithelium. Both concentrations of AP caused a skewed sex ratio, significantly reducing the percentage of males at metamorphosis. The effects of AP on metamorphosis and thyroid function were reversed during the 28-d nontreatment recovery period. We conclude that AP inhibits thyroid activity and alters gonadal differentiation in developing X. laevis. These effects were observed at concentrations at or below concentrations reported in surface waters contaminated with ammonium perchlorate, suggesting that this contaminant may pose a threat to normal development and growth in natural amphibian populations.

  5. Parasites of the African clawed frog, Xenopus laevis, in southern California, U.S.A

    USGS Publications Warehouse

    Kuperman, Boris I.; Matey, Victoria E.; Fisher, Richard N.; Ervin, Edward L.; Warburton, Manna L.; Bakhireva, Ludmila; Lehman, Cynthia A.

    2004-01-01

    A total of 230 feral African clawed frogs, Xenopus laevis, from 3 localities in southern California were examined for parasites. The following species were found: 3 species of Protozoa, Nyctotherussp., Balantidium xenopodis, Protoopalina xenopodus; 2 species of Monogenea, Protopolystoma xenopodis, Gyrdicotylus gallieni; 1 species of Digenea, Clinostomum sp. (as metacercariae); 1 species of Cestoda, Cephalochlamys namaquensis; 2 species of Nematoda, Contracaecum sp. (as larvae), Eustrongylides sp. (as larvae); and 1 species of Acanthocephala, Acanthocephalus sp. (as cystacanth). Of these, the protozoans P. xenopodus and B. xenopodis, both monogeneans, and the cestode have an African origin. Contracaecum sp., Eustrongylides sp., and Acanthocephalus sp. have not been previously reported from X. laevis.

  6. Spatiotemporal Development of the Orexinergic (Hypocretinergic) System in the Central Nervous System of Xenopus laevis.

    PubMed

    López, Jesús M; Morales, Lorena; González, Agustín

    2016-01-01

    The present immunohistochemical study represents a detailed spatiotemporal analysis of the localization of orexin-immunoreactive (OX-ir) cells and fibers throughout development in the brain of the anuran amphibian Xenopus laevis, a model frequently used in developmental studies. Anurans undergo remarkable physiological changes during the early life stages, and very little is known about the ontogeny and the localization of the centers that control functions such as appetite and feed ingestion in the developing brain. We examined the onset of the orexinergic system, demonstrated to be involved in appetite regulation, using antibodies against mammalian orexin-A and orexin-B peptides. Simultaneous detection of orexins with other territorial markers was used to assess the precise location of the orexinergic cells in the hypothalamus, analyzed within a segmental paradigm. Double staining of orexins and tyrosine hydroxylase served to evaluate possible interactions with the catecholaminergic systems. At early embryonic stages, the first OX-ir cells were detected in the hypothalamus and, soon after, long descending projections were observed through the brainstem to the spinal cord. As brain development proceeded, the double-staining techniques demonstrated that this OX-ir cell group was located in the suprachiasmatic nucleus within the alar hypothalamus. Throughout larval development, the number of OX-ir cells increased notably and a widespread fiber network that innervated the main areas of the forebrain and brainstem was progressively formed, including innervation in the posterior tubercle and mesencephalon, the locus coeruleus, and the nucleus of the solitary tract where catecholaminergic cells are present. In addition, orexinergic cells were detected in the preoptic area and the tuberal hypothalamus only at late prometamorphic stages. The final distribution pattern, largely similar to that of the adult, was achieved through metamorphic climax. The early expression of

  7. Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

    PubMed Central

    Bement, W M; Capco, D G

    1990-01-01

    Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle. Images PMID:2100203

  8. Accelerated Gene Evolution and Subfunctionalization in thePseudotetraploid Frog Xenopus Laevis

    SciTech Connect

    Hellsten, Uffe; Khokha, Mustafa K.; Grammar, Timothy C.; Harland,Richard M.; Richardson, Paul; Rokhsar, Daniel S.

    2007-03-01

    Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization {approx} 40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.

  9. Xenopus laevis neuronal cell adhesion molecule (nrcam): plasticity of a CAM in the developing nervous system.

    PubMed

    Lokapally, Ashwin; Metikala, Sanjeeva; Hollemann, Thomas

    2017-01-01

    Neuron-glial-related cell adhesion molecule (NRCAM) is a neuronal cell adhesion molecule of the L1 immunoglobulin superfamily, which plays diverse roles during nervous system development including axon growth and guidance, synapse formation, and formation of the myelinated nerve. Perturbations in NRCAM function cause a wide variety of disorders, which can affect wiring and targeting of neurons, or cause psychiatric disorders as well as cancers through abnormal modulation of signaling events. In the present study, we characterize the Xenopus laevis homolog of nrcam. Expression of Xenopus nrcam is most abundant along the dorsal midline throughout the developing brain and in the outer nuclear layer of the retina.

  10. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    PubMed

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  11. Biochemical and Hematologic Reference Intervals for Aged Xenopus laevis in a Research Colony.

    PubMed

    Chang, Angela G; Hu, Jing; Lake, Elizabeth; Bouley, Donna M; Johns, Jennifer L

    2015-09-01

    Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs.

  12. Biochemical and Hematologic Reference Intervals for Aged Xenopus laevis in a Research Colony

    PubMed Central

    Chang, Angela G; Hu, Jing; Lake, Elizabeth; Bouley, Donna M; Johns, Jennifer L

    2015-01-01

    Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs. PMID:26424243

  13. Developmental expression of the fermitin/kindlin gene family in Xenopus laevis embryos.

    PubMed

    Canning, Claire A; Chan, Jessica Sze Ki; Common, John E A; Lane, E Birgitte; Jones, C Michael

    2011-08-01

    Fermitin genes are highly conserved and encode cytocortex proteins that mediate integrin signalling. Fermitin 1 (Kindlin1) is implicated in Kindler syndrome, a human skin blistering disorder. We report the isolation of the three Fermitin orthologs from Xenopus laevis embryos and describe their developmental expression patterns. Fermitin 1 is expressed in the skin, otic and olfactory placodes, pharyngeal arches, pronephric duct, and heart. Fermitin 2 is restricted to the somites and neural crest. Fermitin 3 is expressed in the notochord, central nervous system, cement gland, ventral blood islands, vitelline veins, and myeloid cells. Our findings are consistent with the view that Fermitin 1 is generally expressed in the skin, Fermitin 2 in muscle, and Fermitin 3 in hematopoietic lineages. Moreover, we describe novel sites of Fermitin gene expression that extend our knowledge of this family. Our data provide a basis for further functional analysis of the Fermitin family in Xenopus laevis.

  14. Manipulation and analysis of Xenopus laevis embryos by femtosecond near infrared lasers

    NASA Astrophysics Data System (ADS)

    Gifford, Aliya; Khodaparast, G.; Xu, Y.; Sethi, V.; Meehan, K.; Sible, J.

    2007-03-01

    Given the demand for new and more reliable methodologies for live cell manipulation, we have used a technique (demonstrated earlier by Tirlapur and K"onig, Nature, 418, 290, 2002) using near infrared laser pulses (NIR) to manipulate living cells, specifically, cell of Xenopus laevis embryos, without harming them. In addition nanoparticles such as silica-coated CdSe and CdTe quantum dots are injected into the cell through pores formed by the laser pulses. Due to the highly efficient and size-dependent fluorescence of QDs, they can be used in place of conventional dyes to perform live-cell imaging. In this work, we will discuss our current understanding of NIR lasers and QDs interactions with the Xenopus laevis embryos. The outcome of this project can help us to understand the fundamental phenomena and processes important in biological systems and cellular function.

  15. Forskolin increases cAMP and inhibits progesterone induced meiosis reinitiation in Xenopus laevis oocytes.

    PubMed

    Schorderet-Slatkine, S; Baulieu, E E

    1982-10-01

    The diterpene, forskolin, is a potent and reversible inhibitor of progesterone-induced meiosis in Xenopus laevis oocytes (ED50 of inhibition approximately 3 microM). Forskolin alone increases cAMP concentration in oocytes, but, unlike with cholera toxin treatment, there is no lag phase, and reversibility is obtained by washing the cells. Progesterone decreases the forskolin effect on cAMP accumulation, but cAMP concentration remains above the level observed in oocytes treated with progesterone alone. The data corroborate the previously-established antagonistic effect of cAMP on progesterone-induced meiosis. Preliminary experiments in the presence of a phosphodiesterase inhibitor suggest that, as in other biological systems, forskolin is an activator of adenylate cyclase in xenopus laevis oocytes. Contrary to what is observed when forskolin is present in the incubation medium, no effect of the diterpene is recorded after its injection into oocytes, evoking a site of action at the external side of the membrane.

  16. Determining the optimal developmental stages of Xenopus laevis for initiating exposures to chemicals for sensitively detecting their feminizing effects on gonadal differentiation.

    PubMed

    Li, Yuan-Yuan; Chen, Juan; Qin, Zhan-Fen

    2016-10-01

    Xenopus laevis is an important model for detecting feminizing effects of endocrine disrupting chemicals (EDCs) on amphibians because its genetic males can be induced to phenotypic females by estrogenic chemicals. It is crucial that chemical exposures begin at sensitive developmental stages for gonadal sex-reversal in X. laevis. To determine the optimal stages for initiating exposures, we investigated gonadal sex-reversal induced by low concentrations of 17α-ethinylestradiol (EE2) when exposures were initiated at different stages (3/4, 45/46, 48 and 50) until stage 58. We found that 0.1nM EE2 resulted in 85%, 86%, 43%, and 19% intersex, whereas 1nM EE2 caused 77%, 81%, 17%, and 8% phenotypic females, when genetic male tadpoles were exposed from stages 3/4, 45/46, 48 and 50, respectively. The data show the sensitivity of X. laevis gonads to EE2 at stages 45/46 is similar with that at stages 3/4, but the sensitivity decreases at stage 48 and stage 50, displaying a developmental stage-dependent manner. In another experiment using the offspring of another pair of frogs, we confirmed high sensitivity of X. laevis gonads at stages 45/46 to low concentrations of EE2. Considering that stages 45/46 tadpoles are easier to manipulate and have higher survival rates than earlier embryos, we propose that stages 45/46 are the optimal stages for initiating exposure for detecting feminizing effects of EDCs on gonadal differentiation in X. laevis. The developmental stages for initiating exposures we determined will guarantee the high sensitivity for detecting feminizing effects of EDCs with low estrogenic activities on gonadal differentiation in X. laevis. Also, our study suggests that gonadal differentiation in X. laevis possibly begins at stages 45/46, but not at later stages. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis.

    PubMed

    Tomiyama, Shiori; Nakamachi, Tomoya; Uchiyama, Minoru; Matsuda, Kouhei; Konno, Norifumi

    2015-05-15

    Urotensin II (UII) exhibits diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response via the UII receptor (UTR) in mammals. However, in amphibians the function of the UII-UTR system remains unknown. In the present study, we investigated the potential immune function of UII using leukocytes isolated from the African clawed frog, Xenopus laevis. Stimulation of male frogs with lipopolysaccharide increased mRNA expression of UII and UTR in leukocytes, suggesting that inflammatory stimuli induce activation of the UII-UTR system. Migration assays showed that both UII and UII-related peptide enhanced migration of leukocytes in a dose-dependent manner, and that UII effect was inhibited by the UTR antagonist urantide. Inhibition of Rho kinase with Y-27632 abolished UII-induced migration, suggesting that it depends on the activation of RhoA/Rho kinase. Treatment of isolated leukocytes with UII increased the expression of several cytokine genes including tumor necrosis factor-α, interleukin-1β, and macrophage migration inhibitory factor, and the effects were abolished by urantide. These results suggest that in amphibian leukocytes the UII-UTR system is involved in the activation of leukocyte migration and cytokine gene expression in response to inflammatory stimuli.

  18. An innovative continuous flow system for monitoring heavy metal pollution in water using transgenic Xenopus laevis tadpoles.

    PubMed

    Fini, Jean-Baptiste; Pallud-Mothré, Sophie; Le Mével, Sébastien; Palmier, Karima; Havens, Christopher M; Le Brun, Matthieu; Mataix, Vincent; Lemkine, Gregory F; Demeneix, Barbara A; Turque, Nathalie; Johnson, Paul E

    2009-12-01

    While numerous detection methods exist for environmental heavy metal monitoring, easy-to-use technologies combining rapidity with in vivo measurements are lacking. Multiwell systems exploiting transgenic tadpoles are ideal but require time-consuming placement of individuals in wells. We developed a real-time flow-through system, based on Fountain Flow cytometry, which measures in situ contaminant-induced fluorescence in transgenic amphibian larvae immersed in water samples. The system maintains the advantages of transgenic amphibians, but requires minimal human intervention. Portable and self-contained, it allows on-site measurements. Optimization exploited a transgenic Xenopus laevis bearing a chimeric gene with metal responsive elements fused to eGFP. The transgene was selectively induced by 1 microM Zn(2+). Using this tadpole we show the continuous flow method to be as rapid and sensitive as image analysis. Flow-through readings thus accelerate the overall process of data acquisition and render fluorescent monitoring of tadpoles suitable for on-site tracking of heavy metal pollution.

  19. Cytoskeleton and gravity at work in the establishment of dorso-ventral polarity in the egg of Xenopus laevis

    NASA Astrophysics Data System (ADS)

    Ubbels, Geertje A.; Brom, Tim G.

    The establishment of polarities during early embryogenesis is essential for normal development. Amphibian eggs are appropriate models for studies on embryonic pattern formation. The animal-vegetal axis of the axially symmetrical amphibian egg originates during oogenesis and foreshadows the main body axis of the embryo. The dorso-ventral polarity is epigenetically established before first cleavage. Recent experiments strongly suggest that in the monospermic eggs of the anuran Xenopus laevis both the cytoskeleton and gravity act in the determination of the dorso-ventral polarity. In order to test the role of gravity in this process, eggs will be fertilized under microgravity conditions during the SL-D1 flight in 1985. In a fully automatic experiment container eggs will be kept under well-defined conditions and artificially fertilized as soon as microgravity is reached; eggs and embryos at different stages will then be fixed for later examination. Back on earth the material will be analysed and we will know whether fertilization under microgravity conditions is possible. If so, the relation of the dorso-ventral axis to the former sperm entry point will be determined on the whole embryos; in addition eggs and embryos will be analysed cytologically.

  20. Effect of starvation on Fos and neuropeptide immunoreactivities in the brain and pituitary gland of Xenopus laevis.

    PubMed

    Calle, M; Kozicz, T; van der Linden, E; Desfeux, A; Veening, J G; Barendregt, H P; Roubos, E W

    2006-07-01

    In mammals complex interactions between various brain structures and neuropeptides such as corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1) underlay the control of feeding by the brain. Recently, in the amphibian Xenopus laevis, CRF- and Ucn1-immunoreactivities were shown in the hypothalamic magnocellular nucleus (Mg) and evidence was obtained for their involvement in food intake. To gain a better understanding of the brain structures controlling feeding in X. laevis, the effects of 16 weeks starvation on neurones immunoreactive (ir) to Fos and neuropeptides in various brain structures were quantified. In the Mg, compared to controls, starved animals showed fewer neurones immunopositive for Fos (-55.9%), Ucn1 (-44.0%), cocaine and amphetamine-regulated transcript (CART) (-94.3%) and metenkephalin (ENK) (-65.0%), whereas CRF-ir neurones were 2.1 times more numerous. These differences were mainly apparent in the ventral part of the Mg, followed by the medial and dorsal part of the nucleus. In the neural lobe of the pituitary gland a 22.5% lower optical density of CART-ir was observed. In the four other brain structures investigated, starvation had different effects. The dorsomedial part of the suprachiasmatic nucleus showed 5.9 times more NPY-ir cells and in the ventromedial thalamic area a lower number of NPY-ir cells (-33.6%) was found, whereas the Edinger-Westphal nucleus contained fewer CART-ir cells (-42.2%); no effect of starvation was seen in the ventral hypothalamic nucleus. Our results support the hypothesis that in X. laevis, the Mg plays a pivotal role in feeding-related processes and, moreover, that starvation also has neuropeptide- and brain structure-specific effects in other parts of the brain and in the pituitary gland, suggesting particular roles of these structures and their neuropeptides in physiological adaptation to starvation.

  1. [Vestibular apparatus study of the toad, Xenopus laevis, and rats under prolonged weightlessness].

    PubMed

    Vinnikov, Ia A; Lychakov, D V; Pal'mbakh, L R; Govardovskiĭ, V I; Adanina, V O

    1980-01-01

    Fertilized eggs of the clawed toad Xenopus laevis were placed aboard of orbital laboratory of "Salut-6" spacecraft where they developed for 20 days at temperature 15 degrees C. The larvae were fixed in weightlessness. Light and electronmicroscopic studies revealed undisturbed structure on the saccular and utricular maculae and otolith membranes. Some ultrastructural abnormalities were found in the inner ear of adult rats after 20 days of weightlessness ("Kosmos-936").

  2. Making muscle: Morphogenetic movements and molecular mechanisms of myogenesis in Xenopus laevis.

    PubMed

    Sabillo, Armbien; Ramirez, Julio; Domingo, Carmen R

    2016-03-01

    Xenopus laevis offers unprecedented access to the intricacies of muscle development. The large, robust embryos make it ideal for manipulations at both the tissue and molecular level. In particular, this model system can be used to fate map early muscle progenitors, visualize cell behaviors associated with somitogenesis, and examine the role of signaling pathways that underlie induction, specification, and differentiation of muscle. Several characteristics that are unique to X. laevis include myogenic waves with distinct gene expression profiles and the late formation of dermomyotome and sclerotome. Furthermore, myogenesis in the metamorphosing frog is biphasic, facilitating regeneration studies. In this review, we describe the morphogenetic movements that shape the somites and discuss signaling and transcriptional regulation during muscle development and regeneration. With recent advances in gene editing tools, X. laevis remains a premier model organism for dissecting the complex mechanisms underlying the specification, cell behaviors, and formation of the musculature system.

  3. Histological development of the gonad in juvenile Xenopus laevis

    EPA Science Inventory

    As directed by the Food Quality Protection Act, the US Environmental Protection Agency is developing a screening program for endocrine disrupting compounds. The Larval Amphibian Growth and Development Assay (LAGDA) is a tier II test intended to identify and characterize the adver...

  4. Histological development of the gonad in juvenile Xenopus laevis

    EPA Science Inventory

    As directed by the Food Quality Protection Act, the US Environmental Protection Agency is developing a screening program for endocrine disrupting compounds. The Larval Amphibian Growth and Development Assay (LAGDA) is a tier II test intended to identify and characterize the adver...

  5. Retinal regeneration in the Xenopus laevis tadpole: a new model system

    PubMed Central

    Vergara, M. Natalia

    2009-01-01

    Purpose Retinal regeneration research holds potential for providing new avenues for the treatment of degenerative diseases of the retina. Various animal models have been used to study retinal regeneration over the years, providing insights into different aspects of this process. However the mechanisms that drive this important phenomenon remain to be fully elucidated. In the present study, we introduce and characterize a new model system for retinal regeneration research that uses the tadpole of the African clawed frog, Xenopus laevis. Methods The neural retina was surgically removed from Xenopus laevis tadpoles at stages 51–54, and a heparin-coated bead soaked in fibroblast growth factor 2 (FGF-2) was introduced in the eyes to induce regeneration. Histological and immunohistochemical analyses as well as DiI tracing were performed to characterize the regenerate. A similar surgical approach but with concomitant removal of the anterior portion of the eye was used to assess the capacity of the retinal pigmented epithelium (RPE) to regenerate a retina. Immunohistochemistry for FGF receptors 1 and 2 and phosphorylated extracellular signal-regulated protein kinase (pERK) was performed to start elucidating the intracellular mechanisms involved in this process. The role of the mitogen activated protein kinase (MAPK) pathway was confirmed through a pharmacological approach using the MAPK kinase (MEK) inhibitor U0126. Results We observed that Xenopus laevis tadpoles were able to regenerate a neural retina upon induction with FGF-2 in vivo. The regenerated tissue has the characteristics of a differentiated retina, as assessed by the presence and distribution of different retinal cell markers, and DiI tracing indicated that it is able to form an optic nerve. We also showed that retinal regeneration in this system could take place independently of the presence of the anterior eye tissues. Finally, we demonstrated that FGF-2 treatment induces ERK phosphorylation in the

  6. Characterization of Two Cysteine Transfer RNA Genes from Xenopus Laevis

    DTIC Science & Technology

    1984-07-12

    author hereby certifies that the use of any copyrighted material in the dissertation manuscript entitled: "Characterization of two cysteine tRNA genes...Uniformed Services University of the Health Sciences 11 ABSTRACT Title of Thesis: Characterization of Two Cysteine Transfer RNA Genes from Xenopus...method after constructing a set of deletions and reclonlng into the plasmid pUC 8. The DNA fragment is 1737 bp long and contains two cysteine tRNA genes

  7. Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity.

    PubMed

    Ubbels, G A; Reijnen, M; Meijerink, J; Narraway, J

    1994-01-01

    One assumes that gravity cooperates with the sperm in the establishment of bilateral symmetry in the embryo, particularly in species with yolky eggs. However, only experiments under genuine microgravity can prove this. May 2nd 1988 on the TEXUS-17 Sounding Rocket, eggs of Xenopus laevis became the first vertebrate eggs ever successfully fertilized in Space. Fertilization was done in fully automated hardware; the experiment was successfully repeated and extended in 1989. Here we report a "Space First" from the IML-1 Space Shuttle mission (January 1992): In similar hardware and under microgravity, artificially fertilized Xenopus eggs started embryonic development. Histological fixation was pre-programmed at the time gastrulation would occur on Earth and indeed, gastrulae were fixed. Thus after fertilization in near weightlessness Xenopus embryos do develop bilaterally symmetrically, very probably cued by the sperm alone.

  8. Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity

    NASA Astrophysics Data System (ADS)

    Ubbels, Geertje A.; Reijnen, Mark; Meijerink, Jocelyn; Narraway, Jenny

    1994-08-01

    One assumes that gravity cooperates with the sperm in the establishment of bilateral symmetry in the embryo, particularly in species with yolky eggs. However, only experiments under genuine microgravity can prove this. May 2nd 1988 on the TEXUS-17 Sounding Rocket, eggs of Xenopus laevis became the first vertebrate eggs ever successfully fertilized in Space /1/.Fertilization was done in fully automated hardware; the experiment was successfully repeated and extended in 1989 /2,3/. Here we report a ``Space First'' from the IML-1 Space Shuttle mission (January 1992): In similar hardware and under microgravity, artificially fertilized Xenopus eggs started embryonic development. Histological fixation was pre-programmed at the time gastrulation would occur on Earth and indeed, gastrulae were fixed. Thus after fertilization in near weightlessness Xenopus embryos do develop bilaterally symmetrically, very probably cued by the sperm alone.

  9. Xenopus laevis is a potential alternative model animal species to study reproductive toxicity of phytoestrogens.

    PubMed

    Cong, Lin; Qin, Zhan-Fen; Jing, Xiang-Ning; Yang, Lei; Zhou, Jing-Ming; Xu, Xiao-Bai

    2006-05-10

    This study investigated effects of phytoestrogen quercetin on the gonadal development in Xenopus laevis. X. laevis at Nieuwkoop and Faber stage 46/47 were exposed to 50, 100 and 200 microg/L quercetin till 1 month postmetamorphosis. Gonads from frogs at 1 and 3 months postmetamorphosis were examined in gross morphology and histology. The highest dose of quercetin as well as estradiol (E2) significantly increased the percentages of phenotypic females. Exposure to quercetin at all doses induced abnormal testes with certain ovarian characteristics to some degree in gross morphology, including ovotestes. The abnormality rate exceeded 10% in each quercetin treatment. Histologic examination revealed that some abnormal testes exhibited intersexuality with testicular structure and ovarian structure or oocytes interspersed in testicular structure at 1 month postmetamorphosis. At 3 months postmetamorphosis, testicular abnormalities were more obvious, such as necrosis or apoptosis of spermatogonia, occurrence of developed or undeveloped oocytes, delay of the development of seminiferous tubes without or less late stage spermatocytes. The results have shown that quercetin cannot only feminize but also impair testicular development of X. laevis, i.e. X. laevis is sensitive to phytoestrogen. It is suggested that X. laevis might be an alternative model species to study reproductive toxicity of phytoestrogens.

  10. [Xenopus laevis peroxiredoxins: Gene expression during development and characterization of the enzymes].

    PubMed

    Sharapov, M G; Novoselov, V I; Ravin, V K

    2016-01-01

    Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1-6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10-63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1-6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1-6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.

  11. Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence.

    PubMed Central

    Segil, N; Shrutkowski, A; Dworkin, M B; Dworkin-Rastl, E

    1988-01-01

    As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period. Images Fig. 3. Fig. 4. Fig. 5. PMID:3390159

  12. The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenic Xenopus laevis.

    PubMed

    Lim, Wayland; Neff, Eric S; Furlow, J David

    2004-06-17

    Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

  13. Analysis of HIV-1 Tat effects in Xenopus laevis embryos.

    PubMed

    Venanzi, S; Destrée, O H; Gigliani, F; Battaglia, P A

    1998-01-01

    Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.

  14. Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.

    PubMed

    Burnett, Lindsey A; Sugiyama, Hitoshi; Bieber, Allan L; Chandler, Douglas E

    2011-06-01

    Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior.

  15. The anatomy and development of the claws of Xenopus laevis (Lissamphibia: Anura) reveal alternate pathways of structural evolution in the integument of tetrapods

    PubMed Central

    Maddin, Hillary C; Eckhart, Leopold; Jaeger, Karin; Russell, Anthony P; Ghannadan, Minoo

    2009-01-01

    Digital end organs composed of hard, modified epidermis, generally referred to as claws, are present in mammals and reptiles as well as in several non-amniote taxa such as clawed salamanders and frogs, including Xenopus laevis. So far, only the claws and nails of mammals have been characterized extensively and the question of whether claws were present in the common ancestor of all extant tetrapods is as yet unresolved. To provide a basis for comparisons between amniote and non-amniote claws, we investigated the development, growth and ultrastructure of the epidermal component of the claws of X. laevis. Histological examination of developing claws of X. laevis shows that claw formation is initiated at the tip of the toe by the appearance of superficial cornified cells that are dark brown. Subsequent accumulation of new, proximally extended claw sheath corneocyte layers increases the length of the claw. Histological studies of adult claws show that proliferation of cornifying claw sheath cells occurs along the entire length of the claw-forming epidermis. Living epidermal cells that are converting into the cornified claw sheath corneocytes undergo a form of programmed cell death that is accompanied by degradation of nuclear DNA. Subsequently, the cytoplasm and the nuclear remnants acquire a brown colour by an as-yet unknown mechanism that is likely homologous to the colouration mechanism that occurs in other hard, cornified structures of amphibians such as nuptial pads and tadpole beaks. Transmission electron microscopy revealed that the cornified claw sheath consists of parallel layers of corneocytes with interdigitations being confined to intra-layer contacts and a cementing substance filling the intercorneocyte spaces. Together with recent reports that showed the main molecular components of amniote claws are absent in Xenopus, our data support the hypothesis that claws of amphibians likely represent clade-specific innovations, non-homologous to amniote claws

  16. Microinjected progesterone reinitiates meiotic maturation of Xenopus laevis oocytes.

    PubMed Central

    Tso, J; Thibier, C; Mulner, O; Ozon, R

    1982-01-01

    Microinjection of progesterone dissolved in paraffin oil induces the reinitiation of meiotic maturation in the Xenopus oocyte; 50% maturation is obtained when 50 nl of a 50 microM solution is microinjected into the oocyte. The kinetics of the response to microinjected progesterone are similar to the kinetics of response to externally applied hormone. When an aqueous solution of progesterone is microinjected instead of an oil solution, maturation is never observed, a result which confirms previous work. Leakage of the steroid into the external medium was estimated to range from 1.6 pmol/hr when microinjection was performed in oil to 3.6 pmol/hr when it was performed in aqueous solution. Metabolism of the hormone microinjected in oil is weak (less than 20%) as compared to that after aqueous microinjection (greater than 80%). Progesterone microinjected in oil decreases the cAMP content as does externally applied hormone. We therefore conclude that progesterone acts initially on an intracellular site in order to trigger meiotic maturation of the Xenopus oocyte. PMID:6291050

  17. Biochemical characterization of lysosomes in unfertilized eggs of Xenopus laevis.

    PubMed

    Decroly, M; Goldfinger, M; Six-Tondeur, N

    1979-11-01

    Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.

  18. Xenopus laevis oocytes infected with multi-drug-resistant bacteria: implications for electrical recordings.

    PubMed

    O'Connell, Denice; Mruk, Karen; Rocheleau, Jessica M; Kobertz, William R

    2011-08-01

    The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug-resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3-4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug-resistant bacteria.

  19. Overland movement in African clawed frogs (Xenopus laevis): a systematic review

    PubMed Central

    2016-01-01

    African clawed frogs (Xenopus laevis) are often referred to as ‘purely aquatic’ but there are many publications which suggest extensive overland movements. Previous reviews which considered the topic have not answered the following questions: (1) is there evidence for overland dispersal in native and invasive ranges; (2) what is the range of distances moved overland; (3) when does overland movement occur; and (4) is there evidence of breeding migratory behaviour? A systematic review was chosen to synthesise and critically analyse all literature on the overland movement in Xenopus laevis. Database searches resulted in 57 documents which revealed a paucity of empirical studies, with 28 containing no data, and 19 having anecdotal content. Overwhelming evidence shows that both native and invasive populations of X. laevis move overland, with well documented examples for several other members of the genus (X. borealis, X. gilli, X. muelleri, X. fraseriand X. tropicalis). Reports of distances moved overland were from 40 m to 2 km, with no apparent difference between native and invasive ranges. Overland movements are not confined to wet seasons or conditions, but the literature suggests that moving overland does not occur in the middle of the day. Migrations to temporary water-bodies for breeding have been suggested, but without any corroborating data. PMID:27688972

  20. Overland movement in African clawed frogs (Xenopus laevis): a systematic review.

    PubMed

    Measey, John

    2016-01-01

    African clawed frogs (Xenopus laevis) are often referred to as 'purely aquatic' but there are many publications which suggest extensive overland movements. Previous reviews which considered the topic have not answered the following questions: (1) is there evidence for overland dispersal in native and invasive ranges; (2) what is the range of distances moved overland; (3) when does overland movement occur; and (4) is there evidence of breeding migratory behaviour? A systematic review was chosen to synthesise and critically analyse all literature on the overland movement in Xenopus laevis. Database searches resulted in 57 documents which revealed a paucity of empirical studies, with 28 containing no data, and 19 having anecdotal content. Overwhelming evidence shows that both native and invasive populations of X. laevis move overland, with well documented examples for several other members of the genus (X. borealis, X. gilli, X. muelleri, X. fraseriand X. tropicalis). Reports of distances moved overland were from 40 m to 2 km, with no apparent difference between native and invasive ranges. Overland movements are not confined to wet seasons or conditions, but the literature suggests that moving overland does not occur in the middle of the day. Migrations to temporary water-bodies for breeding have been suggested, but without any corroborating data.

  1. Effects of depleted uranium on survival, growth, and metamorphosis in the african clawed frog (Xenopus laevis)

    USGS Publications Warehouse

    Mitchell, S.E.; Caldwell, C.A.; Gonzales, G.; Gould, W.R.; Arimoto, R.

    2005-01-01

    Embryos (stage 8-47, Nieuwkoop and Faber) of the African clawed frog (Xenopus laevis) were subjected to water-borne depleted uranium (DU) concentrations that ranged from 4.8 to 77.7 mg/Lusing an acute 96-h frog embryo teratogenesis assay-Xenopus (FETAX). In a chronic 64-d assay, X. laevis (from embryo through metamorphosis; stages 8-66) were subjected to concentrations of DU that ranged from 6.2 to 54.3 mg/L Our results indicate DU is a non teratogenic metal. No effects on mortality, malformations, or growth were observed in the 96-h FETAX with concentrations of DU that ranged from 4.8 to 77.7 mg/L From stage 8 to stage 47, X. laevis tadpoles do not actively feed and the gills are not well developed. Thus, uptake of DU was reduced despite exposure to elevated concentrations. The 64-d assay resulted in no concentration response for either mortality or malformations; however, a delay in metamorphosis was observed in tadpoles subjected to elevated DU concentrations (from 13.1 to 54.3 mg/L) compared to tadpoles in both the well-water control and reference. The delay in metamorphosis was likely due to increasing body burden of DU that ranged from 0.98 to 2.82 mg/kg. Copyright?? Taylor & Francis Inc.

  2. Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

    PubMed

    Tassan, Jean-Pierre; Wühr, Martin; Hatte, Guillaume; Kubiak, Jacek

    2017-01-01

    Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

  3. A novel gene, Ami is expressed in vascular tissue in Xenopus laevis.

    PubMed

    Inui, Masafumi; Asashima, Makoto

    2006-08-01

    We report the isolation and expression pattern of a novel gene, Ami in Xenopus laevis. Ami was initially isolated as a highly expressed gene in cardiovascular tissues. The deduced amino acid sequence of Ami was most closely similar to human complement factor D and mouse adipsin in mammals. In adult Xenopus tissues, the transcript of Ami was detected in liver, fat body, lung, gut, vessel, heart, muscle, testis, and ovary, but expression in blood cells or skin was hardly detected. This expression profile was significantly different from that observed for mammalian homologues. Ami transcripts in Xenopus laevis were expressed from the late neurula stage, remained constant until the tadpole stage. The mRNA localized to paraxial regions at the neurula stage and anterior ventral regions at the tailbud stage. From the late tailbud to tadpole stage, expression was detected along the forming blood vessels, including the anterior cardinal veins, posterior cardinal veins, intersomitic veins, dorsal longitudinal anastomosing vessel, dorsal aorta, pronephric sinus, and most prominently around the vascular vitelline network. The expression around the vascular vitelline network demonstrated left-right asymmetry in stage 42 embryo. Comparison with the endothelium marker, Xmsr, suggested that Ami is expressed in endothelial cells.

  4. Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis.

    PubMed

    Scudiero, Rosaria; Tussellino, Margherita; Carotenuto, Rosa

    2015-05-01

    Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys-Cys-Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

  5. Significance of temporal and spectral acoustic cues for sexual recognition in Xenopus laevis

    PubMed Central

    Vignal, Clémentine; Kelley, Darcy

    2006-01-01

    As in many anurans, males of the totally aquatic species, Xenopus laevis, advertise their sexual receptivity using vocalizations. Unusually for anurans, X. laevis females also advertise producing a fertility call that results in courtship duets between partners. Although all X. laevis calls consist of repetitive click trains, male and female calls exhibit sex-specific acoustic features that might convey sexual identity. We tested the significance of the carrier frequency and the temporal pattern of calls using underwater playback experiments in which modified calls were used to evoke vocal responses in males. Since males respond differently to male and female calls, the modification of a key component of sexual identity in calls should change the male's response. We found that a female-like slow call rhythm triggers more vocal activity than a male-like fast rhythm. A call containing both a female-like temporal pattern and a female-like carrier frequency elicits higher levels of courtship display than either feature alone. In contrast, a male-like temporal pattern is sufficient to trigger typical male–male encounter vocalizations regardless of spectral cues. Thus, our evidence supports a role for temporal acoustic cues in sexual identity recognition and for spectral acoustic cues in conveying female attractiveness in X. laevis. PMID:17476767

  6. Tissue distribution of enrofloxacin in African clawed frogs (Xenopus laevis) after intramuscular and subcutaneous administration.

    PubMed

    Felt, Stephen; Papich, Mark G; Howard, Antwain; Long, Tyler; McKeon, Gabriel; Torreilles, Stéphanie; Green, Sherril

    2013-03-01

    As part of an enrofloxacin pharmacokinetic study, concentrations of enrofloxacin and ciprofloxacin (metabolite) were measured in various tissues (brain, heart, kidney, liver, lung, and spleen) collected from treated (subcutaneous delivery, n = 3; intramuscular delivery, n = 3; untreated controls, n = 2) adult female Xenopus laevis by using HPLC. Enrofloxacin was rapidly absorbed after administration by either route and readily diffused into all sampled tissues. Enrofloxacin and ciprofloxacin were present in the tissue samples collected at 8 h. The highest average tissue concentrations for enrofloxacin were found in kidney, with the lowest concentrations in liver. Ciprofloxacin tissue concentrations paralleled but were always lower than those of enrofloxacin for all time points and tissues except brain and kidney. These results, together with previously published pharmacokinetic data and known minimal inhibitory concentrations of common pathogenic bacteria, provide a strong evidence-based rationale for choosing enrofloxacin to treat infectious diseases in X. laevis.

  7. Diagnosis of Aeromonas hydrophila, Mycobacterium species, and Batrachochytrium dendrobatidis in an African Clawed Frog (Xenopus laevis)

    PubMed Central

    Hill, William A; Newman, Shelley J; Craig, Linden; Carter, Christopher; Czarra, Jane; Brown, J Paige

    2010-01-01

    Here we describe diagnosis of concurrent infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a wild female Xenopus laevis captured in Chile and transported to the United States. After approximately 130 d in the laboratory, the frog was presented for dysecdysis and obtundation. After euthanasia, tissues were submitted for histopathologic evaluation and PCR analysis for B. dendrobatidis and Ranavirus. Clinically significant gross lesions included cutaneous ulcerations on the lip, right forelimb, and ventral chest. Microscopic findings included regionally extensive splenic necrosis, diffuse pneumonia, and fibrinous coelomitis all containing intralesional bacteria. PCR analysis yielded positive results for B. dendrobatidis only. Bacterial culture of the ulcerated skin and liver yielded A. hydrophila. Infection with Contracaecum spp. was diagnosed as an incidental finding. To our knowledge, this case is the first report of simultaneous infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a laboratory-maintained X. laevis captured from the wild. PMID:20353698

  8. Generation of a Xenopus laevis F1 albino J strain by genome editing and oocyte host-transfer

    PubMed Central

    Ratzan, Wil; Falco, Rosalia; Salanga, Cristy; Salanga, Matthew; Horb, Marko E.

    2016-01-01

    Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs. PMID:26993591

  9. Xenopus laevis Stromal cell-derived factor 1: conservation of structure and function during vertebrate development.

    PubMed

    Braun, Mike; Wunderlin, Markus; Spieth, Kathrin; Knöchel, Walter; Gierschik, Peter; Moepps, Barbara

    2002-03-01

    Transmembrane signaling of the CXC chemokine stromal cell-derived factor-1 (SDF-1) is mediated by CXCR4, a G protein-coupled receptor initially identified in leukocytes and shown to serve as a coreceptor for the entry of HIV into lymphocytes. Characterization of SDF-1- and CXCR4-deficient mice has revealed that SDF-1 and CXCR4 are of vital developmental importance. To study the role of the SDF-1/CXCR4-chemokine/receptor system as a regulator of vertebrate development, we isolated and characterized a cDNA encoding SDF-1 of the lower vertebrate Xenopus laevis (xSDF-1). Recombinant xSDF-1 was produced in insect cells, purified, and functionally characterized. Although xSDF-1 is only 64-66% identical with its mammalian counterparts, it is indistinguishable from human (h)SDF-1alpha in terms of activating both X. laevis CXCR4 and hCXCR4. Thus, both xSDF-1 and hSDF-1alpha promoted CXCR4-mediated activation of heterotrimeric G(i2) in a cell-free system and induced release of intracellular calcium ions in and chemotaxis of intact lymphoblastic cells. Analysis of the time course of xSDF-1 mRNA expression during Xenopus embryogenesis revealed a tightly coordinated regulation of xSDF-1 and X. laevis CXCR4. xSDF-1 mRNA was specifically detected in the developing CNS, incipient sensory organs, and the embryonic heart. In Xenopus, CXCR4 mRNA appears to be absent from the heart anlage, but present in neural crest cells. This observation suggests that xSDF-1 expressed in the heart anlage may attract cardiac neural crest cells expressing CXCR4 to migrate to the primordial heart to regulate both septation of the cardiac outflow tract and differentiation of the myocardium during early heart development.

  10. Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    PubMed Central

    Dzementsei, Aliaksandr; Schneider, David; Janshoff, Andreas; Pieler, Tomas

    2013-01-01

    Summary The directional migration of primordial germ cells (PGCs) to the site of gonad formation is an advantageous model system to study cell motility. The embryonic development of PGCs has been investigated in different animal species, including mice, zebrafish, Xenopus and Drosophila. In this study we focus on the physical properties of Xenopus laevis PGCs during their transition from the passive to the active migratory state. Pre-migratory PGCs from Xenopus laevis embryos at developmental stages 17–19 to be compared with migratory PGCs from stages 28–30 were isolated and characterized in respect to motility and adhesive properties. Using single-cell force spectroscopy, we observed a decline in adhesiveness of PGCs upon reaching the migratory state, as defined by decreased attachment to extracellular matrix components like fibronectin, and a reduced adhesion to somatic endodermal cells. Data obtained from qPCR analysis with isolated PGCs reveal that down-regulation of E-cadherin might contribute to this weakening of cell-cell adhesion. Interestingly, however, using an in vitro migration assay, we found that movement of X. laevis PGCs can also occur independently of specific interactions with their neighboring cells. The reduction of cellular adhesion during PGC development is accompanied by enhanced cellular motility, as reflected in increased formation of bleb-like protrusions and inferred from electric cell-substrate impedance sensing (ECIS) as well as time-lapse image analysis. Temporal alterations in cell shape, including contraction and expansion of the cellular body, reveal a higher degree of cellular dynamics for the migratory PGCs in vitro. PMID:24285703

  11. Growth and development of tadpoles (Xenopus laevis) exposed to selective serotonin reuptake inhibitors, fluoxetine and sertraline, throughout metamorphosis.

    PubMed

    Conners, Deanna E; Rogers, Emily D; Armbrust, Kevin L; Kwon, Jeong-Wook; Black, Marsha C

    2009-12-01

    Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed drugs that are present in sewage effluents and surface waters. The objective of the present study was to determine whether low environmentally relevant concentrations of the SSRIs fluoxetine and sertraline could impair growth and development in tadpoles of the African clawed frog (Xenopus laevis) and to evaluate if such effects may be caused by a disruption of the neuroendocrine system. Tadpoles were exposed to SSRIs at concentrations of 0.1, 1, and 10 microg/L for 70 d throughout metamorphosis. No effects on deformities were observed. Tadpoles exposed to fluoxetine (10 microg/L) and sertraline (0.1, 1, and 10 microg/L) exhibited reduced growth at metamorphosis. Tadpoles exposed to sertraline (0.1 and 1 microg/L) exhibited an acceleration of development as indicated by an increase in the time to tail resorption. The effects of SSRIs on growth and development in tadpoles were likely driven by reduced food intake. Reduced feeding rates were observed in SSRI-exposed tadpoles, and nutritional status can influence growth and development in amphibians via effects on the neuroendocrine system. Only sertraline was capable of causing developmental toxicity in tadpoles at environmentally relevant concentrations. These data warrant additional research to characterize the risks to human health and wildlife from pharmaceutical exposures.

  12. Manipulation and in vitro maturation of Xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development.

    PubMed

    Miyamoto, Kei; Simpson, David; Gurdon, John B

    2015-02-09

    Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

  13. Effects of Roundup and glyphosate formulations on intracellular transport, microtubules and actin filaments in Xenopus laevis melanophores.

    PubMed

    Hedberg, Daniel; Wallin, Margareta

    2010-04-01

    Glyphosate containing herbicides, such as Roundup, are commonly used and generally considered to be safe. However, some toxic effects are found on amphibians in vivo and human and mouse cells in vitro. In this study the effects of Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were studied on intracellular transport by measuring aggregation capacity in Xenopus laevis melanophores. The chemicals inhibited retrograde transport of melanosomes in the range of 0.5-5mM. Cellular morphology and localization of microtubules and actin filaments were affected as determined by immunocytochemistry. Both glyphosate and Roundup decreased pH in the media. Acidic pH inhibited melanosome transport and altered microtubule and actin morphology in the absence of chemicals, while transport inhibiting concentrations of glyphosate, Roundup and glyphosateisopropylamine disassembled both microtubules and actin filaments. At physiological pH the effects of Roundup decreased whereas glyphosate failed to inhibit transport. Physiological pH decreases glyphosate lipophilicity and its diffusion into the cytoplasm. The Roundup formulation contains surfactants, such as POEA (polyetylated tallow amine) that increases membrane permeability allowing cellular uptake at physiological pH. Our results show that the effects of glyphosate containing compounds are pH-dependent and that they inhibit intracellular transport through disassembly of the cytoskeleton possibly by interfering with intracellular Ca(2+)-balance. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  14. Defined nutrient medium for the in vitro maintenance of Xenopus laevis oocytes.

    PubMed

    Eppig, J J; Dumont, J N

    1976-06-01

    A procedure is described for the isolation and culture of large numbers of follicle cell-free Xenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenace of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morpological and functional characteristics of oocytes for periods up to 3 weeks.

  15. Expressional characterization of mRNA (guanine-7) methyltransferase (rnmt) during early development of Xenopus laevis.

    PubMed

    Lokapally, Ashwin; Metikala, Sanjeeva; Hollemann, Thomas

    2016-01-01

    Methylation of the guanosine cap structure at the 5' end of mRNA is essential for efficient translation of all eukaryotic cellular mRNAs, gene expression and cell viability and promotes transcription, splicing, polyadenylation and nuclear export of mRNA. In the current study, we present the spatial expression pattern of the Xenopus laevis rnmt homologue. A high percentage of protein sequence similarity, especially within the methyltransferase domain, as well as an increased expression in the cells of the transcriptionally active stages, suggests a conserved RNA cap methylation function. Spatial expression analysis identified expression domains in the brain, the retina, the lens, the otic vesicles and the branchial arches.

  16. Stable magnetic field gradient levitation of Xenopus laevis: toward low-gravity simulation.

    PubMed

    Valles, J M; Lin, K; Denegre, J M; Mowry, K L

    1997-08-01

    We have levitated, for the first time, living biological specimens, embryos of the frog Xenopus laevis, using a large inhomogeneous magnetic field. The magnetic field/field gradient product required for levitation was 1430 kG2/cm, consistent with the embryo's susceptibility being dominated by the diamagnetism of water and protein. We show that unlike any other earth-based technique, magnetic field gradient levitation of embryos reduces the body forces and gravity-induced stresses on them. We discuss the use of large inhomogeneous magnetic fields as a probe for gravitationally sensitive phenomena in biological specimens.

  17. The Xenopus laevis ribosomal gene promoter contains a binding site for nuclear factor-1.

    PubMed Central

    Walker, P; Reeder, R H

    1988-01-01

    Nuclear Factor I (NF1) is a DNA binding protein that is known to function in the replication of Adeno virus and also binds to many promoters recognized by RNA polymerase II. We have found that there is also an NF1 binding site within the ribosomal gene promoter from Xenopus laevis as well as in several other promoters recognized by RNA polymerase I. The function of a binding site for a polymerase II transcription factor within a promoter recognized by polymerase I is not known. However, its presence suggests interesting regulatory possibilities. Images PMID:3205719

  18. Influence of 50-Hz electromagnetic field on anurian (Xenopus laevis) metamorphosis.

    PubMed

    Grimaldi, S; Lisi, A; Reiti, S; Manni, V; Ledda, M; Giuliani, L

    2004-10-20

    In this study, we show the effect of a 1-mT magnetic field AC at 50 Hz on Xenopus laevis tadpole populations. In the course of a 65-day exposure to the field, tadpole survival showed a small, but significant, decrease (p < 0.0004), together with a striking parallel 6-day shift in tadpole maturation frequency and a significant impairment of their metamorphosis. Particularly, metamorphosis was successful for 85% of individuals in the unirradiated tadpole population and for 45% of individuals in the irradiated tadpole population, respectively.

  19. Thyroid hormone controls multiple independent programs required for limb development in Xenopus laevis metamorphosis.

    PubMed

    Brown, Donald D; Cai, Liquan; Das, Biswajit; Marsh-Armstrong, Nicholas; Schreiber, Alexander M; Juste, Rejeanne

    2005-08-30

    Thyroid hormone (TH) is required for limb development in Xenopus laevis. Specific cell types in the growing limb were targeted for expression of a dominant negative form of the TH receptor by sperm-mediated transgenesis. Limb muscle development, the innervation of muscle from the spinal cord, and cartilage growth can be inhibited without affecting patterning of the limb or differentiation of other cell types. Remodeling of the skin occurs late in metamorphosis after the limb has formed. The coordination of these independent programs is affected in part by the control that TH exerts over DNA replication in all cell types of the limb.

  20. Arsenic toxicity and HSP70 expression in Xenopus laevis embryos.

    PubMed

    Gornati, Rosalba; Monetti, Claudio; Vigetti, Davide; Bosisio, Stefano; Fortaner, Salvador; Sabbioni, Enrico; Bernardini, Giovanni; Prati, Mariangela

    2002-01-01

    The evaluation of the effect of trace metals on health can be difficult, because of their presence in the environment in various chemical forms. Exposure to arsenic compounds is an example of this complexity, as it can be present in the environment in inorganic and organic forms. The effects of arsenic in vertebrates are complicated by several variables, such as speciation of the element, the exposure route, and the susceptibility of the particular animal species. The embryotoxicity and teratogenicity of three arsenic species - sodium arsenite (NaAsO(2)), disodium hydrogen arsenate (Na(2)HAsO(4)) and dimethylarsinic acid [(CH3)2AsOOH] - were evaluated by the modified frog embryo teratogenic assay on Xenopus (FETAX). We also show how the classical FETAX endpoints, based on morphological and morphometrical analysis, can conveniently be integrated with the study of molecular markers. Possible changes in the expression of the mRNA for the heat-shock protein HSP70, following exposure to NaAsO(2), were examined by using the reverse transcriptase polymerase chain reaction. HSP70 mRNA is strongly induced by arsenic.

  1. Atmospheric pressure plasma accelerates tail regeneration in tadpoles Xenopus laevis

    NASA Astrophysics Data System (ADS)

    Rivie, A.; Martus, K.; Menon, J.

    2017-08-01

    Atmospheric pressure plasma is a partially ionized gas composed of neutral and charged particles, including electrons and ions, as well as reactive oxygen species (ROS). Recently, it is utilized as possible therapy in oncology, sterilization, skin diseases, wound healing and tissue regeneration. In this study we focused on effect of plasma exposure on tail regeneration of tadpoles, Xenopus leavis with special emphasis on role of ROS, antioxidant defenses and morphological features of the regenerate. When amputated region of the tail was exposed to the helium plasma it resulted in a faster rate of growth, elevated ROS and increase in antioxidant enzymes in the regenerate compared to that of untreated control. An increase in nitric oxide (free radical) as well as activity of nitric oxide synthase(s) were observed once the cells of the regeneration blastema - a mass of proliferating cells are ready for differentiation. Microscopically the cells of the regenerate of plasma treated tadpoles show altered morphology and characteristics of cellular hypoxia and oxidative stress. We summarize that plasma exposure accelerates the dynamics of wound healing and tail regeneration through its effects on cell proliferation and differentiation as well as angiogenesis mediated through ROS signaling.

  2. Sodium conductance and the activation potential in Xenopus laevis eggs.

    PubMed

    Peres, A; Mancinelli, E

    1985-09-01

    Experiments have been performed to identify the membrane permeability changes causing activation potential in Xenopus eggs. The eggs were artificially activated either by pricking or by addition of the Ca2+ ionophore A23187 to the bath. Two different ionic currents appear to control the activation potential: (i) a chloride current which develops after a delay of 30 s to 5 min from the activating stimulus and which, in low external chloride, produces a depolarization and, (ii) a voltage-dependent outward current which begins to flow when the membrane potential is more positive than about +20 mV and tends to hyperpolarize the membrane. The chloride current lasts about 3-4 min; the voltage-dependent outward current is present before activation and disappears more slowly than the Cl- current. Changes in external sodium concentration affect the reversal potential of the outward current before and after the development of the inward Cl- current. We suggest that the chloride current has the role of producing a rapid depolarization necessary to block polyspermy, while the voltage-dependent sodium outward current might prevent the depolarization from reaching excessively high values and help the repolarization phase.

  3. Repair of Heteroduplex DNA in Xenopus Laevis Oocytes

    PubMed Central

    Lehman, C. W.; Jeong-Yu, S.; Trautman, J. K.; Carroll, D.

    1994-01-01

    We have hypothesized that the inheritance of heteroallelic markers during recombination of homologous DNAs in Xenopus oocytes is determined by resolution of a heteroduplex intermediate containing multiple single-base mismatches. To test this idea, we prepared synthetic heteroduplexes carrying 8 separate mispairs in vitro and injected them into oocyte nuclei. DNA was recovered and analyzed directly, by Southern blot-hybridization, and indirectly, by cloning individual repair products in bacteria. Mismatch correction was quite efficient in the oocytes; markers on the same strand were commonly co-corrected, indicating a long-patch mechanism; and the distribution of markers was very similar to that obtained by recombination. This supports our interpretation of the recombination outcome in terms of a resection-annealing mechanism. The injected heteroduplexes carried strand breaks (nicks) as a result of their method of preparation. We tested the idea that mismatch correction might be nick-directed by ligating the strands of the heteroduplex substrate to form covalently closed circles. Repair in oocytes was still efficient, and long patches predominated; but the pattern of recovered markers was quite different than with the nicked substrate. This suggests that nicks, when present, do indeed direct repair, but that, in their absence, recognition of specific mismatches governs repair of the ligated heteroduplexes. PMID:7828827

  4. Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis

    PubMed Central

    1987-01-01

    A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta- parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos. PMID:3558484

  5. Fluorescent imaging in vivo of developing blood vessels on the optic tectum of Xenopus laevis.

    PubMed

    Tiedeken, J J; Rovainen, C M

    1991-05-01

    The growth and development of individual living capillaries, venules, and endothelial sprouts on the pial surface of the brain were examined with video microscopy and intravascular FITC-dextran in anesthetized tadpoles of pigment-deficient Xenopus laevis, stages 42-50. The fluorescent tracer, injected intracardially through glass micropipets, was well tolerated by the tadpoles and improved the visibility of vessels compared to transmitted light. Case histories of vascular development on the optic tectum confirmed the sprouting of new capillaries during angiogenesis. The caudal tectum and its vascular domains grew faster than the rostral, but the densities of caudal surface vessels were at least as high as rostral densities, indicating that angiogenesis was well matched to neural development. Internal capillary branches were further elaborated and pial venules increased in diameter in premetamorphic tadpoles and in Xenopus frogs.

  6. Optogenetic Control of Apoptosis in Targeted Tissues of Xenopus laevis Embryos

    PubMed Central

    Jewhurst, Kyle; Levin, Michael; McLaughlin, Kelly A

    2014-01-01

    KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies—particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney. PMID:25374461

  7. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  8. How Xenopus Laevis Replicates DNA Reliably even though Its Origins of Replication are Located and Initiated Stochastically

    NASA Astrophysics Data System (ADS)

    Bechhoefer, John; Marshall, Brandon

    2007-03-01

    DNA replication in Xenopus laevis is extremely reliable, failing to complete before cell division no more than once in 10 000 times; yet replication origin sites are located and initiated stochastically. Using a model based on 1D theories of nucleation and growth and using concepts from extreme-value statistics, we derive the distribution of replication times given a particular initiation function. We show that the experimentally observed initiation strategy for Xenopus laevis meets the reliability constraint and is close to the one that requires the fewest resources of a cell.

  9. Thyroxine-dependent modulations of the expression of the neural cell adhesion molecule N-CAM during Xenopus laevis metamorphosis.

    PubMed

    Levi, G; Broders, F; Dunon, D; Edelman, G M; Thiery, J P

    1990-04-01

    During amphibian metamorphosis, a complete remodeling of the phenotype takes place under complex hormonal control whose final effectors are thyroid hormones. This process implies the activation of coordinated programs of cell death, proliferation, migration, adhesion and differentiation. Inasmuch as the neural cell adhesion molecule N-CAM is thought to play a central role in the control of morphogenetic processes, we have studied by immunohistofluorescence and immunoblots the patterns of expression of N-CAM at different stages of Xenopus laevis metamorphosis. A scan was made of all major organs and appendages. Before the metamorphic climax, all neuronal cell bodies and processes express high levels of N-CAM. During the metamorphic climax, N-CAM expression decreases sharply on the cell bodies and processes of the peripheral nervous system (PNS) but remains high in the central nervous system (CNS). Towards the end of metamorphosis, the PNS and spinal nerves are virtually negative for N-CAM while the CNS is still positive. The optic and olfactory nerves, although myelinated, are still strongly positive for N-CAM. The lens and olfactory epithelia express N-CAM throughout metamorphosis. In the brain. N-CAM is present at all times as three polypeptides of 180, 140, and 120 X 10(3) Mr; before metamorphosis some of the N-CAM is in its polysialylated form. During metamorphosis and the subsequent growth of the animal, the amount of N-CAM decreases gradually. In all polypeptides, the polysialylated form is the first to disappear. Cardiac muscle expresses high level of N-CAM from its first formation throughout metamorphosis; in contrast, the level of N-CAM in skeletal muscle is high in newly formed muscles, but decreases rapidly after myoblast fusion. The liver of adult Xenopus contains large amounts of a 160 X 10(3) polypeptide that is recognized by polyclonal and monoclonal antibodies against N-CAM. cDNA probes of Xenopus brain N-CAM recognize major transcripts of 9.2, 3

  10. Transcription factors and induction in Xenopus laevis embryos.

    PubMed

    Knöchel, W; Kaufmann, E

    1997-04-01

    Studies with amphibian embryos have contributed major insights into the molecular basis of induction processes and the formation of germ layers during vertebrate embryogenesis. Primary signals that have been identified as growth factors or growth factor-related ligands act as inducing factors on their target cells and, by a change of the genetic program, evoke a specification of the cellular differentiation pathways. While at present the signal transduction mechanisms leading from the ligands via cognate receptors to the nuclei are still poorly understood, there is growing information on transcription factors which are activated upon induction. They govern the expression of other regulatory molecules and co-ordinate the expression of cell type-specific structural genes. Meanwhile, it is generally accepted that development and cellular differentiation in all multicellular organisms depends upon a cascade of evolutionarily conserved transcription factors. Striking structural similarities within their DNA-binding domains allow many of these factors to be subdivided into different transcription factor families. Most of the basic knowledge on these factors emerged from the pioneering work done with Drosophila embryos which was greatly facilitated by the availability of numerous mutants. Despite the fact that Drosophila development until the blastoderm stage proceeds in a multinuclear syncytium and thus is significantly different from that in vertebrate organisms, the primary structures of many embryonic transcription factors have been conserved in higher organisms. This especially holds true for the various DNA binding motifs and it facilitated the isolation and characterization of vertebrate homologues to factors previously identified in lower organisms.

  11. Isoflurane anesthesia in the African clawed frog (Xenopus laevis).

    PubMed

    Smith, J M; Stump, K C

    2000-11-01

    Isoflurane is one of the safest and most accepted anesthetic agents for reptiles, birds, and mammals. It has also been used in terrestrial amphibians. The use of inhalation agents in an entirely aquatic frog presents a new dilemma for delivery in contrast to terrestrial species. The African Clawed Frog respires by using both transcutaneous gas exchange and air breathing. These frogs remain submerged for long periods of time, thus making standard inhalation techniques impractical. We tested five methods for delivering isoflurane: 1) bubbling isoflurane and oxygen in the water, 2) intracoelomic injection, 3) subcutaneous injection, 4) intramuscular injection, and 5) topical application. For the topical application, we developed a simple technique by using an absorptive pad with a vapor-barrier backing, saturating the pad with the liquid isoflurane, and placing the pad on the back of the frog while it was confined in a plastic bowl. Although two of the three injectable routes induced anesthesia, only the topical route produced rapid induction with consistent, safe recovery. Bubbling isoflurane with oxygen into water was unsuccessful. Topical application of isoflurane was most successful and appears to be a safe and practical method that can be used as an alternative to tricaine methylsulphonate, hypothermia, or other methods for anesthetizing African Clawed Frogs.

  12. Water transport by GLUT2 expressed in Xenopus laevis oocytes

    PubMed Central

    Zeuthen, Thomas; Zeuthen, Emil; MacAulay, Nanna

    2007-01-01

    The glucose transporter GLUT2 has been shown to also transport water. In the present paper we investigated the relation between sugar and water transport in human GLUT2 expressed in Xenopus oocytes. Sugar transport was determined from uptakes of non-metabolizable glucose analogues, primarily 3-O-methyl-d-glucopyranoside; key experimental results were confirmed using d(+)-glucose. Water transport was derived from changes in oocyte volume monitored at a high resolution (20 pl, 1 s). Expression of GLUT2 induced a sugar permeability, PS, of about 5 × 10−6 cm s−1 and a passive water permeability, Lp, of 5.5 × 10−5 cm s−1. Accordingly, the passive water permeability of a GLUT2 protein is about 10 times higher than its sugar permeability. Both permeabilities were abolished by phloretin. Isosmotic addition of sugar to the bathing solution (replacing mannitol) induced two parallel components of water influx in GLUT2, one by osmosis and one by cotransport. The osmotic driving force arose from sugar accumulation at the intracellular side of the membrane and was given by an intracellular diffusion coefficient for sugar of 10−6 cm2 s−1, one-fifth of the free solution value. The diffusion coefficient was determined in oocytes coexpressing GLUT2 and the water channel AQP1 where water transport was predominantly osmotic. By the cotransport mechanism about 35 water molecules were transported for each sugar molecule by a mechanism within the GLUT2. These water molecules could be transported uphill, against an osmotic gradient, energized by the flux of sugar. This capacity for cotransport is 10 times smaller than that of the Na+-coupled glucose transporters (SGLT1). The physiological role of GLUT2 for intestinal transport under conditions of high luminal sugar concentrations is discussed. PMID:17158169

  13. Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling.

    PubMed

    Morin, Ryan D; Chang, Elbert; Petrescu, Anca; Liao, Nancy; Griffith, Malachi; Chow, William; Kirkpatrick, Robert; Butterfield, Yaron S; Young, Alice C; Stott, Jeffrey; Barber, Sarah; Babakaiff, Ryan; Dickson, Mark C; Matsuo, Corey; Wong, David; Yang, George S; Smailus, Duane E; Wetherby, Keith D; Kwong, Peggy N; Grimwood, Jane; Brinkley, Charles P; Brown-John, Mabel; Reddix-Dugue, Natalie D; Mayo, Michael; Schmutz, Jeremy; Beland, Jaclyn; Park, Morgan; Gibson, Susan; Olson, Teika; Bouffard, Gerard G; Tsai, Miranda; Featherstone, Ruth; Chand, Steve; Siddiqui, Asim S; Jang, Wonhee; Lee, Ed; Klein, Steven L; Blakesley, Robert W; Zeeberg, Barry R; Narasimhan, Sudarshan; Weinstein, John N; Pennacchio, Christa Prange; Myers, Richard M; Green, Eric D; Wagner, Lukas; Gerhard, Daniela S; Marra, Marco A; Jones, Steven J M; Holt, Robert A

    2006-06-01

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization.

  14. Expression profiles of LHbeta, FSHbeta and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles.

    PubMed

    Urbatzka, R; Lorenz, C; Lutz, I; Kloas, W

    2010-09-01

    The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus-pituitary-gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHbeta and FSHbeta mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHbeta and FSHbeta mRNA was observed at late premetamorphosis. A clear raise of LHbeta mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHbeta mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHbeta and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus-pituitary-gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.

  15. Isolation and characterization of two alternatively spliced complementary DNAs encoding a Xenopus laevis angiotensin II receptor.

    PubMed

    Nishimatsu, S; Koyasu, N; Sugaya, T; Ohnishi, J; Yamagishi, T; Murakami, K; Miyazaki, H

    1994-08-02

    We have isolated two cDNAs of 1.7 and 3.0 kb, produced by alternative splicing, that encode a angiotensin II (AII) receptor from a Xenopus laevis heart cDNA library. The two clones had identical coding regions with each other and were found to belong to the G protein-coupled receptor superfamily like the mammalian type 1 AII receptors (AT1); their amino acid sequence was 68.7% homologous with the human AT1 receptor sequence. However, there was a 1.3 kb insertion at the 3'-untranslated region of the longer clone. The insertion contained 9 repeats of an ATTTA motif, suggesting that the two mRNAs undergo distinct post-transcriptional regulation by virtue of a difference in their stability. Although the Xenopus receptor exhibited distinct specificities for AII receptor antagonists compared with mammalian AII receptors, several common characteristics, including the effect of dithiothreitol and guanosine 5'-O-(3-thiotriphosphate), demonstrated that the cloned receptor is a counterpart of the mammalian AT1 receptor. Moreover, the cloned receptor was expressed most abundantly in the Xenopus heart, which is inconsistent with the tissue distribution of mammalian AII receptors. This indicated that the Xenopus heart, unlike that of mammals, plays a major role in the AII-dependent regulation of blood pressure and extracellular fluid volume.

  16. Does Atrazine Influence Larval Development and Sexual Differentiation in Xenopus laevis?

    PubMed Central

    Kloas, Werner; Lutz, Ilka; Springer, Timothy; Krueger, Henry; Wolf, Jeff; Holden, Larry; Hosmer, Alan

    2009-01-01

    Debate and controversy exists concerning the potential for the herbicide atrazine to cause gonadal malformations in developing Xenopus laevis. Following review of the existing literature the U.S. Environmental Protection Agency required a rigorous investigation conducted under standardized procedures. X. laevis tadpoles were exposed to atrazine at concentrations of 0.01, 0.1, 1, 25, or 100 μg/l from day 8 postfertilization (dpf) until completion of metamorphosis or dpf 83, whichever came first. Nearly identical experiments were performed in two independent laboratories: experiment 1 at Wildlife International, Ltd. and experiment 2 at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB). Both experiments employed optimized animal husbandry procedures and environmental conditions in validated flow-through exposure systems. The two experiments demonstrated consistent survival, growth, and development of X. laevis tadpoles, and all measured parameters were within the expected ranges and were comparable in negative control and atrazine-treated groups. Atrazine, at concentrations up to 100 μg/l, had no effect in either experiment on the percentage of males or the incidence of mixed sex as determined by histological evaluation. In contrast, exposure of larval X. laevis to 0.2 μg 17β-estradiol/l as the positive control resulted in gonadal feminization. Instead of an even distribution of male and female phenotypes, percentages of males:females:mixed sex were 19:75:6 and 22:60:18 in experiments 1 and 2, respectively. These studies demonstrate that long-term exposure of larval X. laevis to atrazine at concentrations ranging from 0.01 to 100 μg/l does not affect growth, larval development, or sexual differentiation. PMID:19008211

  17. Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

    NASA Technical Reports Server (NTRS)

    Danilchik, Michael V.; Bedrick, Steven D.; Brown, Elizabeth E.; Ray, Kimberly

    2003-01-01

    In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of

  18. Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

    NASA Technical Reports Server (NTRS)

    Danilchik, Michael V.; Bedrick, Steven D.; Brown, Elizabeth E.; Ray, Kimberly

    2003-01-01

    In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of

  19. The Role of Sdf-1α signaling in Xenopus laevis somite morphogenesis.

    PubMed

    Leal, Marisa A; Fickel, Sarah R; Sabillo, Armbien; Ramirez, Julio; Vergara, Hernando Martínez; Nave, Ceazar; Saw, Daniel; Domingo, Carmen R

    2014-04-01

    Stromal derived factor-1α (sdf-1α), a chemoattractant chemokine, plays a major role in tumor growth, angiogenesis, metastasis, and in embryogenesis. The sdf-1α signaling pathway has also been shown to be important for somite rotation in zebrafish (Hollway et al., 2007). Given the known similarities and differences between zebrafish and Xenopus laevis somitogenesis, we sought to determine whether the role of sdf-1α is conserved in Xenopus laevis. Using a morpholino approach, we demonstrate that knockdown of sdf-1α or its receptor, cxcr4, leads to a significant disruption in somite rotation and myotome alignment. We further show that depletion of sdf-1α or cxcr4 leads to the near absence of β-dystroglycan and laminin expression at the intersomitic boundaries. Finally, knockdown of sdf-1α decreases the level of activated RhoA, a small GTPase known to regulate cell shape and movement. Our results show that sdf-1α signaling regulates somite cell migration, rotation, and myotome alignment by directly or indirectly regulating dystroglycan expression and RhoA activation. These findings support the conservation of sdf-1α signaling in vertebrate somite morphogenesis; however, the precise mechanism by which this signaling pathway influences somite morphogenesis is different between the fish and the frog. Copyright © 2013 Wiley Periodicals, Inc.

  20. The role of Sdf-1α signaling in Xenopus laevis somite morphogenesis

    PubMed Central

    Leal, Marisa A.; Fickel, Sarah R.; Sabillo, Armbien; Ramirez, Julio; Vergara, Hernando Martínez; Nave, Ceazar; Saw, Daniel; Domingo, Carmen R.

    2014-01-01

    Background Stromal derived factor-1α (sdf-1α), a chemoattractant chemokine, plays a major role in tumor growth, angiogenesis, metastasis and in embryogenesis. The sdf-1α signaling pathway has also been shown to be important for somite rotation in zebrafish (Hollway, et al 2007). Given the known similarities and differences between zebrafish and Xenopus laevis somitogenesis, we sought to determine whether the role of sdf-1α is conserved in Xenopus laevis. Results Using a morpholino approach, we demonstrate that knockdown of sdf-1α or its receptor, cxcr4, leads to a significant disruption in somite rotation and myotome alignment. We further show that depletion of sdf-1α or cxcr4 leads to the near absence of β-dystroglycan and laminin expression at the intersomitic boundaries. Finally, knockdown of sdf-1α decreases the level of activated RhoA, a small GTPase known to regulate cell shape and movement. Conclusion Our results show that sdf-1α signaling regulates somite cell migration, rotation and myotome alignment by directly or indirectly regulating dystroglycan expression and RhoA activation. These findings support the conservation of sdf-1α signaling in vertebrate somite morphogenesis; however, the precise mechanism by which this signaling pathway influences somite morphogenesis is different between the fish and the frog. PMID:24357195

  1. In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

    PubMed

    Suzuki, Miyuki; Takagi, Chiyo; Miura, Shinichirou; Sakane, Yuto; Suzuki, Makoto; Sakuma, Tetsushi; Sakamoto, Naoaki; Endo, Tetsuya; Kamei, Yasuhiro; Sato, Yuko; Kimura, Hiroshi; Yamamoto, Takashi; Ueno, Naoto; Suzuki, Ken-ichi T

    2016-04-01

    Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.

  2. Development of Erythroid Progenitors under Erythropoietin Stimulation in Xenopus laevis Larval Liver.

    PubMed

    Okui, Takehito; Hosozawa, Sakiko; Kohama, Satoka; Fujiyama, Shingo; Maekawa, Shun; Muto, Hiroshi; Kato, Takashi

    2016-12-01

    Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre- (NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. β2-globin mRNA expression peaked at day 7 after the start of culture. Larval β2-globin was highly expressed in NF56-derived cells, while adult β2-globinwas detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of eporand gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

  3. Response of larval Xenopus laevis to atrazine: assessment of growth, metamorphosis, and gonadal and laryngeal morphology.

    PubMed

    Carr, James A; Gentles, Angie; Smith, Ernest E; Goleman, Wanda L; Urquidi, Lina J; Thuett, Kerry; Kendall, Ronald J; Giesy, John P; Gross, Tim S; Solomon, Keith R; Van Der Kraak, Glen

    2003-02-01

    Larval Xenopus laevis were exposed to one of four concentrations of atrazine (0, 1, 10, or 25 microg/L, 11 replicate tanks per treatment, 60-65 larvae per replicate) dissolved in an artificial pond water (frog embryo teratogenesis assay- Xenopus [FETAX]) medium beginning 48 h after hatching until the completion of metamorphosis. Separate groups of larvae (six replicate tanks per treatment, 60-65 larvae per replicate) were exposed to estradiol (100 microg/L), dihydrotestosterone (100 microg/L), or ethanol vehicle control dissolved in FETAX medium. None of the treatments affected posthatch mortality, larval growth, or metamorphosis. There were no treatment effects on sex ratios except for estradiol, which produced a greater percentage of female offspring. Exposure to either estradiol or 25 microg atrazine/L increased the incidence of intersex animals based on assessment of gonadal morphology. Atrazine did not reduce the size of the laryngeal dilator muscle, a sexually dimorphic muscle in this species. We conclude that environmentally relevant concentrations of atrazine do not influence metamorphosis or sex ratios and do not inhibit sexually dimorphic larynx growth in X. laevis. The incidence of atrazine-induced intersex animals was small (<5%) and occurred only at the greatest concentration of atrazine tested, a concentration that is rarely observed in surface waters in the United States.

  4. Entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

    SciTech Connect

    Kao, K.R.; Elinson, R.P.

    1988-05-01

    The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland. In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.

  5. Transgenic Xenopus laevis for live imaging in cell and developmental biology.

    PubMed

    Takagi, Chiyo; Sakamaki, Kazuhiro; Morita, Hitoshi; Hara, Yusuke; Suzuki, Makoto; Kinoshita, Noriyuki; Ueno, Naoto

    2013-05-01

    The stable transgenesis of genes encoding functional or spatially localized proteins, fused to fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), is an extremely important research tool in cell and developmental biology. Transgenic organisms constructed with fluorescent labels for cell membranes, subcellular organelles, and functional proteins have been used to investigate cell cycles, lineages, shapes, and polarity, in live animals and in cells or tissues derived from these animals. Genes of interest have been integrated and maintained in generations of transgenic animals, which have become a valuable resource for the cell and developmental biology communities. Although the use of Xenopus laevis as a transgenic model organism has been hampered by its relatively long reproduction time (compared to Drosophila melanogaster and Caenorhabditis elegans), its large embryonic cells and the ease of manipulation in early embryos have made it a historically valuable preparation that continues to have tremendous research potential. Here, we report on the Xenopus laevis transgenic lines our lab has generated and discuss their potential use in biological imaging.

  6. The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis.

    PubMed

    Jenks, Bruce G; Kuribara, Miyuki; Kidane, Adhanet H; Kramer, Bianca M R; Roubos, Eric W; Scheenen, Wim J J M

    2012-07-01

    Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in the mammalian pituitary gland is well documented its functional significance remains obscure. Studies on the pars intermedia of the pituitary of the amphibian Xenopus laevis have shown that BDNF is produced by the neuroendocrine melanotrope cells, its expression is physiologically regulated, and the melanotrope cells themselves express receptors for the neurotrophin. The neurotrophin has been shown to act as an autocrine factor on the melanotrope to promote cell growth and regulate gene expression. In doing so BDNF supports the physiological function of the cell to produce and release α-melanophore-stimulating hormone for the purpose of adjusting the animal's skin color to that of its background.

  7. Pan-African phylogeography of a model organism, the African clawed frog 'Xenopus laevis'.

    PubMed

    Furman, Benjamin L S; Bewick, Adam J; Harrison, Tia L; Greenbaum, Eli; Gvoždík, Václav; Kusamba, Chifundera; Evans, Ben J

    2015-02-01

    The African clawed frog Xenopus laevis has a large native distribution over much of sub-Saharan Africa and is a model organism for research, a proposed disease vector, and an invasive species. Despite its prominent role in research and abundance in nature, surprisingly little is known about the phylogeography and evolutionary history of this group. Here, we report an analysis of molecular variation of this clade based on 17 loci (one mitochondrial, 16 nuclear) in up to 159 individuals sampled throughout its native distribution. Phylogenetic relationships among mitochondrial DNA haplotypes were incongruent with those among alleles of the putatively female-specific sex-determining gene DM-W, in contrast to the expectation of strict matrilineal inheritance of both loci. Population structure and evolutionarily diverged lineages were evidenced by analyses of molecular variation in these data. These results further contextualize the chronology, and evolutionary relationships within this group, support the recognition of X. laevis sensu stricto, X. petersii, X. victorianus and herein revalidated X. poweri as separate species. We also propose that portions of the currently recognized distributions of X. laevis (north of the Congo Basin) and X. petersii (south of the Congo Basin) be reassigned to X. poweri.

  8. Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis.

    PubMed

    Wozniak, Katherine L; Mayfield, Brianna L; Duray, Alexis M; Tembo, Maiwase; Beleny, David O; Napolitano, Marc A; Sauer, Monica L; Wisner, Bennett W; Carlson, Anne E

    2017-01-01

    The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.

  9. Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis).

    PubMed

    Philips, Blythe H; Crim, Marcus J; Hankenson, F Claire; Steffen, Earl K; Klein, Peter S; Brice, Angela K; Carty, Anthony J

    2015-11-01

    Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibacterial efficacy and the gross and microscopic appearance of Xenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone-iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus.

  10. Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis)

    PubMed Central

    Philips, Blythe H; Crim, Marcus J; Hankenson, F Claire; Steffen, Earl K; Klein, Peter S; Brice, Angela K; Carty, Anthony J

    2015-01-01

    Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibacterial efficacy and the gross and microscopic appearance of Xenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone–iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus. PMID:26632790

  11. Inverse Effects on Growth and Development Rates by Means of Endocrine Disruptors in African Clawed Frog Tadpoles ("Xenopus Laevis")

    ERIC Educational Resources Information Center

    Hackney, Zachary Carl

    2007-01-01

    Previous work on fish, frogs, and salamanders, showed the ability for estrogen (EE2) and anthropogenic endocrine disruptors to skew sex ratios and cause hermaphrodism. This study addressed the effects of estrogens on growth and development rates of African clawed frog tadpoles ("Xenopus laevis") during their gender determination stages. The…

  12. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

    SciTech Connect

    Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

    1988-05-03

    DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

  13. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis

    PubMed Central

    Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G. John; Lillo, Francesco; De Villiers, F. André; Vogt, Solveig; De Busschere, Charlotte; Backeljau, Thierry; Rödder, Dennis

    2016-01-01

    By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species’ native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great

  14. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis.

    PubMed

    Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G John; Lillo, Francesco; De Villiers, F André; Vogt, Solveig; De Busschere, Charlotte; Backeljau, Thierry; Rödder, Dennis

    2016-01-01

    By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species' native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great Britain.

  15. Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

    PubMed Central

    Perlman, S. M.; Ford, P. J.; Rosbash, M. M.

    1977-01-01

    Complementary DNA transcribed from adult Xenopus laevis globin mRNA was used to assay ovary RNA from Xenopus for the presence of globin sequences by RNA·cDNA hybridization. These sequences are present at approximately the same concentration as the majority of poly(A)-containing ovary sequences. The sequences are also found at approximately 200,000 copies per cell in poly(A)-containing RNA extracted from mature oocytes. To rule out contamination of the oocytes with somatic cells, two additional experiments were performed. First, RNA isolated from ovulated unfertilized eggs, which are devoid of somatic cells, was also shown to contain the globin sequences. Second, globin mRNA was isolated from Xenopus tadpoles. Adult globin mRNA is free of the tadpole sequence and no homology was detected between adult and tadpoles globin RNA. The ovary was shown to contain tadpole globin RNA at nearly the same concentration as the adult sequences. Thus, the results cannot be explained by contamination with erythroid cells which should contain only the adult sequence. The swimming tadpole, which possesses an active circulatory system, was also assayed for the tadpole and adult globin sequences. Whereas the adult sequences are present at approximately the same concentration as in the mature oocyte, the concentration of the tadpole sequences increases at least 300-fold in the first 3 days following fertilization. PMID:269434

  16. Post-translational Regulation of Hexokinase Function and Protein Stability in the Aestivating Frog Xenopus laevis.

    PubMed

    Childers, Christine L; Storey, Kenneth B

    2016-02-01

    Xenopus laevis endure substantial dehydration which can impose hypoxic stress due to impaired blood flow. Tissues may increase reliance on anaerobic glycolysis for energy production making the regulation of hexokinase (HK) important. We investigated the enzymatic properties and phosphorylation state of purified HK from the muscle of control and dehydrated (30% total body water lost) frogs. Bioinformatic tools were also applied to analyze the structural implication of HK phosphorylation in silico. HK from the muscle of dehydrated frogs showed a significantly higher Vmax (3.4-fold) and Km for glucose (2.4-fold) compared with control HK but the Km for ATP was unaltered. HK from dehydrated frogs also showed greater phosphoserine content (20% increase) and lower phosphothreonine (22% decrease) content compared to control HK. Control HK had a higher melting temperature (Tm = 61.9 °C) than from dehydrated (Tm = 54.2 °C) frogs when thermostability was tested using differential scanning fluorimetry. In silico phosphorylation of a Xenopus HK caused alterations in active site binding, corroborating phosphorylation as the probable mechanism for kinetic regulation. Physiological consequences of dehydration-induced HK phosphorylation appear to facilitate glycolytic metabolism in hypoxic situations. Augmented HK function increases the ability of Xenopus to overcome compromised oxidative phosphorylation associated with ischemia during dehydration.

  17. Flow sensing in developing Xenopus laevis is disrupted by visual cues and ototoxin exposure.

    PubMed

    Simmons, Andrea Megela; Warnecke, Michaela; Vu, Thanh Thao; Smith, Andrew T Stevens

    2015-02-01

    We explored how lateral line cues interact with visual cues to mediate flow sensing behaviors in the nocturnal developing frog, Xenopus laevis, by exposing animals to current flows under different lighting conditions and after exposure to the ototoxin gentamicin. Under dark conditions, Xenopus tadpoles move downstream at the onset of current flow, then turn, and orient toward the direction of the flow with high accuracy. Postmetamorphic froglets also exhibit positive rheotaxis but with less accuracy and longer latency. The addition of discrete light cues to an otherwise dark environment disrupts rheotaxis and positioning. Orientation is less accurate, latency to orient is longer, and animals do not move as far downstream in the presence of light. Compared with untreated tadpoles tested in the dark, tadpoles exposed to gentamicin show less accurate rheotaxis with longer latency and do not move as far downstream in response to flow. These effects are compounded by the presence of light cues. The disruptive effects of light on flow sensing in Xenopus emphasize the disturbances to natural behaviors that may be produced by anthropogenic illumination in nocturnal habitats.

  18. Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development.

    PubMed

    Tecza, Aleksandra; Bugner, Verena; Kühl, Michael; Kühl, Susanne J

    2011-10-01

    pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)-based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross-rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.

  19. Adrenoreceptor-mediated modulation of the spinal locomotor pattern during swimming in Xenopus laevis tadpoles.

    PubMed

    Fischer, H; Merrywest, S D; Sillar, K T

    2001-03-01

    This study focused on the contribution of different adrenoreceptor subtypes to the modulation of fictive swimming activity in a relatively simple, yet intact, lower vertebrate system, the immobilized Xenopus laevis tadpole and explored their possible role in mediating the noradrenergic modulation of spinal motor networks. In Xenopus embryos, near the time of hatching, activation of alpha(1) adrenoreceptors increased the duration of episodes of fictive swimming, whilst in larvae, 24 h after hatching, they were decreased. Activation of alpha(2) adrenoreceptors, however, markedly reduced episode duration at both developmental stages. Cycle periods in both stages were increased by the activation of alpha(1) and/or alpha(2) receptor subclasses, whereas beta adrenoreceptors were not apparently involved in the modulation of cycle periods or the duration of swim episodes. However, both beta and alpha(1) receptor activation decreased the intersegmental delay in the head-to-tail propagation of swimming activity, while alpha(2) receptors did not influence these rostro-caudal delays. Activation of neither alpha, nor beta, receptor subclasses had any consistent effect on the duration of ventral motor bursts. Our findings suggest that noradrenergic modulation of the swim-pattern generator in Xenopus tadpoles is mediated through the activation of alpha and beta adrenoreceptors. In addition, activation of particular receptor subclasses might enable the selective modulation of either the segmental rhythm generating networks, the intersegmental coordination of those networks or control at both levels simultaneously.

  20. STRUCTURE AND FUNCTION OF THE MIDDLE EAR APPARATUS OF THE AQUATIC FROG, XENOPUS LAEVIS

    PubMed Central

    Mason, MJ; Wang, M; Narins, PM

    2010-01-01

    We report the results of anatomical and vibrometric studies of the middle ear of the African clawed frog, Xenopus laevis. The cartilaginous tympanic disk of Xenopus shows pronounced sexual dimorphism, that of male frogs being much larger than that of females, relative to body size. The stapes footplate, however, is not enlarged in males. The cucullaris muscle was found to insert on the stapes in frogs of both sexes. Using laser interferometry to examine the response of middle ear structures to airborne sound, the stapes footplate was found to vibrate close to 180° out-of-phase with the tympanic disk across a range of frequencies, this resembling the relationship between tympanic membrane and footplate movement previously described in ranid frogs. By contrast, whereas there is a pronounced difference in vibration velocity between tympanic membrane and footplate in ranids, the footplate vibration velocity in Xenopus was found to be similar to that of the tympanic disk. This may be interpreted as an adaptation to improve the detection of sound underwater. PMID:20953303

  1. Adipose tissue macrophages develop from bone marrow–independent progenitors in Xenopus laevis and mouse

    PubMed Central

    Hassnain Waqas, Syed F.; Noble, Anna; Hoang, Anh C.; Ampem, Grace; Popp, Manuela; Strauß, Sarah; Guille, Matthew; Röszer, Tamás

    2017-01-01

    ATMs have a metabolic impact in mammals as they contribute to metabolically harmful AT inflammation. The control of the ATM number may have therapeutic potential; however, information on ATM ontogeny is scarce. Whereas it is thought that ATMs develop from circulating monocytes, various tissue-resident Mϕs are capable of self-renewal and develop from BM-independent progenitors without a monocyte intermediate. Here, we show that amphibian AT contains self-renewing ATMs that populate the AT before the establishment of BM hematopoiesis. Xenopus ATMs develop from progenitors of aVBI. In the mouse, a significant amount of ATM develops from the yolk sac, the mammalian equivalent of aVBI. In summary, this study provides evidence for a prenatal origin of ATMs and shows that the study of amphibian ATMs can enhance the understanding of the role of the prenatal environment in ATM development. PMID:28642277

  2. Identification and characterization of a novel intelectin in the digestive tract of Xenopus laevis.

    PubMed

    Nagata, Saburo

    2016-06-01

    The intelectin (Intl) family is a group of secretory lectins in chordates that serve multiple functions, including innate immunity, through Ca(2+)-dependent recognition of carbohydrate chains. Although six Intl family lectins have so far been reported in Xenopus laevis, none have been identified in the intestine. Using a monoclonal antibody to the Xenopus embryonic epidermal lectin (XEEL or Intl-1), I identified cross-reactive proteins in the intestines. The proteins were purified by affinity chromatography on a galactose-Sepharose column and found to be oligomers consisting of N-glycosylated 39 kDa and 40.5 kDa subunit peptides. N-terminal amino acid sequencing of these peptides, followed by cDNA cloning, identified two novel Intls (designated Intl-3 and Intl-4) that showed 59-79% amino acid identities with known Xenopus Intl family proteins. From the amino acid sequence, immunoreactivity, and properties of the recombinant protein, Intl-3 was considered the intestinal lectin identified by the anti-XEEL antibody. The purified Intl-3 protein could potentially bind to Escherichia coli and its lipopolysaccharides (LPS), and to Staphylococcus aureus and its peptidoglycans, depending on Ca(2+). In addition, the Intl-3 protein agglutinated E. coli cells in the presence of Ca(2+). Intraperitoneal injection of LPS increased the intestinal and rectal contents of Intl-3 and XCL-1 (or 35K serum lectin) proteins within three days; however, unlike XCL-1, Intl-3 was detectable in neither the sera nor the other tissues regardless of LPS stimulation. Immunohistochemical analyses revealed accumulation of the Intl-3 protein in mucus secretory granules of intestinal goblet cells. The results of this study suggest that Xenopus Intl-3 is involved in the innate immune protection of the digestive tract against bacterial infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development

    PubMed Central

    Slater, Bethany J.; Liu, Karen J.; Kwan, Matthew D.; Quarto, Natalina; Longaker, Michael T.

    2009-01-01

    Background The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied. Methodology/Principal Findings We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (μCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13. Conclusion A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process. PMID:19156194

  4. Unequal contribution of native South African phylogeographic lineages to the invasion of the African clawed frog, Xenopus laevis, in Europe

    PubMed Central

    Courant, Julien; Herrel, Anthony; Rebelo, Rui; Rödder, Dennis; Measey, G. John; Backeljau, Thierry

    2016-01-01

    Due to both deliberate and accidental introductions, invasive African Clawed Frog (Xenopus laevis) populations have become established worldwide. In this study, we investigate the geographic origins of invasive X. laevis populations in France and Portugal using the phylogeographic structure of X. laevis in its native South African range. In total, 80 individuals from the whole area known to be invaded in France and Portugal were analysed for two mitochondrial and three nuclear genes, allowing a comparison with 185 specimens from the native range. Our results show that native phylogeographic lineages have contributed differently to invasive European X. laevis populations. In Portugal, genetic and historical data suggest a single colonization event involving a small number of individuals from the south-western Cape region in South Africa. In contrast, French invasive X. laevis encompass two distinct native phylogeographic lineages, i.e., one from the south-western Cape region and one from the northern regions of South Africa. The French X. laevis population is the first example of a X. laevis invasion involving multiple lineages. Moreover, the lack of population structure based on nuclear DNA suggests a potential role for admixture within the invasive French population. PMID:26855879

  5. Unequal contribution of native South African phylogeographic lineages to the invasion of the African clawed frog, Xenopus laevis, in Europe.

    PubMed

    De Busschere, Charlotte; Courant, Julien; Herrel, Anthony; Rebelo, Rui; Rödder, Dennis; Measey, G John; Backeljau, Thierry

    2016-01-01

    Due to both deliberate and accidental introductions, invasive African Clawed Frog (Xenopus laevis) populations have become established worldwide. In this study, we investigate the geographic origins of invasive X. laevis populations in France and Portugal using the phylogeographic structure of X. laevis in its native South African range. In total, 80 individuals from the whole area known to be invaded in France and Portugal were analysed for two mitochondrial and three nuclear genes, allowing a comparison with 185 specimens from the native range. Our results show that native phylogeographic lineages have contributed differently to invasive European X. laevis populations. In Portugal, genetic and historical data suggest a single colonization event involving a small number of individuals from the south-western Cape region in South Africa. In contrast, French invasive X. laevis encompass two distinct native phylogeographic lineages, i.e., one from the south-western Cape region and one from the northern regions of South Africa. The French X. laevis population is the first example of a X. laevis invasion involving multiple lineages. Moreover, the lack of population structure based on nuclear DNA suggests a potential role for admixture within the invasive French population.

  6. Neurotransmitter signaling pathways required for normal development in Xenopus laevis embryos: a pharmacological survey screen

    PubMed Central

    Sullivan, Kelly G.; Levin, Michael

    2016-01-01

    Neurotransmitters are not only involved in brain function but are also important signaling molecules for many diverse cell types. Neurotransmitters are widely conserved, from evolutionarily ancient organisms lacking nervous systems through man. Here, we report results from a loss- and gain-of-function survey, using pharmacologic modulators of several neurotransmitter pathways to examine possible roles in normal embryogenesis. Applying reagents targeting the glutamatergic, adrenergic, and dopaminergic pathways to embryos of Xenopus laevis from gastrulation to organogenesis stages, we observed and quantified numerous malformations including craniofacial defects, hyperpigmentation, muscle mispatterning, and miscoiling of the gut. These data implicate several key neurotransmitters in new embryonic patterning roles, reveal novel earlier stages for processes involved in eye development, suggest new targets for subsequent molecular-genetic investigation, and highlight the necessity for in-depth toxicology studies of psychoactive compounds to which human embryos might be exposed during pregnancy. PMID:27060969

  7. Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

    PubMed Central

    Rivera-Acevedo, Ricardo E.; Pless, Stephan A.; Schwarz, Stephan K.W.; Ahern, Christopher A.

    2013-01-01

    Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing a pathway for large and otherwise relatively impermeant molecules. Further, we have shown recently that these nonselective cation channels, when activated by capsaicin, are potently and reversibly blocked by external application of quaternary ammonium compounds and local anesthetics. Here we describe a novel phenomenon in transient receptor potential channel pharmacology whereby their expression levels in Xenopus laevis oocytes, as assessed by the magnitude of macroscopic currents, are negatively correlated with extracellular blocker affinity: small current densities give rise to nanomolar blockade by quaternary ammoniums and this affinity decreases linearly as current density increases. Possible mechanisms to explain these data are discussed in light of similar observations in other channels and receptors. PMID:23428812

  8. Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

    PubMed Central

    Sato, Ken-ichi

    2014-01-01

    Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm)” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal. PMID:25546390

  9. Light conditions affect the roll-induced vestibuloocular reflex in Xenopus laevis tadpoles

    NASA Astrophysics Data System (ADS)

    El-Yamany, Nabil A.

    2008-12-01

    In Xenopus laevis tadpoles, effects of asymmetrical light conditions on the roll-induced vestibuloocular reflex (rVOR) were tested for the developmental period between stage 47 and 49. For comparison, the rVOR was tested in dim- and high-symmetrical light environments. Test parameters were the rVOR gain and rVOR amplitude. Under all light conditions, the rVOR increased from tadpole stage 47 to 49. For all stages, the asymmetrical light field induced the strongest response, the dim light field the weakest one. The response for the left and right eye was identical, even if the tadpoles were tested under asymmetrical light conditions. The experiments can be considered as hints (1) for an age-dependent light sensitivity of vestibular neurons, and (2) for the existence of control systems for coordinated eye movements that has its origin in the proprioceptors of the extraocular eye muscles.

  10. Metabolic Regulation of CaMKII Protein and Caspases in Xenopus laevis Egg Extracts*

    PubMed Central

    McCoy, Francis; Darbandi, Rashid; Chen, Si-Ing; Eckard, Laura; Dodd, Keela; Jones, Kelly; Baucum, Anthony J.; Gibbons, Jennifer A.; Lin, Sue-Hwa; Colbran, Roger J.; Nutt, Leta K.

    2013-01-01

    The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways. PMID:23400775

  11. Titration of four replication factors is essential for the Xenopus laevis midblastula transition.

    PubMed

    Collart, Clara; Allen, George E; Bradshaw, Charles R; Smith, James C; Zegerman, Philip

    2013-08-23

    The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors--Cut5, RecQ4, Treslin, and Drf1--are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

  12. D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

    PubMed

    Soma, Hiroki; Furuya, Ryuji; Kaneko, Ryo; Tsukamoto, Ayaka; Shirasu, Kazumitsu; Tanigawa, Minoru; Nagata, Yoko

    2013-10-01

    We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

  13. Optical coherence tomography for detection of compound action potential in Xenopus Laevis sciatic nerve

    NASA Astrophysics Data System (ADS)

    Troiani, Francesca; Nikolic, Konstantin; Constandinou, Timothy G.

    2016-03-01

    Due to optical coherence tomography (OCT) high spatial and temporal resolution, this technique could be used to observe the quick changes in the refractive index that accompany action potential. In this study we explore the use of time domain Optical Coherence Tomography (TD-OCT) for real time action potential detection in ex vivo Xenopus Laevis sciatic nerve. TD-OCT is the easiest and less expensive OCT technique and, if successful in detecting real time action potential, it could be used for low cost monitoring devices. A theoretical investigation into the order of magnitude of the signals detected by a TD-OCT setup is provided by this work. A linear dependence between the refractive index and the intensity changes is observed and the minimum SNR for which the setup could work is found to be SNR = 2 x 104.

  14. Tail structure is formed when blastocoel roof contacts blastocoel floor in Xenopus laevis.

    PubMed

    Nishihara, Akiha; Hashimoto, Chikara

    2014-04-01

    The tail organizer has been assessed by such transplantation methods as the Einsteck procedure. However, we found that simple wounding of blastocoel roof (BCR) made it possible to form secondary tails without any transplantation in Xenopus laevis. We revealed that the ectopic expression of Xbra was blocked by inhibiting the contact between BCR and blastocoel floor (BCF), and wounding per se seemed to be not directly related to the secondary tail formation. Therefore, the secondary tail might be induced by the contact between BCR and BCF due to the leak of blastocoel fluid from the wound. This secondary tail was similar to the original tail in the expression pattern of tail genes, and in the fact that the inhibition of fibroblast growth factor signaling prevented the secondary tail induction. Our results imply that the secondary tail formation reflects the developmental processes of the original tail, indicating that simple wounding of BCR is useful for the analysis of tail formation in normal development.

  15. Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts.

    PubMed

    Hara, Yuki; Merten, Christoph A

    2015-06-08

    Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion.

  16. Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry

    PubMed Central

    Flachsova, Monika; Sindelka, Radek; Kubista, Mikael

    2013-01-01

    We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the 8 to 32-cell Xenopus laevis embryos to determine when and how asymmetry in the body plan is introduced. We demonstrate that the asymmetry along the animal-vegetal axis in the oocyte is transferred to the daughter cells during early cell divisions. All studied mRNAs are distributed evenly among the set of animal as well as vegetal blastomeres. We find no asymmetry in mRNA levels that might be ascribed to the dorso-ventral specification or the left-right axis formation. We hypothesize that while the animal-vegetal asymmetry is a consequence of mRNA gradients, the dorso-ventral and left-right axes specifications are induced by asymmetric distribution of other biomolecules, probably proteins. PMID:23880666

  17. Planar induction of anteroposterior pattern in the developing central nervous system of Xenopus laevis

    NASA Technical Reports Server (NTRS)

    Doniach, T.; Phillips, C. R.; Gerhart, J. C.

    1992-01-01

    It has long been thought that anteroposterior (A-P) pattern in the vertebrate central nervous system is induced in the embryo's dorsal ectoderm exclusively by signals passing vertically from underlying, patterned dorsal mesoderm. Explants from early gastrulae of the frog Xenopus laevis were prepared in which vertical contact between dorsal ectoderm and mesoderm was prevented but planar contact was maintained. In these, four position-specific neural markers (engrailed-2, Krox-20, XlHbox 1, and XlHbox 6) were expressed in the ectoderm in the same A-P order as in the embryo. Thus, planar signals alone, following a path available in the normal embryo, can induce A-P neural pattern.

  18. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

    PubMed Central

    Strittmatter, S M; Cannon, S C; Ross, E M; Higashijima, T; Fishman, M C

    1993-01-01

    The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction. Images Fig. 1 Fig. 2 PMID:7685122

  19. Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

    PubMed

    Slaby, Sylvain; Lemière, Sébastien; Hanotel, Julie; Lescuyer, Arlette; Demuynck, Sylvain; Bodart, Jean-François; Leprêtre, Alain; Marin, Matthieu

    2016-08-01

    Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

  20. Correlative light and electron microscopy for a free-floating spindle in Xenopus laevis egg extracts.

    PubMed

    Tranfield, Erin M; Heiligenstein, Xavier; Peristere, Ina; Antony, Claude

    2014-01-01

    Cryoimmobilization is an optimal method of preserving sample ultrastructure in electron microscopy studies. However, cryoimmobilization is limited to thin samples and this limitation may necessitate the isolation of the structure of interest. For cellular structures that are found in low number, or only during certain phases of the cell cycle, an added benefit of isolation is the possibility to concentrate the structures. We developed a method to perform correlative light and electron microscopy on infrequent isolated subcellular structures. In this chapter, we will describe our protocol that uses a combination of existing techniques and new solutions for the isolation, identification, cryoimmobilization, targeted ultramicrotomy, and imaging of the free-floating meiotic spindles assembled in Xenopus laevis egg extract. © 2014 Elsevier Ltd. All rights reserved.

  1. Planar induction of anteroposterior pattern in the developing central nervous system of Xenopus laevis

    NASA Technical Reports Server (NTRS)

    Doniach, T.; Phillips, C. R.; Gerhart, J. C.

    1992-01-01

    It has long been thought that anteroposterior (A-P) pattern in the vertebrate central nervous system is induced in the embryo's dorsal ectoderm exclusively by signals passing vertically from underlying, patterned dorsal mesoderm. Explants from early gastrulae of the frog Xenopus laevis were prepared in which vertical contact between dorsal ectoderm and mesoderm was prevented but planar contact was maintained. In these, four position-specific neural markers (engrailed-2, Krox-20, XlHbox 1, and XlHbox 6) were expressed in the ectoderm in the same A-P order as in the embryo. Thus, planar signals alone, following a path available in the normal embryo, can induce A-P neural pattern.

  2. Asymmetric Localization and Distribution of Factors Determining Cell Fate During Early Development of Xenopus laevis.

    PubMed

    Sindelka, Radek; Sidova, Monika; Abaffy, Pavel; Kubista, Mikael

    2017-01-01

    Asymmetric division is a property of eukaryotic cells that is fundamental to the formation of higher life forms. Despite its importance, the mechanism behind it remains elusive. Asymmetry in the cell is induced by polarization of cell fate determinants that become unevenly distributed among progeny cells. So far dozens of determinants have been identified. Xenopus laevis is an ideal system to study asymmetric cell division during early development, because of the huge size of its oocytes and early-stage blastomeres. Here, we present the current knowledge about localization and distribution of cell fate determinants along the three body axes: animal-vegetal, dorsal-ventral, and left-right. Uneven distribution of cell fate determinants during early development specifies the formation of the embryonic body plan.

  3. Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells.

    PubMed

    Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio; Zuñiga, Nikole; Cline, Hollis; Larraín, Juan

    2015-12-15

    Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2(/)3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2(/)3(+) cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2(/)3(+) neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism

  4. [Identification of a Novel Calcium (Ca^(2+))-Activated Chloride Channel Accessory Gene in Xenopus laevis].

    PubMed

    Lee, R M; Jeong, S M

    2016-01-01

    Calcium (Ca^(2+))-activated chloride channel accessories (CLCAs) are putative anion channel-related proteins with diverse physiological functions. Exploring CLCA diversity is important for prediction of gene structure and function. In an effort to identify novel CLCA genes in Xenopus laevis, we successfully cloned and characterized a Xenopus laevis cDNA predicted to encode the xCLCA3 gene. Cloning of xCLCA3 was achieved by computational analysis, rapid amplification of cDNA ends (RACE), and a tissue distribution analysis by semi-quantitative reverse transcription (RT) PCR or real-time PCR. We obtained a 2958 bp xCLCA3 cDNA sequence with an open reading frame encoding 943 amino acids. According to the primary structure analysis, xCLCA3 contains a predicted signal sequence, multiple sites of N-linked (N-) glycosylation, N-myristoylation, PKA, PKC, and casein kinase II phosphorylation sites, five putative hydrophobic segments, and the HExxH metalloprotease motif. Additionally, the transmembrane prediction server yielded a preserved N-terminal CLCA domain and a von Willebrand factor type A domain with one transmembrane domain in the C-terminal region. Expression analysis showed that xCLCA3 is expressed in a number of tissues, with strong expression in the brain, colon, small intestine, lung, kidney, and spleen, and poor expression in the heart and liver. These results suggest that xCLCA3 may be a candidate CLCA family member as well as a metalloprotease, rather than just an ion channel accessory protein.

  5. A comparison of electronic and traditional cigarette butt leachate on the development of Xenopus laevis embryos.

    PubMed

    Parker, Tatiana Tatum; Rayburn, James

    2017-01-01

    Potential developmental toxicities of three different cigarette butt leachates were evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX). Xenopus laevis embryos were exposed to regular cigarette butt (RCB), menthol (MCB) and electronic (ECB) in concentrations ranging from 0 to 4 butts/l for RCB and MCB and 0-10 butts/l for ECB. The embryos were from stage 8 to 11 and were exposed for a 96-h period in static renewal test conditions. Median lethal concentration (LC50), malformation (EC50), non-observed adverse effect concentration (NOAEC), and lowest observed adverse effect concentration (LOAEC) were calculated. Results from these studies suggest that each tested leachate is teratogenic for X. laevis embryos. The lowest LC50 was determined for ECB exposure at 17.9 cigarette butts/L. The LC50 value was the highest with RCB and MCB having LC50 s of approximately 1 cigarette butt/L. There were notable EC50 differences with RCB having the highest and ECB the lowest. The NOAEC and LOAEC levels for RCB and MCB were below 1 cigarette butt/L for both mortality and malformations; over 8 butts/L for ECB mortality and over 4 butts/L for malformations. From these results, we conclude that RCB leachate is the most toxic compound, while MCB leachate has the higher teratogenicity. ECB leachate has the lowest toxic and teratogenic effects on embryos but there were still noticeable effects. The results confirmed that the FETAX assay can be useful in an integrated biological hazard assessment for the preliminary screening for ecological risks of cigarette butts, and electronic cigarettes, in aquatic environment.

  6. Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells

    PubMed Central

    Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio; Zuñiga, Nikole; Cline, Hollis; Larraín, Juan

    2016-01-01

    Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2/3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2/3+ cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2/3+ neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism that is

  7. Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

    SciTech Connect

    Bahouth, S.W.; Malbon, C.C.

    1987-05-01

    Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in the control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.

  8. Characterization of cadmium chloride-induced BiP accumulation in Xenopus laevis A6 kidney epithelial cells.

    PubMed

    Shirriff, Cody S; Heikkila, John J

    2017-01-01

    Endoplasmic reticulum (ER) stress can result in the accumulation of unfolded/misfolded protein in the ER lumen, which can trigger the unfolded protein response (UPR) resulting in the activation of various genes including immunoglobulin-binding protein (BiP; also known as glucose-regulated protein 78 or HSPA5). BiP, an ER heat shock protein 70 (HSP70) family member, binds to unfolded protein, inhibits their aggregation and re-folds them in an ATP-dependent manner. While cadmium, an environmental contaminant, was shown to induce the accumulation of HSP70 in vertebrate cells, less information is available regarding the effect of this metal on BiP accumulation or function. In this study, cadmium chloride treatment of Xenopus laevis A6 kidney epithelial cells induced a dose- and time-dependent increase in BiP, HSP70 and heme oxygenase-1 (HO-1) accumulation. Exposure of cells to a relatively low cadmium concentration at a mild heat shock temperature of 30°C greatly enhanced BiP and HSP70 accumulation compared to cadmium at 22°C. Treatment of cells with the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced cadmium-induced BiP and HSP70 accumulation. Immunocytochemistry revealed that cadmium-induced BiP accumulation occurred in a punctate pattern in the perinuclear region. In some cells treated with cadmium chloride or the proteasomal inhibitor, MG132, large BiP complexes were observed that co-localized with aggregated protein or aggresome-like structures. These BiP/aggresome-like structures were also observed in cells treated simultaneously with cadmium at 30°C or in the presence of buthionine sulfoximine. In amphibians, the association of BiP with unfolded protein and its possible role in aggresome function may be vital in the maintenance of cellular proteostasis.

  9. Ontogeny of hypoxic modulation of cardiac performance and its allometry in the African clawed frog Xenopus laevis.

    PubMed

    Pan, T-C Francis; Burggren, Warren W

    2013-01-01

    The ontogeny of cardiac hypoxic responses, and how such responses may be modified by rearing environment, are poorly understood in amphibians. In this study, cardiac performance was investigated in Xenopus laevis from 2 to 25 days post-fertilization (dpf). Larvae were reared under either normoxia or moderate hypoxia (PO₂ = 110 mmHg), and each population was assessed in both normoxia and acute hypoxia. Heart rate (f(H)) of normoxic-reared larvae exhibited an early increase from 77 ± 1 beats min⁻¹ at 2 dpf to 153 ± 1 beats min⁻¹ at 4 dpf, followed by gradual decreases to 123 ± 3 beats min⁻¹ at 25 dpf. Stroke volume (SV), 6 ± 1 nl, and cardiac output (CO), 0.8 ± 0.1 μl min⁻¹, at 5 dpf both increased by more than 40-fold to 25 dpf with rapid larval growth (~30-fold increase in body mass). When exposed to acute hypoxia, normoxic-reared larvae increased f(H) and CO between 5 and 25 dpf. Increased SV in acute hypoxia, produced by increased end-diastolic volume (EDV), only occurred before 10 dpf. Hypoxic-reared larvae showed decreased acute hypoxic responses of EDV, SV and CO at 7 and 10 dpf. Over the period of 2-25 dpf, cardiac scaling with mass showed scaling coefficients of -0.04 (f(H)), 1.23 (SV) and 1.19 (CO), contrary to the cardiac scaling relationships described in birds and mammals. In addition, f(H) scaling in hypoxic-reared larvae was altered to a shallower slope of -0.01. Collectively, these results indicate that acute cardiac hypoxic responses develop before 5 dpf. Chronic hypoxia at a moderate level can not only modulate this cardiac reflex, but also changes cardiac scaling relationship with mass.

  10. Efficacy of tricaine methanesulfonate (MS-222) as an anesthetic agent for blocking sensory-motor responses in Xenopus laevis tadpoles.

    PubMed

    Ramlochansingh, Carlana; Branoner, Francisco; Chagnaud, Boris P; Straka, Hans

    2014-01-01

    Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222) is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes.

  11. Aqueous leaf extracts display endocrine activities in vitro and disrupt sexual differentiation of male Xenopus laevis tadpoles in vivo.

    PubMed

    Hermelink, Björn; Urbatzka, Ralph; Wiegand, Claudia; Pflugmacher, Stephan; Lutz, Ilka; Kloas, Werner

    2010-09-01

    The occurrence of natural substances acting as endocrine disrupting compounds (EDC) in the environment is to date poorly understood. Therefore, (anti)androgenic and (anti)estrogenic activities of three different aqueous leaf extracts (beech, reed and oak) were analyzed in vitro using yeast androgen and estrogen screen. The most potent extract was selected for in vivo exposure of Xenopus laevis tadpoles to analyze the potential effects on development and reproductive biology of amphibians. Tadpoles were exposed from stage 48 to stage 66 (end of metamorphosis) to aqueous oak leaf extracts covering natural occurring environmental concentrations of dissolved organic carbon. Gene expression analyses of selected genes of the hypothalamus-pituitary-gonad and of the hypothalamus-pituitary-thyroid axis as well as histological investigation of gonads and thyroid glands were used to evaluate endocrine disrupting effects on the reproductive biology and development. Female tadpoles remained unaffected by the exposure whereas males showed severe significant histological alterations of testes at the two highest oak leaf extract concentrations demonstrated by the occurrence of lacunae and oogonia. In addition, a significant elevation of luteinizing hormone beta mRNA expression with increasing extract concentration in male tadpoles indicates an involvement of hypothalamus-pituitary-gonad axis mainly via antiandrogenic activity. These results suggest that antiandrogenic EDC of oak leaf extract are responsible for inducing the observed effects in male tadpoles. The present study demonstrates for the first time that in surface waters, natural occurring oak leaf compounds at environmentally relevant concentrations display antiandrogenic activities and have considerable effects on the endocrine system of anurans affecting sexual differentiation of male tadpoles.

  12. Proteomics analysis of Xenopus laevis gonad tissue following chronic exposure to atrazine.

    PubMed

    Chen, Xiuping; Wang, Jiamei; Zhu, Haojun; Ding, Jiatong; Peng, Yufa

    2015-08-01

    Atrazine is the most commonly detected pesticide contaminant in ground and surface water. Previous studies have shown that atrazine is an endocrine disruptor owing to its adverse effects on the male reproductive system in several vertebrates, but very few molecular mechanisms for these effects have been revealed. In the present study, Xenopus laevis were exposed to 100 ppb of atrazine for 120 d, and then the isobaric tags for relative and absolute quantitation (iTRAQ) technique was used to detect global changes in protein profiles of the testes and ovaries. The results showed that 100 ppb of atrazine exposure adversely affected the growth of X. laevis and did not induce hermaphroditism but delayed or prevented the development of male seminiferous tubules. Proteomic analysis showed that atrazine altered expression of 143 and 121 proteins in the testes and ovaries, respectively, and most of them are involved in cellular and metabolic processes and biological regulation based on their biological processes. In addition, apoptosis, tight junctions, and metabolic pathways were significantly altered in the atrazine-treated gonads. Based on the above results, it is postulated that the reproductive toxicity of atrazine may be the result of disruption of tight junctions and metabolic signaling pathways and/or induction of apoptosis in germ cells. © 2015 SETAC.

  13. Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis.

    PubMed

    Voglmeir, Josef; Laurent, Nicolas; Flitsch, Sabine L; Oelgeschläger, Michael; Wilson, Iain B H

    2015-02-01

    The biosynthesis of mucin-type O-linked glycans in animals is initiated by members of the large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which play important roles in embryogenesis, organogenesis, adult tissue homeostasis and carcinogenesis. Until now, the mammalian forms of these enzymes have been the best characterized. However, two N-acetylgalactosaminyltransferases (xGalNAc-T6 and xGalNAc-T16) from the African clawed frog (Xenopus laevis), which are most homologous to those encoded by the human GALNT6 and GALNT16 (GALNTL1) genes, were shown to have contrasting roles in TGF-β/BMP signaling in embryogenesis. In this study we have examined these two enzymes further and show differences in their in vivo function during X. laevis embyrogenesis as evidenced by in situ hybridization and overexpression experiments. In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling. In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo.

  14. NF2/Merlin is required for the axial pattern formation in the Xenopus laevis embryo.

    PubMed

    Zhu, Xuechen; Min, Zheying; Tan, Renbo; Tao, Qinghua

    2015-11-01

    The NF2 gene product Merlin is a FERM-domain protein possessing a broad tumor-suppressing function. NF2/Merlin has been implicated in regulating multiple signaling pathways critical for cell growth and survival. However, it remains unknown whether NF2/Merlin regulates Wnt/β-catenin signaling during vertebrate embryogenesis. Here we demonstrate that NF2/Merlin is required for body pattern formation in the Xenopus laevis embryo. Depletion of the maternal NF2/Merlin enhances organizer gene expression dependent on the presence of β-catenin, and causes dorsanteriorized development; Morpholino antisense oligo-mediated knockdown of the zygotic NF2/Merlin shifts posterior genes anteriorwards and reduces the anterior development. We further demonstrate that targeted depletion of NF2 in the presumptive dorsal tissues increases the levels of nuclear β-catenin in the neural epithelial cells. Biochemical analyses reveal that NF2 depletion promotes the production of active β-catenin and concurrently decreases the level of N-terminally phosphorylated β-catenin under the stimulation of the endogenous Wnt signaling. Our findings suggest that NF2/Merlin negatively regulates the Wnt/β-catenin signaling activity during the pattern formation in early X. laevis embryos.

  15. CHROMATIN ASSEMBLY AND TRANSCRIPTIONAL CROSS-TALK IN XENOPUS LAEVIS OOCYTE AND EGG EXTRACTS

    PubMed Central

    Wang, Wei-Lin; Shechter, David

    2016-01-01

    Chromatin, primarily a complex of DNA and histone proteins, is the physiological form of the genome. Chromatin is generally repressive for transcription and other information transactions that occur on DNA. A wealth of post-translational modifications on canonical histones and histone variants encode regulatory information to recruit or repel effector proteins on chromatin, promoting and further repressing transcription and thereby form the basis of epigenetic information. During metazoan oogenesis, large quantities of histone proteins are synthesized and stored in preparation for the rapid early cell cycles of development and to elicit maternal control of chromatin assembly pathways. Oocyte and egg cell-free extracts of the frog Xenopus laevis are a compelling model system for the study of chromatin assembly and transcription precisely because they exist in an extreme state primed for rapid chromatin assembly or for transcriptional activity. We show that chromatin assembly rates are slower in X. laevis oocyte than in egg extracts, while conversely only oocyte extracts transcribe template plasmids. We demonstrate that rapid chromatin assembly in egg extracts represses RNA Polymerase II dependent transcription, while pre-binding of TATA-Binding Protein (TBP) to a template plasmid promotes transcription. Our experimental evidence presented here supports a model in which chromatin assembly and transcription are in competition and that the onset of zygotic genomic activation may be in part due to stable transcriptional complex assembly. PMID:27759158

  16. Distribution of Single-Wall Carbon Nanotubes in the Xenopus laevis Embryo after Microinjection

    PubMed Central

    Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

    2016-01-01

    Single-wall carbon nanotubes (SWCNTs) are advanced materials with the potential for a myriad of diverse applications, including biological technologies and largescale usage with the potential for environmental impacts. SWCNTs have been exposed to developing organisms to determine their effects on embryogenesis, and results have been inconsistent arising, in part, from differing material quality, dispersion status, material size, impurity from catalysts, and stability. For this study, we utilized highly purified SWCNT samples with short, uniform lengths (145 ± 17 nm) well dispersed in solution. To test high exposure doses, we microinjected > 500 μg mL-1 SWCNT concentrations into the well-established embryogenesis model, Xenopus laevis, and determined embryo compatibility and sub-cellular localization during development. SWCNTs localized within cellular progeny of the microinjected cells, but heterogeneously distributed throughout the target-injected tissue. Co-registering unique Raman spectral intensity of SWCNTs with images of fluorescently labelled sub-cellular compartments demonstrated that even at the regions of highest SWCNT concentration, there were no gross alterations to sub-cellular microstructures, including filamentous actin, endoplasmic reticulum and vesicles. Furthermore, SWCNTs did not aggregate or localize to the perinuclear sub-cellular region. Combined, these results suggest that purified and dispersed SWCNTs are not toxic to X. laevis animal cap ectoderm and may be suitable candidate materials for biological applications. PMID:26510384

  17. Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

    PubMed Central

    Nagasawa, Kazumichi; Tanizaki, Yuta; Okui, Takehito; Watarai, Atsuko; Ueda, Shinobu; Kato, Takashi

    2013-01-01

    Summary The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism. PMID:24167716

  18. Distribution of single wall carbon nanotubes in the Xenopus laevis embryo after microinjection.

    PubMed

    Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

    2016-04-01

    Single wall carbon nanotubes (SWCNTs) are advanced materials with the potential for a myriad of diverse applications, including biological technologies and large-scale usage with the potential for environmental impacts. SWCNTs have been exposed to developing organisms to determine their effects on embryogenesis, and results have been inconsistent arising, in part, from differing material quality, dispersion status, material size, impurity from catalysts and stability. For this study, we utilized highly purified SWCNT samples with short, uniform lengths (145 ± 17 nm) well dispersed in solution. To test high exposure doses, we microinjected > 500 µg ml(-1) SWCNT concentrations into the well-established embryogenesis model, Xenopus laevis, and determined embryo compatibility and subcellular localization during development. SWCNTs localized within cellular progeny of the microinjected cells, but were heterogeneously distributed throughout the target-injected tissue. Co-registering unique Raman spectral intensity of SWCNTs with images of fluorescently labeled subcellular compartments demonstrated that even at regions of highest SWCNT concentration, there were no gross alterations to subcellular microstructures, including filamentous actin, endoplasmic reticulum and vesicles. Furthermore, SWCNTs did not aggregate and localized to the perinuclear subcellular region. Combined, these results suggest that purified and dispersed SWCNTs are not toxic to X. laevis animal cap ectoderm and may be suitable candidate materials for biological applications.

  19. Hormone-sensitive stages in the sexual differentiation of laryngeal muscle fiber number in Xenopus laevis

    PubMed Central

    Marin, Melanie L.; Tobias, Martha L.

    2012-01-01

    Summary The number of muscle fibers in the vocal organ of the adult male African clawed frog, Xenopus laevis, exceeds that of adult females. This sex difference is the result of rapid fiber addition in males between the end of metamorphosis, post-metamorphic stage 0 (PM0) and PM2. At PM0, male and female frogs have similar numbers of laryngeal muscle fibers. Males then add more muscle fibers than females and achieve an adult value that is 1.7 times the female number. Males castrated at PM0 have the same fiber number as females. Ovariectomy at PM0 does not alter muscle fiber addition in females. Gonadectomy at PM2 has no effect on fiber addition in either sex. Females attain masculine muscle fiber number if their ovaries are replaced with a testis at metamorphosis. Exogenous testosterone treatment at PM0 significantly increases fiber number in females but not in males. Exogenous testosterone given at PM2 has no effect on fiber number in females but decreases fiber number in males. We conclude that the testes are necessary for the marked addition of laryngeal muscle fibers seen in male X. laevis between PM0 and PM2. The masculine pattern of muscle fiber addition can be induced in females provided with a testis. Androgen secretion from the testes most probably accounts for masculinization of laryngeal muscle fiber number. After PM2, androgens are no longer necessary for muscle fiber addition and cannot increase fiber number in females. PMID:2088715

  20. Expression of cardiac sarcolemmal Na sup + -Ca sup 2+ exchange activity in Xenopus laevis oocytes

    SciTech Connect

    Longoni, S.; Coady, M.J.; Ikeda, T.; Philipson, K.D. )

    1988-12-01

    Injection of Xenopus laevis oocytes with rabbit heart poly(A){sup +}RNA results in expression of Na{sup +} inside (Na{sub i}{sup +})-dependent Ca{sup 2+} uptake activity. The activity was measured by first loading the oocytes with Na{sup +} using nystatin and then incubating the oocytes in K{sup +} or Na{sup +} medium containing {sup 45}Ca. The expressed Na{sup +} gradient-dependent Ca{sup 2+} uptake was five to eight times that observed with water-injected oocytes or with poly(A){sup +}RNA-injected oocytes for which the Na{sup +} load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A){sup +}RNA on a sucrose gradient determined that the active message had a size range between 3 and 8 kb. The properties of the Na{sup +} gradient-dependent Ca{sup 2+} uptake indicated that Na{sup +}-Ca{sup 2+} exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na{sup +}-Ca{sup 2+} exchange.

  1. Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

    PubMed

    Shrestha, Bindesh; Sripadi, Prabhakar; Reschke, Brent R; Henderson, Holly D; Powell, Matthew J; Moody, Sally A; Vertes, Akos

    2014-01-01

    Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

  2. FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis.

    PubMed

    Schuff, Maximilian; Siegel, Doreen; Bardine, Nabila; Oswald, Franz; Donow, Cornelia; Knöchel, Walter

    2010-01-15

    Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis.

  3. Expression of spasmolysin (FIM-A.1): an integumentary mucin from Xenopus laevis.

    PubMed

    Hauser, F; Gertzen, E M; Hoffmann, W

    1990-08-01

    In the past, a unique type of precursor for a secretory protein was discovered. It contains a central repetitive domain rich in threonine residues and terminal cysteine-rich domains. Due to striking homologies of these terminal domains with pancreatic spasmolytic polypeptide, originally the name "prepro-spasmolysin" was proposed. Here we show that the mature protein has a MW of about 130 kDa, consisting of about 70% carbohydrate and 30% protein. Similar O-linked glycoproteins have been found in mucins from human intestine. For this and numerous other reasons we decided to rename this glycoprotein "frog integumentary mucin A.1" (FIM-A.1). Furthermore, analysis of the protein with specific antibodies against the predicted C-terminal end indicates that FIM-A.1 is probably not processed at pairs of basic residues. In situ hybridization as well as immunofluorescence studies revealed that FIM-A.1 is expressed and stored exclusively in mature mucous glands of Xenopus laevis skin. Only cone cells at the proximal part of these glands do not synthesize FIM-A.1. In contrast, all other physiologically active peptides from X. laevis skin investigated so far are synthesized in granular glands. A hypothetical function of FIMs for defense against microbial infections is discussed.

  4. Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry

    PubMed Central

    Reschke, Brent R.; Henderson, Holly D.; Powell, Matthew J.; Moody, Sally A.; Vertes, Akos

    2014-01-01

    Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis. PMID:25506922

  5. Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

    PubMed Central

    Duray, Alexis M.; Tembo, Maiwase; Beleny, David O.; Napolitano, Marc A.; Sauer, Monica L.; Wisner, Bennett W.

    2017-01-01

    Background The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Methodology/principal finding Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Conclusions/Significance Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu. PMID:28114360

  6. Waterborne exposure to triadimefon causes thyroid endocrine disruption and developmental delay in Xenopus laevis tadpoles.

    PubMed

    Li, Meng; Li, Shuying; Yao, Tingting; Zhao, Renjie; Wang, Qiangwei; Zhu, Guonian

    2016-08-01

    Triadimefon (TDF) is a triazole-derivative fungicide that is detectable in the environment and target agricultural products, prompting concern over its risk to wildlife and human health. In our study, Nieuwkoop & Faber stage 51 Xenopus laevis tadpoles were exposed to different nominal concentrations TDF (0, 0.112, and 1.12mg/L) for 21 days while the tadpoles were undergoing pre-morphological development. Developmental condition, bioaccumulation and thyroid hormone levels, and mRNA expression of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were examined. Exposure to TDF caused a reduction in developmental rates on pre-metamorphosis of X. laevis. TDF exposure significantly decreased thyroid hormone (T4 and T3) concentrations, indicating thyroid endocrine disruption. The downregulation of thyroglobulin and upregulation of genes related to thyroid hormone metabolism (ugt1ab) might be responsible for the decreased thyroid hormone concentrations. Treatment with TDF also significantly increased mRNA expression of genes involved in thyroid-stimulating hormone as a compensatory mechanism response to decreased thyroid hormone concentrations. Gene expression and in silico ligand docking studies were combined to study the interaction between TDF and thyroid hormone receptor. Results showed that TDF could consequently affect the HPT axis signaling pathway. In addition, bioconcentration of TDF was observed in tadpoles, indicating the bioactivity of this compound. Taken together, the results suggest that TDF alters the HPT axis-related genes and changes thyroid hormone contents in X. laevis tadpoles, thus causing thyroid endocrine disruption and consequently delaying thyroid hormones-dependent metamorphic development.

  7. Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine.

    PubMed

    Sai, Linlin; Dong, Zhihua; Li, Ling; Guo, Qiming; Jia, Qiang; Xie, Lin; Bo, Cunxiang; Liu, Yanzhong; Qu, Binpeng; Li, Xiangxin; Shao, Hua; Ng, Jack C; Peng, Cheng

    2016-09-01

    As a widely used herbicide, atrazine (AZ) has been extensively studied for its adverse effects on the reproductive system, especially feminization in male animals. However, the relationship of gene expression changes and associated toxicological endpoints remains unclear. In this study, developing Xenopus laevis tadpoles were exposed to concentration of AZ at 0.1, 1, 10 or 100 μg/L continuously. Compared with froglets in the control group, there were no significant differences in body length, body weight, liver weight and hepatosomatic index (HSI) of males in groups treated with AZ for 90 d. At 100 μg/L AZ treatment caused a significant reduction of gonad weight and gonadosomatic index (GSI) of males (p < 0.01). In addition, AZ at all dose levels caused testicular degeneration, especially in froglets from the groups with 0.1 and 100 μg/L which exhibited U-shaped dose-response trend. We further investigated the gene expression changes associated with the testicular degeneration induced by AZ. We found that the expression of 1165 genes was significantly altered with 616 upregulated and 549 downregulated compared to the expression profile of the control animals. KEGG analysis showed that genes which were significantly affected by AZ are mainly involved in arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation pathway. Our results show that AZ may affect reproductive and immune systems by interference with the related gene expression changes during the male X. laevis development. The findings may help to clarify the feminization mechanisms of AZ in male X. laevis.

  8. Dehydration triggers differential microRNA expression in Xenopus laevis brain.

    PubMed

    Luu, Bryan E; Storey, Kenneth B

    2015-11-15

    African clawed frogs, Xenopus laevis, although primarily aquatic, have a high tolerance for dehydration, being capable of withstanding the loss of up to 32-35% of total water body water. Recent studies have shown that microRNAs play a role in the response to dehydration by the liver, kidney and ventral skin of X. laevis. MicroRNAs act by modulating the expression of mRNA transcripts, thereby affecting diverse biochemical pathways. In this study, 43 microRNAs were assessed in frog brains comparing control and dehydrated (31.2±0.83% of total body water lost) conditions. MicroRNAs of interest were measured using a modified protocol which employs polyadenylation of microRNAs prior to reverse transcription and qPCR. Twelve microRNAs that showed a significant decrease in expression (to 41-77% of control levels) in brains from dehydrated frogs (xla-miR-15a, -150, -181a, -191, -211, -218, -219b, -30c, -30e, -31, -34a, and -34b) were identified. Genomic analysis showed that the sequences of these dehydration-responsive microRNAs were highly conserved as compared with the comparable microRNAs of mice (91-100%). Suppression of these microRNAs implies that translation of the mRNA transcripts under their control could be enhanced in response to dehydration. Bioinformatic analysis using the DIANA miRPath program (v.2.0) predicted the top two KEGG pathways that these microRNAs collectively regulate: 1. Axon guidance, and 2. Long-term potentiation. Previous studies indicated that suppression of these microRNAs promotes neuroprotective pathways by increasing the expression of brain-derived neurotrophic factor and activating anti-apoptotic pathways. This suggests that similar actions may be triggered in X. laevis brains as a protective response to dehydration.

  9. Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevis.

    PubMed

    Beckham, Yvonne M; Nath, Kimberly; Elinson, Richard P

    2003-01-01

    Eleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3' untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui.

  10. Molecular cloning and expression of prostaglandin F2α receptor isoforms during ovulation in the ovarian follicles of Xenopus laevis.

    PubMed

    Liu, Zhiming; Su, Xiurong; Li, Taiwu; Pan, Daodong; Sena, Johnny; Dhillon, Jasvinder

    2010-11-01

    Prostaglandins F2α levels increase during ovulatory period in Xenopus laevis in response to stimulation by gonadotropins and progesterone. PGF2α exerts its effects on ovulation through interaction with its receptor (FP) in ovaries. Little is known about the characteristics of the FP receptor and its regulation during the ovulatory period in non-mammalian species. In the present study, two isoforms of prostaglandin F receptor (FP A and B) cDNAs were isolated from Xenopus laevis ovarian tissues using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). The cDNAs of FP A and FP B were sequenced. In Xenopus laevis ovary, FP A and B mRNA levels were up-regulated during gonadotropin- and progresterone-induced ovulation in vitro. The mRNA level of FP B was higher than that of FP A. Moreover, FP A and FP B mRNA levels were measured in various tissues including eye, liver, lungs, heart, muscle, ovary, and skin. Overall, FP B mRNA level was approximately 10- to 100-fold higher than that of FP A, except in the muscle and skin where FP A mRNA level was comparable to that of FP B. The results suggest that in Xenopus ovarian follicles FP receptors play an important role during gonadotropin- and progesterone-induced ovulation.

  11. Retinoid-like compounds produced by phytoplankton affect embryonic development of Xenopus laevis.

    PubMed

    Smutná, M; Priebojová, J; Večerková, J; Hilscherová, K

    2017-04-01

    Teratogenic effects, which were remarkably similar to those induced by retinoic acids, have been seen in wild frogs indicating possible source of retinoids in the environment. Recent studies indicate that some cyanobacterial species can contain teratogenic retinoic acids (RAs) and their analogues. Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. The study investigated the effects of exudates (extracellular compounds) of two cyanobacteria species with retinoic-like activity and one algae species on embryonic development of amphibians. The retinoid-like activity determined by in vitro reporter gene assay reached 528ng retinoid equivalents (REQ)/L and 1000ng REQ/L in exudates of Cylindrospermopsis raciborskii and Microcystis aeruginosa, respectively, while algal exudates showed no detectable activity. Total mean of retinoid-like copounds into exudate was 35.6ng ATRA/mil.cells for M.aeruginosa and 6.71ng ATRA/mil.cells for C.raciborskii, respectively. Toxicity tests with amphibian embryos up to 96h of development were carried out according to the standard guide for the Frog Embryo Teratogenesis Assay Xenopus. Lowest observed effect concentrations (LOEC) of malformations (2.5-2.6µg/L REQ) were two times lower than LOEC for ATRA (5µg/L). The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, gut and eyes deformation) and interference with growth in frogs embryos, while such effects were not observed for the algae. Xenopus embryos were also exposed to all-trans retinoic acid (ATRA) in concentration range (1-40µg/L) equivalent to the REQs detected in cyanobacterial exudates. ATRA (10µg/L) caused similar teratogenic phenotypes at corresponding REQs as cyanobacterial exudates. The study confirms the ability of some species of cyanobacteria to produce retinoids naturally and excrete them directly into the environment at concentrations which might have adverse influence on

  12. Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

    PubMed

    Haynes-Gimore, Nikesha; Banach, Maureen; Brown, Edward; Dawes, Ryan; Edholm, Eva-Stina; Kim, Minsoo; Robert, Jacques

    2015-12-15

    Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

  13. Semi-Solid Tumor model in Xenopus laevis/gilli cloned tadpoles for Intravital study of neovascularization, immune cells and melanophore infiltration

    PubMed Central

    Haynes-Gimore, Nikesha; Banach, Maureen; Brown, Edward; Dawes, Ryan; Edholm, Eva-Stina; Kim, Minsoo; Robert, Jacques

    2015-01-01

    Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores. PMID:25601449

  14. A Database of microRNA Expression Patterns in Xenopus laevis

    PubMed Central

    Moxon, Simon; Lopez-Gomollon, Sara; Viaut, Camille; Tomlinson, Matthew L.; Patrushev, Ilya; Gilchrist, Michael J.; Dalmay, Tamas; Dotlic, Dario; Münsterberg, Andrea E.; Wheeler, Grant N.

    2015-01-01

    MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase. PMID:26506012

  15. The embryonic development of Xenopus laevis under a low frequency electric field.

    PubMed

    Boga, Ayper; Binokay, Secil; Emre, Mustafa; Sertdemir, Yasar

    2012-06-01

    The aim of this study was to determine the effects of a low frequency electric field on the early embryonic development of frogs. The embryos of African clawed toads, Xenopus laevis, were exposed to a 20-μA electric current during the cleavage stages. The developmental processes of embryos during and after electric field exposure were monitored for teratogenic effects. All the embryos continuously exposed to the electric field died without undergoing any developmental processes. However, when the embryos were exposed to the electric field for 20-min periods (four times/over 2 d), the embryos developed into both normal tadpoles (70 %) and malformed tadpoles with light edema, reduced pigmentation, or axial anomalies, such as crooked tails. After exposure, the control embryos were at development stage 35.5 (2 d 2 h), while the normal embryos of the assay group were at developmental stage 41(3 d 4 h). There was a 1 d 2 h difference between the two developmental stages, revealing the importance of that time period for embryogenesis. In conclusion, the effects of electric current on Xenopus embryos are dependent on the initial developmental stage and the duration of exposure.

  16. Labeling strategy and signal broadening mechanism of Protein NMR spectroscopy in Xenopus laevis oocytes.

    PubMed

    Ye, Yansheng; Liu, Xiaoli; Chen, Yanhua; Xu, Guohua; Wu, Qiong; Zhang, Zeting; Yao, Chendie; Liu, Maili; Li, Conggang

    2015-06-08

    We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in-cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high-resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. (19) F labeling is more promising than (15) N, (13) C, and (2) H enrichment. We also used (19) F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells.

  17. A Database of microRNA Expression Patterns in Xenopus laevis.

    PubMed

    Ahmed, Ayisha; Ward, Nicole J; Moxon, Simon; Lopez-Gomollon, Sara; Viaut, Camille; Tomlinson, Matthew L; Patrushev, Ilya; Gilchrist, Michael J; Dalmay, Tamas; Dotlic, Dario; Münsterberg, Andrea E; Wheeler, Grant N

    2015-01-01

    MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase.

  18. Expression and functional characterization of Xhmg-at-hook genes in Xenopus laevis.

    PubMed

    Macrì, Simone; Sgarra, Riccardo; Ros, Gloria; Maurizio, Elisa; Zammitti, Salvina; Milani, Ornella; Onorati, Marco; Vignali, Robert; Manfioletti, Guidalberto

    2013-01-01

    High Mobility Group A proteins (HMGA1 and HMGA2) are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook) that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS), including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.

  19. Effect of chronic copper and pentachlorophenol exposure to early life stages of Xenopus laevis

    SciTech Connect

    Fort, D.J.; Stover, E.L.

    1995-12-31

    An evaluation of the effects of low-level copper and pentachlorophenol exposure on various early life stages of the South African clawed frog, Xenopus laevis was performed using stage-specific and long-term continuous exposures. Stage-specific exposure experiments were conducted such that separate subsets of embryos and larvae from the same clutch were exposed to two toxicants, copper and pentachlorophenol, from 0 d to 4 d (standard Frog Embryo Teratagenesis Assay Xenopus [FETAX]), 4 d to 8 d, 8 d to 12 d, and 12 d to 16 d. Results from two separate concentration-response experiments indicated that sensitivity to either toxicant increased in each successive time period. Continuous exposure studies conducted for 60 to 75 days indicated that copper, but not pentachlorophenol induced reduction deficiency malformations of the hind limb at concentrations as low as 0.05 mg/L. Pentachlorophenol concentrations as low as 0.5/{micro}g/L inhibited tail resorption. However, copper did not adversely affect the process of tail resorption. These results indicated that studies evaluating longer-term developmental processes are important in ecological hazard evaluation.

  20. Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis.

    PubMed

    Tsutsumi, Rio; Yamada, Shigehito; Agata, Kiyokazu

    2016-02-01

    A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a "spike." Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs' limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus.

  1. Lateral line-mediated rheotactic behavior in tadpoles of the African clawed frog (Xenopus laevis)

    PubMed Central

    Simmons, Andrea M.; Costa, Lauren M.; Gerstein, Hilary B.

    2005-01-01

    Tadpoles (Xenopus laevis) have a lateral line system whose anatomical structure has been described, but whose functional significance has not been closely examined. These experiments tested the hypothesis that the lateral line system is involved in rheotaxis. Tadpoles in developmental stages 47–56 oriented toward the source of a water current. Orientation was less precise after treatment with cobalt chloride or streptomycin, but was similar to that of untreated animals after exposure to gentamicin. In no current conditions, tadpoles exhibited a characteristic head-down posture by which they held themselves in the water column at an angle around 45°. This body posture became significantly less tilted in the presence of water current. Treatment with cobalt chloride or streptomycin increased the angle of tilt close to that seen in no current conditions, while gentamicin treatment tended to decrease tilt angle. The data are consistent with anatomical and physiological findings that tadpole neuromasts are similar to superficial, but not canal, neuromasts in fishes, and they suggest that the lateral line system is involved in both directional current detection and current-related postural adjustments in Xenopus. PMID:15300386

  2. In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media

    SciTech Connect

    Risley, M.S.; Miller, A.; Bumcrot, D.A.

    1987-05-01

    Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. /sup 3/H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since /sup 3/H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis.

  3. Regulation of respiratory and vocal motor pools in the isolated brain of Xenopus laevis.

    PubMed

    Zornik, Erik; Kelley, Darcy B

    2008-01-16

    The aquatic frog Xenopus laevis uses a complex vocal repertoire during mating and male-male interactions. Calls are produced without breathing, allowing the frogs to vocalize for long periods underwater. The Xenopus vocal organ, the larynx, is innervated by neurons in cranial motor nucleus (n.) IX-X, which contains both vocal (laryngeal) and respiratory (glottal) motor neurons. The primary descending input to n.IX-X comes from the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), located in the rostral hindbrain. We wanted to characterize premotor inputs to respiratory and vocal motor neurons and to determine what mechanisms might be involved in regulating two temporally distinct rhythmic behaviors: breathing and calling. Using isolated brain and larynx preparations, we recorded extracellular activity from the laryngeal nerve and muscles and intracellular activity in laryngeal and glottal motor neurons. Spontaneous nerve activities mimicking respiratory and vocal patterns were observed. DTAM projection neurons (DTAM(IX-X) neurons) provide direct input to glottal and laryngeal motor neurons. Electrical stimulation produced short-latency coordinated activity in the laryngeal nerve. DTAM(IX-X) neurons provide excitatory monosynaptic inputs to laryngeal motor neurons and mixed excitatory and inhibitory inputs to glottal motor neurons. DTAM stimulation also produced a delayed burst of glottal motor neuron activity. Together, our data suggest that neurons in DTAM produce vocal motor output by directly activating laryngeal motor neurons and that DTAM may coordinate vocal and respiratory motor activity.

  4. Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis

    PubMed Central

    Yamada, Shigehito

    2016-01-01

    Abstract A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a “spike.” Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs’ limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus. PMID:27499877

  5. Analysis of the expression of microtubule plus-end tracking proteins (+TIPs) during Xenopus laevis embryogenesis.

    PubMed

    Park, Edmond Changkyun; Lee, Hyeyoon; Hong, Yeonhee; Kim, Mi-Jung; Lee, Zee-Won; Kim, Seung Il; Kim, Soohyun; Kim, Gun-Hwa; Han, Jin-Kwan

    2012-01-01

    Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracellular transport in diverse cellular processes. Microtubule plus-end tracking proteins (+TIPs), a structurally and functionally diverse group of proteins, are specifically accumulated in the microtubule plus end and regulate dynamic microtubule behavior. We characterized the +TIPs, Clip1, p150(glued), Clasp1, Lis1 and Stim1, in Xenopus laevis and report their expression patterns during embryogenesis in this paper. All the five +TIP genes are maternally expressed and have similar expression patterns during Xenopus embryo development. The expression of +TIPs is localized in the animal hemisphere and ectoderm region at early stages of embryonic development. As development progresses to later stages, the ectodermal expression of +TIPs persists in head and neural tube structures. Clasp1, p150(glued) and Lis1 in particular are specifically expressed in the cranial nerves. Importantly, +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs, and our analysis provides insight that could serve as the basis for future research of microtubules in vertebrate development, cell movements during gastrulation and neurogenesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

    SciTech Connect

    Mertz, L.M.; Catt, K.J. )

    1991-10-01

    The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.

  7. Effect of intercalating agents on RNA polymerase I promoter selection in Xenopus laevis.

    PubMed Central

    Pruitt, S C; Reeder, R H

    1984-01-01

    We have analyzed the effect of DNA intercalating agents on the transcription signals from two different Xenopus laevis RNA polymerase I promoters. The transcription signal from the promoter for the 7.5-kilobase rRNA precursor (the gene promoter) is unaffected over a large range of intercalating agent concentrations regardless of whether the template is injected plasmid DNA in oocytes, the amplified endogenous nucleoli of oocytes, or the endogenous chromosomes of cultured Xenopus kidney cells. The transcription signal from a closely related promoter located in the spacer DNA between genes (the spacer promoter) ranges between undetectable to equivalent to the gene promoter signal on different templates. The transcription signal from the spacer promoter is also differentially affected by intercalating agents relative to the gene promoter. Depending on the template, these agents can either increase or decrease the transcription signal from the spacer promoter. Fusions between the gene and spacer promoters demonstrate that intercalating agents affect transcription initiation. One explanation for these results is that the degree of supercoiling of the template DNA can differentially inhibit transcription from the spacer promoters. The different effects of intercalating agents on transcription from the spacer promoters of various templates could then be explained as differences in the degree of supercoiling present on these templates initially. Images PMID:6543244

  8. Gravity-related critical periods in vestibular and tail development of Xenopus laevis.

    PubMed

    Horn, Eberhard R; Gabriel, Martin

    2011-11-01

    Sensory systems are characterized by developmental periods during which they are susceptible to environmental modifications, in particular to sensory deprivation. The experiment, XENOPUS, on Soyuz in 2008 was the fourth space flight experiment since 1993 to explore whether tail and vestibular development of Xenopus laevis has a gravity-related critical period. During this flight, tadpoles were used that had developed either the early hindlimb (stage 47) or forelimb bud (stage 50) at launch of the spacecraft. The results revealed (1) no impact of microgravity on the development of the roll-induced vestibuloocular reflex (rVOR) in both stages and (2) a stage-related sensitivity of tail development to microgravity exposure. These results were combined and compared with observations from space flights on other orbital platforms. The combined data revealed (1) a narrow gravity-related critical period for rVOR development close to the period of the first appearance of the reflex and (2) a longer one for tail development lasting from the early tail bud to the early forelimb bud stage.

  9. Characterization of a novel member of the FGF family, XFGF-20, in Xenopus laevis.

    PubMed

    Koga, C; Adati, N; Nakata, K; Mikoshiba, K; Furuhata, Y; Sato, S; Tei, H; Sakaki, Y; Kurokawa, T; Shiokawa, K; Yokoyama, K K

    1999-08-11

    The cDNA for a novel member of the FGF family (XFGF-20) was isolated from a Xenopus cDNA library prepared at the tailbud stage using as a probe the product of degenerate PCR performed with primers based on mammalian FGF-9s. This cDNA was 1860 bp long, and contained a single open reading frame that encoded 208 amino acid residues. The deduced amino acid sequence contained a motif characteristic of the FGF family and it was similar (73.1% overall homology) to XFGF-9 but differed from XFGF-9 in its amino-terminal region (33.3% homology). XFGF-20 mRNA was expressed only zygotically in embryos at and after the blastula stage, but it was also specifically expressed in the stomach and testis of adults. By contrast, XFGF-9 mRNA was expressed maternally in eggs and in many adult tissues. When XFGF-20 mRNA was overexpressed in early embryos, gastrulation was abnormal and development of anterior structures was suppressed. In such embryos, the expression of the Xbra transcript was suppressed during gastrulation while the expression of the transcripts of cerberus, Siamois, dkk-1, chordin, and Xotx-2 genes was normal. These results suggest that correct expression of XFGF-20 during gastrulation is required for the formation of normal head structures in Xenopus laevis during embryogenesis and that expression of the Xbra gene mediates this phenomenon.

  10. Pattern of calbindin-D28k and calretinin immunoreactivity in the brain of Xenopus laevis during embryonic and larval development.

    PubMed

    Morona, Ruth; González, Agustín

    2013-01-01

    The present study represents a detailed spatiotemporal analysis of the localization of calbindin-D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions.

  11. Using Xenopus laevis retinal and spinal neurons to study mechanisms of axon guidance in vivo and in vitro.

    PubMed

    Erdogan, Burcu; Ebbert, Patrick T; Lowery, Laura Anne

    2016-03-01

    The intricate and precise establishment of neuronal connections in the developing nervous system relies on accurate navigation of growing axons. Since Ramón y Cajal's discovery of the growth cone, the phenomenon of axon guidance has been revealed as a coordinated operation of guidance molecules, receptors, secondary messengers, and responses driven by the dynamic cytoskeleton within the growth cone. With the advent of new and accelerating techniques, Xenopus laevis emerged as a robust model to investigate neuronal circuit formation during development. We present here the advantages of the Xenopus nervous system to our growing understanding of axon guidance.

  12. A novel short anionic antibacterial peptide isolated from the skin of Xenopus laevis with broad antibacterial activity and inhibitory activity against breast cancer cell.

    PubMed

    Li, Siming; Hao, Linlin; Bao, Wanguo; Zhang, Ping; Su, Dan; Cheng, Yunyun; Nie, Linyan; Wang, Gang; Hou, Feng; Yang, Yang

    2016-07-01

    A vastarray of bioactive peptides from amphibian skin secretions is attracting increasing attention due to the growing problem of bacteria resistant to conventional antibiotics. In this report, a small molecular antibacterial peptide, named Xenopus laevis antibacterial peptide-P1 (XLAsp-P1), was isolated from the skin of Xenopus laevis using reversed-phase high-performance liquid chromatography. The primary structure of XLAsp-P1, which has been proved to be a novel peptide by BLAST search in AMP database, was DEDDD with a molecular weight of 607.7 Da analysed by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). The highlight of XLAsp-P1 is the strong in vitro potency against a variety of Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MICs) starting at 10 μg/mL and potent inhibitory activity against breast cancer cell at tested concentrations from 5 to 50 μg/mL. In addition, only 6.2 % of red blood cells was haemolytic when incubated with 64 μg/mL (higher than MICs of all bacterial strain) of XLAsp-P1. The antimicrobial mechanism for this novel peptide was the destruction of the cell membrane investigated by transmission electron microscopy. All these showed that XLAsp-P1 is a novel short anionic antibacterial peptide with broad antibacterial activity and inhibitory activity against breast cancer cell.

  13. The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

    PubMed

    Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

    2014-01-01

    The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

  14. Epithelial-connective tissue interactions induced by thyroid hormone receptor are essential for adult stem cell development in the Xenopus laevis intestine

    PubMed Central

    Hasebe, Takashi; Buchholz, Daniel R.; Shi, Yun-Bo; Ishizuya-Oka, Atsuko

    2012-01-01

    In the amphibian intestine during metamorphosis, stem cells appear and generate the adult absorptive epithelium, analogous to the mammalian one, under the control of thyroid hormone (TH). We have previously shown that the adult stem cells originate from differentiated larval epithelial cells in the Xenopus laevis intestine. To clarify whether TH signaling in the epithelium alone is sufficient for inducing the stem cells, we have now performed tissue recombinant culture experiments, using transgenic X. laevis tadpoles that express a dominant positive TH receptor (dpTR) under a control of heat shock promoter. Wild-type (Wt) or dpTR transgenic (Tg) larval epithelium (Ep) was isolated from the tadpole intestine, recombined with homologous or heterologous non-epithelial tissues (non-Ep), and then cultivated in the absence of TH with daily heat shocks to induce transgenic dpTR expression. Adult epithelial progenitor cells expressing sonic hedgehog became detectable on day 5 in both the recombinant intestine of Tg Ep and Tg non-Ep (Tg/Tg) and that of Tg Ep and Wt non-Ep (Tg/Wt). However, in Tg/Wt intestine, they did not express other stem cell markers such as Musashi-1 and never generated the adult epithelium expressing a marker for absorptive epithelial cells. Our results indicate that, while it is unclear why some larval epithelial cells dedifferentiate into adult progenitor/stem cells, TR-mediated gene expression in the surrounding tissues other than the epithelium is required for them to develop into adult stem cells, suggesting the importance of TH-inducible epithelial-connective tissue interactions in establishment of the stem cell niche in the amphibian intestine. PMID:21280164

  15. Histological microstructure of the claws of the African clawed frog, Xenopus laevis (Anura: Pipidae): implications for the evolution of claws in tetrapods.

    PubMed

    Maddin, Hillary C; Musat-Marcu, Sorin; Reisz, Robert R

    2007-05-15

    Claws are consistent components of amniote anatomy and may thus be implicated in the success of the amniote invasion of land. However, the evolutionary origin of these structures in tetrapods is unclear. Claws are present in certain extant non-amniotes, such as Xenopus laevis, the African clawed frog. The histology of the soft tissue component of the claws of X. laevis is described and compared with the amniote condition in order to gain new information on the question of homology of claws in these two groups based on patterns of keratinization. The X. laevis claw sheath is composed of a localized thickening of the corneous region of the epidermis that envelops the terminal phalanx. Noted differences between the non-cornified layers of the epidermis of the claw and non-claw region are the overall grainier appearance of the cells and an increased abundance of desmosomes in the intermediate spinosus cells. The biochemical identity of the sheath keratin(s) is inferred to be different from that of non-claw region epidermis, based on histological differences and differences in stain affinity between the two regions. The microstructure of the frog claw differs from that of amniotes in several respects, including the lack of a specified zone of growth near the base of the claw. Amphibians and amniotes, therefore, have very different patterns of claw sheath growth. Observations do not support homology of claws on a structural level in these two groups; however, further experimental work may confirm a conserved pattern of cornification in these structures in tetrapods. (c) 2006 Wiley-Liss, Inc.

  16. Developmental diversity of amphibians.

    PubMed

    Elinson, Richard P; del Pino, Eugenia M

    2012-01-01

    The current model amphibian, Xenopus laevis, develops rapidly in water to a tadpole which metamorphoses into a frog. Many amphibians deviate from the X. laevis developmental pattern. Among other adaptations, their embryos develop in foam nests on land or in pouches on their mother's back or on a leaf guarded by a parent. The diversity of developmental patterns includes multinucleated oogenesis, lack of RNA localization, huge non-pigmented eggs, and asynchronous, irregular early cleavages. Variations in patterns of gastrulation highlight the modularity of this critical developmental period. Many species have eliminated the larva or tadpole and directly develop to the adult. The wealth of developmental diversity among amphibians coupled with the wealth of mechanistic information from X. laevis permit comparisons that provide deeper insights into developmental processes.

  17. DEVELOPMENTAL DIVERSITY OF AMPHIBIANS

    PubMed Central

    Elinson, Richard P.; del Pino, Eugenia M.

    2011-01-01

    The current model amphibian, Xenopus laevis, develops rapidly in water to a tadpole which metamorphoses into a frog. Many amphibians deviate from the X. laevis developmental pattern. Among other adaptations, their embryos develop in foam nests on land or in pouches on their mother’s back or on a leaf guarded by a parent. The diversity of developmental patterns includes multinucleated oogenesis, lack of RNA localization, huge non-pigmented eggs, and asynchronous, irregular early cleavages. Variations in patterns of gastrulation highlight the modularity of this critical developmental period. Many species have eliminated the larva or tadpole and directly develop to the adult. The wealth of developmental diversity among amphibians coupled with the wealth of mechanistic information from X. laevis permit comparisons that provide deeper insights into developmental processes. PMID:22662314

  18. Continued Studies on the Effects of Simazine on the Liver Histological Structure and Metamorphosis in the Developing Xenopus laevis.

    PubMed

    Sai, Linlin; Qu, Binpeng; Li, Yan; Jia, Qiang; Bo, Cunxiang; Liu, Yanzhong; Yu, Gongchang; Xie, Lin; Li, Ling; Ng, Jack C; Peng, Cheng

    2016-10-01

    This study continued our previous work (Sai et al. in Bull Environ Contam Toxicol 95:157-163, 2015a) by analysing the effects of simazine on the liver histological structure and metamorphosis in the developing Xenopus laevis. Tadpoles (Nieuwkoop-Faber stage 46) were exposed to simazine at 0.1, 1.2, 11.0 and 100.9 μg/L for 100 days. When tadpoles were exposed to simazine at 11.0 and 100.9 µg/L, an increased mortality and damaged liver tissues were observed together with significant inhibition of percent of X. laevis completing metamorphosis on days 80 and 90 and prolonged time of completing metamorphosis. On the other hand, we found that simazine has no significant effects on liver weight and altered hepatosomatic index. Results of this study may be considered to inform risk assessment of the effects of simazine on the development of X. laevis.

  19. Sexually differentiated, androgen-regulated, larynx-specific myosin heavy-chain isoforms in Xenopus tropicalis; comparison to Xenopus laevis

    PubMed Central

    Baur, Laura A.; Nasipak, Brian T.; Kelley, Darcy B.

    2010-01-01

    We have shown that the sarcoplasmic myosin heavy-chain (MyHC) isoform xtMyHC-101d is highly and specifically expressed in the larynx of the aquatic anuran, Xenopus tropicalis. In male larynges, the predominant MyHC isoform is xtMyHC-101d, while in females, another isoform, xtMyHC-270c, predominates. The X. tropicalis genome has been sequenced in its entirety, and xtMyHC-101d is part of a specific array of xtMyHC genes expressed otherwise in embryonic muscles. The administration of the androgen dihydrotestosterone increases the expression of xtMyHC-101d in juvenile larynges of both sexes. Using ATPase histochemistry, we found that in adults, X. tropicalis male laryngeal muscle contains only fast-twitch fibers, while the female laryngeal muscle contains a mix of fast- and slow-twitch fibers. Juvenile larynges are female-like in fiber type composition (44% slow twitch, 56% fast twitch); androgen treatment increases the percentage of fast-twitch fibers to 86%. xtMyHC-101d predominates in larynges of dihydrotestosterone-treated juveniles but not in larynges of untreated juveniles. We compared the larynx-specific expression of xtMyHC genes in X. tropicalis to the MyHC gene expressed in X. laevis larynx (xlMyHC-LM) by sequencing the entire xlMyHC-LM gene. The androgen-regulated xtMyHC that predominates in the male larynx of X. tropicalis is not the gene phylogenetically most similar to xlMyHC-LM at the nucleotide level but is instead a similar isoform found in the same MyHC array and expressed in the embryonic muscle. PMID:18551305

  20. An improved method for real-time monitoring of membrane capacitance in Xenopus laevis oocytes.

    PubMed Central

    Schmitt, Bernhard M; Koepsell, Hermann

    2002-01-01

    Measurements of membrane capacitance (C(m)) in Xenopus laevis oocytes offer unique experimental possibilities but are difficult to perform with current methods. To improve C(m) measurements in the two-electrode voltage clamp (TEVC) mode, we developed a paired-ramp protocol and tested its performance in a model circuit (with tunable C(m), membrane resistance R(m), and series resistance R(s)) and in Xenopus oocytes. In the cell model and with R(s) = 0 Omega, inaccuracy of C(m) estimates was <1% under widely varying conditions (R(m) ranging from 100 to 2000 kOmega, and C(m) from 50 to 1000 nF). With R(s) > 0 Omega, C(m) was underestimated by a relative error epsilon closely approximated as epsilon approximate 2 x R(s)/(R(s) + R(m)), in keeping with the theoretical prediction. Thus, epsilon may be neglected under standard conditions or, under extreme conditions, corrected for if R(s) is known. Relative imprecision of C(m) estimates was small, independent of R(s), and inversely related to C(m) (<1.5% at 50 nF, <0.4% at 200 nF). Averaging allowed reliable detection of C(m) deviations from 200 nF of 0.1 nF, i.e., 0.05%. In Xenopus oocytes, we could resolve C(m) changes that were small (e.g., DeltaC(m) approximate 2 nF upon 100 muM 8-Br-cAMP), fast (e.g., DeltaC(m)/Deltat approximate 20nF/30s upon 1 muM phorbol myristate acetate (PMA)) or extended and complex (e.g., fast increase, followed by prolonged C(m) decrease upon 1 muM PMA). Rapidly alternating between paired ramps and a second, step protocol allowed quasi-simultaneous monitoring of additional electrical parameters such as R(m), slope conductance g(m), and reversal potential E(rev). Taken together, our method is suited to monitor C(m) in Xenopus oocytes conveniently, with high temporal resolution, accuracy and precision, and in parallel with other electrical parameters. Thus, it may be useful for the study of endo- and exocytosis and of membrane protein regulation and for electrophysiological high

  1. Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

    PubMed

    Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

    2017-01-01

    The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented

  2. Ion Currents Induced by ATP and Angiotensin II in Cultured Follicular Cells of Xenopus laevis

    PubMed Central

    Montiel-Herrera, Marcelino; Zaske, Ana María; García-Colunga, Jesús; Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2011-01-01

    Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at -60 mV revealed that ATP (20-250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at -60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known Fin and Sin-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using wholecell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at -60 mV. The net Ang II response, mediated by the activation of the AT1 receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes. PMID:22083304

  3. Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

    PubMed Central

    Parodi, Jorge; la Paz, Lenin Ochoa-de; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2012-01-01

    Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca2+ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca2+-dependent Cl− currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (Tout) and the serum-activated, oscillatory Cl− currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca2+-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication. PMID:23104436

  4. Exposure to butachlor causes thyroid endocrine disruption and promotion of metamorphosis in Xenopus laevis.

    PubMed

    Li, Shuying; Li, Meng; Wang, Qiangwei; Gui, Wenjun; Zhu, Guonian

    2016-06-01

    Butachlor is extensively applied in rice paddy ecosystem in china, and has been widespread contaminant in the aquatic environment. Here, Xenopus laevis was used for the evaluation of teratogenesis developmental toxicity, and disruption of thyroid system when exposure to different concentrations of butachlor by window phase exposure. Acute toxicity investigation shown that 96 h-LC50 value of butachlor was 1.424 mg L(-1) and 0.962 mg L(-1) for tadpoles (starting from stages 46/47) and embryos (starting from stages 8/9), respectively. Exposure to butachlor caused malformation, including abnormal eye, pericardial edema, enlarged proctodaeum and bent tail. Window phase exposure test indicated that butachlor significantly promote the contents of whole-body thyroid hormones (THs, T3 and T4) at higher levels, indicating thyroid endocrine disruption. At 7 days, exposure to butachlor up-regulated the mRNA expression of genes involved in THs synthesis and metabolism (tshα, tg, tpo and dio1) and THs receptors (trα and trβ). At 14 days, up-regulation of the mRNA expression of genes related to THs synthesis and metabolism (tshα, tshβ, tg, tpo, dio1, dio2 and ttr) and THs receptors (trβ) were also observed after the exposure to butachlor. At 21 days, butachlor up-regulated the mRNA expression of tshα, tg, tpo genes and down-regulated the mRNA expression of tshβ, tg, dio1, ttr and trα genes. These results showed that butachlor could change the mRNA expression of genes involved in the HPT axis and increase whole-body thyroid hormones levels of X. laevis tadpoles in a dose- and time-dependent manner, causing thyroid endocrine disruption and developmental toxicity.

  5. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    PubMed

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes.

  6. Identification of a novel dehydration responsive gene, drp10, from the African clawed frog, Xenopus laevis.

    PubMed

    Biggar, Kyle K; Biggar, Yulia; Storey, Kenneth B

    2015-07-01

    During periods of environmental stress a number of different anuran species employ adaptive strategies to promote survival. Our study found that in response to dehydration (i.e., loss of total body water content), the African clawed frog (Xenopus laevis) increased the expression of a novel gene (drp10) that encodes a structural homolog of the freeze-responsive FR10 protein found in wood frogs. Similar to FR10, the DRP10 protein was found to also contain a highly conserved N-terminal cleavable signal peptide. Furthermore, DRP10 was found to have high structural homology to the available crystal structures of type A and E apolipoproteins in Homo sapiens, and a type IV LS-12 anti-freeze protein in the longhorn sculpin, Myoxocephalus octodecemspinosis. In response to dehydration, the transcript expression of drp10 was found to increase 1.52 ± 0.16-fold and 1.97 ± 0.11-fold in response to medium (15%) and high (30%) dehydration stresses in the liver tissue of X. laevis, respectively, while drp10 expression increased 2.12 ± 0.12-fold and 1.46 ± 0.16-fold in kidney tissue. Although the molecular function of both dehydration-responsive DRP10 and the freeze-responsive FR10 have just begun to be elucidated, it is likely that both are frog-specific proteins that likely share a similar purpose during water-related stresses.

  7. Specific Ligand Binding Domain Residues Confer Low Dioxin Responsiveness to AHR1β of Xenopus laevis

    PubMed Central

    Odio, Camila; Holzman, Sarah A.; Denison, Michael S.; Fraccalvieri, Domenico; Bonati, Laura; Franks, Diana G.; Hahn, Mark E.; Powell, Wade H.

    2013-01-01

    The aryl hydrocarbon receptor (AHR) is a PAS-family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1β associated with low TCDD sensitivity. Substitution of the entire ligand-binding domain (LBD) with the corresponding sequence from mouse AHRb-1 dramatically increased TCDD responsiveness in transactivation assays. To identify amino acid residues responsible, we constructed a comparative model of the AHR1β LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity of similar dimensions to the putative binding cavity of mouse AHRb-1, suggesting the importance of side-chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved β-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, EC50 declined 3-fold. When the mutations were combined, the EC50 declined from 18.6 nM to 0.8 nM, nearly matching mouse AHR for TCDD sensitivity. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2b]carbazole (FICZ). Mutations that increased TCDD sensitivity also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists. PMID:23394719

  8. Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.

    PubMed

    Mawaribuchi, Shuuji; Takahashi, Shuji; Wada, Mikako; Uno, Yoshinobu; Matsuda, Yoichi; Kondo, Mariko; Fukui, Akimasa; Takamatsu, Nobuhiko; Taira, Masanori; Ito, Michihiko

    2017-06-15

    Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Effects of 17 beta-estradiol exposure on Xenopus laevis gonadal histopathology.

    PubMed

    Wolf, Jeffrey C; Lutz, Ilka; Kloas, Werner; Springer, Timothy A; Holden, Larry R; Krueger, Henry O; Hosmer, Alan J

    2010-05-01

    The natural estrogen 17 beta-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 microg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 microg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p<0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment.

  10. Identification and characterization of Xenopus laevis homologs of mammalian TRAF6 and its binding protein TIFA.

    PubMed

    Inoue, Jun-Ichiro; Yagi, Shigenori; Ishikawa, Kosuke; Azuma, Sakura; Ikawa, Shuntaro; Semba, Kentaro

    2005-09-26

    Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) transduces signals from members of the TNFR superfamily and the Toll/IL-1R family, leading to activation of transcription factors such as NFkappaB and AP-1. Genetic disruption of the TRAF6 gene in mice results in various developmental abnormalities during embryogenesis, including osteopetrosis, failure of neural tube closure, defective formation of skin appendices, absence of lymph nodes, and absence of mature thymic epithelial cells. To clarify the effect of TRAF6 in development, we previously identified a TRAF-interacting protein with a forkhead-associated domain (TIFA), which binds and activates TRAF6 upon extracellular stimulation. To understand the physiological roles of TRAF6 and TIFA in early development, we studied these genes in Xenopus laevis. Here, we describe identification of X. laevis homologs of mammalian TRAF6 (XTRAF6) and TIFA (XTIFA). As was the case for the mammalian homologs, overexpression of XTRAF6 or XTIFA activated NFkappaB, whereas XTIFA carrying a mutation that abolishes XTRAF6 binding failed to activate NFkappaB, suggesting that XTIFA activates NFkappaB by binding to XTRAF6. XTIFA and XTRAF6 mRNAs were expressed at similar levels in zygotes from the neurula stage and then increased. Whole-mount in situ hybridization revealed that XTRAF6 mRNA was expressed in the head region and neural tube during the neurula stage, and the expression expanded to the pharyngeal apparatus during the tailbud stage. This localization is consistent with the defective neural tube closure and abnormal thymus organogenesis observed in TRAF6-deficient mice. Our results suggest possible cooperation between XTRAF6 and XTIFA during embryogenesis.

  11. A role for biliverdin IXalpha in dorsal axis development of Xenopus laevis embryos.

    PubMed

    Falchuk, Kenneth H; Contin, Jennifer M; Dziedzic, T Scott; Feng, Zhongling; French, Thayer C; Heffron, Gregory J; Montorzi, Marcelo

    2002-01-08

    The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3-0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IXalpha, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254- or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254- or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with microM amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IXalpha dimethyl ester does not induce recovery. The results define an approach to produce dorsal axis-deficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IXalpha in X. laevis embryogenesis.

  12. The synthetic progestogen, Levonorgestrel, but not natural progesterone, affects male mate calling behavior of Xenopus laevis.

    PubMed

    Hoffmann, Frauke; Kloas, Werner

    2012-05-01

    Worldwide, more than 100 million women use hormonal contraceptives, which act through progestogenic modes of action. These man-made hormones can enter the aquatic environment as they are excreted via feces and urine. Xeno-progestins are able to interfere with the endocrine system of female aquatic vertebrates impairing oogenesis and reproduction. However, data on progestogenic effects on reproductive behavior of male aquatic vertebrates are lacking. To evaluate whether progestins affect the mating behavior of male Xenopus laevis, we exposed male frogs to three environmentally relevant concentrations (10(-7) M, 10(-8) M and 10(-10) M) of the synthetic progestin Levonorgestrel (LNG) and the corresponding natural steroid progesterone (PRG), respectively. LNG at all exposure concentrations increased the proportions of advertisement calling, indicating a sexually aroused state of the males. Furthermore LNG at 10(-7) M decreased the relative proportions of rasping, a call type indicating a sexually unaroused state of the male. PRG, on the other hand, did not affect any of those parameters. Temporal and spectral features of the advertisement call itself were not affected by any of the two exposure treatments. Since LNG exhibits slight androgenic activity, the results suggest that LNG effects on male mate calling behavior of X. laevis are due to its moderate androgenic but not to its progestogenic activities. However, although males' sexual arousal seems to be enhanced by LNG, the adverse effects of LNG on female reproduction presumably outweigh these enhancing effects and LNG exposure nonetheless might result in reduced reproductive success of these animals.

  13. Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis.

    PubMed

    Chen, Xiuping; Wang, Jiamei; Zhu, Haojun; Li, Yunhe; Ding, Jiatong; Peng, Yufa

    2015-01-01

    In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L-1 or 10 μg L-1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development.

  14. Functional and structural effects of amyloid-β aggregate on Xenopus laevis oocytes.

    PubMed

    Parodi, Jorge; Ochoa-de la Paz, Lenin; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2012-10-01

    Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

  15. Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis

    PubMed Central

    Zhu, Haojun; Li, Yunhe; Ding, Jiatong; Peng, Yufa

    2015-01-01

    In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L–1 or 10 μg L–1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development. PMID:26695426

  16. Differential modulation of cytochrome P-450 1A and P-glycoprotein expression by aryl hydrocarbon receptor agonists and thyroid hormone in Xenopus laevis liver and intestine.

    PubMed

    Colombo, Anita; Bonfanti, Patrizia; Orsi, Federica; Camatini, Marina

    2003-04-10

    Several defence mechanisms, such as cytochrome P450 1A (CYP1A) enzymes and P-glycoprotein (Pgp), may influence the intracellular concentration and consequently the toxicity of xenobiotics. The parallel expression of CYP1A and Pgp has been investigated in mammals and, to a lesser extent in fish, in search for evidence of co-ordinated responses to xenobiotic exposure. The aryl hydrocarbon receptor (AHR) agonists are well known CYP1A inducers but some of them resulted not to have a uniquely defined action on Pgp levels in mammalian and fish species. To the best of our knowledge, no detailed studies have been carried out so far on amphibians Xenopus laevis. For this reason, in this work, the time dependent responses of the hepatic CYP1A and Pgp, to the prototypical CYP1A inducers, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in X. laevis have been assessed at the protein level and compared. The responsiveness of Xenopus intestinal Pgp to these compounds has also been analysed, as the epithelial cells lining the lumen of intestine represent another preferential site of Pgp expression. In addition, since the thyroid hormone has been demonstrated to down regulate the mdr gene during Xenopus development and in primary culture of Xenopus intestinal epithelial cells, the effects of 3,3',5-triiodo-L-thyronine (T(3)) on CYP1A and Pgp protein levels have been investigated in adult organisms. Western blot evidenced that a single injection of B(a)P (100 mg/kg), 3MC (20 mg/kg), and TCDD (3 microg/kg) elicited a statistically significant induction of hepatic CYP1A at all time points considered (72, 120 and 168 h) which decreased in time. The same trend of liver CYP1A induction was observed in T(3) treated Xenopus (15 microg/kg). Unlike CYP1A induction, the modulation of hepatic and intestinal Pgp expression exhibits an heterogeneous pattern. The basal levels of hepatic and intestinal Pgp were not statistically significant

  17. Psf2 plays important roles in normal eye development in Xenopus laevis

    PubMed Central

    Walter, Brian E.; Perry, Kimberly J.; Fukui, Lisa; Malloch, Erica L.; Wever, Jason

    2008-01-01

    Purpose Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a “sliding clamp.” Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. Methods A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. Results Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick

  18. Polyunsaturated fatty acids are potent openers of human M-channels expressed in Xenopus laevis oocytes.

    PubMed

    Liin, S I; Karlsson, U; Bentzen, B H; Schmitt, N; Elinder, F

    2016-09-01

    Polyunsaturated fatty acids have been reported to reduce neuronal excitability, in part by promoting inactivation of voltage-gated sodium and calcium channels. Effects on neuronal potassium channels are less explored and experimental data ambiguous. The aim of this study was to investigate anti-excitable effects of polyunsaturated fatty acids on the neuronal M-channel, important for setting the resting membrane potential in hippocampal and dorsal root ganglion neurones. Effects of fatty acids and fatty acid analogues on mouse dorsal root ganglion neurones and on the human KV 7.2/3 channel expressed in Xenopus laevis oocytes were studied using electrophysiology. Extracellular application of physiologically relevant concentrations of the polyunsaturated fatty acid docosahexaenoic acid hyperpolarized the resting membrane potential (-2.4 mV by 30 μm) and increased the threshold current to evoke action potentials in dorsal root ganglion neurones. The polyunsaturated fatty acids docosahexaenoic acid, α-linolenic acid and eicosapentaenoic acid facilitated opening of the human M-channel, comprised of the heteromeric human KV 7.2/3 channel expressed in Xenopus oocytes, by shifting the conductance-vs.-voltage curve towards more negative voltages (by -7.4 to -11.3 mV by 70 μm). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV 7.2/3 channel. These findings suggest that circulating polyunsaturated fatty acids, with a minimum requirement of multiple double bonds and a charged carboxyl group, dampen excitability by opening neuronal M-channels. Collectively, our data bring light to the molecular targets of polyunsaturated fatty acids and thus a possible mechanism by which polyunsaturated fatty acids reduce neuronal excitability. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  19. Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp.

    PubMed Central

    Costa, P F; Emilio, M G; Fernandes, P L; Ferreira, H G; Ferreira, K G

    1989-01-01

    1. A method of estimating absolute ionic permeability coefficients which does not depend on the use of impermeant substitutes is reported. 2. The method is based on a pump leak model of the Xenopus laevis oocyte membrane. The procedure consists of measuring, in the same experiment, the pump current and the currents generated under voltage clamp by the partial substitution of one or two ions at a time. For each experimental condition, the measured currents are substituted in a Goldman-Hodgkin-Katz type equation with two unknowns (the permeability coefficients). The set of equations thus generated enables the computation of all the ionic permeability coefficients. 3. The Xenopus oocyte membrane (stages IV and V, Dumont, 1972) has been found to be permeable to conventional ion substitutes such as N-methyl-D-glucamine (NMG), sulphate, isethionate and gluconate. 4. The values for sodium, potassium and chloride permeability coefficients obtained from sixty-eight pooled experiments were, respectively, 5.44, 17.41 and 1.49 x 10(-8) cm s-1. 5. The diffusional currents for sodium, potassium and chloride computed from the experiments referred to above were, respectively, -1.16, 0.69 and -0.038 microA cm-2. 6. A stoichiometry of the Na+-K+ pump exchange of 3/1.8 was computed. 7. The intracellular concentrations of sodium, potassium and chloride ions, as determined by ion-selective microelectrodes, were, respectively, 10.1 +/- 0.66 mM (n = 12), 109.5 +/- 3.3 mM (n = 13) and 37.7 +/- 1.18 mM (n = 19), corresponding to equilibrium potentials of 61, -95 and -28 mV. 8. Since chloride is not at equilibrium across the membrane, we propose that there is an inward uphill Cl- transport. PMID:2600847

  20. Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

    PubMed Central

    Beyeler, Anna; Rao, Guillaume; Ladepeche, Laurent; Jacques, André; Simmers, John; Le Ray, Didier

    2013-01-01

    During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance

  1. Vestibular lesion-induced developmental plasticity in spinal locomotor networks during Xenopus laevis metamorphosis.

    PubMed

    Beyeler, Anna; Rao, Guillaume; Ladepeche, Laurent; Jacques, André; Simmers, John; Le Ray, Didier

    2013-01-01

    During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance

  2. Alpha-adrenoreceptor activation modulates swimming via glycinergic and GABAergic inhibitory pathways in Xenopus laevis tadpoles.

    PubMed

    Merrywest, Simon D; Fischer, Hanno; Sillar, Keith T

    2002-01-01

    This study focuses upon the network pathways underlying the adrenoreceptor-mediated modulation of fictive swimming in the immobilized Xenopus laevis tadpole. As shown recently, noradrenaline (NA) increases cycle periods while simultaneously reducing the rostrocaudal delay in head-to-tail firing and the duration of swimming episodes. Furthermore, both swimming frequency and duration are reduced by selective pharmacological activation of alpha1- and/or alpha2-adrenoreceptors, while alpha1-receptor activation also reduces rostrocaudal delays. We show that NA could still modulate aspects of swimming after blocking either glycine or GABA(A) receptors with strychnine and bicuculline, respectively. Furthermore, after prior application of NA, strychnine could counteract noradrenergic effects on cycle periods and rostrocaudal delays, while bicuculline could counteract effects on cycle periods, suggesting that these two fast inhibitory pathways are both involved in the NA modulation of swimming. In addition, blocking glycine receptors reduced the effects of alpha1-receptors on cycle periods and delays, while blocking GABA(A) receptors had no effect. Blocking either glycine or GABA(A) receptors, however, lessened the reduction in swimming frequency by alpha2-receptors. In addition, pre-application of bicuculline prevented a reduction in episode durations by NA, alpha1- and alpha2-receptors. Our findings suggest that the noradrenergic modulation of Xenopus swimming is mediated via alpha-adrenoreceptors interacting with both glycinergic and GABAergic inhibitory pathways. Both alpha1- and alpha2-receptor activation influences the GABAergic pathway controlling the duration of swimming episodes and is involved in the glycinergic modulation of the swimming rhythm and its longitudinal co-ordination, with alpha2-receptors additionally affecting swimming frequency through GABAergic pathways.

  3. Gamete interactions in Xenopus laevis: identification of sperm binding glycoproteins in the egg vitelline envelope.

    PubMed

    Tian, J; Gong, H; Thomsen, G H; Lennarz, W J

    1997-03-10

    A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.

  4. The pharmacokinetics of enrofloxacin in adult African clawed frogs (Xenopus laevis).

    PubMed

    Howard, Antwain M; Papich, Mark G; Felt, Stephen A; Long, Charles T; McKeon, Gabriel P; Bond, Emmitt S; Torreilles, Stéphanie L; Luong, Richard H; Green, Sherril L

    2010-11-01

    Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 μg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 μg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 μg/mL, 915.85 mL/kg, and 47.42 μg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.

  5. Identification of fucosylated glycoconjugates in Xenopus laevis testis by lectin histochemistry.

    PubMed

    Valbuena, Galder; Madrid, Juan Francisco; Hernández, Francisco; Sáez, Francisco José

    2010-08-01

    Glycoconjugates play roles in many physiological and pathological processes. Previous works have shown important functions mediated by glycans in spermatogenesis, and the carbohydrate composition of testis has been studied by several approaches, including lectin-histochemical methods. However, the testis of Xenopus laevis, an animal model extensively employed in biochemical, cell and developmental research, has not yet been analysed. The aim of this work was to carry out a histochemical study of the fucose (Fuc)-containing glycoconjugates of Xenopus testis by means of lectins, combined with deglycosylation pretreatments. Four Fuc-binding lectins were used: orange peel (Aleuria aurantia) lectin (AAL), gorse seed (Ulex europaeus) agglutinin-I (UEA-I), fresh water eel (Anguilla anguilla) agglutinin (AAA), and asparagus pea (Lotus tetragonolobus) agglutinin (LTA), each recognizing different forms of fucosylated glycans. Labelling with UEA-I, which preferably binds Fucalpha(1,2) containing oligosaccharides, did not show any appreciable staining. LTA, specific for Fucalpha(1,3), and AAA, which binds Fucalpha(1,2), labelled spermatocytes and spermatids, but no labelling was seen when the histochemical procedure was carried out after either beta-elimination (which removes O-linked oligosaccharides) or incubation with PNGase F (which removes N-linked oligosaccharides), suggesting that fucosylated glycans are of both N- and O-linked types. AAL, which has its highest affinity to Fucalpha(1,6), but also recognizes Fucalpha(1,2) and Fucalpha(1,3), labelled the whole testis, and the staining remained when the histochemical method was performed after either beta-elimination or incubation with PNGase F. Labelling with AAL could be explained by the fact that this lectin could be binding to diverse fucosylated glycans in N- and O-glycans, and even in glycolipids. The importance of these glycans is discussed.

  6. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  7. Expanding the genetic toolkit in Xenopus: Approaches and Opportunities for Human Disease Modeling

    PubMed Central

    Tandon, Panna; Conlon, Frank; Furlow, J. David; Horb, Marko E.

    2016-01-01

    The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations. PMID:27109192

  8. Urotensin II receptor (UTR) exists in hyaline chondrocytes: a study of peripheral distribution of UTR in the African clawed frog, Xenopus laevis.

    PubMed

    Konno, Norifumi; Fujii, Yuya; Imae, Haruka; Kaiya, Hiroyuki; Mukuda, Takao; Miyazato, Mikiya; Matsuda, Kouhei; Uchiyama, Minoru

    2013-05-01

    Urotensin II (UII) and UII-related peptide (URP) exhibit diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response through UII receptor (UTR), which is expressed in the central nervous system and peripheral tissues of fish and mammals. In amphibians, only UII has been identified. As the first step toward elucidating the actions of UII and URP in amphibians, we cloned and characterized URP and UTR from the African clawed frog Xenopus laevis. Functional analysis showed that treatment of UII or URP with Chinese hamster ovary cells transfected with the cloned receptor increased the intracellular calcium concentration in a concentration-dependent manner, whereas the administration of the UTR antagonist urantide inhibited UII- or URP-induced Ca(2+) mobilization. An immunohistochemical study showed that UTR was expressed in the splenocytes and leukocytes isolated from peripheral blood, suggesting that UII and URP are involved in the regulation of the immune system. UTR was also localized in the apical membrane of the distal tubule of the kidney and in the transitional epithelial cells of the urinary bladder. This result supports the view that the UII/URP-UTR system plays an important role in osmoregulation of amphibians. Interestingly, immunopositive labeling for UTR was first detected in the chondrocytes of various hyaline cartilages (the lung septa, interphalangeal joint and sternum). The expression of UTR was also observed in the costal cartilage, tracheal cartilages, and xiphoid process of the rat. These novel findings probably suggest that UII and URP mediate the formation of the cartilaginous matrix. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Dynamics of background adaptation in Xenopus laevis: role of catecholamines and melanophore-stimulating hormone.

    PubMed

    van Zoest, I D; Heijmen, P S; Cruijsen, P M; Jenks, B G

    1989-10-01

    The pars intermedia of the pituitary gland in Xenopus laevis secretes alpha-melanophore-stimulating hormone (alpha-MSH), which causes dispersion of pigment in dermal melanophores in animals on a black background. In the present study we have determined plasma levels of alpha-MSH in animals undergoing adaptation to white and black backgrounds. Plasma values of black-adapted animals were high and decreased rapidly after transfer to a white background, as did the degree of pigment dispersion in dermal melanophores. Plasma MSH values of white-adapted animals were below the detection limit of our radioimmunoassay. Transfer of white animals to a black background resulted in complete dispersion of melanophore pigment within a few hours, but plasma MSH levels remained low for at least 24 hr. This discrepancy between plasma MSH and degree of pigment dispersion suggested the involvement of an additional factor for stimulating dispersion. Results of in vitro and in vivo experiments with receptor agonists and antagonists indicated that a beta-adrenergic mechanism, functioning at the level of the melanophore, is involved in the stimulation of pigment dispersion during the early stages of background adaptation.

  10. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    PubMed

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B

    1990-11-01

    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

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

    PubMed Central

    Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

    2014-01-01

    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. PMID:24523664

  12. Global realized niche divergence in the African clawed frog Xenopus laevis.

    PubMed

    Rödder, Dennis; Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G J; Lillo, Francesco; De Villiers, F A; De Busschere, Charlotte; Backeljau, Thierry

    2017-06-01

    Although of crucial importance for invasion biology and impact assessments of climate change, it remains widely unknown how species cope with and adapt to environmental conditions beyond their currently realized climatic niches (i.e., those climatic conditions existing populations are exposed to). The African clawed frog Xenopus laevis, native to southern Africa, has established numerous invasive populations on multiple continents making it a pertinent model organism to study environmental niche dynamics. In this study, we assess whether the realized niches of the invasive populations in Europe, South, and North America represent subsets of the species' realized niche in its native distributional range or if niche shifts are traceable. If shifts are traceable, we ask whether the realized niches of invasive populations still contain signatures of the niche of source populations what could indicate local adaptations. Univariate comparisons among bioclimatic conditions at native and invaded ranges revealed the invasive populations to be nested within the variable range of the native population. However, at the same time, invasive populations are well differentiated in multidimensional niche space as quantified via n-dimensional hypervolumes. The most deviant invasive population are those from Europe. Our results suggest varying degrees of realized niche shifts, which are mainly driven by temperature related variables. The crosswise projection of the hypervolumes that were trained in invaded ranges revealed the south-western Cape region as likely area of origin for all invasive populations, which is largely congruent with DNA sequence data and suggests a gradual exploration of novel climate space in invasive populations.

  13. Dynamic properties of calcium-activated chloride currents in Xenopus laevis oocytes

    PubMed Central

    M. De la Fuente, Ildefonso; Malaina, Iker; Pérez-Samartín, Alberto; Boyano, María Dolores; Pérez-Yarza, Gorka; Bringas, Carlos; Villarroel, Álvaro; Fedetz, María; Arellano, Rogelio; Cortes, Jesus M.; Martínez, Luis

    2017-01-01

    Chloride is the most abundant permeable anion in the cell, and numerous studies in the last two decades highlight the great importance and broad physiological role of chloride currents mediated anion transport. They participate in a multiplicity of key processes, as for instance, the regulation of electrical excitability, apoptosis, cell cycle, epithelial secretion and neuronal excitability. In addition, dysfunction of Cl− channels is involved in a variety of human diseases such as epilepsy, osteoporosis and different cancer types. Historically, chloride channels have been of less interest than the cation channels. In fact, there seems to be practically no quantitative studies of the dynamics of chloride currents. Here, for the first time, we have quantitatively studied experimental calcium-activated chloride fluxes belonging to Xenopus laevis oocytes, and the main results show that the experimental Cl− currents present an informational structure characterized by highly organized data sequences, long-term memory properties and inherent “crossover” dynamics in which persistent correlations arise at short time intervals, while anti-persistent behaviors become dominant in long time intervals. Our work sheds some light on the understanding of the informational properties of ion currents, a key element to elucidate the physiological functional coupling with the integrative dynamics of metabolic processes. PMID:28198817

  14. Characterization of tweety gene (ttyh1-3) expression in Xenopus laevis during embryonic development

    PubMed Central

    Rabe, Brian A.; Huyck, Ryan W.; Williams, Cheyenne C.; Saha, Margaret S.

    2015-01-01

    The tweety family of genes encodes large-conductance chloride channels and has been implicated in a wide array of cellular processes including cell division, cell adhesion, regulation of calcium activity, and tumorigenesis, particularly in neuronal cells. However, their expression patterns during early development remain largely unknown. Here, we describe the spatial and temporal patterning of ttyh1, ttyh2, and ttyh3 in Xenopus laevis during early embryonic development. Ttyh1 and ttyh3 are initially expressed at the late neurula stage are and primarily localized to the developing nervous system; however ttyh1 and ttyh3 both show transient expression in the somites. By swimming tadpole stages, all three genes are expressed in the brain, spinal cord, eye, and cranial ganglia. While ttyh1 is restricted to proliferative, ventricular zones, ttyh3 is primarily localized to postmitotic regions of the developing nervous system. Ttyh2, however, is strongly expressed in cranial ganglia V, VII, IX and X. The differing temporal and spatial expression patterns of ttyh1, ttyh2, and ttyh3 suggest that they may play distinct roles throughout embryonic development. PMID:25541457

  15. The Effect of Plasma Exposure on Tail Regeneration of Tadpoles Xenopus Laevis

    NASA Astrophysics Data System (ADS)

    June, Joyce; Rivie, Adonis; Ezuduemoih, Raphael; Menon, Jaishri; Martus, Kevin

    2014-03-01

    Wound healing requires a balanced combination of nutrients and growth factors for healing and tissue regeneration. The effect of plasma exposure on tail regeneration of tadpoles, Xenopus laevis is investigated. The exposure of the wound to the helium plasma immediately followed the amputation of 40% of the tail. Amputation of the tail initiates regeneration of spinal cord, muscle, notochord, skin and connective tissues. By 24 h, the wound was covered by wound epithelium and blastema was formed by day 5. There was increased angiogenesis in plasma exposed tail regenerate compared to the control following 5 d post amputation. Observed was an increase in NO production in the regenerate of plasma exposed tadpoles was derived from increased activity of nNOS and iNOS. Western blot analysis for vascular endothelial growth factor showed stronger bands for the protein in amputated tadpoles of both the groups. Analysis of the composition and characteristics of the plasma using optical emission spectroscopy indicates excited state species consisting of N2, N2+,and OH is present in the plasma. This study was supported, in part, by the NSF Grant 1040108.

  16. Developmental changes in head movement kinematics during swimming in Xenopus laevis tadpoles.

    PubMed

    Hänzi, Sara; Straka, Hans

    2017-01-15

    During the post-embryonic developmental growth of animals, a number of physiological parameters such as locomotor performance, dynamics and behavioural repertoire are adjusted to match the requirements determined by changes in body size, proportions and shape. Moreover, changes in movement parameters also cause changes in the dynamics of self-generated sensory stimuli, to which motion-detecting sensory systems have to adapt. Here, we examined head movements and swimming kinematics of Xenopus laevis tadpoles with a body length of 10-45 mm (developmental stage 46-54) and compared these parameters with fictive swimming, recorded as ventral root activity in semi-intact in vitro preparations. Head movement kinematics was extracted from high-speed video recordings of freely swimming tadpoles. Analysis of these locomotor episodes indicated that the swimming frequency decreased with development, along with the angular velocity and acceleration of the head, which represent self-generated vestibular stimuli. In contrast, neither head oscillation amplitude nor forward velocity changed with development despite the ∼3-fold increase in body size. The comparison between free and fictive locomotor dynamics revealed very similar swimming frequencies for similarly sized animals, including a comparable developmental decrease of the swimming frequency. Body morphology and the motor output rhythm of the spinal central pattern generator therefore develop concurrently. This study thus describes development-specific naturalistic head motion profiles, which form the basis for more natural stimuli in future studies probing the vestibular system. © 2017. Published by The Company of Biologists Ltd.

  17. Temperature-independent energy expenditure in early development of the African clawed frog Xenopus laevis.

    PubMed

    Nagano, Yatsuhisa; Ode, Koji L

    2014-08-01

    The thermal dissipation of activated eggs and embryos undergoing development from cleavage to the tailbud stage of the African clawed frog Xenopus laevis was measured as a function of incubation time at temperatures ranging from T = 288.2 K to 295.2 K, using a high-precision isothermal calorimeter. A23187-mediated activation of mature eggs induced stable periodic thermal oscillations lasting for 8-34 h. The frequency agreed well with the cell cycle frequency of initial cleavages at the identical temperature. In the developing embryo, energy metabolism switches from embryonic to adult features during gastrulation. The thermal dissipation after gastrulation fit well with a single modified Avrami equation, which has been used for modeling crystal-growth. Both the oscillation frequency of the activated egg and the growth rate of the embryo strongly depend on temperature with the same apparent activation energy of approximately 87 kJ mole(-1). This result suggests that early development proceeds as a single biological time, attributable to a single metabolic rate. A temperature-independent growth curve was derived by scaling the thermogram to the biological time, indicating that the amount of energy expenditure during each developmental stage is constant over the optimal temperature range.

  18. Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis.

    PubMed

    Zhang, Li; Salom, David; He, Jianhua; Okun, Alex; Ballesteros, Juan; Palczewski, Krzysztof; Li, Ning

    2005-11-08

    G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogeneous quality in the vertebrate retina. We used transgenic Xenopus laevis to convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogeneously glycosylated. Ligand and [(35)S]GTPgammaS binding assays of the 5HT(1A) and EDG(1) GPCRs confirmed that they were properly folded and functional. 5HT(1A)R was highly purified by taking advantage of the rhodopsin C-terminal immunoaffinity tag common to all GPCR constructs. We have also developed an automated system that can generate hundreds of transgenic tadpoles per day. This expression approach could be extended to other animal model systems and become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies.

  19. Dynamic properties of calcium-activated chloride currents in Xenopus laevis oocytes.

    PubMed

    M De la Fuente, Ildefonso; Malaina, Iker; Pérez-Samartín, Alberto; Boyano, María Dolores; Pérez-Yarza, Gorka; Bringas, Carlos; Villarroel, Álvaro; Fedetz, María; Arellano, Rogelio; Cortes, Jesus M; Martínez, Luis

    2017-02-13

    Chloride is the most abundant permeable anion in the cell, and numerous studies in the last two decades highlight the great importance and broad physiological role of chloride currents mediated anion transport. They participate in a multiplicity of key processes, as for instance, the regulation of electrical excitability, apoptosis, cell cycle, epithelial secretion and neuronal excitability. In addition, dysfunction of Cl(-) channels is involved in a variety of human diseases such as epilepsy, osteoporosis and different cancer types. Historically, chloride channels have been of less interest than the cation channels. In fact, there seems to be practically no quantitative studies of the dynamics of chloride currents. Here, for the first time, we have quantitatively studied experimental calcium-activated chloride fluxes belonging to Xenopus laevis oocytes, and the main results show that the experimental Cl(-) currents present an informational structure characterized by highly organized data sequences, long-term memory properties and inherent "crossover" dynamics in which persistent correlations arise at short time intervals, while anti-persistent behaviors become dominant in long time intervals. Our work sheds some light on the understanding of the informational properties of ion currents, a key element to elucidate the physiological functional coupling with the integrative dynamics of metabolic processes.

  20. Characterization of CXC-type chemokine molecules in early Xenopus laevis development.

    PubMed

    Goto, Toshiyasu; Michiue, Tatsuo; Ito, Yuzuru; Asashima, Makoto

    2013-01-01

    Chemokine molecules play important roles in the immune system. However, several chemokine molecules are expressed during early development before the immune system is established. Using reverse transcription–polymerase chain reaction (RT-PCR) and overexpression of chemokine molecules, we identified and characterized Xenopus laevis CXC-type chemokine ligands (XCXCL13L1, XCXCL13L2, XCXCLa, XCXCLb, XCXCLd, and XCXCLe) and receptors (XCXCR1/2, XCXCR3, XCXCR5, XCXCR6, and XCXCRa) during early development. The CXC-type ligands have low identity with genes for human CXC ligands (CXCL). With the exception of XCXCRa, the CXC receptors (CXCR) identified in the present study had high (40%–65%) identity with human CXCR genes. Although the expression patterns for the CXCL and CXCR genes differed, transcript levels for all genes were very low during early embryogenesis. Overexpression of XCXCL13L1, XCXCL13L2, XCXCLa, XCXCR3, XCXCR6, and XCXCRa interfered with gastrulation and neural fold closure. The results of the present study suggest that several chemokine molecules are related to cell movements during early morphogenesis.

  1. Diverse functions of kindlin/fermitin proteins during embryonic development in Xenopus laevis.

    PubMed

    Rozario, Tania; Mead, Paul E; DeSimone, Douglas W

    2014-08-01

    The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development.

  2. An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis.

    PubMed

    Buisson, Nicolas; Sirour, Cathy; Moreau, Nicole; Denker, Elsa; Le Bouffant, Ronan; Goullancourt, Aline; Darribère, Thierry; Bello, Valérie

    2014-12-01

    Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells.

  3. The Effect of Plasma on Tail Regeneration of Tadpoles Xenopus Laevis

    NASA Astrophysics Data System (ADS)

    June, Joyce; Amadi, Chima; Menon, Jaishri; Martus, Kevin

    2013-03-01

    Healthy wounds require a balanced combination of nutrients and growth factors for healing and tissue regeneration. Nitric oxide, (NO), is also crucial in wound healing processes and linked with production of several cytokines, interaction with other free radicals and influence on microcirculation. Hypothesize is that exposure to plasma will affect wound healing and tail regeneration in tadpoles Xenopus laevis and plasma induced endogenous NO production may have an important role to play at the cellular level. Tail amputation was immediately followed by exposure of the wound to the helium plasma. For histological features, blastema (growing regenerate) was fixed in 4% neutral buffer formalin for paraffin sections. In situ staining for NO was carried out 5 days post amputation. The rate of the regenerating tail was proportional to the plasma exposure time at the expense of metamorphic rate. Histological features show that the tadpoles exposed to the plasma had a higher level of cellular proliferation and microvasculature in blastema. In situ staining for NO indicated its increased endogenous production compared to the control. These findings suggest that accelerated wound healing and tail regeneration following exposure to the plasma may be due to its direct effect on cell proliferation and increased NO production which may be involved in microvascularization. This study was supported, in part, by the NSF Grant 1040108

  4. Mortality and morbidity in African clawed frogs (Xenopus laevis) associated with construction noise and vibrations.

    PubMed

    Felt, Stephen A; Cowan, Andrea M; Luong, Richard; Green, Sherril L

    2012-03-01

    In Spring 2008, 175 adult female Xenopus laevis were exposed to construction-related vibrations that caused overt water rippling in the frog tanks. The 3 affected tanks were custom-built static, 300-gal 'pond-style' tanks that sat on the floor of the housing room. The water in the tank developed visible ripples as a result of the vibrations transmitted through the floor during jack-hammering in an adjacent room that was approximately 10 ftaway. All frogs in the tanks displayed buoyancy problems, excessive air gulping, and skin sloughing; ultimately 7 frogs died. In addition, these 7 animals were bloated, and 5 of these 7 had regurgitated and everted their stomach and distal esophagus into the oral cavity, resulting in airway obstruction and death. Gross pathologic findings included regurgitation and eversion of the stomach of the distal portion of the esophagus into the oral cavity, obstruction of the airway, and lung overinflation. No significant histologic lesions were observed. Construction vibrations transmitted through the water appeared to have disrupted the mechanoreceptive function of the lateral line system, resulting in overstimulation of the noxious feeding response, regurgitation, and eversion of the stomach and distal esophagus into the oral cavity and subsequent suffocation due to airway obstruction. After immediate cessation of the jack-hammering and relocation of the remaining frogs, no additional morbidities or mortalities occurred.

  5. Temperature-independent energy expenditure in early development of the African clawed frog Xenopus laevis

    NASA Astrophysics Data System (ADS)

    Nagano, Yatsuhisa; Ode, Koji L.

    2014-08-01

    The thermal dissipation of activated eggs and embryos undergoing development from cleavage to the tailbud stage of the African clawed frog Xenopus laevis was measured as a function of incubation time at temperatures ranging from T = 288.2 K to 295.2 K, using a high-precision isothermal calorimeter. A23187-mediated activation of mature eggs induced stable periodic thermal oscillations lasting for 8-34 h. The frequency agreed well with the cell cycle frequency of initial cleavages at the identical temperature. In the developing embryo, energy metabolism switches from embryonic to adult features during gastrulation. The thermal dissipation after gastrulation fit well with a single modified Avrami equation, which has been used for modeling crystal-growth. Both the oscillation frequency of the activated egg and the growth rate of the embryo strongly depend on temperature with the same apparent activation energy of approximately 87 kJ mole-1. This result suggests that early development proceeds as a single biological time, attributable to a single metabolic rate. A temperature-independent growth curve was derived by scaling the thermogram to the biological time, indicating that the amount of energy expenditure during each developmental stage is constant over the optimal temperature range.

  6. Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

    PubMed

    Raymond Delpech, Valérie; Ihara, Makoto; Coddou, Claudio; Matsuda, Kazuhiko; Sattelle, David B

    2003-11-01

    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha7, chicken alpha4beta2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta2 and ALS/beta2. No agonist action of NTX (0.1-100 microM) was observed on chicken alpha7, chicken alpha4beta2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha7) or heteromeric (alpha4beta2) nicotinic AChRs. Block by NTX of the chicken alpha7, chicken alpha4beta2 and the SAD/beta2 and ALS/beta2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.

  7. Brain-derived neurotrophic factor (BDNF) expression in normal and regenerating olfactory epithelium of Xenopus laevis.

    PubMed

    Frontera, Jimena Laura; Cervino, Ailen Soledad; Jungblut, Lucas David; Paz, Dante Agustín

    2015-03-01

    Olfactory epithelium has the capability to continuously regenerate olfactory receptor neurons throughout life. Adult neurogenesis results from proliferation and differentiation of neural stem cells, and consequently, olfactory neuroepithelium offers an excellent opportunity to study neural regeneration and the factors involved in the maintenance and regeneration of all their cell types. We analyzed the expression of BDNF in the olfactory system under normal physiological conditions as well as during a massive regeneration induced by chemical destruction of the olfactory epithelium in Xenopus laevis larvae. We described the expression and presence of BDNF in the olfactory epithelium and bulb. In normal physiological conditions, sustentacular (glial) cells and a few scattered basal (stem) cells express BDNF in the olfactory epithelium as well as the granular cells in the olfactory bulb. Moreover, during massive regeneration, we demonstrated a drastic increase in basal cells expressing BDNF as well as an increase in BDNF in the olfactory bulb and nerve. Together these results suggest an important role of BDNF in the maintenance and regeneration of the olfactory system.

  8. Diverse functions of kindlin/fermitin proteins during embryonic development in Xenopus laevis

    PubMed Central

    Rozario, Tania; Mead, Paul E.; DeSimone, Douglas W.

    2014-01-01

    The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development. PMID:25173804

  9. Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.

    PubMed

    Stout, Alina; D'Amico, Salvatore; Enzenbacher, Tiffany; Ebbert, Patrick; Lowery, Laura Anne

    2014-09-07

    Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8).

  10. Tail regeneration in Xenopus laevis as a model for understanding tissue repair.

    PubMed

    Tseng, A-S; Levin, M

    2008-09-01

    Augmentation of regenerative ability is a powerful strategy being pursued for the biomedical management of traumatic injury, cancer, and degeneration. While considerable attention has been focused on embryonic stem cells, it is clear that much remains to be learned about how somatic cells may be controlled in the adult organism. The tadpole of the frog Xenopus laevis is a powerful model system within which fundamental mechanisms of regeneration are being addressed. The tadpole tail contains spinal cord, muscle, vasculature, and other terminally differentiated cell types and can fully regenerate itself through tissue renewal--a process that is most relevant to mammalian healing. Recent insight into this process has uncovered fascinating molecular details of how a complex appendage senses injury and rapidly repairs the necessary morphology. Here, we review what is known about the chemical and bioelectric signals underlying this process and draw analogies to evolutionarily conserved pathways in other patterning systems. The understanding of this process is not only of fundamental interest for the evolutionary and cell biology of morphogenesis, but will also generate information that is crucial to the development of regenerative therapies for human tissues and organs.

  11. Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles ( Xenopus laevis)

    NASA Astrophysics Data System (ADS)

    Sebastian, C.; Horn, E.

    2001-01-01

    In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1) the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) μg induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.

  12. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  13. Effects of dietary exposure of polycyclic musk HHCB on the metamorphosis of Xenopus laevis.

    PubMed

    Pablos, María Victoria; Jiménez, María Ángeles; San Segundo, Laura; Martini, Federica; Beltrán, Eulalia; Fernández, Carlos

    2016-06-01

    The compound 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[γ]-2-benzopyrane (HHCB; galaxolide, Chemical Abstracts Service number 1222-05-5) is a synthetic musk used extensively as a fragrance in many consumer products and classified as an emerging pollutant. The ecotoxicological information available for HHCB addresses exposure via water, but this compound is frequently adsorbed into particulate matter. The goal of the present study was to assess the effects of dietary exposure to several environmentally relevant HHCB concentrations adsorbed in food during Xenopus laevis metamorphosis. The authors sought to determine if such exposure to this synthetic musk resulted in histological changes in the thyroid gland in conjunction with changes in development (staging, timing to metamorphosis), body weight, and length. Developmental acceleration on day 14, together with hypertrophy of the thyroid follicular epithelium in tadpoles, suggested a possible agonistic effect of HHCB, which would have been compensated after metamorphosis by regulatory mechanisms to maintain homeostasis. Further research into the potential thyroid-related mechanisms of action of HHCB should be conducted. Environ Toxicol Chem 2016;35:1428-1435. © 2015 SETAC. © 2015 SETAC.

  14. Gonadal development of larval male Xenopus laevis exposed to atrazine in outdoor microcosms

    USGS Publications Warehouse

    Jooste, A.M.; Du Preez, L.H.; Carr, J.A.; Giesy, J.P.; Gross, T.S.; Kendall, R.J.; Smith, E.E.; Van Der Kraak, G. L.; Solomon, K.R.

    2005-01-01

    The potential effects of atrazine on gonadal development in metamorphs and subadults of the African clawed frog (Xenopus laevis) were studied under conditions of natural photoperiod and temperatures in outdoor microcosms from August 2002 to June 2003 in South Africa. Triplicate 1100 L microcosms for each nominal concentration of 0.0, 1, 10, and 25 ??g of atrazine/L were used. Measured atrazine concentrations varied <25% throughout the study, and no atrazine was detected in the control microcosms. Tadpoles developed well at all concentrations. On the basis of histological examination of testes of recently metamorphosed stage 66 frogs, 57% of the individuals in the reference group exhibited testicular oocytes as compared with 57, 59, and 39% of the 1, 10, and 25 ??g/L atrazine groups, respectively. The average prevalence of testicular oocytes for all of the treatments including the controls was 54% in a single testis, while, in 35% of individuals, testicular oocytes were observed in both testes. The number of testicular oocytes per individual ranged from 0 to 58 with means of 9.5, 9.8, 8.5, and 11.1 for the 0.0, 1, 10, and 25 ??g of atrazine/L groups, respectively. Ten months after metamorphosis, another subset of juveniles was examined, and the maximum number of testicular oocytes observed was five in one animal. The presence of testicular oocytes was not related to exposure to atrazine and may be a natural phenomenon during ontogeny. ?? 2005 American Chemical Society.

  15. The polymorphic integumentary mucin B.1 from Xenopus laevis contains the short consensus repeat.

    PubMed

    Probst, J C; Hauser, F; Joba, W; Hoffmann, W

    1992-03-25

    The frog integumentary mucin B.1 (FIM-B.1), discovered by molecular cloning, contains a cysteine-rich C-terminal domain which is homologous with von Willebrand factor. With the help of the polymerase chain reaction, we now characterize a contiguous region 5' to the von Willebrand factor domain containing the short consensus repeat typical of many proteins from the complement system. Multiple transcripts have been cloned, which originate from a single animal and differ by a variable number of tandem repeats (rep-33 sequences). These different transcripts probably originate solely from two genes and are generated presumably by alternative splicing of an huge array of functional cassettes. This model is supported by analysis of genomic FIM-B.1 sequences from Xenopus laevis. Here, rep-33 sequences are arranged in an interrupted array of individual units. Additionally, results of Southern analysis revealed genetic polymorphism between different animals which is predicted to be within the tandem repeats. A first investigation of the predicted mucins with the help of a specific antibody against a synthetic peptide determined the molecular mass of FIM-B.1 to greater than 200 kDa. Here again, genetic polymorphism between different animals is detected.

  16. Tradeoffs between somatic and gonadal investments during development in the African clawed frog (Xenopus laevis).

    PubMed

    McCoy, Krista A; McCoy, Michael W; Amick, Alison; Guillette, Louis J; St Mary, Colette M

    2007-11-01

    Tradeoffs between time to and size at metamorphosis occur in many organisms with complex life histories. The ability to accelerate metamorphosis can increase survival to the next life stage, but the resulting smaller size at metamorphosis is often associated with lower post-metamorphic survival or reduced fecundity of adults. Reduced fecundity is thought to be because of reduced energy reserves, longer time to maturity, or reduced capacity to carry eggs or compete for mates. This pattern could also be explained by a shift in allocation to somatic growth that further retards the growth or development of reproductive tissues. The main goal of this study was to determine if the relationship between growth and development of somatic and gonadal tissues depends on environmental conditions. We address this question through two experiments in which we quantify the development and growth of the body and gonads of Xenopus laevis reared in different resource environments. First, tadpoles were reared communally and development and growth were evaluated over time. Restricted food reduced somatic and gonadal growth rate, but did not affect the developmental rate of either tissue type. Second, tadpoles were reared individually and evaluated at metamorphosis. Restricted food reduced somatic development and growth, but only influenced size, and not developmental stage of testes at metamorphosis. This work demonstrates that environmental conditions influence tradeoffs between growth and development of somatic and gonadal tissues, apparently in a sex-specific manner. These tradeoffs may contribute to phenotypic correlations between small size and reduced fitness.

  17. Induction of maturation in Xenopus laevis oocytes by a steroid linked to a polymer

    PubMed Central

    Godeau, J. Francois; Schorderet-Slatkine, Sabine; Hubert, Patrick; Baulieu, Etienne-Emile

    1978-01-01

    A progesterone analog has been covalently linked via an amide bond to polyethylene oxide (molecular weight, 20,000). This macromolecular steroid molecule displays the biological activity of progesterone in inducing meiotic maturation when incubated with Xenopus laevis oocytes (stage VI) in vitro. Its efficiency (half-maximum effective concentration, 30 μM) is approximately 10 times lower than that of its low molecular weight homolog (3 μM). Control experiments with polyethylene oxide and an estradiol derivative (up to 1 mM) assessed the specificity of the progesterone macromolecular analog. Uptake experiments using radioactive derivatives revealed a small (if not negligible) intake of the macromolecular progesterone analog by the oocytes compared to that of free steroids, and no parallelism was found between radioactivity incorporation and effect. The possibility of cleavage of the macromolecular derivative during the incubation was ruled out. Furthermore, injection of the polymer-linked progesterone into the oocytes did not induce maturation. These observations suggest that the macromolecular progesterone analog itself is responsible for the biological effect and that the presence of this compound inside the cell is neither necessary nor sufficient for triggering reinitiation of meiosis. These conclusions are in agreement with the proposal that interaction with the plasma membrane of the oocyte is necessary for progesterone action in this particular system, in contrast to the case of somatic cells which have intracellular steroid receptors. Images PMID:276879

  18. Hepatic confinement of newly produced erythrocytes caused by low-temperature exposure in Xenopus laevis.

    PubMed

    Maekawa, Shun; Iemura, Hitomi; Kuramochi, Yuko; Nogawa-Kosaka, Nami; Nishikawa, Hironori; Okui, Takehito; Aizawa, Youichi; Kato, Takashi

    2012-09-01

    Diminished erythrocyte count and erythropoiesis have been reported during hypothermia in some ectothermic animals. In this study, the African clawed frog, Xenopus laevis, was used to investigate the cause of hypothermia-induced anemia. We developed a new model of hypothermia at 5°C and monitored blood cell count and erythropoiesis on several days. Erythrocyte count declined by 30% on the first day following cold exposure (5°C) and mRNA expression of hemeoxygenase-1 was enhanced 10-fold; accumulation of iron as a result of heme degradation was observed in the liver. One day after low-temperature exposure, erythropoietin mRNA expression was elevated in the liver and lung compared with that at normal temperature (22°C) by qRT-PCR analysis. Examination of liver sections (i.e. the erythropoietic organ) showed an increase in o-dianisidine-positive erythrocytes in the hepatic sinusoid 5 days after the onset of low-temperature exposure compared with normal liver. Peripheral erythrocyte count remained low, indicating that newly produced erythrocytes did not migrate from the liver to the circulation during hypothermia. In conclusion, this study reveals hypothermic anemia as being associated with hepatic erythrocyte destruction; prolonged anemia during low-temperature exposure is concomitant with newly produced erythrocytes being confined to the liver and may lead to new insights into vertebrate hematopoiesis.

  19. Designation of the anterior/posterior axis in pregastrula Xenopus laevis.

    PubMed

    Lane, M C; Sheets, M D

    2000-09-01

    A new fate map for mesodermal tissues in Xenopus laevis predicted that the prime meridian, which runs from the animal pole to the vegetal pole through the center of Spemann's organizer, is the embryo's anterior midline, not its dorsal midline (M. C. Lane and W. C. Smith, 1999, Development 126, 423-434). In this report, we demonstrate by lineage labeling that the column 1 blastomeres at st. 6, which populate the prime meridian, give rise to the anterior end of the embryo. In addition, we surgically isolate and culture tissue centered on this meridian from early gastrulae. This tissue forms a patterned head with morphologically distinct ventral and dorsal structures. In situ hybridization and immunostaining reveal that the cultured heads contain the anterior tissues of all three germ layers, correctly patterned. Regardless of how we dissect early gastrulae along meridians running from the animal to the vegetal pole, both the formation of head structures and the expression of anterior marker genes always segregate with the prime meridian passing through Spemann's organizer. The prime meridian also gives rise to dorsal, axial mesoderm, but not uniquely, as specification tests show that dorsal mesoderm arises in fragments of the embryo which exclude the prime meridian. These results support the hypothesis that the midline that bisects Spemann's organizer is the embryo's anterior midline. Copyright 2000 Academic Press.

  20. Characterization of highly and moderately repetitive 500 bp Eco RI fragments from Xenopus laevis DNA.

    PubMed Central

    Hummel, S; Meyerhof, W; Korge, E; Knöchel, W

    1984-01-01

    Three different types of repetitive Eco RI fragments, which comigrate within a visible band of approximately 500 bp at gel electrophoresis of Xenopus laevis DNA Eco RI digests have been cloned and sequenced. These sequences are designated as Repetitive Eco RI Monomers: REM 1, REM 2 and REM 3. The sequences contain direct repeats, inverted repeats and palindromic elements. Genomic organization of the most abundant sequence (REM 1; 0.4% of total DNA) is that of an interspersed sequence. REM 2 (0.08%) is partly organized as an interspersed element and partly found in tandem arrangement, whereas REM 3 (0.02%) represents the tandemly repeated monomeric unit of a satellite DNA. In situ hybridization has shown that REM 1 and REM 2 sequences are found on most chromosomes, REM 1 being preferentially located on specific chromosomal loci. REM 3 is located near the centromere region of only one chromosome pair (presumably number 1). Hybridization of Northern blots from RNAs of different developmental stages revealed that REM 1, REM 2 and REM 3 sequences are transcribed and that transcription is under developmental control. Images PMID:6330690

  1. Male-male clasping may be part of an alternative reproductive tactic in Xenopus laevis.

    PubMed

    Rhodes, Heather J; Stevenson, Rachel J; Ego, Courtney L

    2014-01-01

    Male Xenopus laevis frogs have been observed to clasp other males in a sustained, amplectant position, the purpose of which is unknown. We examined three possible hypotheses for this counter-intuitive behavior: 1) clasping males fail to discriminate the sex of the frogs they clasp; 2) male-male clasping is an aggressive or dominant behavior; or 3) that males clasp other males to gain proximity to breeding events and possibly engage in sperm competition. Our data, gathered through a series of behavioral experiments in the laboratory, refute the first two hypotheses. We found that males did not clasp indiscriminately, but showed a sex preference, with most males preferentially clasping a female, but a proportion preferentially clasping another male. Males that clasped another male when there was no female present were less likely to "win" reproductive access in a male-male-female triad, indicating that they did not establish dominance through clasping. However, those males did gain proximity to oviposition by continued male-male clasping in the presence of the female. Thus, our findings are consistent with, but cannot confirm, the third hypothesis of male-male clasping as an alternative reproductive tactic.

  2. Electroencephalographic and physiologic changes after tricaine methanesulfonate immersion of African clawed frogs (Xenopus laevis).

    PubMed

    Lalonde-Robert, Vanessa; Desgent, Sébastien; Duss, Sandra; Vachon, Pascal

    2012-01-01

    The objective of this study was to determine electroencephalographic and complementary physiologic changes in Xenopus leavis frogs after bath immersion in MS222. We also evaluated the addition of sodium pentobarbital injected intracoelomi- cally 2 h after MS222 immersion to achieve euthanasia. Frogs (n = 9) weighing 105.5 ± 8.4 g (mean ± 1 SD) were immersed in MS222 at either 1 or 3 g/L until anesthesia was achieved; a conductive stainless steel screw then was implanted in the skull on top of the outer pial surface of the brain. Frogs were immersed again in MS222 at the same concentration as previously, and electroencephalograms, heart rate, oxygen saturation, and respiratory movements were recorded. Amplitude and mean frequency of the electroencephalographic signal were evaluated at 15-min intervals until a flat-line signal was achieved. At 2 h after induction, frogs were injected intracoelomically with sodium pentobarbital (0.5 mL; 240 mg/mL) to accelerate euthanasia. Immersion of frogs in 1 or 3 g/L of MS222 depressed cerebral activity within 30 min without a significant effect on cardiac function. Intracoelomic injection of sodium pentobarbital at 2 h after MS222 administration rapidly (3.2 ± 1.7 min) induced cardiac arrest. In conclusion, immersion in MS222 can be used for the collection of organs from X. laevis frogs, but the addition of pentobarbital is required to achieve euthanasia.

  3. Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

    SciTech Connect

    Schmalzing, G.; Eckard, P.; Kroener, S.P.; Passow, H. )

    1990-01-01

    During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by (gamma-32P)ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from (3H)ouabain bound to the cell surface before maturation could be phosphorylated with inorganic (32P)phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

  4. Neuronal degeneration and regeneration induced by axotomy in the olfactory epithelium of Xenopus laevis.

    PubMed

    Cervino, Ailen S; Paz, Dante A; Frontera, Jimena L

    2017-07-18

    The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific- neuronal death through apoptosis between 24 and 48h post-injury. In concordance, there was a progressive decrease of the mature-ORN marker OMP until it was completely absent 72h post-injury. On the other hand, neurogenesis was evident 48h post-injury by an increase in the number of proliferating basal cells as well as NCAM-180- GAP-43+ immature neurons. Mature ORNs were replenished 21 days post-injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol, 2017. © 2017 Wiley Periodicals, Inc.

  5. Meckel's cartilage in Xenopus laevis during metamorphosis: a light and electron microscope study.

    PubMed Central

    Thomson, D A

    1986-01-01

    Meckel's cartilage, in Xenopus laevis prior to metamorphosis, is a tissue exhibiting very large lacunae, separated by thin rims of matrix, presenting a net-like appearance, similar to that of cartilage in invertebrates. The cells on the periphery of the tissue are rather more flattened, and more closely packed. On the lateral aspects of the cartilage distinct columns of apparently dividing cells are evident. During metamorphic climax, the amount of matrix separating the lacunae increases, with an associated decrease in lacunar size, and some of the deeper cells develop cilia, which are not seen either before or after climax. By the end of metamorphic climax there is a considerable increase in the amount of matrix present in the tissue, while many cells at all depths in the cartilage show the presence of lysosome-like structures, possibly associated with the changing shape of the cartilage. Intramembranous ossification is proceeding around Meckel's cartilage, but there is no evidence of endochondral ossification up to the end of metamorphosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 Fig. 17 Fig. 18 Fig. 19 PMID:3693112

  6. Comparative development of end-plate currents in two muscles of Xenopus laevis.

    PubMed Central

    Kullberg, R; Owens, J L

    1986-01-01

    The development of miniature end-plate currents (m.e.p.c.s) was studied in the superior oblique and interhyoideus muscles of Xenopus laevis. An analysis of m.e.p.c. decays shows that each muscle possesses its own characteristic programme of end-plate current development. In the superior oblique, the exponential decay constants of m.e.p.c.s were initially about 3 ms; they declined within half a day to 1 ms and remained at that value for six weeks. They then gradually became longer, reaching a mean value of 1.7 ms at late metamorphosis. In the interhyoideus, m.e.p.c. decay constants were initially about 6 ms. They declined in less than one day to a mean value of 2.6 ms and remained there for the following seven weeks. Upon completion of metamorphosis, the decay constants underwent a further decrease to about 1 ms. In both muscles, the changes in m.e.p.c. decays were correlated with developmental changes in muscle contraction speeds, as measured by maximum twitch frequencies. The above changes in end-plate currents in the superior oblique and interhyoideus muscles are discussed in terms of the development of acetylcholine receptor channel gating and acetylcholinesterase activity. PMID:3018234

  7. Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis

    PubMed Central

    1983-01-01

    We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF. PMID:6345556

  8. Biosynthesis, processing, and control of release of melanotropic peptides in the neurointermediate lobe of Xenopus laevis

    PubMed Central

    1977-01-01

    The neurointermediate lobes of dark-adapted toads Xenopus laevis were incubated for 30 min in [3H]arginine and then "chased" for various time periods. By use of this pulse-chase paradigm there were detected 10 trichloroacetic acid (TCA)-precipitable peptides separated on acid-urea polyacrylamide gels and one TCA-soluble peptide separated by high- voltage electrophoresis (pH 4.9) with melanotropic activity. Each of these peptides had a different degree of melanocyte stimulating hormone (MSH) activity as revealed by the Anolis skin bioassay. Three of these TCA-precipitable peptides comigrated with ACTH, beta-lipotrophin, and alpha-MSH on acid-urea gels. Evidence suggesting a precursor-product mode of biosynthesis of the melanotropic peptides is presented. 7 of the 10 TCA-precipitable peptides and the one TCA-soluble peptide with melanotropic activity were released into the medium. The half-time of release of the TCA-precipitable peptides was about 2 h, whereas the half-time of TCA-soluble peptide release was about 30 min. The release of these peptides was inhibited by 5 X 10(-5) M dopamine. Dopamine inhibition of release did not appear to affect the biosynthesis of the melanotropic peptides, but did appear to enhance the degradation of the newly synthesized TCA-soluble peptide in the tissue. White adaptation of the toads greatly decreased the biosynthesis of all of the TCA- precipitable melanotropic peptides. PMID:894250

  9. Subcellular in vivo 1H MR spectroscopy of Xenopus laevis oocytes.

    PubMed

    Lee, Seung-Cheol; Cho, Jee-Hyun; Mietchen, Daniel; Kim, Young-Sook; Hong, Kwan Soo; Lee, Chulhyun; Kang, Dongmin; Park, Ki Deok; Choi, Byong-Seok; Cheong, Chaejoon

    2006-03-01

    In vivo magnetic resonance (MR) spectra are typically obtained from voxels whose spatial dimensions far exceed those of the cells they contain. This study was designed to evaluate the potential of localized MR spectroscopy to investigate subcellular phenomena. Using a high magnetic field and a home-built microscopy probe with large gradient field strengths, we achieved voxel sizes of (180 microm)3. In the large oocytes of the frog Xenopus laevis, this was small enough to allow the recording of the first compartment-selective in vivo MR spectra from the animal and vegetal cytoplasm as well as the nucleus. The two cytoplasmic regions differed in their lipid contents and NMR lineshape characteristics-differences that are not detectable with whole-cell NMR techniques. In the nucleus, the signal appeared to be dominated by water, whereas other contributions were negligible. We also used localized spectroscopy to monitor the uptake of diminazene acturate, an antitrypanosomal agent, into compartments of a single living oocyte. The resulting spectra from the nucleus and cytoplasm revealed different uptake kinetics for the two components of the drug and demonstrate that MR technology is on the verge of becoming a tool for cell biology.

  10. Subcellular In Vivo 1H MR Spectroscopy of Xenopus laevis Oocytes

    PubMed Central

    Lee, Seung-Cheol; Cho, Jee-Hyun; Mietchen, Daniel; Kim, Young-Sook; Hong, Kwan Soo; Lee, Chulhyun; Kang, Dongmin; Park, Ki Deok; Choi, Byong-Seok; Cheong, Chaejoon

    2006-01-01

    In vivo magnetic resonance (MR) spectra are typically obtained from voxels whose spatial dimensions far exceed those of the cells they contain. This study was designed to evaluate the potential of localized MR spectroscopy to investigate subcellular phenomena. Using a high magnetic field and a home-built microscopy probe with large gradient field strengths, we achieved voxel sizes of (180 μm)3. In the large oocytes of the frog Xenopus laevis, this was small enough to allow the recording of the first compartment-selective in vivo MR spectra from the animal and vegetal cytoplasm as well as the nucleus. The two cytoplasmic regions differed in their lipid contents and NMR lineshape characteristics—differences that are not detectable with whole-cell NMR techniques. In the nucleus, the signal appeared to be dominated by water, whereas other contributions were negligible. We also used localized spectroscopy to monitor the uptake of diminazene acturate, an antitrypanosomal agent, into compartments of a single living oocyte. The resulting spectra from the nucleus and cytoplasm revealed different uptake kinetics for the two components of the drug and demonstrate that MR technology is on the verge of becoming a tool for cell biology. PMID:16361348

  11. Triclosan and anuran metamorphosis: no effect on thyroid-mediated metamorphosis in Xenopus laevis.

    PubMed

    Fort, Douglas J; Rogers, Robert L; Gorsuch, Joseph W; Navarro, Lisa T; Peter, Robert; Plautz, James R

    2010-02-01

    Nieuwkoop and Faber stage 51 Xenopus laevis larvae were exposed for 21 days to four different concentrations of triclosan (TCS): <0.2 (control), 0.6, 1.5, 7.2, or 32.3 microg TCS/l. Primary endpoints were survival, hind limb length, body length (whole; snout to vent), developmental stage, wet whole body weight, and thyroid histology. Thyroid hormone (TH) concentrations were determined in whole thyroid and plasma samples from stage-matched exposure day 21 specimens. TH receptor-beta (TRbeta) expression was measured in stage-matched tail fin tissue samples collected at exposure days 0 and 21. Reduced larval growth occurred at exposure day 21 with 1.5 microg/l treatment. Larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters. Thyroid histology was not affected by TCS, and thyroxine (T4) levels in thyroid glands or plasma were not different from controls. A concentration-dependent increase in TRbeta expression in exposure day 21 larvae was not detected. However, increased expression was found in stage-matched larvae exposed to 1.5 or 7.2 microg TCS/l. Our study indicates that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model.

  12. The mucosubstance coating the pneumonocytes in the lungs of Xenopus laevis and Lacerta viridis.

    PubMed

    Meban, C

    1975-01-01

    The layer of mucosubstance that is associated with the free surface membranes of the pneumonocytes in the lungs of the toad Xenopus laevis and the lizard Lacerta viridis was demonstrated by electron microscopy using iron oxide stain. The form and staining reactions of the mucosubstance layer were similar in both animals. In electron micrographs the mucosubstance was represented by a band of densely stained material (25-50 nm thick) which coated the entire free surface of the pneumonocytes. It appeared to be firmly attached to the outer leaflet of the superficial plasma membrane. Short lengths of osmiophilic membranes, presumed to be fragments of pulmonary surfactant, were often observed lying free in the air spaces but they did not show any affinity for iron stain. Incubation of lung sections in a solution of neuraminidase produced a marked decrease in the intensity of the surface staining; no change was detected after incubation in trypsin, papain, hyaluronidase, N-acetyl cysteine, or phosphate buffer. It is, therefore, concluded that the pneumonocyte surface coat consists mainly of a sialomucin.

  13. Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

    PubMed Central

    Beyert, N; Reichenberger, S; Peters, M; Hartung, M; Göttlich, B; Goedecke, W; Vielmetter, W; Pfeiffer, P

    1994-01-01

    Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt. Images PMID:8202366

  14. Long-term starvation in Xenopus laevis Daudin--II. Effects on several organs.

    PubMed

    Merkle, S; Hanke, W

    1988-01-01

    1. The effect of starvation for 12 months on organo-somatic indices, glycogen, protein and water contents of several organs and the Na+/K+ ratio in muscle was studied in the South African clawed toad Xenopus laevis Daudin. 2. The liver- and ovary-somatic index were reduced by 30 and 70% of the initial value after 12 months. Fat bodies had disappeared after approximately 6 months of starvation. The indices of heart and kidney were not changed. 3. Glycogen concentration of the liver, ovaries and muscle were depleted nearly totally during the first half of the experimental time, whereas glycogen in the kidney seemed to be unaffected. 4. Protein concentration increased in the liver, decreased in the muscle and remained constant in the kidney. 5. Starvation caused an increase of the water concentration of the whole animal and different organs, especially at the end of the experiment. 6. The Na+/K+ ratio of the muscle increased significantly after 6 months of starvation and reached a maximum after 10 months.

  15. Platelet derived growth factor B gene expression in the Xenopus laevis developing central nervous system.

    PubMed

    Giannetti, Kety; Corsinovi, Debora; Rossino, Cristina; Appolloni, Irene; Malatesta, Paolo; Ori, Michela

    2016-01-01

    Platelet-derived growth factor B (PDGF-B) belongs to the mitogen and growth factor family and like the other members it has many roles in cell differentiation, proliferation and migration during development, adult life and in pathological conditions. Among them it has been observed that aberrant PDGF signalling is frequently linked to glioma development and progression, and Pdgf-b over-expression in mouse neural progenitors leads to the formation of gliomas. Despite this evidence, the mechanisms underlying PDGF-B driven tumorigenesis and its role during brain development are not fully understood. In order to contribute to clarifying possible new roles of pdgf-b signalling, we present here the embryonic gene expression pattern of pdgf-b, so far unknown in early vertebrate development. By using Xenopus laevis as a model system we performed qRT-PCR and whole mount in situ hybridization. Pdgf-b mRNA is expressed in discrete regions of the developing central nervous system, in the cranial nerve placodes and in the notochord. We also compared the gene expression of pdgf-b with that of its receptor pdgfr-α suggesting so far unsuspected roles for this signalling pathway during the development of specific embryonic structures.

  16. Microfluidic platform for electrophysiological studies on Xenopus laevis oocytes under varying gravity levels.

    PubMed

    Schaffhauser, Daniel F; Andrini, Olga; Ghezzi, Chiara; Forster, Ian C; Franco-Obregón, Alfredo; Egli, Marcel; Dittrich, Petra S

    2011-10-21

    Voltage clamp measurements reveal important insights into the activity of membrane ion channels. While conventional voltage clamp systems are available for laboratory studies, these instruments are generally unsuitable for more rugged operating environments. In this study, we present a non-invasive microfluidic voltage clamp system developed for the use under varying gravity levels. The core component is a multilayer microfluidic device that provides an immobilisation site for Xenopus laevis oocytes on an intermediate layer, and fluid and electrical connections from either side of the cell. The configuration that we term the asymmetrical transoocyte voltage clamp (ATOVC) also permits electrical access to the cytosol of the oocyte without physical introduction of electrodes by permeabilisation of a large region of the oocyte membrane so that a defined membrane patch can be voltage clamped. The constant low level air pressure applied to the oocyte ensures stable immobilisation, which is essential for keeping the leak resistance constant even under varying gravitational forces. The ease of oocyte mounting and immobilisation combined with the robustness and complete enclosure of the fluidics system allow the use of the ATOVC under extreme environmental conditions, without the need for intervention by a human operator. Results for oocytes over-expressing the epithelial sodium channel (ENaC) obtained under laboratory conditions as well as under conditions of micro- and hypergravity demonstrate the high reproducibility and stability of the ATOVC system under distinct mechanical scenarios.

  17. Development of Functional Sex Differences in the Larynx of Xenopus laevis

    PubMed Central

    Tobias, Martha L.; Marin, Melanie L.; Kelley, Darcy B.

    2012-01-01

    Three laryngeal properties associated with the production of masculine song—laryngeal muscle tension, fiber twitch type, and fiber recruitment—are markedly sexually dimorphic in adult Xenopus laevis frogs. To elucidate the pattern of sexual differentiation, tension and fiber recruitment in male and female larynges and fiber twitch type in male larynges were examined throughout postmetamorphic development. Masculinization of male laryngeal properties begins early in postmetamorphic development and continues until adulthood. In contrast, tension and fiber recruitment in females do not change after the end of metamorphosis. Laryngeal muscle tension and fiber type are gradually and progressively masculinized; the temporal pattern of masculinization is very similar for these properties. Fiber recruitment, on the other hand, appears to masculinize in a stepwise manner. Masculinization of all three properties is highly correlated with larynx weight in males. We have used this relation to divide postmetamorphic development into seven stages associated with key events in sexual differentiation. This staging scheme provides an important experimental tool for studying the hormonal regulation of sexual differentiation, the subject of the accompanying paper. PMID:1879611

  18. Platanna (Xenopus laevis) as a test organism for determining the embryotoxic effects of environmental chemicals

    SciTech Connect

    Dumpert, K.; Zietz, E.

    1984-02-01

    It has been successfully demonstrated that platanna (Xenopus laevis) allows the artificial induction of spawning at any time during the year. The number of eggs collected from a female ranged between 500 and 2400, the fertilization rate varying between 10 and 85%. When unaffected by chemicals, the embryonic development of the larvae took between 8 and 30 weeks. Di(2-ethylhexyl) phthalate (DEHP), methylmercury chloride, and the thalidomide analog EM 12 were used for the experiments described. DEHP at a concentration of 2 ppm retarded the development of the larvae and caused reduced pigmentation of the tadpoles. Methylmercury chloride has been found to have teratogenic and embryolethal effects at a concentration as low as 0.01 ppm. The following teratogenic effects have been determined: bent tails of the larvae, retarded development of the filter system, disturbed osmotic regulation, deranged positional and spatial orientation. EM 12 has been proven to have embryolethal effects at concentrations around 100 ppm. At lower concentrations this substance has teratogenic effects, i.e., it interferes in various ways with the development of the limbs.

  19. Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis.

    PubMed

    Tanida, Hiroaki; Tahara, Eiji; Mochizuki, Miwa; Yamane, Yukiko; Ryoji, Masaru

    2005-12-01

    Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function.

  20. Analgesic effects of meloxicam, morphine sulfate, flunixin meglumine, and xylazine hydrochloride in African-clawed frogs (Xenopus laevis).

    PubMed

    Coble, Dondrae J; Taylor, Douglas K; Mook, Deborah M

    2011-05-01

    We evaluated analgesic use and analgesiometry in aquatic African-clawed frogs (Xenopus laevis). We used the acetic acid test (AAT) to assess the analgesic potential of systemic xylazine hydrochloride, meloxicam, flunixin meglumine, and morphine sulfate after injection into the dorsal lymph sac. Flunixin meglumine provided better analgesia than did the other drugs, most evident at 5 and 9 h after administration. Because the AAT was associated with the development of dermal lesions, we discontinued use of this assay and chose the Hargreaves test as an alternative method of measuring nociception in Xenopus. This assay is commonly performed in rodents, but its efficacy in an aquatic species such as Xenopus was unknown prior to this study. We found that the Hargreaves test was an effective measure of nociception in Xenopus, and we used it to evaluate the effectiveness of the nonopiod agents xylazine hydrochloride, meloxicam, and flunixin meglumine both in the absence of surgery and after surgical oocyte harvest. Similar to findings from the AAT, flunixin meglumine provided better analgesia in the Hargreaves test than did the other agents when analyzed in the absence of surgical intervention. Results were equivocal after oocyte harvest. Although surgical oocyte harvest is a common procedure in Xenopus, and currently there are no published recommendations for analgesia after this invasive surgery. Future studies are needed to clarify the efficacy of nonsteroidal antiinflammatory drugs for that purpose.

  1. Analgesic Effects of Meloxicam, Morphine Sulfate, Flunixin Meglumine, and Xylazine Hydrochloride in African-Clawed Frogs (Xenopus laevis)

    PubMed Central

    Coble, Dondrae J; Taylor, Douglas K; Mook, Deborah M

    2011-01-01

    We evaluated analgesic use and analgesiometry in aquatic African-clawed frogs (Xenopus laevis). We used the acetic acid test (AAT) to assess the analgesic potential of systemic xylazine hydrochloride, meloxicam, flunixin meglumine, and morphine sulfate after injection into the dorsal lymph sac. Flunixin meglumine provided better analgesia than did the other drugs, most evident at 5 and 9 h after administration. Because the AAT was associated with the development of dermal lesions, we discontinued use of this assay and chose the Hargreaves test as an alternative method of measuring nociception in Xenopus. This assay is commonly performed in rodents, but its efficacy in an aquatic species such as Xenopus was unknown prior to this study. We found that the Hargreaves test was an effective measure of nociception in Xenopus, and we used it to evaluate the effectiveness of the nonopiod agents xylazine hydrochloride, meloxicam, and flunixin meglumine both in the absence of surgery and after surgical oocyte harvest. Similar to findings from the AAT, flunixin meglumine provided better analgesia in the Hargreaves test than did the other agents when analyzed in the absence of surgical intervention. Results were equivocal after oocyte harvest. Although surgical oocyte harvest is a common procedure in Xenopus, and currently there are no published recommendations for analgesia after this invasive surgery. Future studies are needed to clarify the efficacy of nonsteroidal antiinflammatory drugs for that purpose. PMID:21640031

  2. Translational control of the Xenopus laevis connexin-41 5'-untranslated region by three upstream open reading frames.

    PubMed

    Meijer, H A; Dictus, W J; Keuning, E D; Thomas, A A

    2000-10-06

    The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5'-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5'-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stage Xenopus embryos. The translational efficiency of the wild-type Cx41 5'-UTR was only 2% compared with that of the beta-globin 5'-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5'-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5'-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency in Xenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

  3. Sexually Dimorphic Expression of a Laryngeal-Specific, Androgen-Regulated Myosin Heavy Chain Gene during Xenopus laevis Development

    PubMed Central

    Catz, Diana S.; Fischer, Leslie M.; Moschella, Maria C.; Tobias, Martha L.

    2012-01-01

    Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue. PMID:1426643

  4. Molecular cloning and expression analysis of PDK family genes in Xenopus laevis reveal oocyte-specific PDK isoform.

    PubMed

    Terazawa, Yumiko; Tokmakov, Alexander A; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2005-12-30

    Pyruvate dehydrogenase kinase (PDK) inactivates the multienzyme mitochondrial pyruvate dehydrogenase complex by the phosphorylation of three seryl residues in the pyruvate dehydrogenase moiety, and thus plays an important role in the control of glucose homeostasis. Genetically and biochemically distinct PDK family isozymes have been identified in mammalian species. In the present study, we demonstrate that the complete family of expressed PDK family genes in the tissues of the African clawed frog, Xenopus laevis, consists of four members, which are divided into two evolutionary groups. Xenopus PDKs (xPDKs) share an overall homology of about 70% to the human isoforms of PDK. The abundance of mRNAs for the four xPDK isoforms was analyzed by the real-time reverse transcriptase PCR technique in the various tissues of Xenopus laevis, including heart, lung, spleen, liver, kidney, skin, testis, oocytes, and eggs. Our data suggest that one of the xPDK isozymes can be referred to as an oocyte-specific xPDK. Functional differences between the xPDK isoforms are discussed, based on their different tissue-specific distributions and phylogenetic similarities to human PDKs.

  5. Xenopus laevis tadpoles can regenerate neural retina lost after physical excision but cannot regenerate photoreceptors lost through targeted ablation.

    PubMed

    Lee, Damian C; Hamm, Lisa M; Moritz, Orson L

    2013-03-13

    To determine whether the Xenopus laevis retina is capable of regenerating photoreceptor cells lost through apoptotic cell death in an inducible transgenic X. laevis model of retinitis pigmentosa (RP). Acute rod photoreceptor apoptosis was induced in transgenic X. laevis expressing drug-inducible caspase 9. We subsequently monitored the ability of the retina to regenerate lost photoreceptors in the absence of drug, and in combination with physical injury or ectopic supplementation of basic fibroblast growth factor (FGF2). Direct activation of caspase 9 in rod photoreceptors resulted in the initiation of apoptosis and complete removal of rod photoreceptors within 4 days. Photoreceptors lost by apoptosis were not replaced over a 4-week recovery time frame. In contrast, physical disruption of rod-ablated retina was repaired by the end of a 3-week time frame, but did not result in rod photoreceptor regeneration other than at the site of injury. Furthermore, ectopic supplementation of FGF2 did not stimulate regeneration of photoreceptors lost by apoptosis. However, FGF2 supplementation increased the rate of regeneration of retina (including rod photoreceptors) in eyes from which retinal tissue was surgically removed. In the X. laevis retina, rod photoreceptors that undergo drug-induced caspase-9-mediated apoptosis are permanently lost and do not regenerate. In contrast, the neural retina (including rod photoreceptors) can regenerate in injured or retinectomized eyes, and this regeneration is promoted by supplementation with FGF2. However, FGF2 does not promote regeneration of rod photoreceptors that are selectively lost by apoptosis.

  6. Thyroid hormones in the skeletogenesis and accessory sources of endogenous hormones in Xenopus laevis (Amphibia; Anura) ontogeny: Experimental evidence.

    PubMed

    Smirnov, S V; Vassilieva, A B

    2014-03-01

    Skeletal development was studied in normal and goitrogen-treated Xenopus laevis tadpoles reared under thyroid hormone (TH) deficiency. Early stages of skeletal development proceed similarly in both groups. Later stages are retarded or completely arrested in goitrogen-treated tadpoles. After goitrogen-treated tadpoles were transferred into pure water or into a medium containing both goitrogen and exogenous TH, tadpoles resumed development. Consequently, late stages of skeletogenesis are TH-dependent and TH-induced. Athyroid X. laevis "giant tadpoles" described in literature differ from goitrogen-arrested tadpoles in that they have features which require TH to appear. The appearance of TH-depended features in giant tadpoles indicates the occurrence of the additional sources of TH other than thyroid gland.

  7. Comparison of diazinon toxicity to embryos of Xenopus laevis and Danio rerio; degradation of diazinon in water.

    PubMed

    Modra, Helena; Vrskova, Dagmar; Macova, Stanislava; Kohoutkova, Jana; Hajslova, Jana; Haluzova, Ivana; Svobodova, Zdenka

    2011-06-01

    The aim of this study was to determine the toxic effect of diazinon (organophosphate insecticide) to embryos of Xenopus laevis and Danio rerio. The 96-h LC₅₀ values showed higher toxicity of diazinon for X. leavis in standard solution (9.84 mg/L) compared to the pond water (12.64 mg/L). Teratogenic index for diazinon was 1.3 and 1.6, respectively. The 96-h LC₅₀ diazinon values demonstrated similar sensitivity of embryos D. rerio (8.21-9.34 mg/L) and X. laevis in standard test solutions. Our results reflect that direct application of diazinon into the water can be associated with significant risks to aquatic organisms.

  8. The non-methylated DNA-binding function of Kaiso is not required in early Xenopus laevis development

    PubMed Central

    Ruzov, Alexey; Savitskaya, Ekaterina; Hackett, Jamie A.; Reddington, James P.; Prokhortchouk, Anna; Madej, Monika J.; Chekanov, Nikolai; Li, Minghui; Dunican, Donncha S.; Prokhortchouk, Egor; Pennings, Sari; Meehan, Richard R.

    2009-01-01

    Summary Mammalian forms of the transcription repressor, Kaiso, can reportedly bind methylated DNA and non-methylated CTGCNA motifs. Here we compare the DNA-binding properties of Kaiso from frog, fish and chicken and demonstrate that only the methyl-CpG-binding function of Kaiso is evolutionarily conserved. We present several independent experimental lines of evidence that the phenotypic abnormalities associated with xKaiso-depleted Xenopus laevis embryos are independent of the putative CTGCNA-dependent DNA-binding function of xKaiso. Our analysis suggests that xKaiso does not play a role in the regulation of either xWnt11 or Siamois, key signalling molecules in the Wnt pathway during X. laevis gastrulation. The major phenotypic defects associated with xKaiso depletion are premature transcription activation before the mid-blastula transition and concomitant activation of a p53-dependent cell-death pathway. PMID:19158185

  9. Effects of GSM-like radiofrequency irradiation during the oogenesis and spermiogenesis of Xenopus laevis.

    PubMed

    Boga, Ayper; Emre, Mustafa; Sertdemir, Yasar; Uncu, İbrahim; Binokay, Secil; Demirhan, Osman

    2016-07-01

    We aimed to evaluate the effect of GSM-like radiofrequency electromagnetic radiation (RF-EMR) on the oogenesis, and spermiogenesis of Xenopus laevis, and so the development of the embryos obtained from Normal Females+Normal Males (i.e. "N(F)+N(M)"); Normal Females+RF-exposed Males (i.e. "N(F)+RF(M)"); RF-exposed Female+Normal Male (i.e. "RF(F)+N(M)"); and RF-exposed Female+RF-exposed Male (i.e. "RF(F)+RF(M)". Various, assessments were performed to determine potential teratogenic effects and mortality, body growth and behavior on first generation embryos. After exposing adults frogs of both sexes to 900MHz RF-EMR (at 1.0W/kg) for 8h a day over a 5-week period, the embryos' specific energy absorption rate (SAR) was calculated. In our present study (control group; 2.2% abnormal, 0.0% dead); with the N(F)+RF(M) combination, the long-term exposure of adult males to GSM-like radiation at 900MHz (RF: 2W) for 5 week/8h/day resulted in normal, abnormal and dead embryo ratios of 88.3%, 3.3% and 8.3%, respectively (p<0.001). In the RF(F)+N(M) combination, long-term exposure (5 week/8h/day) of adult females led to normal, abnormal and dead embryo ratios of 76.7%, 11.7%, and 11.7%, respectively (p<0.001). And in the RF(F)+RF(M) combination, long-term exposure (5 week/8h/day) of both adult males and females led to normal, abnormal and dead embryo ratios of 73.3%, 11.7%, and 15%, respectively (p<0.001). With the exception RF(F)+RF(M) group (p<0.001), no significant changes were observed on body growth (lengths) in comparison to the control group. It was also observed that the offspring of female adult Xenopus exposed to RF-EMR during oogenesis exhibited a more aggressive behavior compared to the control group. Cell phones radiation can thus lead to detrimental effects in humans' male and female reproductive cells.

  10. Thyroid Hormone-Regulated Wnt5a/Ror2 Signaling Is Essential for Dedifferentiation of Larval Epithelial Cells into Adult Stem Cells in the Xenopus laevis Intestine

    PubMed Central

    Ishizuya-Oka, Atsuko; Kajita, Mitsuko; Hasebe, Takashi

    2014-01-01

    Background and Aims Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis. Methods/Findings Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation. Significance Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology. PMID:25211363

  11. Alterations in gene expression levels provide early indicators of chemical stress during Xenopus laevis embryo development: A case study with perfluorooctane sulfonate (PFOS).

    PubMed

    San-Segundo, Laura; Guimarães, Laura; Fernández Torija, Carlos; Beltrán, Eulalia M; Guilhermino, Lúcia; Pablos, María Victoria

    2016-05-01

    In the present study, Xenopus laevis embryos were exposed to a range of perfluorooctane sulfonate (PFOS) concentrations (0, 0.5, 6, 12, 24, 48 and 96mg/L) for 96h in laboratorial conditions to establish toxicity along with possible gene expression changes. Mortality and deformities were monitored daily and head-tail length was measured at the end of the assay as an indicator of growth. At 24 and 96h post-exposure (hpe), the mRNA expression levels of the genetic markers involved in general stress responses (hsp70, hsp47, crh-a and ucn1), oxidative stress (cat.2 and sod), lipid metabolism (ppard) and apoptosis (tp53 and bax) were analyzed by RT-qPCR. Malformations were significantly higher in the embryos exposed to the highest PFOS concentration (41.8% to 56.4%) compared to controls (5.5%) at 48, 72 and 96hpe. Growth inhibition was observed in the embryos exposed to PFOS concentrations≥48mg/L. At 24 hpe, a statistically significant up-regulation of genes hsp70, hsp47, ppard, tp53 and bax in relation to controls was found. Similar responses were found for genes hsp70, hsp47, crh-a, ucn1, sod and ppard at 96 hpe. Alterations in the mRNA expression levels indicated both a stress response to PFOS exposure during X. laevis embryo development, and alterations in the regulation of oxidative stress, apoptosis, and differentiation. These molecular alterations were detected at an earlier exposure time or at lower concentrations than those producing developmental toxicity. Therefore, these sensitive warning signals could be used together with other biomarkers to supplement alternative methods (i.e. the frog embryo test) for developmental toxicity safety evaluations, and as tools in amphibian risk assessments for PFOS and its potential substitutes.

  12. Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity

    PubMed Central

    1992-01-01

    High resistance epithelia express on their apical side an amiloride- sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride- sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613- 10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function. PMID:1334959

  13. Embryonic Expression and Evolution of Duplicated E-Protein Genes in Xenopus Laevis: Parallels with Ancestral E-Protein Genes

    PubMed Central

    Shain, D. H.; Neuman, T.; Zuber, M. X.

    1997-01-01

    E-proteins comprise a subfamily of helix-loop-helix transcription factors that have been identified in arthropods and several chordate taxa. In mammals, there are three classes of E-protein genes (E2A, E2-2, and HEB) that encode related, and often interchangeable, gene products. We have determined that the clawed frog Xenopus laevis contains twice the number of transcriptionally active E-protein genes when compared with other vertebrate species. Based upon genomic Southern blots and nucleotide sequence comparisons, it is likely that the additional X. laevis genes arose from tetraploidization. During embryogenesis, XE2A (homologue of mammalian E2A) transcripts were broadly expressed in anterior and posterior regions of the embryo while homologues of E2-2 (XE2.2) and HEB (XE1.2) appeared in vertebrate-specific structures including the pineal gland, olfactory bulb, and brachial arches. A phylogenetic analysis of these genes and other known metazoan E-proteins suggests that there were two periods of marked E-protein gene expansion; one that predated the radiation of vertebrates, and the other that coincided with Xenopus tetraploidization. Both of these periods were characterized by the rapid evolution of E2-2 and HEB-class genes, but not of E2A. We propose that the former genes acquired new or specialized roles during early chordate evolution and also more recently in Xenopus, as reflected by the stereotypic expression patterns of these genes during X. laevis development. PMID:9136023

  14. Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system.

    PubMed

    Banach, Maureen; Edholm, Eva-Stina; Robert, Jacques

    2017-06-15

    A large family of highly related and clustered Xenopus nonclassical MHC class Ib (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X. laevis tadpoles, the absence of XNC10.1 and Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in F0 generation of X. laevis. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Characterization of the hypothalamus of Xenopus laevis during development. I. The alar regions.

    PubMed

    Domínguez, Laura; Morona, Ruth; González, Agustín; Moreno, Nerea

    2013-03-01

    The patterns of expression of a set of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the alar hypothalamus of Xenopus laevis throughout development. Combined immunohistochemical and in situ hybridization techniques were used for the identification of subdivisions and their boundaries. The alar hypothalamus was located rostral to the diencephalon in the secondary prosencephalon and represents the rostral continuation of the alar territories of the diencephalon and brainstem, according to the prosomeric model. It is composed of the supraoptoparaventricular (dorsal) and the suprachiasmatic (ventral) regions, and limits dorsally with the preoptic region, caudally with the prethalamic eminence and the prethalamus, and ventrally with the basal hypothalamus. The supraoptoparaventricular area is defined by the orthopedia (Otp) expression and is subdivided into rostral and caudal portions, on the basis of the Nkx2.2 expression only in the rostral portion. This region is the source of many neuroendocrine cells, primarily located in the rostral subdivision. The suprachiasmatic region is characterized by Dll4/Isl1 expression, and was also subdivided into rostral and caudal portions, based on the expression of Nkx2.1/Nkx2.2 and Lhx1/7 exclusively in the rostral portion. Both alar regions are mainly connected with subpallial areas strongly implicated in the limbic system and show robust intrahypothalamic connections. Caudally, both regions project to brainstem centers and spinal cord. All these data support that in terms of topology, molecular specification, and connectivity the subdivisions of the anuran alar hypothalamus possess many features shared with their counterparts in amniotes, likely controlling similar reflexes, responses, and behaviors. Copyright © 2012 Wiley Periodicals, Inc.

  16. Williams Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis

    PubMed Central

    Barnett, Chris; Yazgan, Oya; Kuo, Hui-Ching; Malakar, Sreepurna; Thomas, Trevor; Fitzgerald, Amanda; Harbour, Billy; Henry, Jonathan J.; Krebs, Jocelyn E.

    2012-01-01

    Williams Syndrome Transcription Factor (WSTF) is one of ~25 haplodeficient genes in patients with the complex developmental disorder Williams Syndrome (WS). WS results in visual/spatial processing defects, cognitive impairment, unique behavioral phenotypes, characteristic “elfin” facial features, low muscle tone and heart defects. WSTF exists in several chromatin remodeling complexes and has roles in transcription, replication, and repair. Chromatin remodeling is essential during embryogenesis, but WSTF’s role in vertebrate development is poorly characterized. To investigate the developmental role of WSTF, we knocked down WSTF in Xenopus laevis embryos using a morpholino that targets WSTF mRNA. BMP4 shows markedly increased and spatially aberrant expression in WSTF-deficient embryos, while SHH, MRF4, PAX2, EPHA4 and SOX2 expression are severely reduced, coupled with defects in a number of developing embryonic structures and organs. WSTF-deficient embryos display defects in anterior neural development. Induction of the neural crest, measured by expression of the neural crest-specific genes SNAIL and SLUG, is unaffected by WSTF depletion. However, at subsequent stages WSTF knockdown results in a severe defect in neural crest migration and/or maintenance. Consistent with a maintenance defect, WSTF knockdowns display a specific pattern of increased apoptosis at the tailbud stage in regions corresponding to the path of cranial neural crest migration. Our work is the first to describe a role for WSTF in proper neural crest function, and suggests that neural crest defects resulting from WSTF haploinsufficiency may be a major contributor to the pathoembryology of WS. PMID:22691402

  17. Low concentrations of metal mixture exposures have adverse effects on selected biomarkers of Xenopus laevis tadpoles.

    PubMed

    Yologlu, Ertan; Ozmen, Murat

    2015-11-01

    Polluted ecosystems may contain mixtures of metals, such that the combinations of metals, even in low concentrations, may cause adverse effects. In the present study, we focused on toxic effects of mixtures of selected metals, the LC50 values, and also their safety limit in aquatic systems imposed by the European legislation using a model organism. Xenopus laevis tadpoles were used as test organisms. They were exposed to metals or their combinations due to 96-h LC50 values. Glutathione S-transferase (GST), glutathione reductase (GR), acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione peroxidase (GPx), and catalase (CAT) levels were evaluated. Metallothionein concentrations were also determined. The LC50s for Cd, Pb, and Cu were calculated as 5.81mg AI/L, 123.05mg AI/L, and 0.85mg AI/L, respectively. Low lethality ratios were observed with unary exposure of each metal in lower concentrations. Double or triple combinations of LC50 and LC50/2 concentrations caused 100% lethality with Cd+Cu and Pb+Cd+Cu mixtures, while the Pb+Cu mixture also caused high lethal ratios. The selected enzyme activities were significantly affected by metals or mixtures, and dose-related effects were determined. The metallothionein levels generally increased as related to concentration in unary metals and mixtures. Acceptable limit values of unary metals and mixtures did not significantly change metallothionein levels. The results suggest that oxidative stress-related mechanisms are involved in the toxicity induced by selected metals with combinations of very low concentrations.

  18. Characterization of the hypoth