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Sample records for amphibian xenopus laevis

  1. The unique myelopoiesis strategy of the amphibian Xenopus laevis.

    PubMed

    Yaparla, Amulya; Wendel, Emily S; Grayfer, Leon

    2016-10-01

    Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs' liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species. PMID:27234705

  2. Plasticity of lung development in the amphibian, Xenopus laevis

    PubMed Central

    Rose, Christopher S.; James, Brandon

    2013-01-01

    Summary Contrary to previous studies, we found that Xenopus laevis tadpoles raised in normoxic water without access to air can routinely complete metamorphosis with lungs that are either severely stunted and uninflated or absent altogether. This is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions can be raised to forego this function without adverse effects. This study compared lung development in untreated, air-deprived (AD) and air-restored (AR) tadpoles and frogs using whole mounts, histology, BrdU labeling of cell division and antibody staining of smooth muscle actin. We also examined the relationship of swimming and breathing behaviors to lung recovery in AR animals. Inhibition and recovery of lung development occurred at the stage of lung inflation. Lung recovery in AR tadpoles occurred at a predictable and rapid rate and correlated with changes in swimming and breathing behavior. It thus presents a new experimental model for investigating the role of mechanical forces in lung development. Lung recovery in AR frogs was unpredictable and did not correlate with behavioral changes. Its low frequency of occurrence could be attributed to developmental, physical and behavioral changes, the effects of which increase with size and age. Plasticity of lung inflation at tadpole stages and loss of plasticity at postmetamorphic stages offer new insights into the role of developmental plasticity in amphibian lung loss and life history evolution. PMID:24337117

  3. 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

  4. 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

  5. Examination of an amphibian-based assay using the larvae of Xenopus laevis and Ambystoma mexicanum.

    PubMed

    Saka, Masahiro

    2003-05-01

    Semistatic acute toxicity tests of amphibian larvae (Xenopus laevis and Ambystoma mexicanum) were conducted at different developmental stages and by different methods to establish a simple amphibian-based assay. Test substance was pentachlorophenol sodium salt (PCP-Na). The endpoint was mortality and the 24-, 48-, 72-, and 96-h LC50 values were calculated by probit analysis. Interspecific differences in larval responses were not clear. Larval sensitivity tended to increase with larval age. Newly hatched larvae were most resistant to PCP-Na. During the tests of well-developed larvae, concentrations of dissolved oxygen and PCP-Na in the test solutions greatly dropped owing to uptake by the larvae. Therefore, middle-developed (2-week-old) larvae were most suitable for the test. Toxicity tests for volatile substances would be also possible using 2-week-old larvae in closed vessels. Test individuals should be kept individually to avoid the effects of poisonous skin secretions released from dead larvae. PMID:12706392

  6. 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. PMID:20869532

  7. Colony-Stimulating Factor-1-Responsive Macrophage Precursors Reside in the Amphibian (Xenopus laevis) Bone Marrow Rather than the Hematopoietic Sub-Capsular Liver

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2013-01-01

    Macrophage precursors originate from, and undergo lineage commitment within designated sites of hematopoiesis, such as the mammalian bone marrow. These cells subsequently differentiate in response to stimulation with macrophage colony-stimulating factor (CSF-1). The amphibian bone marrow, unlike that of mammals, has been overlooked as a source of leukocyte precursors in favor of the liver sub-capsular region, where hematopoiesis occurs in anurans. Here we report that the bone marrow rather than the liver periphery provides macrophage progenitors to the amphibian Xenopus laevis. We identified the amphibian CSF-1, examined its gene expression in developing and virally infected X. laevis and produce it in recombinant form (rXlCSF-1). This rXlCSF-1 did not bind or elicit proliferation/differentiation of sub-cortical liver cells. Surprisingly, a sub-population of bone marrow cells engaged this growth factor and formed rXlCSF-1-concentration-dependant colonies in semi-solid medium. Furthermore, rXlCSF-1-treated bone marrow (but not liver) cultures comprised of cells with characteristic macrophage morphology and high gene expression of the macrophage marker, colony stimulating factor-1 receptor (CSF-1R). Together, our findings indicate that in contrast to all other vertebrates studied to date, Xenopus committed macrophage precursors populations are not present in the central site of hematopoiesis, but reside in the bone marrow. PMID:23485675

  8. The Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on the Mortality and Growth of Two Amphibian Species (Xenopus laevis and Pseudacris triseriata)

    PubMed Central

    Collier, Alex; Orr, Lowell; Morris, Julie; Blank, James

    2008-01-01

    We observed a slight drop in the growth of Xenopus laevis and Pseudacris triseriata larvae following acute exposure (24–48 h) during egg development to three concentrations of TCDD (0.3, 3.0, 30.0 μg/l). Our exposure protocol was modeled on a previous investigation that was designed to mimic the effects of maternal deposition of TCDD. The doses selected were consistent with known rates of maternal transfer between mother and egg using actual adult body burdens from contaminated habitats. Egg and embryonic mortality immediately following exposure increased only among 48 h X. laevis treatments. Control P. triseriata and X. laevis completed metamorphosis more quickly than TCDD-treated animals. The snout-vent length of recently transformed P. triseriata did not differ between treatments although controls were heavier than high-dosed animals. Likewise, the snout-vent length and weight of transformed X. laevis did not differ between control and TCDD treatments. These findings provide additional evidence that amphibians, including P. triseriata and X. laevis are relatively insensitive to acute exposure to TCDD during egg and embryonic development. Although the concentrations selected for this study were relatively high, they were not inconsistent with our current understanding of bioaccumulation via maternal transfer. PMID:19151431

  9. Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay.

    PubMed

    Pollock, K; Gosálvez, J; Arroyo, F; López-Fernández, C; Guille, M; Noble, A; Johnston, S D

    2015-11-01

    The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n=3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1h (T1) and 24h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r=0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay. PMID:25482041

  10. 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

    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 (LC(50)) 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

  11. 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

  12. Occludin and hydromineral balance in Xenopus laevis.

    PubMed

    Chasiotis, Helen; Kelly, Scott P

    2009-01-01

    To investigate the response of the tight junction (TJ) protein occludin to environmental change in an anuran amphibian, we examined occludin tissue distribution, immunolocalization and alterations in mRNA expression in African clawed frogs (Xenopus laevis) acclimated to brackish water (BW) conditions (from freshwater to 2 per thousand, 5 per thousand or 10 per thousand salt water). Occludin mRNA is widely expressed in Xenopus and is abundant in tissues involved in regulating salt and water balance, such as the gastrointestinal (GI) tract, kidney and urinary bladder. Immunohistochemical analyses revealed strong occludin immunolabelling in the apicolateral region of epithelia lining the GI tract and mRNA expression increased along the longitudinal axis of the gut. In kidney tissue, occludin was differentially expressed on the luminal side of the nephron tubule, appearing in the distal tubules and collecting ducts only. In response to BW acclimation, Xenopus exhibited a significant loss of tissue water as well as salinity-dependent elevations in serum osmolality as a result of increased urea levels followed by elevated serum Na(+) and Cl(-) levels. Tissue-specific alterations in the ionomotive enzyme Na(+),K(+)-ATPase were also observed in Xenopus in response to BW acclimation. Most notably, Na(+),K(+)-ATPase activity in the rectum increased in response to elevated environmental salt concentrations while renal activity decreased. Furthermore, acclimation to BW caused tissue-specific and salinity-dependent alterations in occludin mRNA expression within select Xenopus osmoregulatory organs. Taken together, these studies suggest that alterations in occludin, in conjunction with active transport processes, may contribute to amphibian hydromineral homeostasis during environmental change. PMID:19112148

  13. 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

  14. Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests).

    PubMed

    Mouchet, F; Gauthier, L; Mailhes, C; Jourdain, M J; Ferrier, V; Triffault, G; Devaux, A

    2006-02-15

    The management of contaminated soils and wastes is a matter of considerable human concern. The present study evaluates the genotoxic potential of aqueous extracts of two soils (leachates) and of bottom ash resulting from municipal solid waste incineration (MSWIBA percolate), using amphibian larvae (Xenopus laevis). Soil A was contaminated by residues of solvents and metals and Soil B by polycyclic aromatic hydrocarbons and metals. MSWIBA was predominantly contaminated by metals. Two genotoxic endpoints were analysed in circulating erythrocytes taken from larvae: clastogenic and/or aneugenic effects (micronucleus induction) after 12 days of exposure and DNA-strand-breaking potency (comet assay) after 1 and 12 days of exposure. In addition, in vitro bacterial assays (Mutatox and Ames tests) were carried out and the results were compared with those of the amphibian test. Physicochemical analyses were also taken into account. Results obtained with the amphibians established the genotoxicity of the aqueous extracts and the comet assay revealed that they were genotoxic from the first day of exposure. The latter test could thus be considered as a genotoxicity-screening tool. Although genotoxicity persisted after 12 days' exposure, DNA damage decreased overall between days 1 and 12 in the MSWIBA percolate, in contrast to the soil leachates. Bacterial tests detected genotoxicity only for the leachate of soil A (Mutatox). The results confirm the ecotoxicological relevance of the amphibian model and underscore the importance of bioassays, as a complement to physico-chemical data, for risk evaluation. PMID:16442436

  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. Comparative toxicity of methidathion and glyphosate on early life stages of three amphibian species: Pelophylax ridibundus, Pseudepidalea viridis, and Xenopus laevis.

    PubMed

    Güngördü, Abbas

    2013-09-15

    The assessments of pesticide toxicity on nontarget organisms have largely been focused on the determination of median lethal concentration (LC50) values using single/laboratory species. Although useful, these studies cannot describe the biochemical mechanisms of toxicity and also cannot explain the effects of pesticides on natural species. In this study, the toxic effects of glyphosate and methidathion were evaluated comparatively on early developmental stages of 3 anurans-2 natural (Pelophylax ridibundus, Pseudepidalea viridis) and 1 laboratory species (Xenopus laevis). The 96-h LC50 values for methidathion and glyphosate were determined as 25.7-19.6 mg active ingredient (AI)/L for P. viridis, 27.4-22.7 mg AI/L for P. ridibundus, and 15.3-5.05 mg AI/L for X. laevis tadpoles. Furthermore, as early signs of intoxication, glutathione S-transferase (GST), acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione reductase, lactate dehydrogenase, and aspartate aminotrasferase were assayed in 4-day-old tadpoles after 96-h pesticide exposure. The GST induction after 3.2mg AI/L methidathion exposure was determined to be 173%, 83%, and 38% of control, and the AChE inhibition for the same dose was determined to be 86%, 96%, and 30% of control for P. ridibundus, P. viridis, and X. laevis, respectively. Unlike the application of methidathion, all enzyme activities showed statistically significant increases on glyphosate exposure compared to controls. However, these increases in enzyme activities were not shown to be parallel with the increase of concentration. The levels of increases of GST and AChE were determined to be 111% and 31% for P. ridibundus, 13% and 51% for P. viridis, and 15% and 36% for X. laevis after 3.2mg AI/L glyphosate exposure, respectively. The findings of the study suggest that the most sensitive species to pesticide exposure is X. laevis. The selected biomarker enzymes AChE, CaE, and GST are useful in understanding the toxic mechanisms of these

  17. Preliminary validation of a short-term morphological assay to evaluate adverse effects on amphibian metamorphosis and thyroid function using Xenopus laevis.

    PubMed

    Fort, D J; Rogers, R L; Morgan, L A; Miller, M F; Clark, P A; White, J A; Paul, R R; Stover, E L

    2000-01-01

    Short-term static-renewal studies were performed on Xenopus laevis embryos with 16 selected test materials from day 50 (stage 60) to day 64 (stage 66) (14-day test) to evaluate effects on tail resorption and thyroid function. Of the 16 test materials, nine were found to inhibit significantly the rate of tail resorption, four were found to stimulate metamorphosis and three had no appreciable effect on the rate of metamorphosis. In an effort to determine if the morphological effects observed were related to alteration in thyroid activity, measurement of triiodothyronine (T3) in the test organisms and coadministration studies using thyroxine (agonist) or propylthiouracil (antagonist) were performed based on the morphological response noted during tail resorption. Of the nine compounds found to inhibit the rate of tail resorption, six were found to reduce the levels of T3. In each case, the inhibitory response could be at least partially alleviated by the co-administration of thyroxine. Larvae exposed to the four stimulatory agents had somewhat elevated levels of T3 and were responsive to propylthiouracil antagonism. These results suggest that 12 of the 14 compounds tested in this study that altered the rate of tail resorption did so via the thyroid axis. Overall, the X. laevis model appeared to be a suitable system for evaluating the impact of environmental agents and chemical products on thyroid function. PMID:11139173

  18. 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.

  19. Replication of ribosomal DNA in Xenopus laevis.

    PubMed

    Bozzoni, I; Baldari, C T; Amaldi, F; Buongiorno-Nardelli, M

    1981-09-01

    The study of the localization of the replication origins of rDNA in Xenopus laevis has been approached by two different methods. 1. The DNA of X. laevis larvae was fractionated by CsCl gradient centrifugation in bulk and ribosomal DNA and examined in the electron microscope. In bulk DNA, clusters of microbubbles, which are related with the origins of replication, appear to be spaced along the DNA molecules at intervals comparable with the size of the 'average' replicon of X. laevis. In ribosomal DNA, the distance between adjacent clusters is much shorter and corresponds to the size of the rDNA repeating unit. When ribosomal DNA was submitted to digestion with restriction enzymes (Eco RI and HindIII) the microbubbles are observed in the non-transcribed spacer-containing fragment. 2. Cultured cells of X. laevis were synchronized by mitotic selection and incubated with 5-fluoro-2-deoxyuridine for a time longer than the G1 phase. This treatment synchronizes the replicons and allows them to start replicating very slowly. It was thus possible to obtain a preferential labelling of the regions containing the origins. The analysis by gel electrophoresis of the Eco Ri-digested rDNA showed that the radioactivity was preferentially incorporated in the fragments which contain the non-transcribed spacer. The results of these two approaches indicate that the rRNA gene cluster consists of multiple units of replication, possibly one per gene unit. Furthermore they show that the origins of replication are localized into the non-transcribed spacer. PMID:7297565

  20. Facial Transplants in Xenopus laevis Embryos

    PubMed Central

    Sive, Hazel

    2014-01-01

    Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals. PMID:24748020

  1. Effects of tributyltin on metamorphosis and gonadal differentiation of Xenopus laevis at environmentally relevant concentrations.

    PubMed

    Shi, Huahong; Zhu, Pan; Guo, Suzhen

    2014-05-01

    Tributyltin (TBT), a well known endocrine disruptor, has high teratogenicity to embryos of amphibian (Xenopus tropicalis). An amphibian metamorphosis assay (AMA) and a complete AMA (CAMA) were conducted for TBT. In AMA, the body weight, the snout-to-vent length and the hind limb length of X. laevis tadpoles were decreased in tributyltin chloride (TBTCl; 12.5-200 ng/L) treatment groups after 7 days exposure. TBT greatly retarded the development of tadpoles, decreased the number of follicle and induced thyroid follicle cell hyperplasia after 19 days exposure. In CAMA, 10 and 100 ng/L TBTCl led to various malformations of gonad, including intersex, segmental aplasia and multiple ovary cavities of X. laevis following exposure from stages 46 to stage 66. The sex ratio was male-biased in TBT treatment groups. These results suggest that TBT delayed the metamorphosis, inhibited the growth of tadpoles and disrupted the gonadal differentiation of X. laevis at environmentally relevant concentrations. PMID:22903176

  2. Metaphase protein phosphorylation in Xenopus laevis eggs.

    PubMed Central

    Lohka, M J; Kyes, J L; Maller, J L

    1987-01-01

    Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior. Images PMID:3821728

  3. 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

  4. Trichodina xenopodus, a ciliated protozoan, in a laboratory-maintained Xenopus laevis.

    PubMed

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

    2013-08-01

    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

  5. Thyroid disruption effects of environmental level perfluorooctane sulfonates (PFOS) in Xenopus laevis.

    PubMed

    Cheng, Yan; Cui, Yuan; Chen, Hui-ming; Xie, Wen-ping

    2011-11-01

    Perfluorooctane sulfonate (PFOS), one of the emerging persistent organic pollutants (POPs), has caused growing international concern especially related to the potential disruption in the development and function of thyroid system. Xenopus laevis is an amphibian species widely used as a suitable amphibian model for thyroid disruption research. To study the thyroid disruption effects related to PFOS exposure at environmental low levels, X. laevis tadpoles were exposed to 0.1, 1, 10 and 100 μg/l PFOS in water respectively from stage 46/47 to stage 62. The results showed that the time to metamorphosis (presented by forelimb emergence, FLE) did not significantly change with PFOS exposure, but exhibited an increasing trend (except for 10 μg/l exposure). Partial colloid depletion was observed for PFOS exposure, but no significant histological abnormality was observed in treatment groups. In addition, PFOS exposure resulted in up-regulation of thyroid hormone-regulated genes-thyroid receptor beta A (TRβA), basic transcription element-binding protein (BTEB) and type II deiodinase (DI2) mRNA expression, presented as an inverted U-shaped dose response pattern. However, the mRNA expression of type III deiodinase (DI3) remained unaffected compared with the control. These results demonstrated that PFOS might disrupt the thyroid system in X. laevis tadpoles regarding FLE changes and regulation alternation of thyroid hormone-regulated genes. Our study has raised new concerns for possible thyroid disruption of PFOS in amphibians at environmental relevant levels. PMID:21809121

  6. Toward defining the phosphoproteome of Xenopus laevis embryos

    PubMed Central

    McGivern, Jered V.; Swaney, Danielle L.; Coon, Joshua J.; Sheets, Michael D.

    2010-01-01

    Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. PMID:19384857

  7. 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.

  8. 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...

  9. Melatonin deacetylation: retinal vertebrate class distribution and Xenopus laevis tissue distribution.

    PubMed

    Grace, M S; Cahill, G M; Besharse, J C

    1991-09-13

    Deacetylation is a rapid clearance mechanism for ocular melatonin. We have studied the distribution of retinal melatonin deacetylase activity among vertebrate classes. Exogenous radiolabeled melatonin is metabolized by ocular tissue prepared from the amphibian Xenopus laevis, the reptile Anolis carolinensis, the teleost fish Carassius auratus, and the bird Gallus domesticus. In contrast, we were unable to detect ocular melatonin breakdown in rat or pig. In each species exhibiting ocular melatonin breakdown, melatonin is first deacetylated to 5-methoxytryptamine, which is deaminated, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Deacetylation of melatonin is inhibited by eserine (physostigmine), causing a reduction in the levels of all 3 metabolites. Deamination of 5-methoxytryptamine is inhibited by the monoamine oxidase inhibitor pargyline, such that 5-methoxyindoleacetic acid and 5-methoxytryptophol levels are decreased while levels of 5-methoxytryptamine are increased. Incubation with the deacetylase inhibitor eserine increases endogenous melatonin levels in Xenopus and Carassius eyecups, indicating that endogenous melatonin is metabolized via the deacetylase. We also studied the tissue distribution of the deacetylase in Xenopus laevis. Melatonin deacetylation occurs in retina, retinal pigment epithelium, and skin, all of which are sites of melatonin action. These results indicate that among non-mammalian vertebrates, deacetylation is a common clearance mechanism for ocular melatonin, and may degrade melatonin at other sites of action as well. Melatonin deacetylation may help regulate local melatonin concentration, and generates other biologically active methoxyindoles. PMID:1782560

  10. Studying MAP Kinase pathways during early development of Xenopus laevis.

    PubMed

    Keren, Aviad; Bengal, Eyal

    2010-01-01

    The following chapter describes several methods involved in the detection of MAPK activities and phosphorylated proteins during early development of Xenopus laevis. The Xenopus embryo provides a powerful platform for biochemical studies. We describe here basic methods of embryo manipulations such as egg fertilization, embryo growth and maintenance, microinjection of capped RNA and antisense morpholino oligonucleotides (AMOs), and isolation of explants. In addition, we describe methods to detect phosphorylated proteins, to analyze kinase activity, and to interfere with signaling pathways. Immunohistochemical staining performed on whole embryos or on tissue sections is an additional method for the detection of phosphorylated proteins in the developing embryo. Approaches to activate or inhibit MAPK activities including the ectopic expression of mutated isoforms of MAPK kinase, or the incubation of embryo explants with pharmacological inhibitors are described. Finally, we describe an in vitro kinase assay specifically designed for the Xenopus embryo. PMID:20811998

  11. Serum Clinical Biochemical and Hematologic Reference Ranges of Laboratory-Reared and Wild-Caught Xenopus laevis

    PubMed Central

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

    2011-01-01

    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). PMID:22330708

  12. Color and intensity discrimination in Xenopus laevis tadpoles.

    PubMed

    Rothman, Gabriel R; Blackiston, Douglas J; Levin, Michael

    2016-09-01

    Investigations into the physiology of Xenopus laevis have the potential to greatly accelerate biomedical research, especially concerning neural plasticity and sensory systems, but are limited by the lack of available information on behavioral learning in this species. Here, we attempt to lay the foundations for a behavioral assay in Xenopus that can be used in conjunction with biological manipulations. We tested cohorts of Xenopus tadpoles across four light-mediated active-avoidance experiments, using either wavelength or intensity as the salient discriminative cue. In the wavelength task, we determine a baseline learning rate and characterize retention of learning, identifying active extinction effects as far more potent than the passage of time in the loss of behavior. In the intensity task, we examine the effects of varying differences between the discriminative stimuli on acquisition and extinction and identify a critical range of intensity differences where learning changes. The results of our experiments demonstrate that Xenopus is a tractable model organism for cognitive research and can learn a variety of associative tasks in the laboratory settings. These data reveal new aspects of the Xenopus larval visual processing system and facilitate future research between cognitive methods and biological/chemical manipulations to study mechanisms of brain structure and function. PMID:27146661

  13. B-lymphocyte populations in Xenopus laevis.

    PubMed

    Hadji-Azimi, I; Coosemans, V; Canicatti, C

    1990-01-01

    Two-color immunofluorescence technique was used to show the development and distribution of surface mu- cytoplasmic mu+ (s mu- c mu+) pre-B, s mu+ B- and s mu+ cIg+ plasma cells in metamorphic, postmetamorphic, and adult Xenopus. Generation of pre-B cells was evident in hematopoietic liver and spleen, but not in bone marrow, thymus, and duodenal mucosa. Surface immunoglobulin positive small lymphocytes were the most abundant in the spleen while plasma cells were detected in the thymus, duodenal mucosa, spleen, and liver. We had shown previously the appearance of s mu- c mu+ pre-B cells in the liver of Xenopus larvae at developmental stage 46 and later at stage 49 in the spleen. The frequency of pre-B cells dropped to zero at stage 58, the climax of metamorphosis. Pre-B cells start to reappear slowly as a second wave, at stage 60 through early postmetamorphic life in the liver and spleen. The percentage of surface Ig+ (sIg+) cells in the spleen of developing animals from stage 60 onward is comparable to that observed in adult life. In adult animals, the periphery of the liver continues to be active in hematopoiesis and contains some IgM producing plasma cells and rare sIg+ small lymphocytes while the pre-B cells are almost nonexistent in this region. The spleen, which is also active in some hematopoiesis, constitutes the main site of B-cell differentiation. Three ontogenic stages of pre-B, B-, and plasma cells are present in this organ. Pre-B and plasma cells are of low density and heterogeneous in size while small sIg+ B lymphocytes are of high density and much more homogeneous in size. The bone marrow in these lower anuran amphibia is rudimentary and is not a lymphopoietic tissue; in adult animals it is active only in differentiation of neutrophilic granulocytes. PMID:2338158

  14. Endocannabinoid system in Xenopus laevis development: CB1 receptor dynamics.

    PubMed

    Migliarini, Beatrice; Beatrice, Migliarini; Marucci, Gabriella; Gabriella, Marucci; Ghelfi, Francesca; Francesca, Ghelfi; Carnevali, Oliana; Oliana, Carnevali

    2006-04-01

    This study investigates for the first time the dynamics of endocannabinoid system appearance during low vertebrate Xenopus laevis development. We observed that the CB1 gene started to be expressed during the organogenesis period (+/- 1 dpf, st. 28) and expression persisted throughout the three further stages analyzed. Attention was focused on the localization of the CB1 messenger that was found both at the central level (in romboencephalon and in olfactory placods) and at the peripheral level (in the gastrointestinal tract) at +/- 3 dpf (st. 41), +/- 4 dpf (st. 46) and +/- 12 dpf (st. 49). We also considered the synthesis of CB1 protein that occurred from st. 41 onwards and, from this stage, we tested the receptor functionality in response to anandamide using cytosensor microphysiometry. CB1 functionality increased with development at both central and peripheral level. These data provide sufficient evidence to encourage further analysis on endocannabinoid physiological roles during embryonic and larval X. laevis growth. PMID:16519888

  15. Purification and characterization of Xenopus laevis topoisomerase II.

    PubMed Central

    Benedetti, P; Baldi, M I; Mattoccia, E; Tocchini-Valentini, G P

    1983-01-01

    We have purified to apparent homogeneity a type II DNA topoisomerase from Xenopus laevis oocyte nuclei (germinal vesicles, or GV). The most pure preparations contain a single polypeptide of 175,000 daltons as determined by SDS-gel electrophoresis. The enzyme changes the linking number of DNA circles in steps of two and reversibly knots or catenates DNA rings. No gyrase activity is detectable and ATP is required. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:10872324

  16. 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.

  17. 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.

  18. Distinct patterns of endosulfatase gene expression during Xenopus laevis limb development and regeneration

    PubMed Central

    Wang, Yi‐Hsuan

    2015-01-01

    Abstract The heparan sulfate 6‐O‐endosulfatases sulf1 and sulf2 regulate multiple cellular processes and organ development. Sulfs modulate a range of heparan‐sulfate‐dependent extracellular pathways, including the fibroblast growth factor, bone morphogenetic protein, and wingless/wnt signaling pathways. Known patterns of sulf transcript expression together with functional experiments have implicated the sulfs in chondrogenesis and muscle regeneration in mammals. Here, we describe the expression patterns of Xenopus laevis sulf1 and sulf2 in developing forelimbs and hindlimbs and demonstrate novel expression of the sulf transcripts in the regenerating hindlimbs, with prominent sulf2 expression in the proliferating blastema and transient expression of sulf1 in the redeveloping apical epidermal ridge. These findings further suggest involvement of the sulfs in successful limb regeneration in amphibians.

  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. PMID:26528898

  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. PMID:26492040

  1. 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. PMID:26294036

  2. Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis.

    PubMed

    Korte, Joseph J; Sternberg, Robin M; Serrano, Jose A; Thoemke, Kara R; Moen, Scott M; Lillegard, Kathryn E; Hornung, Michael W; Tietge, Joseph E; Degitz, Sigmund J

    2011-05-01

    Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-β antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy. PMID:21354158

  3. Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis.

    PubMed

    Cohen, Sarit; Méchali, Marcel

    2002-12-01

    Instability and plasticity of telomeric DNA, which includes extrachromosomal DNA, are usually correlated with the absence of telomerase and with abnormal growth of mammalian cells. Here, we show the formation of extrachromosomal circular DNA of telomeric repeats (tel-eccDNA) during the development of Xenopus laevis. Tel-eccDNA is double-stranded relaxed circles composed of the vertebrate consensus telomeric repeats [TTAGGG](n). Its size varies from <2 to >20 kb and it comprises up to 10% of the total cellular telomere content of the early embryo (pre-MBT stage). The amount of tel-eccDNA is reduced in later developmental stages and in adult tissues. Using a cell-free system derived from Xenopus egg extracts, we show that tel-eccDNA can be formed de novo from the telomere chromosomal tracts of sperm nuclei and naked DNA in a replication-independent manner. These results reveal an unusual plasticity of telomeric DNA during normal development of Xenopus. PMID:12446568

  4. 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.

  5. 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

  6. The structure and development of Xenopus laevis cornea.

    PubMed

    Hu, Wanzhou; Haamedi, Nasrin; Lee, Jaehoon; Kinoshita, Tsutomu; Ohnuma, Shin-ichi

    2013-11-01

    The African clawed frog, Xenopus laevis, is a widely used model organism for tissue development. We have followed the process of corneal development closely in Xenopus and examined the corneal ultrastructure at each stage during its formation. Xenopus cornea development starts at stage 25 from a simple embryonic epidermis overlying the developing optic vesicle. After detachment of the lens placode which takes place around stage 30, cranial neural crest cells start to invade the space between the lens and the embryonic epidermis to construct the corneal endothelium. At stage 41, a second wave of migratory cells containing presumptive keratocytes invades the matrix leading to the formation of inner cornea and outer cornea. Three-dimensional electron microscopic examination shows that a unique cell mass, the stroma attracting center, connects the two layers like the center pole of a tent. After stage 48, many secondary stromal keratocytes individually migrate to the center and form the stroma layer. At stage 60, the stroma space is largely filled by collagen lamellae and keratocytes, and the stroma attracting center disappears. At early metamorphosis, the embryonic epithelium gradually changes to the adult corneal epithelium, which is covered by microvilli. Around stage 62 the embryonic epithelium thickens and a massive cell death is observed in the epithelium, coinciding with eyelid opening. After metamorphosis, the frog cornea has attained the adult structure of three cellular layers, epithelium, stroma, and endothelium, and two acellular layers between the cellular layers, namely the Bowman's layer and Descemet's membrane. After initial completion, Xenopus cornea, in particular the stroma, continues to thicken and enlarge throughout the lifetime of the animal. In the adult, a p63 positive limbus-like wavy structure is observed at the peripheral edge of the cornea. Proliferation analysis shows that the basal corneal epithelial cells actively divide and there are a

  7. 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. PMID:18471902

  8. 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...

  9. 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. PMID:22802115

  10. 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...

  11. A transgenic Xenopus laevis reporter model to study lymphangiogenesis.

    PubMed

    Ny, Annelii; Vandevelde, Wouter; Hohensinner, Philipp; Beerens, Manu; Geudens, Ilse; Diez-Juan, Antonio; Brepoels, Katleen; Plaisance, Stéphane; Krieg, Paul A; Langenberg, Tobias; Vinckier, Stefan; Luttun, Aernout; Carmeliet, Peter; Dewerchin, Mieke

    2013-01-01

    The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening. PMID:24143274

  12. 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. PMID:26073702

  13. Effects of 17α-trenbolone and melengestrol acetate on Xenopus laevis growth, development, and survival.

    PubMed

    Finch, Bryson E; Blackwell, Brett R; Faust, Derek R; Wooten, Kimberly J; Maul, Jonathan D; Cox, Stephen B; Smith, Philip N

    2013-02-01

    The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB and 10 ng/L MGA + 100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout-vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA + 500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout-vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development. PMID:22890510

  14. NMDAR-Dependent Control of Call Duration in Xenopus laevis

    PubMed Central

    Katzen, Abraham W.; Rhodes, Heather J.; Yamaguchi, Ayako

    2010-01-01

    Many rhythmic behaviors, such as locomotion and vocalization, involve temporally dynamic patterns. How does the brain generate temporal complexity? Here, we use the vocal central pattern generator (CPG) of Xenopus laevis to address this question. Isolated brains can elicit fictive vocalizations, allowing us to study the CPG in vitro. The X. laevis advertisement call is temporally modulated; calls consist of rhythmic click trills that alternate between fast (∼60 Hz) and slow (∼30 Hz) rates. We investigated the role of two CPG nuclei—the laryngeal motor nucleus (n.IX–X) and the dorsal tegmental area of the medulla (DTAM)—in setting rhythm frequency and call durations. We discovered a local field potential wave in DTAM that coincides with fictive fast trills and phasic activity that coincides with fictive clicks. After disrupting n.IX–X connections, the wave persists, whereas phasic activity disappears. Wave duration was temperature dependent and correlated with fictive fast trills. This correlation persisted when wave duration was modified by temperature manipulations. Selectively cooling DTAM, but not n.IX–X, lengthened fictive call and fast trill durations, whereas cooling either nucleus decelerated the fictive click rate. The N-methyl-d-aspartate receptor (NMDAR) antagonist dAPV blocked waves and fictive fast trills, suggesting that the wave controls fast trill activation and, consequently, call duration. We conclude that two functionally distinct CPG circuits exist: 1) a pattern generator in DTAM that determines call duration and 2) a rhythm generator (spanning DTAM and n.IX–X) that determines click rates. The newly identified DTAM pattern generator provides an excellent model for understanding NDMAR-dependent rhythmic circuits. PMID:20393064

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

    PubMed Central

    Ng-Sui-Hing, Ng-Kwet-Leok A.; Rabe, Brian A.; Lewis, Brittany B.; Saha, Margaret S.

    2012-01-01

    The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18. While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45. Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of

  16. Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue.

    PubMed

    McDonough, Molly J; Allen, Chelsea E; Ng-Sui-Hing, Ng-Kwet-Leok A; Rabe, Brian A; Lewis, Brittany B; Saha, Margaret S

    2012-01-01

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

  17. 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.

  18. Comparative functional analysis of rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters suggest differential regulations.

    PubMed

    Goswami, Moloy T; Desai, Kartiki V; Kondaiah, Paturu

    2003-07-01

    We have carried out a comparative functional analysis of the rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-beta1 is low as compared to TGF-beta5 promoter in XTC cells but comparable in A6 cells, while TGF-beta5 promoter shows nearly negligible activity as compared to TGF-beta5 promoter in all the tested mammalian cell lines. Moreover, TGF-beta5 promoter is found to be repressed in XTC cells on treatment with TGF-beta5 protein. Thus, the regulation of TGF-beta1 and TGF-beta5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-beta1 and TGF-beta5 proteins, TGF-beta1 and TGF-beta5 genes have distinct functions in their respective species. PMID:12962305

  19. 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.

  20. 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. PMID:25698756

  1. Dehydration mediated microRNA response in the African clawed frog Xenopus laevis.

    PubMed

    Wu, Cheng-Wei; Biggar, Kyle K; Storey, Kenneth B

    2013-10-25

    Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure >30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37±6, 64±8, and 80±4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05±0.45, 2.11±0.08, and 1.36±0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40±0.09, 1.31±0.05, and 2.17±0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35±0.05 and 1.74±0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression. PMID:23958654

  2. 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.

  3. Characterization of nuclear protein kinases of Xenopus laevis oocytes

    SciTech Connect

    Leiva, L.; Gonzalez, C.; Allende, C.; Allende, J.

    1986-05-01

    Xenopus laevis oocytes contain large nuclei (germinal vesicles) that can be isolated in very pure form and which permit the study of enzymatic activities present in these organelles. Incubation of pure oocyte nuclear homogenates with /sup 32/P in a buffered solution containing 5 mM MgCl/sub 2/ results in the phosphorylation of a large number of proteins by endogenous protein kinases. This phosphorylation is not affected by the addition of cyclic nucleotides or calcium ion and calmodulin. On the other hand the nuclear kinases are considerably stimulated by spermine and spermidine and strongly inhibited by heparin (10 ..mu..g/ml). Addition of exogenous protein substrates shows that the major oocyte kinases are very active with casein and phosvitin as substrates but do not phosphorylate histones or protamines. DEAE-Sephadex chromatography of the nuclear extract fractionates the casein phosphorylating activity in two main peaks. The first peak is not retained on the column equilibrated with 0.1 M NH/sub 2/SO/sub 4/ and uses exclusively ATP as phosphate donor and is insensitive to polyamines or heparin. The second peak which corresponds to 70% of the casein phosphorylation elutes at 0.27 M NH/sub 2/SO/sub 4/ and uses both ATP and GTP as phosphate donors and is greatly stimulated by polyamines and completely inhibited by 10 ..mu..g/ml heparin. On this evidence the authors conclude that the major protein kinase peak corresponds to casein kinase type II which has been found in mammalian nuclei.

  4. Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

    PubMed Central

    Robert, Jacques; Grayfer, Leon; Edholm, Eva-Stina; Ward, Brian; De Jesús Andino, Francisco

    2014-01-01

    Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals. PMID:25390636

  5. 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

  6. 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

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

    PubMed

    Peña-Villalobos, Isaac; Narváez, Cristóbal; Sabat, Pablo

    2016-01-01

    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

  8. Regional expression of Pax7 in the brain of Xenopus laevis during embryonic and larval development

    PubMed Central

    Bandín, Sandra; Morona, Ruth; Moreno, Nerea; González, Agustín

    2013-01-01

    Pax7 is a member of the highly conserved Pax gene family that is expressed in restricted zones of the central nervous system (CNS) during development, being involved in early brain regionalization and the maintenance of the regional identity. Using sensitive immunohistochemical techniques we have analyzed the spatiotemporal pattern of Pax7 expression in the brain of the anuran amphibian Xenopus laevis, during development. Pax7 expression was first detected in early embryos in the basal plate of prosomere 3, roof and alar plates of prosomere 1 and mesencephalon, and the alar plate of rhombomere 1. As development proceeded, Pax7 cells were observed in the hypothalamus close to the catecholaminergic population of the mammillary region. In the diencephalon, Pax7 was intensely expressed in a portion of the basal plate of prosomere 3, in the roof plate and in scattered cells of the thalamus in prosomere 2, throughout the roof of prosomere 1, and in the commissural and juxtacommissural domains of the pretectum. In the mesencephalon, Pax7 cells were localized in the optic tectum and, to a lesser extent, in the torus semicircularis. The rostral portion of the alar part of rhombomere 1, including the ventricular layer of the cerebellum, expressed Pax7 and, gradually, some of these dorsal cells were observed to populate ventrally the interpeduncular nucleus and the isthmus (rhombomere 0). Additionally, Pax7 positive cells were found in the ventricular zone of the ventral part of the alar plate along the rhombencephalon and the spinal cord. The findings show that the strongly conserved features of Pax7 expression through development shared by amniote vertebrates are also present in the anamniote amphibians as a common characteristic of the brain organization of tetrapods. PMID:24399938

  9. 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.

  10. 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. PMID:25192837

  11. 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.

  12. 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

  13. 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...

  14. 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 ...

  15. 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. PMID:24737764

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

    PubMed Central

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

    2014-01-01

    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. PMID:24737764

  17. Adaptive plasticity of spino-extraocular motor coupling during locomotion in metamorphosing Xenopus laevis.

    PubMed

    von Uckermann, Géraldine; Lambert, François M; Combes, Denis; Straka, Hans; Simmers, John

    2016-04-15

    During swimming in the amphibian ITALIC! Xenopus laevis, efference copies of rhythmic locomotor commands produced by the spinal central pattern generator (CPG) can drive extraocular motor output appropriate for producing image-stabilizing eye movements to offset the disruptive effects of self-motion. During metamorphosis, ITALIC! X. laevisremodels its locomotor strategy from larval tail-based undulatory movements to bilaterally synchronous hindlimb kicking in the adult. This change in propulsive mode results in head/body motion with entirely different dynamics, necessitating a concomitant switch in compensatory ocular movements from conjugate left-right rotations to non-conjugate convergence during the linear forward acceleration produced during each kick cycle. Here, using semi-intact or isolated brainstem/spinal cord preparations at intermediate metamorphic stages, we monitored bilateral eye motion along with extraocular, spinal axial and limb motor nerve activity during episodes of spontaneous fictive swimming. Our results show a progressive transition in spinal efference copy control of extraocular motor output that remains adapted to offsetting visual disturbances during the combinatorial expression of bimodal propulsion when functional larval and adult locomotor systems co-exist within the same animal. In stages at metamorphic climax, spino-extraocular motor coupling, which previously derived from axial locomotor circuitry alone, can originate from both axial and ITALIC! de novohindlimb CPGs, although the latter's influence becomes progressively more dominant and eventually exclusive as metamorphosis terminates with tail resorption. Thus, adaptive interactions between locomotor and extraocular motor circuitry allows CPG-driven efference copy signaling to continuously match the changing spatio-temporal requirements for visual image stabilization throughout the transitional period when one propulsive mechanism emerges and replaces another. PMID:27103674

  18. Ultrastructural and neurochemical architecture of the pituitary neural lobe of Xenopus laevis.

    PubMed

    van Wijk, Diane C W A; Meijer, Kari H; Roubos, Eric W

    2010-09-01

    The melanotrope cell in the amphibian pituitary pars intermedia is a model to study fundamental aspects of neuroendocrine integration. They release alpha-melanophore-stimulating hormone (alphaMSH), under the control of a large number of neurochemical signals derived from various brain centers. In Xenopus laevis, most of these signals are produced in the hypothalamic magnocellular nucleus (Mg) and are probably released from neurohemal axon terminals in the pituitary neural lobe, to stimulate alphaMSH-release, causing skin darkening. The presence in the neural lobe of at least eight stimulatory factors implicated in melanotrope cell control has led us to investigate the ultrastructural architecture of this neurohemal organ, with particular attention to the diversity of neurohemal axon terminals and their neurochemical contents. Using regular electron microscopy, we here distinguish six types of neurohemal axon terminal, on the basis of the size, shape and electron-density of their secretory granule contents. Subsequently, we have identified the neurochemical contents of these terminal types by immuno-electron microscopy and antisera raised against not only the 'classical' neurohormones vasotocin and mesotocin but also brain-derived neurotrophic factor, cocaine- and amphetamine-regulated transcript peptide, corticotropin-releasing factor, metenkephalin, pituitary adenylyl cyclase-activating polypeptide, thyrotropin-releasing hormone and urocortin-1. This has revealed that each terminal type possesses a unique set of neurochemical messengers, containing at least four, but in some cases up to eight messengers. These results reveal the potential of the Mg/neural lobe system to release a wide variety of neurochemical messengers in a partly co-ordinated and partly differential way to control melanotrope cell activity as well as ion and water balance regulatory organs, in response to various, continuously changing, environmental stimuli. PMID:20067800

  19. 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....

  20. Xenopus laevis: an ideal experimental model for studying the developmental dynamics of neural network assembly and sensory-motor computations.

    PubMed

    Straka, Hans; Simmers, John

    2012-04-01

    The amphibian Xenopus laevis represents a highly amenable model system for exploring the ontogeny of central neural networks, the functional establishment of sensory-motor transformations, and the generation of effective motor commands for complex behaviors. Specifically, the ability to employ a range of semi-intact and isolated preparations for in vitro morphophysiological experimentation has provided new insights into the developmental and integrative processes associated with the generation of locomotory behavior during changing life styles. In vitro electrophysiological studies have begun to explore the functional assembly, disassembly and dynamic plasticity of spinal pattern generating circuits as Xenopus undergoes the developmental switch from larval tail-based swimming to adult limb-based locomotion. Major advances have also been made in understanding the developmental onset of multisensory signal processing for reactive gaze and posture stabilizing reflexes during self-motion. Additionally, recent evidence from semi-intact animal and isolated CNS experiments has provided compelling evidence that in Xenopus tadpoles, predictive feed-forward signaling from the spinal locomotor pattern generator are engaged in minimizing visual disturbances during tail-based swimming. This new concept questions the traditional view of retinal image stabilization that in vertebrates has been exclusively attributed to sensory-motor transformations of body/head motion-detecting signals. Moreover, changes in visuomotor demands associated with the developmental transition in propulsive strategy from tail- to limb-based locomotion during metamorphosis presumably necessitates corresponding adaptive alterations in the intrinsic spinoextraocular coupling mechanism. Consequently, Xenopus provides a unique opportunity to address basic questions on the developmental dynamics of neural network assembly and sensory-motor computations for vertebrate motor behavior in general. PMID:21834082

  1. An RNA molecule copurifies with RNase P activity from Xenopus laevis oocytes.

    PubMed Central

    Doria, M; Carrara, G; Calandra, P; Tocchini-Valentini, G P

    1991-01-01

    Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related. Images PMID:1710353

  2. 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.

  3. Neurogenic development of the auditory areas of the midbrain and diencephalon in the Xenopus laevis and evolutionary implications.

    PubMed

    Zeng, Shao Ju; Tian, CuiPing; Zhang, XinWen; Zuo, Ming Xue

    2008-04-24

    To study whether the core-versus-shell pattern of neurogenesis occurred in the mesencephalic and diencephalic auditory areas of amniotes also appears in the amphibian, [(3)H]-thymidine was injected into tadpoles at serial developmental stages of Xenopus laevis. Towards the end of metamorphism, [(3)H]-thymidine labeling was examined and led to two main observations: 1) neuron generation in the principal nucleus (Tp) started at stage 50, and peaked at stage 53, whereas it began at stage 48.5, and peaked around stage 49 in the other two mesencephalic auditory areas, the laminar nucleus (Tl) and the magnocellular nucleus (Tmc). 2) Neuron generation appeared at stage 40, and peaked around stage 52 in the posterior thalamic nucleus (P) and the central thalamic nucleus (C). Our study revealed that, like the cores of mesencephalic auditory nuclei in amniotes, Tp showed differences from Tl and Tmc in the onset and the peak of neurogenesis. However, such differences did not occur in the P and C. Our neurogenetic data were consistent with anatomical and physiological reports indicating a clear distinction between the mesencephalic, but not the diencephalic auditory areas of the amphibian. Our data are helpful to get insights into the organization of auditory nuclei and its evolution in vertebrates. PMID:18346715

  4. Temperature-sensitive intermediate filament assembly. Alternative structures of Xenopus laevis vimentin in vitro and in vivo.

    PubMed

    Herrmann, H; Eckelt, A; Brettel, M; Grund, C; Franke, W W

    1993-11-01

    In assembly assays of intermediate filaments (IFs) from vimentin of the amphibian species Xenopus laevis we have observed the formation of so far unknown structures at temperatures above 28 degrees C. Upon assembly in vitro at temperatures above 34 degrees C massive aggregates, partly with a protofilamentous substructure, were found and their formation correlated with drastically reduced end-viscosity. Large spheroidal, dense aggregates with a complex suborganization were also seen to form at 37 degrees C in the cytoplasm of living mammalian cells devoid of endogenous vimentin upon transfection with cDNA encoding the amphibian vimentin, and this was also true for vimentin forced to accumulate in the nucleoplasm by the introduction of a "nuclear localization signal". Upon shift from the non-permissive (37 degrees C) to the permissive (28 degrees C) temperature, such aggregates of non-IF vimentin structures gradually disappeared and a normal-looking IF meshwork formed. The results, which are discussed in relation to other structures assembled by IF proteins, indicate a marked thermosensitivity in the amino acid sequence of the vimentin which seems to have been reduced during evolution of warm-blooded animals. They further show that members of the multigene gene family of IF proteins can occur in structures totally different from IFs. PMID:8230211

  5. 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. PMID:19943663

  6. Generation of the germ layers along the animal-vegetal axis in Xenopus laevis.

    PubMed

    Yasuo, H; Lemaire, P

    2001-01-01

    After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis. PMID:11291851

  7. Usage of the three termination codons in a single eukaryotic cell, the Xenopus laevis oocyte.

    PubMed Central

    Bienz, M; Kubli, E; Kohli, J; deHenau, S; Huez, G; Marbaix, G; Grosjean, H

    1981-01-01

    Oocytes from Xenopus laevis were injected with purified amber (UAG), ochre (UAA), and opal (UGA) suppressor tRNAs from yeasts. The radioactively labeled proteins translated from the endogenous mRNAs were then separated on two-dimensional gels. All three termination codons are used in a single cell, the Xenopus laevis oocyte. But a surprisingly low number of readthrough polypeptides were observed from the 600 mRNAs studied in comparison to uninjected oocytes. The experimental data are compared with the conclusions obtained from the compilation of all available termination sequences on eukaryotic and prokaryotic mRNAs. This comparison indicates that the apparent resistance of natural termination codons against readthrough, as observed by the microinjection experiments, cannot be explained by tandem or very close second stop codons. Instead it suggests that specific context sequences around the termination codons may play a role in the efficiency of translation termination. Images PMID:7024919

  8. Tolerance to Isoflurane Does Not Occur in Developing Xenopus laevis Tadpoles

    PubMed Central

    Milutinovic, Pavle S.; Zhao, Jing

    2009-01-01

    Introduction Tolerance is observed for a variety of central nervous system depressants including ethanol, which is an anesthetic, but has not been convincingly demonstrated for a potent halogenated volatile anesthetic. Failure to demonstrate tolerance to these agents may be the result of inadequate exposure to anesthetic. In this study, we exposed Xenopus laevis tadpoles to surgical anesthetic concentrations of isoflurane for one week. Methods Xenopus laevis tadpoles were produced by in vitro fertilization, and exposed to isoflurane (0.59%, 0.98%, 1.52%) or oxygen for one week starting from the time of fertilization. Results Changes in anesthetic EC50 were small and not in a consistent direction. Control animals had an anesthetic EC50 of 0.594% ± 0.003% isoflurane. Tadpoles exposed to 1.52% isoflurane had a lower EC50 than controls (by 16%), while tadpoles raised under 0.59 and 0.98% isoflurane had higher EC50s than control (by 4.7% and 7.4%, respectively). Conclusion We provide the first description of week-long exposures of vertebrates to surgical anesthetic concentrations of isoflurane, and the first report of such exposures in developing vertebrates. Tolerance to isoflurane does not occur in developing Xenopus laevis tadpoles. Taken together with studies in other organisms, the development of tolerance to ethanol but not isoflurane suggests that mechanisms these drugs share probably do not account for the development of tolerance. PMID:19095846

  9. 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...

  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. PMID:27239855

  11. The roles of Bcl-xL in modulating apoptosis during development of Xenopus laevis

    PubMed Central

    Johnston, Jillian; Chan, Robert; Calderon-Segura, Maria; McFarlane, Sarah; Browder, Leon W

    2005-01-01

    Background Apoptosis is a common and essential aspect of development. It is particularly prevalent in the central nervous system and during remodelling processes such as formation of the digits and in amphibian metamorphosis. Apoptosis, which is dependent upon a balance between pro- and anti-apoptotic factors, also enables the embryo to rid itself of cells damaged by gamma irradiation. In this study, the roles of the anti-apoptotic factor Bcl-xL in protecting cells from apoptosis were examined in Xenopus laevis embryos using transgenesis to overexpress the XR11 gene, which encodes Bcl-xL. The effects on developmental, thyroid hormone-induced and γ-radiation-induced apoptosis in embryos were examined in these transgenic animals. Results Apoptosis was abrogated in XR11 transgenic embryos. However, the transgene did not prevent the apoptotic response of tadpoles to thyroid hormone during metamorphosis. Post-metamorphic XR11 frogs were reared to sexual maturity, thus allowing us to produce second-generation embryos and enabling us to distinguish between the maternal and zygotic contributions of Bcl-xL to the γ-radiation apoptotic response. Wild-type embryos irradiated before the mid-blastula transition (MBT) underwent normal cell division until reaching the MBT, after which they underwent massive, catastrophic apoptosis. Over-expression of Bcl-xL derived from XR11 females, but not males, provided partial protection from apoptosis. Maternal expression of XR11 was also sufficient to abrogate apoptosis triggered by post-MBT γ-radiation. Tolerance to post-MBT γ-radiation from zygotically-derived XR11 was acquired gradually after the MBT in spite of abundant XR11 protein synthesis. Conclusion Our data suggest that Bcl-xL is an effective counterbalance to proapoptotic factors during embryonic development but has no apparent effect on the thyroid hormone-induced apoptosis that occurs during metamorphosis. Furthermore, post-MBT apoptosis triggered by irradiation before the

  12. PACSIN2 regulates cell adhesion during gastrulation in Xenopus laevis.

    PubMed

    Cousin, Hélène; Desimone, Douglas W; Alfandari, Dominique

    2008-07-01

    We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin beta1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated alpha5beta1 integrin and cytoskeleton strength during cell movement. PMID:18495106

  13. 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.

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

    PubMed Central

    O'Connell, Denice; Mruk, Karen; Rocheleau, Jessica M.

    2011-01-01

    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. PMID:21788613

  15. Functional expression of murine multidrug resistance in Xenopus laevis oocytes

    SciTech Connect

    Castillo, G.; Vera, J.C.; Rosen, O.M. ); Yang, Chiaping Huang; Horwitz, S.B. )

    1990-06-01

    The development of multidrug resistance (MDR) is associated with the overproduction of a plasma membrane glycoprotein, P glycoprotein. Here the authors report the functional expression of a member of the murine MDR family of proteins and show that Xenopus oocytes injected with RNA encoding the mouse mdr1b P glycoprotein develop a MDR-like phenotype. Immunological analysis indicated that oocytes injected with the mdr1b RNA synthesized a protein with the size and immunological characteristics of the mouse mdr1b P glycoprotein. These oocytes exhibited a decreased accumulation of ({sup 3}H)vinblastine and showed an increased capacity to extrude the drug compared to control oocytes not expressing the P glycoprotein. In addition, competition experiments indicated that verapamil, vincristine, daunomycin, and quinidine, but not colchicine, can overcome the rapid drug efflux conferred by the expression of the mouse P glycoprotein.

  16. Effects of ZnO nanomaterials on Xenopus laevis growth and development.

    PubMed

    Nations, Shawna; Long, Monique; Wages, Mike; Canas, Jaclyn; Maul, Jonathan D; Theodorakis, Chris; Cobb, George P

    2011-02-01

    The objectives of this study were to quantify uptake and developmental effects of zinc oxide nanomaterials (nano-ZnO) on Xenopus laevis throughout the metomormosis process. To accomplish this, X. laevis were exposed to aqueous suspensions of 40-100 nm nano-ZnO beginning in-ovo and proceeding through metamorphosis. Nanomaterials were dispersed via sonication methods into reconstituted moderately hard water test solutions. A flow-through system was utilized to decrease the likelihood of depletion in ZnO concentration. Exposure to 2 mg/L nano-ZnO significantly increased mortality incidence to 40% and negatively affected metamorphosis of X. laevis. Tadpoles exposed to 2 mg/L nano-ZnO developed slower as indicated by tadpoles with an average stage of 56 at the conclusion of the study which was significantly lower than the control tadpole stages. No tadpoles exposed to 2 mg/L of nano-ZnO completed metamorphosis by the conclusion of the study. Tadpoles exposed to 0.125 mg/L nano-ZnO experienced faster development along with larger body measurements indicating that low dose exposure to nano-ZnO can stimulate growth and metamorphosis of X. laevis. PMID:20801509

  17. Integument structure and function in juvenile Xenopus laevis with disrupted thyroid balance.

    PubMed

    Carvalho, Edison S M; Fuentes, Juan; Power, Deborah M

    2011-12-01

    The skin is the largest organ in the body and is a barrier between the internal and external environment. The present study evaluates how PTU, a goitrogen, that is used to treat hyperthyroidism affects the structure and electrical properties of the frog (Xenopus laevis) skin. The results are considered in the context of the two-membrane model established in the seminal work of Ussing and collegues in the 1940s and 1950s. In vitro experiments with skin from Xenopus adults revealed that PTU can act directly on skin and causes a significant increase (p<0.05, One-way ANOVA) in short circuit current (Isc) via an amiloride-insensitive mechanism. Juvenile Xenopus exposed to waterborne PTU (5 mg/L) had a significantly bigger and more active thyroid gland (p<0.01, Student's t-test) than control Xenopus. The bioelectric properties of skin taken from Xenopus juveniles treated with PTU in vivo had a lower Isc, (3.05±0.4, n=13) and Rt (288.2±39.5) than skin from control Xenopus (Isc, 4.19±1.14, n=14; Rt, 343.3±43.3). A histological assessment of skin from PTU treated Xenopus juveniles revealed the epidermis was significantly thicker (p<0.01, Student's t-test) and had a greater number of modified exocrine glands (p<0.01, Student's t-test) in the dermis compared to control skin. Modifications in skin structure are presumably the basis for its changed bioelectric properties and the study highlights a site of action for environmental chemicals which has been largely neglected. PMID:21963960

  18. Isolation of Chlamydia psittaci from naturally infected African clawed frogs (Xenopus laevis).

    PubMed Central

    Wilcke, B W; Newcomer, C E; Anver, M R; Simmons, J L; Nace, G W

    1983-01-01

    An inclusion-forming agent was isolated from the livers of commercially raised African clawed frogs (Xenopus laevis) involved in an epizootic of high morbidity and mortality. Original isolation was made in McCoy cells. This agent was identified as Chlamydia psittaci based on the formation of typical intracytoplasmic inclusions which developed within 48 h, were not stained by iodine, and were resistant to sulfadiazine. The isolate from one particular frog (designated as strain 178) was further studied and found to be lethal for 7-day-old embryonated chicken eggs after intra-yolk sac inoculation. This strain was demonstrated not to be pathogenic for mice when inoculated intraperitoneally. The cell culture isolate of C. psittaci was transmitted to uninfected X. laevis, causing disease and death. Images PMID:6347897

  19. 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

  20. 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

  1. Sterility and Gene Expression in Hybrid Males of Xenopus laevis and X. muelleri

    PubMed Central

    Malone, John H.; Chrzanowski, Thomas H.; Michalak, Pawel

    2007-01-01

    Background Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri). Methodology/Principal Findings Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. Conclusions/Significance Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing

  2. Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

    PubMed Central

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

    2015-01-01

    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. PMID:25742326

  3. Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

    PubMed Central

    Hanocq, F.; Kirsch-Volders, M.; Hanocq-Quertier, J.; Baltus, E.; Steinert, G.

    1972-01-01

    High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets. Images PMID:4113867

  4. The developmental expression of two Xenopus laevis steel homologues, Xsl-1 and Xsl-2.

    PubMed

    Martin, Benjamin L; Harland, Richard M

    2004-12-01

    We have investigated the expression patterns of two Xenopus laevis genes with similarity to mouse steel (sl) using whole-mount in situ hybridization. Areas of overlapping expression include the epidermis, stomodeum, proctodeum, and otic placodes. Xsl-1 is specifically expressed in the pronephros, neural tube, gil arches, and a superficial region of the somite, while Xsl-2 is in the ganglion and facial nerve projecting to visceral arch 3, the mandibular arch, and the clefts of anterior somites. Thus, both transcripts exhibit partially overlapping expression patterns, but their tissue distribution differs significantly from that of sl expression in the mouse and chicken. PMID:15567720

  5. Removal of 2',3'-dideoxynucleotide residues from injected DNA in Xenopus laevis oocytes.

    PubMed

    Legerski, R J; Penkala, J E; Peterson, C A; Wright, D A

    1990-07-01

    Dideoxynucleotides have proved to be potent differential inhibitors of DNA polymerases in vitro and in vivo. Used extensively in studies of DNA repair and replication, they have more recently been used as antiviral agents particularly in treating patients for acquired immunodeficiency syndrome (AIDS). Once incorporated, these sugar-modified analogues prevent the further extension of the polynucleotide chain because of the absence of a 3'-hydroxyl group. We demonstrated that, upon injection into Xenopus laevis oocytes, 2',3'-dideoxynucleotides are efficiently removed from plasmid DNA preterminated with these analogues allowing subsequent closure by ligation. The removal process is not sensitive to aphidicolin but is quantitatively inhibited by novobiocin. PMID:2366792

  6. Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

    PubMed Central

    Dominguez, I; Diaz-Meco, M T; Municio, M M; Berra, E; García de Herreros, A; Cornet, M E; Sanz, L; Moscat, J

    1992-01-01

    A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways. Images PMID:1508183

  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. Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts.

    PubMed

    Groen, Aaron C; Mitchison, Timothy J

    2016-01-01

    For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro. PMID:27193841

  9. 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

  10. 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

  11. The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos.

    PubMed

    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 (K.R. Kao, Y. Masui, and R.P. Elinson, 1986, Nature (London) 322, 371-373). 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. PMID:3282938

  12. 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. PMID:26914410

  13. Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.

    PubMed

    Jelaso, Anna M; Lehigh-Shirey, Elisabeth; Means, Jay; Ide, Charles F

    2003-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that pose global ecological and human health problems. Although it is well established that PCBs are associated with a variety of adverse health effects in wildlife and in humans, it is often difficult to determine direct cause-and-effect relationships between exposure and specific health outcomes. In this study, gene expression signatures were used to relate exposure to PCBs with altered physiological responses and/or specific health effects. Real-time PCR was used to measure gene expression levels for 10 genes in Xenopus laevis tadpoles (18 days postfertilization, PF) after acute exposure (2 days) to the PCB mixture Aroclor 1254. Specific gene expression signatures correlated with exposure and were predictive of adverse health effects. Exposure to low levels of Aroclor 1254 (5-50 ppb) significantly increased expression of six genes, independent of any health effects; exposure to midlevel concentrations (300-400 ppb) significantly decreased expression levels of two genes, NGF and beta-actin, prior to the onset of observable health effects; exposure to higher doses (500-700 ppb) significantly decreased NGF and beta-actin expression concomitant with the appearance of gross morphological abnormalities, behavioral deficits, and a statistically significant decrease in survival. This study expands upon our previous work that demonstrated an age-dependent susceptibility to Aroclor 1254 in Xenopus laevis tadpoles and that defined specific gene expression signatures as useful bioindicators of exposure and as predictors of overt or impending health effects. PMID:12874807

  14. 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

  15. Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

    PubMed Central

    Swick, A G; Janicot, M; Cheneval-Kastelic, T; McLenithan, J C; Lane, M D

    1992-01-01

    Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary. Images PMID:1542676

  16. Gene expression analysis of the ovary of hybrid females of Xenopus laevis and X. muelleri

    PubMed Central

    2008-01-01

    Background Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis) and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri). In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females. Results We find a similar pattern of asymmetry in expression compared to males in that there were more genes differentially expressed between hybrids and X. muelleri compared to hybrids and X. laevis. We also found a dramatic increase in the number of misexpressed genes with hybrid females having about 20 times more genes misexpressed in ovaries compared to testes of hybrid males and therefore the match between phenotype and expression pattern is not supported. Conclusion We discuss these intriguing findings in the context of reproductive isolation and suggest that divergence in female expression may be involved in sterility of hybrid males due to the inherent sensitivity of spermatogenesis as defined by the faster male evolution hypothesis for Haldane's rule. PMID:18331635

  17. Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption.

    PubMed

    Fini, J B; Le Mével, S; Palmier, K; Darras, V M; Punzon, I; Richardson, S J; Clerget-Froidevaux, M S; Demeneix, B A

    2012-10-01

    Thyroid hormone (TH) is essential for vertebrate brain development. Most research on TH and neuronal development focuses on late development, mainly the perinatal period in mammals. However, in human infants neuromotor development correlates best with maternal TH levels in the first trimester of pregnancy, suggesting that TH signaling could affect early brain development. Studying TH signaling in early embryogenesis in mammals is experimentally challenging. In contrast, free-living embryos, such as Xenopus laevis, permit physiological experimentation independent of maternal factors. We detailed key elements of TH signaling: ligands, receptors (TR), and deiodinases during early X. laevis development, before embryonic thyroid gland formation. Dynamic profiles for all components were found. Between developmental stages 37 and 41 (~48 h after hatching, coincident with a phase of continuing neurogenesis) significant increases in T(3) levels as well as in mRNA encoding deiodinases and TR occurred. Exposure of embryos at this developmental stage for 24 h to either a TH antagonist, NH-3, or to tetrabromobisphenol A, a flame retardant and known TH disruptor, differentially modulated the expression of a number of TH target genes implicated in neural stem cell function or neural differentiation. Moreover, 24-h exposure to either NH-3 or tetrabromobisphenol A diminished cell proliferation in the brain. Thus, these data show first, that TH signaling exerts regulatory roles in early X. laevis neurogenesis and second, that this period represents a potential window for endocrine disruption. PMID:22968643

  18. Thrombopoietin induces production of nucleated thrombocytes from liver cells in Xenopus laevis

    PubMed Central

    Tanizaki, Yuta; Ichisugi, Megumi; Obuchi-Shimoji, Miyako; Ishida-Iwata, Takako; Tahara-Mogi, Ayaka; Meguro-Ishikawa, Mizue; Kato, Takashi

    2015-01-01

    The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated thrombocytes instead of platelets, the features of nucleated thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO (xlTPO). Hepatic and splenic cells were cultured in liquid suspension with recombinant xlTPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X. laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xlTPO produced mature, spindle-shaped thrombocytes that were activated by thrombin, thereby altering their morphology. XlTPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xlTPO enhanced viability of peripheral thrombocytes, indicating the xlTPO-Mpl pathway stimulates anti-apoptotic in peripheral thrombocytes. The development of thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200). PMID:26687619

  19. 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. PMID:26632790

  20. 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

  1. 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…

  2. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  3. 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.

  4. 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

  5. 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

  6. 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. PMID:26797504

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

    PubMed

    Mason, Mj; Wang, M; Narins, Pm

    2009-01-01

    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

  8. Nucleolin from Xenopus laevis: cDNA cloning and expression during development.

    PubMed

    Caizergues-Ferrer, M; Mariottini, P; Curie, C; Lapeyre, B; Gas, N; Amalric, F; Amaldi, F

    1989-03-01

    Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis. PMID:2656405

  9. Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts

    PubMed Central

    Banaszynski, Laura A.; Allis, C. David; Shechter, David

    2010-01-01

    Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract (“NPE”); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system. PMID:20051265

  10. Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis.

    PubMed

    Pastori, R L; Moskaitis, J E; Buzek, S W; Schoenberg, D R

    1991-04-01

    Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver. PMID:1922078

  11. DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

    PubMed Central

    La Volpe, A; Taggart, M; McStay, B; Bird, A

    1983-01-01

    We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function. Images PMID:6310495

  12. 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. PMID:26855011

  13. Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis.

    PubMed

    Yamashiro, Sawako; Noguchi, Tatsuhiko; Mabuchi, Issei

    2003-05-01

    Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells. PMID:12673597

  14. 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

  15. 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. PMID:26855879

  16. Remyelination by Resident Oligodendrocyte Precursor Cells in a Xenopus laevis Inducible Model of Demyelination.

    PubMed

    Sekizar, Sowmya; Mannioui, Abdelkrim; Azoyan, Loris; Colin, Catherine; Thomas, Jean-Léon; Du Pasquier, David; Mallat, Michel; Zalc, Bernard

    2015-01-01

    We have generated a Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of the myelin basic protein regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by the green fluorescent protein reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. The NTR enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Ablation of oligodendrocytes by MTZ treatment of the tadpole induced demyelination, and here we show that myelin debris are subsequently eliminated by microglial cells. After cessation of MTZ treatment, remyelination proceeded spontaneously. We questioned the origin of remyelinating cells. Our data suggest that Sox10+ oligodendrocyte precursor cells (OPCs), which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination, and this required only minimal (if any) cell division of OPCs. © 2015 S. Karger AG, Basel. PMID:25896276

  17. In vivo pump-probe optical coherence tomography imaging in Xenopus laevis.

    PubMed

    Carrasco-Zevallos, Oscar; Shelton, Ryan L; Kim, Wihan; Pearson, Jeremy; Applegate, Brian E

    2015-01-01

    Currently, optical coherence tomography (OCT), is not capable of obtaining molecular information often crucial for identification of disease. To enable molecular imaging with OCT, we have further developed a technique that harnesses transient changes in light absorption in the sample to garner molecular information. A Fourier-domain Pump-Probe OCT (PPOCT) system utilizing a 532 nm pump and 830 nm probe has been developed for imaging hemoglobin. Methylene blue, a biological dye with well-know photophysics, was used to characterize the system before investigating the origin of the hemoglobin PPOCT signal. The first in vivo PPOCT images were recorded of the vasculature in Xenopus laevis. The technique was shown to work equally well in flowing and nonflowing vessels. Furthermore, PPOCT was compared with other OCT extensions which require flow, such as Doppler OCT and phase-variance OCT. PPOCT was shown to better delineate tortuous vessels, where nodes often restrict Doppler and phase-variance reconstruction. PMID:24282110

  18. Pre-meiotic transformation of germplasm-related structures during male gamete differentiation in Xenopus laevis.

    PubMed

    Reunov, Arkadiy A; Reunova, Yulia A

    2016-02-01

    To highlight the ultrastructural features of transformation occurring with germplasm-related structures (GPRS), the spermatogenic cells of Xenopus laevis were studied by transmission electron microscopy and quantitative analysis. It was determined that in spermatogonia and spermatocytes, the compact germinal granules underwent fragmentation into particles comparable with inter-mitochondrial cement (IMC). Fragments of IMC agglutinated some cell mitochondria and resulted in the creation of mitochondrial clusters. Clustered mitochondria responded with loss of their membranes that occurred by the twisting of membranous protrusions around themselves until multi-layered membranes were formed. The mitochondrial affinity of multi-layered membranes was proven by an immunopositive test for mitochondrial dihydrolipoamide acetyltransferase. As a consequence of mitochondrial membrane twisting, the naked mitochondrial cores appeared and presumably underwent dispersion, which is the terminal stage of GPRS transformation. As no GPRS were observed in spermatids and sperm, it was assumed that these structures are functionally assigned to early stages of meiotic differentiation. PMID:25511532

  19. Subchronic and chronic developmental effects of copper oxide (CuO) nanoparticles on Xenopus laevis.

    PubMed

    Nations, Shawna; Long, Monique; Wages, Mike; Maul, Jonathan D; Theodorakis, Christopher W; Cobb, George P

    2015-09-01

    Metal oxide nanoparticles, such as copper oxide (CuO), are mass produced for use in a variety of products like coatings and ceramics. Acute exposure to CuO nanoparticles has caused toxicity to many aquatic organisms, yet there is no information on the effect of prolonged CuO nanomaterial exposures. This study examined effects of chronic exposure to CuO nanoparticles on Xenopus laevis growth and development. Experiments included a 14 d subchronic exposure and a 47 d chronic exposure throughout metamorphosis. The subchronic exposure caused mortality in all tested CuO concentrations, and significant growth effects occurred after exposure to 2.5 mg L(-1) CuO. Chronic exposure to 0.3 mg L(-1) CuO elicited significant mortality and affected the rate of metamorphosis. Exposure to lower concentrations of CuO stimulated metamorphosis and growth, indicating that low dose exposure can have hormetic effects. PMID:25950410

  20. 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.

  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. 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.

  3. 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

  4. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

    PubMed Central

    Kaeser, Gwendolyn E.; Rabe, Brian A.; Saha, Margaret S.

    2011-01-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABAA and metabotropic GABAB receptors. During the development of the nervous system GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABAA and GABAB receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABAA α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. PMID:21384470

  5. 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. PMID:27218424

  6. Efficacy of Tricaine Methanesulfonate (MS-222) as an Anesthetic Agent for Blocking Sensory-Motor Responses in Xenopus laevis Tadpoles

    PubMed Central

    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. PMID:24984086

  7. Thyroid hormone activates Wnt/β-catenin signaling involved in adult epithelial development during intestinal remodeling in Xenopus laevis.

    PubMed

    Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Ishizuya-Oka, Atsuko

    2016-08-01

    During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including β-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear β-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling. PMID:27068920

  8. 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.

  9. Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy.

    PubMed

    Sadaghiani, B; Thiébaud, C H

    1987-11-01

    The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level. PMID:3666314

  10. 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

  11. 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

  12. A Ca2+-activated channel from Xenopus laevis oocyte membranes reconstituted into planar bilayers.

    PubMed Central

    Young, G P; Young, J D; Deshpande, A K; Goldstein, M; Koide, S S; Cohn, Z A

    1984-01-01

    Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These channels are activated by Ca2+ at micromolar concentration but not by Mg2+, Zn2+, or Mn2+, even at millimolar concentrations. Decreasing Ca2+ concentration to less than 1 microM decreases the time of channel opening until channels close completely in the absence of Ca2+ and in the presence of EGTA. I- and Br- are more permeable through this channel than Cl-. The time during which the channels remain open is also voltage-dependent, with the channels switching off at higher voltages in both polarities. Single-channel activity shows a conductance of 380 pS in 1 M NaCl and 1 mM CaCl2, with an average open lifetime of 1.5 s at 40 mV. Similar channels are found in different stages of oocyte maturation. These observations support the hypothesis that an increase in oocyte-free Ca2+ activates directly these channels, and the resultant Cl- efflux forms the ionic basis for the fertilization potential in X. laevis. PMID:6089180

  13. Prediction of Functionally Important Phospho-Regulatory Events in Xenopus laevis Oocytes

    PubMed Central

    Johnson, Jeffrey R.; Santos, Silvia D.; Johnson, Tasha; Pieper, Ursula; Strumillo, Marta; Wagih, Omar; Sali, Andrej; Krogan, Nevan J.; Beltrao, Pedro

    2015-01-01

    The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 2636 phosphosites. We used structural information and phosphoproteomic data for 13 other species in order to predict functionally important phospho-regulatory events. We found that the degree of conservation of phosphosites across species is predictive of sites with known molecular function. In addition, we predicted kinase-protein interactions for a set of cell-cycle kinases across all species. The degree of conservation of kinase-protein interactions was found to be predictive of functionally relevant regulatory interactions. Finally, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. Our analysis suggests that a small class of phosphosites occurs in positions that have the potential to regulate protein conformation. PMID:26312481

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

    PubMed Central

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

    2015-01-01

    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. PMID:25447273

  15. 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. PMID:26510384

  16. 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

  17. 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. PMID:27289584

  18. 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. PMID:27288644

  19. 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. PMID:25601449

  20. 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.

  1. 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.

  2. Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system.

    PubMed

    Sinzelle, L; Vallin, J; Coen, L; Chesneau, A; Du Pasquier, D; Pollet, N; Demeneix, B; Mazabraud, A

    2006-12-01

    Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure. PMID:16957880

  3. 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

    2015-06-01

    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. PMID:25965532

  4. 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.

  5. A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

    PubMed Central

    Viczian, Andrea S.; Zuber, Michael E.

    2014-01-01

    Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once. PMID:24962702

  6. sox4 And sox11 Function during Xenopus laevis Eye Development

    PubMed Central

    Metzig, Marlen; Tao, Si; Hollemann, Thomas; Kühl, Michael; Kühl, Susanne J.

    2013-01-01

    SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development. PMID:23874955

  7. Regulation of Respiratory and Vocal Motor Pools in the Isolated Brain of Xenopus laevis

    PubMed Central

    Zornik, Erik; Kelley, Darcy B.

    2010-01-01

    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 (DTAMIX–X neurons) provide direct input to glottal and laryngeal motor neurons. Electrical stimulation produced short-latency coordinated activity in the laryngeal nerve. DTAMIX–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. PMID:18199762

  8. A simple behavioral assay for testing visual function in Xenopus laevis.

    PubMed

    Viczian, Andrea S; Zuber, Michael E

    2014-01-01

    Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once. PMID:24962702

  9. 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. PMID:27499877

  10. 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

  11. 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. PMID:22723004

  12. 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

  13. 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. PMID:26952034

  14. RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    PubMed Central

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K.; Menssen, Ruth; Wolf, Dieter H.; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  15. Use of large-scale expression cloning screens in the Xenopus laevis tadpole to identify gene function.

    PubMed

    Grammer, T C; Liu, K J; Mariani, F V; Harland, R M

    2000-12-15

    We have conducted an expression cloning screen of approximately 50, 000 cDNAs from a tadpole stage Xenopus laevis cDNA library to functionally identify genes affecting a wide range of cellular and developmental processes. Fifty-seven cDNAs were isolated for their ability to alter gross tadpole morphology or the expression patterns of tissue-specific markers. Thirty-seven of the cDNAs have not been previously described for Xenopus, and 15 of these show little or no similarity to sequences in the NCBI database. The screen and the identified genes are presented in this paper to demonstrate the power, ease, speed, and flexibility of expression cloning in the X. laevis embryo. Future screens such as this one can be done on a larger scale and will complement the sequence-based screens and genome-sequencing projects which are producing a large body of novel genes without ascribed functions. PMID:11112324

  16. DNA sequence of the Xenopus laevis mitochondrial heavy and light strand replication origins and flanking tRNA genes.

    PubMed Central

    Wong, J F; Ma, D P; Wilson, R K; Roe, B A

    1983-01-01

    We have determined the primary structure of the two regions of the Xenopus laevis mitochondrial genome which encompass the origins of heavy (H) and light (L) strand replication. The first segment, which consists of 2398 nucleotides, contains the displacement loop (D-loop), the tRNA genes for threonine, proline and phenylalanine, the origin of H-strand replication, and the promoters of H- and L-strand transcription. The second segment, which consists of 447 nucleotides, contains the L-strand replication origin flanked by the tRNA genes for tryptophan, alanine, asparagine, cysteine, and tyrosine. A comparison of the sequences of the Xenopus laevis mitochondrial L-strand replication origin region and the eight tRNA genes with their counterparts from the mammalian mitochondrial genomes reveals that these regions are quite homologous, while its D-loop region shows only slight homology with those of the mammalian mitochondrial genomes. PMID:6308566

  17. 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

  18. Molecular cloning and sequencing of mRNAs coding for minor adult globin polypeptides of Xenopus laevis.

    PubMed Central

    Knöchel, W; Meyerhof, W; Hummel, S; Grundmann, U

    1983-01-01

    Globin mRNA was isolated from immature red blood cells of an adult Xenopus laevis female. mRNA/cDNA hybrids were integrated in the Pst I cleavage site of pBR 322 by G/C tailing, and cloned in Escherichia coli strain HB 101. By restriction site analysis as well as hybridization behaviour we identified two clones coding for minor adult alpha and beta globin chains. Nucleotide sequence analysis and derived amino acid sequences are presented. PMID:6298748

  19. The morphology and attachment of Protopolystoma xenopodis (Monogenea: Polystomatidae) infecting the African clawed frog Xenopus laevis

    PubMed Central

    Theunissen, Maxine; Tiedt, Louwrens; Du Preez, Louis H.

    2014-01-01

    The African clawed frog Xenopus laevis (Anura: Pipidae) is host to more than 25 parasite genera encompassing most of the parasitic invertebrate groups. Protopolystoma xenopodis Price, 1943 (Monogenea: Polystomatidae) is one of two monogeneans infecting X. laevis. This study focussed on the external morphology of different developmental stages using scanning electron microscopy, histology and light microscopy. Eggs are released continuously and are washed out when the frog urinates. After successful development, an active swimming oncomiracidium leaves the egg capsule and locates a potential post-metamorphic clawed frog. The oncomiracidium migrates to the kidney where it attaches and starts to feed on blood. The parasite then migrates to the urinary bladder where it reaches maturity. Eggs are fusiform, about 300 μm long, with a smooth surface and are operculated. Oncomiracidia are elongated and cylindrical in shape, with an oval posterior cup-shaped haptor that bears a total of 20 sclerites; 16 marginal hooklets used for attachment to the kidney of the host and two pairs of hamulus primordia. Cilia from the 64 ciliated cells enable the oncomiracidium to swim for up to 24 h when the cilia subsequently curl up, become non-functional and are shed from the body. The tegument between the ciliated cells bears a series of sensory papillae. The body of the mature parasite is elongated and pyriform and possesses an opisthaptor armed with three pairs of suckers and two pairs of falciform hooks to ensure a firm grip on the flexible internal surface of the urinary bladder. PMID:24823278

  20. 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

    2002-01-01

    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. PMID:11782548

  1. Regulation of adenovirus transcription by an Ela gene in microinjected Xenopus laevis oocytes

    SciTech Connect

    Jones, N.C.; Richter, J.D.; Weeks, D.L.; Smith, L.D.

    1983-12-01

    The regulation of adenovirus type 5 gene expression by the E1a gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the E1a gene were coinjected into oocyte nuclei. The presence of the E1a gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the E1a gene requires the removal of intron sequences by RNA splicing. The E1a gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary E1a transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the E1a mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the E1a protein may supplant the E1a plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

  2. 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. PMID:25277091

  3. 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. PMID:26971167

  4. 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

  5. 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

  6. The Pharmacokinetics of Enrofloxacin in Adult African Clawed Frogs (Xenopus laevis)

    PubMed Central

    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

    2010-01-01

    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. PMID:21205443

  7. Intracellular glycosylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis

    SciTech Connect

    Gottlieb, T.A.; Wallace, R.A.

    1982-01-01

    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. No apparent role of the rough endoplasmic reticulum in this process was demonstrable. Kinetics of the intracellular translocation of glycosylated vitellogenin indicate that the galactosylated intermediate is secreted more rapidly than the glucosamine-labeled precursor. This was corroborated by measuring the rates of accumulation of various pulse-labeled forms of vitellogenin in the chase medium. In addition, a negligible amount of mannose was incorporated into intracellular or secreted vitellogenin. The antibiotic tunicamycin was shown to inhibit (/sup 3/H) glucosamine incorporation into microsomal vitellogenin by 70% without any significant effect on the synthesis of the protein backbone. In addition, nonglycosylated vitellogenin showed normal secretion kinetics. After suitable pretreatment with the antibiotic followed by a labelling period in tunicamycin-free medium, mannose was still not incorporated into vitellogenin, whereas glucosamine behaved in a typical manner. In contrast to this finding, gas-liquid chromatography of the alditol acetate derivatives of the neutral hexoses of vitellogenin showed that mannose was indeed a major component of the vitellogenin oligosaccharide side chain. These preliminary results indicate that the oligosaccharide component of vitellogenin in Xenopus laevis 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 the subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.

  8. FMRP Regulates Neurogenesis In Vivo in Xenopus laevis Tadpoles1,2,3

    PubMed Central

    Faulkner, Regina L.; Wishard, Tyler J.; Thompson, Christopher K.; Liu, Han-Hsuan

    2014-01-01

    Abstract Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction; however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles that express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or overexpression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS. PMID:25844398

  9. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

    PubMed

    Bates, Ryan C; Fees, Colby P; Holland, William L; Winger, Courtney C; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J

    2014-02-01

    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 min 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 specifically 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

  10. 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

  11. 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

  12. Histochemical Analyses of Biliary Development During Metamorphosis of Xenopus laevis Tadpoles.

    PubMed

    Ueno, Tomoya; Ishihara, Akinori; Yagi, Shinomi; Koike, Toru; Yamauchi, Kiyoshi; Shiojiri, Nobuyoshi

    2015-01-01

    In mammalian liver development, intrahepatic biliary morphogenesis takes place in periportal, but not in pericentral, regions. Liver progenitor cells transiently form epithelial plate structures and then intrahepatic bile ducts around the portal veins under the influence of the mesenchyme. The present study was undertaken to histochemically examine normal biliary development and its dependence on the action of the thyroid hormone triiodothyronine (T3) in Xenopus laevis tadpoles. In these tadpoles, the development of hepatic ducts and intrahepatic biliary ducts commenced along the portal veins at NF stages 48-50 and stages 50-52, respectively, when the blood concentration of thyroid hormone may be still low. Some periportal hepatocytes expressed carbamoylphosphate synthase I and SOX9, which are hepatocyte and biliary cell markers, respectively, suggesting that periportal hepatocytes give rise to biliary epithelial cells. Periportal biliary cells did not form ductal plates, nor was the periportal mesenchyme well developed as seen in fetal mouse livers. jag1 mRNA was moderately expressed in cells of portal veins and biliary epithelial cells, and notch1 and notch2 mRNAs were weakly detectable in biliary epithelial cells during metamorphosis as seen in developing mammalian livers. These results suggest that Notch signaling plays a decisive role in biliary cell differentiation and morphogenesis of Xenopus tadpoles. Anti-thyroid agent treatment of the tadpoles resulted in delayed biliary morphogenesis, suggesting that biliary development may depend on T3. However, T3 treatment of the tadpoles did not enhance biliary development. Thus, T3 may act positively on biliary development at a very low concentration. PMID:25660701

  13. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

    PubMed

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu; Serrano, Elba E

    2011-10-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology. PMID:21959846

  14. 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

    2014-01-01

    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). PMID:25225829

  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. 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

  17. Sensory physiology, anatomy and immunohistochemistry of Rohon-Beard neurones in embryos of Xenopus laevis.

    PubMed Central

    Clarke, J D; Hayes, B P; Hunt, S P; Roberts, A

    1984-01-01

    Rohon-Beard neurones show substance P-like immunoactivity in their somas and in their centrally projecting axons. Peripherally, the morphology of their free nerve endings within the trunk skin has been shown using horseradish peroxidase staining. The excitation of Rohon-Beard neurones by natural and electrical stimulation of the skin has been examined using intracellular micro-electrodes in the late embryo of Xenopus laevis. Rohon-Beard cells are sensitive to transient, local indentation of the trunk skin, responding with one or a few impulses. They adapt rapidly to repeated stimulation. They can also be excited by a brief current pulse to the skin. They are not sensitive to slow indentation of the skin, nor are they excited by epithelial action potentials. The responses to skin stimulation are not abolished by a Ringer solution containing 12 mM-Mg2+ and only 0.5 mM-Ca2+. Intracellularly evoked action potentials in single Rohon-Beard cells are sometimes sufficient to evoke sustained episodes of fictive swimming. The results indicate that Rohon-Beard cells are responsible for detecting light touch stimuli to the embryo's body and for initiating swimming in response to this stimulus. Images PLATE 2 PLATE 1 PMID:6201612

  18. 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.

  19. Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis.

    PubMed Central

    Imai, Y; Davey, J; Kawagishi-Kobayashi, M; Yamamoto, M

    1997-01-01

    The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h- cells but not in h+ cells. h- cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids, with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homolog of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. The isolation of a vertebrate gene encoding farnesyl cysteine carboxyl methyltransferase opens the way to in-depth studies of the role of methylation in a large body of proteins, including Ras superfamily proteins. PMID:9032282

  20. 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

  1. Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones

    PubMed Central

    Stout, Alina; D’Amico, Salvatore; Enzenbacher, Tiffany; Ebbert, Patrick; Lowery, Laura Anne

    2014-01-01

    Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. 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 polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. 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 types6-8. PMID:25225829

  2. 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. PMID:26472276

  3. 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.

  4. Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis

    PubMed Central

    Schweidenback, Caterina T.H.; Emerman, Amy B.; Jambhekar, Ashwini

    2015-01-01

    ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, double-stranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR. PMID:25519486

  5. 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.

  6. Involvement of sperm proteases in the binding of sperm to the vitelline envelope in Xenopus laevis.

    PubMed

    Kubo, Hideo; Kotani, Masaharu; Yamamoto, Yukio; Hazato, Tadahiko

    2008-01-01

    Sperm binding to the vitelline envelope in dejellied Xenopus laevis eggs was effectively inhibited by inhibitors for trypsin (soybean trypsin inhibitor and p-toluenesulfonyl-L-lysine chloroethyl ketone) and aminopeptidase B (o-phenanthroline, bestatin, and arphamenine B). Likewise, synthetic 4-methylcoumaryl-7-amide (MCA) substrates (t-butoxycarbonyl-GlyArgArg-MCA, benzyloxycarbonyl-ArgArg-MCA, and Arg-MCA) inhibited binding. Consistently, when jellied eggs were inseminated in the presence of these substrates or inhibitors for proteases, fertilization was effectively blocked. The medium in which live sperm or the sperm membrane fraction were suspended exhibited hydrolyzing activities against the synthetic substrates mentioned above, and these activities were effectively inhibited by the protease inhibitors. Ultracentrifugal fractionation of the sperm suspension following induction of the acrosome reaction by a calcium ionophore, A23187, indicated that a considerable amount of the total tryptic and aminopeptidase B activity was released into the medium. On this occasion, part of the tryptic and aminopeptidase B activity was definitely estimated to be discharged in association with a vesiculated membrane, supporting the notion that the proteases involved in binding to the vitelline envelope are present on the sperm plasma membrane. PMID:18275249

  7. Cell specialization in the epithelium of the small intestine of feeding Xenopus laevis tadpoles.

    PubMed Central

    Marshall, J A; Dixon, K E

    1978-01-01

    The intestinal epithelium of feeding Xenopus laevis tadpoles was studied using light microscope, electron microscope and autoradiographic techniques. The wall of the small intestine differs from that of most other vertebrates studied in that it lacks villous-like folds. A single prominent longitudinal fold, the typhlosole, forms about stage 49, and a series of shallow longitudinal epithelial pleats is also present in some animals. The morphology of the epithelial cells indicates that there are no differences between the cells in their degree of specialization. Three epithelial cell types were recognized: principal cells, gland cells and endocrine cells, making up about 65%, 15% and 1%, respectively, of all cells present, while approximately 20% of the cells in the epithelium are lymphocytes, 1% appear to be discharged gland cells, and 2% degenerating cells. No Paneth, caveolated or undifferentiated cells were identified. The findings are discussed in relation to other studies on cell proliferation and on nuclear transplantation. Images Figs. 3-4 Figs. 5-6 Figs. 7-8 Fig. 9 Fig. 10 Fig. 11 PMID:649494

  8. 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. PMID:25078857

  9. Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

    PubMed

    Boggavarapu, Rajendra; Hirschi, Stephan; Harder, Daniel; Meury, Marcel; Ucurum, Zöhre; Bergeron, Marc J; Fotiadis, Dimitrios

    2016-01-01

    This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail. PMID:27485339

  10. 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.

  11. 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

  12. 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. PMID:22776127

  13. 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

  14. 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. PMID:16302973

  15. Peripheral myelin of Xenopus laevis: role of electrostatic and hydrophobic interactions in membrane compaction.

    PubMed

    Luo, XiaoYang; Cerullo, Jana; Dawli, Tamara; Priest, Christina; Haddadin, Zaid; Kim, Angela; Inouye, Hideyo; Suffoletto, Brian P; Avila, Robin L; Lees, Jonathan P B; Sharma, Deepak; Xie, Bo; Costello, Catherine E; Kirschner, Daniel A

    2008-04-01

    P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of X. laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0's putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0's adhesion role and the

  16. 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. PMID:26802562

  17. Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

    PubMed Central

    2014-01-01

    Background Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). Results We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are

  18. 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. PMID:27017260

  19. Response of amphibian egg non-yolk cytoplasm to gravity orientation

    NASA Technical Reports Server (NTRS)

    Smith, R. C.; Neff, A. W.; Malacinski, G. M.

    1985-01-01

    In order to study amphibian egg cytoplasmic organization and egg symmetrization at the molecular level, a library of seventeen monoclonal antibodies (MoAbs) against Xenopus laevis non-yolk egg proteins was produced. Several of these MoAbs react with non-yolk cytoplasmic antigens which are unevenly distributed in the fertile Xenopus egg.

  20. 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

  1. Embryotoxic effects of environmental chemicals: tests with the South African clawed toad (Xenopus laevis)

    SciTech Connect

    Dumpert, K.

    1987-06-01

    In the course of the investigations reported below, it was shown that p-chloroaniline has a lethal effect on the embryos of Xenopus laevis at a concentration of 100 ppm and is development inhibiting (teratogenic) at concentrations of 1 and 10 ppm, respectively. In the case of aniline, a significant development-inhibiting effect was observed at a concentration as low as 1 ppm. A toxic effect was caused by concentrations between 30 and 40 ppm during embryogenesis and by concentrations above 40 ppm during larval development. A very conspicuous finding was an inhibiting effect of 20 to 40 ppm aniline on pigmentation during embryogenesis and of a concentration as low as 1 ppm on the body size of the young toads. In the case of potassium dichromate, it was possible to barely detect a weak development-inhibiting effect during embryogenesis but no development-retarding effect during larval development. Toxic effects of potassium dichromate occurred during embryogenesis at concentrations of 5 and 7.5 ppm and during the larval development at concentrations above 10 ppm. Sodium dodecylbenzenesulfonic acid at a concentration of 50 ppm was found to have such a strong embryolethal effect that 80% of the eggs showed no cell division at all and the remaining 20% developed to only the bicellular stage. A teratogenic effect of this substance was not observed. Phenol, too, was found to be toxic at a concentration of 50 ppm; in contrast to sodium dodecylbenzenesulfonic acid, however, it did not show any lethal effect on the embryos but it did on the tadpoles, mainly in the first stages of larval development. Lower concentrations of phenol (5 and 10 ppm) had a nonsignificant inhibiting effect on the growth of the larvae. A teratogenic effect of phenol was not detected.

  2. Trialkyltin Rexinoid-X Receptor Agonists Selectively Potentiate Thyroid Hormone Induced Programs of Xenopus laevis Metamorphosis.

    PubMed

    Mengeling, Brenda J; Murk, Albertinka J; Furlow, J David

    2016-07-01

    The trialkyltins tributyltin (TBT) and triphenyltin (TPT) can function as rexinoid-X receptor (RXR) agonists. We recently showed that RXR agonists can alter thyroid hormone (TH) signaling in a mammalian pituitary TH-responsive reporter cell line, GH3.TRE-Luc. The prevalence of TBT and TPT in the environment prompted us to test whether they could also affect TH signaling. Both trialkyltins induced the integrated luciferase reporter alone and potentiated TH activation at low doses. Trimethyltin, which is not an RXR agonist, did not. We turned to a simple, robust, and specific in vivo model system of TH action: metamorphosis of Xenopus laevis, the African clawed frog. Using a precocious metamorphosis assay, we found that 1nM TBT and TPT, but not trimethyltin, greatly potentiated the effect of TH treatment on resorption phenotypes of the tail, which is lost at metamorphosis, and in the head, which undergoes extensive remodeling including gill loss. Consistent with these responses, TH-induced caspase-3 activation in the tail was enhanced by cotreatment with TBT. Induction of a transgenic reporter gene and endogenous collagenase 3 (mmp13) and fibroblast-activating protein-α (fap) genes were not induced by TBT alone, but TH induction was significantly potentiated by TBT. However, induction of other TH receptor target genes such as TRβ and deiodinase 3 by TH were not affected by TBT cotreatment. These data indicate that trialkyltins that can function as RXR agonists can selectively potentiate gene expression and resultant morphological programs directed by TH signaling in vivo. PMID:27167774

  3. Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

    PubMed Central

    Danker, T.; Schillers, H.; Storck, J.; Shahin, V.; Krämer, B.; Wilhelmi, M.; Oberleithner, H.

    1999-01-01

    Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ± 0.34 S/cm2). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 × 106 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically “open” state with a mean single NPC electrical conductance of 1.7 ± 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism. PMID:10557355

  4. Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs.

    PubMed

    Jelaso, Anna M; DeLong, Cari; Means, Jay; Ide, Charles F

    2005-05-01

    Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that contribute to worldwide health problems. Despite data associating PCBs with adverse health effects, decisions to clean up contaminated sites remain controversial. Cleanup decisions are typically based on risk assessment methods that are not sensitive enough to detect subtle changes in health. We have recently shown that gene expression signatures can serve as sensitive molecular biomarkers of exposure and related health effects. Our initial studies were carried out with developing Xenopus laevis tadpoles that were exposed to the PCB mixture Aroclor 1254 (A1254) for 2 days. A1254 was dissolved in dimethyl sulfoxide and added to the aquarium water for rapid loading of PCBs into the tadpole tissue. These studies showed that increases in the expression of specific genes occurred independent of adverse health effects, and decreases in specific genes correlated with the appearance of observable health effects, including decreased survival and gross morphological and behavioral abnormalities. In this report, we extend our previous work to test the use of gene expression signatures as biomarkers in frogs exposed to PCBs through the diet from early tadpole stages through metamorphosis. This work showed that chronic low-dose exposure to A1254 (24 ppm) in food produced tissue levels of 17 ppm and increased gene expression of nerve growth factor and proopiomelanocortin independent of adverse health effects. Exposure to higher doses of A1254 (200 ppm) produced tissue levels of 80 ppm and increased expression of p450 1A1, also, independent of adverse health effects. This work provides further evidence for the use of gene expression changes as biomarkers of exposure to PCBs. PMID:15721885

  5. CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

    PubMed

    Palma, Alejandra G; Galizia, Luciano; Kotsias, Basilio A; Marino, Gabriela I

    2016-05-01

    Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases. PMID:26888038

  6. Prolonged vestibular stimulation induces homeostatic plasticity of the vestibulo-ocular reflex in larval Xenopus laevis.

    PubMed

    Dietrich, Haike; Straka, Hans

    2016-07-01

    Vestibulo-ocular reflexes (VOR) stabilise retinal images during head/body motion in vertebrates by generating spatio-temporally precise extraocular motor commands for corrective eye movements. While VOR performance is generally robust with a relatively stable gain, cerebellar circuits are capable of adapting the underlying sensory-motor transformation. Here, we studied cerebellum-dependent VOR plasticity by recording head motion-induced lateral rectus and superior oblique extraocular motor discharge in semi-intact preparations of Xenopus laevis tadpoles. In the absence of visual feedback, prolonged sinusoidal rotation caused either an increase or decrease of the VOR gain depending on the motion stimulus amplitude. The observed changes in extraocular motor discharge gradually saturated after 20 min of constant rotation and returned to baseline in the absence of motion stimulation. Furthermore, plastic changes in lateral rectus and superior oblique motor commands were plane-specific for horizontal and vertical rotations, respectively, suggesting that alterations are restricted to principal VOR connections. Comparison of multi- and single-unit activity indicated that plasticity occurs in all recorded units of a given extraocular motor nucleus. Ablation of the cerebellum abolished motoneuronal gain changes and prevented the induction of plasticity, thus demonstrating that both acquisition and retention of this type of plasticity require an intact cerebellar circuitry. In conclusion, the plane-specific and stimulus intensity-dependent modification of the VOR gain through the feed-forward cerebellar circuitry represents a homeostatic plasticity that likely maintains an optimal working range for the underlying sensory-motor transformation. PMID:27152983

  7. Long-range gap junctional signaling controls oncogene-mediated tumorigenesis in Xenopus laevis embryos

    PubMed Central

    Chernet, Brook T.; Fields, Chris; Levin, Michael

    2015-01-01

    In addition to the immediate microenvironment, long-range signaling may be an important component of cancer. Molecular-genetic analyses have implicated gap junctions—key mediators of cell-cell communication—in carcinogenesis. We recently showed that the resting voltage potential of distant cell groups is a key determinant of metastatic transformation and tumor induction. Here, we show in the Xenopus laevis model that gap junctional communication (GJC) is a modulator of the long-range bioelectric signaling that regulates tumor formation. Genetic disruption of GJC taking place within tumors, within remote host tissues, or between the host and tumors significantly lowers the incidence of tumors induced by KRAS mutations. The most pronounced suppression of tumor incidence was observed upon GJC disruption taking place farther away from oncogene-expressing cells, revealing a role for GJC in distant cells in the control of tumor growth. In contrast, enhanced GJC communication through the overexpression of wild-type connexin Cx26 increased tumor incidence. Our data confirm a role for GJC in tumorigenesis, and reveal that this effect is non-local. Based on these results and on published data on movement of ions through GJs, we present a quantitative model linking the GJC coupling and bioelectrical state of cells to the ability of oncogenes to initiate tumorigenesis. When integrated with data on endogenous bioelectric signaling during left-right patterning, the model predicts differential tumor incidence outcomes depending on the spatial configurations of gap junction paths relative to tumor location and major anatomical body axes. Testing these predictions, we found that the strongest influence of GJ modulation on tumor suppression by hyperpolarization occurred along the embryonic left-right axis. Together, these data reveal new, long-range aspects of cancer control by the host's physiological parameters. PMID:25646081

  8. Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

    PubMed Central

    Bellier, S; Dubois, M F; Nishida, E; Almouzni, G; Bensaude, O

    1997-01-01

    Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells. PMID:9032270

  9. Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

    PubMed

    Music, Ena; Khan, Saad; Khamis, Imran; Heikkila, John J

    2014-11-01

    The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats. PMID:25064141

  10. Bacteriophage φC31 Integrase Mediated Transgenesis in Xenopus laevis for Protein Expression at Endogenous Levels

    NASA Astrophysics Data System (ADS)

    Allen, Bryan G.; Weeks, Daniel L.

    Bacteriophage φC31 inserts its genome into that of its host bacterium via the integrase enzyme which catalyzes recombination between a phage attachment site (attP) and a bacterial attachment site (attB). Integrase requires no accessory factors, has a high efficiency of recombination, and does not need perfect sequence fidelity for recognition and recombination between these attachment sites. These imperfect attachment sites, or pseudo-attachment sites, are present in many organisms and have been used to insert transgenes in a variety of species. Here we describe the φC31 integrase approach to make transgenic Xenopus laevis embryos.

  11. Expression of Exogenous mRNA in Xenopus laevis Embryos for the Study of Cell Cycle Regulation

    NASA Astrophysics Data System (ADS)

    Sible, Jill C.; Wroble, Brian N.

    The microinjection of mRNA that is transcribed and capped in vitro into fertilized eggs and embryos of Xenopus laevis provides a powerful means for discovering the function of proteins during early development. Proteins may be overexpressed for a gain-of-function effect or exogenous protein function may be compromised by the microinjection of mRNA encoding “dominant-negative” proteins. This methodology is particularly suited for the investigation of the regulation of the cell cycle, checkpoints, and apoptosis in early development.

  12. Intracellular microRNA profiles form in the Xenopus laevis oocyte that may contribute to asymmetric cell division.

    PubMed

    Sidova, Monika; Sindelka, Radek; Castoldi, Mirco; Benes, Vladimir; Kubista, Mikael

    2015-01-01

    Asymmetric distribution of fate determinants within cells is an essential biological strategy to prepare them for asymmetric division. In this work we measure the intracellular distribution of 12 maternal microRNAs (miRNA) along the animal-vegetal axis of the Xenopus laevis oocyte using qPCR tomography. We find the miRNAs have distinct intracellular profiles that resemble two out of the three profiles we previously observed for mRNAs. Our results suggest that miRNAs in addition to proteins and mRNAs may have asymmetric distribution within the oocyte and may contribute to asymmetric cell division as cell fate determinants. PMID:26059897

  13. Intracellular microRNA profiles form in the Xenopus laevis oocyte that may contribute to asymmetric cell division

    PubMed Central

    Sidova, Monika; Sindelka, Radek; Castoldi, Mirco; Benes, Vladimir; Kubista, Mikael

    2015-01-01

    Asymmetric distribution of fate determinants within cells is an essential biological strategy to prepare them for asymmetric division. In this work we measure the intracellular distribution of 12 maternal microRNAs (miRNA) along the animal-vegetal axis of the Xenopus laevis oocyte using qPCR tomography. We find the miRNAs have distinct intracellular profiles that resemble two out of the three profiles we previously observed for mRNAs. Our results suggest that miRNAs in addition to proteins and mRNAs may have asymmetric distribution within the oocyte and may contribute to asymmetric cell division as cell fate determinants. PMID:26059897

  14. Early changes in the ultrastructure of the pars intermedia of the pituitary of Xenopus laevis after change of background color.

    PubMed

    Volcanes, B D; Weatherhead, B

    1976-01-01

    Stereological analysis of the secretory cells of the pars intermedia of Xenopus laevis over a period of 3 days following the transfer of animals from a white to a black background has revealed that significant alterations in the ultrastructural appearance of these cells can be detected 8 h after the transfer. In particular, changes in the secretory granules and the rough endoplasmic reticulum were found to correlate well with previous reports concerning the melanocyte-stimulating hormone (MSH) content and the capacity for protein synthesis of the pars intermedia. PMID:1028946

  15. Exposure to 3,3',5-triiodothyronine affects histone and RNA polymerase II modifications, but not DNA methylation status, in the regulatory region of the Xenopus laevis thyroid hormone receptor βΑ gene.

    PubMed

    Kasai, Kentaro; Nishiyama, Norihito; Izumi, Yushi; Otsuka, Shunsuke; Ishihara, Akinori; Yamauchi, Kiyoshi

    2015-11-01

    Thyroid hormones (THs) play a critical role in amphibian metamorphosis, during which the TH receptor (TR) gene, thrb, is upregulated in a tissue-specific manner. The Xenopus laevis thrb gene has 3 TH response elements (TREs) in the 5' flanking regulatory region and 1 TRE in the exon b region, around which CpG sites are highly distributed. To clarify whether exposure to 3,3',5-triiodothyronine (T3) affects histone and RNA polymerase II (RNAPII) modifications and the level of DNA methylation in the 5' regulatory region, we conducted reverse transcription-quantitative polymerase chain reaction, bisulfite sequencing and chromatin immunoprecipitation assay using X. laevis cultured cells and premetamorphic tadpoles treated with or without 2 nM T3. Exposure to T3 increased the amount of the thrb transcript, in parallel with enhanced histone H4 acetylation and RNAPII recruitment, and probably phosphorylation of RNAPII at serine 5, in the 5' regulatory and exon b regions. However, the 5' regulatory region remained hypermethylated even with exposure to T3, and there was no significant difference in the methylation status between DNAs from T3-untreated and -treated cultured cells or tadpole tissues. Our results demonstrate that exposure to T3 induced euchromatin-associated epigenetic marks by enhancing histone acetylation and RNAPII recruitment, but not by decreasing the level of DNA methylation, in the 5' regulatory region of the X. laevis thrb gene. PMID:26417689

  16. Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

    SciTech Connect

    Aranguren, Xabier L.; Beerens, Manu; Vandevelde, Wouter; Dewerchin, Mieke; Carmeliet, Peter; Luttun, Aernout

    2011-06-24

    Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.

  17. Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector.

    PubMed

    Sugiyama, Shin-Ichiro; Miyoshi, Hiroyuki; Yamauchi, Kiyoshi

    2005-12-01

    We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals. PMID:16102758

  18. De novo Transcriptome Assemblies of Rana (Lithobates) catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes

    PubMed Central

    Birol, Inanc; Behsaz, Bahar; Hammond, S. Austin; Kucuk, Erdi; Veldhoen, Nik; Helbing, Caren C.

    2015-01-01

    In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates) catesbeiana) and the African clawed frog (Xenopus laevis). We used high throughput RNA sequencing (RNA-seq) data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences. PMID:26121473

  19. Nucleotide sequence of the L1 ribosomal protein gene of Xenopus laevis: remarkable sequence homology among introns.

    PubMed Central

    Loreni, F; Ruberti, I; Bozzoni, I; Pierandrei-Amaldi, P; Amaldi, F

    1985-01-01

    Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5' end of this gene has been mapped within a 20-pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60-nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post-transcriptional level and it involves the block of the normal splicing of some intron sequences. Images Fig. 3. Fig. 5. PMID:3841512

  20. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

    PubMed Central

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-01-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  1. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    PubMed

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  2. A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

    PubMed

    Edens, Lisa J; Levy, Daniel L

    2016-01-01

    A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies. PMID:27584618

  3. Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.

    PubMed

    Heyer, M; Müller-Berger, S; Romero, M F; Boron, W F; Frömter, E

    1999-08-01

    The rat kidney Na+-HCO3- cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3- as permeable ions. The current carried by rkNBC (INBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4, 4'-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3- to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 micromol/l) blocks INBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3- concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3- concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP

  4. N-Glycans in Xenopus laevis testis characterised by lectin histochemistry.

    PubMed

    Valbuena, Galder; Madrid, Juan Francisco; Martínez de Ubago, María; Gómez-Santos, Laura; Alonso, Edurne; Díaz-Flores, Lucio; Sáez, Francisco J

    2016-03-01

    Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The β-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these

  5. The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos

    PubMed Central

    Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J. B.

    2015-01-01

    Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders. PMID:26580070

  6. Metamorphosis-induced changes in the coupling of spinal thoraco-lumbar motor outputs during swimming in Xenopus laevis.

    PubMed

    Beyeler, Anna; Métais, Charles; Combes, Denis; Simmers, John; Le Ray, Didier

    2008-09-01

    Anuran metamorphosis includes a complete remodeling of the animal's biomechanical apparatus, requiring a corresponding functional reorganization of underlying central neural circuitry. This involves changes that must occur in the coordination between the motor outputs of different spinal segments to harmonize locomotor and postural functions as the limbs grow and the tail regresses. In premetamorphic Xenopus laevis tadpoles, axial motor output drives rostrocaudally propagating segmental myotomal contractions that generate propulsive body undulations. During metamorphosis, the anterior axial musculature of the tadpole progressively evolves into dorsal muscles in the postmetamorphic froglet in which some of these back muscles lose their implicit locomotor function to serve exclusively in postural control in the adult. To understand how locomotor and postural systems interact during locomotion in juvenile Xenopus, we have investigated the coordination between postural back and hindlimb muscle activity during free forward swimming. Axial/dorsal muscles, which contract in bilateral alternation during undulatory swimming in premetamorphic tadpoles, change their left-right coordination to become activated in phase with bilaterally synchronous hindlimb extensions in locomoting juveniles. Based on in vitro electrophysiological experiments as well as specific spinal lesions in vivo, a spinal cord region was delimited in which propriospinal interactions are directly responsible for the coordination between leg and back muscle contractions. Our findings therefore indicate that dynamic postural adjustments during adult Xenopus locomotion are mediated by local intraspinal pathways through which the lumbar generator for hindlimb propulsive kicking provides caudorostral commands to thoracic spinal circuitry controlling the dorsal trunk musculature. PMID:18596184

  7. Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis

    PubMed Central

    Mills, Elizabeth A.; Davis, Chung-ha O.; Bushong, Eric A.; Boassa, Daniela; Kim, Keun-Young; Ellisman, Mark H.; Marsh-Armstrong, Nicholas

    2015-01-01

    Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event. PMID:26240339

  8. Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis.

    PubMed

    Mills, Elizabeth A; Davis, Chung-ha O; Bushong, Eric A; Boassa, Daniela; Kim, Keun-Young; Ellisman, Mark H; Marsh-Armstrong, Nicholas

    2015-08-18

    Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event. PMID:26240339

  9. Electrophysiological Characterization of Na,K-ATPases Expressed in Xenopus laevis Oocytes Using Two-Electrode Voltage Clamping.

    PubMed

    Hilbers, Florian; Poulsen, Hanne

    2016-01-01

    The transport of three Na(+) per two K(+) means that the Na,K-ATPase is electrogenic, and though the currents generated by the ion pump are small compared to ion channel currents, they can be measured using electrophysiology, both steady-state pumping and individual steps in the transport cycle. Various electrophysiological techniques have been used to study the endogenous pumps of the squid giant axon and of cardiac myocytes from for example rabbits. Here, we describe the characterization of heterologously expressed Na,K-ATPases using two-electrode voltage clamping (TEVC) and oocytes from the Xenopus laevis frog as the model cell. With this system, the effects of particular mutations can be studied, including the numerous mutations that in later years have been found to cause human diseases. PMID:26695042

  10. Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors.

    PubMed Central

    Otsuka, A; de Paolis, A; Tocchini-Valentini, G P

    1981-01-01

    A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes. The RNase preparation is essentially free of contaminating RNase. A quantitative assay for RNase XlaI was developed, and the reaction products were characterized. RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini). Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences. Images PMID:6765601