A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis.

PubMed

Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-05-11

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc-Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad-Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development.

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis

PubMed Central

Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-01-01

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc–Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad–Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development. PMID:28492553

Building the Future: Post-transcriptional Regulation of Cell Fate Decisions Prior to the Xenopus Midblastula Transition.

PubMed

Sheets, Michael D

2015-01-01

In all animals, a critical period in early development is when embryonic cells switch from relying solely upon maternally deposited RNAs and proteins to relying upon molecules encoded by the zygotic genome. Xenopus embryos have served as a model for examining this switch, as well as the maternally controlled stages that prepare for it. In Xenopus, the robust activation of zygotic transcription occurs at the 12th cleavage division and is referred to as the midblastula transition (MBT). Prior to MBT, gene expression is regulated by post-transcriptional events including mRNA and protein localization, protein post-translational modification, and mRNA translation. After the MBT, appropriate transcriptional regulation of the zygotic genome becomes critical and predominates. However, it is important to realize that the first key cell fate decisions that have profound impacts on development occur prior to the MBT and these are governed by regulating the expression of maternally deposited regulatory mRNAs and proteins. In this chapter, I will discuss post-transcriptional mechanisms that function during the maternal stages of Xenopus development with an emphasis on mechanisms known to directly modulate cell fate decisions. Emerging approaches and technologies that will help better understand this phase of development will also be discussed. © 2015 Elsevier Inc. All rights reserved.

Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification.

PubMed

Oh, Denise; Houston, Douglas W

2017-12-15

The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development. Copyright © 2017 Elsevier Inc. All rights reserved.

CELL SEGREGATION, MIXING, AND TISSUE PATTERN IN THE SPINAL CORD OF THE XENOPUS LAEVIS NEURULA

PubMed Central

Davidson, Lance A.; Keller, Raymond E.

2014-01-01

Background During Xenopus laevis neurulation, neural ectodermal cells of the spinal cord are patterned at the same time that they intercalate mediolaterally and radially, moving within and between two cell layers. Curious if these rearrangements disrupt early cell identities, we lineage-traced cells in each layer from neural plate stages to the closed neural tube, and used in situ hybridization to assay gene expression in the moving cells. Results Our biotin- and fluorescent labeling of deep and superficial cells reveals that mediolateral intercalation does not disrupt cell cohorts, in other words it is conservative. However, outside the midline notoplate, later radial intercalation does displace superficial cells dorsoventrally, radically disrupting cell cohorts. The tube roof is composed almost exclusively of superficial cells, including some displaced from ventral positions; gene expression in these displaced cells must now be surveyed further. Superficial cells also flank the tube’s floor, which is, itself, almost exclusively composed of deep cells. Conclusions Our data provide: 1) a fate map of superficial- and deep-cell positions within the Xenopus neural tube, 2) the paths taken to these positions, and 3) preliminary evidence of re-patterning in cells carried out of one environment and into another, during neural morphogenesis. PMID:23813905

Xenopus egg extract: A powerful tool to study genome maintenance mechanisms.

PubMed

Hoogenboom, Wouter S; Klein Douwel, Daisy; Knipscheer, Puck

2017-08-15

DNA repair pathways are crucial to maintain the integrity of our genome and prevent genetic diseases such as cancer. There are many different types of DNA damage and specific DNA repair mechanisms have evolved to deal with these lesions. In addition to these repair pathways there is an extensive signaling network that regulates processes important for repair, such as cell cycle control and transcription. Despite extensive research, DNA damage repair and signaling are not fully understood. In vitro systems such as the Xenopus egg extract system, have played, and still play, an important role in deciphering the molecular details of these processes. Xenopus laevis egg extracts contain all factors required to efficiently perform DNA repair outside a cell, using mechanisms conserved in humans. These extracts have been used to study several genome maintenance pathways, including mismatch repair, non-homologous end joining, ICL repair, DNA damage checkpoint activation, and replication fork stability. Here we describe how the Xenopus egg extract system, in combination with specifically designed DNA templates, contributed to our detailed understanding of these pathways. Copyright © 2017. Published by Elsevier Inc.

Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig.

PubMed

Liu, Ying; Ostrup, Olga; Li, Rong; Li, Juan; Vajta, Gábor; Kragh, Peter M; Schmidt, Mette; Purup, Stig; Hyttel, Poul; Klærke, Dan; Callesen, Henrik

2014-08-01

In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

Rotation in Xenopus laevis embryos during the second cell cycle.

PubMed

Starodubov, Sergey M; Golychenkov, Vladimir A

2009-01-01

Using time-lapse video recording and comparing successive digital images, we found that 38% of Xenopus laevis embryos (n=118) exhibited rotation during the second cell cycle. This rotation, which we term the second rotation, started approximately during the appearance of the first cleavage furrow and proceeded clockwise or counterclockwise around the vertical axis. Rotations lasted for 5-30 minutes, i.e. up to the beginning of the third cell cycle. The mean rotation angle was 36.4 degrees, with a maximum rotation of 77 degrees. No mortality was observed among the embryos exhibiting rotation. The second rotation was observed to be similar to the well-known fertilization rotation which takes place during the first cell cycle. The possible nature and significance of the second rotation are discussed.

Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

PubMed

Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

2016-05-01

Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

Dynamic Properties of Electrotonic Coupling between Cells of Early Xenopus Embryos

PubMed Central

DiCaprio, R. A.; French, A. S.; Sanders, E. J.

1974-01-01

Frequency response functions were measured between the cells of Xenopus laevis embryos during the first two cleavage stages. Linear systems theory was then used to produce electronic models which account for the electrical behavior of the systems. Coupling between the cells may be explained by models which have simple resistive elements joining each cell to its neighbors. The vitelline, or fertilization, membrane which surrounds the embryos has no detectable resistance to the passage of electric current. The electrical properties of the four-cell embryo can only be explained by the existence of individual junctions linking each pair of cells. This arrangement suggests that electrotonic coupling is important in the development of the embryos, at least until the four-cell stage. ImagesFIGURE 5FIGURE 14FIGURE 15 PMID:19431351

The unexpected teratogenicity of RXR antagonist UVI3003 via activation of PPARγ in Xenopus tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Jingmin

2017-01-01

The RXR agonist (triphenyltin, TPT) and the RXR antagonist (UVI3003) both show teratogenicity and, unexpectedly, induce similar malformations in Xenopus tropicalis embryos. In the present study, we exposed X. tropicalis embryos to UVI3003 in seven specific developmental windows and identified changes in gene expression. We further measured the ability of UVI3003 to activate Xenopus RXRα (xRXRα) and PPARγ (xPPARγ) in vitro and in vivo. We found that UVI3003 activated xPPARγ either in Cos7 cells (in vitro) or Xenopus embryos (in vivo). UVI3003 did not significantly activate human or mouse PPARγ in vitro; therefore, the activation of Xenopus PPARγ by UVI3003more » is novel. The ability of UVI3003 to activate xPPARγ explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos. Our results indicate that activating PPARγ leads to teratogenic effects in Xenopus embryos. More generally, we infer that chemicals known to specifically modulate mammalian nuclear hormone receptors cannot be assumed to have the same activity in non-mammalian species, such as Xenopus. Rather they must be tested for activity and specificity on receptors of the species in question to avoid making inappropriate conclusions. - Highlights: • UVI3003 is a RXRs antagonist and shows teratogenicity to Xenopus embryos. • UVI3003 activated xPPARγ either in Cos7 cells or Xenopus embryos. • UVI3003 did not activate human or mouse PPARγ in Cos7 cells. • Activating PPARγ leads to teratogenic effects in Xenopus embryos.« less

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Inference of genetic network of Xenopus frog egg: improved genetic algorithm.

PubMed

Wu, Shinq-Jen; Chou, Chia-Hsien; Wu, Cheng-Tao; Lee, Tsu-Tian

2006-01-01

An improved genetic algorithm (IGA) is proposed to achieve S-system gene network modeling of Xenopus frog egg. Via the time-courses training datasets from Michaelis-Menten model, the optimal parameters are learned. The S-system can clearly describe activative and inhibitory interaction between genes as generating and consuming process. We concern the mitotic control in cell-cycle of Xenopus frog egg to realize cyclin-Cdc2 and Cdc25 for MPF activity. The proposed IGA can achieve global search with migration and keep the best chromosome with elitism operation. The generated gene regulatory networks can provide biological researchers for further experiments in Xenopus frog egg cell cycle control.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

PubMed

Nakayama, Takuya; Nakajima, Keisuke; Cox, Amanda; Fisher, Marilyn; Howell, Mary; Fish, Margaret B; Yaoita, Yoshio; Grainger, Robert M

2017-06-15

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of

Transforming Growth Factor Beta (TGFβ) Is Produced by and Influences the Proliferative Response of Xenopus laevis Lymphocytes

PubMed Central

Haynes, Laura

1993-01-01

Both TGF/β2 and 5 have been described in the South African clawed frog Xenopus laevis and have been cloned from the tadpole-derived fibroblast cell line, XTC. Because TGFβ has such a profound inhibitory effect on the mammalian immune system, this study was performed to determine whether TGFβ: (a) has any in vitro effects on the growth of Xenopus lymphoblasts, and (b) is produced by mitogen-activated Xenopus lymphocytes. Following stimulation with mitogen or alloantigen, T lymphocytes from Xenopus secrete a T-cell growth factor (TCGF) that is functionally homologous to mammalian interleukin-2 (IL-2). Both recombinant human TGFβ1 and Xenopus TGFβ5 inhibit TCGF-induced proliferation of Xenopus splenic blasts and this inhibition can be reversed with anti-pan TGFβ antiserum. The Xenopus mitogen-induced saturated ammonium sulfate precipitated TCGF-containing supernatant (SAS TCGF SN) also contains latent TGFβ as assayed on mink lung fibroblasts and Xenopus splenic blasts, and experiments utilizing anti-TGFβ antiserum showed that only TGFβ5 is present in this supernatant. PMID:8281035

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

PubMed

Suzuki, Ken-Ichi T; Suzuki, Miyuki; Shigeta, Mitsuki; Fortriede, Joshua D; Takahashi, Shuji; Mawaribuchi, Shuuji; Yamamoto, Takashi; Taira, Masanori; Fukui, Akimasa

2017-06-15

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation. Copyright © 2016 Elsevier Inc. All rights reserved.

Seeing the future: using Xenopus to understand eye regeneration.

PubMed

Tseng, Ai-Sun

2017-01-01

Studies of Xenopus eye development have contributed considerably to the understanding of vertebrate neurogenesis, including eye field specification, cell fate determination and identification of genes critical for eye formation. This knowledge has served as a solid foundation for cellular and molecular examinations of the robust regenerative capacity of the Xenopus eye. The retina, lens, and the optic nerve are capable of regeneration after injury in both larval and adult stages. Here, we discuss the current models for studying eye regeneration in Xenopus and their potential applications for providing insights into human eye diseases. As Xenopus has many of the same tools that are available for other regeneration models, we thus highlight the distinct strengths and versatility of this organism that make it especially suited for extrapolating and testing strategies aimed at promoting regeneration and repair in eye tissues. Furthermore, we outline a promising future for the use of new techniques and approaches to address outstanding questions in understanding eye regeneration. © 2017 Wiley Periodicals, Inc.

Host-defense peptides from skin secretions of the octoploid frogs Xenopus vestitus and Xenopus wittei (Pipidae): insights into evolutionary relationships.

PubMed

Mechkarska, Milena; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert; Michalak, Katarzyna; Michalak, Pawel; Conlon, J Michael

2014-09-01

The primary structures of host-defense peptides have proved useful in elucidating the evolution history of frogs. Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from the octoploid frogs, Xenopus vestitus (Kivu clawed frog) and Xenopus wittei (De Witte's clawed frog) in the family Pipidae. Structural characterization demonstrated that the X. vestitus peptides belong to the magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 4 peptides), xenopsin-precursor fragment (XPF; 1 peptide), and caerulein-precursor fragment (CPF; 5 peptides) families. The X. wittei peptides comprise magainin (4 peptides), PGLa (1 peptide), XPF (2 peptides), and CPF (7 peptides). In addition, secretions from both species contain caerulein, identical to the peptide from Xenopus laevis, but X. wittei secretions contains the novel peptide [R4K]xenopsin. The variability in the numbers of paralogs in each peptide family indicates a selective silencing of the host-defense peptide genes following the polyploidization events. The primary structures of the peptides provide insight into phylogenetic relationships among the octoploid Xenopus frogs. The data support a sister-group relationship between X. vestitus and Xenopus lenduensis, suggestive of bifurcating speciation after allopolyploidization, whereas X. wittei is more closely related to the Xenopus amieti-Xenopus andrei group suggesting a common tetraploid ancestor. Consistent with previous data, the CPF peptides showed the highest growth inhibitory activity against bacteria with CPF-W6 (GIGSLLAKAAKLAAGLV.NH2) combining high antimicrobial potency against Staphylococcus aureus (MIC=4 μM) with relatively low hemolytic activity (LC50=190 μM). Copyright © 2014 Elsevier Inc. All rights reserved.

Live imaging of targeted cell ablation in Xenopus: a new model to study demyelination and repair

PubMed Central

Kaya, F.; Mannioui, A.; Chesneau, A.; Sekizar, S.; Maillard, E.; Ballagny, C.; Houel-Renault, L.; Du Pasquier, D.; Bronchain, O.; Holtzmann, I.; Desmazieres, A.; Thomas, J.-L.; Demeneix, B. A.; Brophy, P. J.; Zalc, B.; Mazabraud, A.

2012-01-01

Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the central nervous system (CNS). In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair. PMID:22973012

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

PubMed Central

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

2016-01-01

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

What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia.

PubMed

Walentek, Peter; Quigley, Ian K

2017-01-01

Over the past years, the Xenopus embryo has emerged as an incredibly useful model organism for studying the formation and function of cilia and ciliated epithelia in vivo. This has led to a variety of findings elucidating the molecular mechanisms of ciliated cell specification, basal body biogenesis, cilia assembly, and ciliary motility. These findings also revealed the deep functional conservation of signaling, transcriptional, post-transcriptional, and protein networks employed in the formation and function of vertebrate ciliated cells. Therefore, Xenopus research can contribute crucial insights not only into developmental and cell biology, but also into the molecular mechanisms underlying cilia related diseases (ciliopathies) as well as diseases affecting the ciliated epithelium of the respiratory tract in humans (e.g., chronic lung diseases). Additionally, systems biology approaches including transcriptomics, genomics, and proteomics have been rapidly adapted for use in Xenopus, and broaden the applications for current and future translational biomedical research. This review aims to present the advantages of using Xenopus for cilia research, highlight some of the evolutionarily conserved key concepts and mechanisms of ciliated cell biology that were elucidated using the Xenopus model, and describe the potential for Xenopus research to address unresolved questions regarding the molecular mechanisms of ciliopathies and airway diseases. © 2017 Wiley Periodicals, Inc.

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

PubMed

Green, Yangsook Song; Vetter, Monica L

2011-10-01

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Adult-type myogenesis of the frog Xenopus laevis specifically suppressed by notochord cells but promoted by spinal cord cells in vitro.

PubMed

Yamane, Hitomi; Ihara, Setsunosuke; Kuroda, Masaaki; Nishikawa, Akio

2011-08-01

Larval-to-adult myogenic conversion occurs in the dorsal muscle but not in the tail muscle during Xenopus laevis metamorphosis. To know the mechanism for tail-specific suppression of adult myogenesis, response character was compared between adult myogenic cells (Ad-cells) and larval tail myogenic cells (La-cells) to a Sonic hedgehog (Shh) inhibitor, notochord (Nc) cells, and spinal cord (SC) cells in vitro. Cyclopamine, an Shh inhibitor, suppressed the differentiation of cultured Ad (but not La) cells, suggesting the significance of Shh signaling in promoting adult myogenesis. To test the possibility that Shh-producing axial elements (notochord and spinal cord) regulate adult myogenesis, Ad-cells or La-cells were co-cultured with Nc or SC cells. The results showed that differentiation of Ad-cells were strongly inhibited by Nc cells but promoted by SC cells. If Ad-cells were "separately" co-cultured with Nc cells without direct cell-cell interactions, adult differentiation was not inhibited but rather promoted, suggesting that Nc cells have two roles, one is a short-range suppression and another is a long-range promotion for adult myogenesis. Immunohistochemical analysis showed both notochord and spinal cord express the N-terminal Shh fragment throughout metamorphosis. The "spinal cord-promotion" and long-range effect by Nc cells on adult myogenesis is thus involved in Shh signaling, while the signaling concerning the short-range "Nc suppression" will be determined by future studies. Interestingly, these effects, "Nc suppression" and "SC promotion" were not observed for La-cells. Situation where the spinal cord/notochord cross-sectional ratio is quite larger in tadpole trunk than in the tail seems to contribute to trunk-specific promotion and tail-specific suppression of adult myogenesis during Xenopus metamorphosis.

Dynamic expression of the LAP family of genes during early development of Xenopus tropicalis.

PubMed

Yang, Qiutan; Lv, Xiaoyan; Kong, Qinghua; Li, Chaocui; Zhou, Qin; Mao, Bingyu

2011-10-01

The leucine-rich repeats and PDZ (LAP) family of genes are crucial for the maintenance of cell polarity as well as for epithelial homeostasis and tumor suppression in both vertebrates and invertebrates. Four members of this gene family are known: densin, erbin, scribble and lano. Here, we identified the four members of the LAP gene family in Xenopus tropicalis and studied their expression patterns during embryonic development. The Xenopus LAP proteins show a conserved domain structure that is similar to their homologs in other vertebrates. In Xenopus embryos, these genes were detected in animal cap cells at the early gastrula stage. At later stages of development, they were widely expressed in epithelial tissues that are highly polar in nature, including the neural epithelia, optic and otic vesicles, and in the pronephros. These data suggest that the roles of the Xenopus LAP genes in the control of cell polarity and morphogenesis are conserved during early development. Erbin and lano show similar expression patterns in the developing head, suggesting potential functional interactions between the two molecules in vivo.

XMAP310: A Xenopus Rescue-promoting Factor Localized to the Mitotic Spindle

PubMed Central

Andersen, Søren S.L.; Karsenti, Eric

1997-01-01

To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289–1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5–10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985–993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation. PMID:9362515

Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

PubMed

Seigfried, Franziska A; Dietmann, Petra; Kühl, Michael; Kühl, Susanne J

2018-06-01

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development. Copyright © 2018 Elsevier B.V. All rights reserved.

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells*

PubMed Central

Jevtić, Predrag; Edens, Lisa J.; Li, Xiaoyang; Nguyen, Thang; Chen, Pan; Levy, Daniel L.

2015-01-01

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change. PMID:26429910

Participation of Xenopus Elr-type Proteins in Vegetal mRNA Localization during Oogenesis*

PubMed Central

Arthur, Patrick K.; Claussen, Maike; Koch, Susanne; Tarbashevich, Katsiaryna; Jahn, Olaf; Pieler, Tomas

2009-01-01

Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes. PMID:19458392

Activation of muscle-specific actin genes in Xenopus development by an induction between animal and vegetal cells of a blastula.

PubMed

Gurdon, J B; Fairman, S; Mohun, T J; Brennan, S

1985-07-01

Muscle gene expression is induced a few hours after vegetal cells of a Xenopus blastula are placed in contact with animal cells that normally develop into epidermis and nerve cells. We have used a muscle-specific actin gene probe to determine the timing of gene activation in animal-vegetal conjugates. Muscle actin RNA is first transcribed in a minority of animal cells at a stage equivalent to late gastrula. The time of muscle gene activation is determined by the developmental stage of the responding (animal) cells, and not by the time when cells are first placed in contact. The minimal cell contact time required for induction is between 1 1/2 and 2 1/2 hr, and the minimal time for gene activation after induction is 5-7 hr.

Polystyrene nanoparticles affect Xenopus laevis development

NASA Astrophysics Data System (ADS)

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

2015-02-01

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

High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis.

PubMed

Weinmann-Dorsch, C; Grummt, F

1985-09-01

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

PubMed

Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

2014-07-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells

PubMed Central

Reifenberger, Matthew S.; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Eaton, Douglas C.; Alli, Abdel A.

2014-01-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na+ channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. PMID:24829507

Tumor immunology viewed from alternative animal models—the Xenopus story

PubMed Central

Banach, Maureen; Robert, Jacques

2017-01-01

a) Purpose of review Nonmammalian comparative animal models are important not only to gain fundamental evolutionary understanding of the complex interactions of tumors with the immune system, but also to better predict the applicability of novel immunotherapeutic approaches to humans. After reviewing recent advances in developing alternative models, we focus on the amphibian Xenopus laevis and its usefulness in deciphering the perplexing roles of MHC class I-like molecules and innate (i)T cells in tumor immunity. b) Recent findings Experiments using MHC-defined inbred and cloned animals, tumor cell lines, effective reagents, sequenced genomes, and adapted gene editing techniques in Xenopus, have revealed that the critical involvement of class I-like molecules and iT cells in tumor immunity has been conserved during evolution. c) Summary Comparative studies with the X. laevis tumor immunity model can contribute to the development of better and more efficient cancer immunotherapies. PMID:28944105

Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 mRNAmore » 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.« less

Chromosome Banding in Amphibia. XXXII. The Genus Xenopus (Anura, Pipidae).

PubMed

Schmid, Michael; Steinlein, Claus

2015-01-01

Mitotic chromosomes of 16 species of the frog genus Xenopus were prepared from kidney and lung cell cultures. In the chromosomes of 7 species, high-resolution replication banding patterns could be induced by treating the cultures with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession, and in 6 of these species the BrdU/dT-banded chromosomes could be arranged into karyotypes. In the 3 species of the clade with 2n = 20 and 4n = 40 chromosomes (X. tropicalis, X. epitropicalis, X. new tetraploid 1), as well as in the 3 species with 4n = 36 chromosomes (X. laevis, X. borealis, X. muelleri), the BrdU/dT-banded karyotypes show a high degree of homoeology, though differences were detected between these groups. Translocations, inversions, insertions or sex-specific replication bands were not observed. Minor replication asynchronies found between chromosomes probably involve heterochromatic regions. BrdU/dT replication banding of Xenopus chromosomes provides the landmarks necessary for the exact physical mapping of genes and repetitive sequences. FISH with an X. laevis 5S rDNA probe detected multiple hybridization sites at or near the long-arm telomeric regions in most chromosomes of X. laevis and X. borealis, whereas in X. muelleri, the 5S rDNA sequences are located exclusively at the long-arm telomeres of a single chromosome pair. Staining with the AT base pair-specific fluorochrome quinacrine mustard revealed brightly fluorescing heterochromatic regions in the majority of X. borealis chromosomes which are absent in other Xenopus species.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development

PubMed Central

Fox, Catherine A.; Dowdle, Megan E.; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

2017-01-01

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented

Expression of pluripotency factors in larval epithelia of the frog Xenopus: Evidence for the presence of cornea epithelial stem cells

PubMed Central

Perry, Kimberly J.; Thomas, Alvin G.; Henry, Jonathan J.

2013-01-01

Understanding the biology of somatic stem cells in self renewing tissues represents an exciting field of study, especially given the potential to harness these cells for tissue regeneration and repair in treating injury and disease. The mammalian cornea contains a population of basal epithelial stem cells involved in cornea homeostasis and repair. Research has been restricted to mammalian systems and little is known about the presence or function of these stem cells in other vertebrates. Therefore, we carried out studies to characterize frog cornea epithelium. Careful examination shows that the Xenopus larval cornea epithelium consists of three distinct layers that include an outer epithelial layer and underlying basal epithelium, in addition to a deeper fibrous layer that contains the main sensory nerve trunks that give rise to numerous branches that extend into these epithelia. These nerves convey sensory and presumably also autonomic innervation to those tissues. The sensory nerves are all derived as branches of the trigeminal nerve/ganglion similar to the situation encountered in mammals, though there appear to be some potentially interesting differences, which are detailed in this paper. We show further that numerous pluripotency genes are expressed by cells in the cornea epithelium, including: sox2, p63, various oct4 homologs, c-myc, klf4 and many others. Antibody localization revealed that p63, a well known mammalian epithelial stem cell marker, was localized strictly to all cells in the basal cornea epithelium. c-myc, was visualized in a smaller subset of basal epithelial cells and adjacent stromal tissue predominately at the periphery of the cornea (limbal zone). Finally, sox2 protein was found to be present throughout all cells of both the outer and basal epithelia, but was much more intensely expressed in a distinct subset of cells that appeared to be either multinucleate or possessed multi-lobed nuclei that are normally located at the periphery of the

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

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

PubMed Central

Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian

2012-01-01

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication. PMID:22521908

Further Characterization of an Interleukin-2-1Ike Cytokine Produced by Xenopus Laevis T Lymphocytes

PubMed Central

Haynes, Laura

1993-01-01

A T-cell growth factor (TCGF) is produced by antigen- or mitogen-stimulated T lymphocytes from the South African clawed frog Xenopus laevis. This study further defines the physical and biological properties of this cytokine and demonstrates that TCGF is biochemically similar to mammalian interleukin-2 (IL-2). Biologically active TCGF eluted from SDS-PAGE displays a Mr of 16 kD and lectin-affinity chromatography indicates that the three-dimensionmal configuration of carbohydrates on TCGF and human IL-2 is similar. Secretion of TCGF is detectable 1 day after stimulation of splenocytes with the T-cell mitogen phytohemagglutinin (PHA) and peaks following 2 to 3 days of stimulation. Finally, despite the biological and physical similarities between Xenopus TCGF and mammalian IL-2, anti-human IL-2 monoclonal antibodies do not recognize Xenopus TCGF. PMID:8281036

Three new members of the RNP protein family in Xenopus.

PubMed Central

Good, P J; Rebbert, M L; Dawid, I B

1993-01-01

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system. Images PMID:8451200

Determination of notochord cells of Xenopus laevis.

PubMed

Zeng, M B

1993-12-01

In amphibians, numerous works of influences of the notochord on neighbouring tissues have been accumulated. However, on the contrary, scarcely any work is known about how the notochord is influenced by its neighbouring tissues and how it is determined. By using the experimental method of explantation and culturing in vitro, how the notochord is determined in the early development and whether the neighbouring tissues exert influences on it have been investigated. The results showed that the determination of notochord is a progressive process and the presumptive notochord of Xenopus appears to be a very good material to study influences of neighbouring tissues on the determination of the notochord.

Light responses in rods of vitamin A-deprived Xenopus.

PubMed

Solessio, Eduardo; Umino, Yumiko; Cameron, David A; Loew, Ellis; Engbretson, Gustav A; Knox, Barry E; Barlow, Robert B

2009-09-01

Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration. The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry. The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)). Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

The nervus terminalis in larval and adult Xenopus laevis.

PubMed

Hofmann, M H; Meyer, D L

1989-09-25

Nervus terminalis (nt) projections were studied by HRP injections into one nostril in adult Xenopus and in Xenopus tadpoles. Central nt targets are: medial septum, preoptic nucleus, nucleus of the anterior commissure, and hypothalamus (mainly ipsilaterally). In Xenopus tadpoles, additional fibers reach the ipsilateral dorsal thalamus and the mesencephalic tegmentum, bilaterally; furthermore, hypothalamic projections are bilateral. Xenopus tadpole nt connections resemble those of adult urodeles more closely than the projections of frogs. However, Xenopus tadpoles lack nt innervation of the medial septum.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 occurrencemore » of prolactin specific binding sites in Xenopus laevis choroid plexus cells.« less

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

PubMed

Silva, Ana Cristina; Filipe, Mário; Steinbeisser, Herbert; Belo, José António

2011-05-01

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior–posterior patterning and left–right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

Cadherin-11 Mediates Contact Inhibition of Locomotion during Xenopus Neural Crest Cell Migration

PubMed Central

Becker, Sarah F. S.; Mayor, Roberto; Kashef, Jubin

2013-01-01

Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC. PMID:24392028

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis.

PubMed

Grimaldi, Annalisa; Tettamanti, Gianluca; Martin, Benjamin L; Gaffield, William; Pownall, Mary E; Hughes, Simon M

2004-07-01

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of

The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.

PubMed Central

Minshull, J; Golsteyn, R; Hill, C S; Hunt, T

1990-01-01

Cyclins play a key role in the induction of mitosis. In this paper we report the isolation of a cyclin A cDNA clone from Xenopus eggs. Its cognate mRNA encodes a protein that shows characteristic accumulation and destruction during mitotic cell cycles. The cyclin A polypeptide is associated with a protein that cross-reacts with an antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the cyclin A-cdc2 complex exhibits protein kinase activity that oscillates with the cell cycle. This kinase activity rises more smoothly than that of the cyclin B-cdc2 complexes and reaches a peak earlier in the cell cycle; indeed, cyclin A is destroyed before nuclear envelope breakdown. None of the cyclin-cdc2 complexes show simple relationships between the concentration of the cyclin moiety and the kinase activity. All three cyclin associated kinases (A, B1 and B2) phosphorylate identical sites on histones with the consensus XSPXK/R, although they show significant differences in their substrate preferences. We discuss possible models for the different roles of the A- and B-type cyclins in the control of cell division. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2143983

Developing Xenopus Embryos Recover by Compacting and Expelling Single-Wall Carbon Nanotubes

PubMed Central

Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

2015-01-01

Single-wall carbon nanotubes are high aspect ratio nanomaterials that are being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties, and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single-wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 μm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one-to-two cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call “boluses”. Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. PMID:26153061

Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

PubMed

Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

2016-04-01

Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. Copyright © 2015 John Wiley & Sons, Ltd.

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis

PubMed Central

Okada, Morihiro; Miller, Thomas C.; Fu, Liezhen

2015-01-01

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis. PMID:26086244

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis.

PubMed

Okada, Morihiro; Miller, Thomas C; Fu, Liezhen; Shi, Yun-Bo

2015-09-01

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

PubMed Central

Bjerke, Maureen A.; Dzamba, Bette; Wang, Chong; DeSimone, Douglas W.

2014-01-01

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity. PMID:25127991

Amphibian (Xenopus sp.) iodothyronine deiodinase ...

EPA Pesticide Factsheets

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

MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3.

PubMed

Mimura, Satoru; Kubota, Yumiko; Takisawa, Haruhiko

2018-01-01

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

Xenopus Bicaudal-C Is Required for the Differentiation of the Amphibian Pronephros

PubMed Central

Tran, Uyen; Mary Pickney, L.; Duygu Özpolat, B.; Wessely, Oliver

2007-01-01

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which - when disrupted - results in PKD. PMID:17521625

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90 degrees before first cleavage

NASA Technical Reports Server (NTRS)

Huang, S.; Johnson, K. E.; Wang, H. Z.

1998-01-01

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90 degrees with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35-38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90 degrees rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90 degrees, but the fate of the blastomeres did not simply show a 90 degrees switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position: (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.

Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing.

PubMed

Popov, Ivan K; Kwon, Taejoon; Crossman, David K; Crowley, Michael R; Wallingford, John B; Chang, Chenbei

2017-06-15

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases

PubMed Central

Kashef, Jubin; Köhler, Almut; Kuriyama, Sei; Alfandari, Dominique; Mayor, Roberto; Wedlich, Doris

2009-01-01

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function. PMID:19528317

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.

PubMed

Kashef, Jubin; Köhler, Almut; Kuriyama, Sei; Alfandari, Dominique; Mayor, Roberto; Wedlich, Doris

2009-06-15

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.

FTIR Study of the Photoactivation Process of Xenopus (6-4) Photolyase†

PubMed Central

Yamada, Daichi; Zhang, Yu; Iwata, Tatsuya; Hitomi, Kenichi; Getzoff, Elizabeth D.; Kandori, Hideki

2012-01-01

Photolyases (PHRs) are blue-light activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The FAD chromophore of PHRs has four different redox states: oxidized (FADox), anion radical (FAD•−), neutral radical (FADH•) and fully reduced (FADH−). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV-visible spectroscopy to study the detailed photoactivation process of Xenopus (6-4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FADox is converted to semiquinone via light-induced one-electron and one-proton transfers, and then to FADH− by light-induced one-electron transfer. We successfully trapped FAD•− at 200 K, where electron transfer occurs, but proton transfer does not. UV-visible spectroscopy following 450-nm illumination of FADox at 277 K defined the FADH•/FADH− mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic C=O stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested from UV-visible and FTIR analysis of FAD•− at 200 K. Spectral analysis of amide-I vibrations revealed structural perturbation of the protein’s β-sheet during initial electron transfer (FAD•− formation), transient increase in α-helicity during proton transfer (FADH• formation) and reversion to the initial amide-I signal following subsequent electron transfer (FADH− formation). Consequently, in (6-4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH− did not perturb α-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of the present FTIR observations. PMID:22747528

Morphology of the caudal spinal cord in Rana (Ranidae) and Xenopus (Pipidae) tadpoles.

PubMed

Nishikawa, K; Wassersug, R

1988-03-08

Using a variety of neuroanatomical and histological techniques, we compare the spinal cord and peripheral nerve distribution in the tails of larvae from Xenopus laevis and three species of Rana. The relatively large, postsacral spinal cord of Xenopus contains abundant motoneurons and their axons. Spinal nerves exit from the spinal cord in a regular array, one nerve per myotome, from the cervical region to near the end of the tail. Somata of motoneurons innervating caudal myotomes are found along the entire length of the tail. In contrast, the caudal cord of Rana is reduced to a filum terminale consisting of little more than an ependymal tube; spinal nerves to all caudal myotomes leave the cord in the sacral region and reach their motor targets via a cauda equina and caudal plexus. Motoneuron cell bodies innervating caudal myotomes are found only in the sacral region. The Rana larval pattern is similar to that of adult frogs and mammals, whereas the Xenopus larval pattern is more like that of salamanders and reptiles. These gross neuroanatomical differences are not due to differences in the size or developmental stage of the tadpoles, but instead are associated with differences in the swimming behavior of the larvae. The presence of motoneurons in the caudal spinal cord of Xenopus may provide local intermyotomal control within the tail; the elongated topography of the cord appears to permit finer, rostral-to-caudal regulation of neuromuscular activity. The Rana spinal cord, on the other hand--with motoneurons clustered anteriorly--may produce concurrent firing of adjacent ipsilateral myotomes, but at the expense of fine intermyotomal regulation. The fact that nerves in the tail of Xenopus enter and exit from the spinal cord locally, as opposed to far anteriorly as in Rana, means that for tadpoles of the same size, reflex arc lengths are many times shorter in Xenopus.

Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats.

PubMed

Gaitán-Peñas, Héctor; Pusch, Michael; Estévez, Raúl

2018-03-02

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

Anosmin-1 is essential for neural crest and cranial placodes formation in Xenopus.

PubMed

Bae, Chang-Joon; Hong, Chang-Soo; Saint-Jeannet, Jean-Pierre

2018-01-15

During embryogenesis vertebrates develop a complex craniofacial skeleton associated with sensory organs. These structures are primarily derived from two embryonic cell populations the neural crest and cranial placodes, respectively. Neural crest cells and cranial placodes are specified through the integrated action of several families of signaling molecules, and the subsequent activation of a complex network of transcription factors. Here we describe the expression and function of Anosmin-1 (Anos1), an extracellular matrix protein, during neural crest and cranial placodes development in Xenopus laevis. Anos1 was identified as a target of Pax3 and Zic1, two transcription factors necessary and sufficient to generate neural crest and cranial placodes. Anos1 is expressed in cranial neural crest progenitors at early neurula stage and in cranial placode derivatives later in development. We show that Anos1 function is required for neural crest and sensory organs development in Xenopus, consistent with the defects observed in Kallmann syndrome patients carrying a mutation in ANOS1. These findings indicate that anos1 has a conserved function in the development of craniofacial structures, and indicate that anos1-depleted Xenopus embryos represent a useful model to analyze the pathogenesis of Kallmann syndrome. Copyright © 2017. Published by Elsevier Inc.

Islet-1 is required for ventral neuron survival in Xenopus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Yu; Zhao, Shuhua; Li, Jiejing

Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in the nervous system in Xenopus embryos. Knock-down of Xisl-1 by specific morpholino leads to severe developmental defects, including eye and heart failure. Staining with the neuronal markers N-tubulin and Xisl-1 itself reveals that the motor neurons and a group of ventral interneurons are lost in the Xisl-1 morphants. Terminal dUTP nick-end labeling (TUNEL) analysis shows that Xisl-1 morpholino injection induces extensive apoptosismore » in the ventral neural plate, which can be largely inhibited by the apoptosis inhibitor M50054. We also find that over-expression of Xisl-1 is able to promote cell proliferation and induce Xstat3 expression in the injected side, suggesting a potential role for Xisl-1 in the regulation of cell proliferation in co-operation with the Jak-Stat pathway.« less

XENOPUS LAEVIS: A CULTURING AND REARING PROTOCOL

EPA Science Inventory

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

Spinal cord regeneration in Xenopus tadpoles proceeds through activation of Sox2-positive cells

PubMed Central

2012-01-01

Background In contrast to mammals, amphibians, such as adult urodeles (for example, newts) and anuran larvae (for example, Xenopus) can regenerate their spinal cord after injury. However, the cellular and molecular mechanisms involved in this process are still poorly understood. Results Here, we report that tail amputation results in a global increase of Sox2 levels and proliferation of Sox2+ cells. Overexpression of a dominant negative form of Sox2 diminished proliferation of spinal cord resident cells affecting tail regeneration after amputation, suggesting that spinal cord regeneration is crucial for the whole process. After spinal cord transection, Sox2+ cells are found in the ablation gap forming aggregates. Furthermore, Sox2 levels correlated with regenerative capabilities during metamorphosis, observing a decrease in Sox2 levels at non-regenerative stages. Conclusions Sox2+ cells contribute to the regeneration of spinal cord after tail amputation and transection. Sox2 levels decreases during metamorphosis concomitantly with the lost of regenerative capabilities. Our results lead to a working hypothesis in which spinal cord damage activates proliferation and/or migration of Sox2+ cells, thus allowing regeneration of the spinal cord after tail amputation or reconstitution of the ependymal epithelium after spinal cord transection. PMID:22537391

Susceptibility of early life stages of Xenopus laevis to cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 surfacemore » 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.« less

Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis.

PubMed

Kageura, H

1990-12-01

To examine the spatial distribution of dorsal determinants in the early embryos of Xenopus laevis, individual cells from the 32-cell embryo were transplanted into the same tier of the ventral side of a synchronous recipient. Their abilities to initiate a secondary embryo were measured by the incidence of secondary embryos and by the length of the secondary axis relative to the primary embryo. The ability was found to be localized in all cells (A1, B1, C1, and D1) of the dorsal most column and in the vegetal cells (C2 and D2) of the dorsolateral column. Transplanted C1 (subequatorial) cells caused the highest incidence of a secondary embryo and the average relative length of the secondary embryo was also greatest. Effectiveness decreased in the order: D1, B1, D2, C2, and A1. When these results were compared with Dale and Slack's fate map of the 32-cell embryo, it was concluded that the distribution of dorsal determinants is unique and does not coincide with the prospective regions for any tissues, though it is somewhat similar to the prospective region of dorsal endoderm or notochord. From these results it seems that dorsal determinants do not determine a particular tissue in an embryo but rather the "dorsal" region of an embryo.

Membrane junctions in Xenopus eggs: their distribution suggests a role in calcium regulation.

PubMed

Gardiner, D M; Grey, R D

1983-04-01

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions' distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.

Hyperinnervation improves Xenopus laevis limb regeneration.

PubMed

Mitogawa, Kazumasa; Makanae, Aki; Satoh, Akira

2018-01-15

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Ovarian hyperstimulation syndrome in gonadotropin-treated laboratory South African clawed frogs (Xenopus laevis).

PubMed

Green, Sherril L; Parker, John; Davis, Corrine; Bouley, Donna M

2007-05-01

Ovarian hyperstimulation syndrome (OHS) is a rare but sometimes fatal iatrogenic complication of ovarian stimulation associated with the administration of exogenous gonadotropins to women undergoing treatment for infertility. Laboratory Xenopus spp are commonly treated with human chorionic gonadotropin (hCG) to stimulate ovulation and optimize the number of oocytes harvested for use in biomedical research. Here we report cases of OHS in 2 gonadotropin-treated laboratory Xenopus laevis. After receiving hCG, the frogs developed severe subcutaneous accumulation of fluid, coelomic distention, and whole-body edema and were unable to dive, although they continued to eat and swim. At postmortem examination, extensive subcutaneous edema was present; ascites and massive numbers of free-floating eggs were found in the coelomic cavity and in aberrant locations: around the heart-sac and adhered to the liver capsule. Whole-body edema, gross enlargement of the ovaries, ascites, and abdominal distention are findings comparable to those observed in women with OHS. The pathophysiology of OHS is thought to be related to hormonally induced disturbances of vasoactive mediators, one of which may be vascular endothelial growth factor secreted by theca and granulosa cells. We know of no other report describing OHSlike symptoms in gonadotropin-treated frogs, and the cases described here are 2 of the 3 we have observed at our respective institutions over the last 6 y. According to these results, OHS appears to be rare in gonadotropin-treated laboratory Xenopus. However, the condition should be included in the differential diagnosis for the bloated frog.

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

PubMed

Leininger, Elizabeth C; Kelley, Darcy B

2015-01-01

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

XBtg2 is required for notochord differentiation during early Xenopus development.

PubMed

Sugimoto, Kaoru; Hayata, Tadayoshi; Asashima, Makoto

2005-09-01

The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochord tissue, which did not undergo vacuolation. The XBtg2-depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5-D-4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

PubMed Central

Wei, Shuo; Xu, Guofeng; Bridges, Lance C.; Williams, Phoebe; Nakayama, Takuya; Shah, Anoop; Grainger, Robert M.; White, Judith M.; DeSimone, Douglas W.

2012-01-01

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in X. tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus. PMID:22227340

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

PubMed

Taniguchi, Yuka; Watanabe, Kenji; Mochii, Makoto

2014-06-18

Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole

PubMed Central

2014-01-01

Background Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. Results In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. Conclusion As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans. PMID:24941877

Slowed Relaxation in Fatigued Skeletal Muscle Fibers of Xenopus and Mouse

PubMed Central

Westerblad, Håkan; Lännergren, Jan; Allen, David G.

1997-01-01

Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors down-stream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2+-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics. PMID:9089444

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

PubMed

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

2010-02-01

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

The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication

PubMed Central

Liao, Shuren; Graham, Jeanine; Yan, Hong

2000-01-01

The Bloom's syndrome gene (BLM) plays a pivotal role in the maintenance of genomic stability in somatic cells. It encodes a DNA helicase (BLM) of the RecQ family, but the exact function of BLM remains elusive. To study this question, we have cloned the BLM homolog of the frog Xenopus laevis (xBLM) and have raised antibodies to it. Immunodepletion of xBLM from a Xenopus egg extract severely inhibits the replication of DNA in reconstituted nuclei. Moreover, the inhibition can be rescued by the addition of the recombinant xBLM protein. These results provide the first direct evidence that BLM plays an important role in DNA replication, suggesting that Bloom's syndrome may be the consequence of defective DNA replication. PMID:11040210

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

PubMed

Shibata, M; Shinga, J; Yasuhiko, Y; Kai, M; Miura, K; Shimogori, T; Kashiwagi, K; Igarashi, K; Shiokawa, K

1998-07-01

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

Tissue-tissue interaction-triggered calcium elevation is required for cell polarization during Xenopus gastrulation.

PubMed

Shindo, Asako; Hara, Yusuke; Yamamoto, Takamasa S; Ohkura, Masamichi; Nakai, Junichi; Ueno, Naoto

2010-02-02

The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates

Developmental toxicity of p,p'-dichlorodiphenyltrichloroethane, 2,4,6-trinitrotoluene, their metabolites, and benzo[a]pyrene in Xenopus laevis embryos.

PubMed

Saka, Masahiro

2004-04-01

Since 1995, high incidences of deformed frogs have been documented in Kitakyushu, Japan. In this area, relatively high concentrations of DDT, trinitrotoluene (TNT), their metabolites (p,p'-dichlorodiphenyldichloroethylene [DDE], p,p'-dichlorodiphenyldichloroethane [DDD], 2-amino-4,6-dinitrotoluene [2ADNT], and 4-amino-2,6-dinitrotoluene [4ADNT]), and benzo[a]pyrene [BaP]) have been identified from field samples. I used a standardized assay with Xenopus laevis embryos (frog embryo teratogenesis assay--Xenopus, FETAX) to examine the developmental toxicity of these compounds. Both DDE and BaP were considered nearly nontoxic in embryonic development because they induced low (< 10%) mortality and malformation incidence even at the highest concentrations tested (DDE, 393 microM; BaP, 13.2 microM). The DDD (96-h median lethal concentration [LC50] = 44.1 microM, 96-h median effective concentration [EC50] for malformation = 14.9 microM) was more lethal and teratogenic than its parent compound, DDT (96-h LC50 = 101 microM, 96-h EC50 = 41.5 microM). Predominant symptoms observed were axial malformations (DDT and DDD) and irregular gut coiling (DDT). However, DDT and DDD should not act as major lethal or teratogenic toxicants in the aquatic environment within a short-term exposure via water because their 96-h LC50 and 96-h EC50 values were extremely high, considering their low solubility in water. The TNT (96-h LC50 = 16.7 microM) was more lethal than 2ADNT (96-h LC50 = 166 microM) or 4ADNT (96-h LC50 = 115 microM). Although 4ADNT (96-h EC50 = 85.8 microM) induced various tadpole malformations, it was a weak teratogen compared with TNT (96-h EC50 = 9.78 microM) and 2ADNT (96-h EC50 = 16.9 microM). The most typical malformations observed were axial malformations, eye abnormalities (TNT), edema, and irregular gut coiling (2ADNT and 4ADNT). The 96-h LC50 and 96-h EC50 values of TNT, 2ADNT, and 4ADNT were lower than their saturated concentrations in water. Therefore, these

Embryonic wound healing by apical contraction and ingression in Xenopus laevis.

PubMed

Davidson, Lance A; Ezin, Akouavi M; Keller, Ray

2002-11-01

We have characterized excisional wounds in the animal cap of early embryos of the frog Xenopus laevis and found that these wounds close accompanied by three distinct processes: (1) the assembly of an actin purse-string in the epithelial cells at the wound margin, (2) contraction and ingression of exposed deep cells, and (3) protrusive activity of epithelial cells at the margin. Microsurgical manipulation allowing fine control over the area and depth of the wound combined with videomicroscopy and confocal analysis enabled us to describe the kinematics and challenge the mechanics of the closing wound. Full closure typically occurs only when the deep, mesenchymal cell-layer of the ectoderm is left intact; in contrast, when deep cells are removed along with the superficial, epithelial cell-layer of the ectoderm, wounds do not close. Actin localizes to the superficial epithelial cell-layer at the wound margin immediately after wounding and forms a contiguous "purse-string" in those cells within 15 min. However, manipulation and closure kinematics of shaped wounds and microsurgical cuts made through the purse-string rule out a major force-generating role for the purse-string. Further analysis of the cell behaviors within the wound show that deep, mesenchymal cells contract their apical surfaces and ingress from the exposed surface. High resolution time-lapse sequences of cells at the leading edge of the wound show that these cells undergo protrusive activity only during the final phases of wound closure as the ectoderm reseals. We propose that assembly of the actin purse-string works to organize and maintain the epithelial sheet at the wound margin, that contraction and ingression of deep cells pulls the wound margins together, and that protrusive activity of epithelial cells at the wound margin reseals the ectoderm and re-establishes tissue integrity during wound healing in the Xenopus embryonic ectoderm. Copyright 2002 Wiley-Liss, Inc.

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

PubMed

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

2010-01-01

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

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

PubMed

Moore, Kathryn B; Logan, Mary A; Aldiri, Issam; Roberts, Jacqueline M; Steele, Michael; Vetter, Monica L

2018-05-01

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program. Copyright © 2018 Elsevier Inc. All rights reserved.

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation

PubMed Central

Balashova, Olga A.; Visina, Olesya

2017-01-01

Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor 1 (Folr1; also known as FRα) impairs neural tube formation and leads to NTDs. Folr1 knockdown in neural plate cells only is necessary and sufficient to induce NTDs. Folr1-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model in which the folate receptor interacts with cell adhesion molecules, thus regulating the apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism could unveil novel cellular and molecular events mediated by folate and lead to new ways of preventing NTDs. PMID:28255006

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be; Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be; Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com

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 ofmore » 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.« less

Relocation of mitochondria to the prospective dorsal marginal zone during Xenopus embryogenesis

NASA Technical Reports Server (NTRS)

Yost, H. J.; Phillips, C. R.; Boore, J. L.; Bertman, J.; Whalon, B.; Danilchik, M. V.

1995-01-01

Dorsal-ventral axis formation in Xenopus laevis begins with a cytoplasmic rotation during the first cell cycle and culminates in a series of cell interactions and movements during gastrulation and neurulation that lead to the formation of dorsal-anterior structures. Evidence reported here indicates that mitochondria are differentially redistributed along the prospective dorsal-ventral axis as a consequence of the cortical-cytoplasmic rotation during the first cell cycle. This finding reinvigorates a possibility that has been considered for many years: asymmetries in cytoplasmic components and metabolic activities contribute to the development of morphological asymmetries.

Wise retained in the endoplasmic reticulum inhibits Wnt signaling by reducing cell surface LRP6.

PubMed

Guidato, Sonia; Itasaki, Nobue

2007-10-15

The Wnt signaling pathway is tightly regulated by extracellular and intracellular modulators. Wise was isolated as a secreted protein capable of interacting with the Wnt co-receptor LRP6. Studies in Xenopus embryos revealed that Wise either enhances or inhibits the Wnt pathway depending on the cellular context. Here we show that the cellular localization of Wise has distinct effects on the Wnt pathway readout. While secreted Wise either synergizes or inhibits the Wnt signals depending on the partner ligand, ER-retained Wise consistently blocks the Wnt pathway. ER-retained Wise reduces LRP6 on the cell surface, making cells less susceptible to the Wnt signal. This study provides a cellular mechanism for the action of Wise and introduces the modulation of cellular susceptibility to Wnt signals as a novel mechanism of the regulation of the Wnt pathway.

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

PubMed

Wahli, W; Martinez, E; Corthésy, B; Cardinaux, J R

1989-01-01

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin

Maternal syntabulin is required for dorsal axis formation and is a germ plasm component in Xenopus.

PubMed

Colozza, Gabriele; De Robertis, Edward M

2014-07-01

In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates. Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

A Matter of the Heart: The African Clawed Frog Xenopus as a Model for Studying Vertebrate Cardiogenesis and Congenital Heart Defects

PubMed Central

Hempel, Annemarie; Kühl, Michael

2016-01-01

The African clawed frog, Xenopus, is a valuable non-mammalian model organism to investigate vertebrate heart development and to explore the underlying molecular mechanisms of human congenital heart defects (CHDs). In this review, we outline the similarities between Xenopus and mammalian cardiogenesis, and provide an overview of well-studied cardiac genes in Xenopus, which have been associated with congenital heart conditions. Additionally, we highlight advantages of modeling candidate genes derived from genome wide association studies (GWAS) in Xenopus and discuss commonly used techniques. PMID:29367567

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

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 themore » 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.« less

Xenopus: An Emerging Model for Studying Congenital Heart Disease

PubMed Central

Kaltenbrun, Erin; Tandon, Panna; Amin, Nirav M.; Waldron, Lauren; Showell, Chris; Conlon, Frank L.

2011-01-01

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well-studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes. PMID:21538812

Further Development and Validation of the frog Embryo Teratogenesis Assay - Xenopus (FETAX)

DTIC Science & Technology

1991-02-28

abnormalities.39 40 The teratogenic effects of serotonin in the laboratory rat include anophthalmia , hydrocephalus, exencephaly, omphalocoele and vacuolization...kinky tail. ZnSO4 in Xenopus, should be tested in parallel with hemangioma. anophthalmia and scoliosis). Skeletal a metabolic activation system to show...teratogenic effects of 0 serotonin in the laboratory rat include anophthalmia , hydrocephalus, exencephaly, omphalocele and vacuolization of myocardial cells.41

The Nedd4 binding protein 3 is required for anterior neural development in Xenopus laevis.

PubMed

Kiem, Lena-Maria; Dietmann, Petra; Linnemann, Alexander; Schmeisser, Michael J; Kühl, Susanne J

2017-03-01

The Fezzin family member Nedd4-binding protein 3 (N4BP3) is known to regulate axonal and dendritic branching. Here, we show that n4bp3 is expressed in the neural tissue of the early Xenopus laevis embryo including the eye, the brain and neural crest cells. Knockdown of N4bp3 in the Xenopus anterior neural tissue results in severe developmental impairment of the eye, the brain and neural crest derived cranial cartilage structures. Moreover, we demonstrate that N4bp3 depletion leads to a significant reduction of both eye and brain specific marker genes and reduced neural crest cell migration. Finally, we demonstrate an impact of N4bp3 deficiency on cell apoptosis and proliferation. Our studies indicate that N4bp3 is required for early anterior neural development of vertebrates. This is in line with a study implicating that genetic disruption of N4BP3 in humans might be related to neurodevelopmental disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression.

PubMed

Miedlich, Susanne U; Taya, Manisha; Young, Melissa Rasar; Hammes, Stephen R

2017-06-15

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

E-cadherin is required for cranial neural crest migration in Xenopus laevis.

PubMed

Huang, Chaolie; Kratzer, Marie-Claire; Wedlich, Doris; Kashef, Jubin

2016-03-15

The cranial neural crest (CNC) is a highly motile and multipotent embryonic cell population, which migrates directionally on defined routes throughout the embryo, contributing to facial structures including cartilage, bone and ganglia. Cadherin-mediated cell-cell adhesion is known to play a crucial role in the directional migration of CNC cells. However, migrating CNC co-express different cadherin subtypes, and their individual roles have yet to be fully explored. In previous studies, the expression of individual cadherin subtypes has been analysed using different methods with varying sensitivities, preventing the direct comparison of expression levels. Here, we provide the first comprehensive and comparative analysis of the expression of six cadherin superfamily members during different phases of CNC cell migration in Xenopus. By applying a quantitative RT-qPCR approach, we can determine the copy number and abundance of each expressed cadherin through different phases of CNC migration. Using this approach, we show for the first time expression of E-cadherin and XB/C-cadherin in CNC cells, adding them as two new members of cadherins co-expressed during CNC migration. Cadherin co-expression during CNC migration in Xenopus, in particular the constant expression of E-cadherin, contradicts the classical epithelial-mesenchymal transition (EMT) model postulating a switch in cadherin expression. Loss-of-function experiments further show that E-cadherin is required for proper CNC cell migration in vivo and also for cell protrusion formation in vitro. Knockdown of E-cadherin is not rescued by co-injection of other classical cadherins, pointing to a specific function of E-cadherin in mediating CNC cell migration. Finally, through reconstitution experiments with different E-cadherin deletion mutants in E-cadherin morphant embryos, we demonstrate that the extracellular domain, but not the cytoplasmic domain, of E-cadherin is sufficient to rescue CNC cell migration in vivo

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

PubMed

Semino, C E; Specht, C A; Raimondi, A; Robbins, P W

1996-05-14

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morin, R D; Chang, E; Petrescu, A

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 betweenmore » 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.« less

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

PubMed

Zhang, Zheng Z; Pannunzio, Nicholas R; Lu, Zhengfei; Hsu, Ellen; Yu, Kefei; Lieber, Michael R

2015-10-01

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

NASA Astrophysics Data System (ADS)

Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

2002-10-01

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to -aminobutyric acid, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

Hoxa2 knockdown in Xenopus results in hyoid to mandibular homeosis.

PubMed

Baltzinger, Mireille; Ori, Michela; Pasqualetti, Massimo; Nardi, Irma; Rijli, Filippo M

2005-12-01

The skeletal structures of the face and throat are derived from cranial neural crest cells (NCCs) that migrate from the embryonic neural tube into a series of branchial arches (BAs). The first arch (BA1) gives rise to the upper and lower jaw cartilages, whereas hyoid structures are generated from the second arch (BA2). The Hox paralogue group 2 (PG2) genes, Hoxa2 and Hoxb2, show distinct roles for hyoid patterning in tetrapods and fishes. In the mouse, Hoxa2 acts as a selector of hyoid identity, while its paralogue Hoxb2 is not required. On the contrary, in zebrafish Hoxa2 and Hoxb2 are functionally redundant for hyoid arch patterning. Here, we show that in Xenopus embryos morpholino-induced functional knockdown of Hoxa2 is sufficient to induce homeotic changes of the second arch cartilage. Moreover, Hoxb2 is downregulated in the BA2 of Xenopus embryos, even though initially expressed in second arch NCCs, similar to mouse and unlike in zebrafish. Finally, Xbap, a gene involved in jaw joint formation, is selectively upregulated in the BA2 of Hoxa2 knocked-down frog embryos, supporting a hyoid to mandibular change of NCC identity. Thus, in Xenopus Hoxa2 does not act redundantly with Hoxb2 for BA2 patterning, similar to mouse and unlike in fish. These data bring novel insights into the regulation of Hox PG2 genes and hyoid patterning in vertebrate evolution and suggest that Hoxa2 function is required at late stages of BA2 development. Copyright 2005 Wiley-Liss, Inc.

Functional and structural effects of amyloid-β aggregate on Xenopus laevis oocytes.

PubMed

Parodi, Jorge; Ochoa-de la Paz, Lenin; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2012-10-01

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

PubMed Central

Long, E O; Gross, N; Wake, C T; Mach, J P; Carrel, S; Accolla, R; Mach, B

1982-01-01

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821356

Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis

PubMed Central

Field, Christine M.; Pelletier, James F.; Mitchison, Timothy J.

2017-01-01

We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen, and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ~11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers, and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues. PMID:28065319

Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl

PubMed Central

2014-01-01

Background To gain insight into what differences might restrict the capacity for limb regeneration in Xenopus froglets, we used High Performance Liquid Chromatography (HPLC)/double mass spectrometry to characterize protein expression during fibroblastema formation in the amputated froglet hindlimb, and compared the results to those obtained previously for blastema formation in the axolotl limb. Results Comparison of the Xenopus fibroblastema and axolotl blastema revealed several similarities and significant differences in proteomic profiles. The most significant similarity was the strong parallel down regulation of muscle proteins and enzymes involved in carbohydrate metabolism. Regenerating Xenopus limbs differed significantly from axolotl regenerating limbs in several ways: deficiency in the inositol phosphate/diacylglycerol signaling pathway, down regulation of Wnt signaling, up regulation of extracellular matrix (ECM) proteins and proteins involved in chondrocyte differentiation, lack of expression of a key cell cycle protein, ecotropic viral integration site 5 (EVI5), that blocks mitosis in the axolotl, and the expression of several patterning proteins not seen in the axolotl that may dorsalize the fibroblastema. Conclusions We have characterized global protein expression during fibroblastema formation after amputation of the Xenopus froglet hindlimb and identified several differences that lead to signaling deficiency, failure to retard mitosis, premature chondrocyte differentiation, and failure of dorsoventral axial asymmetry. These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb. PMID:25063185

Restriction of the Xenopus DEADSouth mRNA to the primordial germ cells is ensured by multiple mechanisms.

PubMed

Yamaguchi, Takeshi; Kataoka, Kensuke; Watanabe, Kenji; Orii, Hidefumi

2014-02-01

DEADSouth mRNA encoding the RNA helicase DDX25 is a component of the germ plasm in Xenopus laevis. We investigated the mechanisms underlying its specific mRNA expression in primordial germ cells (PGCs). Based on our previous findings of several microRNA miR-427 recognition elements (MREs) in the 3' untranslated region of the mRNA, we first examined whether DEADSouth mRNA was degraded by miR-427 targeting in somatic cells. Injection of antisense miR-427 oligomer and reporter mRNA for mutated MREs revealed that DEADSouth mRNA was potentially degraded in somatic cells via miR-427 targeting, but not in PGCs after the mid-blastula transition (MBT). The expression level of miR-427 was very low in PGCs, which probably resulted in the lack of miR-427-mediated degradation. In addition, the DEADSouth gene was expressed zygotically after MBT. Thus, the predominant expression of DEADSouth mRNA in the PGCs is ensured by multiple mechanisms including zygotic expression and prohibition from miR-427-mediated degradation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems.

PubMed

McDowell, Gary S; Philpott, Anna

2018-05-16

The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts. © 2019 Cold Spring Harbor Laboratory Press.

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

NASA Technical Reports Server (NTRS)

Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

2000-01-01

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

( sup 125 I)Bolton-Hunter neuropeptide-Y-binding sites on folliculo-stellate cells of the pars intermedia of Xenopus laevis: A combined autoradiographic and immunocytochemical study

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Rijk, E.P.; Cruijsen, P.M.; Jenks, B.G.

1991-02-01

It has previously been established that neuropeptide-Y (NPY) is a potent inhibitor of alpha MSH release from the pars intermedia of the amphibian Xenopus laevis. The location of binding sites for NPY in the pars intermedia of the pituitary has now been studied with light microscopic autoradiography, using a dispersed cell labeling method with the specific NPY receptor ligand ({sup 125}I)Bolton-Hunter NPY. The majority of radioactive labeling was associated with folliculo-stellate cells; the percentage of labeling as well as the mean number of grains were approximately 5 times higher for folliculo-stellate cells than for melanotropes. An excess of nonlabeled NPYmore » drastically reduced radiolabeling of folliculo-stellate cells, but had no effect on the degree of labeling of melanotropes. These results show that folliculo-stellate cells of X. laevis possess specific binding sites for NPY and indicate that NPY exerts its inhibitory action on the release of alpha MSH in an indirect fashion, by acting on the folliculo-stellate cells.« less

Activation of Sox3 Gene by Thyroid Hormone in the Developing Adult Intestinal Stem Cell During Xenopus Metamorphosis

PubMed Central

Sun, Guihong; Fu, Liezhen; Wen, Luan

2014-01-01

The maturation of the intestine into the adult form involves the formation of adult stem cells in a thyroid hormone (T3)-dependent process in vertebrates. In mammals, this takes place during postembryonic development, a period around birth when the T3 level peaks. Due to the difficulty of manipulating late-stage, uterus-enclosed embryos, very little is known about the development of the adult intestinal stem cells. Interestingly, the remodeling of the intestine during the T3-dependent amphibian metamorphosis mimics the maturation of mammalian intestine. Our earlier microarray studies in Xenopus laevis revealed that the transcription factor SRY (sex-determining region Y)-box 3 (Sox3), well known for its involvement in neural development, was upregulated in the intestinal epithelium during metamorphosis. Here, we show that Sox3 is highly and specifically expressed in the developing adult intestinal progenitor/stem cells. We further show that its induction by T3 is independent of new protein synthesis, suggesting that Sox3 is directly activated by liganded T3 receptor. Thus, T3 activates Sox3 as one of the earliest changes in the epithelium, and Sox3 in turn may facilitate the dedifferentiation of the larval epithelial cells into adult stem cells. PMID:25211587

Conservation and divergence of ADAM family proteins in the Xenopus genome

PubMed Central

2010-01-01

Background Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. Results Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. Conclusions Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease

Hybridization between the African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin secretions of female offspring.

PubMed

Mechkarska, Milena; Meetani, Mohammed; Michalak, Pawel; Vaksman, Zalman; Takada, Koji; Conlon, J Michael

2012-09-01

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Mueller's clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin-2 from X. laevis by a single amino acid substitution (Gly(13)→Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment. Copyright © 2012 Elsevier Inc. All rights reserved.

Modeling human craniofacial disorders in Xenopus

PubMed Central

Dubey, Aditi; Saint-Jeannet, Jean-Pierre

2017-01-01

Purpose of Review Craniofacial disorders are among the most common human birth defects and present an enormous health care and social burden. The development of animal models has been instrumental to investigate fundamental questions in craniofacial biology and this knowledge is critical to understand the etiology and pathogenesis of these disorders. Recent findings The vast majority of craniofacial disorders arise from abnormal development of the neural crest, a multipotent and migratory cell population. Therefore, defining the pathogenesis of these conditions starts with a deep understanding of the mechanisms that preside over neural crest formation and its role in craniofacial development. Summary This review discusses several studies using Xenopus embryos to model human craniofacial conditions, and emphasizes the strength of this system to inform important biological processes as they relate to human craniofacial development and disease. PMID:28255527

Xwnt-8 and lithium can act upon either dorsal mesodermal or neurectodermal cells to cause a loss of forebrain in Xenopus embryos

NASA Technical Reports Server (NTRS)

Fredieu, J. R.; Cui, Y.; Maier, D.; Danilchik, M. V.; Christian, J. L.

1997-01-01

When Xenopus gastrulae are made to misexpress Xwnt-8, or are exposed to lithium ions, they develop with a loss of anterior structures. In the current study, we have characterized the neural defects produced by either Xwnt-8 or lithium and have examined potential cellular mechanisms underlying this anterior truncation. We find that the primary defect in embryos exposed to lithium at successively earlier stages during gastrulation is a progressive rostral to caudal deletion of the forebrain, while hindbrain and spinal regions of the CNS remain intact. Misexpression of Xwnt-8 during gastrulation produces an identical loss of forebrain. Our results demonstrate that lithium and Wnts can act upon either prospective neural ectodermal cells, or upon dorsal mesodermal cells, to cause a loss of anterior pattern. Specifically, ectodermal cells isolated from lithium- or Wnt-exposed embryos are unable to form anterior neural tissue in response to inductive signals from normal dorsal mesoderm. In addition, although dorsal mesodermal cells from lithium- or Wnt-exposed embryos are specified properly, and produce normal levels of the anterior neural inducing molecules noggin and chordin, they show a greatly reduced capacity to induce anterior neural tissue in conjugated ectoderm. Taken together, our results are consistent with a model in which Wnt- or lithium-mediated signals can induce either mesodermal or ectodermal cells to produce a dominant posteriorizing morphogen which respecifies anterior neural tissue as posterior.

ZWY Sex Determination in Xenopus tropicalis

EPA Science Inventory

Most vertebrate species with described genetic sex determination are either male (XY) or female (ZW) heterogametic. To date, studies with Xenopus species indicate that members of this genus operate under a ZW sex determination system. We used two different approaches and demonst...

The Xenopus Tgfbi is required for embryogenesis through regulation of canonical Wnt signalling.

PubMed

Wang, Feng; Hu, Wanzhou; Xian, Jian; Ohnuma, Shin-ichi; Brenton, James D

2013-07-01

Tgfbi, a fasciclin family extracellular matrix protein, has various roles in human diseases from corneal dystrophies to cancer. However, the molecular mechanisms that underlie its functions are poorly understood. Here, we studied the role of Tgfbi during Xenopus embryogenesis. During gastrulation and immediately after, Xtgfbi is expressed at developmentally important signaling centers including the dorsal marginal zone, notochord and floorplate. Xtgfbi knockdown by anti-sense morpholinos causes defective organizer induction, patterning and differentiation of muscle, neuron and neural crests, similar to suppression of canonical Wnt signaling. In Xenopus embryos and animal caps as well as DLD-1 cells, we show that Tgfbi is strongly required for the full activation of the canonical Wnt pathway by promoting phosphorylation of GSK3β and consequently enhancing the stabilization and nuclear localization of β-catenin. Further analysis shows that Tgfbi is likely to promote GSK3β phosphorylation through integrin-linked kinase. Copyright © 2013 Elsevier Inc. All rights reserved.

Early development of Xenopus embryos is affected by simulated gravity

NASA Technical Reports Server (NTRS)

Yokota, Hiroki; Neff, Anton W.; Malacinski, George M.

1994-01-01

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) 'morphological patterning' such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) 'the timing of embryonic events' such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.

Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis

PubMed Central

Hasebe, Takashi; Fu, Liezhen; Heimeier, Rachel A.; Das, Biswajit; Ishizuya-Oka, Atsuko; Shi, Yun-Bo

2013-01-01

Background Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells. Methodology/Principal Findings The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium. Conclusions/Significance Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells. PMID:23383234

Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues

EPA Science Inventory

African clawed frog Xenopus sp. has been used extensively for developmental biology and toxicology research. Xenopus (Silurana) tropicalis has been coveted more recently for genomics research because its diploid genome has been sequenced. Amid concerns of environmental pollutants...

Vision Drives Correlated Activity without Patterned Spontaneous Activity in Developing Xenopus Retina

PubMed Central

Demas, James A.; Payne, Hannah; Cline, Hollis T.

2011-01-01

Developing amphibians need vision to avoid predators and locate food before visual system circuits fully mature. Xenopus tadpoles can respond to visual stimuli as soon as retinal ganglion cells (RGCs) innervate the brain, however, in mammals, chicks and turtles, RGCs reach their central targets many days, or even weeks, before their retinas are capable of vision. In the absence of vision, activity-dependent refinement in these amniote species is mediated by waves of spontaneous activity that periodically spread across the retina, correlating the firing of action potentials in neighboring RGCs. Theory suggests that retinorecipient neurons in the brain use patterned RGC activity to sharpen the retinotopy first established by genetic cues. We find that in both wild type and albino Xenopus tadpoles, RGCs are spontaneously active at all stages of tadpole development studied, but their population activity never coalesces into waves. Even at the earliest stages recorded, visual stimulation dominates over spontaneous activity and can generate patterns of RGC activity similar to the locally correlated spontaneous activity observed in amniotes. In addition, we show that blocking AMPA and NMDA type glutamate receptors significantly decreases spontaneous activity in young Xenopus retina, but that blocking GABAA receptor blockers does not. Our findings indicate that vision drives correlated activity required for topographic map formation. They further suggest that developing retinal circuits in the two major subdivisions of tetrapods, amphibians and amniotes, evolved different strategies to supply appropriately patterned RGC activity to drive visual circuit refinement. PMID:21312343

Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.

PubMed

Hirsch, Nicolas; Zimmerman, Lyle B; Gray, Jessica; Chae, Jeiwook; Curran, Kristen L; Fisher, Marilyn; Ogino, Hajime; Grainger, Robert M

2002-12-01

For over a century, amphibian embryos have been a source of significant insight into developmental mechanisms, including fundamental discoveries about the process of induction. The recently developed transgenesis for Xenopus offers new approaches to these poorly understood processes, particularly when undertaken in the quickly maturing species Xenopus tropicalis, which greatly facilitates establishment of permanent transgenic lines. Several X. tropicalis transgenic lines have now been generated, and experiments demonstrating the value of these lines to study induction in embryonic tissue recombinants and explants are presented here. A revised protocol for transgenesis in X. tropicalis resulting in a significant increase in the percentage of transgenic animals that reach adulthood is presented, as well as improvements in tadpole and froglet husbandry, which have facilitated the raising of large numbers of adults. Working transgenic populations have been rapidly expanded, and some transgenes have been bred to homozygosity. Established lines include those bearing the promoter regions of Pax-6, Otx-2, Rx, and EF1alpha coupled to fluorescent reporter genes. Multireporter lines combining, in a single animal, up to three gene promoters coupled to different fluorescent reporters have also been established. The value of X. tropicalis transgenic lines for the study of induction is demonstrated by showing activation of Pax-6 by noggin treatment of Pax-6/GFP transgenic animal caps, illustrating how reporter lines allow a rapid, in vivo assay for an inductive response. An experiment showing lens induction in gamma-crystallin/GFP transgenic lens ectoderm when it is recombined with mouse optic vesicle demonstrates conservation of inducing signals from amphibians and mammals. It also shows how the warmer culture temperatures tolerated by X. tropicalis embryos can be used in assays of factors produced by mammalian cells and tissues. The many applications of transgenic reporter

Mechanics of Fluid-Filled Interstitial Gaps. II. Gap Characteristics in Xenopus Embryonic Ectoderm.

PubMed

Barua, Debanjan; Parent, Serge E; Winklbauer, Rudolf

2017-08-22

The ectoderm of the Xenopus embryo is permeated by a network of channels that appear in histological sections as interstitial gaps. We characterized this interstitial space by measuring gap sizes, angles formed between adjacent cells, and curvatures of cell surfaces at gaps. From these parameters, and from surface-tension values measured previously, we estimated the values of critical mechanical variables that determine gap sizes and shapes in the ectoderm, using a general model of interstitial gap mechanics. We concluded that gaps of 1-4 μm side length can be formed by the insertion of extracellular matrix fluid at three-cell junctions such that cell adhesion is locally disrupted and a tension difference between cell-cell contacts and the free cell surface at gaps of 0.003 mJ/m 2 is generated. Furthermore, a cell hydrostatic pressure of 16.8 ± 1.7 Pa and an interstitial pressure of 3.9 ± 3.6 Pa, relative to the central blastocoel cavity of the embryo, was found to be consistent with the observed gap size and shape distribution. Reduction of cell adhesion by the knockdown of C-cadherin increased gap volume while leaving intracellular and interstitial pressures essentially unchanged. In both normal and adhesion-reduced ectoderm, cortical tension of the free cell surfaces at gaps does not return to the high values characteristic of the free surface of the whole tissue. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Prx-1 expression in Xenopus laevis scarless skin-wound healing and its resemblance to epimorphic regeneration.

PubMed

Yokoyama, Hitoshi; Maruoka, Tamae; Aruga, Akio; Amano, Takanori; Ohgo, Shiro; Shiroishi, Toshihiko; Tamura, Koji

2011-12-01

Despite a strong clinical need for inducing scarless wound healing, the molecular factors required to accomplish it are unknown. Although skin-wound healing in adult mammals often results in scarring, some amphibians can regenerate injured body parts, even an amputated limb, without it. To understand the mechanisms of perfect skin-wound healing in regenerative tetrapods, we studied the healing process in young adult Xenopus "froglets" after experimental skin excision. We found that the excision wound healed completely in Xenopus froglets, without scarring. Mononuclear cells expressing a homeobox gene, prx1, accumulated under the new epidermis of skin wounds on the limb and trunk and at the regenerating limb. In transgenic Xenopus froglets expressing a reporter for the mouse prx1 limb-specific enhancer, activity was seen in the healing skin and in the regenerating limb. Comparable activity did not accompany skin-wound healing in adult mice. Our results suggest that scarless skin-wound healing may require activation of the prx1 limb enhancer, and competence to activate the enhancer is probably a prerequisite for epimorphic regeneration, such as limb regeneration. Finally, the induction of this prx1 enhancer activity may be useful as a reliable marker for therapeutically induced scarless wound healing in mammals.

Intracellular pH in early Xenopus embryos: its effect on current flow between blastomeres.

PubMed Central

Turin, L; Warner, A E

1980-01-01

1. Electrophysiological techniques were used to monitor the flow of electric current from one cell to the next in Xenopus laevis embryos between the 4-cell and early blastula stages of development. Intracellular pH and blastocoel pH were determined using pH-sensitive micro-electrodes. 2. The resting intracellular pH was 7.74+/-0.02 (S.E. of mean, n = 29); there were no systematic differences between developmental stages. Blastocoel cavity pH was 8.4+/-0.06 (S.E. of mean, n = 10). The intracellular buffer value was 18 m-equiv. H+/pH unit per litre. 3. In embryos treated with bicarbonate buffered Holtfreter solution equilibrated with 100% CO2 the intracellular pH fell to 6.3+/-0.17 (S.D., n = 8). The membrane potential fell and the input resistance increased. The size of the effect on membrane potential and input resistance varied. 4. From the 32-cell stage onwards current flow from one cell to the next was abolished when the intracellular pH fell to below 6.5; the effect was rapid in onset and completely reversible. At cleavage stages of development lowering intracellular pH with CO2 had no effect on current flow from cell to cell. 5. The relationship between intracellular pH and current flow from cell to cell was sigmoid and covered between 0.2 and 0.4 pH units. The pH at which current flow was completely abolished ranged from 6.85 to 6.4. 6. Alterations in extraembryonic pH over the range 5.8-7.5 had no effect on any parameter measured. 7. We conclude that lowering the intracellular pH increases the resistance of both non-junctional junctional membranes. The data do not allow us to extract the pH junctional conductance relationship. 8. Variations in intracellular pH may provide a useful tool for the study of the functional role of direct cell to cell communication in both adult organs and early embryos. PMID:6770084

OCT imaging of craniofacial anatomy in xenopus embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Deniz, Engin; Jonas, Stephan M.; Griffin, John; Hooper, Michael C.; Choma, Michael A.; Khokha, Mustafa K.

2016-03-01

The etiology of craniofacial defects is incompletely understood. The ability to obtain large amounts of gene sequence data from families affected by craniofacial defects is opening up new ways to understand molecular genetic etiological factors. One important link between gene sequence data and clinical relevance is biological research into candidate genes and molecular pathways. We present our recent research using OCT as a nondestructive phenotyping modality of craniofacial morphology in Xenopus embryos, an important animal model for biological research in gene and pathway discovery. We define 2D and 3D scanning protocols for a standardized approach to craniofacial imaging in Xenopus embryos. We define standard views and planar reconstructions for visualizing normal anatomy and landmarks. We compare these views and reconstructions to traditional histopathology using alcian blue staining. In addition to being 3D, nondestructive, and having much faster throughout, OCT can identify craniofacial features that are lost during traditional histopathological preparation. We also identify quantitative morphometric parameters to define normative craniofacial anatomy. We also note that craniofacial and cardiac defects are not infrequently present in the same patient (e.g velocardiofacial syndrome). Given that OCT excels at certain aspects of cardiac imaging in Xenopus embryos, our work highlights the potential of using OCT and Xenopus to study molecular genetic factors that impact both cardiac and craniofacial development.

Activin-like signal activates dorsal-specific maternal RNA between 8- and 16-cell stages of Xenopus.

PubMed

Hainski, A M; Moody, S A

1996-01-01

In many animals the dorsal-ventral axis forms by an initial localization of maternal molecules, which then regulate the spatial location of signals that directly influence the expression of axis-specific fates. Several recent studies have demonstrated that dorsal-animal blastomeres of the Xenopus morula (8-32 cells) are biased toward dorsal fates prior to mesoderm inductive signaling. In this study we ask whether the dorsal bias is the result of autonomous expression of maternal molecules specifically localized within dorsal cells or of early activating signals. It was found that although 16-cell dorsal-animal blastomeres (D1.1) can differentiate into dorsal tissues when cultured alone, the 8-cell mothers (D1) can not. Likewise, although RNA extracted from D1.1 can induce an extra dorsal axis when injected into vegetal blastomeres, RNA extracted from D1 can not. However, D1 does express dorsal tissues if co-cultured with dorsal-vegetal cells or with culture medium containing a mixture of activins (PIF-medium). Furthermore, short-term culture of D1 in PIF-medium enables the D1 RNA to induce an ectopic dorsal axis. Ventral-animal blastomeres also can express dorsal axial tissues when co-cultured with dorsal-vegetal blastomeres or in PIF-medium, but the RNA from the activin-treated ventral cells cannot induce ectopic dorsal axes. These studies demonstrate that there are maternal RNAs that, shortly after fertilization, are present only in the dorsal-animal region. They do not act cell autonomously, but require an activin-like signal. These RNAs may function by increasing the responsiveness of dorsal-animal blastomeres to the mesoderm inductive signals present in both the morula and the blastula.

Bisphenol A causes malformation of the head region in embryos of Xenopus laevis and decreases the expression of the ESR-1 gene mediated by Notch signaling.

PubMed

Imaoka, Susumu; Mori, Tomohiro; Kinoshita, Tsutomu

2007-02-01

Bisphenol A (BpA) is widely used in industry and dentistry. Its effects on the embryonic development of Xenopus laevis were investigated. Xenopus embryos at stage 10.5 were treated with BpA. Developmental abnormalities were observed at stage 35; malformation of the head region including eyes and scoliosis. The expression of several markers of embryonic development was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The pan-neural marker SOX-2, the neural stem cell marker nrp-1, the mesodermal marker MyoD, and the endodermal marker sox17alpha, were used. Although the expression of marker genes was not changed by treatment with BpA, that of Pax-6, a key regulator of the morphogenesis of the eyes, was decreased by BpA. Pax-6 is a downstream factor of Notch signaling. So, the expression of a typical Notch-dependent factor, ESR-1, was investigated in the presence of BpA. The expression of ESR-1 was efficiently suppressed by BpA. In whole mount in situ hybridization (WISH), Pax-6 was expressed in the central nervous system and eyes. The expression was lost completely on treatment with BpA. The expression of ESR-1 in the central nervous system and eyes also disappeared with BpA treatment. Injection of the intracellular domain of Notch efficiently recovered ESR-1 expression in the presence of BpA although injection of a ligand for notch, Delta, did not. These results suggest that BpA decreased the expression of ESR-1 by disrupting the Notch signal.

Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs.

PubMed

Tokmakov, Alexander A; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2010-05-01

Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.

RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

PubMed

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.

Apoptosis regulates notochord development in Xenopus.

PubMed

Malikova, Marina A; Van Stry, Melanie; Symes, Karen

2007-11-15

The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed.

Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

PubMed Central

Tuma, M. Carolina; Zill, Andrew; Le Bot, Nathalie; Vernos, Isabelle; Gelfand, Vladimir

1998-01-01

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles. PMID:9852150

Application of reaction-diffusion models to cell patterning in Xenopus retina. Initiation of patterns and their biological stability.

PubMed

Shoaf, S A; Conway, K; Hunt, R K

1984-08-07

We have examined the behavior of two reaction-diffusion models, originally proposed by Gierer & Meinhardt (1972) and by Kauffman, Shymko & Trabert (1978), for biological pattern formation. Calculations are presented for pattern formation on a disc (approximating the geometry of a number of embryonic anlagen including the frog eye rudiment), emphasizing the sensitivity of patterns to changes in initial conditions and to perturbations in the geometry of the morphogen-producing space. Analysis of the linearized equations from the models enabled us to select appropriate parameters and disc size for pattern growth. A computer-implemented finite element method was used to solve the non-linear model equations reiteratively. For the Gierer-Meinhardt model, initial activation (varying in size over two orders of magnitude) of one point on the disc's edge was sufficient to generate the primary gradient. Various parts of the disc were removed (remaining only as diffusible space) from the morphogen-producing cycle to investigate the effects of cells dropping out of the cycle due to cell death or malfunction (single point removed) or differentiation (center removed), as occur in the Xenopus eye rudiment. The resulting patterns had the same general shape and amplitude as normal gradients. Nor did a two-fold increase in disc size affect the pattern-generating ability of the model. Disc fragments bearing their primary gradient patterns were fused (with gradients in opposite directions, but each parallel to the fusion line). The resulting patterns generated by the model showed many similarities to results of "compound eye" experiments in Xenopus. Similar patterns were obtained with the model of Kauffman's group (1978), but we found less stability of the pattern subject to simulations of central differentiation. However, removal of a single point from the morphogen cycle (cell death) did not result in any change. The sensitivity of the Kauffman et al. model to shape perturbations is

Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis.

PubMed

Moriyama, Yuki; Ohata, Yoshihisa; Mori, Shoko; Matsukawa, Shinya; Michiue, Tatsuo; Asashima, Makoto; Kuroda, Hiroki

2011-01-28

Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period. Copyright © 2010 Elsevier Inc. All rights reserved.

Expression of the mammalian calcium signaling response to Trypanosoma cruzi in Xenopus laevis oocytes.

PubMed

Leite, M F; Moyer, M S; Andrews, N W

1998-04-01

Infective stages of the protozoan parasite Trypanosoma cruzi contain a soluble factor that induces elevation in the intracellular free Ca2+ concentration ([Ca2+]i) of mammalian cells. The process is pertussis toxin (PTx)-sensitive, and involves phospholipase C (PLC) activation, inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ release from intracellular stores (Tardieux I, et al. J Exp Med 1994;179:1017-1022; Rodriguez A, et al. J Cell Biol 1995;129:1263-1273). We now report that a molecule exposed on the surface of the target cells is required to trigger the signaling cascade, and that a response with identical characteristics can be induced in Xenopus laevis oocytes injected with mRNA from normal rat kidney (NRK) fibroblasts. Xenopus oocytes do not show an endogenous response to the trypomastigote Ca2+ signaling factor, but a vigorous response in the form of a propagating Ca2+ wave is expressed after injection of NRK cell mRNA. As previously demonstrated for mammalian cells, the response is inhibited when injected oocytes are pretreated with PTx, implicating Galphai or Galphao trimeric G-proteins, and with thapsigargin, which depletes intracellular Ca2+ stores. Moreover, the [Ca2+]i transients triggered by the T. cruzi soluble factor in mRNA-injected oocytes are blocked by the same inhibitors of the parasite oligopeptidase B that abolish the [Ca2+]i response in NRK cells (Burleigh B, Andrews NW. J Biol Chem 1995;270:5172-5180; Burleigh BA et al. J Cell Biol 1997;136:609-620). The NRK mRNA fraction that induces expression of the [Ca2+]i response to the T. cruzi signaling factor contains messages from 1.5 to 2.0 kb, a size range consistent with the family of seven-transmembrane G-protein-coupled receptors.

Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis

PubMed Central

Wen, Luan; Fu, Liezhen; Guo, Xiaogang; Chen, Yonglong; Shi, Yun-Bo

2015-01-01

Histone methylations have been implicated to play important roles in diverse cellular processes. Of particular interest is the methylation of histone H3K79, which is catalyzed by an evolutionarily conserved methyltransferase, disruptor of telomeric silencing (Dot1)-like (Dot1L). To investigate the role of Dot1L during vertebrate development, we have generated a Dot1L-specific transcription activator-like effector nuclease (TALEN) nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We show that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis because normally feeding tadpoles were formed, consistent with the lack of maternal Dot1L expression. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings suggest that Dot1L and H3K79 methylation play an important role for tadpole growth and development prior to metamorphosis into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of 2 separate phases of vertebrate development with distinct requirements for epigenetic modifications.—Wen, L., Fu, L., Guo, X., Chen, Y., Shi, Y.-B. Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis. PMID:25366346

Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

PubMed

Miyata, Sairi; Yada, Tomotaka; Ishikawa, Natsuko; Taheruzzaman, Kazi; Hara, Ryohei; Matsuzaki, Takashi; Nishikawa, Akio

2017-03-01

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T 3 ) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T 3 . In larval tail cells, cell count was 76% lower in the presence of T 3 , and IGF-1 did not promote proliferation and differentiation in T 3 -containing medium. In larval dorsal cells, cell count was also lower in the presence of T 3 , but IGF-1 enhanced proliferation and differentiation in T 3 -containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T 3 and helps accelerate dorsal muscle remodeling during metamorphosis.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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. Wemore » 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.« less

Regulation of early Xenopus development by ErbB signaling

PubMed Central

Nie, Shuyi; Chang, Chenbei

2008-01-01

ErbB signaling has long been implicated in cancer formation and progression and is shown to regulate cell division, migration and death during tumorigenesis. The functions of the ErbB pathway during early vertebrate embryogenesis, however, are not well understood. Here we report characterization of ErbB activities during early frog development. Gain-of-function analyses show that EGFR, ErbB2 and ErbB4 induce ectopic tumor-like cell mass that contains increased numbers of mitotic cells. Both the muscle and the neural markers are expressed in these ectopic protrusions. ErbBs also induce mesodermal markers in ectodermal explants. Loss-of-function studies using carboxyl terminal-truncated dominant-negative ErbB receptors demonstrate that blocking ErbB signals leads to defective gastrulation movements and malformation of the embryonic axis with a reduction in the head structures in early frog embryos. These data, together with the observation that ErbBs are expressed early during frog embryogenesis, suggest that ErbBs regulate cell proliferation, movements and embryonic patterning during early Xenopus development. PMID:16258939

Lectins and substitution for helper function in anti-hapten responses in Xenopus laevis.

PubMed

Clothier, R H; James, H S; Ruben, L N; Balls, M

1984-08-01

Substitution by lectins for the carrier-priming requirement in thymus-dependent, antigen-binding responses in Xenopus laevis has been examined. Concanavalin A (Con A) was found to substitute for carrier priming in control, early-thymectomized and adult-thymectomized animals, but not in animals given a single, high dose of N-methyl-N-nitrosourea, which has a permanent effect on certain thymus-dependent functions in this species. Lipopolysaccharide and other lectins, such as peanut agglutinin and wheat germ agglutinin, were unable to substitute for carrier priming. These effects of Con A are discussed in terms of substitution via amplifier T cells or a helper T cell subset.

Expression patterns of Xenopus FGF receptor-like 1/nou-darake in early Xenopus development resemble those of planarian nou-darake and Xenopus FGF8.

PubMed

Hayashi, Shuichi; Itoh, Mari; Taira, Sumiko; Agata, Kiyokazu; Taira, Masanori

2004-08-01

Fibroblast growth factors (FGFs) mediate many cell-to-cell signaling events during early development. Nou-darake (ndk), a gene encoding an FGF receptor (FGFR)-like molecule, was found to be highly and specifically expressed in the head region of the planarian Dugesia japonica, and its functional analyses provided strong molecular evidence for the existence of a brain-inducing circuit based on the FGF signaling pathway. To analyze the role of ndk during vertebrate development, we isolated the Xenopus ortholog of ndk, the vertebrate FGFR-like 1 gene (XFGFRL1). Expression of XFGFRL1/Xndk was first detected in the anterior region at the late gastrula stage and dramatically increased at the early neurula stage in an overall anterior mesendodermal region, including the prechordal plate, paraxial mesoderm, anterior endoderm, and archenteron roof. This anterior expression pattern resembles that of ndk in planarians, suggesting that the expression of FGFRL1/ndk is conserved in evolution between these two distantly diverged organisms. During the tail bud stages, XFGFRL1/Xndk expression was detected in multiple regions, including the forebrain, eyes, midbrain-hindbrain boundary, otic vesicles, visceral arches, and somites. In many of these regions, XFGFRL1/Xndk was coexpressed with XFGF8, indicating that XFGFRL1/Xndk is a member of the XFGF8 synexpression group, which includes sprouty, sef, and isthmin. Copyright 2004 Wiley-Liss, Inc.

In vivo time-lapse imaging of cell proliferation and differentiation in the optic tectum of Xenopus laevis tadpoles

PubMed Central

Bestman, Jennifer E.; Lee-Osbourne, Jane; Cline, Hollis T.

2012-01-01

We analyzed the function of neural progenitors in the developing CNS of Xenopus laevis tadpoles using in vivo time-lapse confocal microscopy to collect images through the tectum at intervals of 2 to 24 hours over 3 days. Neural progenitor cells were labeled with fluorescent protein reporters based on expression of endogenous Sox2 transcription factor. With this construct, we identified Sox2-expressing cells as radial glia and as a component of the progenitor pool of cells in the developing tectum that gives rise to neurons and other radial glia. Lineage analysis of individual radial glia and their progeny demonstrated that less than 10% of radial glia undergo symmetric divisions resulting in two radial glia, while the majority of radial glia divide asymmetrically to generate neurons and radial glia. Time-lapse imaging revealed the direct differentiation of radial glia into neurons. Although radial glia may guide axons as they navigate to superficial tectum, we find no evidence that radial glia function as a scaffold for neuronal migration at early stages of tectal development. Over three days, the number of labeled cells increased 20%, as the fraction of radial glia dropped and the proportion of neuronal progeny increased to approximately 60% of the labeled cells. Tadpoles provided with short-term visual enhancement generated significantly more neurons, with a corresponding decrease in cell proliferation. Together these results demonstrate that radial glial cells are neural progenitors in the developing optic tectum and reveal that visual experience increases the proportion of neurons generated in an intact animal. PMID:22113462

Peptidomic analysis of the extensive array of host-defense peptides in skin secretions of the dodecaploid frog Xenopus ruwenzoriensis (Pipidae).

PubMed

Coquet, Laurent; Kolodziejek, Jolanta; Jouenne, Thierry; Nowotny, Norbert; King, Jay D; Conlon, J Michael

2016-09-01

The Uganda clawed frog Xenopus ruwenzoriensis with a karyotype of 2n=108 is one of the very few vertebrates with dodecaploid status. Peptidomic analysis of norepinephrine-stimulated skin secretions from this species led to the isolation and structural characterization of 23 host-defense peptides belonging to the following families: magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 6 peptides), xenopsin precursor fragment (XPF; 3 peptides), caerulein precursor fragment (CPF; 8 peptides), and caerulein precursor fragment-related peptide (CPF-RP; 3 peptides). In addition, the secretions contained caerulein, identical to the peptide from Xenopus laevis, and two peptides that were identified as members of the trefoil factor family (TFF). The data indicate that silencing of the host-defense peptide genes following polyploidization has been appreciable and non-uniform. Consistent with data derived from comparison of nucleotide sequences of mitochrondrial and nuclear genes, cladistic analyses based upon the primary structures of the host-defense peptides provide support for an evolutionary scenario in which X. ruwenzoriensis arose from an allopolyploidization event involving an octoploid ancestor of the present-day frogs belonging to the Xenopus amieti species group and a tetraploid ancestor of Xenopus pygmaeus. Copyright © 2016 Elsevier Inc. All rights reserved.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

PubMed Central

Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

2015-01-01

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis

PubMed Central

Lee, Ra Mi; Ryu, Rae Hyung; Jeong, Seong Won; Oh, Soo Jin; Huang, Hue; Han, Jin Soo; Lee, Chi Ho; Lee, C. Justin; Jan, Lily Yeh

2011-01-01

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA. PMID:21826170

Patterned femtosecond-laser ablation of Xenopus laevis melanocytes for studies of cell migration, wound repair, and developmental processes

PubMed Central

Mondia, Jessica P.; Adams, Dany S.; Orendorff, Ryan D.; Levin, Michael; Omenetto, Fiorenzo G.

2011-01-01

Ultrafast (femtosecond) lasers have become an important tool to investigate biological phenomena because of their ability to effect highly localized tissue removal in surgical applications. Here we describe programmable, microscale, femtosecond-laser ablation of melanocytes found on Xenopus laevis tadpoles, a technique that is applicable to biological studies in development, regeneration, and cancer research. We illustrate laser marking of individual melanocytes, and the drawing of patterns on melanocyte clusters to help track their migration and/or regeneration. We also demonstrate that this system can upgrade scratch tests, a technique used widely with cultured cells to study cell migration and wound healing, to the more realistic in vivo realm, by clearing a region of melanocytes and monitoring their return over time. In addition, we show how melanocyte ablation can be used for loss-of-function experiments by damaging neighboring tissue, using the example of abnormal tail regeneration following localized spinal cord damage. Since the size, shape, and depth of melanocytes vary as a function of tadpole age and melanocyte location (head or tail), an ablation threshold chart is given. Mechanisms of laser ablation are also discussed. PMID:21833375

Patterned femtosecond-laser ablation of Xenopus laevis melanocytes for studies of cell migration, wound repair, and developmental processes.

PubMed

Mondia, Jessica P; Adams, Dany S; Orendorff, Ryan D; Levin, Michael; Omenetto, Fiorenzo G

2011-08-01

Ultrafast (femtosecond) lasers have become an important tool to investigate biological phenomena because of their ability to effect highly localized tissue removal in surgical applications. Here we describe programmable, microscale, femtosecond-laser ablation of melanocytes found on Xenopus laevis tadpoles, a technique that is applicable to biological studies in development, regeneration, and cancer research. We illustrate laser marking of individual melanocytes, and the drawing of patterns on melanocyte clusters to help track their migration and/or regeneration. We also demonstrate that this system can upgrade scratch tests, a technique used widely with cultured cells to study cell migration and wound healing, to the more realistic in vivo realm, by clearing a region of melanocytes and monitoring their return over time. In addition, we show how melanocyte ablation can be used for loss-of-function experiments by damaging neighboring tissue, using the example of abnormal tail regeneration following localized spinal cord damage. Since the size, shape, and depth of melanocytes vary as a function of tadpole age and melanocyte location (head or tail), an ablation threshold chart is given. Mechanisms of laser ablation are also discussed.

Accelerated Gene Evolution and Subfunctionalization in thePseudotetraploid Frog Xenopus Laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hellsten, Uffe; Khokha, Mustafa K.; Grammar, Timothy C.

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.

Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis.

PubMed

Fukuzawa, Toshihiko

2010-10-01

Unusual light-reflecting pigment cells, "white pigment cells", specifically appear in the periodic albino mutant (a(p) /a(p)) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores.

Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins.

PubMed

Dorr, Ricardo; Ozu, Marcelo; Parisi, Mario

2007-04-15

Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

Evolution of Heat Sensors Drove Shifts in Thermosensation between Xenopus Species Adapted to Different Thermal Niches*

PubMed Central

Saito, Shigeru; Ohkita, Masashi; Saito, Claire T.; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

2016-01-01

Temperature is one of the most critical environmental factors affecting survival, and thus species that inhabit different thermal niches have evolved thermal sensitivities suitable for their respective habitats. During the process of shifting thermal niches, various types of genes expressed in diverse tissues, including those of the peripheral to central nervous systems, are potentially involved in the evolutionary changes in thermosensation. To elucidate the molecular mechanisms behind the evolution of thermosensation, thermal responses were compared between two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) adapted to different thermal environments. X. laevis was much more sensitive to heat stimulation than X. tropicalis at the behavioral and neural levels. The activity and sensitivity of the heat-sensing TRPA1 channel were higher in X. laevis compared with those of X. tropicalis. The thermal responses of another heat-sensing channel, TRPV1, also differed between the two Xenopus species. The species differences in Xenopus TRPV1 heat responses were largely determined by three amino acid substitutions located in the first three ankyrin repeat domains, known to be involved in the regulation of rat TRPV1 activity. In addition, Xenopus TRPV1 exhibited drastic species differences in sensitivity to capsaicin, contained in chili peppers, between the two Xenopus species. Another single amino acid substitution within Xenopus TRPV1 is responsible for this species difference, which likely alters the neural and behavioral responses to capsaicin. These combined subtle amino acid substitutions in peripheral thermal sensors potentially serve as a driving force for the evolution of thermal and chemical sensation. PMID:27022021

The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

PubMed

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

2015-11-01

In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport. Copyright © 2015 Elsevier Inc. All rights reserved.

Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis

PubMed Central

2010-01-01

Unusual light-reflecting pigment cells, “white pigment cells”, specifically appear in the periodic albino mutant (ap/ap) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores. PMID:20859642

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

PubMed

Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

2016-08-01

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Transdifferentiation from cornea to lens in Xenopus laevis depends on BMP signalling and involves upregulation of Wnt signalling

PubMed Central

2011-01-01

Background Surgical removal of the lens from larval Xenopus laevis results in a rapid transdifferention of central corneal cells to form a new lens. The trigger for this process is understood to be an induction event arising from the unprecedented exposure of the cornea to the vitreous humour that occurs following lens removal. The molecular identity of this trigger is unknown. Results Here, we have used a functional transgenic approach to show that BMP signalling is required for lens regeneration and a microarray approach to identify genes that are upregulated specifically during this process. Analysis of the array data strongly implicates Wnt signalling and the Pitx family of transcription factors in the process of cornea to lens transdifferentiation. Our analysis also captured several genes associated with congenital cataract in humans. Pluripotency genes, in contrast, were not upregulated, supporting the idea that corneal cells transdifferentiate without returning to a stem cell state. Several genes from the array were expressed in the forming lens during embryogenesis. One of these, Nipsnap1, is a known direct target of BMP signalling. Conclusions Our results strongly implicate the developmental Wnt and BMP signalling pathways in the process of cornea to lens transdifferentiation (CLT) in Xenopus, and suggest direct transdifferentiation between these two anterior eye tissues. PMID:21896182

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

PubMed

Miyamoto, Kei; Suzuki, Ken-Ichi T; 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.

Thyroid Hormone‐Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis

PubMed Central

Fujimoto, Kenta; Kajita, Mitsuko; Fu, Liezhen; Shi, Yun‐Bo; Ishizuya‐Oka, Atsuko

2016-01-01

Abstract In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real‐time reverse transcription‐polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up‐regulated during both natural and TH‐induced metamorphosis in a tissue‐specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up‐regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ‐secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH‐induced up‐regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2017;35:1028–1039 PMID:27870267

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

PubMed Central

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

2006-01-01

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization. PMID:16672307

Xenopus as a Model Organism for Birth Defects – Congenital Heart Disease and Heterotaxy

PubMed Central

Duncan, Anna R.; Khokha, Mustafa K.

2016-01-01

Congenital heart disease is the leading cause of birth defects, affecting 9 out of 1000 newborns each year. A particularly severe form of congenital heart disease is heterotaxy, a disorder of left-right development. Despite aggressive surgical management, patients with heterotaxy have poor survival rates and severe morbidity due to their complex congenital heart disease. Recent genetic analysis of affected patients has found novel candidate genes for heterotaxy although their underlying mechanisms remain unknown. In this review, we discuss the importance and challenges of birth defects research including high locus heterogeneity and few second alleles that make defining disease causality difficult. A powerful strategy moving forward is to analyze these candidate genes in a high-throughput human disease model. Xenopus is ideal for these studies. We present multiple examples demonstrating the power of Xenopus in discovery new biology from the analysis of candidate heterotaxy genes such as GALNT11, NEK2 and BCOR. These genes have diverse roles in embryos and have led to a greater understanding of complex signaling pathways and basic developmental biology. It is our hope that the mechanistic analysis of these candidate genes in Xenopus enabled by next generation sequencing of patients will provide clinicians with a greater understanding of patient pathophysiology allowing more precise and personalized medicine, to help them more effectively in the future. PMID:26910255

Full-grown oocytes from Xenopus laevis resume growth when placed in culture

PubMed Central

Wallace, Robin A.; Misulovin, Ziva; Etkin, Laurence D.

1981-01-01

When most full-grown, follicle cell-invested oocytes from Xenopus laevis are placed in an appropriate culture medium, they resume growth and remain physiologically healthy for at least 2-3 weeks. Rates of growth by full-grown oocytes in vitro generally approximate and can even exceed the most rapid growth rate achieved by vitellogenic oocytes in vivo. Resumption of oocyte growth can be correlated with the loss of investing follicle cells, which under normal conditions appear to interfere with vitellogenin and nutrient access to the oocyte. The final size reached by the oocyte within the ovary is thus not an intrinsic property of the oocyte but is extrinsically imposed by the somatic environment. Images PMID:16593019

A glyphosate micro-emulsion formulation displays teratogenicity in Xenopus laevis.

PubMed

Bonfanti, Patrizia; Saibene, M; Bacchetta, R; Mantecca, P; Colombo, A

2018-02-01

Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup ® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup ® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup ® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup ® Power 2.0 may be related to the improved efficacy in delivering

The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis ▿

PubMed Central

Collart, Clara; Christov, Christo P.; Smith, James C.; Krude, Torsten

2011-01-01

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery. PMID:21791613

Abelson Interactor 1 (Abi1) and Its Interaction with Wiskott-Aldrich Syndrome Protein (Wasp) Are Critical for Proper Eye Formation in Xenopus Embryos*

PubMed Central

Singh, Arvinder; Winterbottom, Emily F.; Ji, Yon Ju; Hwang, Yoo-Seok; Daar, Ira O.

2013-01-01

Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process. PMID:23558677

Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites.

PubMed

Yasuhiko, Yukuto; Shiokawa, Koichiro; Mochizuki, Toshio; Asashima, Makoto; Yokoyama, Takahiko

2006-04-01

The homozygous inv (inversion of embryonic turning) mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs (Xinv-1 and Xinv-2) of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites (IQ motifs) are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs of Xenopus inv and mouse inv proteins may regulate their function in different ways.

Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes

PubMed Central

Karpinka, J. Brad; Fortriede, Joshua D.; Burns, Kevin A.; James-Zorn, Christina; Ponferrada, Virgilio G.; Lee, Jacqueline; Karimi, Kamran; Zorn, Aaron M.; Vize, Peter D.

2015-01-01

Xenbase (http://www.xenbase.org), the Xenopus frog model organism database, integrates a wide variety of data from this biomedical model genus. Two closely related species are represented: the allotetraploid Xenopus laevis that is widely used for microinjection and tissue explant-based protocols, and the diploid Xenopus tropicalis which is used for genetics and gene targeting. The two species are extremely similar and protocols, reagents and results from each species are often interchangeable. Xenbase imports, indexes, curates and manages data from both species; all of which are mapped via unique IDs and can be queried in either a species-specific or species agnostic manner. All our services have now migrated to a private cloud to achieve better performance and reliability. We have added new content, including providing full support for morpholino reagents, used to inhibit mRNA translation or splicing and binding to regulatory microRNAs. New genomes assembled by the JGI for both species and are displayed in Gbrowse and are also available for searches using BLAST. Researchers can easily navigate from genome content to gene page reports, literature, experimental reagents and many other features using hyperlinks. Xenbase has also greatly expanded image content for figures published in papers describing Xenopus research via PubMedCentral. PMID:25313157

Effects of 4-tert-octylphenol on Xenopus tropicalis in a Long Term Exposure

EPA Science Inventory

Endocrine disrupting chemicals that activate the estrogen receptor are routinely detected in the environment and are a concern for the health of both exposed humans and indigenous wildlife. We exposed the western clawed frog (Xenopus tropicalis) to the weak estrogen octylphenol f...

Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes.

PubMed

Ito, Y; Yokoyama, S; Higashida, H

1992-05-22

Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.

Thyroid Hormone-Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis.

PubMed

Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Fu, Liezhen; Shi, Yun-Bo; Ishizuya-Oka, Atsuko

2017-04-01

In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real-time reverse transcription-polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up-regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ-secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2017;35:1028-1039. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Characterization of Pax3 and Sox10 transgenic Xenopus laevis embryos as tools to study neural crest development.

PubMed

Alkobtawi, Mansour; Ray, Heather; Barriga, Elias H; Moreno, Mauricio; Kerney, Ryan; Monsoro-Burq, Anne-Helene; Saint-Jeannet, Jean-Pierre; Mayor, Roberto

2018-03-06

The neural crest is a multipotent population of cells that originates a variety of cell types. Many animal models are used to study neural crest induction, migration and differentiation, with amphibians and birds being the most widely used systems. A major technological advance to study neural crest development in mouse, chick and zebrafish has been the generation of transgenic animals in which neural crest specific enhancers/promoters drive the expression of either fluorescent proteins for use as lineage tracers, or modified genes for use in functional studies. Unfortunately, no such transgenic animals currently exist for the amphibians Xenopus laevis and tropicalis, key model systems for studying neural crest development. Here we describe the generation and characterization of two transgenic Xenopus laevis lines, Pax3-GFP and Sox10-GFP, in which GFP is expressed in the pre-migratory and migratory neural crest, respectively. We show that Pax3-GFP could be a powerful tool to study neural crest induction, whereas Sox10-GFP could be used in the study of neural crest migration in living embryos. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Genome of the Western Clawed Frog Xenopus tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hellsten, Uffe; Harland, Richard M.; Gilchrist, Michael J.

2009-10-01

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with humanmore » and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.« less

Xenopus laevis in Developmental and Molecular Biology.

ERIC Educational Resources Information Center

Dawid, Igor B.; Sargent, Thomas D.

1988-01-01

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

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

Atmospheric pressure plasma accelerates tail regeneration in tadpoles Xenopus laevis

NASA Astrophysics Data System (ADS)

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

2017-08-01

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

Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

PubMed Central

Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2014-01-01

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

The Xenopus Maternal-to-Zygotic Transition from the Perspective of the Germline.

PubMed

Yang, Jing; Aguero, Tristan; King, Mary Lou

2015-01-01

In Xenopus, the germline is specified by the inheritance of germ-plasm components synthesized at the beginning of oogenesis. Only the cells in the early embryo that receive germ plasm, the primordial germ cells (PGCs), are competent to give rise to the gametes. Thus, germ-plasm components continue the totipotent potential exhibited by the oocyte into the developing embryo at a time when most cells are preprogrammed for somatic differentiation as dictated by localized maternal determinants. When zygotic transcription begins at the mid-blastula transition, the maternally set program for somatic differentiation is realized. At this time, genetic control is ceded to the zygotic genome, and developmental potential gradually becomes more restricted within the primary germ layers. PGCs are a notable exception to this paradigm and remain transcriptionally silent until the late gastrula. How the germ-cell lineage retains full potential while somatic cells become fate restricted is a tale of translational repression, selective degradation of somatic maternal determinants, and delayed activation of zygotic transcription. © 2015 Elsevier Inc. All rights reserved.

Xenopus laevis oocyte maturation is affected by metal chlorides.

PubMed

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

2015-08-01

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

Gene Expression in Pre-MBT Embryos and Activation of Maternally-Inherited Program of Apoptosis to be Executed at around MBT as a Fail-Safe Mechanism in Xenopus Early Embryogenesis

PubMed Central

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Uchiyama, Hiroaki; Kuroyanagi, Shinsaku; Takai, Jun-Ichi; Takahashi, Senji; Kajitani, Masayuki; Kaito, Chikara; Sekimizu, Kazuhisa; Takayama, Eiji; Igarashi, Kazuei; Hara, Hiroshi

2008-01-01

S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethione (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation of caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastocoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extents of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a �

Delayed fertilization of anuran amphibian (Xenopus) eggs leads to reduced numbers of primordial germ cells

NASA Technical Reports Server (NTRS)

Wakahara, M.; Neff, A. W.; Malacinski, G. M.

1984-01-01

Several media were tested for the extent to which they promoted high fertilization efficiencies in ovulated, stripped Xenopus eggs. One medium was selected for maintaining eggs in a 'delayed fertilization' (DelF) condition. DelF eggs displayed several unusual characteristics, including shift of the center of gravity, prominent sperm entrance site, and occasional polyspermy. The frequency of normal pattern formation varied according to the length of time eggs were maintained in the DelF condition. Various developmental abnormalities were observed during gastrulation, neurulation, and organogenesis. Most abnormalities appeared, however, to be related to morphogenesis of the endoderm. Primordial germ cell (PGC) development was examined in DelF eggs which displayed normal external morphological features at the swimming tadpole stage. PGC counts were usually normal in short-duration (eg, 5 hr) DelF eggs, but frequently substantially reduced or completely diminished in longer-duration (eg, 25h) tadpoles. Six spawnings were compared and shown to exhibit considerable variability in fertility, morphogenesis, and PGC development. Yolk platelet shifts and developmental parameters were examined in two additional spawnings. The subcortical cytoplasm in which the germ plasm is normally localized appeared to be disrupted in longer duration DelF eggs. That observation may account for low PGC counts in DelF tadpoles.

G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/β-catenin signaling and are essential for head formation in Xenopus.

PubMed

Miyagi, Asuka; Negishi, Takefumi; Yamamoto, Takamasa S; Ueno, Naoto

2015-11-01

Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/β-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/β-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/β-catenin signaling by promoting β-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/β-catenin signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes.

PubMed

Karpinka, J Brad; Fortriede, Joshua D; Burns, Kevin A; James-Zorn, Christina; Ponferrada, Virgilio G; Lee, Jacqueline; Karimi, Kamran; Zorn, Aaron M; Vize, Peter D

2015-01-01

Xenbase (http://www.xenbase.org), the Xenopus frog model organism database, integrates a wide variety of data from this biomedical model genus. Two closely related species are represented: the allotetraploid Xenopus laevis that is widely used for microinjection and tissue explant-based protocols, and the diploid Xenopus tropicalis which is used for genetics and gene targeting. The two species are extremely similar and protocols, reagents and results from each species are often interchangeable. Xenbase imports, indexes, curates and manages data from both species; all of which are mapped via unique IDs and can be queried in either a species-specific or species agnostic manner. All our services have now migrated to a private cloud to achieve better performance and reliability. We have added new content, including providing full support for morpholino reagents, used to inhibit mRNA translation or splicing and binding to regulatory microRNAs. New genomes assembled by the JGI for both species and are displayed in Gbrowse and are also available for searches using BLAST. Researchers can easily navigate from genome content to gene page reports, literature, experimental reagents and many other features using hyperlinks. Xenbase has also greatly expanded image content for figures published in papers describing Xenopus research via PubMedCentral. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Prolonged in vivo imaging of Xenopus laevis.

PubMed

Hamilton, Paul W; Henry, Jonathan J

2014-08-01

While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS-222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Developmental Dynamics 243:1011-1019, 2014. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Transcriptional regulators in the Hippo signaling pathway control organ growth in Xenopus tadpole tail regeneration.

PubMed

Hayashi, Shinichi; Ochi, Haruki; Ogino, Hajime; Kawasumi, Aiko; Kamei, Yasuhiro; Tamura, Koji; Yokoyama, Hitoshi

2014-12-01

The size and shape of tissues are tightly controlled by synchronized processes among cells and tissues to produce an integrated organ. The Hippo signaling pathway controls both cell proliferation and apoptosis by dual signal-transduction states regulated through a repressive kinase cascade. Yap1 and Tead, transcriptional regulators that act downstream of the Hippo signaling kinase cascade, have essential roles in regulating cell proliferation. In amphibian limb or tail regeneration, the local tissue outgrowth terminates when the correct size is reached, suggesting that organ size is strictly controlled during epimorphic organ-level regeneration. We recently demonstrated that Yap1 is required for the regeneration of Xenopus tadpole limb buds (Hayashi et al., 2014, Dev. Biol. 388, 57-67), but the molecular link between the Hippo pathway and organ size control in vertebrate epimorphic regeneration is not fully understood. To examine the requirement of Hippo pathway transcriptional regulators in epimorphic regeneration, including organ size control, we inhibited these regulators during Xenopus tadpole tail regeneration by overexpressing a dominant-negative form of Yap (dnYap) or Tead4 (dnTead4) under a heat-shock promoter in transgenic animal lines. Each inhibition resulted in regeneration defects accompanied by reduced cell mitosis and increased apoptosis. Single-cell gene manipulation experiments indicated that Tead4 cell-autonomously regulates the survival of neural progenitor cells in the regenerating tail. In amphibians, amputation at the proximal level of the tail (deep amputation) results in faster regeneration than that at the distal level (shallow amputation), to restore the original-sized tail with similar timing. However, dnTead4 overexpression abolished the position-dependent differential growth rate of tail regeneration. These results suggest that the transcriptional regulators in the Hippo pathway, Tead4 and Yap1, are required for general vertebrate

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...

Genetic screens for mutations affecting development of Xenopus tropicalis.

PubMed

Goda, Tadahiro; Abu-Daya, Anita; Carruthers, Samantha; Clark, Matthew D; Stemple, Derek L; Zimmerman, Lyle B

2006-06-01

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

PubMed

Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

1998-01-08

In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Developing Xenopus Laevis as a Model to Screen Drugs for Fragile X Syndrome

DTIC Science & Technology

2013-10-01

several candidate treatments for Fragile X Syndrome have gone to clinical trials. Though promising, no treatment has yet been approved. This sad ...Xenopus laevis tadpoles. J Comp Neurol 520, 401-433. Dong, W., Lee, R.H., Xu, H., Yang, S., Pratt, K.G., Cao, V., Song , Y.K., Nurmikko, A., and

Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis

PubMed Central

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

2012-01-01

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

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

The cloning and characterization of a localized maternal transcript in Xenopus laevis whose zygotic counterpart is detected in the CNS.

PubMed

Reddy, B A; Kloc, M; Etkin, L D

1992-12-01

We have cloned a cDNA (xlan4) from a Xenopus laevis oocyte cDNA library whose cognate mRNA is localized in the animal pole region of full grown oocytes. The cDNA can be translated in vitro to produce a predicted size protein of 35 kDa and, is also expressed in E. coli as a fusion protein. The conceptual protein encoded by the xlan4 cDNA is 17.5% proline rich and possesses several PEST sequences found in proteins with short half-lives. The xlan4 mRNA is 2.6 kb and during early development its titer decreases until the neurula stage after which it begins to reaccumulate. Northern blots on dissected embryos and in situ hybridization revealed that the zygotic expression is limited to the dorsal axial structures consisting primarily of the CNS. UV irradiation of the vegetal pole region immediately following fertilization that produces ventralized embryos results in a loss of zygotic xlan4 expression. In the adult, xlan4 mRNA is limited primarily to the brain. The presence of this mRNA in animal pole region which contributes to the future neural cell lineages suggests that this gene product may function either in the specification of neural cell types or in a neural specific function.

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

Pattern formation in early embryogenesis of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mglinets, V.A.

1995-07-01

Establishment of egg polarity, separation of germ layers, and the appearance of animal-vegetal, dorsoventral, and anteroposterior axes in Xenopus laevis embryos are considered. The control of these processes by gene coding for growth factors, protooncogens, and homeobox-containing genes is also been reviewed.

A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole.

PubMed

Sato, Kentaro; Umesono, Yoshihiko; Mochii, Makoto

2018-01-15

Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp). The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC. We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-β or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation. These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of a New Decision Tree to Rapidly Screen Chemical Estrogenic Activities of Xenopus laevis.

PubMed

Wang, Ting; Li, Weiying; Zheng, Xiaofeng; Lin, Zhifen; Kong, Deyang

2014-02-01

During the last past decades, there is an increasing number of studies about estrogenic activities of the environmental pollutants on amphibians and many determination methods have been proposed. However, these determination methods are time-consuming and expensive, and a rapid and simple method to screen and test the chemicals for estrogenic activities to amphibians is therefore imperative. Herein is proposed a new decision tree formulated not only with physicochemical parameters but also a biological parameter that was successfully used to screen estrogenic activities of the chemicals on amphibians. The biological parameter, CDOCKER interaction energy (Ebinding ) between chemicals and the target proteins was calculated based on the method of molecular docking, and it was used to revise the decision tree formulated by Hong only with physicochemical parameters for screening estrogenic activity of chemicals in rat. According to the correlation between Ebinding of rat and Xenopus laevis, a new decision tree for estrogenic activities in Xenopus laevis is finally proposed. Then it was validated by using the randomly 8 chemicals which can be frequently exposed to Xenopus laevis, and the agreement between the results from the new decision tree and the ones from experiments is generally satisfactory. Consequently, the new decision tree can be used to screen the estrogenic activities of the chemicals, and combinational use of the Ebinding and classical physicochemical parameters can greatly improves Hong's decision tree. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

PubMed Central

Cahu, Julie; Olichon, Aurelien; Hentrich, Christian; Schek, Henry; Drinjakovic, Jovana; Zhang, Cunjie; Doherty-Kirby, Amanda; Lajoie, Gilles; Surrey, Thomas

2008-01-01

Background Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. Methodology/Principal Findings We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. Conclusions/Significance These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein. PMID:19079595

Xenopus in Space and Time: Fossils, Node Calibrations, Tip-Dating, and Paleobiogeography.

PubMed

Cannatella, David

2015-01-01

Published data from DNA sequences, morphology of 11 extant and 15 extinct frog taxa, and stratigraphic ranges of fossils were integrated to open a window into the deep-time evolution of Xenopus. The ages and morphological characters of fossils were used as independent datasets to calibrate a chronogram. We found that DNA sequences, either alone or in combination with morphological data and fossils, tended to support a close relationship between Xenopus and Hymenochirus, although in some analyses this topology was not significantly better than the Pipa + Hymenochirus topology. Analyses that excluded DNA data found strong support for the Pipa + Hymenochirus tree. The criterion for selecting the maximum age of the calibration prior influenced the age estimates, and our age estimates of early divergences in the tree of frogs are substantially younger than those of published studies. Node-dating and tip-dating calibrations, either alone or in combination, yielded older dates for nodes than did a root calibration alone. Our estimates of divergence times indicate that overwater dispersal, rather than vicariance due to the splitting of Africa and South America, may explain the presence of Xenopus in Africa and its closest fossil relatives in South America.

Confocal Imaging of Early Heart Development in Xenopus laevis

PubMed Central

Kolker, Sandra J.; Tajchman, Urszula; Weeks, Daniel L.

2013-01-01

Xenopus laevis provides a number of advantages for studies on cardiovascular development. The embryos are fairly large, easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24–46 are presented. PMID:10644411

Functional Reintegration of Sensory Neurons and Transitional Dendritic Reduction of Mitral/Tufted Cells during Injury-Induced Recovery of the Larval Xenopus Olfactory Circuit.

PubMed

Hawkins, Sara J; Weiss, Lukas; Offner, Thomas; Dittrich, Katarina; Hassenklöver, Thomas; Manzini, Ivan

2017-01-01

Understanding the mechanisms involved in maintaining lifelong neurogenesis has a clear biological and clinical interest. In the present study, we performed olfactory nerve transection on larval Xenopus to induce severe damage to the olfactory circuitry. We surveyed the timing of the degeneration, subsequent rewiring and functional regeneration of the olfactory system following injury. A range of structural labeling techniques and functional calcium imaging were performed on both tissue slices and whole brain preparations. Cell death of olfactory receptor neurons and proliferation of stem cells in the olfactory epithelium were immediately increased following lesion. New olfactory receptor neurons repopulated the olfactory epithelium and once again showed functional responses to natural odorants within 1 week after transection. Reinnervation of the olfactory bulb (OB) by newly formed olfactory receptor neuron axons also began at this time. Additionally, we observed a temporary increase in cell death in the OB and a subsequent loss in OB volume. Mitral/tufted cells, the second order neurons of the olfactory system, largely survived, but transiently lost dendritic tuft complexity. The first odorant-induced responses in the OB were observed 3 weeks after nerve transection and the olfactory network showed signs of major recovery, both structurally and functionally, after 7 weeks.

Functional Reintegration of Sensory Neurons and Transitional Dendritic Reduction of Mitral/Tufted Cells during Injury-Induced Recovery of the Larval Xenopus Olfactory Circuit

PubMed Central

Hawkins, Sara J.; Weiss, Lukas; Offner, Thomas; Dittrich, Katarina; Hassenklöver, Thomas; Manzini, Ivan

2017-01-01

Understanding the mechanisms involved in maintaining lifelong neurogenesis has a clear biological and clinical interest. In the present study, we performed olfactory nerve transection on larval Xenopus to induce severe damage to the olfactory circuitry. We surveyed the timing of the degeneration, subsequent rewiring and functional regeneration of the olfactory system following injury. A range of structural labeling techniques and functional calcium imaging were performed on both tissue slices and whole brain preparations. Cell death of olfactory receptor neurons and proliferation of stem cells in the olfactory epithelium were immediately increased following lesion. New olfactory receptor neurons repopulated the olfactory epithelium and once again showed functional responses to natural odorants within 1 week after transection. Reinnervation of the olfactory bulb (OB) by newly formed olfactory receptor neuron axons also began at this time. Additionally, we observed a temporary increase in cell death in the OB and a subsequent loss in OB volume. Mitral/tufted cells, the second order neurons of the olfactory system, largely survived, but transiently lost dendritic tuft complexity. The first odorant-induced responses in the OB were observed 3 weeks after nerve transection and the olfactory network showed signs of major recovery, both structurally and functionally, after 7 weeks. PMID:29234276

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos.

PubMed

Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra

2010-01-01

Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

Adenosine A1 receptors modulate high voltage-activated Ca2+ currents and motor pattern generation in the Xenopus embryo

PubMed Central

Brown, Paul; Dale, Nicholas

2000-01-01

Adenosine causes voltage- and non-voltage-dependent inhibition of high voltage-activated (HVA) Ca2+ currents in Xenopus laevis embryo spinal neurons. As this inhibition can be blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and mimicked by N6-cyclopentyladenosine (CPA) it appears to be mediated by A1 receptors. Agents active at A2 receptors either were without effect or could be blocked by DPCPX. AMP had no agonist action on these receptors. By using ω-conotoxin GVIA we found that adenosine inhibited an N-type Ca2+ current as well as a further unidentified HVA current that was insensitive to dihydropyridines, ω-agatoxin TK and ω-conotoxin MVIIC. Both types of current were subject to voltage- and non-voltage-dependent inhibition. We used CPA and DPCPX to test whether A1 receptors regulated spinal motor pattern generation in spinalized Xenopus embryos. DPCPX caused a near doubling of, while CPA greatly shortened, the length of swimming episodes. In addition, DPCPX slowed, while CPA greatly speeded up, the rate of run-down of motor activity. Our results demonstrate a novel action of A1 receptors in modulating spinal motor activity. Furthermore they confirm that adenosine is produced continually throughout swimming episodes and acts to cause the eventual termination of activity. PMID:10856119

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.

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Reproductive Maturation of the Tropical Clawed Frog, Xenopus tropicalis

EPA Science Inventory

The model species Xenopus tropicalis is being widely used in developmental biology and amphibian toxicology studies. In order to increase our understanding of the role of steroid hormones in maturation in this species, we collected baseline reproductive data from metamorphosis t...

Host-defense and trefoil factor family peptides in skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae).

PubMed

Conlon, J Michael; Mechkarska, Milena; Kolodziejek, Jolanta; Leprince, Jérôme; Coquet, Laurent; Jouenne, Thierry; Vaudry, Hubert; Nowotny, Norbert; King, Jay D

2015-10-01

Peptidomic analysis of norepinephrine-stimulated skin secretions from the octoploid Mawa clawed frog Xenopus boumbaensis Loumont, 1983 led to the identification and characterization of 15 host-defense peptides belonging to the magainin (two peptides), peptide glycine-leucine-amide (PGLa; three peptides), xenopsin precursor fragment (XPF; three peptides), caerulein precursor fragment (CPF; two peptides), and caerulein precursor fragment-related peptide (CPF-RP; five peptides) families. In addition, caerulein and three peptides with structural similarity to the trefoil factor family (TFF) peptides, xP2 and xP4 from Xenopus laevis were also present in the secretions. Consistent with data from comparisons of the nucleotides sequence of mitochondrial and nuclear genes, the primary structures of the peptides suggest a close phylogenetic relationship between X. boumbaensis and the octoploid frogs Xenopus amieti and Xenopus andrei. As the three species occupy disjunct ranges within Cameroon, it is suggested that they diverged from a common ancestor by allopatric speciation. Copyright © 2015 Elsevier Inc. All rights reserved.

Developing Laryngeal Muscle of Xenopus laevis as a Model System: Androgen-Driven Myogenesis Controls Fiber Type Transformation

PubMed Central

Nasipak, Brian; Kelley, Darcy B.

2014-01-01

The developmental programs that contribute to myogenic stem cell proliferation and muscle fiber differentiation control fiber numbers and twitch type. In this study, we describe the use of an experimental model system—androgen-regulated laryngeal muscle of juvenile clawed frogs, Xenopus laevis—to examine the contribution of proliferation by specific populations of myogenic stem cells to expression of the larynx-specific myosin heavy chain isoform, LM. Androgen treatment of juveniles (Stage PM0) resulted in up-regulation of an early (Myf-5) and a late (myogenin) myogenic regulatory factor; the time course of LM up-regulation tracked that of myogenin. Myogenic stem cells stimulated to proliferate by androgen include a population that expresses Pax-7, a marker for the satellite cell myogenic stem cell population. Since androgen can switch muscle fiber types from fast to slow even in denervated larynges, we developed an ex vivo culture system to explore the relation between proliferation and LM expression. Cultured whole larynges maintain sensitivity to androgen, increasing in size and LM expression. Blockade of cell proliferation with cis-platin prevents the switch from slow to fast twitch muscle fibers as assayed by ATPase activity. Blockade of cell proliferation in vivo also resulted in inhibition of LM expression. Thus, both in vivo and ex vivo, inhibition of myogenic stem cell proliferation blocks androgen-induced LM expression and fiber type switching in juveniles. PMID:21954146

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

PubMed

Hara, Yuki; Merten, Christoph A

2015-06-08

Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion. Copyright © 2015 Elsevier Inc. All rights reserved.

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

PubMed Central

2012-01-01

Background Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations. Results Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres. Conclusions Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms. PMID:22369050

Planar induction of convergence and extension of the neural plate by the organizer of Xenopus.

PubMed

Keller, R; Shih, J; Sater, A K; Moreno, C

1992-03-01

This paper demonstrates that convergence and extension within the neural plate of Xenopus laevis are regulated by planar inductive interactions with the adjacent Spemann organizer. The companion article (Keller et al.: Developmental Dynamics 193:199-217, 1992) showed that the prospective hindbrain and spinal cord occupy a very short and very wide area just above the Spemann organizer in the early gastrula and that these regions converge and extend greatly during gastrulation and neurulation, using a sequence of radial and mediolateral cell intercalations. In this article, we show that "planar" contact of these regions with the organizer at their vegetal edge until stage 11 is sufficient to induce convergence and extension, after which their convergence and extension become autonomous. Grafts of the organizer in planar contact with uninduced ectodermal tissues induce these ectodermal tissues to converge and extend by a planar inductive signal from the organizer. Labeling of the inducing or responding tissues confirms that only planar interactions occur. Neural convergence and extension are actually hindered in explants deliberately constructed so that vertical interactions occur. These results show unambiguously that the Spemann organizer induces the extraordinary and precocious convergence and extension movements of the Xenopus neural plate by planar interactions acting over short distances.

Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moriyama, Yuki; Ohata, Yoshihisa; Mori, Shoko

Research highlights: {yields} Does famous anti-aging drug rapamycin work from the beginning of life? The answer is yes. {yields} This study shows that developmental speed of frog embryo was dose-dependently decreased by rapamycin treatment. {yields} In additions, morphogenetic effects such as less pigmentations and gut malformation are occurred by rapamycin. -- Abstract: Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood.more » Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.« less

Xenopus Zic3 controls notochord and organizer development through suppression of the Wnt/β-catenin signaling pathway.

PubMed

Fujimi, Takahiko J; Hatayama, Minoru; Aruga, Jun

2012-01-15

Zic3 controls neuroectodermal differentiation and left-right patterning in Xenopus laevis embryos. Here we demonstrate that Zic3 can suppress Wnt/β-catenin signaling and control development of the notochord and Spemann's organizer. When we overexpressed Zic3 by injecting its RNA into the dorsal marginal zone of 2-cell-stage embryos, the embryos lost mesodermal dorsal midline structures and showed reduced expression of organizer markers (Siamois and Goosecoid) and a notochord marker (Xnot). Co-injection of Siamois RNA partially rescued the reduction of Xnot expression caused by Zic3 overexpression. Because the expression of Siamois in the organizer region is controlled by Wnt/β-catenin signaling, we subsequently examined the functional interaction between Zic3 and Wnt signaling. Co-injection of Xenopus Zic RNAs and β-catenin RNA with a reporter responsive to the Wnt/β-catenin cascade indicated that Zic1, Zic2, Zic3, Zic4, and Zic5 can all suppress β-catenin-mediated transcriptional activation. In addition, co-injection of Zic3 RNA inhibited the secondary axis formation caused by ventral-side injection of β-catenin RNA in Xenopus embryos. Zic3-mediated Wnt/β-catenin signal suppression required the nuclear localization of Zic3, and involved the reduction of β-catenin nuclear transport and enhancement of β-catenin degradation. Furthermore, Zic3 co-precipitated with Tcf1 (a β-catenin co-factor) and XIC (I-mfa domain containing factor required for dorsoanterior development). The findings in this report produce a novel system for fine-tuning of Wnt/β-catenin signaling. Copyright © 2011. Published by Elsevier Inc.

Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes.

PubMed

Oron, Y; Gillo, B; Gershengorn, M C

1988-06-01

Xenopus laevis oocytes are giant cells suitable for studies of plasma membrane receptors and signal transduction pathways because of their capacity to express receptors after injection of heterologous mRNA. We studied depolarizing chloride currents evoked by acetylcholine (AcCho) in native oocytes ("intrinsic AcCho response"), by thyrotropin-releasing hormone (TRH) in oocytes injected with pituitary (GH3) cell RNA ("acquired TRH response"), and by AcCho in oocytes injected with rat brain RNA ("acquired AcCho response"). We found differences in the latencies and patterns of these responses and in the responsiveness to these agonists when applied to the animal or vegetal hemisphere, even though all of the responses are mediated by the same signal transduction pathway. The common intrinsic response to AcCho is characterized by minimal latency (0.86 +/- 0.05 sec), a rapid, transient depolarization followed by a distinct prolonged depolarization, and larger responses obtained after AcCho application at the vegetal rather than the animal hemisphere. By contrast, the acquired responses to TRH and AcCho are characterized by much longer latencies, 9.3 +/- 1.0 and 5.5 +/- 0.8 sec, respectively, and large rapid depolarizations followed by less distinct prolonged depolarizations. The responsiveness on the two hemispheres to TRH and AcCho in mRNA-injected oocytes is opposite to that for the common intrinsic AcCho response in that there is a much greater response when agonist is applied at the animal rather than the vegetal hemisphere. We suggest that the differences in these responses are caused by differences in the intrinsic properties of these receptors. Because different receptors appear to be segregated in the same oocyte in distinct localizations, Xenopus oocytes may be an important model system in which to study receptor sorting in polarized cells.

Left-Right Asymmetric Morphogenesis in the Xenopus Digestive System

USGS Publications Warehouse

Muller, Jennifer K.; Prather, D.R.; Nascone-Yoder, N. M.

2003-01-01

The morphogenetic mechanisms by which developing organs become left-right asymmetric entities are unknown. To investigate this issue, we compared the roles of the left and right sides of the Xenopus embryo during the development of anatomic asymmetries in the digestive system. Although both sides contribute equivalently to each of the individual digestive organs, during the initial looping of the primitive gut tube, the left side assumes concave topologies where the right side becomes convex. Of interest, the concave surfaces of the gut tube correlate with expression of the LR gene, Pitx2, and ectopic Pitx2 mRNA induces ectopic concavities in a localized manner. A morphometric comparison of the prospective concave and convex surfaces of the gut tube reveals striking disparities in their rate of elongation but no significant differences in cell proliferation. These results provide insight into the nature of symmetry-breaking morphogenetic events during left-right asymmetric organ development. ?? 2003 Wiley-Liss, Inc.

Isolation of Xenopus frizzled-10A and frizzled-10B genomic clones and their expression in adult tissues and embryos.

PubMed

Moriwaki, J; Kajita, E; Kirikoshi, H; Koike, J; Sagara, N; Yasuhiko, Y; Saitoh, T; Hirai, M; Katoh, M; Shiokawa, K

2000-11-19

Frizzled genes, encoding WNT receptors, play key roles in cell fate determination. Here, we isolated two Xenopus frizzled genes (Xfz10A and Xfz10B), probably reflecting pseudotetraploidy in Xenopus. Xfz10A (586 amino acids) and Xfz10B (580 amino acids) both encoded by a single exon, consisted of the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Xfz10A and Xfz10B were 97.0% identical at the amino acid level, and Xfz10B was 100% identical to previously reported Xfz9, yet Xfz10A was 85.3% and 62.4% identical to FZD10 and FZD9, respectively. Xfz10 mRNA appeared as 3.4 kb in adult tissues and embryos. RT-PCR analyses revealed the expression of more Xfz10A mRNA in stomach, kidney, eye, skeletal muscle, and skin, and more Xfz10B mRNA in heart and ovary, but in embryos, two mRNAs were equally expressed from the blastula stage with their peak expression at the late gastrula stage. The main site of Xfz10 mRNA expression was neural fold at the neurula stage and the dorsal region of midbrain, hindbrain, and spinal cord at the tadpole stage. These results suggest that Xfz10 has important roles in neural tissue formation. Copyright 2000 Academic Press.

The role of Mixer in patterning the early Xenopus embryo.

PubMed

Kofron, Matt; Wylie, Chris; Heasman, Janet

2004-05-01

The transcription factor VegT, is required in early Xenopus embryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm, was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.

The fungicide benomyl inhibits differentiation of neural tissue in the Xenopus embryo and animal cap explants.

PubMed

Yoon, Chun-Sik; Jin, Jung-Hyo; Park, Joo-Hung; Youn, Hyun-Joo; Cheong, Seon-Woo

2003-10-01

The toxic effect of benomyl on the embryogenesis of Xenopus laevis was investigated, and the tissues most affected by benomyl were identified. The toxicity of benomyl at various concentrations (5-20 microM) was tested with the Xenopus frog embryo teratogenesis assay (FETAX), used with slight modification. All test embryos subjected to 20 microM of benomyl died, and exposure to 10 and 15 microM benomyl produced growth inhibition and 11 types of severe external malformations. Histological examination of the test embryos showed dysplasia of the brain, eyes, intestine, otic vesicle, and muscle and swelling of the pronephric ducts and integuments. Among the tissues and organs affected, malformation of neural tissue was the most severe. The presumptive ectoderm isolated from st. 9 embryo was cultured in 10 ng/mL of activin A to induce neural tissue and mesoderm. When it was cultured with 10 ng/mL of activin A in the presence of 1 and 10 microM of benomyl, neural tissue induction was inhibited more severely than that of any other tissue. The gene expression of cultivated explants was investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay in order to study the inhibition of neural tissue by benomyl. The results showed that with increasing benomyl concentration, the expression of the neural-specific marker NCAM (neural cell adhesion molecule), was more strongly inhibited than the muscle-specific marker muscle actin. Electron micrographs of test explants showed many residual yolk platelets and mitochondrial degeneration. In the present investigation the most severe toxic effects of benomyl were seen in the nerve tissues of the Xenopus embryo. This inhibition of neural development may have been caused by the inhibition of the assembly of neural microtubules and by the effect of benomyl on neuronal proliferation and migration. Copyright 2003 Wiley Periodicals, Inc.

DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALMUS-PITUARY-THYROID AXIS IN XENOPUS LAEVIS

EPA Science Inventory

As recommended by the Endocrine Disrupter Screening and Testing Program Advisory Committee (EDSTAC), the US EPA has been developing a screening test capable of detecting effects of Endocrine Disrupting Chemicals (EDCS) on the hypothalamus-pituatary-thyroid (HPT) axis in Xenopus l...

DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALAMUS-PITUITARY-THYROID AXIS IN XENOPUS LAEVIS

EPA Science Inventory

As recommended by the Endocrine Disruptor Screening and Testing Program Advisory Committee (EDSTAC), the USEPA has been developing a screening test capable of detecting effects of Endocrine Disrupting Chemicals (EDCs) on the hypothalamus-pituitary-thyroid (HPT) axis in Xenopus la...

DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALAMUS-PITUATARY-THYROID AXIS IN XENOPUS LAEVIS

EPA Science Inventory

As recommended by the Endocrine Disruptor Screening and Testing Program Advisory Committee (EDSTAC), the USEPA has been developing a screening test capable of detecting effects of Endocrine Disrupting Chemicals (EDCs) on the hypothalamus-pituitary-thyroid (HPT) axis in Xenopus la...

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

PubMed Central

Sullivan, Kelly G.; Levin, Michael

2016-01-01

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

Psf2 plays important roles in normal eye development in Xenopus laevis

PubMed Central

Walter, Brian E.; Perry, Kimberly J.; Fukui, Lisa; Malloch, Erica L.; Wever, Jason

2008-01-01

Purpose Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a “sliding clamp.” Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. Methods A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. Results Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick

Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.

PubMed

Campbell, James H; Heikkila, John J

2018-04-23

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl 2 . In the present study, CdCl 2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl 2 -induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl 2 -induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl 2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures. Copyright © 2018 Elsevier Inc. All rights reserved.

Establishment of polarities in the oocyte of Xenopus laevis: the provisional axial symmetry of the full-grown oocyte of Xenopus laevis.

PubMed

Ubbels, G A

1997-04-01

We aimed at understanding of formation and function of the "Nieuwkoop Centre" in embryonic pattern formation. Discussed are data on genesis of cytoplasmic localizations in ovarian oocytes, transient modifications of cytoskeletal structures creating cytoplasmic asymmetries in fertilized eggs, the axis determining "vegetal cortical rotation" and fate of distinct cells, as shown by injection of specific molecular markers into particular blastomeres at specific times. Egg rotation and centrifugation suggested that sperm that gravity cooperate in symmetrization of the axially symmetrical anuran egg. After fertilization in space or in a fast rotating clinostate, axis formation and embryonic development were normal although the blastocoel was transiently abnormal. Normal tadpoles came back on Earth after ovulation, fertilization and culture in space. They metamorphosed normally and got healthy Earth-born F1 offspring. We conclude that neither sperm nor gravity are required for determination of the bilateral symmetry in the embryo of Xenopus laevis. In normal development sperm and gravity, either alone or in collaboration, may overrule an initial bilaterality inherent to, the full-grown oocyte, residing in some still unidentified component(s)/or mechanisms.

Protein 4.1 and its interaction with other cytoskeletal proteins in Xenopus laevis oogenesis.

PubMed

Carotenuto, Rosa; Petrucci, Tamara C; Correas, Isabel; Vaccaro, Maria C; De Marco, Nadia; Dale, Brian; Wilding, Martin

2009-06-01

In human red blood cells, protein 4.1 (4.1R) is an 80-kDa polypeptide that stabilizes the spectrin-actin network and anchors it to the plasma membrane. In non-erythroid cells there is a great variety of 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, which localize at various intracellular sites, including the nucleus. We studied protein 4.1R distribution in relation to beta-spectrin, actin and cytokeratin during Xenopus oogenesis. Immunoprecipitation experiments indicate that at least two isoforms of protein 4.1R are present in Xenopus laevis oocytes: a 56-kDa form in the cytoplasm and a 37-kDa form in the germinal vesicle (GV). Antibodies to beta-spectrin reveal two bands of 239 and 100 kDa in the cytoplasm. Coimmunoprecipitation experiments indicate that both the 37- and 56-kDa isoforms of protein 4.1R associate with the 100-kDa isoform of beta-spectrin. Moreover, the 56-kDa form coimmunoprecipitates with a cytokeratin of the same molecular weight. Confocal immunolocalization shows that protein 4.1R distribution is in the peripheral cytoplasm, in the mitochondrial cloud (MC) and in the GV of previtellogenic oocytes. In the cytoplasm of vitellogenic oocytes, a loose network of fibers stained by the anti-protein 4.1R antibody spreads across the cytoplasm. beta-Spectrin has a similar distribution. Protein 4.1R was found to colocalize with actin in the cortex of oocytes in the form of fluorescent dots. Double immunolocalization of protein 4.1R and cytokeratin depicts two separate networks that overlap throughout the whole cytoplasm. Protein 4.1R filaments partially colocalize with cytokeratin in both the animal and vegetal hemispheres. We hypothesize that protein 4.1R could function as a linker protein between cytokeratin and the actin-based cytoskeleton.

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells

PubMed Central

Kennedy, Allyson E.; Kandalam, Suraj; Olivares-Navarrete, Rene

2017-01-01

Since electronic cigarette (ECIG) introduction to American markets in 2007, vaping has surged in popularity. Many, including women of reproductive age, also believe that ECIG use is safer than traditional tobacco cigarettes and is not hazardous when pregnant. However, there are few studies investigating the effects of ECIG exposure on the developing embryo and nothing is known about potential effects on craniofacial development. Therefore, we have tested the effects of several aerosolized e-cigarette liquids (e-cigAM) in an in vivo craniofacial model, Xenopus laevis, as well as a mammalian neural crest cell line. Results demonstrate that e-cigAM exposure during embryonic development induces a variety of defects, including median facial clefts and midface hypoplasia in two of e-cigAMs tested e-cigAMs. Detailed quantitative analyses of the facial morphology revealed that nicotine is not the main factor in inducing craniofacial defects, but can exacerbate the effects of the other e-liquid components. Additionally, while two different e-cigAMs can have very similar consequences on facial appearances, there are subtle differences that could be due to the differences in e-cigAM components. Further assessment of embryos exposed to these particular e-cigAMs revealed cranial cartilage and muscle defects and a reduction in the blood supply to the face. Finally, the expression of markers for vascular and cartilage differentiation was reduced in a mammalian neural crest cell line corroborating the in vivo effects. Our work is the first to show that ECIG use could pose a potential hazard to the developing embryo and cause craniofacial birth defects. This emphasizes the need for more testing and regulation of this new popular product. PMID:28957438

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells.

PubMed

Kennedy, Allyson E; Kandalam, Suraj; Olivares-Navarrete, Rene; Dickinson, Amanda J G

2017-01-01

Since electronic cigarette (ECIG) introduction to American markets in 2007, vaping has surged in popularity. Many, including women of reproductive age, also believe that ECIG use is safer than traditional tobacco cigarettes and is not hazardous when pregnant. However, there are few studies investigating the effects of ECIG exposure on the developing embryo and nothing is known about potential effects on craniofacial development. Therefore, we have tested the effects of several aerosolized e-cigarette liquids (e-cigAM) in an in vivo craniofacial model, Xenopus laevis, as well as a mammalian neural crest cell line. Results demonstrate that e-cigAM exposure during embryonic development induces a variety of defects, including median facial clefts and midface hypoplasia in two of e-cigAMs tested e-cigAMs. Detailed quantitative analyses of the facial morphology revealed that nicotine is not the main factor in inducing craniofacial defects, but can exacerbate the effects of the other e-liquid components. Additionally, while two different e-cigAMs can have very similar consequences on facial appearances, there are subtle differences that could be due to the differences in e-cigAM components. Further assessment of embryos exposed to these particular e-cigAMs revealed cranial cartilage and muscle defects and a reduction in the blood supply to the face. Finally, the expression of markers for vascular and cartilage differentiation was reduced in a mammalian neural crest cell line corroborating the in vivo effects. Our work is the first to show that ECIG use could pose a potential hazard to the developing embryo and cause craniofacial birth defects. This emphasizes the need for more testing and regulation of this new popular product.

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

PubMed

Sommerville, John; Brumwell, Craig L; Politz, Joan C Ritland; Pederson, Thoru

2005-03-15

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

Developing Xenopus Laevis as a Model to Screen Drugs for Fragile X Syndrome

DTIC Science & Technology

2014-06-01

demonstrated the capacity to rescue the decreased FMRP expression by gene delivery. We characterized an innate visually-guided avoidance behavior in tadpoles ... tadpole is a unique model system that allows easy access to the nervous system at early stages of development, is amenable to in vivo gene...established quantitative in vivo imaging methods to knockdown and assay synthesis of FMRP in Xenopus tadpole brains. We also established 2 behavioral

Survey of the vestibulum, and behavior of Xenopus laevis larvae developed during a 7-days space flight.

PubMed

Briegleb, W; Neubert, J; Schatz, A; Klein, T; Kruse, B

1986-01-01

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D-1-Mission). The further evaluation of the "weightless-larvae" revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the l-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

NASA Astrophysics Data System (ADS)

Briegleb, W.; Neubert, J.; Schatz, A.; Klein, T.; Kruse, B.

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission). The further evaluation of the ``weightless-larvae'' revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes.

PubMed

Noh, S J; Kim, M J; Shim, S; Han, J K

1998-08-01

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural

The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

PubMed

Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

2014-01-01

The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

Asymmetric Distribution of Metals in the Xenopus Laevis Oocyte: a Synchrotron X-Ray Fluorescence Microprobe Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popescu, B.F.Gh.; Belak, Z.R.; Ignatyev, K.

2009-06-04

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was alsomore » concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.« less

Asymmetri Distribution of Metals in the Xenopus Laevis Oocyte: a Synchrotron X-Ray Fluorescence Microprobe Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popescu, B.F.G.; Belak, Z.R.; Ignatyev, K.

2009-04-29

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was alsomore » concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.« less

A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements.

PubMed

Guimond, A; Moss, T

1999-02-01

We have used a differential cloning approach to isolate ribosomal/non-ribosomal frontier sequences from Xenopus laevis. A ribosomal intergenic spacer sequence (IGS) was cloned and shown not to be physically linked with the ribosomal locus. This ribosomal orphon contained the IGS sequences found immediately downstream of the 28S gene and included an array of enhancer repetitions and a non-functional spacer promoter. The orphon sequence was flanked by a member of the novel 'Frt' low copy repetitive element family. Three individual Frt repeats were sequenced and all members of this family were shown to lie clustered at two chromosomal sites, one of which contained the ribosomal orphon. One of the Frt elements contained an insertion of 297 bp that showed extensive homology to sequences within at least three other Xenopus genes. Each homology region was flanked by members of the T2 family of short interspersed repetitive elements, (SINEs), and by its target insertion sequence, suggesting multiple translocation events. The data are discussed in terms of the evolution of the ribosomal gene locus.

Transient Early Embryonic Expression of Nkx2-5 Mutations Linked to Congenital Heart Defects in Human Causes Heart Defects in Xenopus laevis

PubMed Central

Bartlett, Heather L.; Sutherland, Lillian; Kolker, Sandra J.; Welp, Chelsea; Tajchman, Urszula; Desmarais, Vera; Weeks, Daniel L.

2007-01-01

Nkx2-5 is a homeobox containing transcription factor that is conserved and expressed in organisms that form hearts. Fruit flies lacking the gene (tinman) fail to form a dorsal vessel, mice that are homozygous null for Nkx2-5 form small, deformed hearts, and several human cardiac defects have been linked to dominant mutations in the Nkx2-5 gene. The Xenopus homologs (XNkx2-5) of two truncated forms of Nkx2-5 that have been identified in humans with congenital heart defects were used in the studies reported here. mRNAs encoding these mutations were injected into single cell Xenopus embryos, and heart development was monitored. Our results indicate that the introduction of truncated XNkx2-5 variants leads to three principle developmental defects. The atrial septum and the valve of the atrioventricular canal were both abnormal. In addition, video microscopic timing of heart contraction indicated that embryos injected with either mutant form of XNkx2-5 have conduction defects. PMID:17685485

Cortical Isolation from Xenopus laevis Oocytes and Eggs.

PubMed

Sive, Hazel L; Grainger, Robert M; Harland, Richard M

2007-06-01

INTRODUCTIONIn Xenopus laevis, the cortex is the layer of gelatinous cytoplasm that lies just below the plasma membrane of the egg. Rotation of the cortex relative to the deeper cytoplasm soon after fertilization is intimately linked to normal dorsal axis specification. The cortex can be dissected from the egg to analyze its composition and activity or to clone associated RNAs. This protocol describes a procedure for isolating the vegetal cortex of the fertilized egg.

Origin and voltage dependence of asparagine-induced depolarization in intestinal cells of Xenopus embryo.

PubMed Central

Bergman, C; Bergman, J

1985-01-01

The kinetics and voltage dependence of asparagine (Asn)-induced depolarization in endoderm cells from Xenopus laevis embryos were analysed using current-clamp techniques. The depolarization is assumed to reflect the activation of an amino acid membrane carrier; it is accompanied by a slight increase in membrane resistance and cannot be explained by only the electrogenic character of the Asn carrier. It is proposed that the Asn depolarization arises, at least in part, from the decrease of the permeability ratio PK/PNa indirectly associated with the Na-coupled amino acid uptake. At room temperature (20-23 degrees C) the Asn response develops according to a single exponential function whose time constant is correlated with the final level of depolarization. Both amplitude and rise time of the depolarization are sensitive to variations of membrane potential and changes in Asn or Na external concentrations. Lowering the temperature decreases the amplitude of the Asn depolarization and increases its rise time with a Q10 factor of two; the kinetics remain of the Michaelis-Menten type, with a marked decrease in delta Emax and no change in Km. When the holding potential is altered by depolarizing and hyperpolarizing currents, the Asn response varies according to a bell-shaped characteristic presenting an optimum near the normal resting level. Membrane depolarizations induced by Na/K-pump inhibitors or high external K concentrations reduce the size of the Asn response; repolarizing the cell by current injection does not reverse the inhibitory effect of external K ions. Hyperpolarizing the membrane with a K-free Ringer solution increases the amplitude of the Asn response. In all these cases a decrease in delta Emax accounts for the apparent voltage sensitivity of the carrier mechanism. When induced by alterations of [K]o, an additional change in Km is observed, suggesting a K/Na-competitive inhibition of the Asn carrier. The results are discussed in terms of the amino acid

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination.

PubMed

Snedden, Donald D; Bertke, Michelle M; Vernon, Dominic; Huber, Paul W

2013-07-01

The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

PubMed

Peter, A B; Schittny, J C; Niggli, V; Reuter, H; Sigel, E

1991-08-01

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo.

PubMed

Nagel, M; Winklbauer, R

1999-05-01

The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

PubMed

Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

2010-11-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

PubMed Central

Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

2010-01-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. PMID:20925114

Relating cell shape and mechanical stress in a spatially disordered epithelium using a vertex-based model

PubMed Central

Nestor-Bergmann, Alexander; Goddard, Georgina; Woolner, Sarah; Jensen, Oliver E

2018-01-01

Abstract Using a popular vertex-based model to describe a spatially disordered planar epithelial monolayer, we examine the relationship between cell shape and mechanical stress at the cell and tissue level. Deriving expressions for stress tensors starting from an energetic formulation of the model, we show that the principal axes of stress for an individual cell align with the principal axes of shape, and we determine the bulk effective tissue pressure when the monolayer is isotropic at the tissue level. Using simulations for a monolayer that is not under peripheral stress, we fit parameters of the model to experimental data for Xenopus embryonic tissue. The model predicts that mechanical interactions can generate mesoscopic patterns within the monolayer that exhibit long-range correlations in cell shape. The model also suggests that the orientation of mechanical and geometric cues for processes such as cell division are likely to be strongly correlated in real epithelia. Some limitations of the model in capturing geometric features of Xenopus epithelial cells are highlighted. PMID:28992197

Effects of 4-ter-Octylphenol on Xenopus tropicalis in a Long Term Exposure

DTIC Science & Technology

2011-03-17

Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per...subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1 . REPORT DATE 17...STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT We exposed Xenopus tropicalis to 1 , 3.3, 11 and 36 ug/L

Musculocontractural Ehlers–Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin

PubMed Central

Gouignard, Nadège; Maccarana, Marco; Strate, Ina; von Stedingk, Kristoffer; Malmström, Anders

2016-01-01

ABSTRACT Of all live births with congenital anomalies, approximately one-third exhibit deformities of the head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers–Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused by dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 in connective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1 is important for the generation of isolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial–mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo. Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and

Musculocontractural Ehlers-Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin.

PubMed

Gouignard, Nadège; Maccarana, Marco; Strate, Ina; von Stedingk, Kristoffer; Malmström, Anders; Pera, Edgar M

2016-06-01

Of all live births with congenital anomalies, approximately one-third exhibit deformities of the head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers-Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused by dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 in connective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1 is important for the generation of isolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial-mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in

Nondestructive Imaging of Internal Structures of Frog (Xenopus laevis) Embryos by Shadow-Projection X-Ray Microtomography

NASA Astrophysics Data System (ADS)

Aoki, Sadao; Yoneda, Ikuo; Nagai, Takeharu; Ueno, Naoto; Murakami, Kazuo

1994-04-01

Nondestructive high-resolution imaging of frog ( Xenopus laevis) embryos has been developed by X-ray microtomography. Shadow-projection X-ray microtomography with a brilliant fine focus laboratory X-ray source could image fine structures of Xenopus embryos which were embedded in paraffin wax. The imaging system enabled us to not only distinguish endoderm from ectoderm at the gastrula stage, but also to obtain a cross-section view of the tail bud embryo showing muscle, notochord and neural tube without staining. Furthermore, the distribution of myosin was also imaged in combination with whole-mount immunohistochemistry.

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.

Structure and expression of the Xenopus retinoblastoma gene.

PubMed

Destrée, O H; Lam, K T; Peterson-Maduro, L J; Eizema, K; Diller, L; Gryka, M A; Frebourg, T; Shibuya, E; Friend, S H

1992-09-01

We have cloned a Xenopus homology (XRb1) of the human retinoblastoma susceptibility gene. DNA sequence analysis shows that the XRb1 gene product is highly conserved in many regions. The leucine repeat motif and many of the potential cdc2 phosphorylation sites, as well as potential sites for other kinases, are retained. The region of the protein homologous to the SV40 T antigen binding site and the basic region directly C-terminal to the E1A binding site are all conserved. XRb1 gene expression at the RNA level was studied by Northern blot analysis. Transcripts of 4.2 and 10-kb are present as maternal RNA stores in the oocyte. While the 4.2-kb product is stable until at least the mid-blastula stage, the 10-kb transcript is selectively degraded. Between stages 11 and 13 the 10-kb transcript reappears and also a minor product of approximately 11 kb becomes apparent. Both the 4.2- and the 10-kb transcripts remain present until later stages of development and are also present in all adult tissues examined, although at differing levels. Antibodies raised against human p105Rb which recognize the protein product of the XRb1 gene, pXRb1, detect the Xenopus 99-kDa protein prior to the mid-blastula stage, but at lower levels than at later stages in development.

Persistent fibrosis, hypertrophy and sarcomere disorganisation after endoscopy-guided heart resection in adult Xenopus

PubMed Central

Girardot, Fabrice; Péricard, Louise; Demeneix, Barbara A.; Coen, Laurent; Chai, Norin

2017-01-01

Models of cardiac repair are needed to understand mechanisms underlying failure to regenerate in human cardiac tissue. Such studies are currently dominated by the use of zebrafish and mice. Remarkably, it is between these two evolutionary separated species that the adult cardiac regenerative capacity is thought to be lost, but causes of this difference remain largely unknown. Amphibians, evolutionary positioned between these two models, are of particular interest to help fill this lack of knowledge. We thus developed an endoscopy-based resection method to explore the consequences of cardiac injury in adult Xenopus laevis. This method allowed in situ live heart observation, standardised tissue amputation size and reproducibility. During the first week following amputation, gene expression of cell proliferation markers remained unchanged, whereas those relating to sarcomere organisation decreased and markers of inflammation, fibrosis and hypertrophy increased. One-month post-amputation, fibrosis and hypertrophy were evident at the injury site, persisting through 11 months. Moreover, cardiomyocyte sarcomere organisation deteriorated early following amputation, and was not completely recovered as far as 11 months later. We conclude that the adult Xenopus heart is unable to regenerate, displaying cellular and molecular marks of scarring. Our work suggests that, contrary to urodeles and teleosts, with the exception of medaka, adult anurans share a cardiac injury outcome similar to adult mammals. This observation is at odds with current hypotheses that link loss of cardiac regenerative capacity with acquisition of homeothermy. PMID:28278282

Deep cytoplasmic rearrangements in ventralized Xenopus embryos

NASA Technical Reports Server (NTRS)

Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

1993-01-01

Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

Lateral mobility of plasma membrane lipids in Xenopus eggs: regional differences related to animal/vegetal polarity become extreme upon fertilization.

PubMed

Dictus, W J; van Zoelen, E J; Tetteroo, P A; Tertoolen, L G; de Laat, S W; Bluemink, J G

1984-01-01

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (approximately 50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 X 10(-8) cm2/sec) in the animal than in the vegetal plasma membrane (D = 7.6 X 10(-8) cm2/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (greater than 100X) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D less than 10(-10) cm2/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 X 10(-8) cm2/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.

Mirror-image duplication of the primary axis and heart in Xenopus embryos by the overexpression of Msx-1 gene.

PubMed

Chen, Y; Solursh, M

1995-10-01

The Msx-1 gene (formerly known as Hox-7) is a member of a discrete subclass of homeobox-containing genes. Examination of the expression pattern of Msx-1 in murine and avian embryos suggests that this gene may be involved in the regionalization of the medio-lateral axis during earlier development. We have examined the possible functions of Xenopus Msx-1 during early Xenopus embryonic development by overexpression of the Msx-1 gene. Overexpression of Msx-1 causes a left-right mirror-image duplication of primary axial structures, including notochord, neural tube, somites, suckers, and foregut. The embryonic developing heart is also mirror-image duplicated, including looping directions and polarity. These results indicate that Msx-1 may be involved in the mesoderm formation as well as left-right patterning in the early Xenopus embryonic development.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

PubMed Central

Yu, Ling; Helms, My N.; Yue, Qiang; Eaton, Douglas C.

2008-01-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ∼3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells.

PubMed

Yu, Ling; Helms, My N; Yue, Qiang; Eaton, Douglas C

2008-11-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that

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...

Competition and feeding ecology in two sympatric Xenopus species (Anura: Pipidae)

PubMed Central

Vogt, Solveig; de Villiers, F. André; Ihlow, Flora; Rödder, Dennis

2017-01-01

The widespread African clawed frog (Xenopus laevis) occurs in sympatry with the IUCN Endangered Cape platanna (Xenopus gilli) throughout its entire range in the south-western Cape, South Africa. In order to investigate aspects of the interspecific competition between populations of X. laevis and X. gilli, an assessment of their niche differentiation was conducted through a comprehensive study on food composition and trophic niche structure at two study sites: the Cape of Good Hope (CoGH) and Kleinmond. A total of 399 stomach contents of X. laevis (n = 183) and X. gilli (n = 216) were obtained together with samples of available prey to determine food preferences using the Electivity index (E*), the Simpson’s index of diversity (1 − D), the Shannon index (H′), and the Pianka index (Ojk). Xenopus gilli diet was more diverse than X. laevis, particularly in Kleimond where the Shannon index was nearly double. Both species were found to consume large amounts of tadpoles belonging to different amphibian species, including congeners, with an overall higher incidence of anurophagy than previously recorded. However, X. laevis also feeds on adult X. gilli, thus representing a direct threat for the latter. While trophic niche overlap was 0.5 for the CoGH, it was almost 1 in Kleinmond, suggesting both species utilise highly congruent trophic niches. Further, subdividing the dataset into three size classes revealed overlap to be higher in small frogs in both study sites. Our study underlines the importance of actively controlling X. laevis at sites with X. gilli in order to limit competition and predation, which is vital for conservation of the south-western Cape endemic. PMID:28439453

Kinematics of gray crescent formation in Xenopus eggs: the displacement of subcortical cytoplasm relative to the egg surface.

PubMed

Vincent, J P; Oster, G F; Gerhart, J C

1986-02-01

Specification of the amphibian dorso-ventral axis takes place in the period between fertilization and first cleavage when the gray crescent forms. In the course of gray crescent formation, the egg reorganizes its periphery by a movement for which two descriptions have been given. According to the "rotation hypothesis," which was originated and supported for Rana eggs, the entire egg cortex rotates by an arc of 30 degrees relative to the stationary subcortical cytoplasm, leaving the crescent as a zone of altered coloration. The "contraction hypothesis" on the other hand, which was proposed for Xenopus and Rana eggs, asserts that there is a cortical contraction focused at the sperm entry point that leads to stretching of the opposite equatorial zone at which the crescent appears. We have reinvestigated the case of Xenopus eggs by imprinting one kind of fluorescent dye pattern (Nile blue) onto the subcortical cytoplasm and another kind (fluorescein-lectin) onto the egg surface. When the egg surface is held fixed by embedding the egg in gelatin, two major movements of the subcortical cytoplasm are observable. First, starting at time 0.3 (30% of the time between fertilization and first cleavage), the animal hemisphere subcortical cytoplasm converges toward a point, while the vegetal hemisphere is quiescent. This convergence continues with decreasing strength until approximately 0.8 of the first cell cycle. Second, at 0.45, an overall rotation of the animal and vegetal subcortical cytoplasm commences, superimposed on the animal hemisphere convergence. By 0.8-0.9 the rotation is complete, having accomplished a 30 degrees displacement of the subcortical cytoplasm relative to the surface. This rotation reliably locates the future dorsal midline of the embryo at the meridian on which the displacement of the subcortical cytoplasm is greatest in a vegetal direction. In normal unembedded eggs, when the egg surface is free to move, it rotates 30 degrees relative to the

Induction of mortality and malformation in Xenopus laevis embryos by water sources associated with field frog deformities.

PubMed Central

Burkhart, J G; Helgen, J C; Fort, D J; Gallagher, K; Bowers, D; Propst, T L; Gernes, M; Magner, J; Shelby, M D; Lucier, G

1998-01-01

Water samples from several ponds in Minnesota were evaluated for their capacity to induce malformations in embryos of Xenopus laevis. The FETAX assay was used to assess the occurrence of malformations following a 96-hr period of exposure to water samples. These studies were conducted following reports of high incidences of malformation in natural populations of frogs in Minnesota wetlands. The purpose of these studies was to determine if a biologically active agent(s) was present in the waters and could be detected using the FETAX assay. Water samples from ponds with high incidences of frog malformations (affected sites), along with water samples from ponds with unaffected frog populations (reference sites), were studied. Initial experiments clearly showed that water from affected sites induced mortality and malformation in Xenopus embryos, while water from reference sites had little or no effect. Induction of malformation was dose dependent and highly reproducible, both with stored samples and with samples taken at different times throughout the summer. The biological activity of the samples was reduced or eliminated when samples were passed through activated carbon. Limited evidence from these samples indicates that the causal factor(s) is not an infectious organism nor are ion concentrations or metals responsible for the effects observed. Results do indicate that the water matrix has a significant effect on the severity of toxicity. Based on the FETAX results and the occurrence of frog malformations observed in the field, these studies suggest that water in the affected sites contains one or more unknown agents that induce developmental abnormalities in Xenopus. These same factors may contribute to the increased incidence of malformation in native species. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9831545

Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus, Pipidae) from West and Central Africa

PubMed Central

Evans, Ben J.; Carter, Timothy F.; Greenbaum, Eli; Gvoždík, Václav; Kelley, Darcy B.; McLaughlin, Patrick J.; Pauwels, Olivier S. G.; Portik, Daniel M.; Stanley, Edward L.; Tinsley, Richard C.; Tobias, Martha L.; Blackburn, David C.

2015-01-01

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups). PMID:26672747

An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis.

PubMed

Buisson, Nicolas; Sirour, Cathy; Moreau, Nicole; Denker, Elsa; Le Bouffant, Ronan; Goullancourt, Aline; Darribère, Thierry; Bello, Valérie

2014-12-01

Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells. © 2014. Published by The Company of Biologists Ltd.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

2008-05-02

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

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

A Homolog of Subtilisin-Like Proprotein Convertase 7 Is Essential to Anterior Neural Development in Xenopus

PubMed Central

Senturker, Sema; Thomas, John Terrig; Mateshaytis, Jennifer; Moos, Malcolm

2012-01-01

Background Subtilisin-like Proprotein Convertase 7 (SPC7) is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the bone morphogenetic protein (BMP) family of signaling molecules. Other SPCs are known to be required during embryonic development but corresponding data regarding SPC7 have not been reported previously. Methodology/Principal Findings We demonstrated that Xenopus SPC7 (SPC7) was expressed predominantly in the developing brain and eye, throughout the neural plate initially, then more specifically in the lens and retina primordia as development progressed. Since no prior functional information has been reported for SPC7, we used gain- and loss-of-function experiments to investigate the possibility that it may also convey patterning or tissue specification information similarly to Furin, SPC4, and SPC6. Overexpression of SPC7 was without effect. In contrast, injection of SPC7 antisense morpholino oligonucleotides (MO) into a single blastomere at the 2- or 4-cell stage produced marked disruption of head structures; anophthalmia was salient. Bilateral injections suppressed head and eye formation completely. In parallel with suppression of eye and brain development by SPC7 knockdown, expression of early anterior neural markers (Sox2, Otx2, Rx2, and Pax6) and late eye-specific markers (β-Crystallin and Opsin), and of BMP target genes such as Tbx2 and Tbx3, was reduced or eliminated. Taken together, these findings suggest a critical role for SPC7–perhaps, at least in part, due to activation of one or more BMPs–in early patterning of the anterior neural plate and its derivatives. Conclusion/Significance SPC7 is required for normal development of the eye and brain, possibly through processing BMPs, though other potential substrates cannot be excluded. PMID:22761776

Host-defense peptides from skin secretions of Fraser's clawed frog Xenopus fraseri (Pipidae): Further insight into the evolutionary history of the Xenopodinae.

PubMed

Conlon, J Michael; Mechkarska, Milena; Kolodziejek, Jolanta; Nowotny, Norbert; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert

2014-12-01

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus fraseri Boulenger, 1905 (Pipidae) led to identification of 13 host-defense peptides. The primary structures of the peptides demonstrate that they belong to the magainin (3 peptides), peptide glycine-leucine-amide, PGLa (4 peptides), and xenopsin-precursor fragment, XPF (2 peptides) families, first identified in Xenopus laevis, together with caerulein precursor fragment-related peptides, CPF-RP (4 peptides), first identified in Silurana tropicalis. In addition, the secretions contain a molecular variant of xenopsin displaying the substitution Arg(4)→Lys compared with X. laevis xenopsin and peptide glycine-tyrosine-amide (PGYa) (GRIIPIYPEFERVFA KKVYPLY.NH2) whose function is unknown. The most potent antimicrobial peptide identified is CPF-RP-F1 (GFGSVLGKALKFGANLL.NH2) with MIC=12.5μM against Staphylococcus aureus and 50μM against Escherichia coli. On the basis of similarities in morphology and advertisement calls, X. fraseri has been placed in a species group that includes the octoploids Xenopus amieti and Xenopus andrei, and the tetraploid Xenopus pygmaeus. Cladistic analyses based upon the primary structures of magainin, PGLa, and CPF-RP peptides support a close evolutionary relationship between X. fraseri, X. amieti and X. andrei but suggest a more distant relationship with X. pygmaeus. Copyright © 2014 Elsevier Inc. All rights reserved.

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

EPA Science Inventory

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

A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes

PubMed Central

Englund, Ulrika H.; Gertow, Jens; Kågedal, Katarina; Elinder, Fredrik

2014-01-01

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na+ current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na+-channel blockers tetrodotoxin (1 µM) and amiloride (10 µM), while the Ca2+-channel blocker verapamil (50 µM) in the bath solution completely blocked the current. The intracellular Na+ concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na+ whith either K+ or Choline+. Prevention of this influx of Na+ also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na+ increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na+ channel is up-regulated during apoptosis and that influx of Na+ is a crucial step in the apoptotic process in Xenopus oocytes. PMID:24586320

Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation

PubMed Central

Tang, Guo-Qing; Maxwell, E. Stuart

2008-01-01

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus. PMID:18032731

Genes encoding Xenopus laevis Ig L chains: Implications for the evolution of [kappa] and [lambda] chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zezza, D.J.; Stewart, S.E.; Steiner, L.A.

1992-12-15

Xenopus laevis Ig contain two distinct types of L chains, designated [rho] or L1 and [sigma] or L2. The authors have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and Cmore » gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian [kappa] chains than to those of mammalian [lambda] chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J[kappa]. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V[kappa]. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5[prime]-flanking region, but diverges in sequence 5[prime] to position [minus]95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3[prime]-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3[prime]-ends, as in V[kappa]. Taken together, the data suggest that Xenopus L1 L chain genes are members of the [kappa] gene family. 80 refs., 9 figs.« less

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

PubMed Central

Minami, Ryosuke; Shimada, Masumi; Yokosawa, Hideyoshi; Kawahara, Hiroyuki

2007-01-01

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development. PMID:17428197

Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

PubMed

Nakazaki, Yuta; Tsuyama, Takashi; Azuma, Yutaro; Takahashi, Mikiko; Tada, Shusuke

2017-09-02

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Friend of GATA (FOG) Interacts with the Nucleosome Remodeling and Deacetylase Complex (NuRD) to Support Primitive Erythropoiesis in Xenopus laevis

PubMed Central

Mimoto, Mizuho S.; Christian, Jan L.

2012-01-01

Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect. PMID:22235346

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

PubMed

Strausfeld, U P; Howell, M; Descombes, P; Chevalier, S; Rempel, R E; Adamczewski, J; Maller, J L; Hunt, T; Blow, J J

1996-06-01

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Label-free real-time imaging of myelination in the Xenopus laevis tadpole by in vivo stimulated Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Hu, Chun-Rui; Zhang, Delong; Slipchenko, Mikhail N.; Cheng, Ji-Xin; Hu, Bing

2014-08-01

The myelin sheath plays an important role as the axon in the functioning of the neural system, and myelin degradation is a hallmark pathology of multiple sclerosis and spinal cord injury. Electron microscopy, fluorescent microscopy, and magnetic resonance imaging are three major techniques used for myelin visualization. However, microscopic observation of myelin in living organisms remains a challenge. Using a newly developed stimulated Raman scattering microscopy approach, we report noninvasive, label-free, real-time in vivo imaging of myelination by a single-Schwann cell, maturation of a single node of Ranvier, and myelin degradation in the transparent body of the Xenopus laevis tadpole.

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

PubMed

Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

2014-11-01

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas. © 2014 Proks et al.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

PubMed Central

Petry, Sabine; Groen, Aaron C.; Ishihara, Keisuke; Mitchison, Timothy J.; Vale, Ronald D.

2013-01-01

Summary The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance. PMID:23415226

Native serotonin 5-HT3 receptors expressed in Xenopus oocytes differ from homopentameric 5-HT3 receptors.

PubMed

van Hooft, J A; Kreikamp, A P; Vijverberg, H P

1997-09-01

Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT3 receptor (5-HT3R) agonists 2-methyl-5-HT, dopamine, and m-chlorophenylbiguanide on 5-HT3R native to N1E-115 cells and on homopentameric 5-HT3R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT3R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT3R-A(L) and 5-HT3R-A(S) receptors expressed in oocytes (4-8%). m-Chlorophenylbiguanide does not distinguish between 5-HT3R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT3R native to differentiated N1E-115 cells is conserved when poly(A)+ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT3R subunits, N1E-115 cells express additional proteins involved in 5-HT3R function.

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.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

PubMed

Corthésy, B; Cardinaux, J R; Claret, F X; Wahli, W

1989-12-01

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Analysis of Craniocardiac Malformations in Xenopus using Optical Coherence Tomography

PubMed Central

Deniz, Engin; Jonas, Stephan; Hooper, Michael; N. Griffin, John; Choma, Michael A.; Khokha, Mustafa K.

2017-01-01

Birth defects affect 3% of children in the United States. Among the birth defects, congenital heart disease and craniofacial malformations are major causes of mortality and morbidity. Unfortunately, the genetic mechanisms underlying craniocardiac malformations remain largely uncharacterized. To address this, human genomic studies are identifying sequence variations in patients, resulting in numerous candidate genes. However, the molecular mechanisms of pathogenesis for most candidate genes are unknown. Therefore, there is a need for functional analyses in rapid and efficient animal models of human disease. Here, we coupled the frog Xenopus tropicalis with Optical Coherence Tomography (OCT) to create a fast and efficient system for testing craniocardiac candidate genes. OCT can image cross-sections of microscopic structures in vivo at resolutions approaching histology. Here, we identify optimal OCT imaging planes to visualize and quantitate Xenopus heart and facial structures establishing normative data. Next we evaluate known human congenital heart diseases: cardiomyopathy and heterotaxy. Finally, we examine craniofacial defects by a known human teratogen, cyclopamine. We recapitulate human phenotypes readily and quantify the functional and structural defects. Using this approach, we can quickly test human craniocardiac candidate genes for phenocopy as a critical first step towards understanding disease mechanisms of the candidate genes. PMID:28195132

Cloning of an origin of DNA replication of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, S.; Taylor, J.H.

1980-09-01

DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the (/sup 3/H)-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 timesmore » as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated (/sup 3/H)thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment.« less

Sequential Turnovers of Sex Chromosomes in African Clawed Frogs (Xenopus) Suggest Some Genomic Regions Are Good at Sex Determination

PubMed Central

Furman, Benjamin L. S.; Evans, Ben J.

2016-01-01

Sexual differentiation is fundamentally important for reproduction, yet the genetic triggers of this developmental process can vary, even between closely related species. Recent studies have uncovered, for example, variation in the genetic triggers for sexual differentiation within and between species of African clawed frogs (genus Xenopus). Here, we extend these discoveries by demonstrating that yet another sex determination system exists in Xenopus, specifically in the species Xenopus borealis. This system evolved recently in an ancestor of X. borealis that had the same sex determination system as X. laevis, a system which itself is newly evolved. Strikingly, the genomic region carrying the sex determination factor in X. borealis is homologous to that of therian mammals, including humans. Our results offer insights into how the genetic underpinnings of conserved phenotypes evolve, and suggest an important role for cooption of genetic building blocks with conserved developmental roles. PMID:27605520

Examining the contents of isolated Xenopus germinal vesicles.

PubMed

Gall, Joseph G; Wu, Zheng'an

2010-05-01

One can manually isolate the giant oocyte nucleus or germinal vesicle (GV) of Xenopus from a living oocyte with nothing more complicated than jewelers' forceps and a dissecting microscope. Similarly, one can remove the nuclear envelope by hand and allow the lampbrush chromosomes and other nuclear organelles to spread on a microscope slide. After centrifugation, the nuclear contents adhere tightly to the slide, where they can be subjected to immunostaining or fluorescent in situ hybridization for visualization by conventional or confocal microscopy. Preparations of isolated GV contents reveal details of nuclear structure that are almost impossible to attain by more conventional techniques.

Wnt signalling pathway parameters for mammalian cells.

PubMed

Tan, Chin Wee; Gardiner, Bruce S; Hirokawa, Yumiko; Layton, Meredith J; Smith, David W; Burgess, Antony W

2012-01-01

Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters

Development of the Zebra Danio Model: Carcinogenesis and Gene Transfer Studies

DTIC Science & Technology

1996-09-01

J., and Enomoto, M. (1988). Liver cell carcinomas in the medaka (Oryzias latipes) induced by methylazoxymethanol-acetate. J. Comp. Path. 98, 441-452...accelerate steroid- induced cell division in Xenopus oocytes (Sadler et al., 1986). More recently, ras p21 has been implicated in the transduction of a... induced cell division in Xenopus laevis oocytes. Mol Cell Biol 6:719-722. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A

[Antitumor effect of recombinant Xenopus laevis vascular endothelial growth factor (VEGF) as a vaccine combined with adriamycin on EL4 lymphoma in mice].

PubMed

Niu, Ting; Liu, Ting; Jia, Yong-Qian; Liu, Ji-Yan; Wu, Yang; Hu, Bing; Tian, Ling; Yang, Li; Kan, Bing; Wei, Yu-Quan

2005-09-01

To explore the antitumor effect of immunotherapy with recombinant Xenopus laevis vascular endothelial growth factor (xVEGF) as a vaccine combined with adriamycin on lymphoma model in mice. EL4 lymphoma model was established in C57BL/6 mice. Mice were randomized into four groups: combination therapy, adriamycin alone, xVEGF alone and normal saline (NS) groups, and then were given relevant treatments. The growth of tumor, the survival rate of tumor-bearing mice, and the potential toxicity of regimens above were observed. Anti-VEGF antibody-producing B cells (APBCs) were detected by enzyme-linked immunospot (ELISPOT) assay. In addition, microvessel density (MVD) of tumor was detected by immunohistochemistry, and tumor cell apoptosis was also detected by TUNEL staining. The tumor volumes of mice were significantly smaller in combination group than those in other three groups (P < 0.05). Complete regression of tumor was observed in 3 of 10 mice in combination group. Forty-eight days after inoculation of tumor cells, the survival rate of mice was significantly higher in combination group than in NS group (P < 0.01). The anti-VEGF APBC count in combination group or xVEGF group was significantly higher, compared with that in adriamycin group or NS group (P < 0.01). MVD in tumor tissues was significantly lower in combination group than those in other three groups (P < 0.05). Moreover, tumor cell apoptosis was significantly higher in combination group than those in other three groups (P < 0.05). In this experimental study, the use of xVEGF vaccine and adriamycin as a combination of immunotherapy with chemotherapy has sucessfully produced synergistic antitumor effect on lymphoma in mice.

Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts

PubMed Central

Shimamoto, Yuta; Kapoor, Tarun M.

2014-01-01

SUMMARY To explain how micron-sized cellular structures generate and respond to forces we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based set-up to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multi-mode imaging system allows tracking of microtubules, chromosomes and needle tips. This set-up can be used to analyze the viscoelastic properties of the spindle on time-scales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 days. PMID:22538847

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos

PubMed Central

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-01

AIM: To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. METHODS: Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. RESULTS: In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos.

PubMed

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-14

To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a

Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis

PubMed Central

Roco, Álvaro S.; Olmstead, Allen W.; Degitz, Sigmund J.; Amano, Tosikazu; Zimmerman, Lyle B.; Bullejos, Mónica

2015-01-01

Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis’ sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis’ sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology. PMID:26216983

Environmental estrogens alter early development in Xenopus laevis.

PubMed

Bevan, Cassandra L; Porter, Donna M; Prasad, Anita; Howard, Marthe J; Henderson, Leslie P

2003-04-01

A growing number of environmental toxicants found in pesticides, herbicides, and industrial solvents are believed to have deleterious effects on development by disrupting hormone-sensitive processes. We exposed Xenopus laevis embryos at early gastrula to the commonly encountered environmental estrogens nonylphenol, octylphenol, and methoxychlor, the antiandrogen, p,p-DDE, or the synthetic androgen, 17 alpha-methyltestosterone at concentrations ranging from 10 nM to 10 microM and examined them at tailbud stages (approximately 48 hr of treatment). Exposure to the three environmental estrogens, as well as to the natural estrogen 17 beta-estradiol, increased mortality, induced morphologic deformations, increased apoptosis, and altered the deposition and differentiation of neural crest-derived melanocytes in tailbud stage embryos. Although neural crest-derived melanocytes were markedly altered in embryos treated with estrogenic toxicants, expression of the early neural crest maker Xslug, a factor that regulates both the induction and subsequent migration of neural crest cells, was not affected, suggesting that the disruption induced by these compounds with respect to melanocyte development may occur at later stages of their differentiation. Co-incubation of embryos with the pure antiestrogen ICI 182,780 blocked the ability of nonylphenol to induce abnormalities in body shape and in melanocyte differentiation but did not block the effects of methoxychlor. Our data indicate not only that acute exposure to these environmental estrogens induces deleterious effects on early vertebrate development but also that different environmental estrogens may alter the fate of a specific cell type via different mechanisms. Finally, our data suggest that the differentiation of neural crest-derived melanocytes may be particularly sensitive to the disruptive actions of these ubiquitous chemical contaminants.

Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

PubMed

Lepetit, D; Pasquet, S; Olive, M; Thézé, N; Thiébaud, P

2000-01-01

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

Evaluation of microtransplantation of rat brain neurolemma into Xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage-sensitive ion channels.

PubMed

Murenzi, Edwin; Toltin, Abigail C; Symington, Steven B; Morgan, Molly M; Clark, John M

2017-05-01

Microtransplantation of mammalian brain neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Na v 1.1-Na v 1.9) was determined in neurolemma using automated western blotting, with the predominant isoforms expressed being Na v 1.2 and Na v 1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in

Assessment of laryngeal muscle and testicular cell types in Xenopus laevis (Anura Pipidae) inhabiting maize and non-maize growing areas of South Africa

USGS Publications Warehouse

Smith, E.E.; Du Preez, L.H.; Gentles, A.; Solomon, K.R.; Tandler, B.; Carr, J.A.; Van Der Kraak, G. L.; Kendall, R.J.; Giesy, J.P.; Gross, T.S.

2005-01-01

We tested the hypothesis that adult African clawed frogs (Xenopus laevis) inhabiting water bodies in maize-growing areas (MGA) of South Africa would exhibit differences in testicular structure compared to frogs from water bodies in non-maize-growing areas (NMGA) in the same locale. Adults of both sexes were collected during the autumn of 2002 in South Africa, and stereological analytical techniques were used to quantify the distribution of testicular cell types. In addition, total laryngeal mass was used as a gauge of secondary sex differences in animals from MGA and NMGA study sites. Evaluation of the total laryngeal mass revealed that there were no statistically significant differences between X. laevis of the same sex from the NMGA and MGA sites. Mean percent fractional-volume values for seminiferous tubule distribution of testicular cell types of mature X. laevis, ranged from 3-4% for spermatogonia, 26-28% for spermatocytes, 54-57% for spermatozoa, and 14-15% for other cells types. The mean percent volume for blood vessels ranged from 0.3-0.4%. These values did not differ significantly between frogs from NMGA and MGA areas. Collectively, these data demonstrated no differences in gonadal and laryngeal development in X. laevis collected in South Africa from MGA and NMGA areas and that there is little evidence for an effect of agricultural chemicals used in maize production functioning as endocrine disrupters in this species. Screening of X. laevis testes revealed a small incidence of Stage 1 testicular oocytes in adult male frogs collected from the NMGA (3%) and MGA (2%).

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 ...

Lidocaine preferentially inhibits the function of purinergic P2X7 receptors expressed in Xenopus oocytes.

PubMed

Okura, Dan; Horishita, Takafumi; Ueno, Susumu; Yanagihara, Nobuyuki; Sudo, Yuka; Uezono, Yasuhito; Minami, Tomoko; Kawasaki, Takashi; Sata, Takeyoshi

2015-03-01

Lidocaine has been widely used to relieve acute pain and chronic refractory pain effectively by both systemic and local administration. Numerous studies reported that lidocaine affects several pain signaling pathways as well as voltage-gated sodium channels, suggesting the existence of multiple mechanisms underlying pain relief by lidocaine. Some extracellular adenosine triphosphate (ATP) receptor subunits are thought to play a role in chronic pain mechanisms, but there have been few studies on the effects of lidocaine on ATP receptors. We studied the effects of lidocaine on purinergic P2X3, P2X4, and P2X7 receptors to explore the mechanisms underlying pain-relieving effects of lidocaine. We investigated the effects of lidocaine on ATP-induced currents in ATP receptor subunits, P2X3, P2X4, and P2X7 expressed in Xenopus oocytes, by using whole-cell, two-electrode, voltage-clamp techniques. Lidocaine inhibited ATP-induced currents in P2X7, but not in P2X3 or P2X4 subunits, in a concentration-dependent manner. The half maximal inhibitory concentration for lidocaine inhibition was 282 ± 45 μmol/L. By contrast, mepivacaine, ropivacaine, and bupivacaine exerted only limited effects on the P2X7 receptor. Lidocaine inhibited the ATP concentration-response curve for the P2X7 receptor via noncompetitive inhibition. Intracellular and extracellular N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) and benzocaine suppressed ATP-induced currents in the P2X7 receptor in a concentration-dependent manner. In addition, repetitive ATP treatments at 5-minute intervals in the continuous presence of lidocaine revealed that lidocaine inhibition was use-dependent. Finally, the selective P2X7 receptor antagonists Brilliant Blue G and AZ11645373 did not affect the inhibitory actions of lidocaine on the P2X7 receptor. Lidocaine selectively inhibited the function of the P2X7 receptor expressed in Xenopus oocytes. This effect may be caused by acting on sites in the ion

hmmr mediates anterior neural tube closure and morphogenesis in the frog Xenopus.

PubMed

Prager, Angela; Hagenlocher, Cathrin; Ott, Tim; Schambony, Alexandra; Feistel, Kerstin

2017-10-01

Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Variants of the Xenopus laevis ribosomal transcription factor xUBF are developmentally regulated by differential splicing.

PubMed

Guimond, A; Moss, T

1992-07-11

XUBF is a Xenopus ribosomal transcription factor of the HMG-box family which contains five tandemly disposed homologies to the HMG1 & 2 DNA binding domains. XUBF has been isolated as a protein doublet and two cDNAs encoding the two molecular weight variants have been characterised. The major two forms of xUBF identified differ by the presence or absence of a 22 amino acid segment lying between HMG-boxes 3 and 4. Here we show that the mRNAs for these two forms of xUBF are regulated during development and differentiation over a range of nearly 20 fold. By isolating two of the xUBF genes, it was possible to show that both encoded the variable 22 amino acid segment in exon 12. Oocyte splicing assays and the sequencing of PCR-generated cDNA fragments, demonstrated that the transcripts from one of these genes were differentially spliced in a developmentally regulated manner. Transcripts from the second gene were found to be predominantly or exclusively spliced to produce the lower molecular weight form of xUBF. Expression of a high molecular weight form from yet a third gene was also detected. Although the intron-exon structures of the Xenopus and mouse UBF genes were found to be essentially identical, the differential splicing of exon 8 found in mammals, was not detected in Xenopus.

Modulation of K+ currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation

PubMed Central

Brown, Paul; Dale, Nicholas

2002-01-01

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K+ currents by 10 ± 3 %. UTP and the analogues α,β-methylene-ATP and 2-methylthio-ATP also inhibited K+ currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K+ currents could be inhibited by ADP (9 ± 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that α,β-methylene-ADP, often used to block the ecto-5′-nucleotidase, also inhibited voltage-gated K+ currents (7 ± 2.3 %). This inhibition was occluded by ADP, suggesting that α,β-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5′-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, α,β-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of α,β-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K+ currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation. PMID:11986373

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

PubMed Central

Shu, Hong-Jin; Bracamontes, John; Taylor, Amanda; Wu, Kyle; Eaton, Megan M; Akk, Gustav; Manion, Brad; Evers, Alex S; Krishnan, Kathiresan; Covey, Douglas F; Zorumski, Charles F; Steinbach, Joe Henry; Mennerick, Steven

2012-01-01

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. PMID:21950777

Anxa4 Genes are Expressed in Distinct Organ Systems in Xenopus laevis and tropicalis But are Functionally Conserved

PubMed Central

Massé, Karine L; Collins, Robert J; Bhamra, Surinder; Seville, Rachel A

2007-01-01

Anxa4 belongs to the multigenic annexin family of proteins which are characterized by their ability to interact with membranes in a calcium-dependent manner. Defined as a marker for polarized epithelial cells, Anxa4 is believed to be involved in many cellular processes but its functions in vivo are still poorly understood. Previously, we cloned Xanx4 in Xenopus laevis (now referred to as anxa4a) and demonstrated its role during organogenesis of the pronephros, providing the first evidence of a specific function for this protein during the development of a vertebrate. Here, we describe the strict conservation of protein sequence and functional domains of anxa4 during vertebrate evolution. We also identify the paralog of anxa4a, anxa4b and show its specific temporal and spatial expression pattern is different from anxa4a. We show that anxa4 orthologs in X. laevis and tropicalis display expression domains in different organ systems. Whilst the anxa4a gene is mainly expressed in the kidney, Xt anxa4 is expressed in the liver. Finally, we demonstrate Xt anxa4 and anxa4a can display conserved function during kidney organogenesis, despite the fact that Xt anxa4 transcripts are not expressed in this domain. This study highlights the divergence of expression of homologous genes during Xenopus evolution and raises the potential problems of using X. tropicalis promoters in X. laevis. PMID:19279706

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models.

PubMed

Naert, Thomas; Van Nieuwenhuysen, Tom; Vleminckx, Kris

2017-01-01

The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models. We believe that due to particular advantageous characteristics, outlined within this review, GEXM will become a valuable alternative animal model for modeling human cancer. Furthermore, we provide perspectives of how GEXM will be used as a platform for elucidation of novel therapeutic targets and for preclinical drug validation. Finally, we also discuss some future prospects on how the recent expansions and adaptations of the CRISPR/Cas9 toolbox might influence and push forward X. tropicalis cancer research. © 2017 Wiley Periodicals, Inc.

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

PubMed Central

Guedon, G; Sovia, D; Ebel, J P; Befort, N; Remy, P

1985-01-01

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:4092696

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

PubMed

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Planar Cell Polarity Pathway Genes and Risk for Spina Bifida

PubMed Central

Wen, Shu; Zhu, Huiping; Lu, Wei; Mitchell, Laura E.; Shaw, Gary M.; Lammer, Edward J.; Finnell, Richard H.

2009-01-01

Spina bifida, a neural tube closure defect (NTD) involving the posterior portion of what will ultimately give rise to the spinal cord, is one of the most common and serious birth defects. The etiology of spina bifida is thought to be multi-factorial and involve multiple interacting genes and environmental factors. The causes of this congenital malformation remain largely unknown. However, several candidate genes for spina bifida have been identified in lower vertebrates, including the planar cell polarity (PCP) genes. We used data from a case-control study conducted in California to evaluate the association between variation within several key PCP genes and the risk of spina bifida. The PCP genes included in this study were the human homologues of the Xenopus genes Flamingo, Strabismus, Prickle, Dishevelled and Scrib, two of the homologues of Xenopus Wnt genes, WNT5A and WNT11, and two of the homologues of Xenopus Frizzled, FZD3 and FZD6. None of the 172 SNPs that were evaluated were significantly associated with spina bifida in any racial/ethnic group after correction for multiple testing. However, several SNPs in the PRICKLE2 gene had unadjusted p value<0.01. In conclusion our results, though largely negative, suggest that the PRICKLE2 gene may potentially modify the risk of spina bifida and deserves further investigation. PMID:20101694

Planar cell polarity pathway genes and risk for spina bifida.

PubMed

Wen, Shu; Zhu, Huiping; Lu, Wei; Mitchell, Laura E; Shaw, Gary M; Lammer, Edward J; Finnell, Richard H

2010-02-01

Spina bifida, a neural tube closure defect (NTD) involving the posterior portion of what will ultimately give rise to the spinal cord, is one of the most common and serious birth defects. The etiology of spina bifida is thought to be multi-factorial and involve multiple interacting genes and environmental factors. The causes of this congenital malformation remain largely unknown. However, several candidate genes for spina bifida have been identified in lower vertebrates, including the planar cell polarity (PCP) genes. We used data from a case-control study conducted in California to evaluate the association between variation within several key PCP genes and the risk of spina bifida. The PCP genes included in this study were the human homologs of the Xenopus genes Flamingo, Strabismus, Prickle, Dishevelled, and Scrib, two of the homologs of Xenopus Wnt genes, WNT5A and WNT11, and two of the homologs of Xenopus Frizzled, FZD3 and FZD6. None of the 172 SNPs that were evaluated were significantly associated with spina bifida in any racial/ethnic group after correction for multiple testing. However, several SNPs in the PRICKLE2 gene had unadjusted P-value <0.01. In conclusion, our results, though largely negative, suggest that the PRICKLE2 gene may potentially modify the risk of spina bifida and deserves further investigation. Copyright 2010 Wiley-Liss, Inc.

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...

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

PubMed Central

Köttgen, Michael; Löffler, Thomas; Jacobi, Christoph; Nitschke, Roland; Pavenstädt, Hermann; Schreiber, Rainer; Frische, Sebastian; Nielsen, Søren; Leipziger, Jens

2003-01-01

Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl– secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K+ channels. We therefore speculated that a basolateral P2Y6 receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y6 receptor using crypt [Ca2+]i measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y6elevates cAMP, we coexpressed the P2Y1 or P2Y6 receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl– currents. In oocytes expressing the P2Y1 receptor, ATP transiently activated the endogenous Ca2+-activated Cl– current, but not CFTR. In contrast, in oocytes expressing the P2Y6receptor, UDP transiently activated the Ca2+-activated Cl– current and subsequently CFTR. CFTR Cl– currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y6 receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca2+]i and cAMP. In support of these data P2Y6 receptor stimulation differentially activates CFTR in Xenopus oocytes. PMID:12569163

Antioxidant metabolism in Xenopus laevis embryos is affected by stratospheric balloon flight.

PubMed

Rizzo, Angela M; Rossi, Federica; Zava, Stefania; Montorfano, Gigliola; Adorni, Laura; Cotronei, Vittorio; Zanini, Alba; Berra, Bruno

2007-07-01

To test the effects of low levels of radiation from space on living organisms, we flew Xenopus laevis embryos at different stages of development on a stratospheric balloon (BI.R.BA mission). After recovery, different parameters were analyzed to assess the effects of flight, with particular regard to oxidative stress damage. Because of failed temperature control during flight, the flight shielded embryos (FC) could not be used for biochemical or morphological comparisons. In contrast, the incubation conditions (i.e. temperature, containers, volumes) for the flight embryos (F) were parallel to those for the ground controls. Mortality data show that younger embryos (16 h) flown on the balloon (F) are more sensitive to radiation exposure than older ones (40 h and 6 days). Exposure during flight lowered the antioxidant potential in all embryos, particularly older ones. These preliminary data demonstrate that flight on a stratospheric balloon might affect antioxidant metabolism, though it is not yet possible to correlate these results with low radiation exposure during flight.

Calcium dependent current recordings in Xenopus laevis oocytes in microgravity

NASA Astrophysics Data System (ADS)

Wuest, Simon L.; Roesch, Christian; Ille, Fabian; Egli, Marcel

2017-12-01

Mechanical unloading by microgravity (or weightlessness) conditions triggers profound adaptation processes at the cellular and organ levels. Among other mechanisms, mechanosensitive ion channels are thought to play a key role in allowing cells to transduce mechanical forces. Previous experiments performed under microgravity have shown that gravity affects the gating properties of ion channels. Here, a method is described to record a calcium-dependent current in native Xenopus laevis oocytes under microgravity conditions during a parabolic flight. A 3-voltage-step protocol was applied to provoke a calcium-dependent current. This current increased with extracellular calcium concentration and could be reduced by applying extracellular gadolinium. The custom-made ;OoClamp; hardware was validated by comparing the results of the 3-voltage-step protocol to results obtained with a well-established two-electrode voltage clamp (TEVC). In the context of the 2nd Swiss Parabolic Flight Campaign, we tested the OoClamp and the method. The setup and experiment protocol worked well in parabolic flight. A tendency that the calcium-dependent current was smaller under microgravity than under 1 g condition could be observed. However, a conclusive statement was not possible due to the small size of the data base that could be gathered.

Tol2 transposon-mediated transgenesis in Xenopus tropicalis.

PubMed

Hamlet, Michelle R Johnson; Yergeau, Donald A; Kuliyev, Emin; Takeda, Masatoshi; Taira, Masanori; Kawakami, Koichi; Mead, Paul E

2006-09-01

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.

Synchronization modulation of Na/K pumps on Xenopus oocytes

NASA Astrophysics Data System (ADS)

Liang, Pengfei; Mast, Jason; Chen, Wei

We developed a new technique named synchronization modulation to electrically synchronize and modulate the Na/K pump molecules by a specially designed oscillating electric field. This technique is based on the theory of energy-trap in quantum physics as well as the concept of electronic synchrotron accelerator. As a result, the Na-transports are all entrapped into the positive half-cycle of the applied electric field and consequently, all of the K-transports are entrapped into the negative half cycle of the field. To demonstrate the process of the pump synchronization and modulation, we use Xenopus oocytes as a platform and introduce two-electrode whole-cell voltage clamp in measurement of pump current. Practically, we first synchronize the pump molecules running at the same pace (rate and phase) by a specially designed oscillation electric field. Then, we carefully maintain the pump synchronization status and gradually change the field frequency (decrease and increase) to modulate the pump molecules to newer pumping rate. The result shows a separation of the inward K current from the outward Na current, and about 10 time increase of the total (inward plus outward) pump current from the net outward current from the random paced pump molecules. Also, the ratio of the modulated total pump current with synchronized total pump current is consistent with the ratio of their field frequencies.

HNF1(beta) is required for mesoderm induction in the Xenopus embryo.

PubMed

Vignali, R; Poggi, L; Madeddu, F; Barsacchi, G

2000-04-01

XHNF1(&bgr;) is a homeobox-containing gene initially expressed at the blastula stage in the vegetal part of the Xenopus embryo. We investigated its early role by functional ablation, through mRNA injection of an XHNF1(beta)/engrailed repressor fusion construct (XHNF1(beta)/EngR). Dorsal injections of XHNF1(beta)/EngR mRNA abolish dorsal mesoderm formation, leading to axial deficiencies; ventral injections disrupt ventral mesoderm formation without affecting axial development. XHNF1(beta)/EngR phenotypic effects specifically depend on the DNA-binding activity of its homeodomain and are fully rescued by coinjection of XHNF1(beta) mRNA. Vegetal injection of XHNF1(beta)/EngR mRNA blocks the mesoderm-inducing ability of vegetal explants. Both B-Vg1 and VegT maternal determinants trigger XHNF1(beta) expression in animal caps. XHNF1(beta)/EngR mRNA blocks B-Vg1-mediated, but not by eFGF-mediated, mesoderm induction in animals caps. However, wild-type XHNF1(beta) mRNA does not trigger Xbra expression in animal caps. We conclude that XHNF1(beta) function is essential, though not sufficient, for mesoderm induction in the Xenopus embryo.

Differential inhibition of N and P/Q Ca2+ currents by 5-HT1A and 5-HT1D receptors in spinal neurons of Xenopus larvae

PubMed Central

Sun, Qian-Quan; Dale, Nicholas

1998-01-01

In whole-cell patch clamp recordings made from non-sensory neurons acutely isolated from the spinal cord of Xenopus (stage 40–42) larvae, two forms of inhibition of the high voltage-activated (HVA) Ca2+ currents were produced by 5-HT. One was voltage dependent and associated with both slowing of the activation kinetics and shifting of the voltage dependence of the HVA currents. This inhibition was relieved by strong depolarizing prepulses. A second form of inhibition was neither associated with slowing of the activation kinetics nor relieved by depolarizing prepulses and was thus voltage independent. In all neurons examined, 5-HT (1 μM) reversibly reduced 34 ± 1.6 % (n = 102) of the HVA Ca2+ currents. In about 40 % of neurons, the inhibition was totally voltage independent. In another 5 %, the inhibition was totally voltage dependent. In the remaining neurons, inhibition was only partially (by around 40 %) relieved by a large depolarizing prepulse, suggesting that in these, the inhibition consisted of both voltage-dependent and -independent components. By using selective channel blockers, we found that 5-HT acted on both N- and P/Q-type channels. However, whereas the inhibition of P/Q-type currents was only voltage independent, the inhibition of N-type currents had both voltage-dependent and -independent components. The effects of 5-HT on HVA Ca2+ currents were mediated by 5-HT1A and 5-HT1D receptors. The 5-HT1A receptors not only preferentially caused voltage-independent inhibition, but did so by acting mainly on the ω-agatoxin-IVA-sensitive Ca2+ channels. In contrast, the 5-HT1D receptor produced both voltage-dependent and -independent inhibition and was preferentially coupled to ω-conotoxin-GVIA sensitive channels. This complexity of modulation may allow fine tuning of transmitter release and calcium signalling in the spinal circuitry of Xenopus larvae. PMID:9625870

Identification of Germ Plasm-Associated Transcripts by Microarray Analysis of Xenopus Vegetal Cortex RNA

PubMed Central

Cuykendall, Tawny N.; Houston, Douglas W.

2011-01-01

RNA localization is a common mechanism for regulating cell structure and function. Localized RNAs in Xenopus oocytes are critical for early development, including germline specification by the germ plasm. Despite the importance of these localized RNAs, only approximately 25 have been identified and fewer are functionally characterized. Using microarrays, we identified a large set of localized RNAs from the vegetal cortex. Overall, our results indicate a minimum of 275 localized RNAs in oocytes, or 2–3% of maternal transcripts, which are in general agreement with previous findings. We further validated vegetal localization for 24 candidates and further characterized three genes expressed in the germ plasm. We identified novel germ plasm expression for reticulon 3.1, exd2 (a novel exonuclease-domain encoding gene), and a putative noncoding RNA. Further analysis of these and other localized RNAs will likely identify new functions of germ plasm and facilitate the identification of cis-acting RNA localization elements. PMID:20503379

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sweigert, S.E.; Carroll, D.

1990-11-01

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detectedmore » directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.« less

Effects of dietary exposure of polycyclic musk HHCB on the metamorphosis of Xenopus laevis.

PubMed

Pablos, María Victoria; Jiménez, María Ángeles; San Segundo, Laura; Martini, Federica; Beltrán, Eulalia; Fernández, Carlos

2016-06-01

The compound 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[γ]-2-benzopyrane (HHCB; galaxolide, Chemical Abstracts Service number 1222-05-5) is a synthetic musk used extensively as a fragrance in many consumer products and classified as an emerging pollutant. The ecotoxicological information available for HHCB addresses exposure via water, but this compound is frequently adsorbed into particulate matter. The goal of the present study was to assess the effects of dietary exposure to several environmentally relevant HHCB concentrations adsorbed in food during Xenopus laevis metamorphosis. The authors sought to determine if such exposure to this synthetic musk resulted in histological changes in the thyroid gland in conjunction with changes in development (staging, timing to metamorphosis), body weight, and length. Developmental acceleration on day 14, together with hypertrophy of the thyroid follicular epithelium in tadpoles, suggested a possible agonistic effect of HHCB, which would have been compensated after metamorphosis by regulatory mechanisms to maintain homeostasis. Further research into the potential thyroid-related mechanisms of action of HHCB should be conducted. Environ Toxicol Chem 2016;35:1428-1435. © 2015 SETAC. © 2015 SETAC.

Localization, structure and polymorphism of two paralogous Xenopus laevis mitochondrial malate dehydrogenase genes.

PubMed

Tlapakova, Tereza; Krylov, Vladimir; Macha, Jaroslav

2005-01-01

Two paralogous mitochondrial malate dehydrogenase 2 (Mdh2) genes of Xenopus laevis have been cloned and sequenced, revealing 95% identity. Fluorescence in-situ hybridization (FISH) combined with tyramide amplification discriminates both genes; Mdh2a was localized into chromosome q3 and Mdh2b into chromosome q8. One kb cDNA probes detect both genes with 85% accuracy. The remaining signals were on the paralogous counterpart. Introns interrupt coding sequences at the same nucleotide as defined for mouse. Restriction polymorphism has been detected in the first intron of Mdh2a, while the individual variability in intron 6 of Mdh2b gene is represented by an insertion of incomplete retrotransposon L1Xl. Rates of nucleotide substitutions indicate that both genes are under similar evolutionary constraints. X. laevis Mdh2 genes can be used as markers for physical mapping and linkage analysis.

Identification of Gender-specific Transcripts by Microarray in Gonad Tissue of Larval and Juvenile Xenopus tropicalis

EPA Science Inventory

Amphibian model species Xenopus tropicalis is currently being utilized by EPA in the development of a standardized in vivo reproductive toxicity assay. Perturbations to the hypothalamic-pituitary-gonadal axis from exposure to endocrine disrupting compounds during larval develop...

In Vivo Analysis of the Neurovascular Niche in the Developing Xenopus Brain

PubMed Central

Li, Jianli

2017-01-01

Abstract The neurovascular niche is a specialized microenvironment formed by the interactions between neural progenitor cells (NPCs) and the vasculature. While it is thought to regulate adult neurogenesis by signaling through vascular-derived soluble cues or contacted-mediated cues, less is known about the neurovascular niche during development. In Xenopus laevis tadpole brain, NPCs line the ventricle and extend radial processes tipped with endfeet to the vascularized pial surface. Using in vivo labeling and time-lapse imaging in tadpoles, we find that intracardial injection of fluorescent tracers rapidly labels Sox2/3-expressing NPCs and that vascular-circulating molecules are endocytosed by NPC endfeet. Confocal imaging indicates that about half of the endfeet appear to appose the vasculature, and time-lapse analysis of NPC proliferation and endfeet-vascular interactions suggest that proliferative activity does not correlate with stable vascular apposition. Together, these findings characterize the neurovascular niche in the developing brain and suggest that, while signaling to NPCs may occur through vascular-derived soluble cues, stable contact between NPC endfeet and the vasculature is not required for developmental neurogenesis. PMID:28795134

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

EPA Science Inventory

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

ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype.

PubMed

Miglionico, Rocchina; Ostuni, Angela; Armentano, Maria Francesca; Milella, Luigi; Crescenzi, Elvira; Carmosino, Monica; Bisaccia, Faustino

2017-01-01

Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype. Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype. To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21 Cip p53 independent; and 4) downregulation of lamin A/C. These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.

Stimulation of the amino acid transporter SLC6A19 by JAK2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhavsar, Shefalee K.; Hosseinzadeh, Zohreh; Merches, Katja

Highlights: Black-Right-Pointing-Pointer The amino acid transporter SLC6A19 is upregulated by Janus kinase-2 JAK2. Black-Right-Pointing-Pointer The {sup V617F}JAK2 mutant, causing myeloproliferative disease, is more effective. Black-Right-Pointing-Pointer JAK2 inhibitor AG490 reverses stimulation of SLC6A19 by {sup V617F}JAK2. Black-Right-Pointing-Pointer JAK2 enhances SLC6A19 protein insertion into the cell membrane. Black-Right-Pointing-Pointer SLC6A19 may contribute to amino acid uptake into {sup V617F}JAK2 expressing tumor cells. -- Abstract: JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation {sup V617F}JAK2 mutant is found in the majority of myeloproliferativemore » diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na{sup +} coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, {sup V617F}JAK2 or inactive {sup K882E}JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I{sub le}), which was significantly increased following coexpression of JAK2 or {sup V617F}JAK2, but not by coexpression of {sup K882E}JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 {mu}M) resulted in a gradual decline of I{sub le}. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I{sub le} following inhibition of carrier insertion by brefeldin A (5 {mu}M) was

A nuclear F-actin scaffold stabilizes ribonucleoprotein droplets against gravity in large cells.

PubMed

Feric, Marina; Brangwynne, Clifford P

2013-10-01

The size of a typical eukaryotic cell is of the order of ∼10 μm. However, some cell types grow to very large sizes, including oocytes (immature eggs) of organisms from humans to starfish. For example, oocytes of the frog Xenopus laevis grow to a diameter ≥1 mm. They have a correspondingly large nucleus (germinal vesicle) of ∼450 μm in diameter, which is similar to smaller somatic nuclei, but contains a significantly higher concentration of actin. The form and structure of this nuclear actin remain controversial, and its potential mechanical role within these large nuclei is unknown. Here, we use a microrheology and quantitative imaging approach to show that germinal vesicles contain an elastic F-actin scaffold that mechanically stabilizes these large nuclei against gravitational forces, which are usually considered negligible within cells. We find that on actin disruption, ribonucleoprotein droplets, including nucleoli and histone locus bodies, undergo gravitational sedimentation and fusion. We develop a model that reveals how gravity becomes an increasingly potent force as cells and their nuclei grow larger than ∼10 μm, explaining the requirement for a stabilizing nuclear F-actin scaffold in large Xenopus oocytes. All life forms are subject to gravity, and our results may have broad implications for cell growth and size control.

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development.

PubMed

Webb, Sarah E; Miller, Andrew L

2006-11-01

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming

Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-β Antagonist, Controls Early Neuroectoderm Specification in Xenopus

PubMed Central

Xu, Suhong; Cheng, Feng; Liang, Juan; Wu, Wei; Zhang, Jian

2012-01-01

Dorsal–ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification. PMID:22448144

Axial elongation in mouse embryos involves mediolateral cell intercalation behavior in the paraxial mesoderm

NASA Astrophysics Data System (ADS)

Yen, WeiWei; Burdsal, Carol; Periasamy, Ammasi; Sutherland, Ann E.

2006-02-01

The cell mechanical and signaling pathways involved in gastrulation have been studied extensively in invertebrates and amphibians, such as Xenopus, and more recently in non-mammalian vertebrates such as zebrafish and chick. However, because culturing mouse embryos extra-utero is very difficult, this fundamental process has been least characterized in the mouse. As the primary mammalian model for genetics, biochemistry, and the study of human disease and birth defects, it is important to investigate how gastrulation proceeds in murine embryos. We have developed a method of using 4D multiphoton excitation microscopy and extra-utero culture to visualize and characterize the morphogenetic movements in mouse embryos dissected at 8.5 days of gestation. Cells are labeled by expression of an X chromosome-linked enhanced green fluorescent protein (EGFP) transgene. This method has provided a unique approach, where, for the first time, patterns of cell behavior in the notochord and surrounding paraxial mesoderm can be visualized and traced quantitatively. Our observations of mouse embryos reveal both distinct differences as well as striking similarities in patterned cell motility relative to other vertebrate models such as Xenopus, where axial extension is driven primarily by mediolateral oriented cell behaviors in the notochord and paraxial somitic mesoderm. Unlike Xenopus, the width of the mouse notochord remains the same between 4-somite stage and 8-somite stage embryos. This implies the mouse notochord plays a lesser role in driving axial extension compared to Xenopus, although intercalation may occur where the anterior region of the node becomes notochordal plate. In contrast, the width of mouse paraxial mesoderm narrows significantly during this period and cells within the paraxial mesoderm are both elongated and aligned perpendicular to the midline. In addition, these cells are observed to intercalate, consistent with a role for paraxial mesoderm in driving convergence

Fox (forkhead) genes are involved in the dorso-ventral patterning of the Xenopus mesoderm.

PubMed

El-Hodiri, H; Bhatia-Dey, N; Kenyon, K; Ault, K; Dirksen, M; Jamrich, M

2001-01-01

Fox (forkhead/winged helix) genes encode a family of transcription factors that are involved in embryonic pattern formation, regulation of tissue specific gene expression and tumorigenesis. Several of them are transcribed during Xenopus embryogenesis and are important for the patterning of ectoderm, mesoderm and endoderm. We have isolated three forkhead genes that are activated during gastrulation and play an important role in the dorso-ventral patterning of the mesoderm. XFKH1 (FoxA4b), the first vertebrate forkhead gene to be implicated in embryonic pattern formation, is expressed in the Spemann-Mangold organizer region and later in the embryonic notochord. XFKH7, the Xenopus orthologue of the murine Mfh1(Foxc2), is expressed in the presomitic mesoderm, but not in the notochord or lateral plate mesoderm. Finally, XFD-13'(FoxF1b)1 is expressed in the lateral plate mesoderm, but not in the notochord or presomitic mesoderm. Expression pattern and functional experiments indicate that these three forkhead genes are involved in the dorso-ventral patterning of the mesoderm.

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

USGS Publications Warehouse

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

2004-01-01

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

The Pesticide Malathion Disrupts "Xenopus" and Zebrafish Embryogenesis: An Investigative Laboratory Exercise in Developmental Toxicology

ERIC Educational Resources Information Center

Chemotti, Diana C.; Davis, Sarah N.; Cook, Leslie W.; Willoughby, Ian R.; Paradise, Christopher J.; Lom, Barbara

2006-01-01

Malathion is an organophosphorus insecticide, which is often sprayed to control mosquitoes. When applied to aquatic habitats, malathion can also influence the embryogenesis of non-target organisms such as frogs and fish. We modified the frog embryo teratogen assay in "Xenopus" (FETAX), a standard toxicological assay, into an investigative…

Rapid Gynogenetic Mapping of Xenopus tropicalis Mutations to Chromosomes

PubMed Central

Khokha, Mustafa K.; Krylov, Vladimir; Reilly, Michael J.; Gall, Joseph G.; Bhattacharya, Dipankan; Cheung, Chung Yan J.; Kaufman, Sarah; Lam, Dang Khoa; Macha, Jaroslav; Ngo, Catherine; Prakash, Neha; Schmidt, Philip; Tlapakova, Tereza; Trivedi, Toral; Tumova, Lucie; Abu-Daya, Anita; Geach, Timothy; Vendrell, Elisenda; Ironfield, Holly; Sinzelle, Ludivine; Sater, Amy K.; Wells, Dan E.; Harland, Richard M.; Zimmerman, Lyle B.

2010-01-01

Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations (Grammer et al., 2005; Noramly et al., 2005; Goda et al., 2006). Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed Fluorescence In Situ Hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. PMID:19441086

Breeding based remobilization of Tol2 transposon in Xenopus tropicalis.

PubMed

Lane, Maura A; Kimber, Megan; Khokha, Mustafa K

2013-01-01

Xenopus is a powerful model for studying a diverse array of biological processes. However, despite multiple methods for transgenesis, relatively few transgenic reporter lines are available and commonly used. Previous work has demonstrated that transposon based strategies are effective for generating transgenic lines in both invertebrate and vertebrate systems. Here we show that the Tol2 transposon can be remobilized in the genome of X. tropicalis and passed through the germline via a simple breeding strategy of crossing transposase expressing and transposon lines. This remobilization system provides another tool to exploit transgenesis and opens new opportunities for gene trap and enhancer trap strategies.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

PubMed

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-Lin; Zhang, Xiao-Gang; Dawe, Gavin S; Xiao, Zhi-Cheng

2016-12-23

Amyloid precursor protein (APP), commonly associated with Alzheimer's disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Na v 1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

PubMed Central

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

2016-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

Reactivation of larval keratin gene (krt62.L) in blastema epithelium during Xenopus froglet limb regeneration.

PubMed

Satoh, Akira; Mitogawa, Kazumasa; Saito, Nanami; Suzuki, Miyuki; Suzuki, Ken-Ichi T; Ochi, Haruki; Makanae, Aki

2017-12-15

Limb regeneration is considered a form of limb redevelopment because of the molecular and morphological similarities. Forming a regeneration blastema is, in essence, creating a developing limb bud in an adult body. This reactivation of a developmental process in a mature body is worth studying. Xenopus laevis has a biphasic life cycle that involves distinct larval and adult stages. These distinct developmental stages are useful for investigating the reactivation of developmental processes in post-metamorphic frogs (froglets). In this study, we focused on the re-expression of a larval gene (krt62.L) during Xenopus froglet limb regeneration. Recently renamed krt62.L, this gene was known as the larval keratin (xlk) gene, which is specific to larval-tadpole stages. During limb regeneration in a froglet, krt62.L was re-expressed in a basal layer of blastema epithelium, where adult-specific keratin (Krt12.6.S) expression was also observable. Nerves produce important regulatory factors for amphibian limb regeneration, and also play a role in blastema formation and maintenance. The effect of nerve function on krt62.L expression could be seen in the maintenance of krt62.L expression, but not in its induction. When an epidermis-stripped limb bud was grafted in a froglet blastema, the grafted limb bud could reach the digit-forming stage. This suggests that krt62.L-positive froglet blastema epithelium is able to support the limb development process. These findings imply that the developmental process is locally reactivated in an postmetamorphic body during limb regeneration. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Single olfactory organ associated with prosencephalic malformation and cyclopia in a Xenopus laevis tadpole.

PubMed

Magrassi, L; Graziadei, P P

1987-06-02

A cyclops Xenopus laevis tadpole with a single olfactory organ is described. At a stage comparable to 48, the telencephalon was severely atrophic and only the region where the olfactory fibres terminated appeared to have the cytoarchitecture of the olfactory bulb. In this animal the central nervous system (CNS) appeared normally developed only posterior to the preoptic area. The hypothesis of a diencephalic origin of the region where the olfactory fibres terminated is discussed in the light of our previous results of olfactory placode transplantation. By analogy between this case and other malformations (cyclopia, holoprosencephaly) in higher vertebrates and humans, the need is emphasized for a more precise anatomical description of the olfactory input in related malformations.

Block by Extracellular Divalent Cations of Drosophila Big Brain Channels Expressed in Xenopus Oocytes

PubMed Central

Yanochko, Gina M.; Yool, Andrea J.

2004-01-01

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain (bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca2+ and Ba2+ but not by Mg2+ in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu274) and aspartate (Asp253) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu71) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu71 to Asp introduced a new sensitivity to block by extracellular Mg2+; substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca2+, suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination. PMID:14990474

Behavioral Repertoire of Xenopus tropicalis: Baseline Female-male Interactions during Spawning Events and Male Vocal Communication

EPA Science Inventory

The aquatic frog, Xenopus tropicalis, is being developed for use as a model amphibian species for inclusion in the EPA’s Endocrine Disruptor Screening Program. Current toxicity test designs do not incorporate measures of fecundity due to high variability in the responses of frog...

Evolution of the B7 family: co-evolution of B7H6 and NKp30, identification of a new B7 family member, B7H7, and of B7's historical relationship with the MHC.

PubMed

Flajnik, Martin F; Tlapakova, Tereza; Criscitiello, Michael F; Krylov, Vladimir; Ohta, Yuko

2012-08-01

The B7 family of genes is essential in the regulation of the adaptive immune system. Most B7 family members contain both variable (V)- and constant (C)-type domains of the immunoglobulin superfamily (IgSF). Through in silico screening of the Xenopus genome and subsequent phylogenetic analysis, we found novel genes belonging to the B7 family, one of which is the recently discovered B7H6. Humans and rats have a single B7H6 gene; however, many B7H6 genes were detected in a single large cluster in the Xenopus genome. The B7H6 expression patterns also varied in a species-specific manner. Human B7H6 binds to the activating natural killer receptor, NKp30. While the NKp30 gene is single-copy and maps to the MHC in most vertebrates, many Xenopus NKp30 genes were found in a cluster on a separate chromosome that does not harbor the MHC. Indeed, in all species so far analyzed from sharks to mammals, the number of NKp30 and B7H6 genes correlates well, suggestive of receptor-ligand co-evolution. Furthermore, we identified a Xenopus-specific B7 homolog (B7HXen) and revealed its close linkage to B2M, which we have demonstrated previously to have been originally encoded in the MHC. Thus, our study provides further proof that the B7 precursor was included in the proto MHC. Additionally, the comparative analysis revealed a new B7 family member, B7H7, which was previously designated in the literature as an unknown gene, HHLA2.

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

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

PubMed

Afroze, Syeda H; Uddin, M Nasir; Cao, Xiaobo; Asea, Alexzander; Gizachew, Dawit

2011-08-01

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

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

Plasticity of lung development in the amphibian, Xenopus laevis.

PubMed

Rose, Christopher S; James, Brandon

2013-12-15

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.

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.

Mouse A6-positive hepatic oval cells derived from embryonic stem cells.

PubMed

Yin, Dong-zhi; Cai, Ji-ye; Zheng, Qi-chang; Chen, Zheng-wei; Zhao, Jing-xian; Yuan, You-neng

2014-02-01

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.