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1.  Embryonic Expression and Function of the Xenopus Ink4d Cyclin D-Dependent Kinase Inhibitor 
Here we report the cloning and functional characterization of the cyclin D-dependent kinase 4 and 6 (Cdk4/6) inhibitory protein Cdkn2d/p19Ink4d of Xenopuslaevis (Xl-Ink4d). Xl-Ink4d is the only Ink4 family gene highly expressed during Xenopus development and its transcripts were detected maternally and during neurulation. The Xl-Ink4d protein has 63% identity to mouse and human Cdkn2d/p19Ink4d and its function as a negative regulator of cell cycle traverse is evolutionary conserved. Indeed, Xl-lnk4d can functionally substitute for mouse Cdkn2d in binding to mouse Cdk4 and inhibiting cyclin-D1-dependent CDK4 kinase activity. Further, enforced expression of Xl-lnk4d arrests mouse fibroblasts in the G1 phase of the cell cycle. These findings indicate that CDKN2d/p19Ink4d is conserved through vertebrate evolution and suggest Xl-lnk4d may contribute to the development of Xenopuslaevis.
PMCID: PMC4192657  PMID: 25309971
Xenopuslaevis; Cyclin-dependent kinase inhibitor; Ink4d; Cdkn2d; Cell cycle
2.  Transgenesis in Xenopus using the Sleeping Beauty transposon system 
Transposon-based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon. SB has been used extensively in human cell lines and in whole animal vertebrate model systems such as the mouse, rat and zebrafish. In this study, we describe the use of SB in the diploid frog Xenopus tropicalis to generate stable transgenic lines. SB transposon transgenes integrate into the X. tropicalis genome by a non-canonical process and are passed through the germline. We compare the activity of SB in this model organism with that of Tol2, a hAT (hobo, Ac1, TAM)-like transposon system.
PMCID: PMC2848081  PMID: 19517568
Transposon; Xenopus tropicalis; Xenopus laevis; Sleeping Beauty; SB10; SB11; transgenics; green fluorescent protein; GFP
3.  CDK9/cyclin complexes modulate endoderm induction by direct interaction with Mix.3/mixer 
Mix-related homeodomain proteins are involved in endoderm formation in the early vertebrate embryo. We used a yeast two-hybrid screen to identify proteins that interact with Mix.3/mixer to regulate endoderm induction. We demonstrate that cyclin dependent kinase 9 (CDK9) interacts with the carboxyl terminal domain of Mix.3. CDK9 is the catalytic subunit of the PTEF-b transcription elongation complex that phosphorylates the C-terminal domain of RNA polymerase II to promote efficient elongation of nascent transcripts. Using whole embryo transcription reporter and animal pole explant assays, we show that Mix.3 activity is regulated by CDK9/cyclin complexes. Co-expression of cyclin T2 and cyclin K had different effects on Mix.3 transcriptional activity and endoderm induction. Our data suggests that binding of CDK9, and the recruitment of different cyclin partners, can modulate the endoderm inducing activity of Mix.3 during embryonic development.
PMCID: PMC2832930  PMID: 19347956
Mix.3; mixer; CDK9; cyclin T2; cyclin K; P-TEFb; endoderm; Xenopus laevis
4.  Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting Agent 
PLoS Biology  2012;10(8):e1001379.
Analysis of a genetic module repurposed between yeast and vertebrates reveals that a common antifungal medication is also a potent vascular disrupting agent.
Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.
Author Summary
Yeast cells and vertebrate blood vessels would not seem to have much in common. However, we have discovered that during the course of evolution, a group of proteins whose function in yeast is to maintain cell walls has found an alternative use in vertebrates regulating angiogenesis. This remarkable repurposing of the proteins during evolution led us to hypothesize that, despite the different functions of the proteins in humans compared to yeast, drugs that modulated the yeast pathway might also modulate angiogenesis in humans and in animal models. One compound seemed a particularly promising candidate for this sort of approach: thiabendazole (TBZ), which has been in clinical use as a systemic antifungal and deworming treatment for 40 years. Gratifyingly, our study shows that TBZ is indeed able to act as a vascular disrupting agent and an angiogenesis inhibitor. Notably, TBZ also slowed tumor growth and decreased vascular density in human tumors grafted into mice. TBZ’s historical safety data and low cost make it an outstanding candidate for translation to clinical use as a complement to current anti-angiogenic strategies for the treatment of cancer. Our work demonstrates how model organisms from distant branches of the evolutionary tree can be exploited to arrive at a promising new drug.
PMCID: PMC3423972  PMID: 22927795
5.  Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis 
Mobile DNA  2011;2:15.
The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization.
Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'.
In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.
PMCID: PMC3271037  PMID: 22115366
6.  Transposon transgenesis in Xenopus 
Methods (San Diego, Calif.)  2010;51(1):92-100.
Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs.
PMCID: PMC2879075  PMID: 20211730
Sleeping Beauty; Tol2; transposon; transgenesis; Xenopus laevis; Xenopus tropicalis
7.  The Genome of the Western Clawed Frog Xenopus tropicalis 
Science (New York, N.Y.)  2010;328(5978):633-636.
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 human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.
PMCID: PMC2994648  PMID: 20431018
8.  Remobilization of Tol2 transposons in Xenopus tropicalis 
The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.
To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis.
In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.
PMCID: PMC2848417  PMID: 20096115
9.  Manipulating the Xenopus genome with transposable elements 
Genome Biology  2007;8(Suppl 1):S11.
The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.
PMCID: PMC2106847  PMID: 18047688

Results 1-9 (9)