A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.     

High-throughput transgenesis in Xenopus using I-SceI meganuclease

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.     

I-SceI meganuclease mediates highly efficient transgenesis in fish

The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.     

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

In the rapidly developing, diploid amphibian Xenopus tropicalis, genetics can be married to the already powerful tools of the amphibian system to overcome a disability that has hampered Xenopus laevis as a model organism: the difficulties inherent in conducting genetic analyses in a tetraploid organism with a longer generation time. We describe here a gynogenetic screen to uncover naturally occurring recessive mutations in wild X. tropicalis populations, a procedure that is both faster and easier than conventional genetic screens traditionally employed in model organisms to dissect early developmental pathways. During the first round of our screen, gynogenetic diploids from over 160 females comprising four different wild-caught populations were examined. Forty-two potential mutant phenotypes were isolated during this round of gynogenesis. From this group, we describe 10 lines that have genetically heritable recessive mutations. A wide range of developmental defects were obtained in this screen, encompassing effects limited to individual organs as well phenotypes characterized by more global changes in tadpole body morphology. The frequency of recessive mutations detected in our screen appears lower than that seen in other vertebrate genetic screens, but given constraints on the screening procedure used here, is likely to be consistent with rates seen in other animals, and clearly illustrates how wild-caught animals can be a productive source of developmental mutations for experimental study. The development of genetic strategies for the Xenopus system, together with new genomic resources, existing technologies for transgenesis, and other means for manipulating gene expression, as well as the power of performing embryonic manipulations, will provide an impressive set of tools for resolving complex cell and developmental phenomena in the future.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

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.     

Spatially and temporally regulated alpha6 integrin cleavage during Xenopus laevis development

The α6 integrin is essential for early nervous system development in Xenopus laevis. We have previously reported a uPA cleaved form of integrin α6 (α6p), in invasive human prostate cancer tissue, whose presence correlates with increased migration and invasive capacity. We now report that α6 is cleaved during the normal development of Xenopus in a spatially and temporally controlled manner. In addition, unlike normal mammalian tissues, which lack α6p, the major form of the α6 integrin present in adult Xenopus is α6p. The protease responsible for the cleavage in mammals, uPA, is not involved in the cleavage of Xenopus α6. Finally, overexpression of a mammalian α6 mutant which cannot be cleaved leads to developmental abnormalities suggesting a potential role for the cleavage in development.     

The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis

While limb regeneration has been extensively studied in amphibians, little is known about the initial events in limb formation in metamorphosing anurans. The small secreted integrin ligand nephronectin (npnt) is necessary for development of the metanephros in mouse. Although expressed in many tissues, its role in other developmental processes is not well-studied. Here we show that a transgene insertion that disrupts this gene ablates forelimb formation in Xenopus tropicalis. Our results suggest a novel role for integrin signalling in limb development, and represent the first insertional phenotype to be cloned in amphibians.     

Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.     

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.     

Identification of mutants in inbred Xenopus tropicalis

Xenopus tropicalis offers the potential for genetic analysis in an amphibian. In order to take advantage of this potential, we have been inbreeding strains of frogs for future mutagenesis. While inbreeding a population of Nigerian frogs, we identified three mutations in the genetic background of this strain. These mutations are all recessive embryonic lethals. We show that multigenerational mutant analysis is feasible and demonstrate that mutations can be identified, propagated, and readily characterized using hybrid, dihybrid, and even trihybrid crosses. In addition, we are optimizing conditions to raise frogs rapidly and present our protocols for X. tropicalis husbandry. We find that males mature faster than females (currently 4 versus 6 months to sexual maturity). Here we document our progress in developing X. tropicalis as a genetic model organism and demonstrate the utility of the frog to study the genetics of early vertebrate development.     

Endocannabinoid system in Xenopus laevis development: CB1 receptor dynamics

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

Identification of the blood group Lewis(a) determinant in the oviducal mucins of Xenopus tropicalis

The amphibian Xenopus tropicalis appears an increasingly appealing model for both genetic and developmental biology studies, compared to the related species Xenopus laevis. Study of the glycosylation pattern of its secreted glycoproteins revealed that this species synthesizes large amounts of Lewis(a) epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of (1)H-nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewis(a)-bearing O-linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(beta 1-3)GlcNAc(beta 1-6)GalNAc-ol. These structural data suggested the emergence of an alpha 1,4-fucosyltransferase activity in animals outside the primate lineage. On this basis, the screening of a X. tropicalis GenBank database with human Lewis-fucosyltransferase sequences revealed the occurrence of a putative fucosyltransferase gene that presented an unusual acceptor motif.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

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