Katanin contributes to interspecies spindle length scaling in Xenopus

Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which, unlike X. laevis, lacks an inhibitory phosphorylation site in the katanin p60 catalytic subunit. Katanin inhibition lengthened spindles in both species. In X. tropicalis, k-fiber MT bundles that connect to chromosomes at their kinetochores extended through spindle poles, disrupting them. In both X. tropicalis extracts and the spindle simulation, a balance between k-fiber number and MT depolymerization is required to maintain spindle morphology. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady-state structure.     

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.     

Toward an unbiased evolutionary platform for unraveling Xenopus developmental gene networks

The availability of both the Xenopus tropicalis genome and the soon to be released Xenopus laevis genome provides a solid foundation for Xenopus developmental biologists. The Xenopus community has presently amassed expression data for ～2,300 genes in the form of published images collected in the Xenbase, the principal Xenopus research database. A few of these genes have been examined in both X. tropicalis and X. laevis and the cross-species comparison has been proven invaluable for studying gene function. A recently published work has yielded developmental expression profiles for the majority of Xenopus genes across fourteen developmental stages spanning the blastula, gastrula, neurula, and the tail-bud. While this data was originally queried for global evolutionary and developmental principles, here we demonstrate its general use for gene-level analyses. In particular, we present the accessibility of this dataset through Xenbase and describe biases in the characterized genes in terms of sequence and expression conservation across the two species. We further indicate the advantage of examining coexpression for gene function discovery relating to developmental processes conserved across species. We suggest that the integration of additional large-scale datasets--comprising diverse functional data--into Xenbase promises to provide a strong foundation for researchers in elucidating biological processes including the gene regulatory programs encoding development.     

Identification of novel microRNAs in Xenopus laevis metaphase II arrested eggs

Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in Xenopus laevis. We characterize 115 miRNAs that have been described either in Xenopus tropicalis (85), X. laevis (9), or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. In addition, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identify target miRNAs for future functional studies.     

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.     

Evolution of the Xenopus piggyBac transposon family TxpB: domesticated and untamed strategies of transposon subfamilies

A new family, termed TxpB, of DNA transposons belonging to the piggyBac superfamily was found in 3 Xenopus species (Xenopus tropicalis, Xenopus laevis, and Xenopus borealis). Two TxpB subfamilies of Kobuta and Uribo1 were found in all the 3 species, and another subfamily termed Uribo2 was found in X. tropicalis. Molecular phylogenetic analyses of their open reading frames (ORFs) revealed that TxpB transposons have been maintained for over 100 Myr. Both the Uribo1 and the Uribo2 ORFs were present as multiple copies in each genome, and some of them were framed by terminal inverted repeat sequences. In contrast, all the Kobuta ORFs were present as a single copy in each genome and exhibited high evolutionary conservation, suggesting domestication of Kobuta genes by the host. Genomic insertion polymorphisms of the Uribo1 and Uribo2 transposons (nonautonomous type) were observed in a single species of X. tropicalis, indicating recent transposition events. Transfection experiments in cell culture revealed that an expression vector construct for the intact Uribo2 ORF caused precise excision of a nonautonomous Uribo2 element from the target vector construct but that for the Kobuta ORF did not. The present results support our viewpoint that some Uribo2 members are naturally active autonomous transposons, whereas Kobuta members may be domesticated by hosts.     

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

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

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.     

Construction of BAC library for the amphibian Xenopus tropicalis

Xenopus tropicalis has become an alternative model to the amphibian Xenopus laevis because it is better suited for genetic and genomic studies. We have constructed a genomic BAC library consisting of over 100,000 clones from sperm of Xenopus tropicalis. Analysis by pulsed field gel electrophoresis of representative BAC clones indicated the average size of insert DNA to be 100 kb, and we estimated the library covers 6 times the Xenopus tropicalis genome of 1.7 x 10(9) base pairs. To evaluate the BAC library, we attempted to isolate BAC clones which contain a protocadherin gamma (Pcdh gamma) gene and found that the isolated BAC clones are assembled as two separate contigs. This result suggests the presence of at least two clusters for the Pcdh gamma gene in the genome of X. tropicalis.     

Vitellogenin genes and their products in closely and distantly related species of Xenopus

Plasma vitellogenins from two closely related species of Xenopus, X. laevis and X. borealis, and a more ancient species, X. tropicalis, exhibited the same size on gel electrophoresis and were immunologically related. Partial peptide maps of 125I-labelled plasma vitellogenins, however, revealed marked differences in th structure and organisation of vitellogenin in the three Xenopus species. Northern blot hybridisation of liver RNA from oestrogen-treated males and females, probed with cloned vitellogenin cDNA, revealed the presence of mRNA of the same size in the three species of Xenopus, which was absent in untreated male liver. Cell-free translation of total liver RNA showed the presence of functional mRNA coding for vitellogenin subunit of the same size (Mr congruent to 210,000). Restriction endonuclease digestion patterns of genomic DNA from the three Xenopus species, using cloned X. laevis vitellogenin cDNA as the hybridisation probe, revealed significant differences in the organisation of these genes, which occur at a higher multiplicity in X. laevis and X. borealis than in X. tropicalis. Thus, despite a high degree of conservation of size, overall sequence and immunological identity of vitellogenin genes and their products in the three species of Xenopus, there is a substantial structural rearrangement during evolution of Xenopus within this multigene family.     

Cross-fertilization and structural comparison of egg extracellular matrix glycoproteins from Xenopus laevis and Xenopus tropicalis

While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm-egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent approximately 85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.     

Bleomycin-induced DNA damage and repair in Xenopus laevis and Xenopus tropicalis

Microgel cell electrophoresis has been used with various species to measure breakage of DNA and DNA repair following exposure to the radiomimetic antibiotic, bleomycin. With humans, a high degree of DNA damage is considered to be predictive of cancer susceptibility. Non-isogeneic Xenopus laevis, the South African clawed toad, rarely develop spontaneous or induced cancers. Here, we investigate bleomycin-induced DNA damage and repair in splenic lymphocytes of this species to test consistency with cancer predictability. As X. laevis is pseudotetraploid in nature, while Xenopus tropicalis is diploid, we additionally explore the effect of polyploidy on DNA damage and repair in these vertebrates. The results show that higher doses of bleomycin are required to induce comparable levels of DNA damage in both Xenopus species, than in humans. X. tropicalis, the diploid, is more bleomycin-sensitive than is X. laevis. Additionally, repair rates of damaged DNA of X. laevis lymphocytes are more rapid than those of X. tropicalis, although both are hours slower than human leukocytes. While no data exist on cancer susceptibility in X. tropicalis, the results suggest greater susceptibility to cancer than X. laevis, but less than in humans. Thus, polyploidy serves as a protection against DNA damage and allows more rapid repair.     

Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis

Now that transgenic strains of Xenopus laevis and X. tropicalis can be generated efficiently and with genomic sequence resources available for X. tropicalis, early amphibian development can be studied using integrated biochemical and genetic approaches. However, housing large numbers of animals generated during genetic screens or produced as novel transgenic lines presents a considerable challenge. We describe a method for cryopreserving Xenopus sperm that should facilitate low maintenance, long-term storage of male gametes. By optimising the cryoprotectant, the rates of cooling and thawing, and conditions for fertilisation, sperm from the equivalent of one-eighth of a X. laevis testis or of two X. tropicalis testes have been cryopreserved and used to fertilise eggs of both species after thawing. Sperm undergo a substantial loss of viability during a freeze-thaw cycle, but sufficient survive to fertilise eggs. Gametes of mutagenised frogs are being stored in connection with a screen for developmental mutations.     

Manipulating the <Emphasis Type="Italic">Xenopus</Emphasis>genome with transposable elements

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.     

Genetic and genomic prospects for Xenopus tropicalis research

Research using Xenopus laevis has made enormous contributions to our understanding of vertebrate development, control of the eukaryotic cell cycle and the cytoskeleton. One limitation, however, has been the lack of systematic genetic studies in Xenopus to complement molecular and cell biological investigations. Work with the closely related diploid frog Xenopus tropicalis is beginning to address this limitation. Here, we review the resources that will make genetic studies using X. tropicalis a reality.     

Nuclear size is regulated by importin α and Ntf2 in Xenopus

The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling.