Dact-4 is a Xenopus laevis Spemann organizer gene related to the Dapper/Frodo antagonist of ß-catenin family of proteins.

Dact/Dapper/Frodo members belong to an evolutionarily conserved family of Dishevelled-binding proteins present in mammals, birds, amphibians and fishes that are involved in the regulation of Wnt and TGF-ß signaling. In addition to the three established genes (Dact1-3) that compose the Dact family, a fourth paralogue group of related proteins has been recently identified and named Dact-4. Interestingly, Dact-4 is the most rapidly evolving gene of the entire family, as it displays very low homology with other Dact proteins and has lost key conserved domains. Dact-4 is not present in mammals, but weakly conserved homologs were found in reptiles and fishes. Recent RNAseq from our group identified new genes specifically expressed in the Xenopus laevis Spemann organizer. Among these, LOC100170590 mRNA encoded a protein sharing weak homology with a coelacanth Dact-like protein member. Here, by analyzing protein phylogeny and synteny, we show that this organizer gene corresponds to Dact-4. We report that Dact-4 is expressed in the Xenopus blastula pre-organizer region in addition to the gastrula organizer, as well as in placodes, eyes, neural tube, presomitic mesoderm and pronephros. Dact-4-Flag microinjection experiments suggest it is a nucleocytoplasmic protein, as are the other Dact paralogues.

Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation.

Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M-1 s-1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation-an established molecular correlate of impaired enzyme activity-in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50-60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.

Xenopus Interferon Complex: Inscribing the Amphibiotic Adaption and Species-Specific Pathogenic Pressure in Vertebrate Evolution?

Several recent studies have revealed previously unknown complexity of the amphibian interferon (IFN) system. Being unique in vertebrate animals, amphibians not only conserve and multiply the fish-like intron-containing IFN genes, but also rapidly evolve amniote-like intronless IFN genes in each tested species. We postulate that the amphibian IFN system confers an essential model to study vertebrate immune evolution in molecular and functional diversity to cope with unprecedented pathophysiological requirement during terrestrial adaption. Studies so far have ascribed a potential role of these IFNs in immune regulation against intracellular pathogens, particularly viruses; however, many knowledge gaps remain elusive. Based on recent reports about IFN's multifunctional properties in regulation of animal physiological and defense responses, we interpret that amphibian IFNs may evolve novel function pertinent to their superior molecular diversity. Such new function revealed by the emerging studies about antifungal and developmental regulation of amphibian IFNs will certainly promote our understanding of immune evolution in vertebrates to address current pathogenic threats causing amphibian decline.

ADAMTS9, a member of the ADAMTS family, in Xenopus development.

Extracellular matrix (ECM) remodeling by metalloproteinases is crucial during development. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling. The human family includes 19 members. In this study we identified the 19 members of the ADAMTS family in Xenopus laevis and Xenopus tropicalis. Gene identification and a phylogenetic study revealed strong conservation of the ADAMTS family and contributed to a better annotation of the Xenopus genomes. Expression of the entire ADAMTS family was studied from early stages to tadpole stages of Xenopus, and detailed analysis of ADAMTS9 revealed expression in many structures during organogenesis such as neural crest (NC) derivative tissues, the pronephros and the pancreas. Versican, a matrix component substrate of ADAMTS9 shows a similar expression pattern suggesting a role of ADAMTS9 in the remodeling of the ECM in these structures by degradation of versican.

Linker histones: novel insights into structure-specific recognition of the nucleosome.

Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing the wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures, influencing nucleosome spacing on DNA, and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while highlighting the remaining questions.

Evolution and functions of Oct4 homologs in non-mammalian vertebrates.

PouV class transcription factor Oct4/Pou5f1 is a central regulator of indefinite pluripotency in mammalian embryonic stem cells (ESCs) but also participates in cell lineage specification in mouse embryos and in differentiating cell cultures. The molecular basis for this versatility, which is shared between Oct4 and its non-mammalian homologs Pou5f1 and Pou5f3, is not yet completely understood. Here, I review the current understanding of the evolution of PouV class transcription factors and discuss equivalent and diverse roles of Oct4 homologs in pluripotency, differentiation, and cell behavior in different vertebrate embryos. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin.

Identification and Bioinformatics Analyses of the Basic Helix-loop-helix Transcription Factors in Xenopus laevis.

Xenopus laevis is a long established model organism for developmental, behavioral and neurological studies. Herein, an updated genome-wide survey was conducted using the ongoing genome project of Xenopus laevis and 106 non-redundant Basic Helix-Loop-Helix (bHLH) genes were identified in the Xenopus laevis genome databases. Gene Ontology (GO) enrichment statistics showed 51 significant GO annotations of biological processes and molecular functions and 5 significant KEGG pathways and a number of Xenopus laevis bHLH genes play significant role in specific development or special physiology processes like the development processes of muscle and eye and other organs. Furthermore, each sub-group of the bHLH family has its special gene functions except for the common GO term categories. Molecular phylogenetic analyses revealed that among these identified bHLH proteins, 105 sequences could classified into 39 families with 46, 25, 10, 5, 16 and 3 members in the corresponding high-order groups A, B, C, D, E and F, respectively with an addition bHLH member categorized as an orphan. The present study provides much useful information for further researches on Xenopus laevis.

A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis.

Xenopus laevis (XLA) is an allotetraploid species which appears to have undergone whole-genome duplication after the interspecific hybridization of 2 diploid species closely related to Silurana/Xenopus tropicalis (XTR). Previous cDNA fluorescence in situ hybridization (FISH) experiments have identified 9 sets of homoeologous chromosomes in X. laevis, in which 8 sets correspond to chromosomes 1-8 of X. tropicalis (XTR1-XTR8), and the last set corresponds to a fusion of XTR9 and XTR10. In addition, recent X. laevis genome sequencing and BAC-FISH experiments support this physiological relationship and show no gross chromosome translocation in the X. laevis karyotype. Therefore, for the benefit of both comparative cytogenetics and genome research, we here propose a new chromosome nomenclature for X. laevis based on the phylogenetic relationship and chromosome length, i.e. XLA1L, XLA1S, XLA2L, XLA2S, and so on, in which the numbering of XLA chromosomes corresponds to that in X. tropicalis and the postfixes 'L' and 'S' stand for 'long' and 'short' chromosomes in the homoeologous pairs, which can be distinguished cytologically by their relative size. The last chromosome set is named XLA9L and XLA9S, in which XLA9 corresponds to both XTR9 and XTR10, and hence, to emphasize the phylogenetic relationship to X. tropicalis, XLA9_10L and XLA9_10S are also used as synonyms.

Pan-African phylogeography of a model organism, the African clawed frog 'Xenopus laevis'.

The African clawed frog Xenopus laevis has a large native distribution over much of sub-Saharan Africa and is a model organism for research, a proposed disease vector, and an invasive species. Despite its prominent role in research and abundance in nature, surprisingly little is known about the phylogeography and evolutionary history of this group. Here, we report an analysis of molecular variation of this clade based on 17 loci (one mitochondrial, 16 nuclear) in up to 159 individuals sampled throughout its native distribution. Phylogenetic relationships among mitochondrial DNA haplotypes were incongruent with those among alleles of the putatively female-specific sex-determining gene DM-W, in contrast to the expectation of strict matrilineal inheritance of both loci. Population structure and evolutionarily diverged lineages were evidenced by analyses of molecular variation in these data. These results further contextualize the chronology, and evolutionary relationships within this group, support the recognition of X. laevis sensu stricto, X. petersii, X. victorianus and herein revalidated X. poweri as separate species. We also propose that portions of the currently recognized distributions of X. laevis (north of the Congo Basin) and X. petersii (south of the Congo Basin) be reassigned to X. poweri.

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae).

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianusAhl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensisPerret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.

Involvement of XZFP36L1, an RNA-binding protein, in Xenopus neural development.

Xenopus ZFP36L1 (zinc finger protein 36, C3H type-like 1) belongs to the ZFP36 family of RNA-binding proteins, which contains two characteristic tandem CCCH-type zinc-finger domains. The ZFP36 proteins can bind AU-rich elements in 3' untranslated regions of target mRNAs and promote their turnover. However, the expression and role of ZFP36 genes during neural development in Xenopus embryos remains largely unknown. The present study showed that Xenopus ZFP36L1 was expressed at the dorsal part of the forebrain, forebrain-midbrain boundary, and midbrain-hindbrain boundary from late neurula stages to tadpole stages of embryonic development. Overexpression of XZFP36L1 in Xenopus embryos inhibited neural induction and differentiation, leading to severe neural tube defects. The function of XZP36L1 requires both its zinc finger and C terminal domains, which also affect its subcellular localization. These results suggest that XZFP36L1 is likely involved in neural development in Xenopus and might play an important role in post-transcriptional regulation.

Population-specific incidence of testicular ovarian follicles in Xenopus laevis from South Africa: a potential issue in endocrine testing.

The African clawed frog (Xenopus laevis) has been identified as an appropriate sentinel for testing endocrine activity of existing chemicals in North America and Europe. Some reports suggest that the herbicide, atrazine (CAS Number [1912-24-9]) causes ovarian follicles to form in the testes of this frog. X. laevis collected from North East (NE) sites in South Africa had testicular ovarian follicles, irrespective of exposure to atrazine, while frogs from Southwest Western (SW) Cape region sites had none. Phylogenetic analysis of mitochondrial and nuclear genes indicates that frogs from the SW Cape are evolutionarily divergent from those from NE South Africa and the rest of sub-Saharan Africa. These findings provide a possible explanation for why conflicting results have been reported concerning the impact of atrazine on amphibian sexual differentiation and highlight the importance of understanding taxonomic status of the experimental animal. Even in common laboratory animals, there is a need for their correct taxonomic characterization before their use in tests for endocrine disruption.

Xenopus cDNA microarray identification of genes with endodermal organ expression.

The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ-related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior regions. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major organs, revealed 915 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development.

Ancestry influences the fate of duplicated genes millions of years after polyploidization of clawed frogs (Xenopus).

Allopolyploid species form through the fusion of two differentiated genomes and, in the earliest stages of their evolution, essentially all genes in the nucleus are duplicated. Because unique mutations occur in each ancestor prior to allopolyploidization, duplicate genes in these species potentially are not interchangeable, and this could influence their genetic fates. This study explores evolution and expression of a simple duplicated complex--a heterodimer between RAG1 and RAG2 proteins in clawed frogs (Xenopus). Results demonstrate that copies of RAG1 degenerated in different polyploid species in a phylogenetically biased fashion, predominately in only one lineage of closely related paralogs. Surprisingly, as a result of an early deletion of one RAG2 paralog, it appears that in many species RAG1/RAG2 heterodimers are composed of proteins that were encoded by unlinked paralogs. If the tetraploid ancestor of extant species of Xenopus arose through allopolyploidization and if recombination between paralogs was rare, then the genes that encode functional RAG1 and RAG2 proteins in many polyploid species were each ultimately inherited from different diploid progenitors. These observations are consistent with the notion that ancestry can influence the fate of duplicate genes millions of years after duplication, and they uncover a dimension of natural selection in allopolyploid genomes that is distinct from other genetic phenomena associated with polyploidization or segmental duplication.

Transgenic Xenopus laevis strain expressing cre recombinase in muscle cells.

For reproducible analyses of gene function in Xenopus, the use of transgenic strains is a promising approach but has limitations when investigating factors interfering with development. Therefore, inducible systems are attractive alternatives, and a binary system based on recombinases is a most versatile approach. We have shown previously that Cre and FLP recombinases are active in Xenopus laevis and can induce a silent reporter gene in a corresponding reporter strain. Here, we describe the establishment of the transgenic Xenopus laevis strain A7 expressing Cre recombinase under the control of the muscle-specific cardiac actin promoter. Upon crossing to several distinct reporter strains, A7 is able to induce EYFP, DsRed2, or LacZ reporter genes in a muscle-specific manner. This first Cre-expressing strain allows conditional activation of any gene of interest in muscle cells and, thus, opens up the use of recombinases as a new experimental strategy in Xenopus.

Geographic and within-population structure in variable resistance to parasite species and strains in a vertebrate host.

Host resistance to parasites and parasite infectivity may be subject to significant genetically determined variation within species. However, relatively little is known of how this variability is structured in natural vertebrate populations and their macroparasites. A laboratory experiment on host susceptibility-parasite infectivity variation in a wildlife host-parasite system (subspecies of the anuran X. laevis and their polystome flatworms), including 33 pairwise allopatric and sympatric host-parasite combinations (three parasite geographical isolates x 11 host full-sibling families, n=600), revealed a complex pattern of infection success. Results amongst host sibships from different localities suggested that infection success was subject to a highly significant locality x parasite isolate interaction. Within localities, a highly significant sibship x isolate interaction also occurred in one of two groups of sibships examined. The existence of such interactions suggests a potential for frequency-dependent, Red Queen-like selection. Interaction between locality and isolate was partly due to higher infection levels in sympatric combinations, consistent with a general pattern of host-specific adaptation. However, some allopatric combinations produced unpredictably high infection levels, resulting in very asymmetrical cross-infectivity patterns (where the reciprocal cross-infections produced negligible infection). This phylogeographically structured host-parasite system may, therefore, sometimes generate local parasite strains with high infectivity to allopatric hosts. Secondary contact between populations could thus result in significant, and unequal, transfer of parasites.

On the origin of and phylogenetic relationships among living amphibians.

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.

The phylogenetic utility of cytochrome b: lessons from bufonid frogs.

The mitochondrial cytochrome b gene is widely used in systematic studies to resolve divergences of many different ages. To investigate phylogenetic relationships among frogs of the large family Bufonidae, and to explore the utility of cytochrome b for this purpose, approximately one-third of the gene was sequenced from representatives of this group. Samples were chosen to represent a range of divergence levels within Bufonidae: (1) deep (= old), among species from around the world; (2) middle, among North American species; and (3) shallow (= young), within a single species group (the Bufo boreas group). The inferred amino acid sequences of cytochrome b are highly similar in these frogs although most pairwise comparisons of the nucleotide sequences are 15-20% different. Consequently insufficient information is available to generate robust phylogenetic hypotheses for the older divergences; silent differences are saturated and yet almost no informative replacement differences exist. Among the younger divergences, silent differences are not saturated and some resolution is possible. These results show that (1) the amino acid sequence of cytochrome b evolves differently in Bufonidae than expected based on other vertebrates (2) it consequently provides surprisingly little information about old divergences in Bufonidae, and (3) phylogenetic studies applying particular genes to new groups should begin with preliminary surveys of exemplar taxa representing the range of divergence times within the group to estimate the likely phylogenetic utility of that gene in that group.