ABSTRACT
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.
Subject(s)
Sex Chromosomes , Xenopus/genetics , Animals , Female , Male , Sex Determination ProcessesABSTRACT
Small RNA control of gene expression is critical for developmental processes in vertebrate embryos. To determine the dynamics of small RNA expression and to uncover novel small RNAs in the early vertebrate embryo, we performed high-throughput sequencing of all small RNAs in Xenopus tropicalis embryos at three developmental time points and in dissected halves of gastrula embryos. This analysis allowed us to identify novel microRNAs and we show that microRNA expression is highly dynamic and spatially localized in early embryos. In addition, we have developed a microRNA prediction pipeline and demonstrate that it has the power to predict new miRNAs that are experimentally detectable in frogs, mice, and humans. By combining the small RNA sequencing with mRNA profiling at the different developmental stages, we identify a new class of small noncoding RNAs that we name siteRNAs, which align in clusters to introns of protein-coding genes. We show that siteRNAs are derived from remnants of transposable elements present in the introns. We find that genes containing clusters of siteRNAs are transcriptionally repressed as compared with all genes. Furthermore, we show that this is true for individual genes containing siteRNA clusters, and that these genes are enriched in specific repressive histone modifications. Our data thus suggest a new mechanism of siteRNA-mediated gene silencing in vertebrates, and provide an example of how mobile elements can affect gene regulation.
Subject(s)
DNA Transposable Elements , Introns , MicroRNAs/genetics , MicroRNAs/metabolism , Xenopus Proteins/genetics , Xenopus/embryology , Xenopus/genetics , Animals , Embryo, Nonmammalian , Gastrula/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Silencing , High-Throughput Nucleotide Sequencing , Histones/metabolism , Humans , Mice , Sequence Analysis, RNA , Xenopus laevis/embryology , Xenopus laevis/geneticsABSTRACT
Sarcomere structure underpins structural integrity, signaling, and force transmission in the muscle. In embryos of the frog Xenopus tropicalis, muscle contraction begins even while sarcomerogenesis is ongoing. To determine whether contractile activity plays a role in sarcomere formation in vivo, chemical tools were used to block acto-myosin contraction in embryos of the frog X. tropicalis, and Z-disc assembly was characterized in the paralyzed dicky ticker mutant. Confocal and ultrastructure analysis of paralyzed embryos showed delayed Z-disc formation and defects in thick filament organization. These results suggest a previously undescribed role for contractility in sarcomere maturation in vivo.
Subject(s)
Actins/metabolism , Multiprotein Complexes/metabolism , Muscle Contraction , Sarcomeres/metabolism , Aminobenzoates/pharmacology , Anesthetics/pharmacology , Animals , Muscle Contraction/drug effects , Sarcomeres/ultrastructure , XenopusABSTRACT
BACKGROUND: Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly. RESULTS: We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly. CONCLUSION: We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds.
Subject(s)
Chromosomes/genetics , High-Throughput Nucleotide Sequencing/methods , Lasers , Microdissection , Sequence Analysis, DNA/methods , Animals , Chromosome Mapping , Genomics , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization , Xenopus/geneticsABSTRACT
A decade after the human genome sequence, most vertebrate gene functions remain poorly understood, limiting benefits to human health from rapidly advancing genomic technologies. Systematic in vivo functional analysis is ideally suited to the experimentally accessible Xenopus embryo, which combines embryological accessibility with a broad range of transgenic, biochemical, and gain-of-function assays. The diploid X. tropicalis adds loss-of-function genetics and enhanced genomics to this repertoire. In the last decade, diverse phenotypes have been recovered from genetic screens, mutations have been cloned, and reverse genetics in the form of TILLING and targeted gene editing have been established. Simple haploid genetics and gynogenesis and the very large number of embryos produced streamline screening and mapping. Improved genomic resources and the revolution in high-throughput sequencing are transforming mutation cloning and reverse genetic approaches. The combination of loss-of-function mutant backgrounds with the diverse array of conventional Xenopus assays offers a uniquely flexible platform for analysis of gene function in vertebrate development.
Subject(s)
Xenopus/genetics , Animals , Chromosome Mapping , Evolution, Molecular , Genetic Association Studies , Genetic Testing , Humans , Mutation , Organogenesis , Xenopus/embryologyABSTRACT
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.
Subject(s)
Extracellular Matrix Proteins/metabolism , Forelimb/embryology , Gene Expression Regulation, Developmental/physiology , Signal Transduction/physiology , Xenopus/embryology , Animals , DNA Primers/genetics , Extracellular Matrix Proteins/genetics , Gene Silencing , Genotype , In Situ Hybridization , Reverse Transcriptase Polymerase Chain Reaction , T-Box Domain Proteins/metabolism , TransgenesABSTRACT
BACKGROUND: The X and Y sex chromosomes are conspicuous features of placental mammal genomes. Mammalian sex chromosomes arose from an ordinary pair of autosomes after the proto-Y acquired a male-determining gene and degenerated due to suppression of X-Y recombination. Analysis of earlier steps in X chromosome evolution has been hampered by the long interval between the origins of teleost and amniote lineages as well as scarcity of X chromosome orthologs in incomplete avian genome assemblies. RESULTS: This study clarifies the genesis and remodelling of the Eutherian X chromosome by using a combination of sequence analysis, meiotic map information, and cytogenetic localization to compare amniote genome organization with that of the amphibian Xenopus tropicalis. Nearly all orthologs of human X genes localize to X. tropicalis chromosomes 2 and 8, consistent with an ancestral X-conserved region and a single X-added region precursor. This finding contradicts a previous hypothesis of three evolutionary strata in this region. Homologies between human, opossum, chicken and frog chromosomes suggest a single X-added region predecessor in therian mammals, corresponding to opossum chromosomes 4 and 7. A more ancient X-added ancestral region, currently extant as a major part of chicken chromosome 1, is likely to have been present in the progenitor of synapsids and sauropsids. Analysis of X chromosome gene content emphasizes conservation of single protein coding genes and the role of tandem arrays in formation of novel genes. CONCLUSIONS: Chromosomal regions orthologous to Therian X chromosomes have been located in the genome of the frog X. tropicalis. These X chromosome ancestral components experienced a series of fusion and breakage events to give rise to avian autosomes and mammalian sex chromosomes. The early branching tetrapod X. tropicalis' simple diploid genome and robust synteny to amniotes greatly enhances studies of vertebrate chromosome evolution.
Subject(s)
X Chromosome/genetics , Xenopus/genetics , Animals , Evolution, Molecular , Humans , Mammals/genetics , Synteny/geneticsABSTRACT
Translation elongation isoform eEF1A1 has a pivotal role in protein synthesis and is almost ubiquitously expressed. In mice and rats that transcription of the gene encoding eEF1A1 is downregulated to undetectable levels in muscle after weaning; eEF1A1 is then replaced by a separately encoded but closely related isoform eEF1A2, which has only previously been described in mammals. We now show that not only is eEF1A2 conserved in non-mammalian vertebrate species, but the down-regulation of eEF1A1 protein in muscle is preserved in Xenopus, with the protein being undetectable by adulthood. Interestingly, though, this down-regulation is controlled post-transcriptionally, and levels of full-length eEF1A1 mRNA remain similar to those of eEF1A2. The switching off of eEF1A1 in muscle is therefore sufficiently important to have evolved through the use of repression operating at different levels in different species. The 3'UTR of eEF1A1 is highly conserved and contains predicted binding sites for several miRNAs, suggesting a possible method for controlling of expression. We suggest that isoform switching may have evolved because of a need for certain cell types to modify the well-established non-canonical functions of eEF1A1.
Subject(s)
Muscle, Skeletal/metabolism , Peptide Chain Elongation, Translational , Peptide Elongation Factor 1/metabolism , Xenopus laevis/metabolism , 3' Untranslated Regions , Amino Acid Sequence , Animals , Conserved Sequence , Down-Regulation , Evolution, Molecular , Female , Mice , Molecular Sequence Data , Muscle Development/genetics , Muscle, Skeletal/cytology , Muscle, Skeletal/growth & development , Peptide Elongation Factor 1/genetics , Rats , Xenopus laevis/genetics , Xenopus laevis/growth & developmentABSTRACT
Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.
Subject(s)
Cardiac Myosins/genetics , Codon, Nonsense , Myocardial Contraction/genetics , Myosin Heavy Chains/genetics , Xenopus Proteins/genetics , Xenopus/genetics , Animals , Cardiac Myosins/metabolism , Cardiac Myosins/physiology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Female , Gene Expression Regulation, Developmental , Heart/embryology , Heart/physiology , Immunoblotting , Immunohistochemistry , In Situ Hybridization , Male , Myocardial Contraction/physiology , Myocardium/metabolism , Myosin Heavy Chains/metabolism , Myosin Heavy Chains/physiology , Reverse Transcriptase Polymerase Chain Reaction , Xenopus/embryology , Xenopus/growth & development , Xenopus Proteins/metabolism , Xenopus Proteins/physiologyABSTRACT
BACKGROUND: The protein components of mature skeletal muscle have largely been characterized, but the mechanics and sequence of their assembly during normal development remain an active field of study. Chaperone proteins specific to sarcomeric myosins have been shown to be necessary in zebrafish and invertebrates for proper muscle assembly and function. RESULTS: The Xenopus tropicalis mutation dicky ticker results in disrupted skeletal muscle myofibrillogenesis, paralysis, and lack of heartbeat, and maps to a missense mutation in the muscle-specific chaperone unc45b. Unc45b is known to be required for folding the head domains of myosin heavy chains, and mutant embryos fail to incorporate muscle myosin into sarcomeres. Mutants also show delayed polymerization of alpha-actinin-rich Z-bodies into the Z-disks that flank the myosin-containing A-band. CONCLUSIONS: The dicky ticker phenotype confirms that a requirement for myosin-specific chaperones is conserved in tetrapod sarcomerogenesis, and also suggests a novel role for myosin chaperone function in Z-body maturation.
Subject(s)
Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation, Missense , Xenopus/embryology , Amino Acid Sequence , Amino Acid Substitution , Animals , Genes, Lethal , Molecular Sequence Data , Muscle Development , Myofibrils/metabolism , Myosins/metabolism , Sarcomeres/metabolism , Sequence Alignment , Xenopus/metabolism , ZebrafishABSTRACT
Bio-electrosprays, a recently pioneered direct cell engineering approach, have been demonstrated to handle living cells including stem cells for the development of active specialized and unspecialized microenvironments. This electric field driven technique is currently undergoing vigorous development where the technique is racing towards possible clinical utility. Although this direct cell engineering approach has been elucidated to have no significant effects on the processed cells from a molecular level upwards, the technique needs to demonstrate its potential for use with whole organisms (multi-cellular systems). We believe this is mandatory for whole organisms such as model embryos; developing multi-cellular biological structures are sensitive systems and could possibly be prone to a wide range of embryological disruptions during their dynamic development, post-treatment. Therefore our studies presented herein have investigated the effects on embryos in terms of their structure, function and biological integrity post-bio-electrospraying in comparison to several controls. Our investigations demonstrate the absence of any detectable gross effects on the embryos from a genetic level upwards on post-treated embryos. In fact, these studies clearly elucidate no significant disruptions on the dynamic development of these treated embryos in comparison to those respective controls, thus validating the utility of bio-electrosprays for the careful handling of dynamically developing multi-cellular organisms.
Subject(s)
Models, Animal , Xenopus/embryology , Animals , Specimen Handling/methods , Tissue Culture TechniquesABSTRACT
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.
Subject(s)
Genetic Testing/methods , Mutation , Xenopus/genetics , Animal Diseases/genetics , Animals , Congenital Abnormalities/genetics , Embryo, Nonmammalian/physiology , Female , Genetic Complementation Test , Genomics , Mutagens , Ovum/physiology , Phenotype , Xenopus/embryology , Xenopus/growth & developmentABSTRACT
Analysis of gene function in vertebrates is facilitated by gain-of-function studies, such as injection of synthetic mRNA in amphibian embryos. This approach is hampered by lack of spatial and temporal control of expression of the introduced gene product. An additional level of control is obtained by nuclear-transfer-mediated transgenesis, but functional analyses are complicated by variability and background abnormalities in primary transgenic embryos. The GAL4/UAS system permits establishment of stable lines and elimination of nuclear-transfer-associated abnormalities, through generation of separate UAS-'effector' and GAL4 'transactivator' transgenic lines. When the GAL4 DNA-binding domain is combined with a steroid hormone ligand-binding domain, this system allows full temporal regulation of transgene expression by introduction of an exogenous steroid analogue, the progesterone antagonist RU486. We show here that by crossing stable Xenopus tropicalis transgenic lines, one bearing a UAS-enhanced cyan fluorescent protein (ECFP) reporter construct, and the other with a GAL4-progesterone receptor fusion driven by a retina-specific promoter, reporter expression in the resulting embryos can be induced with RU486 in a tissue-specific manner. These results suggest that the inducible binary system, in which the target gene expression can be controlled in a stage- and tissue-specific pattern, should be readily applicable for gene function studies at all stages of development.
Subject(s)
Xenopus/embryology , Xenopus/genetics , Animals , Animals, Genetically Modified , DNA-Binding Proteins , Female , Gene Expression Regulation, Developmental/drug effects , Genes, Reporter , Green Fluorescent Proteins , Luminescent Proteins/genetics , Male , Microinjections , Mifepristone/pharmacology , Phenotype , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , Recombinant Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Tissue Distribution , Transcription Factors/genetics , Transcriptional Activation/drug effects , Xenopus Proteins/geneticsABSTRACT
Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged ("homomorphic") sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used "RAD-tags" and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.
Subject(s)
Evolution, Molecular , Sex Chromosomes/genetics , Xenopus/genetics , Animals , Female , Gene Expression , Genotype , MaleABSTRACT
The pipid frog Xenopus tropicalis has emerged as a powerful new model system for combining genetic and genomic analysis of tetrapod development with robust embryological, molecular, and biochemical assays. Its early development closely resembles that of its well-understood relative X. laevis, from which techniques and reagents can be readily transferred. In contrast to the tetraploid X. laevis, X. tropicalis has a compact diploid genome with strong synteny to those of amniotes. Recently, advances in high-throughput sequencing together with solution-hybridization whole-exome enrichment technology offer powerful strategies for cloning novel mutations as well as reverse genetic identification of sequence lesions in specific genes of interest. Further advantages include the wide range of functional and molecular assays available, the large number of embryos/meioses produced, and the ease of haploid genetics and gynogenesis. The addition of these genetic tools to X. tropicalis provides a uniquely flexible platform for analysis of gene function in vertebrate development.
Subject(s)
Xenopus/genetics , Animal Husbandry , Animals , Chromosome Mapping , Cold-Shock Response , Cryopreservation , DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel , Embryo, Nonmammalian/physiology , Exons , Female , Fertilization , Genetic Association Studies , Genetic Linkage , Genetic Markers , Genetic Testing , Haploidy , Karyotyping , Male , Mutagenesis , Mutation , Phenotype , Polymorphism, Genetic , Reverse Genetics , Sequence Analysis, DNA , Silver Staining , SpermatozoaABSTRACT
Xenopus tropicalis combine the advantages of X. laevis, for example using explants and targeted gain of function, with the ability to take classical genetics approaches to answering cell and developmental biology questions making it arguably the most versatile of the model organisms. Against this background, husbandry of X. tropicalis is less well developed than for its larger, more robust relative. Here we describe the methods used to keep and breed these frogs successfully.
Subject(s)
Animal Husbandry/methods , Xenopus/physiology , Animal Feed , Animal Husbandry/instrumentation , Animals , Buffers , Chorionic Gonadotropin/administration & dosage , Copulation , Female , Male , Oocytes/cytology , Ovulation , Reproductive Control Agents/administration & dosage , Salts/chemistry , Sex Differentiation , Transportation , Water PurificationABSTRACT
The diploid pipid frog Xenopus tropicalis has recently emerged as a powerful new model system for combining genetic and genomic analysis of tetrapod development with embryological and biochemical assays. Its early development closely resembles that of its well-understood tetraploid relative Xenopus laevis, from which techniques and reagents can be readily transferred, but its compact genome is highly syntenic with those of amniotes. Genetic approaches are facilitated by the large number of embryos produced and the ease of haploid genetics and gynogenesis.
Subject(s)
Genetic Techniques , Xenopus/growth & development , Xenopus/genetics , Animals , Breeding , Centromere/genetics , Cryopreservation , DNA, Complementary/genetics , Embryo, Nonmammalian/metabolism , Female , Fertilization in Vitro , Genetic Linkage , Genetic Markers/genetics , Genomics , Genotype , Haploidy , Karyotyping , Male , Mutagenesis , Mutation , Phenotype , Polymorphism, Single Nucleotide/genetics , Reproduction, Asexual , Silver Staining , Spermatozoa/metabolism , Tissue and Organ Harvesting , Xenopus/embryology , Xenopus/physiologyABSTRACT
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 more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial 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.
Subject(s)
Genome , Sequence Analysis, DNA , Xenopus/genetics , Animals , Chickens/genetics , Chromosome Mapping , Chromosomes/genetics , Computational Biology , Conserved Sequence , DNA Transposable Elements , DNA, Complementary , Embryo, Nonmammalian/metabolism , Evolution, Molecular , Expressed Sequence Tags , Gene Duplication , Genes , Humans , Phylogeny , Synteny , Vertebrates/genetics , Xenopus/embryology , Xenopus Proteins/geneticsABSTRACT
Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.
Subject(s)
Chytridiomycota/physiology , Gene Expression Profiling , Mycoses/genetics , Temperature , Xenopus/genetics , Xenopus/microbiology , Animals , Female , Models, Biological , Oligonucleotide Array Sequence Analysis , Reproducibility of Results , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. 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. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.