ABSTRACT
Traumatic Brain Injury (TBI) is a global driver of disability, and we currently lack effective therapies to promote neural repair and recovery. TBI is characterized by an initial insult, followed by a secondary injury cascade, including inflammation, excitotoxicity, and glial cellular response. This cascade incorporates molecular mechanisms that represent potential targets of therapeutic intervention. In this study, we investigate the response to focal impact injury to the optic tectum of Xenopus laevis tadpoles. This injury disrupts the blood-brain barrier, causing edema, and produces deficits in visually-driven behaviors which are resolved within one week. Within 3 h, injured brains show a dramatic transcriptional activation of inflammatory cytokines, upregulation of genes associated with inflammation, and recruitment of microglia to the injury site and surrounding tissue. Shortly afterward, astrocytes undergo morphological alterations and accumulate near the injury site, and these changes persist for at least 48 h following injury. Genes associated with astrocyte reactivity and neuroprotective functions also show elevated levels of expression following injury. Since our results demonstrate that the response to focal impact injury in Xenopus resembles the cellular alterations observed in rodents and other mammalian models, the Xenopus tadpole offers a new, scalable vertebrate model for TBI.
Subject(s)
Astrocytes , Brain Injuries, Traumatic , Animals , Astrocytes/metabolism , Brain Injuries, Traumatic/metabolism , Disease Models, Animal , Inflammation/metabolism , Larva , Mammals , Mice , Mice, Inbred C57BL , Microglia/metabolism , Neuroinflammatory Diseases , Xenopus laevisABSTRACT
To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.
Subject(s)
Eye/embryology , Eye/metabolism , Gene Expression Regulation, Developmental , MicroRNAs/genetics , RNA Interference , Xenopus laevis/embryology , Xenopus laevis/genetics , Animals , Eye Proteins/genetics , Eye Proteins/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Genetic Association Studies , Loss of Function Mutation , Organogenesis/genetics , Phenotype , Protein Binding , Receptor Protein-Tyrosine Kinases/genetics , Reproducibility of Results , Transcription Factors/genetics , Transcription Factors/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolismABSTRACT
BACKGROUND/AIM: Phosphaplatin platinum (IV) (RRD4) complex has exceptional antitumor properties. The aim of this study was to investigate the effects and the mechanism of action of free and liposome-encapsulated RRD4 in breast cancer. MATERIALS AND METHODS: Liposome-encapsulated RRD4 prepared by thin-film dehydration: hydration and free RRD4 were tested in vivo and in vitro against 4T1 breast cancer cells. Cell proliferation, migration and viability were determined. Tissue and cell production and expression of pigment epithelium-derived factor (PEDF) were assessed by ELISA and western blot. 4T1 cells treated with PEDF siRNA were evaluated for viability and apoptosis. RESULTS: RRD4 inhibited tumor growth and prevented distant metastasis. Liposome formulation enhanced this therapeutic benefit without increasing toxicity and prolonged RRD4 retention in tumor tissues. In vitro, RRD4 induced 4T1 apoptosis through up-regulation of FAS, BAX, and PUMA, and down-regulation of BCL2. RRD4 facilitates a FAS-intrinsic signaling mechanism. PEDF up-regulation represents another antitumor mechanism associated with this phosphaplatin compound. DISCUSSION: Free RRD4 or formulated into liposomes, are excellent candidates for adjuvant therapy against breast tumor growth and metastasis.
Subject(s)
Antineoplastic Agents/pharmacology , Eye Proteins/metabolism , Liposomes/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Nerve Growth Factors/metabolism , Organoplatinum Compounds/pharmacology , Serpins/metabolism , Animals , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Eye Proteins/genetics , Female , Gene Knockdown Techniques , Liposomes/administration & dosage , Liposomes/chemistry , Mammary Neoplasms, Experimental/metabolism , Mice, Inbred BALB C , Nerve Growth Factors/genetics , Organoplatinum Compounds/administration & dosage , Serpins/genetics , Up-Regulation/drug effectsABSTRACT
Model animals are crucial to biomedical research. Among the commonly used model animals, the amphibian, Xenopus, has had tremendous impact because of its unique experimental advantages, cost effectiveness, and close evolutionary relationship with mammals as a tetrapod. Over the past 50 years, the use of Xenopus has made possible many fundamental contributions to biomedicine, and it is a cornerstone of research in cell biology, developmental biology, evolutionary biology, immunology, molecular biology, neurobiology, and physiology. The prospects for Xenopus as an experimental system are excellent: Xenopus is uniquely well-suited for many contemporary approaches used to study fundamental biological and disease mechanisms. Moreover, recent advances in high throughput DNA sequencing, genome editing, proteomics, and pharmacological screening are easily applicable in Xenopus, enabling rapid functional genomics and human disease modeling at a systems level.
Subject(s)
Developmental Disabilities/genetics , Disease Models, Animal , Drug Evaluation, Preclinical , Xenopus/genetics , Animals , Developmental Disabilities/physiopathology , Gene Editing , Genomics , Humans , Mammals , ProteomicsABSTRACT
The establishment of cell lineages occurs via a dynamic progression of gene regulatory networks (GRNs) that underlie developmental commitment and differentiation. To investigate how microRNAs (miRs) function in this process, we compared miRs and miR targets at the initiation of the two major ectodermal lineages in Xenopus. We used next-generation sequencing to identify over 170 miRs expressed in midgastrula ectoderm expressing either noggin or a constitutively active BMP receptor, reflecting anterior neural or epidermal ectoderm, respectively; 125 had not previously been identified in Xenopus. We identified the locations of the pre-miR sequences in the X. laevis genome. Neural and epidermal ectoderm express broadly similar sets of miRs. To identify targets of miR-dependent translational control, we co-immunoprecipitated Argonaute-Ribonucleoprotein (Ago-RNP) complexes from early neural and epidermal ectoderm and sequenced the associated RNA. The Ago-RNP RNAs from these tissues represent overlapping, yet distinct, subsets of genes. Moreover, the profile of Ago-RNP associated genes differs substantially from the profile of total RNAs in these tissues. We generated target predictions for the "high confidence" Ago-RNP RNAs using the identified ectodermal miRs; These RNAs generally had target sites for multiple miRs. Oct4 orthologues, as well as many of their previously identified transcriptional targets, are represented in the Ago-RNP pool in both tissues, suggesting that miR-dependent regulation contributes to the downregulation of the oct4 gene regulatory network and the reduction in ectodermal pluripotency.
Subject(s)
Ectoderm/metabolism , Epidermis/embryology , MicroRNAs/genetics , Neural Plate/metabolism , RNA, Messenger/genetics , Xenopus laevis/embryology , Animals , Cell Differentiation , Embryo, Nonmammalian/metabolism , Epidermis/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Regulatory Networks , MicroRNAs/biosynthesis , Microinjections , Phenotype , RNA, Messenger/administration & dosage , RNA, Messenger/biosynthesis , Xenopus Proteins/genetics , Xenopus laevis/geneticsABSTRACT
Small RNAs from early neural (i.e., Noggin-expressing, or NOG) and epidermal (expressing a constitutively active BMP4 receptor, CABR) ectoderm in Xenopus laevis were sequenced to identify microRNAs (miRs) expressed in each tissue. Argonaute-associated mRNAs were isolated and sequenced to identify genes that are regulated by microRNAs in these tissues. Interactions between these ectodermal miRs and selected miR-regulated mRNAs were predicted using the PITA algorithm; PITA predictions for over 600 mRNAs are presented. All sequencing data are available at NCBI (NCBI Bioproject Accession number: PRJNA325834). This article accompanies the manuscript "MicroRNAs and ectodermal specification I. Identification of miRs and miR-targeted mRNAs in early anterior neural and epidermal ectoderm" (V.V. Shah, B. Soibam, R.A. Ritter, A. Benham, J. Oomen, A.K. Sater, 2016) [1].
ABSTRACT
MicroRNAs (miRNAs) are known to play diverse roles in the regulation of vertebrate development. To investigate miRNA-target mRNA relationships in embryonic development, we have carried out small-RNA sequencing to identify miRNAs expressed in the early gastrula of Xenopus laevis. We identify a total of 180 miRNAs, and we have identified the locations of the miRNA precursor sequences in the X. laevis genome. Of these miRNAs, 141 represent miRs previously identified in Xenopus tropicalis. Alignment to human miRNAs led to the identification of 39 miRNAs that have not previously been described for Xenopus. We have also used a biochemical approach to isolate mRNAs that are associated with the RNA-Induced Silencing Complex (RISC) in early gastrulae and thus candidate targets of miRNA-dependent regulation. Interrogation of this RISC-associated mRNA pool by RT-PCR indicates that a number of genes essential for early patterning and specification may be under regulation by miRNAs. Smad1 transcripts are associated with the RISC; target prediction algorithms identify a single miRNA-binding site for miR-26, which is common to the 3'UTRs of Smad1a and Smad1b. Disruption of the interaction between miR-26 and the Smad1 3'UTR via a Target Protector Morpholino Oligonucleotide (TPMO) leads to a 2-fold increase in Smad1 protein accumulation, moderate increases in the expression of BMP4/Smad1 target genes, and a reduction in organizer gene expression, as well as a partially ventralized phenotype in approximately 25% of embryos. Overexpression of miR-26 resulted in moderately decreased expression of Smad1-dependent genes and an expansion of the region expressing the Organizer gene not1. Our findings indicate that interactions between miR-26 and the Smad1 3'UTR modulate Smad1 function in the establishment of axial patterning; they also establish a foundation for the functional analysis of miRNAs and their regulatory interactions during gastrulation.
Subject(s)
Gastrula/metabolism , Gene Expression Regulation, Developmental , MicroRNAs/metabolism , Smad1 Protein/genetics , Xenopus Proteins/genetics , Xenopus/embryology , Xenopus/genetics , 3' Untranslated Regions/genetics , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Base Sequence , Embryo, Nonmammalian/metabolism , Embryonic Development/genetics , Gastrula/embryology , Immunoprecipitation , MicroRNAs/genetics , Molecular Sequence Data , Phenotype , Protein Binding/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Smad1 Protein/metabolism , Xenopus Proteins/metabolismABSTRACT
Exostosin 1 (EXT1) is a glycosyltransferase that contributes to the biosynthesis of heparan sulfate proteoglycans (HSPG). Loss of ext1 function leads to the human genetic disorder hereditary multiple exostoses (HME) and inhibits development in mouse, zebrafish and Drosophila. In Xenopus, loss of maternal EXT1 leads to impaired wnt11 signaling, resulting in a loss of dorsal embryonic development (Tao et al., 2005), but the functions of zygotic ext1 have not been elucidated. In this study, morpholino oligonucleotides were used to generate a zygotic partial loss of function for ext1, in order to evaluate the requirements for ext1 function in gastrulation and paracrine signaling. Transcriptional profiling was carried out by microarray. Validation and subsequent analyses of gene expression were performed using Q-RT-PCR and in situ hybridization. Western blots were used to assess paracrine signaling pathway activity. Introduction of ext1 MO led to gastrulation defects, which were partially rescued by co-injection of ext1 mRNA. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos identified several developmentally significant genes that are dependent upon Ext1 function, including brachyury (Xbra). In addition, decreased Ext1 was shown to reduce the level of Wnt8 and BMP4 signaling and disrupt ventral-specific gene expression. Ext1 function is required for maintenance of normal levels of BMP and wnt, as well as their target genes. In addition, expression of xbra and the establishment of ventral mesoderm depend upon normal levels of Ext1. These findings suggest that ext1-dependent synthesis of HSPG is critical for wnt and BMP signaling, mesodermal identity, and ventral pattern.
Subject(s)
Body Patterning/physiology , Bone Morphogenetic Proteins/metabolism , Gastrula/metabolism , Gene Expression Regulation, Developmental , Morpholinos/pharmacology , N-Acetylglucosaminyltransferases/antagonists & inhibitors , Xenopus laevis/metabolism , Animals , Biomarkers/metabolism , Blotting, Western , Bone Morphogenetic Proteins/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Gastrula/embryology , Gastrula/pathology , Gene Expression Profiling , Humans , In Situ Hybridization , Mice , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Oligonucleotide Array Sequence Analysis , Paracrine Communication , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Wnt Proteins/genetics , Wnt Proteins/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Xenopus laevis/geneticsABSTRACT
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
FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-ß Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.
Subject(s)
Bone Morphogenetic Protein 4/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Fibroblast Growth Factors/physiology , MAP Kinase Kinase Kinases/physiology , Signal Transduction , Smad1 Protein/metabolism , Xenopus Proteins/metabolism , Animals , Down-Regulation , Immunohistochemistry , In Situ Hybridization , Real-Time Polymerase Chain Reaction , Xenopus/embryologyABSTRACT
BACKGROUND: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. RESULTS: Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. CONCLUSIONS: In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.
ABSTRACT
We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132cM in length, and 4 smaller linkage groups between 7 and 40cM. The total effective size of the map is 1658cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.
Subject(s)
Chromosome Mapping/methods , Genetic Markers/genetics , Spectral Karyotyping/methods , Xenopus/genetics , Animals , Chromosome Banding , Genome/genetics , Genotype , Internet , Minisatellite Repeats/genetics , Polymorphism, Genetic , Xenopus Proteins/geneticsABSTRACT
Planaria are simple flatworms with an extraordinary ability to regenerate missing body parts. This makes them a unique model system for the study of regeneration. Extending an earlier chemical screen, Beane et al. (2011) now reveal a role for H+/K+ ATPase and membrane depolarization in anterior regeneration in planaria.
ABSTRACT
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
The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Photoreceptor Cells/metabolism , Rhodopsin/genetics , Rod Opsins/genetics , Xenopus Proteins/genetics , Animals , Embryo, Nonmammalian/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Homeodomain Proteins/genetics , Retinaldehyde , Rhodopsin/metabolism , Rod Opsins/metabolism , Xenopus Proteins/metabolism , Xenopus laevisABSTRACT
We studied the expression of FGF receptor 3 (FGFR3) mRNA throughout early development of Xenopus laevis by RT-PCR and in situ hybridization. RT-PCR shows that FGFR3 mRNA is localized within the gastrula; regionalized staining is detected by the neural plate stage and continues throughout embryonic development. Strong expression is seen in developing neural structures, especially in the forebrain and hindbrain, including the developing eyes, and in lateral mesoderm. Comparison of these data with previous reports of FGF expression in this species suggests possible FGF-FGFR3 interactions. The pattern of FGFR3 expression appears to be strongly conserved among vertebrate embryos.
Subject(s)
Gene Expression Regulation, Developmental , Receptor, Fibroblast Growth Factor, Type 3/genetics , Animals , Gastrula/metabolism , Prosencephalon/embryology , Prosencephalon/metabolism , RNA, Messenger/metabolism , Receptor, Fibroblast Growth Factor, Type 3/biosynthesis , Rhombencephalon/embryology , Rhombencephalon/metabolism , Xenopus laevis/embryologyABSTRACT
BACKGROUND: The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency. RESULTS: To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis. CONCLUSIONS: In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.
Subject(s)
DNA Transposable Elements , Mutagenesis, Insertional/methods , Xenopus/genetics , Animals , Embryo, Nonmammalian/metabolism , Germ-Line Mutation , Models, Animal , Xenopus/embryologyABSTRACT
Catenins of the p120 subclass display an array of intracellular localizations and functions. Although the genetic knockout of mouse delta-catenin results in mild cognitive dysfunction, we found severe effects of its depletion in Xenopus. delta-catenin in Xenopus is transcribed as a full-length mRNA, or as three (or more) alternatively spliced isoforms designated A, B and C. Further structural and functional complexity is suggested by three predicted and alternative translation initiation sites. Transcript analysis suggests that each splice isoform is expressed during embryogenesis, with the B and C transcript levels varying according to developmental stage. Unlike the primarily neural expression of delta-catenin reported in mammals, delta-catenin is detectable in most adult Xenopus tissues, although it is enriched in neural structures. delta-catenin associates with classical cadherins, with crude embryo fractionations further revealing non-plasma-membrane pools that might be involved in cytoplasmic and/or nuclear functions. Depletion of delta-catenin caused gastrulation defects, phenotypes that were further enhanced by co-depletion of the related p120-catenin. Depletion was significantly rescued by titrated p120-catenin expression, suggesting that these catenins have shared roles. Biochemical assays indicated that delta-catenin depletion results in reduced cadherin levels and cell adhesion, as well as perturbation of RhoA and Rac1. Titrated doses of C-cadherin, dominant-negative RhoA or constitutively active Rac1 significantly rescued delta-catenin depletion. Collectively, our experiments indicate that delta-catenin has an essential role in amphibian development, and has functional links to cadherins and Rho-family GTPases.
Subject(s)
Cadherins/metabolism , Catenins/genetics , Catenins/metabolism , Gastrula/metabolism , Xenopus laevis/embryology , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Alternative Splicing , Animals , Base Sequence , Cell Adhesion , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gastrula/ultrastructure , Gastrulation/physiology , Gene Knockout Techniques , Molecular Sequence Data , Neurulation/physiology , Xenopus laevis/metabolism , Delta CateninABSTRACT
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.