Expression of enzymes involved in thyroid hormone metabolism during the early development of Xenopus tropicalis

BACKGROUND INFORMATION: There are significant indications that amphibians require TH (thyroid hormones) prior to their involvement in the regulation of metamorphosis and before the development of a functional thyroid. RESULTS: In order to investigate the potential role for TH in pre-metamorphic Xenopus tropicalis we have cloned cDNAs for, and analysed the expression of, TPO (thyroid peroxidase), 5'DII (type II iodothyronine deiodinase) and 5DIII (type III iodothyronine deiodinase), enzymes involved in TH metabolism. Zygotic expression of TPO was detected in neurula stage embryos. Expression was observed in the notochord and later in the thyroid. The notochord was also a common site of expression for 5'DII and 5DIII. Other sites of 5'DII expression are the otic vesicles, retina, liver, blood-forming region, branchial arches and brain. 5DIII is also expressed in the brain, retina, liver, developing pro-nephros, blood-forming region and branchial arches. Embryos exposed to the TPO inhibitor methimazole showed a distinctive dose-dependent phenotype of a crimped notochord and shortened axis, together with alterations in (125)I(-) uptake. CONCLUSIONS: These data suggest a novel extrathyroidal role for TH during early development, and support the proposal that embryos require thyroid signalling for normal development prior to metamorphosis.     

Decoding development in Xenopus tropicalis

Xenopus tropicalis is rapidly being adopted as a model organism for developmental biology research and has enormous potential for increasing our understanding of how embryonic development is controlled. In recent years there has been a well-organized initiative within the Xenopus community, funded largely through the support of the National Institutes of Health in the US, to develop X. tropicalis as a new genetic model system with the potential to impact diverse fields of research. Concerted efforts have been made both to adapt established methodologies for use in X. tropicalis and to develop new techniques. A key resource to come out of these efforts is the genome sequence, produced by the US Department of Energy's Joint Genome Institute and made freely available to the community in draft form for the past three years. In this review, we focus on how advances in X. tropicalis genetics coupled with the sequencing of its genome are likely to form a foundation from which we can build a better understanding of the genetic control of vertebrate development and why, when we already have other vertebrate genetic models, we should want to develop genetic analysis in the frog.     

Expression of ribosomal-protein genes in Xenopus laevis development

Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 2-5), become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs.     

Multiple noggins in vertebrate genome: cloning and expression of noggin2 and noggin4 in Xenopus laevis

Noggin is a neural inducer secreted by cells of the Spemann organizer. A single noggin gene was identified until very recently in all tested vertebrates. The only exception was zebrafish, in which two close homologs of noggin, named noggin1 and noggin3, and one gene more diverged from them, noggin2, were cloned. Nevertheless, finding of three zebrafish noggins was attributed exclusively to specific genomic duplications in the fish evolutionary branch. However, very recently it was shown that Xenopus tropicalis have additional noggin homolog, called noggin2 [Fletcher, R.B., Watson, A.L., Harland, R.M. (2004). Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod. Gene Expr. Patterns 5, 225-230], which indicates at least two independent noggin genes in vertebrate phylum. Now we report identification of two novel noggin homologs in each of so evolutionary distant species as Xenopus laevis, chicken and fugu. One of these noggins is ortholog of the X. tropicalis and zebrafish noggin2, whereas another, named noggin4, was not known previously. In the X. laevis embryos, the expression of noggin2 very resembles that of its counterpart in X. tropicalis: it begins with neurulation at the anterior margin of the neural plate and, afterward, continues mainly in the forebrain and dorsal hindbrain. At the same time, noggin4 is expressed starting from the beginning of gastrulation, throughout the ectoderm, with a local expression maximum in the prospective anterior neurectoderm. Later, it is widely expressed on the dorsal side of embryo, including neural tube, eyes, otic vesicles, cranial placodes, branchial arches, and somites. The data presented here demonstrate that the vertebrate phylum contains at least three distinct noggin genes.     

Developmental expression patterns of fosl genes in Xenopus tropicalis

Fos-like antigens (Fosl) including Fosl1 and Fosl2 exclusively heterodimerize with Jun members to form AP-1 complex, thereby participating in various cellular progresses including cell cycle regulation. However, expression patterns of these two genes during embryonic development remains largely unknown. In the present study, both temporal and spatial expression patterns of fosl1 and fosl2 were examined during embryonic development of Xenopus tropicalis. Real-time quantitative PCR results showed that the expression of the two genes was increased from stage 2 to stage 42. However, expression level of fosl1 is much higher than that of fosl2 at stage 42. Whole-mount in situ hybridization showed that fosl1 was expressed in eyes, branchial arch, notochord, otic vesicle, and liver. However, fosl2 was expressed in lung primordium from stage 34 to stage 38, in addition to the moderate expression in eyes and branchial arch at stage 42. Thus, the developmental expression patterns of these two fosl genes is different in Xenopus embryos. These results provide a basis for further functional study of these two genes.     

Down syndrome cell adhesion molecule is important for early development in Xenopus tropicalis

The Down syndrome cell adhesion molecule (DSCAM) is an Ig containing cell adhesion molecule with remarkable structural conservation throughout metazoans. In insects, DSCAM has 38,000 potential isoforms that convey axon guidance, fasciculation, and dendrite morphogenesis during neurodevelopment. In vertebrates, DSCAM is expressed throughout the nervous system and seems to also mediate proper axonal guidance and synaptogenesis without the isoform diversity found in insects. Differences in DSCAM function among several vertebrate species complicate the understanding of an evolutionarily conserved role during embryogenesis. We take advantage of the frog developmental model Xenopus tropicalis to study DSCAM function in early development by expression analysis and morpholino‐mediated knockdown. Our results indicate that DSCAM is expressed early in development and restricted to the head and nervous system. Knockdown of protein expression results in early morphogenetic phenotypes characterized by failed gastrulation and improper posterior neural tube closure. Our results reveal a specific, fundamental role of DSCAM in early morphogenetic movements, presumably through its well‐known role in homophilic cell adhesion. genesis 52:849–857, 2014. © 2014 Wiley Periodicals, Inc.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

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.     

Deep ancestry of mammalian X chromosome revealed by comparison with the basal tetrapod Xenopus tropicalis

ABSTRACT: 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 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 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.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

Expression and subcellular localization of X-ATM during early Xenopus development

ATM, the gene mutated in ataxia telangiectasia, is a protein essential for handling DNA strand breaks. We recently isolated the Xenopus homologue of ATM, X-ATM and we report here the detailed expression pattern of the protein and the mRNA during early Xenopus development. During the cleavage stages, ATM protein was concentrated in and around the nuclei of all cells with low levels of expression also detected in the cytoplasm. Following neurulation, increased protein levels were detected in the nuclei of developing somites and in the central nervous system. Areas of high protein expression correlated with areas of increased mRNA expression which was detected in the nuclei of somites and the developing lens. Received: 2 December 1999 / Accepted: 4 February 2000     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.