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#27546377   2016/08/29 Save this To Up

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis.

Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

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#27318088   2016/08/07 Save this To Up

Capsaicin inhibits the Wnt/β-catenin signaling pathway by down-regulating PP2A.

Xenopus embryo serves as an ideal model for teratogenesis assays to examine the effects of any substances on the cellular processes critical for early development and adult tissue homeostasis. In our chemical library screening with frog embryo, capsaicin was found to repress the Wnt/β-catenin signaling. Depending on the stages at which embryos became exposed to capsaicin, it could disrupt formation of dorsal or posterior body axis of embryo, which is associated with inhibition of maternal or zygotic Wnt signal in early development. In agreement with these phenotypes, capsaicin suppressed the expression of Wnt target genes such as Siamois and Chordin in the organizer region of embryo and in Wnt signals-stimulated tissue explants. In addition, the cellular level of β-catenin, a key component of Wnt pathway, was down-regulated in capsaicin-treated embryonic cells. Unlike wild-type β-catenin, its non-phosphorylatable mutant in which serine and threonine residues phosphorylated by GSK3 are substituted with alanine was not destabilized by capsaicin, indicative of the effect of this chemical on the phosphorylation status of β-catenin. In support of this, capsaicin up-regulated the level of GSK3- or CK1-phosphorylated β-catenin, concomitantly lowering that of its de-phosphorylated version. Notably, capsaicin augmented the phosphorylation of a phosphatase, PP2A at tyrosine 307, suggesting its repression of the enzymatic activity of the phosphatase. Furthermore, capsaicin still enhanced β-catenin phosphorylation in cells treated with a GSK3 inhibitor, LiCl but not in those treated with a phosphatase inhibitor, okadaic acid. Together, these results indicate that capsaicin inhibits the patterning of the dorso-ventral and anterior-posterior body axes of embryo by repressing PP2A and thereby down-regulating the Wnt/β-catenin signaling.

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#27098915   2016/06/09 Save this To Up

Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq).

Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 5'- and 3'-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA.

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#27016259   2016/03/26 Save this To Up

Genome-wide analysis of dorsal and ventral transcriptomes of the Xenopus laevis gastrula.

RNA sequencing has allowed high-throughput screening of differential gene expression in many tissues and organisms. Xenopus laevis is a classical embryological and cell-free extract model system, but its genomic sequence had been lacking due to difficulties arising from allotetraploidy. There is currently much excitement surrounding the release of the completed X. laevis genome (version 9.1) by the Joint Genome Institute (JGI), which provides a platform for genome-wide studies. Here we present a deep RNA-seq dataset of transcripts expressed in dorsal and ventral lips of the early Xenopus gastrula embryo using the new genomic information, which was further annotated by blast searches against the human proteome. Overall, our findings confirm previous results from differential screenings using other methods that uncovered classical dorsal genes such as Chordin, Noggin and Cerberus, as well as ventral genes such as Sizzled, Ventx, Wnt8 and Bambi. Complete transcriptome-wide tables of mRNAs suitable for data mining are presented, which include many novel dorsal- and ventral-specific genes. RNA-seq was very quantitative and reproducible, and allowed us to define dorsal and ventral signatures useful for gene set expression analyses (GSEA). As an example of a new gene, we present here data on an organizer-specific secreted protein tyrosine kinase known as Pkdcc (protein kinase domain containing, cytoplasmic) or Vlk (vertebrate lonesome kinase). Overexpression experiments indicate that Pkdcc can act as a negative regulator of Wnt/ β-catenin signaling independently of its kinase activity. We conclude that RNA-Seq in combination with the X. laevis complete genome now available provides a powerful tool for unraveling cell-cell signaling pathways during embryonic induction.

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#26862700   2016/02/11 Save this To Up

Functional Cloning Using a Xenopus Oocyte Expression System.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

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#26212133   2015/08/13 Save this To Up

Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening.

CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency.

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#25285446   2014/10/07 Save this To Up

Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

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#24104353   2013/12/06 Save this To Up

Esculetin suppresses proliferation of human colon cancer cells by directly targeting β-catenin.

The Wnt pathway is a promising therapeutic and preventive target in various human cancers. The transcriptional complex of β-catenin-T-cell factor (Tcf), a key mediator of canonical Wnt signaling, has been implicated in human colon cancer development. Current treatment of colon cancer depends on traditional cytotoxic agents with limited effects. Therefore, the identification of natural compounds that can disrupt the β-catenin-TcF complex to suppress cancer cell growth with fewer adverse side effects is needed. To identify compounds that inhibit the association between β-catenin and Tcf, we used computer docking to screen a natural compound library. Esculetin, also known as 6,7-dihydroxycoumarin, is a derivative of coumarin and was identified as a potential small-molecule inhibitor of the Wnt-β-catenin pathway. We then evaluated the effect of esculetin on the growth of various human colon cancer cell lines and its effect on Wnt-β-catenin signaling in cells and in an embryonic model. Esculetin disrupted the formation of the β-catenin-Tcf complex through direct binding with the Lys312, Gly307, Lys345, and Asn387 residues of β-catenin in colon cancer cells. In addition, esculetin effectively decreased viability and inhibited anchorage-independent growth of colon cancer cells. Esculetin potently antagonized the cellular effects of β-catenin-dependent activity, and in vivo treatment with esculetin suppressed tumor growth in a colon cancer xenograft mouse model. Our data indicate that the interaction between esculetin and β-catenin inhibits the formation of the β-catenin-Tcf complex, which could contribute to esculetin's positive therapeutic and preventive effects against colon carcinogenesis.

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#23340479   2013/05/17 Save this To Up

Evolutionary conservation of the oocyte transcriptome among vertebrates and its implications for understanding human reproductive function.

Cross-phylum and cross-species comparative transcriptomic analyses provide an evolutionary perspective on how specific tissues use genomic information. A significant mRNA subset present in the oocytes of most vertebrates is stabilized or stored for post-LH surge use. Since transcription is arrested in the oocyte before ovulation, this RNA is important for completing maturation and sustaining embryo development until zygotic genome activation. We compared the human oocyte transcriptome with an oocyte-enriched subset of mouse, bovine and frog (Xenopus laevis) genes in order to evaluate similarities between species. Graded temperature stringency hybridization on a multi-species oocyte cDNA array was used to measure the similarity of preferentially expressed sequences to the human oocyte library. Identity analysis of 679 human orthologs compared with each identified official gene symbol found in the subtractive (somatic-oocyte) libraries comprising our array revealed that bovine/human similarity was greater than mouse/human or frog/human similarity. However, based on protein sequence, mouse/human similarity was greater than bovine/human similarity. Among the genes over-expressed in oocytes relative to somatic tissue in Xenopus, Mus and Bos, a high level of conservation was found relative to humans, especially for genes involved in early embryonic development.

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#23287019   2013/05/31 Save this To Up

Three calcium-sensitive genes, fus, brd3 and wdr5, are highly expressed in neural and renal territories during amphibian development.

Numerous Ca(2+) signaling events have been associated with early development of vertebrate embryo, from fertilization to organogenesis. In Xenopus laevis, Ca(2+) signals are key regulators in the earliest steps of the nervous system development. If neural determination is one of the best-characterized examples of the role of Ca(2+) during embryogenesis, increasing literature supports a determining role of organogenesis and differentiation. In blastula the cells of the presumptive ectoderm (animal caps) are pluripotent and can be induced toward neural fate with an intracellular increase of free Ca(2+) triggered by caffeine. To identify genes that are transcribed early upon Ca(2+) stimuli and involved in neural determination, we have constructed a subtractive cDNA library between neuralized and non-neuralized animal caps. Here we present the expression pattern of three new Ca(2+)-sensitive genes: fus (fused in sarcoma), brd3 (bromodomain containing 3) and wdr5 (WD repeat domain 5) as they all represent potential regulators of the transcriptional machinery. Using in situ hybridization we illustrated the spatial expression pattern of fus, brd3 and wdr5 during early developmental stages of Xenopus embryos. Strikingly, their domains of expression are not restricted to neural territories. They all share a specific expression throughout renal organogenesis which has been found to rely also on Ca(2+) signaling. This therefore highlights the key function of Ca(2+) target genes in specific territories during early development. We propose that Ca(2+) signaling through modulation of fus, brd3 and wdr5 expressions can control the transcription machinery to achieve proper embryogenesis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

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