Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray

Cytoplasmic control of the adenylation state of mRNAs is a critical post-transcriptional process involved in the regulation of mRNAs stability and translational efficiency. The early development of Xenopus laevis has been a major model for the study of such regulations. We describe here a microarray analysis to identify mRNAs that are regulated by changes in their adenylation state during oogenesis and early development of the diploid frog Xenopus tropicalis. The microarray data were validated using qRT–PCR and direct analysis of the adenylation state of endogenous maternal mRNAs during the period studied. We identified more than 500 mRNAs regulated at the post-transcriptional level among the 3000 mRNAs potentially detected by the microarray. The mRNAs were classified into nine different adenylation behavior categories. The various adenylation profiles observed during oocyte maturation and early development and the analyses of 3′-untranslated region sequences suggest that previously uncharacterized sequence elements control the adenylation behavior of the newly identified mRNAs. These data should prove useful in identifying mRNAs with important functions during oocyte maturation and early development

Post-transcriptional regulation in Xenopus embryos: role and targets of EDEN-BP.

International audienceEDEN (embryo deadenylation element)-dependent deadenylation is a regulatory process that was initially identified in Xenopus laevis early embryos and was subsequently shown to exist in Drosophila oocytes. Recent data showed that this regulatory process is required for somitic segmentation in Xenopus. Inactivation of EDEN-BP (EDEN-binding protein) causes severe segmentation defects, and the expression of segmentation markers in the Notch signalling pathway is disrupted. We showed that the mRNA encoding XSu(H) (Xenopus suppressor of hairless), a protein central to the Notch pathway, is regulated by EDEN-BP. Our data also indicate that other segmentation RNAs are targets for EDEN-BP. To identify new EDEN-BP targets, a microarray analysis has been undertaken

Heparanase 2, mutated in urofacial syndrome, mediates peripheral neural development in Xenopus

Urofacial syndrome (UFS; previously Ochoa syndrome) is an autosomal recessive disease characterized by incomplete bladder emptying during micturition. This is associated with a dyssynergia in which the urethral walls contract at the same time as the detrusor smooth muscle in the body of the bladder. UFS is also characterized by an abnormal facial expression upon smiling, and bilateral weakness in the distribution of the facial nerve has been reported. Biallelic mutations in HPSE2 occur in UFS. This gene encodes heparanase 2, a protein which inhibits the activity of heparanase. Here, we demonstrate, for the first time, an in vivo developmental role for heparanase 2. We identified the Xenopus orthologue of heparanase 2 and showed that the protein is localized to the embryonic ventrolateral neural tube where motor neurons arise. Morpholino-induced loss of heparanase 2 caused embryonic skeletal muscle paralysis, and morphant motor neurons had aberrant morphology including less linear paths and less compactly-bundled axons than normal. Biochemical analyses demonstrated that loss of heparanase 2 led to upregulation of fibroblast growth factor 2/phosphorylated extracellular signal-related kinase signalling and to alterations in levels of transcripts encoding neural- and muscle-associated molecules. Thus, a key role of heparanase 2 is to buffer growth factor signalling in motor neuron development. These results shed light on the pathogenic mechanisms underpinning the clinical features of UFS and support the contention that congenital peripheral neuropathy is a key feature of this disorder

Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis

<p>Abstract</p> <p>Background</p> <p>The western African clawed frog <it>Xenopus tropicalis </it>is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as <it>Xenopus laevis </it>but is diploid and has a smaller genome of 1.7 Gbp. Therefore <it>X. tropicalis </it>is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in <it>X. tropicalis</it>.</p> <p>Results</p> <p>A gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers.</p> <p>Conclusion</p> <p>These cDNA sequences permit <it>in silico </it>cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study <it>X. tropicalis </it>biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.</p

Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis

The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of the human protein CUG-BP1/CELF1. Five mRNAs, encoding cell cycle regulators and a protein involved in the notch pathway, have been identified as being deadenylated by EDEN/EDEN-BP. To identify new EDEN-BP targets, we immunoprecipitated EDEN-BP/mRNA complexes from Xenopus tropicalis egg extracts. We identified 153 mRNAs as new binding targets for EDEN-BP using microarrays. Sequence analyses of the 3′ untranslated regions of the newly identified EDEN-BP targets reveal an enrichment in putative EDEN sequences. EDEN-BP binding to a subset of the targets was confirmed both in vitro and in vivo. Among the newly identified targets, Cdk1, a key player of oocyte maturation and cell cycle progression, is specifically targeted by its 3′ UTR for an EDEN-BP-dependent deadenylation after fertilization

Etudes des transcriptomes de Xenopus tropicalis au cours de la métamorphose

La métamorphose des amphibiens est un ensemble de programmes développementaux complexes initiés par un signal unique, les hormones thyroïdiennes et ses récepteurs qui sont des facteurs de transcription. Ce processus implique de nombreux évènements cellulaires incluant l apoptose ainsi que la prolifération et la différenciation cellulaire. Ainsi, la métamorphose des amphibiens est un modèle de choix pour étudier la fonction des hormones thyroïdiennes in vivo. De nombreuses études se sont concentrées sur les aspects physiologiques et morphologiques de la métamorphose, mais la régulation génétique permettant ces changements reste méconnue. Nous avons décidé d étudier les modifications de l expression génique par une approche de génomique utilisant des puces à ADN. Le modèle Xenopus laevis classiquement utilisé présente des désavantages dûs à son génome. Ainsi, le modèle proche Xenopus tropicalis a été préféré. Un répertoire de transcrits spécifiques du système nerveux chez Xenopus tropicalis a été constitué par construction et séquençage de banques d ADN complémentaires. Ce répertoire de transcrits, combiné aux autres données de séquences a ensuite été utilisé pour construire une puce à ADN de type oligonucléotides longs représentant 2900 transcrits impliqués dans différents processus biologiques ou dont la fonction reste encore inconnue. Ce microréseau a été utilisé pour étudier les modifications des transcriptomes de trois organes (i.e. la queue, le foie et le système nerveux central) pendant la métamorphose. Les transcrits identifiés sont impliqués dans de nombreux processus biologiques comme la régulation de la transcription, la signalisation Wnt ou le cycle cellulaire. La comparaison de ces trois répertoires montre que ces transcrits sont régulés de manière ubiquitaire ou tissu spécifique. Nous avons ensuite utilisé la séquence du génome de X.tropicalis pour identifier des cibles directes des hormones thyroïdiennes parmi les transcrits régulés. Les données d expression produites vont par ailleurs être utilisées pour inférer le réseau de régulation transcriptionnel qui est mis en place à la métamorphose.Amphibian metamorphosis is a set of complex developmental processes triggered by a unique signal, thyroid hormones and its receptors that are transcription factors. This process involves various cellular events implicating apoptosis, cell proliferation and differentiation. Hence, amphibian metamorphosis is a very good model to study the role of thyroid hormones in vivo. Numerous studies focused on physiological or morphological aspects of metamorphosis but genetic regulation triggering these changes is still unknown. We choose a genomic approach to study the variations of gene expression during metamorphosis by using microarrays. The classical amphibian model, Xenopus laevis, is not amenable to perform genomic studies due to the redundancy of its genome. To avoid this problem, we use the close species Xenopus tropicalis. A set of specific transcripts of the central nervous system was first constituted by partial cDNA sequencing. This set of sequences combined to the others sequences data existing in Xenopus tropicalis was then used to build a microarray representing 2900 different transcripts implicated in known biological processes or still functionally uncharacterized. This microarray was then used to study the modifications of gene expression in three different organs (i.e. central nervous system, tail and liver) during metamorphosis. Identified transcripts are involved in various biological processes like transcriptional regulation, Wnt signalling or cell cycle. Comparison of the three sets of differentially expressed transcripts shows that these transcripts are regulated in a tissue-specific or ubiquitous manner. Genomic sequence of X.tropicalis was then used to characterize the direct targets of thyroid hormones among the regulated transcripts. In addition, these expression data will be used to infer the transcriptional regulatory network developed during metamorphosis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Modelling of TH-dependent regulation of tadpole tail resorption

International audienceTail resorption observed at the time of amphibian metamorphosis is controlled by the thyroid hormone (TH). The inherent regulation network is complex and involves an important number of different factors. Consequently, global understanding of this biological process needs elaborate experiments. However, these experiments may be difficult to realize because of the need to manipulate in space and in time gene expression and hormonal treatments. Hence, we first modelled and simulated the biological process using Hybrid Functional Petri Nets. This powerful formalism offers a number of useful features and flexibility. Curves obtained in silico by simulations are in agreement with those observed in vivo and in vitro. Our modelling approach led us to ask pertinent biological questions, from which new hypotheses for experimental testing have emerged