Fierro AC et al. (2007), Exploring nervous system transcriptomes during ...

XB-ART-35901

BMC Genomics 2007 May 16;8:118. doi: 10.1186/1471-2164-8-118.

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

Fierro AC , Thuret R , Coen L , Perron M , Demeneix BA , Wegnez M , Gyapay G , Weissenbach J , Wincker P , Mazabraud A , Pollet N .

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The western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis 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 X. tropicalis. 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. These cDNA sequences permit in silico 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 X. tropicalis 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.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: adgrl1 agap2 ahsg apoa1 arf3 armh4 arrb1 ass1 cacna2d1 ccdc136 cel.2 cela1 cntnap1 col5a3 cpb1 ctrb2 diras2 dmtn dnajb2 eif2a eif2ak3 epha8 fibcd1 frat1 frzb gnrh2 gps2 gria4 hba2 hbg2 kif1a klc1 krt12.1 krt78.7 laptm4b map2 map6 mogat2 myb myh8 myl3 ndufa6 nptx2 nrsn1 nrxn1 phyhip plp1 prickle2 rab40b rpl3l serpina3 slc7a7 stom tf tmem59l umodl1 ybx3

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	Figure 1. Assessment of normalization effectiveness. Histogram showing the percentage of sequence matching each oligonucleotide used in the procedure of normalization by hybridization. Bars represent percentages of positive clones calculated before (grey bars) and after normalization (black bars). Data before normalization were obtained after partial sequencing of 1,989 cDNAs from xthr and 1,694 cDNAs from xtbs. Note the relatively high abundance of cell-type specific transcript such as gamma crystallin (crystallinG1) or neurogranin (underlined on the figure).
	Figure 2. Added-value of xthr and xtbs 5'ESTs. 5' cDNA sequences were compared to 4,908 complete X. tropicalis cDNA sequences from XGC and Sanger Institute. When an EST matched unambiguously (>95% id over more than 50 nt on the same orientation) one of these cDNAs, the position of its first residue (X axis) was plotted as a function of the cDNA size (Y axis). Each dot represents the result of an alignment. A position of 0 on the x axis indicates identical 5' ends between the EST and cDNA. Negative values indicate that the EST extends further 5', positive values superior to the cDNA length indicate that the EST extends further 3', and positive values inferior to the cDNA size indicate the 5' EST position relative to the cDNA.
	Figure 3. Digital expression profiles of X. tropicalis transcripts differentially expressed at metamorphosis. Each line gives the expression profile of a given transcript represented by a UniGene cluster. The expression is deduced from counting the occurence of ESTs derived from a given cDNA library. The level of expression is colour coded in blue shades, dark blue means evidence for high levels of transcripts and white means no evidence for expression. On the left, clusters of expression profiles are delineated by a vertical bar labelled with the associated characteristic domain of expression. On the right, the cluster name and its annotation (i.e. the corresponding gene product description as deduced from sequence similarity analysis) are given. Each column corresponds to a category of tissue or stage of development: 8 adult tissues and 6 stages of development. Note that a given category may correspond to several cDNA libraries. Here, only clusters for which evidence of differential expression were used to build the matrix of expression. This matrix was analysed by hierarchical clustering on the expression profile dimension using CLUSTER 3.0 as described in the methods section.
	Figure 4. Digital expression profiles of X. laevis transcripts differentially expressed at metamorphosis. Using the same representation as in Fig. 3 three clusters are depicted that are associated with differential expression at metamorphosis (top cluster), tadpole stage (middle) and in the forming limb (down).

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