Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis
- Ana C Fierro†1, 2, 5Email author,
- Raphaël Thuret†1, 2,
- Laurent Coen3,
- Muriel Perron1, 2,
- Barbara A Demeneix3,
- Maurice Wegnez1, 2,
- Gabor Gyapay4,
- Jean Weissenbach4,
- Patrick Wincker4,
- André Mazabraud1, 2 and
- Nicolas Pollet1, 2, 5Email author
© Fierro et al; licensee BioMed Central Ltd. 2007
Received: 17 November 2006
Accepted: 16 May 2007
Published: 16 May 2007
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.
Xenopus tropicalis is now an anuran amphibian reference genome for vertebrate comparative genomics. It presents the same advantages as Xenopus laevis but has a smaller genome of 1.7 Gbp and a shorter generation time . Moreover, while X. laevis is an allotetraploid derived from an allopolyploidization event, X. tropicalis is diploid [2, 3]. Even though phylogenetic studies indicate that 30 to 50 MY evolution separate the two species [3, 4], it has been shown that most methods and resources developed for X. laevis can be readily applied to X. tropicalis . Thus, the genome of X. tropicalis was selected to explore amphibian genome characteristics by whole-genome shotgun sequencing .
Working on X. laevis constitutes a challenge when dealing with large-scale transcriptomics, such as microarrays experiments or systematic cDNA sequencing. This is because some X. laevis genes are present as diploids, while others form pairs of paralogs (also called "pseudoalleles") that have been conserved with various degrees of divergence, generally less than 10% . On a genomic scale, recent data has led to the estimation of 12% as the minimal fraction of paralogous gene pairs kept after allotetraploidization . However, this estimate is based on the application of strict and conservative criteria: less than 98% nucleotidic similarity and 93% mean similarity between paralogs. Therefore, it is likely that more than 12% of paralogs are indeed active genes in X. laevis. Moreover, such pairs of genes may have distinct expression patterns . An estimated 14% of paralogs show distinct expression profiles based on EST counts . Given these complications, it follows that the X. tropicalis genome is more amenable to systematic transcriptome surveys than that of X. laevis
Transcriptome analysis relies heavily on cDNA analysis. Collections of cDNA sequences have multiple uses for the molecular geneticist. They can be used to establish transcript catalogues [9–11] and to provide experimental evidence when building gene models from genomic sequence, particularly for 5' and 3' untranslated sequences . Further, they can be used to provide global views on genome expression in a given cell type by the estimation of the abundance of the different mRNA species (through signatures as in ) and therefore can help decipher physiological roles played by a given gene product. Finally, partial cDNA sequences (ESTs) are used to identify full-length clones containing the entire open-reading frame for each transcript .
We initiated an EST program so as to provide a functional genomics resource for X. tropicalis containing sequences from the highest possible number of genes expressed in the nervous system. We report the construction of such a gene index and its assessment after the collection of 48.785 partial cDNA sequences. These ESTs are estimated to represent 6,000 genes that were annotated through sequence similarity searches, protein domain searches and Gene Ontology functional classification. Gene expression profiles were derived from EST counts and used to evidence transcripts differentially expressed at metamorphic stages of development. A set of polymorphic intragenic microsatellite markers was deduced from the analysis of ESTs derived from distinct strains of X. tropicalis. We expect that this resource will be valuable for further molecular genetics experiments.
Results and discussion
Construction of cDNA libraries and normalization
Two X. tropicalis cDNA libraries were constructed for this project. The first, designated xthr, was derived from dissected retinas and heads of young tadpoles (Nieuwkoop and Faber st. 25–35). About 500 retinas were dissected from stage 32 X. tropicalis embryos, a stage where differentiating retinal neurons are getting organized into layers. Because these retinas yielded only few polyA+ RNA, the library was enriched by the addition of mRNA from heads of embryos of the same developmental stage. The second library, designated xtbs, was made from central nervous systems of metamorphosing tadpoles. Brains and spinal cords were dissected from tadpoles between stage 58 and 64, the period covering the whole of Xenopus metamorphosis. To build the library, and with the aim of respecting the relative proportion of nervous tissue obtained at the different stages, samples for six animals were pooled for each stage between 58–61 and three animals for each stage between 62–64. All these tissues were combined and the mRNA extracted for preparation of the xtbs library. The SMART technology (Clontech) was used to enrich the representation of full-length cDNA clones (defined here as a copy of the transcript sequences between the 5' cap and a polyA tail).
Xenopus tropicalis EST project statistics
xthr and xtbs
Number of sequences reads obtained
Number of clone sequences obtained
Number of valid sequences
Number of clones with valid sequences
Number of clones with 5' and 3' EST
Number of clones with 5' EST only
Number of clones with 3' EST only
Average trimmed EST length
Number of contigs
Number of contigs groups
Number of contigs grouped
Number of unique contigs
Nulmber of clones in contigs
Number of singletons
Number of clones in singletons
Number of putative transcripts
Max. assembled sequence length
Average assembled sequence length
Max. assembled sequence size
Average assembled sequence size
Number of contigs containing
We analyzed these sequences with PHRAP  to build contigs out of the overlapping and redundant sequences (Table 1). A total of 31,767 sequences were assembled into 8,756 contigs. These were further grouped by virtue of clone links into 6,547 unique groups (scaffolds). Taking into account the 2,982 singletons issued from 2,304 clones, a total of 9,693 transcripts sequences were identified. We compared our results to the global clustering of all X. tropicalis ESTs (including ours) by the UniGene pipeline and the DFCI Gene Index. In UniGene, our set of ESTs belong to 7,778 groups made of between 1 and 220 clones. Similarly, The DFCI Xenopus tropicalis Gene Index clustered these ESTs in 9,350 TCs and 1,160 singletons.
The majority of clusters (66%) contained three or less ESTs. Only 11 contigs were composed of more than 100 sequences (See Additional file 2) and the largest contig contained 159 sequences. Most of the corresponding gene products (23/50) are ribosomal proteins, the other being proteins involved in basic cellular processes (tubulin, elongation factor 1 alpha). Two noteworthy exceptions are myelin basic protein (contig8746) and metallothionein (contig8708), for which transcripts are found almost exclusively in the nervous sytem.
The sequence redundancy (number of ESTs/cluster) of xthr and xtbs libraries was compared to other X. tropicalis cDNA libraries represented in dbEST (See Additional file 3). A statistically significant difference at the 1% level of significance indicates that the complexity is higher for adult-type cDNA libraries, whether or not a size fractionation was performed. Amongst cDNA libraries prepared from embryonic or larval stages of development, the complexity of the xtbs library ranks first, while the complexity of the xthr library is close to the mean value.
Sequences were assembled into contigs of up to 3 kb in size (hsp90 transcript, Contig 8575, See Additional file 4), but the mean contig length of 745 bp indicated that most of them cover only parts of the cognate transcript sequence.
Another way to assess the fraction of clones likely to be full-length has been described by Gilchrist et al. . Using this method on all X. tropicalis cDNA sequences (version Xt6 ) xthr and xtbs libraries we found to contain respectively 42% and 37% of full-length clones (MJ Gilchrist, personal communication). The mean fraction of full-length clones across all libraries is 18%. Therefore, we conclude that our normalization procedure did not impair the proportion of full-length clones compared to non-normalized libraries.
In order to further analyse our dataset, we compared our contigs to ENSEMBL predicted transcripts. Altogether, 4,437 contigs (52%) and 1,423 singletons (48%) matched 4,083 transcripts from 3,703 ENSEMBL predicted genes (15%). The extent of the underclustering of our ESTs was estimated from these numbers and used to calculate that our whole EST set represents about 6,000 genes. We conclude that our cDNA sequence collection significantly improved annotation of the X. tropicalis genome sequence. Similarly, we compared our dataset to 2,402 X. tropicalis RefSeq mRNA sequences. We found that 2,230 contigs (26%) and 484 (16%) singletons matched 1,342 RefSeq entries (56%). These figures suggest that further extensive sequencing of putative full-length cDNA clones from our collection will be of great use in order to cover the entire Xenopus gene set.
We next estimated the proportion of our cDNA sequences representing mRNA molecules produced by a splicing event and hence most likely to correspond to physiological products. We used "exonerate" to compute alignments between cDNA and genomic sequences. We retained only the alignments satisfying the thresholds of 95% identity and 90% coverage. Evidence of splicing was found for 5,025 contigs (65%) out of 7,718 contigs aligned to the genome. From the 2,693 contigs left, only 274 are significantly similar to a protein sequence and it is likely that the others represent 3' untranslated regions, often encoded by a single exon in vertebrates.
Next, two complimentary methods were used to find evidence for alternative splicing. Using genomic sequences (see Methods) we predicted 111 cases of alternative splicing, including conserved ones such as Clathrin light chain (contig7735 and contig8467, See Additional file 5). Using alignments on protein and genomic sequences we found 58 cases (such as elrD represented by contig7817, See Additional file 6).
Results of sequence comparisons
X. laevis reg. entries
X. laevis UniGene
X. tropicalis UniGene
X. tropicalis genome
X. tropicalis cDNA
70 – 90%
90 – 95%
GO Molecular function classification.
Gene ontology term
All molecular function terms
-->calcium ion binding
-->nucleic acid binding
---->transcription factor activity
--->translation factor activity, nucleic acid binding
--->cytoskeletal protein binding
--->phosphoprotein phosphatase activity
--->protein kinase activity
->chaperone regulator activity
->enzyme regulator activity
->nutrient reservoir activity
->signal transducer activity
->structural molecule activity
->transcription regulator activity
->translation regulator activity
-->electron transporter activity
-->ion channel activity
-->neurotransmitter transporter activity
->triplet codon-amino acid adaptor activity
Several known genes specifically expressed in the eye were identified, including different crystallins (beta, gamma and mu isoforms), vsx1 (visual system homeobox 1), pax6 (paired-box protein 6), rdgb (retinal degeneration B homolog), rgr (RPE-retinal G protein-coupled receptor). Well-characterised central nervous system specific genes were identified as well, notably elrC, mbp (myelin basic protein), plp (myelin proteolipid protein 1). The corresponding cDNAs will provide useful differentiation markers for X. tropicalis.
A significant number of the contigs (37%) had no significant similarities to previously described genes, and may represent transcribed pseudogenes, non-coding RNA sequences and undescribed genes. Indeed, comparing our sequences to non-coding RNA sequences (microRNA from RFAM, or ncRNA from the H-INV datasets) we found 2 microRNA precursors (contig7127 and 7850 encoding mir-9-1 and mir-124a respectively) as well as E3 (Contig2965) and 5SN4 (Contig5668) snoRNAs. Contig7127 (452 nt) is derived from the assembly of 6 ESTs derived from 3 distinct cDNA clones of the xtbs library. The alignment of contig7127 sequence on X. tropicalis genome sequence reveals 100% identity and indicates that one splicing event is required. Thus, contig7127 represents a bona fide neural transcript of the mir-9-1 gene. Contig7850 (800 nt) is derived from the assembly of 10 ESTs derived from 6 cDNA clones (one from xtbs and 5 from xthr libraries). Four of these clones are identical and characterized by a 409 bp cDNA, while two are longer and have their 3' ends ESTs as singletons but mapping to the same scaffold region.
Other collections of X. tropicalis ESTs are ongoing [8, 17, 19] using a variety of cDNA libraries made from adult tissues or embryos at different stages of development. Hence, we undertook an in silico analysis of gene expression profiles estimated from EST counts .
In a first analysis, we searched transcripts identified by ESTs derived predominantly from our cDNA libraries. We identified 99 and 238 cDNAs found prominently in the heads and retinas of tailbuds or brain and spinal cord of tadpole, respectively (See Additional file 8 and Additional file 9) and 25 clones found predominantly in both structures. These clones are likely to represent genes differentially expressed in the retina or the central or peripheral nervous system during metamorphosis. The study of these genes in Xenopus could well improve our knowledge on CNS development and function in vertebrates.
Since there are currently ten times more ESTs in cDNA libraries derived from metamorphic stages of development in X. laevis than in X. tropicalis, we did a similar survey of the expression profiles of transcripts in X. laevis.
Highly expressed metamorphic transcripts.
actin alpha 1 skeletal muscle
actin alpha 1 skeletal muscle
eukaryotic translation factor 1 alpha, somatic form
creatine kinase, muscle
tail, limb, tailbud
Calcium ATPase at 60A, cardiac muscle
myosin, heavy polypeptide 13, skeletal muscle
collagen, type I, alpha 1
eukaryotic translation elongation factor 2
actin alpha skeletal muscle
actin cytoplasmic 1
actin cytoplasmic 1
freeze tolerance-associated protein FR47
limb, thymus, spleen_PHA
ribosomal protein L3
fast skeletal troponin C beta
limb, tailbud, tadpole
myosin light chain 1, fast skeletal muscle isoform
actin alpha cardiac
solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5
limb, tadpole, olfactory epith.
acidic ribosomal protein P0
ribosomal protein L4
procollagen, type I, alpha 2
guanine nucleotide binding protein, beta 2, related sequence 1
tropomyosin 1 alpha chain
myosin, heavy polypeptide 4, skeletal muscle
serum albumin 74 kDa
tropomyosin beta chain, skeletal muscle
We then carried out an in silico reconstruction of the transcriptional profile of X. laevis metamorphic genes using the IDEG suite of statistical tests. We removed clusters composed of less than 10 ESTs, leading to a set of 3,599 UniGene clusters. The GT test ranked profiles in three categories: strong (167 clusters with GT > 0.66) medium (1,300 clusters with 0.33 < GT < 0.66) or weak (2,132 cluster with GT < 0.33) differential expression.
A larval beta chain of globin is among the metamorphic cluster together with an alpha chain, an indication of the relevance of our analysis. The comparison of Xl.56714 EST sequences with known proteins shows that they resemble cell surface receptors of the SLAM (Signalling Lymphocytic Activation Molecule) family. The SLAM receptors regulate immune cell activation. Indeed, it is known that immune system remodelling is a major event of metamorphosis . The gene corresponding to Xl.56714 is expressed in metamorphic tadpoles (including tail and intestine) as well as in the adult kidney. However we could not detect significant similarities to any known gene sequences or proteins. Alpha-1 antichymotrypsin (a plasma protease inhibitor) is highly expressed during metamorphosis and found in adult liver. This correlates with the associated stress condition that occurs during tadpole transformations. The gene encoding alpha-2-HS-glycoprotein (also named fetuin) steadily increases in expression from tailbud stage up to metamorphosis. This gene product is secreted in plasma and plays a physiological role during mammalian fetal development, especially in mineralization and growth. A known Xenopus gene encoding a small peptide named PYLa is found exclusively in a cDNA library prepared from stage 62 tadpoles. As for the preprocaerulein transcripts in X. tropicalis, the PYLa transcripts are abundant in metamorphic stages, with ESTs found in limbs and whole tadpoles. Both caerulein and PYLa peptides may be secreted from skin glands and exert antimicrobial activities. This finding corroborates a previous report on caerulein expression . Remarkably, skin glands are known to express a cocktail of signalling peptides, including neuropeptides such as xenopsin, thyrotropin-releasing hormone and PGLa. Whether these peptides play specific roles in the context of metamorphosis is unknown. The cluster Xl.24674 corresponds to a gene resembling uromodulin. Corresponding transcripts are found in metamorphic intestine and whole tadpole. In mammals, uromodulin is excreted in urine and plays a role in the cellular defense response. A gene encoding a stomatin homolog is highly expressed in intestine during metamorphosis. Stomatin is a membrane protein regulating cation exchange and cytoskeletal attachment. Among the genes represented in the metamorphic limb cluster are a keratin and two clusters (Xl.57017 and Xl.57064) annotated as lacking significant similarities to known proteins. In the tadpole cluster, a carboxyl ester lipase is found expressed in tadpole and in metamorphic intestine. Mucin 2 is another gut protein highly expressed in tadpole, as well as carboxypeptidase.
Taken together, these expression profiles, based on EST counts, reveals certain genes that are up-regulated during metamorphosis, possible targets of the thyroid hormones signalling pathway.
The cDNA sequences we produced are derived from the Adiopodoume strain of X. tropicalis, originating from the Ivory Coast. The genomic and most other cDNA sequencing efforts are made on the N strain from Nigeria or a distinct IC strain from Ivory Coast. We therefore looked for polymorphisms that could be used in genetic mapping experiments or to discriminate with mutations obtained from ENU mutagenesis. We identified 8 SNPs derived from mitochondrial genes, three of which (snp1A, snp2A, snp6G) are specific to the Adiopodoume strain (See Additional file 11). The presence of shared alleles for 5 SNPs indicates the close relationship with the N strain as already reported by Evans et al. 2004. We searched for novel polymorphism markers made of di, tri, tetra and pentanucleotide sequence repeats present in our EST collection. We found from two to ten alleles in 225 markers derived from 212 contigs/ESTs clusters. A subset of 107 markers are potential highly informative since two or more alleles are observed at high frequencies (See Additional file 12). The dinucleotide repeat AT and TA are the most common, accounting for 137 markers. These intragenic markers should be useful once placed on a genetic linkage map.
This dataset will provide an invaluable tool for exon definition when the X. tropicalis genome sequence is finally determined. The results presented here are available through a database on our web site . Users can carry out BLAST and other searches based on GO classification, InterproScan results, and expression information. The cDNA sequences have been deposited with Genbank/EMBL/DDBJ (accession numbers CN072222 – CN121006) and clones are available upon request.
Large-scale cDNA sequencing has provided invaluable resources to decipher vertebrate genome structure and function. Recent studies on cDNA sequencing with deep coverage provide fundamental knowledge on the complexity of transcriptomes in mammals . Here, we provide information on the transcript sequence and expression of an estimated 6,000 genes in X. tropicalis. A web resource  is available with associated annotations. The genetic resources stemming from the cDNA sequencing project described here can be used in diverse research projects, including vertebrate comparative genomics, studies on evolution and development, cell biology and developmental genetics. More specifically, retinogenesis and remodelling of the central nervous sytem during metamorphosis will benefit from this cDNA resource.
We are currently undertaking full cDNA insert sequencing for a set of non-redundant clones, as well as characterizing their expression using a whole-mount in situ hybridization screen [28, 29]. The genomic resources developed to study X. tropicalis biology are crucial to explore amphibian physiology and genetics, this model system providing excellent characteristics for addressing key questions related to anuran metamorphosis and its associated regulatory processes.
Embryo and tissue dissection
Embryos of Xenopus tropicalis Adiopodoumé strain were obtained from parents issued of the Geneva collection .
From each of the two libraries made, 58,368 clones were picked, arrayed in microtiter plates and gridded on high-density nylon filters. A sample of 1,989 cDNA clones from the xthr library and 1,694 clones from xtbs were partially sequenced to obtain 4,120 ESTs. This step provided a quality assessment of the two libraries, showing the absence of clones of bacterial origin and few ribosomal (0.45% in xthr, 0% in xtbs) and mitochondrial contaminants (1.2 and 3.6% in xthr and xtbs, respectively). This procedure provided information on overrepresented clones, which were then removed before further sequencing of up to 30,000 clones. These ESTs were grouped into 1,985 clusters.
Normalization of cDNA libraries
Two pools of 25 and 28 oligonucleotides probes of 35 nt in length were labelled using Terminal Transferase (New England Biolabs) and P33-dATP (Amersham). Labelled oligonucleotides were hybridised on two high-density filters representing the xthr and xtbs libraries as in . Positive clones were identified using X-Digitize software on images acquired using a phosphorimager.
High-throughput sequencing, assembling
The reactions were performed with a Big-Dye terminator cycle sequencing kit and analyzed by ABI-3700 and ABI-3730. Sequences were base-called using PHRED, then trimmed using LUCY and custom perl scripts. Sequences less than 100 bp were discarded, as well as those identified to be derived from ribosomal and mitochondrial RNAs. PHRAP was used to assemble the sequences taking into account quality scores, further clustering was obtained by scaffolding using mate-pairs informations. We retained scaffolds only if two clone links were available (excepting singletons) and if the orientation of the reads was consistent.
Repetitive sequences were masked using CENSOR. Contigs and singletons were used as queries in BLASTX and BLASTN searches of Swissprot, Uniprot, Unigene (rel 70) and Xenopus tropicalis JGI assembly v4.1 databases on INFOBIOGEN server. The february 2005 release of ENSEMBL was used. Framefinder was used to identify coding sequences, and protein domains were searched using INTERPROSCAN. The results of sequence assembly, scaffolding and annotation were loaded on a custom-made mySQL database. Web interface was developed using PHP scripts.
Assessment of the fraction of clones likely to be full-length
5'ESTs or contigs were compared to full-length cDNAs using BLASTN. Alignments longer than 50 nt and exhibiting more than 95% identities were selected. Overlap between query and subject sequences was scored only when the alignment encompassed up to the last nucleotide at the 5' or 3' end of the sequences.
Alignments between cDNA and genomic sequences (masked) were computed using exonerate. Evidence for alternative splicing was found when two contigs were aligned to the same genomic region with at least 95% mean overlap but with a different number of exons.
Alignments between cDNA and UNIREF protein sequences were computed using BLASTX. Alignments characterized by a gap (at least 10 aa) introduced in the contig sequence were retrieved. Alternative splicing was considered present if the contig sequence including the gap could be aligned to the genomic sequence.
EST counts were downloaded from UniGene (release 70 for X. laevis and 32 for X. tropicalis). Relevant profiles were extracted using custom perl scripts. GT test was run using the IDEG6 software . Single hierarchical clustering was performed using Cluster 3.0 software . We used absolute correlation similarity metrics followed by complete clustering on mean centered gene expression profiles. Results were visualised using TreeView.
Mitochondrial SNPs were identified on a collection of mitochondrial ESTs downloaded from dbEST, JGI  and from our own set. These ESTs were assembled using CAP3 and analyzed using visualization software . Microsatellites were identified using custom perl scripts.
We thank L. Du Pasquier for the gift of X. tropicalis animals and his continuous support. This research was funded by grants from l'Association pour la Recherche contre le Cancer, le Centre National de la Recherche Scientifique, le Ministère de l'Education, de la Recherche (French Xenopus Stock Center) et de la Technologie and the University of Paris Sud.
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