Faunes F et al. (2009), Identification of novel transcripts with differ...

XB-ART-39086

Genome Biol 2009 Feb 11;102:R15. doi: 10.1186/gb-2009-10-2-r15.

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Identification of novel transcripts with differential dorso-ventral expression in Xenopus gastrula using serial analysis of gene expression.

Faunes F , Sánchez N , Castellanos J , Vergara IA , Melo F , Larraín J .

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Recent evidence from global studies of gene expression indicates that transcriptomes are more complex than expected. Xenopus has been typically used as a model organism to study early embryonic development, particularly dorso-ventral patterning. In order to identify novel transcripts involved in dorso-ventral patterning, we compared dorsal and ventral transcriptomes of Xenopus tropicalis at the gastrula stage using serial analysis of gene expression (SAGE). Of the experimental tags, 54.5% were confidently mapped to transcripts and 125 showed a significant difference in their frequency of occurrence between dorsal and ventral libraries. We selected 20 differentially expressed tags and assigned them to specific transcripts using bioinformatics and reverse SAGE. Five mapped to transcripts with known dorso-ventral expression and the frequency of appearance for these tags in each library is in agreement with the expression described by other methods. The other 15 tags mapped to transcripts with no previously described asymmetric expression along the dorso-ventral axis. The differential expression of ten of these novel transcripts was validated by in situ hybridization and/or RT-PCR. We can estimate that this SAGE experiment provides a list of at least 86 novel transcripts with differential expression along the dorso-ventral axis. Interestingly, the expression of some novel transcripts was independent of beta-catenin. Our SAGE analysis provides a list of novel transcripts with differential expression in the dorso-ventral axis and a large number of orphan tags that can be used to identify novel transcripts and to improve the current annotation of the X. tropicalis genome.

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Species referenced: Xenopus tropicalis
Genes referenced: abr bcr cd63 chrd foxa4 id2 nap1l1 smagp sox11 szl tmco1 txnrd3 ventx1 znf706 znf706l
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	Figure 3. Verification of the differential expression of X. tropicalis transcripts identified by SAGE. (a) Total RNA was obtained from dorsal (DMZ) and ventral (VMZ) explants isolated from gastrula stage X. tropicalis. RT-PCR was performed using specific primers for each transcript. DV01 (pintallavis), DV03 (vent-1.1), chordin and sizzled were included as controls. (b) X. tropicalis embryos at stage 10 (a-i, a'-i'), and stages 18-20 (a"-i") were processed for in situ hybridization with specific probes for each transcript. (a'-i') Hemi-sections from embryos at the gastrula stage. (a-i, a'-i') Dorsal to the left and (a"-i") anterior is up. The frequency of occurrence in each library is indicated in parentheses below the name for each transcript (tag frequency in dorsal library; tag frequency in ventral library).
	Figure 4. Verification of the differential expression of X. laevis homologues. (a) Total RNA was isolated from dorsal (DMZ) and ventral (VMZ) explants at the gastrula stage. RT-PCR was performed using specific primers for each transcript and different cDNA concentrations (serial dilutions of cDNA, 1:1, 1:2 and 1:4). Chordin was included as control. Reverse transcription in the absence (-RT) or presence (+RT) of reverse transcriptase for specificity of cDNA amplification. (b) X. laevis embryos at stage (st.) 10 (a, e, i, m, q; hemi-sections, dorsal to the left), stage 12 (b, c, f, g, j, k, n, o, r, s; anterior is up) and stages 18-20 (d, h, l, p, t; anterior is up) were processed for in situ hybridization with specific probes for each transcript. Stage 12 embryos are pictured from both sides relative to the blastopore to illustrate its asymmetric expression. Numbers under each transcript correspond to the frequency of occurrence in each SAGE library (tag frequency in dorsal library; tag frequency in ventral library).
	Figure 5. Effect of Wnt signaling on expression of novel transcripts. X. tropicalis embryos were injected at the two-cell stage with control and β-catenin morpholinos and total RNA was isolated at the gastrula stage. RT-PCR was performed by using specific primers for selected transcripts (serial dilutions of cDNA, 1:1, 1:2 and 1:4). Only transcripts for which a dorso-ventral expression difference was detected by RT-PCR were analyzed. Chordin was included as a positive control of a gene dependent on β-catenin. PCR in the absence (-) or presence of cDNA (+RT) from embryos injected with control (MoCo) and β-catenin (Moßcat) morpholinos.
	XB-GENEPAGE-5871172 ( hypothetical protein LOC100127312) gene expression in Xenopus tropicalis embryos, NF stage 10, as assayed by in situ hybridization, dorsal left.
	abr (active BCR-related) gene expression in Xenopus tropicalis embryos, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior up.
	tmco1 (transmembrane and coiled-coil domains 1) gene expression in a Xenopus tropicalis embryo, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior up.
	txnrd3 (thioredoxin reductase 3) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization. dorsal view, anterior up.
	znf706.2 (zinc finger protein 706, gene 2) gene expression in a Xenopus laevis embryo, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior up.
	Figure 1. Tag-mapping of experimental tags to X. tropicalis genome and transcript databases. All different experimental tags (23,766 tags) were mapped first to the genome of X. tropicalis and those without a match (311 tags) were discarded from further analysis. The remaining experimental tags that presented one or more matches to the genome (23,455 tags; 100%) were then mapped to the Ensembl modified database, and only those tags found in the first or second positions from the 3'-end of the RNA sequence or belonging to the polyA-next category (see Materials and methods for details) were selected and reported as mapping to this transcript database (5,615 tags; 23.9%; red). The remaining tags that did not exhibit a match to the transcripts in the Ensembl modified database (17,840; 76.1%) were then searched with the same restraints mentioned above in the joint set composed of the NCBI (mRNAs), Unigene (clusters of mRNAs and ESTs) and Gurdon databases (clusters of ESTs). A total of 7,172 tags (30.6%) were found to match to positions 1, 2 or poly-A next in the transcripts from this set (green). The remaining tags without a match to these databases (10,668; 45.5%) were then re-mapped against the complete set of transcripts (a complete joint set of RNAs composed of Ensembl, NCBI, Unigene and Gurdon databases), but with the restraint that the mapping must occur to position 3 or above in a transcript. A total of 5,011 tags (21.4%) that fulfilled these conditions were obtained (blue). The remaining 5,657 (24.1%) tags mapped to the genome, but did not map to any known transcript (yellow).
	Figure 2. Comparison of the normalized frequencies of tag occurrence between dorsal and ventral SAGE libraries. Tag frequencies were normalized with respect to the total tags in each library (31,175 total dorsal tags and 32,047 total ventral tags), grouped according to their ratio of frequency of occurrence in both libraries and plotted against the counts of tags in each category. The number of tags is indicated inside each bar. Expected tags for known genes with a role in dorso-ventral patterning and control genes are indicated for each category. For these genes, the frequency of occurrence in each library is indicated in parentheses (tag frequency in dorsal library; tag frequency in ventral library).

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Abu-Daya, Zygotic nucleosome assembly protein-like 1 has a specific, non-cell autonomous role in hematopoiesis. 2005, Pubmed, Xenbase