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Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization.
Fig. 1. Structure and expression of the vg1 gene cluster. (A) Schematic diagrams of the vg1 gene cluster in X. tropicalis (XTR) and X. laevis (XLA). Arrowheads indicate the direction and positions of genes. Distances in the schematic are proportional to the nucleotide lengths of the genes, except for vg1p.S which contains a partial exon 1. Magenta and blue arrowheads indicate complete vg1 genes and the vg1 pseudogene, respectively. Orange and blue bars indicate the region covered by BAC full-sequences and scaffold70461 which were originally constructed in the v7.1 assembly. (B) cDNA FISH analysis of the vg1 gene. Hybridization signals on PI-stained chromosomes XLA1L and XLA1S (upper) are shown with their Hoechst-stained patterns (lower). The subchromosomal location of the vg1 gene is shown on the Hoechst idiogram of XLA1L. (C) RNA-seq expression profile of vg1 genes during oogenesis and early development. oo12, oo34 and oo56 indicate oocyte stage 1/2, 3/4 and 5/6, respectively.
Fig. 2. Structure of the vg1 pseudogene vg1p.S. (A) Schematic diagram of the vg1p.S structure in comparison with that of vg1-e3.L. vg1p.S lacks exon 2 and the splice donor/acceptor sites. The exon 1 region of vg1p.S shows 92% identity with that of vg1-e3.L. (B) Comparison of the exon 1 nucleotide sequences of vg1-e3.L and vg1p.S. vg1p.S contains a stop codon near the end of exon 1 that resulted from an insertion and a deletion (blue boxes). The intron 1 of vg1-e3.L is underlined. vg1p.S also contains a serine residue at position 20 of the deduced protein product.
Fig. 3. Alignment of Vg1 protein sequences. Deduced protein sequences of vg1-e1.L to -e3.L, vg1_AY838794.1 ( Birsoy et al., 2006), vg1_BC090232.1 (IMAGE cDNA clone) and vg1_M18055.1 ( Rebagliati et al., 1985) were aligned by the Clustal W method. Serine or proline residues at position 20 of the deduced protein products are indicated. The proteolytic processing site is shown by a double underline. Asterisks indicate seven conserved cysteine residues in the mature domain which are shared with other TGF-β family proteins.
Fig. 4. Alignment of the 3′-untranslated regions of vg1 genes. The 340 bp sequence of the 3′-untranslated regions of vg1 genes were aligned by the Clustal W method. Boxes indicate consensus repeat motifs important for vegetal localization of vg1 transcripts. Three VM1 motifs were identified by Gautreau et al. (1997). In addition, two upstream VM1 motifs overlap with E1, and other motifs (E2–E4) involved in the process of vg1 RNA localization are also shown ( Deshler et al., 1997).
Fig. 5. Structure and expression of the nodal3 gene cluster. (A) Schematic diagrams of the nodal3 gene clusters of X. tropicalis (XTR) and X. laevis (XLA). Arrowheads indicate the direction and positions of genes. Distances in the schematic are proportional to the nucleotide lengths of genes. Magenta and blue arrowheads indicate complete nodal3 genes and nodal3 pseudogenes, respectively. An orange bar indicates the region covered by the fosmid full-sequence. (B) cDNA FISH analysis of the nodal3 gene. Hybridization signals on PI-stained chromosomes 3L and 3S (upper) are shown with their Hoechst-stained patterns (lower). The subchromosomal location of nodal3 is shown on the Hoechst idiogram of XLA3L. (C) RNA-seq expression profile of nodal3 genes during oogenesis and early development. oo12, oo34 and oo56 indicate oocyte stage 1/2, 3/4 and 5/6, respectively.
Fig. 6. Alignment of Nodal3 protein sequences. (A) X. laevis Nodal3 protein sequences were aligned by the Clustal W method. The box indicates the deduced signal peptide region. Note that Nodal3p1.L lacks this signal peptide and is unlikely to be functional. The proteolytic processing site is shown by a double underline. Asterisks indicate five conserved cysteine residues in the mature domain which are shared with other TGF-β family proteins. (B) Phylogenetic tree of X. laevis and X. tropicalis Nodal3 proteins. Nodal1 and Nodal2 proteins were also included in the analysis. Evolutionary history was inferred using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary analyses were conducted in MEGA6 ( Tamura et al., 2013).
