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The transcription factor, VegT, is the meso-endodermal determinant in Xenopus laevis. We examined VegT orthologs from several anuran amphibians and the urodele amphibian, the Mexican axolotl. In addition to the conserved T-box, the DNA-binding domain, the orthologs share a conserved 57 amino acid domain at the C-terminal. Most striking is a 17-nucleotide (nt) sequence near the 3' end of the open reading frame. The 17 nts are absolutely conserved among the anurans, whose last common ancestor lived 200 million years ago. As an initial test of the function of the 17 nts, 27 or 49 amino acids, which include the six amino acids coded by the 17 (+1) nts, were deleted from the C-terminal of VegT. These truncated VegT's retained some transcriptional activity, indicating that the 17 nts are not absolutely required for this function. The function of the highly conserved 17 nts is unknown. Two possibilities are that the conserved 17 nts interact with the cytoskeleton or that they are a target for regulation by a microRNA.
Fig. 1 Amphibian VegT clones. This figure shows the relationship between the open-reading frame (ORF) of VegT from Xenopus laevis and the ORFs of our clones of VegT orthologs from other amphibians. Incomplete ORFs are indicated by a dashed line. The orthologs are aligned relative to the VegT T-box, amino acids 40–230, and the numbers with each clone refer to the VegT amino acid position. The asparagine (N) at amino acid 157 distinguishes the VegT T-box from
the Brachyury T-box (Conlon et al., 2001). The percent within the T-box is the percent amino acid identity to the VegT T-box. The final 15 amino acids (441–455) are conserved, with differences of only 1–2 amino acids, among the five amphibians for which sequences are available. Species abbreviations are Rana pipiens (Rp), Eleutherodactylus coqui (Ec), Colostethus machalilla (Cm), Gastrotheca riobambae (Gr), Pipa pipa (Pp), Ambystoma mexicanum (Am). GenBank numbers for the new sequences are CmVegT (DQ400351), GrVegT (DQ388611), PpVegT (DQ363258).
Fig. 2 Conservation of the VegT C-terminal. (A) When the six anuran VegTs are compared, there is a high degree of conservation of the 57 C-terminal amino acids. The sequence MSDWSQ (441–6) (light gray) corresponds to a 17 nt sequence, which is identical among the four known anuran sequences and was used to clone GrVegT and PpVegT. (B) In comparison of the C-terminal 57 amino acids of VegT with orthologs of non-anurans, there is a four amino acid insertion relative to VegT at position 407 (dark gray). The amino acids corresponding to the MSDWSQ in VegT are boxed (light gray) as in (A) for comparison. See Knezevic et al. (1997) for information on the chicken ortholog Ch-Tbx6L and Griffin et al. (1998) for the zebrafish ortholog Spadetail.
Fig. 3 Activity of a VegT deletion construct. RNAs were prepared from VegT cDNA and a deletion construct (VegTDC49), and these RNAs were tested in the Xenopus animal cap assay. Expression of mesodermal (Xbra, Xwnt8), endodermal (Sox17a, Mix1), signaling (Derriere), and organizer (Goosecoid, Chordin) markers was detected by RT-PCR. VegTDC49 RNA led to transcription of the same genes as VegT RNA at 750 pg/embryo, with lower activity at 150 pg/embryo. Similar results were obtained from a second VegTDC49 RNA preparation and from VegTDC27 RNA. EF1a served as a loading control. Water, embryos injected with water; Embryo, whole uninjected embryos. ( RT), sample without reverse transcriptase.
