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Orthologues of Brachyury, a subfamily of T-box transcription factors, specify distinct cell types in different metazoan phyla, suggesting that the function of these genes has changed through the course of evolution. To investigate this evolutionary process, we have compared the activities of Brachyury orthologues from all major phyla in a single cellular context, the pluripotent Xenopus laevis animal cap. In this assay, an ancestral function is revealed: most orthologues, including the Hydra protein, mimic the action of endogenous Xenopus Brachyury, in that they induce mesoderm but not endoderm. Orthologues from Drosophila and ascidians, however, display an additional derived property, represented in our assay by the induction of endoderm. Misexpression of chimeric versions of Brachyury reveals that the C-terminal half of the protein is important for the strength of the induced response but not for its specificity. In contrast, amino acids located within the T-domain and in a short N-terminal peptide are involved in restricting the activity of Brachyury proteins to induction of mesoderm and not endoderm. Possession of this N-terminal motif is correlated with early circumblastoporal expression of Brachyury orthologues. We propose that restriction of Brachyury activity by this motif plays a conserved role in the control of Bilaterian gastrulation.
Fig. 1. Quantitative analysis of endoderm and striated muscle induction in response to misexpression of different Brachyury orthologues. Histogram showing
the percentage of animal caps positive for somitic muscle (grey) or endoderm (black) upon misexpression of the indicated Brachyury orthologues or of the
positive control VegT. Above each bar is shown the total number of tested explants. The range of nanograms of injected mRNA per embryo is indicated.
Dt., Deuterostomians; Pt., Protostomians; Cn, Cnidarians; H.r., Halocynthia roretzi; C.i., Ciona intestinalis.
Fig. 2. The Brachyury proteins induce different types of terminal differentiation in the Xenopus laevis animal cap assay. Dissected animal caps were cultured
until the equivalent of the tailbud stage and assayed for the presence of endoderm (dark blue staining) and subsequently for somitic muscle (purple staining).
Results are shown for a control embryo (A), and for animal caps dissected from uninjected embryos (B), or from embryos injected with the following doses
of synthetic mRNAs: (C) 0.2 ng of VegT mRNA; (D) 1.7 ng of Xbra mRNA; (E) 3.3 ng of Xbra mRNA; (F) 3.4 ng of ntl mRNA; (G) 1 ng of T mRNA;
(H) 1 ng of AmBra1 mRNA; (I) 0.2 ng of As-T mRNA; (J) 0.06 ng of CiBra mRNA; (K) 1 ng of SpBra mRNA; (L) 1 ng of PfBra mRNA; (M) 1 ng of
PdBra mRNA; (N) 0.3 ng of Trg mRNA; (O) 6 ng of HyBra1 mRNA. Scale bars represent 400 m in (A), and 200 m in (B–O).
Fig. 3. Equal protein amounts of Xbra and As-T proteins trigger different biological responses. (A) Embryos were injected animally at the two-cell stage with
the indicated amount of synthetic mRNA coding either for the Xbra-FLAG or the AsT-FLAG protein. At stage 7, animal hemispheres where dissected and
processed by Western blotting with an anti-FLAG antibody. (B–H) Pictures of stage 35 animal caps assayed by in situ hybridisation for the presence of the
endodermin transcript. The embryos were injected with 250 pg (B), 10 pg (C), 3 pg (D), or 1 pg (E) of the Xbra-FLAG mRNA; or with 30 pg (G) and 10
pg (H) of the AsT-FLAG mRNA. (F) Explants dissected from uninjected embryos. Arrowheads and arrow in (C) show animal caps fully or partly
differentiated as epidermis, respectively. The scale bar in (F) represents 400 m in (B–H).
Fig. 4. C-terminal domain swapping experiments. In (A) and (B), the chimeric constructs are drawn as boxes where numbers refer to the amino acid sequence
used from either Xbra, HyBra1, or As-T. (A) Histograms summarising four independent experiments, and indicating the percentage of muscle positive
explants (grey) dissected from uninjected embryos (lane 1) or upon misexpression of synthetic mRNA coding for Xbra (lane 2, 3.3 ng), HyBra1 (lane 3, 2.5–5
ng), XH (lane 4, 2–6 ng), and HX (lane 5, 0.5–4 ng). Above each bar is shown the total number of tested explants. (B) Histograms summarising five
independent experiments. The grey and black bars indicate the percentage of muscle and endoderm positive caps, respectively. Numbers above these bars
indicate the total number of explants assayed for both markers. Results are shown for animal caps dissected from uninjected embryos (lane 1), or dissected
from embryos injected with: 1.7 ng of Xbra mRNA (lane 2); 0.2 ng of As-T mRNA (lane 3); the XA mRNA (lane 4, 0.02–0.05 ng; lane 5, 0.1 ng; lane 6,
0.3–1 ng); and the AX mRNA (lane 7, 0.02 ng; lane 8, 0.09–0.125 ng; lane 9, 0.25–1 ng).
Fig. 5. Endodermal determinants are contained both within the T-box and within its upstream domain. (A) Sequence alignment of the N-terminal amino acids
of Brachyury orthologues and VegT. For clarity, the first 37 amino acids of Drosophila (D.m.) Brachyury have been omitted. H.r., Halocynthia roretzi; C.i.,
Ciona intestinalis; C.s., Ciona savignyi; the mollusc sequence is from Patella vulgata (Lartillot et al., 2002), and the chaetognath sequence is from
Paraspadella gotoi (Takada et al., 2002). Conserved residues are shaded. The arrow shows the site of fusion used in the chimeras. The tree on the left indicates
the phylogenetic relationships between the animals from which these orthologues have been isolated. The blue and red colours refer, respectively, to an
induction of mesoderm only, or of both mesoderm and endoderm, in the animal cap assay. The branch leading to the chaetognaths is dotted to indicate that
their exact phylogenetic position is unclear. (B) Drawings of the chimeras. The numbers around A and X represent the amino acid sequence position used
from As-T and Xbra, respectively. The T-domains are shaded grey. (C) The histograms summarise three independent experiments, and show the percentages
of endoderm positive caps. The numbers of assayed samples are noted above each bar. Injected doses of mRNA are: Xbra, 2 ng; As-T, 0.06–0.1 ng; XAX,
0.1–0.3 ng; AXX, 0.1–0.7 ng; and XAA 0.03 ng. (D) Pictures of explants assayed for the presence of endoderm, and dissected from embryos injected with
2 ng of Xbra mRNA, 0.1 ng of XAX mRNA, 0.2 ng of AXX mRNA, and 0.03 ng of XAA mRNA. The scale bar represents 200 m.
