1. Volume 8, Issue 4, Pages 599-610 (April 2005)
Functional Specificity of the Xenopus T-Domain Protein Brachyury Is Conferred by Its Ability to Interact with Smad1 
Nigel J. Messenger, Christin Kabitschke, Robert Andrews, Donna Grimmer, Ricardo Núñez Miguel, Tom L. Blundell, James C. Smith, Fiona C. Wardle 
Developmental Cell 
Volume 8, Issue 4, Pages (April 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 The N Terminus of Xbra Restricts Its Inducing Activity
(A) XbraK149N does not induce expression of goosecoid. Xenopus embryos were injected with the indicated RNA into the animal pole region at the one cell stage or left uninjected. Animal cap explants were isolated at late blastula stage 9 and cultured until sibling embryos reached early gastrula stage RNA prepared from these animal caps was assayed by real-time RT-PCR.
(B) With one exception (Hydra), the HLL(S/N)AV(E/Q)XE(M/L)XXGSEKGD sequence is specific to Brachyury orthologs that are unable to induce endoderm. N-terminal amino acid sequences of different Brachyury orthologs are grouped according to their abilities to induce just mesoderm in isolated animal pole regions (Meso) or endoderm plus mesoderm (E + M). The N-terminal sequence of VegT, which induces endoderm plus mesoderm, is also shown. The leucine residue at the end of the sequence (L; highlighted in bold) is common to the T domains of all the listed proteins. The HLL(S/N)AV(E/Q)XE(M/L)XXGSEKGD domain is highlighted in blue. This part of the figure, and the data contained within, is based on Figure 5A of Marcellini et al., 2003.
(C) Structures of Xbra and VegT and the creation of the chimeric protein VX.
(D) Mutation of the HLL(S/N)AV(E/Q)XE(M/L)XXGSEKGD sequence of Xbra or replacement of this domain with the equivalent region of VegT allows Xbra to activate goosecoid. Xenopus embryos were injected and assayed by real-time RT-PCR as described above.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2
Activation of goosecoid by VegT and Xbra Does Not Require Intervening Protein Synthesis
(A and B) Xbra, like VegT, can activate expression of goosecoid in the presence of cycloheximide (CHI). Xenopus embryos were injected with the indicated RNAs into the animal pole region at the one cell stage or left uninjected. Animal cap explants were isolated at late blastula stage 9, dexamethasone (Dex) and CHI were added as indicated, and caps were cultured until sibling embryos reached early gastrula stage RNA prepared from these animal caps was assayed by real-time RT-PCR. We note that the fold induction of goosecoid by VegT-GR in these experiments is less than that observed in response to wild-type (wt) VegT in Figure 1.
(C) VegT does not act on the upstream promoter region of goosecoid. Oocytes were injected with VegT RNA along with the luciferase goosecoid promoter or Der promoter construct (as a positive control). The experiment was carried out in triplicate. Bars represent SEM.
(D) Xbra causes transient expression of goosecoid. Animal caps from embryos injected as above were harvested at 15 min intervals after addition of Dex and assayed by real-time RT-PCR as described above.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Xbra Activates Xom, a Repressor of goosecoid Expression; Loss of Xom Function Allows Xbra to Induce goosecoid
(A) Expression of Xom is induced by Xbra, but not by VegT. Animal caps isolated from control embryos or embryos injected with RNA encoding Xbra or VegT were cultured to early gastrula stage 10 and assayed for expression of BMP-4, Vent-1, or Xom.
(B) Xbra binds to the Xom promoter. ChIP assays were performed with preimmune serum (lane 3) or an anti-Xbra antibody (lane 4) and Xenopus tropicalis gastrula stage embryos. DNA was PCR amplified by using primers designed against the promoters of Bix and eFGF (positive controls), Xag1 and β-actin (negative controls), and Xom. The promoter regions of Xom, Bix, and eFGF are enriched in the anti-Xbra chromatin sample compared with the sample treated with preimmune serum, whereas Xag1 and β-actin promoter regions are not enriched. Lanes 1 and 2 show 0.5 ng and 0.1 ng of input chromatin that was PCR amplified as an additional control.
(C) Activation of Xbra-GR at midgastrula stage 11, but not at early gastrula stage 10, causes induction of goosecoid. Animal caps derived from control embryos or from embryos injected with RNA encoding hormone-inducible versions of Xbra or VegT were treated with Dex at the indicated stage and cultured for 2 hr before being assayed for expression of goosecoid.
(D) Coexpression of Xbra with an antisense morpholino oligonucleotide directed against Xom allows activation of goosecoid. Animal caps derived from control embryos or from embryos injected with RNA encoding Xbra or VegT, in the presence or absence of an antisense MO directed against Xom, were dissected at late blastula stage 9 and cultured to early gastrula stage 10.5 before being assayed for expression of goosecoid.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
Xbra Interacts with XSmad1, but Not XSmad2; Interaction Requires BMP Signaling
In (A–C) and (E), embryos were injected at the one cell stage with RNA encoding the indicated tagged proteins. They were collected at early gastrula stage 10.5, and lysates were either subjected immediately to SDS PAGE and Western blotting with the indicated antibody, as an indication of protein loading, or were first immunoprecipitated as indicated. In (D), analyses used uninjected embryos.
(A) Xbra (lane 2), but not VegT (lane 5), coimmunoprecipitates with XSmad1. Coimmunoprecipitation does not occur with XbraHLL→AAA (lane 3) or with VX (lane 4), but XSmad1 does coimmunoprecipitate with HSmad4 (lane 6).
(B) Xbra does not coimmunoprecipitate with XSmad2 (lane 2).
(C) When BMP signaling is abrogated by using a truncated BMP receptor (tBR) the interaction of Xbra with XSmad1 is abolished (compare lanes 3 and 8).
(D) Endogenous Xbra interacts with endogenous Smad1. Embryo extracts were subjected to immunoprecipitation and Western blotting by using the indicated antisera, as described in the Experimental Procedures. Xbra, like Smad4, interacts with endogenous Smad1.
(D) Versions of Smad1 in which Ser378 is mutated to Asn, or in which Tyr336 and Tyr343 are mutated to Asp, do not interact with Xbra.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5
The N Terminus of Xbra Interacts with the C-Terminal 50 Amino Acids of the XSmad1 MH2 Domain and Allows One to Create a Structural Model of the XSmad1-Xbra N-Terminal Complex
(A) Diagrams of the constructs used in the yeast two-hybrid assay.
(B) Growth of transformed yeast on +His plates.
(C) Growth of transformed yeast on −His +35 mM 3AT plates. Growth indicates that the proteins in question interact with each other.
(D) Growth of transformed yeast on +0.2% 5FOA plates. Growth indicates that no interaction occurs.
(E) Key to the experiment.
(F) One subunit of the XSmad1 trimer (blue) is shown complexed with the Xbra N terminus (red). The N and C termini are marked. The expanded regions show residues involved in significant interactions. Residues from the Xbra N terminus are represented with bonds in red, and those from XSmad1 are represented with bonds in blue. Figure generated with MOLSCRIPT (Kraulis, 1991) and Raster3D (Merrit and Murph, 1994).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
Interference with the Interaction between Xbra and XSmad1 Allows Xbra to Activate goosecoid and Causes Anteriorization of the Developing Embryo
(A) Coexpression of the N-terminal 47 amino acids of Xbra allows wt Xbra to induce expression of goosecoid. Xenopus embryos were injected with RNA encoding Xbra (400 pg) or Xbra(1–47) (1 ng) or both. Animal cap explants were isolated at late blastula stage 9 and cultured until sibling embryos reached early gastrula stage RNA prepared from these animal caps was assayed by real-time RT-PCR.
(B) The N-terminal region of Xbra elevates goosecoid expression in isolated ventral marginal zone tissue. Ventral marginal zone regions derived from control embryos or embryos injected ventrally at the four-cell stage with 4 ng RNA encoding wt (HLLSAVE) or mutated (AAASAVE or HLLSGAE) versions of Xbra(1–47) were collected at early gastrula stage 10.5 and assayed by real-time RT-PCR for expression of goosecoid. Note that wt Xbra(1–47), but not the mutated versions, causes upregulation of goosecoid. goosecoid expression in an isolated dorsal marginal zone is shown for comparison purposes.
(C–F). Embryos injected ventrally with wt or mutated versions of Xbra(1–47), as described in (B), were subjected to in situ hybridization by using a probe specific for goosecoid. Note that wt Xbra(1–47), but not the mutated versions, causes upregulation of goosecoid (arrow), albeit in only three cases out of 20.
(G–J) Wt Xbra(1–47) (H), but not the mutated versions (I and J), disrupts normal development (G). Embryos were injected ventrally with wt or mutated versions of Xbra(1–47), as described in (B), and photographed at stage 22.
(K) Embryos injected ventrally with Xbra(1–47), but not the mutated versions, show elevated expression of the anterior and dorsal markers N-CAM and XAG2. Additional experiments revealed upregulation of Otx-2 by just 400 pg of Xbra(1–47) RNA, with even greater elevation by 4 ng RNA. At this higher concentration, however, some elevation of expression was also observed by using the two control constructs (data not shown).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions