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Dev Cell
2005 Apr 01;84:599-610. doi: 10.1016/j.devcel.2005.03.001.
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Functional specificity of the Xenopus T-domain protein Brachyury is conferred by its ability to interact with Smad1.
Messenger NJ
,
Kabitschke C
,
Andrews R
,
Grimmer D
,
Núñez Miguel R
,
Blundell TL
,
Smith JC
,
Wardle FC
.
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Members of the T-box gene family play important and diverse roles in development and disease. Here, we study the functional specificities of the Xenopus T-domain proteins Xbra and VegT, which differ in their abilities to induce gene expression in prospective ectodermal tissue. In particular, VegT induces strong expression of goosecoid whereas Xbra cannot. Our results indicate that Xbra is unable to induce goosecoid because it directly activates expression of Xom, a repressor of goosecoid that acts downstream of BMP signaling. We show that the inability of Xbra to induce goosecoid is imposed by an N-terminal domain that interacts with the C-terminal MH2 domain of Smad1, a component of the BMP signal transduction pathway. Interference with this interaction causes ectopic activation of goosecoid and anteriorization of the embryo. These findings suggest a mechanism by which individual T-domain proteins may interact with different partners to elicit a specific response.
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(17) (1 ng) or both. Animal cap
explants were isolated at late blastula stage
9 and cultured until sibling embryos reached
early gastrula stage 10.5. 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(17) were collected at early gastrula
stage 10.5 and assayed by real-time RT-PCR
for expression of goosecoid. Note that wt
Xbra(17), but not the mutated versions,
causes upregulation of goosecoid. goosecoid
expression in an isolated dorsal marginal
zone is shown for comparison purposes.
(C). Embryos injected ventrally with wt or
mutated versions of Xbra(17), as described
in (B), were subjected to in situ hybridization
by using a probe specific for
goosecoid. Note that wt Xbra(17), but not
the mutated versions, causes upregulation
of goosecoid (arrow), albeit in only three
cases out of 20.
(G) Wt Xbra(17) (H), but not the mutated
versions (I and J), disrupts normal development (G). Embryos were injected ventrally with wt or mutated versions of Xbra(17), as described
in (B), and photographed at stage 22.
(K) Embryos injected ventrally with Xbra(17), 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(17) 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).
