June 15, 2000;
Xbra3 induces mesoderm and neural tissue in Xenopus laevis.
Homologues of the murine Brachyury
gene have been shown to be involved in mesoderm
formation in several vertebrate species. In frogs, the Xenopus Brachyury
, is required for normal formation of posterior mesoderm
. We report the characterisation of a second Brachyury
homologue from Xenopus, Xbra3
, which has levels of identity with mouse Brachyury
similar to those of Xbra
encodes a nuclear protein expressed in mesoderm
in a temporal and spatial manner distinct from that observed for Xbra
expression is induced by mesoderm
-inducing factors and overexpression of Xbra3
can induce mesoderm
formation in animal caps. In contrast to Xbra
is also able to cause the formation of neural tissue
in animal caps. Xbra3
overexpression induces both geminin
, suggesting that Xbra3
can play a role in the earliest stages of neural induction. Xbra3
by an FGF-dependent pathway; a complete switch to anterior
can be effected by the inhibition of FGF signalling. Neither noggin
, nor Xnr3
is induced by Xbra3
to an extent different from their induction by Xbra
nor is BMP4
expression differentially affected.
[+] show captions
FIG. 3. Spatial distribution of Xbra and Xbra3 mRNA. Embryos were hybridised with antisense RNA probes representing regions of the two transcripts which differed sufficiently to ensure specificity for the two gene products (see text). At stage 12, Xbra3 (A, arrowed) is detected weakly around the blastopore in a pattern similar to that seen with Xbra (B). While Xbra message levels decline at stages 25 (C, arrowed) and 35 (D), Xbra3 is maintained at these stages in notochord and tail tip (C, D, arrowed). Hybridisation with an Xbra3 sense transcript gave no signal (C, D, control). A specific antiserum which recognises Xbra3 and not Xbra protein stains the nuclei of notochord cells at stage 35 (E, arrowed). No staining was seen with preimmune serum (not shown).
FIG. 1. Comparison of Xbra3 with other T-domain proteins. (A) Alignment of the predicted polypeptide sequence of Xbra3 with Xbra
(Smith et al., 1991), mouse Brachyury (Mo T; Herrmann et al., 1990), human T (Hu T; Edwards et al., 1996), chick T (Ch T; Kispert et al.,
1995), chick T-box T (Ch TbxT; Knezevic et al., 1997) and zebrafish no-tail (Zf ntl; Schulte-Merker et al., 1994). Dashes have been inserted
to introduce gaps to maximise the alignment. An antiserum specific for Xbra3 was raised against a peptide representing residues 420–431
(overlined). (B) Dendrogram of T-box genes produced by the PHYLIP program. Sequences and SwissProt accession numbers (except where
indicated) are Brachyury (P20293), human T (Hu-T; translated from GenBank; AJ001699), chicken T-box T (TbxT; P79778), Halocynthia
T (As-T; P56158), chicken T (Ch-T; P79777), Drosophila T-related gene (Trg; P55965), Xenopus Brachyury (Xbra; P24781), Xenopus
Brachyury 3 (Xbra3; this work), Hemicentrotus T (Hp-T; Q25113), zebrafish T (Zf-T; Q07998), Xenopus Eomesodermin (Eomes; P79944),
Xenopus Xombi (Xombi; translated from GenBank; S83518), Xenopus Brat (Brat; P87377), chicken T-box L (TbxL; P79779), human T-box
5 (Hu-Tbx5; Q99593), human T-box 3 (Hu-Tbx3; translated from GenBank; AF002228) and Ascidian T (As-T2; O01409; also from
FIG. 2. RT-PCR analysis of the temporal expression patterns of
Xbra and Xbra3. (A) RNA was extracted from embryos at the stages
shown and analysed for the abundance of Xbra and Xbra3 mRNA.
(B) Analysis of Xbra and Xbra3 expression in RNA extracted from
isolated notochords at stages 12 and 24. All assays were controlled
for linearity of the amplification (data not shown).
FIG. 3. Spatial distribution of Xbra and Xbra3 mRNA. Embryos were hybridised with antisense RNA probes representing regions of the
two transcripts which differed sufficiently to ensure specificity for the two gene products (see text). At stage 12, Xbra3 (A, arrowed) is
detected weakly around the blastopore in a pattern similar to that seen with Xbra (B). While Xbra message levels decline at stages 25 (C,
arrowed) and 35 (D), Xbra3 is maintained at these stages in notochord and tail tip (C, D, arrowed). Hybridisation with an Xbra3 sense
transcript gave no signal (C, D, control). A specific antiserum which recognises Xbra3 and not Xbra protein stains the nuclei of notochord
cells at stage 35 (E, arrowed). No staining was seen with preimmune serum (not shown).
FIG. 4. Induction of Xbra and Xbra3 in isolated animal caps by mesoderm-inducing factors. Caps were isolated at stage 9 and treated with
either activin or FGF at concentrations from 50 to 3 z 125 ng/ml. RNA was extracted and analysed by RT-PCR at the stages shown. All
assays were controlled for linearity of the amplification. Analysis at stage 12 shows that both Xbra and Xbra3 are induced by activin and
FGF, with Xbra showing a lower threshold for induction by FGF. At stage 25, a clear difference is seen between Xbra and Xbra3 in the
response to both growth factors. Expression of Xbra3 is clearly maintained at high levels, whereas Xbra transcripts are barely detectable.
FIG. 5. Mesoderm-inducing activity of Xbra3. Embryos were
injected with Xbra3 message at the two-cell stage and RNA was
extracted from caps isolated at the stages indicated and analysed by
RT-PCR. (A) Xbra3 induces mesoderm in an FGF-dependent manner.
Xbra is induced after injection of Xbra3 mRNA or treatment
with FGF in the absence but not in the presence of XFD. (B) Xbra3
induces markers of dorsal mesoderm and neural tissue. While both
Xbra and Xbra3 induce eFGF at stages 12 and 25, only Xbra3
induces cardiac actin and Xnot at stage 25. Induction of NCAM is
seen at stages 12 and 25 only in response to Xbra3. (C) Xbra3
induces the early neurogenic genes geminin and Xngnr-1 in animal
caps isolated from Xbra3-injected embryos at stage 12, whereas
Xbra does not. The cDNA preparations analysed in C are the same
as those analysed in Fig. 7.
FIG. 6. Xbra3 induces posterior neural markers in an FGFdependent
manner. Embryos were injected at the two-cell stage
with the mRNAs indicated and analysed for markers of neural
tissue at stage 25 by RT-PCR. NCAM is induced by Xbra3 in the
presence or absence of XFD. The posterior marker Hoxb-9 is
induced after injection of Xbra3 alone whereas co-injection of XFD
abolishes expression of Hoxb-9 and allows expression of the
anterior marker Xotx2.
FIG. 7. Xbra3 induces early neural genes by a mechanism independent
of known neural inducers. Embryos were injected with
either Xbra or Xbra3 message at the two-cell stage and caps
analysed for known neural-inducing molecules at stage 12 by
RT-PCR. Chordin and Xnr3 are not induced by either Xbra or
Xbra3. Follistatin and noggin mRNAs are induced by both Xbra and
Xbra3 to similar levels at the stages tested. BMP4 expression is not
inhibited by the expression of either Xbra or Xbra3. The cDNA
preparations analysed are the same as those used in Fig. 5C.
FIG. 8. Xbra3 can induce Hoxb-9 expression in a non-cellautonomous
manner. A partial cDNA representing a Hoxb-9
mRNA was cloned from X. borealis and primers which amplified
specifically borealis Hoxb-9 were designed. Caps were isolated
from embryos injected with Xbra3 mRNA and cultured to stage 25
by which time Hoxb-9 expression was detected in both species (A).
Caps from X. laevis embryos injected with Xbra or Xbra3 RNA
were cultured in apposition with caps from uninjected borealis
embryos (see B), and RNA from the total sandwiches was extracted
and amplified using primers specific for X. borealis Hoxb-9. X.
borealis Hoxb-9 was found to be expressed in sandwiches containing
laevis caps expressing Xbra3, but not in sandwiches with
Xbra-expressing caps (C). In (A) and (C), cDNA input to the RT-PCR
was equalised with respect to [32P]GTP incorporation during cDNA
synthesis (see Materials and Methods).