Dev Genes Evol (1999) 209:560–563© Springer-Verlag 1999

EXPRESSION NOTE

Tadayoshi Hayata · Akira Eisaki · Hiroki Kuroda
Makoto Asashima

Expression of Brachyury-like T-box transcription factor,
Xbra3 in Xenopus embryo

Received: 22 March 1999 / Accepted: 26 April 1999

Abstract The Xenopus Brachyury-like Xbra3 gene is a
novel T-box gene that is closely associated with Xenopus
Brachyury. The expression pattern of Xbra3 during development is similar to that of Xbra. During gastrulation
Xbra3 is expressed in the marginal zone, with a gradientof increasing expression from ventral to dorsal. In theearly neurula stage Xbra3 is expressed in the notochord
and posterior mesoderm, but by the tailbud stage its expression is restricted to the forming tailbud and the posterior portion of the notochord.
Key words T-box gene · Brachyury · Early mesoderm ·Notochord tailbud

In early Xenopus development T-box transcription factor
genes, including Xbrachyury (Xbra), Eomesodermin(Eomes), and VegT, function in early mesoderm or endoderm formation (Cunliffe and Smith 1992; Ryan et al.
1996; Zhang and King 1996). Xbra, the Xenopus homologue of mouse Brachyury (T), is expressed in presumptive mesodermal cells around the blastopore in the gastrula stage and is required for notochord and posterior
development (Conlon et al. 1996; Smith et al. 1991).
Eomes is expressed in mesodermal cells in a ventralto-dorsal gradient of increasing concentrations and is reEdited by N. SatohThe nucleotide sequence data reported in this paper have been
submitted to the DDBJ, EMBL, and GenBank Data Libraries(accession no. AB 022680).
T. Hayata · A. Eisaki · H. Kuroda · M. Asashima (✉)
Department of Life Sciences (Biology),
Graduate School of Arts and Sciences, University of Tokyo,
3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japane-mail: cmasa@komaba.ecc.u-tokyo.ac.jp
Tel.: +81-3-54546632, Fax: +81-3-34852904
M. Asashima
Core Research for Evolutional Science and Technology Project,
Japan Science and Technology Cooperation, Tokyo, Japanquired for mesoderm determination and differentiation(Ryan et al. 1996). VegT is localized to the vegetal hemisphere of the egg and expressed in mesoderm in the lategastrula stage. It induces ventral mesoderm, dorsal mesoderm, and endoderm in a concentration-dependent
manner (Zhang and King 1996). Recently it has beenshown that reduction in maternal VegT RNA changes animal cell fate from epidermis and nervous system to epidermis alone, equatorial cell fate from mesoderm to ectoderm, and vegetal cell fate from endoderm to mesoderm and ectoderm (Zhang et al. 1998). These T-box
genes function downstream of the Vg1/activin-like signaling pathway. Activin is a member of the transforming
growth factor-β superfamily and can induce a variety ofmesoderm derivatives and endoderm in animal cap explants in a concentration-dependent fashion in vitro(Asashima et al. 1990; Henry et al. 1996). Therefore
genes that act downstream of the Vg1/activin-like signaling pathway are thought to be important in mesoderm orendoderm.
We used a degenerate PCR strategy to identify novelT-box genes. By comparing the amino acid sequences ofknown T-box genes, such as Xbra, VegT and Eomes, weidentified the consensus sequences “GRRMFP” and“VTAYQN” in the T-domain and constructed degenerate
PCR primers. A fragment containing the T-box wasPCR-amplified from the cDNA of animal cap cells treated with human recombinant activin A. We identified
two novel T-box genes, Xbra3 (accession numberAB022680) and the Xenopus homologue of Tbx2 (accession number AB 023815). Tbx2 will be reported separately. A full-length cDNA clone was isolated from agastrula stage cDNA library using the PCR fragment as aprobe. DNA sequence analysis revealed that the Xbra3cDNA clone consisted of 1818 bp and contained an open
reading frame of 434 amino acids that was closely related to Xbra. The overall amino acid sequence of Xbra3
had 73% identity to Xbra and 90% identity within theT-domain. Recently the results of determination of thecrystallographic structure of the T-domain-DNA complex suggested that Xbra dimerizes to bind palindromic

561
Fig. 1 Comparison of the amino acid sequences of Xbra3 andXbra. Double-underlined T-box
domain; vertical lines identicalamino acids; spaces (dash)have been added to maximize
homology; black circles residues involved in DNA binding;
black background residues involved in dimerization

DNA duplex (Müller and Herrmann 1997). All the amino acid residues involved in dimerization and DNAbinding were conserved in the T-domain of Xbra3(Fig. 1). These findings suggest that Xbra3 has similar
DNA-binding specificity to Xbra, and also indicate that
Xbra3 may not only dimerize with itself but can also heterodimerize with Xbra. Xbra3 possesses 59% identitywith the carboxy-terminal portion of Xbra, which has
transcriptional activity (Conlon et al. 1996), suggestingthat Xbra3 also has transcriptional activity. Because ofthe pseudotetraploidy of Xenopus (Graf and Kobel 1991)
it is possible that Xbra3 is only a pseudoallele of Xbra.
However, the nucleotide sequences of the 5’ and 3’ untranslated regions of Xbra3 have no significant similarity
to those of Xbra (data not shown), and we suggest that
Xbra3 is a novel gene, not a pseudoallele of Xbra suchas Xbra2 (Latinkic et al. 1997).
Whole-mount in situ hybridization was performed todetermine the spatial expression profile of Xbra3 (Fig. 2)
and compare it with Xbra. In the gastrula stage Xbra3
expression seemed to be lower than that of Xbra. Low
levels of Xbra3 expression were specifically detected in
the marginal zone in the early gastrula stage (stage 10.5;
Fig. 2A), and there was a concentration gradient of expression from the dorsal to the ventral side. Xbra was expressed widely throughout the marginal zone (Fig. 2D),
but Xbra3 was expressed in a more restricted fashion. In

the mid-gastrula stage (stage 11), Xbra3 was expressedin axial mesoderm the same as Xbra (arrowhead in Fig.
2B, E). In the early neurula stage (stage 14), Xbra3 expression in the axial mesoderm (future notochord) was
similar to that of Xbra, but Xbra3 expression in axial
mesoderm seemed more intense (Fig. 2C, F). Xbra3 expression in the posterior mesoderm was observed in amore restricted fashion than Xbra expression. By thetailbud stage (stage 31), although most Xbra expression
in the notochord had disappeared, Xbra3 expression persisted in the most posterior part of the notochord (Fig.
2G, H). Xbra was expressed broadly in the forming tailbud, but Xbra3 expression in this region was stronger
and more posterior.
In conclusion, we describe the pattern of expressionof a novel Xenopus T-box gene, Xbra3. However, further
functional analysis is required to determine the developmental role of Brachyury genes in early Xenopus development. It would also be of interest to know whether
Brachyury-like genes (except the Tbx family) are present
in other vertebrates.

562

Fig. 2A–H Comparison of the spatial patterns of expression ofXbra3 and Xbra by whole-mount in situ hybridization. A–C,GXbra3 expression. D–F,H Xbra expression. A,D Vegetal views ofexpression of Xbra3 and Xbra at stage 10.5; top dorsal. B,E Vegetal views of expression of Xbra3 and Xbra at stage11; top dorsal;
arrowheads expression of axial mesoderm. C,F Dorsal views ofneurula embryos (stage 14). G,H Enlarged view of the tailbud of
tailbud stage embryos (stage 31); arrow Xbra3 expression in thenotochord. Whole mount in situ hybridization was performed asdescribed previously (Harland 1991). Full-length Xbra3 antisense
probe was prepared by digesting pXbra3 with BglII and transcribing with T7 RNA polymerase. Xbra3 sense probe was prepared by digesting pXbra3 with XhoI and transcribing with T3
RNA polymerase. Sense probe gave no background signals. Fulllength antisense Xbra probe was prepared by digesting pXT1 (agift from J. C. Smith) with EcoRV and transcribing with T7 RNApolymerase. We also used small probes containing only 3’ UTR ofboth genes. The same results was obtained, except that the UTR
probes gave fainter signals (data not shown)Acknowledgements We are very grateful to Dr. Y. Etoh(Ajinomoto) for giving us the human recombinant activin A.
This work was supported by Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science, Sports, and Culture ofJapan and by CREST (Core Research for Evolutional Science andTechnology) of Japan Science and Technology Corporation.

References
Asashima M, Nakano H, Shimada K, Kinoshita K, Ishii K, Shibai
H, Ueno N (1990) Mesodermal induction in early amphibianembryos by activin A (erythroid differentiation factor). Roux’sArch Dev Biol 200:230–233
Conlon FL, Sedgwick SG, Weston KM, Smith JC (1996) Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsalmesoderm. Development 122:2427–2435
Cunliffe V, Smith JC (1992) Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue. Nature 358:427–430

563
Graf JD, Kobel HR (1991) Genetics of Xenopus laevis. Methods
Cell Biol 36:19–34
Harland RM (1991) In situ hybridization: an improved whole
mount method for Xenopus embryos. Methods Cell Biol 36:
675–685
Henry GL, Brivanlou IH, Kessler DS, Hemmati-Brivanlou A,Melton DA (1996) TGF-β signals and a prepattern in Xenopus
laevis endodermal development. Development 122:1007–1015
Latinkic BV, Umbhauer M, Neal KA, Lerchner W, Smith JC, Cunliffe V (1997) The Xenopus Brachyury promoter is activatedby FGF and low concentrations of activin and suppressed byhigh concentrations of activin and by paired-type homeodomain proteins. Genes Dev 11:3265–3276
Müller CW, Herrmann BG (1997) Crystallographic structure of
the T domain-DNA complex of the Brachyury transcription
factor. Nature 389:884–888

Ryan K, Garrett N, Mitchell A, Gurdon JB (1996) Eomesodermin,a key early gene in Xenopus mesoderm differentiation. Cell 87:
989–1000
Smith JC, Price BM, Green JB, Weigel D, Herrmann BG (1991)
Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67:79–87
Zhang J, King ML (1996) Xenopus VegT RNA is localized to thevegetal cortex during oogenesis and encodes a novel T-box
transcription factor involved in mesodermal patterning. Development 122:4119–4129
Zhang J, Houston DW, King ML, Payne C, Wylie C, Heasman J(1998) The role of maternal VegT in establishing the primary
germ layers in Xenopus embryos. Cell 94:515–524