Download Differential expression of Tbx4 and Tbx5 in Zebrafish fin buds

Mechanisms of Development 87 (1999) 181±184
Gene expression pattern
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Differential expression of Tbx4 and Tbx5 in Zebra®sh ®n buds
Koji Tamura 1, Sayuri Yonei-Tamura 1, Juan Carlos IzpisuÂa Belmonte*
The Salk Institute for Biological Studies, Gene expression Laboratory, 10010 N. Torrey Pines Road, La Jolla, CA 92037-1099, USA
Received 12 February 1999; received in revised form 25 May 1999; accepted 25 May 1999
Abstract
In here we report the identi®cation of two new members of the T-box gene family, zf-tbx5 and zf-tbx4, from the Zebra®sh, Danio rerio. The
amino acid sequences within the T-box domain share high homology with the mouse, chick, and newt orthologs. Whole mount in situ
hybridization revealed speci®c expression of these genes in the eye and Fin buds. zf-tbx5 expression is restricted to the pectoral Fin bud,
whilst zf-tbx4 transcripts are con®ned in the pelvic Fin bud. These results reveal the conserved expression pattern of Tbx5 and Tbx4 during
appendage development in all animal species studied to date. q 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: T-box; Tbx4; Tbx5; Zebra®sh; Gene expression; Embryogenesis; Fin bud
1. Results
It has recently been suggested that two members of the Tbox gene family, Tbx5 and Tbx4, may play an important role
in specifying the identity of the vertebrate limb (GibsonBrown et al., 1996, 1998a; Simon et al., 1997; ; Ohuchi et
al., 1998; Isaac et al., 1998; Logan et al., 1998). To gain a
better understanding of the role of this gene in specifying
limb identity in all animal species, we have isolated the
Tbx5 and Tbx4 genes from the Zebra®sh, Danio rerio.
Fig. 1A shows the amino acid comparison between the
two new Zebra®sh genes and the rest of the Tbx5 and
Tbx4 genes found in other vertebrate species. A phylogenetic tree of the T-box amino acid sequences suggest that zftbx5 and zf-tbx4 belong to the Tbx5 and Tbx4 subfamily
respectively (Fig. 1B).
We have examined the expression patterns of zf-tbx5 and
zf-tbx4 by whole mount in situ hybridization. As has been
previously observed in mouse and chick embryos (Chapman
et al., 1996; Gibson-Brown et al., 1998b; Ohuchi et al.,
1998), zf-tbx5 is expressed in the dorsal optic cup of developing eye (Fig. 2A). At the 31-h stage, when the pectoral Fin
buds have begun bulging outwards, restricted expression of
zf-tbx5 is detected throughout the mesenchyme of the early
Fin buds (Fig. 2C,D,E). These high levels of expression
continue until later stages (Fig. 2F,G). In contrast to zf* Corresponding author. Tel.:11-619-453-4100, ext. 1130/1311;
fax:11-619-455-1349.
E-mail address: [email protected] (J.C.I. Belmonte)
1
These two authors contributed equally to this work.
tbx5, zf-tbx4 transcripts were never detected in the developing pectoral Fin buds (Fig. 2B). Paired pelvic ®ns start to
develop during metamorphosis, at much later stages. zf-tbx5
transcripts were never detected in the pelvic ®ns (Fig. 2H,J)
zf-tbx4 is expressed throughout the entire pelvic Fin buds of
around 6 week old ®sh (Fig. 2I,K).
2. Materials and methods
To isolate Zebra®sh Tbx5, PCR ampli®cation using
degenerate primers for Tbx5 speci®c amino acid sequences
(FADNKWS and THVFPET) within the T-box were
performed with 36-h stage Zebra®sh embryo mRNA.
After cloning into PCR-script (Stratagene) and sequencing,
the PCR fragment was subsequently used to screen a cDNA
library derived from 24-h stage Zebra®sh embryo (Stratagene) at high stringency. A positive clone was obtained,
sequenced and used for in situ hybridization. A partial fragment for Tbx4 was obtained from pelvic Fin bud mRNA
using degenerate primers (for FCDNKWM and THVFPET).
Ampli®ed PCR fragments were cloned, sequenced, and used
for in situ hybridization. Whole mount in situ hybridization
was performed as previously described by Schulte-Merker
et al. (1992). The Zebra®sh Tbx5 and Tbx4 sequences have
been deposited in Genbank with the following accession
numbers: zTbx4: AF152606 and zTbx5: AF152607).
Acknowledgements
This work was supported by JSPS Postdoctoral Fellow-
0925-4773/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0925-477 3(99)00126-4
182
K. Tamura et al. / Mechanisms of Development 87 (1999) 181±184
Fig. 1. Alignment of T-box sequences of Zebra®sh Tbx5 and Tbx4 genes and their putative orthologs in chick, mouse, newt and humans (Agulnik et al., 1996;
Basson et al., 1997; Simon et al., 1997; Isaac et al., 1998). Identities are highlighted and asterisks indicate conserved amino acid speci®cally in the Tbx4
subfamily. (B) Phylogenetic tree of the Tbx5 and Tbx4 amino acid sequences within the T-box region. The values associated with the likelihood of branch
points are indicated as percentiles.
ships for Research Abroad to K.T. and by JSPS Research
Fellowships for Young Scientists to S.Y.T. and by a grant
from National Science Foundation to J.C.I.B. who is a Pew
Scholar.
K. Tamura et al. / Mechanisms of Development 87 (1999) 181±184
183
Fig. 2. Expression of zf-tbx4 and tbx5 in developing eye and Fin buds. (A) Expression of zf-tbx5 in the developing eye of 18-h stage embryo. (B±G) Expression
of zf-tbx4 (B) and zf-tbx5 (C±G) in pectoral Fin bud. (B±E) 31-h, (F,G) 40-h. (E,G) Higher magni®cation of (D,F), respectively. (H±K) Expression of zf-tbx4
(H,I) and zf-tbx5 (J,K) in developing pelvic ®n of 6-week-old ®sh. Black arrowheads indicate zf-tbx4 and zf-tbx5 expressing cells. Red arrowheads outline the
shape of pelvic Fin bud.
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