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Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis
Mechanisms of Development 80 (1999) 219–221
Gene expression pattern
Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis Sayuri Yonei-Tamura1, Koji Tamura1, Tohru Tsukui, Juan Carlos Izpisu´a Belmonte* The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 N. Torrey Pines Road, La Jolla, CA 92037-1099, USA Received 5 October 1998; revised version received 4 November 1998; accepted 6 November 1998
Abstract T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish. Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T-box; Zebrafish; Gene expression; Tbr1; Tbx3; Embryogenesis
1. Results The T-box gene family shares a highly conserved DNA binding motif referred to as the T-box domain (Bollag et al., 1994). Recent studies have revealed that T-box genes are conserved in a wide range of animals (from C. elegans to human) and have critical roles during embryogenesis (reviewed by Papaioannou and Silver, 1998). Here we report the isolation of two members of the T-box gene family from zebrafish. Zebrafish tbr1 (zf-tbr1) shows a 83–97% amino acid identity to the T-box domain of other Tbr1-related genes (Fig. 1A). Expression of zf-tbr1 was restricted to the forebrain (Fig. 2A–F) and was not detected in other regions of the embryo at all stages examined (from 4.5 to 55 h embryos). Zebrafish tbx3 (zf-tbx3) is 89–95% identical to the T-box domain of other Tbx3-related genes (Fig. 1B). Zf-tbx3 was expressed throughout the notochord (Fig. 2H–J). Fig. 2K shows that the expression of zf-tbx3 became restricted in the * Corresponding author. Tel.: +1-619-4534100, ext. 1130 and 1311; fax: +1-619-4551349; e-mail: [email protected] 1 These two authors contributed equally to this work.
caudal region of the notochord by 24-h stage. No staining was observed prior to the 24-h expression in the notochord (see for instance Fig. 2G; 6 h; and Fig. 2L, 30 h). At later stages, zf-tbx3 was expressed in the posterior region of the pectoral fin bud (Fig. 2M,N). The expression of zf-tbr1 in the forebrain seems to be identical to mouse Tbr1 (mu-Tbr1; Bulfone et al., 1995) but different from Xenopus Eomesodermin (x-Eomes, which is closely related to mu-Tbr1; Ryan et al., 1996). Zf-tbx3 was expressed in the notochord, although the other Tbx3-related genes are not expressed there (Bollag et al., 1994; Gibson-Brown et al., 1998).
2. Methods cDNA clones containing the zebrafish tbr1 and tbx3 were Fig. 1. Alignment of T-box sequences of zebrafish T-box genes and their orthologs. (A) Comparison of zf-tbr1 with probable orthologs from Xenopus (x-Eomes; Ryan et al., 1996), mouse (mu-Tbr1; Bulfone et al., 1995) and human (hu-TBR1; Bulfone et al., 1995). (B) Comparison of zf-tbx3 with probable orthologs from chick (ch-Tbx3; Isaac et al., 1998), mouse (mu-Tbx3; Bollag et al., 1994) and human (hu TBX3; Bamshad et al., 1997). Identities are highlighted (A,B).
0925-4773/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S09 25-4773(98)002 19-6
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S. Yonei-Tamura et al. / Mechanisms of Development 80 (1999) 219–221
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Fig. 2. Expression of zebrafish T-box genes during embryogenesis. (A–F) Expression of zf-tbr1. (A,B) 27-h, (C,D) 39 h and (E,F) 55-h embryos. Arrowheads indicate the forebrains (A,C,E). (G–N) Expression of zf-tbx3. (G) 6-h (H) 10-h (I) 12-h, (J) 16-h, (K) 24-h, (L) 30-h, (M) 36-h and (N) 55-h embryos. Black arrowheads indicate the zf-tbx3 expressing region in notochords (H–K) or pectoral fin buds (L–N). White arrowheads indicate outlines of the pectoral fin buds (L–M).
S. Yonei-Tamura et al. / Mechanisms of Development 80 (1999) 219–221
obtained by screening a zebrafish 24-h embryo cDNA (Stratagene) with a 32P-labeled, randomly primed probe derived from a chick Tbx4 cDNA clone (Isaac et al., 1998). Whole mount in situ hybridization was performed according to Schulte-Merker et al. (1992).
Acknowledgements This work was supported by JSPS Research Fellowships for Young Scientists to S.Y.T. and by JSPS Postdoctoral Fellowships for Research Abroad to K.T. and by grants from HFSPO and NIH to J.C.I.B., who is a Pew Scholar. References Bamshad, M., Lin, R.C., Law, D.J., Watkins, W.C., Krakowiak, P.A., Moore, M.E., Franceschini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Bruneau, B.G., Schinzel, A., Seidman, J.G., Seidman, C.E., Jorde, L.B., 1997. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16, 311–315.
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Bollag, R.J., Siegfried, Z., Cebra-Thomas, J.A., Garvey, N., Davison, E.M., Silver, L.M., 1994. An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus. Nat. Genet. 7, 383–389. Bulfone, A., Smiga, S.M., Shimamura, K., Peterson, A., Puelles, L., Rubenstein, J.L., 1995. T-brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex. Neuron 15, 63–78. Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Papaioannou, V.E., 1998. Expression of T-box genes Tbx2-Tbx5 during chick organogenesis. Mech. Dev. 74, 165–169. Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C., Izpisua-Belmonte, J.C., 1998. Tbx genes and limb identity in chick embryo development. Development 125, 867–875. Papaioannou, V.E., Silver, L.M., 1998. The T-box gene family. Bioessays 20, 9–19. Ryan, K., Garrett, N., Mitchell, A., Gurdon, J.B., 1996. Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell 87, 989– 1000. Schulte-Merker, S., Ho, R.K., Herrmann, B.G., Nusslein-Volhard, C., 1992. The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032.
Gene expression pattern
Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis Sayuri Yonei-Tamura1, Koji Tamura1, Tohru Tsukui, Juan Carlos Izpisu´a Belmonte* The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 N. Torrey Pines Road, La Jolla, CA 92037-1099, USA Received 5 October 1998; revised version received 4 November 1998; accepted 6 November 1998
Abstract T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish. Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T-box; Zebrafish; Gene expression; Tbr1; Tbx3; Embryogenesis
1. Results The T-box gene family shares a highly conserved DNA binding motif referred to as the T-box domain (Bollag et al., 1994). Recent studies have revealed that T-box genes are conserved in a wide range of animals (from C. elegans to human) and have critical roles during embryogenesis (reviewed by Papaioannou and Silver, 1998). Here we report the isolation of two members of the T-box gene family from zebrafish. Zebrafish tbr1 (zf-tbr1) shows a 83–97% amino acid identity to the T-box domain of other Tbr1-related genes (Fig. 1A). Expression of zf-tbr1 was restricted to the forebrain (Fig. 2A–F) and was not detected in other regions of the embryo at all stages examined (from 4.5 to 55 h embryos). Zebrafish tbx3 (zf-tbx3) is 89–95% identical to the T-box domain of other Tbx3-related genes (Fig. 1B). Zf-tbx3 was expressed throughout the notochord (Fig. 2H–J). Fig. 2K shows that the expression of zf-tbx3 became restricted in the * Corresponding author. Tel.: +1-619-4534100, ext. 1130 and 1311; fax: +1-619-4551349; e-mail: [email protected] 1 These two authors contributed equally to this work.
caudal region of the notochord by 24-h stage. No staining was observed prior to the 24-h expression in the notochord (see for instance Fig. 2G; 6 h; and Fig. 2L, 30 h). At later stages, zf-tbx3 was expressed in the posterior region of the pectoral fin bud (Fig. 2M,N). The expression of zf-tbr1 in the forebrain seems to be identical to mouse Tbr1 (mu-Tbr1; Bulfone et al., 1995) but different from Xenopus Eomesodermin (x-Eomes, which is closely related to mu-Tbr1; Ryan et al., 1996). Zf-tbx3 was expressed in the notochord, although the other Tbx3-related genes are not expressed there (Bollag et al., 1994; Gibson-Brown et al., 1998).
2. Methods cDNA clones containing the zebrafish tbr1 and tbx3 were Fig. 1. Alignment of T-box sequences of zebrafish T-box genes and their orthologs. (A) Comparison of zf-tbr1 with probable orthologs from Xenopus (x-Eomes; Ryan et al., 1996), mouse (mu-Tbr1; Bulfone et al., 1995) and human (hu-TBR1; Bulfone et al., 1995). (B) Comparison of zf-tbx3 with probable orthologs from chick (ch-Tbx3; Isaac et al., 1998), mouse (mu-Tbx3; Bollag et al., 1994) and human (hu TBX3; Bamshad et al., 1997). Identities are highlighted (A,B).
0925-4773/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S09 25-4773(98)002 19-6
220
S. Yonei-Tamura et al. / Mechanisms of Development 80 (1999) 219–221
1
Fig. 2. Expression of zebrafish T-box genes during embryogenesis. (A–F) Expression of zf-tbr1. (A,B) 27-h, (C,D) 39 h and (E,F) 55-h embryos. Arrowheads indicate the forebrains (A,C,E). (G–N) Expression of zf-tbx3. (G) 6-h (H) 10-h (I) 12-h, (J) 16-h, (K) 24-h, (L) 30-h, (M) 36-h and (N) 55-h embryos. Black arrowheads indicate the zf-tbx3 expressing region in notochords (H–K) or pectoral fin buds (L–N). White arrowheads indicate outlines of the pectoral fin buds (L–M).
S. Yonei-Tamura et al. / Mechanisms of Development 80 (1999) 219–221
obtained by screening a zebrafish 24-h embryo cDNA (Stratagene) with a 32P-labeled, randomly primed probe derived from a chick Tbx4 cDNA clone (Isaac et al., 1998). Whole mount in situ hybridization was performed according to Schulte-Merker et al. (1992).
Acknowledgements This work was supported by JSPS Research Fellowships for Young Scientists to S.Y.T. and by JSPS Postdoctoral Fellowships for Research Abroad to K.T. and by grants from HFSPO and NIH to J.C.I.B., who is a Pew Scholar. References Bamshad, M., Lin, R.C., Law, D.J., Watkins, W.C., Krakowiak, P.A., Moore, M.E., Franceschini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Bruneau, B.G., Schinzel, A., Seidman, J.G., Seidman, C.E., Jorde, L.B., 1997. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16, 311–315.
221
Bollag, R.J., Siegfried, Z., Cebra-Thomas, J.A., Garvey, N., Davison, E.M., Silver, L.M., 1994. An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus. Nat. Genet. 7, 383–389. Bulfone, A., Smiga, S.M., Shimamura, K., Peterson, A., Puelles, L., Rubenstein, J.L., 1995. T-brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex. Neuron 15, 63–78. Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Papaioannou, V.E., 1998. Expression of T-box genes Tbx2-Tbx5 during chick organogenesis. Mech. Dev. 74, 165–169. Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C., Izpisua-Belmonte, J.C., 1998. Tbx genes and limb identity in chick embryo development. Development 125, 867–875. Papaioannou, V.E., Silver, L.M., 1998. The T-box gene family. Bioessays 20, 9–19. Ryan, K., Garrett, N., Mitchell, A., Gurdon, J.B., 1996. Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell 87, 989– 1000. Schulte-Merker, S., Ho, R.K., Herrmann, B.G., Nusslein-Volhard, C., 1992. The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032.