- Email: [email protected]
Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development
Mechanisms of Development 84 (1999) 133±138
Gene expression pattern
Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development Alessandro Bulfone a,*, Salvador Martinez b, Valeria Marigo a, Marilena Campanella a, Andrea Basile a, Nandita Quaderi a, Claudio Gattuso a, John L.R. Rubenstein c, Andrea Ballabio a a
Telethon Institute of Genetics and Medicine (TIGEM), San Raffaele Biomedical Science Park, Via Olgettina, 58, 20132 Milan, Italy b Department of Morphological Sciences, University of Murcia, 30071 Murcia, Spain c Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco 94143-0984, CA, USA Received 11 December 1998; received in revised form 17 February 1999; accepted 27 February 1999
Abstract The members of the T-box gene family share a highly conserved DNA binding domain named the T-domain, and important developmental functions. Here we report the cloning of chicken Tbr1 and of murine and chicken Tbr2 (orthologs of the Xenopus eomesodermin gene), the mapping of the murine Tbr2 to chromosome 9, and their pattern of expression during mouse and chick embryogenesis. Both Tbr 1 and 2 have a restricted and conserved domain of expression in the telencephalic pallium of the two species. Chick Tbr2 has a speci®c and dynamic expression in the gastrulating embryo. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T-box genes; Eomesodermin; Tbr1; Tbr2; Mouse embryo; Chick embryo; Development; Expression pattern; Forebrain; Telencephalon; Cerebral cortex; Cerebellum; Eye; Limb
1. Introduction T-box genes are a family of transcription factors that have a highly conserved DNA binding domain named the Tdomain (Papaioannou and Silver, 1998). T-box genes have important developmental functions during gastrulation (e.g. Brachyury, Wilkinson et al., 1990), mesodermal speci®cation (Tbx6, Chapman and Papaioannou, 1998), limb patterning, (Tbx2-Tbx5, Gibson-Brown et al., 1998; Logan et al., 1998), and brain development (Tbr1, Bulfone et al., 1995; Bulfone et al., 1998; OMB, P¯ugfelder and Heisenberg, 1995). Mutations in hu-TBX3 (Bamshad et al., 1997) and hu-TBX5 (Basson et al., 1997; Li et al., 1997) are responsible for the autosomal dominant developmental syndromes known as Ulnar-Mammary and Holt-Oram, respectively, and the human abnormalities are consistent with the embryonic expression patterns of the mouse orthologs (Chapman et al., 1996; Gibson-Brown et al., 1996). Here we describe the isolation, sequence and expression of chicken Tbr1 (Bulfone et al., 1995) and of murine and chicken Tbr2, a homolog of Tbr1 and the ortholog (Wattler et al., 1998) of the Xenopus eomesodermin gene (Ryan et al., * Corresponding author. Tel.: 1 39-02-21560234; fax: 21560220. E-mail address: [email protected] (A. Bulfone)
1 39-02-
1996, 1998), which has been implicated in gastrulation (Ryan et al., 1996). Both Tbr1 and 2 have a highly restricted pattern of central nervous system expression which is largely limited to the telencephalic pallium. 2. Results The mouse and chicken orthologs of Tbr1 and Tbr2 were identi®ed using degenerate PCR. Comparison of the T-box regions reveals a considerable degree of sequence conservation across species between the chicken and mouse genes (Fig. 1A). At the amino acid level the T-domain of the chicken and mouse Tbr1 and Tbr2 orthologs exhibit 98.5 and 99% identity, respectively, within the T-box domain. The T-domains of Tbr1 and Tbr2 (mouse and chicken) are 87.5% identical; outside of the T-domain there is very little similarity (data not shown). The genomic location of mouse Tbr2 was determined using the Jackson BSS backcross maps from two interspeci®c backcross DNA panels (Rowe et al., 1994), exploiting a Tbr2 PCR ampli®cation size polymorphism between the C57/BL6 and mus spretus genomic DNAs. Tbr2 maps between the markers D9Mit17 and D9Bir15 on distal chromosome 9 (Fig. 1B), where three neurological mutants are known to map: ducky, tippy and spinner.
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00053-2
134
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
Fig. 1. (A) T-box domain alignment of Xenopus Eomesodermin (x-Eomes) with mouse (mu) and chicken (ch) Tbr1 and Tbr2 genes. Amino acid identities (black boxes) and similarities (grey boxes) are indicated. (B) Map of mouse chromosome 9 showing the location of Tbr2.
Tbr2 expression was detected in mouse blastocysts and gastrula embryos (data not shown). Beginning around E10.0, Tbr2 expression was found primarily in the pallial (cortical) parts of the telencephalon (T) (Fig. 2A) which include primordia of the olfactory cortex, neocortex, hippocampus, and parts of the septum and amygdala. It is also expressed in the pallium's connection with the diencephalon (the eminentia thalami, ET) (Fig. 2A). Tbr2 transcripts were present in isolated cells in the ventricular zone (VZ), and more broadly in the postmitotic mantle zone (MZ), where Tbr1 expression is exclusively localized (Fig. 2A,E). As development proceeds (E12.5 and E16.5), there is an increasing number of Tbr2-expressing cells in the VZ, and decreasing expression in the MZ (Fig. 2B,C,I±L). At birth (P0), there is a low level of Tbr2 expression in cortical and hippocampal periventricular cells (Fig. 2D) and a high level in mitral cells of the olfactory bulb and in the anterior olfactory nuclear complex (Fig. 2O and data not shown). The only other areas where Tbr2 expression has been detected are the cerebellum (particularly in the external granular layer at E12.5 in Fig. 2M, at E16.5 in Fig. 2P, and at P0 in the cerebellar germinal trigone in Fig. 2Q), the inner
neuroblast layer of the neural retina at E16.5 (Fig. 2N), and limb mesenchyme (Fig. 2R). Embryonic expression of the chick Tbr1 and Tbr2 was examined from HH (Hamburger and Hamilton) stage 3 to HH28. Tbr1 transcripts could not be detected during gastrulation, whereas Tbr2 is already expressed at HH3 (Fig. 3), it decreases by HH5 with the beginning of primitive streak regression, and then disappears by HH6. At HH3, Tbr2 is strongly expressed in the hypoblast in the most anterior part of the area pellucida (Fig. 3A,C), in the ectoderm anterior and lateral to the primitive streak (Fig. 3A,D±H), and at a lower level in the ingressing mesodermal cells and de®nitive endoderm (Fig. 3A,E±G). By HH4, Tbr2 transcripts are clearly detected in ectodermal and mesenchymal cells along the rostral primitive streak (Fig. 3B,K,L) and also in mesodermal cells anterior to Hensen's node (Fig. 3B,J). At around 5 days of incubation the expression of the two genes are very similar, both spatially and temporally, to the patterns of their mouse orthologs (Fig. 4A,B). Chick Tbr2 is expressed in the pallial derivatives such as the dorsal ventricular ridge (DVR in Fig. 4E) and the cortex (Cx in Fig. 4B,C,E), and in the caudal region of basal nuclei: the
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
135
Fig. 2. Comparison of mouse Tbr2 and Tbr1 brain expression during embryonic development by in situ hybridization on tissue sections. (A±H) Adjacent sagittal sections showing the differential distribution of mTbr1 and mTbr2 transcripts during embryonic development. (A,E) At E10.5, Tbr2 and Tbr1 are coexpressed in the mantle zone (MZ) but Tbr2 is also expressed in cells of the germinative epithelium of the cortical anlage. (B,F) At E12.5, the Tbr2 pattern of expression follows the rostro-caudal (arrow in B) and latero-medial (arrow in I) gradients of differentiation and expression in the mantle is restricted to the deep layers of the incipient cortical plate. (I±M) Set of coronal sections showing Tbr2 expression at E12.5. (N±R) Tbr2 expression in the neural retina at E16.5 (N), in the olfactory bulb at P0 (O), in the developing cerebellum (at E16.5 in P, and P0 in Q), and in the fore limb bud (R, arrow indicates expression in a posterior metacarpal pre-cartilage condensation). Other abbreviations: CGE, caudal ganglionic eminence; CGT, cerebellar germinal trigone; Cp, cerebellar plate; CP, cortical plate; CPU, caudo-putamen; Cx, cortex; DG, dentate gyrus; EGL, external granular layer; ET, eminentia thalami; H, hippocampus; INL, inner neuroblast layer; LGE, lateral ganglionic eminence; M, mesencephalon; MCL, mitral cell layer; MGE, medial ganglionic eminence; ONL, outer neuroblast layer; Rh, rhombencephalon; T, telencephalon; VZ, ventricular zone; WM, white matter.
caudal ganglionic eminence (CGE in Fig. 4C), anlage of the amygdala. At HH25, cTbr2 is expressed in scattered cells which appear to be in the proliferative zone and more diffusely in the MZ, whereas cTbr1 is solely expressed in the MZ (Fig. 4C±F). By HH stage 28, besides being expressed in the proliferative zone, strong cTbr2 expression is concentrated in a layer near the interface of the VZ and MZ (Fig. 4G,H).
3. Methods Mouse and chicken Tbr2 and Tbr1 cDNA clones were obtained by degenerate PCR and by screening embryonic libraries. In situ hybridization was performed according to published protocols (Riddle et al., 1993; Rugarli et al., 1993).
136
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
Fig. 3. Early chick embryonic expression of Tbr2. Whole-mount in situ hybridization of embryos at stages HH3 (A) and HH4 (B). Transverse sections of embryo 4A (C±H) and 4B (I±P) at levels indicated. The location of Hensen's node is indicated (arrowheads in A and B).
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
137
Fig. 4. (A,B) Tbr2 transcript distribution in E12.5 mouse and HH stage 25 chick embryos (sagital sections). (C±H) Comparison of chicken Tbr2 and Tbr1 pallial expression, in sagital sections, at HH stages 25 and 28. Other abbreviations: CGE, caudal ganglionic eminence; Cx, cortex; DVR, dorsal ventricular ridge; EGL, external granular layer; ET, eminentia thalami; Hy, hippocampus; Is, isthmus; M, mesencephalon; MGE, medial ganglionic eminence; MZ, mantle zone; OB, olfactory bulb; OS, optic stalk; P1-4, prosomeres 1-4; S, septum; VZ, ventricular zone; ZL, zona limitans.
Acknowledgements We thank Celia Pardini, Cristina Mocchetti and Micaela Quarto for technical assistance, Andreas Russ for valuable discussions, and Melissa Smith for preparation of this manu-
script. This work was supported by the Italian Telethon Foundation (Grant no. B.37), the Merck Genome Research Institute (Grant no. 37 to A.B.), the EC (Grant no. BMH4CT97-2341 to A.B.), and NINDS Grant no. NS34661-01A1 to J.L.R.R.
138
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
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. Basson, C.T., Bachinsky, D.R., Lin, R.C., Levi, T., Elkins, J.A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T.A., Leblanc-Straceski, J., Renault, B., Kucherlapati, R., Seidman, J.G., Seidman, C.E., 1997. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15, 30±35. Bulfone, A., Smiga, S.M., Shimamura, K., Peterson, A., Puelles, L., Rubenstein, J.L., 1995. T-brain-1: a homolog of Brachyury whose expression de®nes molecularly distinct domains within the cerebral cortex. Neuron 15, 63±78. Bulfone, A., Wang, F., Hevner, R., Anderson, S., Cutforth, T., Chen, S., Meneses, J., Pedersen, R., Axel, R., Rubenstein, J.L.R., 1998. An olfactory sensory map develops in the absence of normal projection neurons or gabaergic interneurons. Neuron 21, 1273±1282. Chapman, D.L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S.I., Gibson-Brown, J.J., Cebra-Thomas, J., Bollag, R.J., Silver, L.M., Papaioannou, V.E., 1996. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206, 379±390. Chapman, D.L., Papaioannou, V.E., 1998. Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391, 695±697. Gibson-Brown, J.J., Agulnik, S.I., Chapman, D.L., Alexiou, M., Garvey, N., Silver, L.M., Papaioannou, V.E., 1996. Evidence of a role for T-box genes in the evolution of limb morphogenesis and the speci®cation of forelimb/hindlimb identity. Mech. Dev. 56, 93±101. Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Niswander, L., Papaioannou, V.E., 1998. Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb speci®cation and development. Development 125, 2499±2509. Li, Q.Y., Newbury-Ecob, R.A., Terrett, J.A., Wilson, D.I., Curtis, A.R., Yi, C.H., Gebuhr, T., Bullen, P.J., Robson, S.C., Strachan, T., Bonnet, D.,
Lyonnet, S., Young, I.D., Raeburn, J.A., Buckler, A.J., Law, D.J., Brook, J.D., 1997. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15, 21±29. Logan, M., Simon, H.G., Tabin, C., 1998. Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity. Development 125, 2825±2835. Papaioannou, V.E., Silver, L.M., 1998. The T-box gene family. Bioessays 20, 9±19. P¯ugfelder, G.O., Heisenberg, M., 1995. Optomotor-blind of Drosophila melanogaster: a neurogenetic approach to optic lobe development and optomotor behaviour. Comp. Biochem. Physiol. A: Physiol. 110, 185± 202. Riddle, R.D., Johnson, R.L., Laufer, E., Tabin, C., 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401±1416. Rowe, L.B., Nadeau, J.H., Turner, R., Frankel, W.N., Letts, V.A., Eppig, J.T., Ko, M.S., Thurston, S.J., Birkenmeier, E.H., 1994. Maps from two interspeci®c backcross DNA panels available as a community genetic mapping resource. Mamm. Genome 5, 253±274. Rugarli, E.I., Lutz, B., Kuratani, S.C., Wawersik, S., Borsani, G., Ballabio, A., Eichele, G., 1993. Expression pattern of the Kallmann syndrome gene in the olfactory system suggests a role in neuronal targeting. Nat. Genet. 4, 19±26. Ryan, K., Garrett, N., Mitchell, A., Gurdon, J.B., 1996. Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell 87, 989± 1000. Ryan, K., Butler, K., Bellefroid, E., Gurdon, J.B., 1998. Xenopus eomesodermin is expressed in neural differentiation. Mech. Dev. 75, 167±170. Wattler, S., Russ, A., Evans, M., Nehls, M., 1998. A combined analysis of genomic and primary protein structure de®nes the phylogenetic relationship of new members of the T-box family. Genomics 48, 24±33. Wilkinson, D.G., Bhatt, S., Herrmann, B.G., 1990. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657± 659.
Gene expression pattern
Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development Alessandro Bulfone a,*, Salvador Martinez b, Valeria Marigo a, Marilena Campanella a, Andrea Basile a, Nandita Quaderi a, Claudio Gattuso a, John L.R. Rubenstein c, Andrea Ballabio a a
Telethon Institute of Genetics and Medicine (TIGEM), San Raffaele Biomedical Science Park, Via Olgettina, 58, 20132 Milan, Italy b Department of Morphological Sciences, University of Murcia, 30071 Murcia, Spain c Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco 94143-0984, CA, USA Received 11 December 1998; received in revised form 17 February 1999; accepted 27 February 1999
Abstract The members of the T-box gene family share a highly conserved DNA binding domain named the T-domain, and important developmental functions. Here we report the cloning of chicken Tbr1 and of murine and chicken Tbr2 (orthologs of the Xenopus eomesodermin gene), the mapping of the murine Tbr2 to chromosome 9, and their pattern of expression during mouse and chick embryogenesis. Both Tbr 1 and 2 have a restricted and conserved domain of expression in the telencephalic pallium of the two species. Chick Tbr2 has a speci®c and dynamic expression in the gastrulating embryo. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T-box genes; Eomesodermin; Tbr1; Tbr2; Mouse embryo; Chick embryo; Development; Expression pattern; Forebrain; Telencephalon; Cerebral cortex; Cerebellum; Eye; Limb
1. Introduction T-box genes are a family of transcription factors that have a highly conserved DNA binding domain named the Tdomain (Papaioannou and Silver, 1998). T-box genes have important developmental functions during gastrulation (e.g. Brachyury, Wilkinson et al., 1990), mesodermal speci®cation (Tbx6, Chapman and Papaioannou, 1998), limb patterning, (Tbx2-Tbx5, Gibson-Brown et al., 1998; Logan et al., 1998), and brain development (Tbr1, Bulfone et al., 1995; Bulfone et al., 1998; OMB, P¯ugfelder and Heisenberg, 1995). Mutations in hu-TBX3 (Bamshad et al., 1997) and hu-TBX5 (Basson et al., 1997; Li et al., 1997) are responsible for the autosomal dominant developmental syndromes known as Ulnar-Mammary and Holt-Oram, respectively, and the human abnormalities are consistent with the embryonic expression patterns of the mouse orthologs (Chapman et al., 1996; Gibson-Brown et al., 1996). Here we describe the isolation, sequence and expression of chicken Tbr1 (Bulfone et al., 1995) and of murine and chicken Tbr2, a homolog of Tbr1 and the ortholog (Wattler et al., 1998) of the Xenopus eomesodermin gene (Ryan et al., * Corresponding author. Tel.: 1 39-02-21560234; fax: 21560220. E-mail address: [email protected] (A. Bulfone)
1 39-02-
1996, 1998), which has been implicated in gastrulation (Ryan et al., 1996). Both Tbr1 and 2 have a highly restricted pattern of central nervous system expression which is largely limited to the telencephalic pallium. 2. Results The mouse and chicken orthologs of Tbr1 and Tbr2 were identi®ed using degenerate PCR. Comparison of the T-box regions reveals a considerable degree of sequence conservation across species between the chicken and mouse genes (Fig. 1A). At the amino acid level the T-domain of the chicken and mouse Tbr1 and Tbr2 orthologs exhibit 98.5 and 99% identity, respectively, within the T-box domain. The T-domains of Tbr1 and Tbr2 (mouse and chicken) are 87.5% identical; outside of the T-domain there is very little similarity (data not shown). The genomic location of mouse Tbr2 was determined using the Jackson BSS backcross maps from two interspeci®c backcross DNA panels (Rowe et al., 1994), exploiting a Tbr2 PCR ampli®cation size polymorphism between the C57/BL6 and mus spretus genomic DNAs. Tbr2 maps between the markers D9Mit17 and D9Bir15 on distal chromosome 9 (Fig. 1B), where three neurological mutants are known to map: ducky, tippy and spinner.
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00053-2
134
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
Fig. 1. (A) T-box domain alignment of Xenopus Eomesodermin (x-Eomes) with mouse (mu) and chicken (ch) Tbr1 and Tbr2 genes. Amino acid identities (black boxes) and similarities (grey boxes) are indicated. (B) Map of mouse chromosome 9 showing the location of Tbr2.
Tbr2 expression was detected in mouse blastocysts and gastrula embryos (data not shown). Beginning around E10.0, Tbr2 expression was found primarily in the pallial (cortical) parts of the telencephalon (T) (Fig. 2A) which include primordia of the olfactory cortex, neocortex, hippocampus, and parts of the septum and amygdala. It is also expressed in the pallium's connection with the diencephalon (the eminentia thalami, ET) (Fig. 2A). Tbr2 transcripts were present in isolated cells in the ventricular zone (VZ), and more broadly in the postmitotic mantle zone (MZ), where Tbr1 expression is exclusively localized (Fig. 2A,E). As development proceeds (E12.5 and E16.5), there is an increasing number of Tbr2-expressing cells in the VZ, and decreasing expression in the MZ (Fig. 2B,C,I±L). At birth (P0), there is a low level of Tbr2 expression in cortical and hippocampal periventricular cells (Fig. 2D) and a high level in mitral cells of the olfactory bulb and in the anterior olfactory nuclear complex (Fig. 2O and data not shown). The only other areas where Tbr2 expression has been detected are the cerebellum (particularly in the external granular layer at E12.5 in Fig. 2M, at E16.5 in Fig. 2P, and at P0 in the cerebellar germinal trigone in Fig. 2Q), the inner
neuroblast layer of the neural retina at E16.5 (Fig. 2N), and limb mesenchyme (Fig. 2R). Embryonic expression of the chick Tbr1 and Tbr2 was examined from HH (Hamburger and Hamilton) stage 3 to HH28. Tbr1 transcripts could not be detected during gastrulation, whereas Tbr2 is already expressed at HH3 (Fig. 3), it decreases by HH5 with the beginning of primitive streak regression, and then disappears by HH6. At HH3, Tbr2 is strongly expressed in the hypoblast in the most anterior part of the area pellucida (Fig. 3A,C), in the ectoderm anterior and lateral to the primitive streak (Fig. 3A,D±H), and at a lower level in the ingressing mesodermal cells and de®nitive endoderm (Fig. 3A,E±G). By HH4, Tbr2 transcripts are clearly detected in ectodermal and mesenchymal cells along the rostral primitive streak (Fig. 3B,K,L) and also in mesodermal cells anterior to Hensen's node (Fig. 3B,J). At around 5 days of incubation the expression of the two genes are very similar, both spatially and temporally, to the patterns of their mouse orthologs (Fig. 4A,B). Chick Tbr2 is expressed in the pallial derivatives such as the dorsal ventricular ridge (DVR in Fig. 4E) and the cortex (Cx in Fig. 4B,C,E), and in the caudal region of basal nuclei: the
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
135
Fig. 2. Comparison of mouse Tbr2 and Tbr1 brain expression during embryonic development by in situ hybridization on tissue sections. (A±H) Adjacent sagittal sections showing the differential distribution of mTbr1 and mTbr2 transcripts during embryonic development. (A,E) At E10.5, Tbr2 and Tbr1 are coexpressed in the mantle zone (MZ) but Tbr2 is also expressed in cells of the germinative epithelium of the cortical anlage. (B,F) At E12.5, the Tbr2 pattern of expression follows the rostro-caudal (arrow in B) and latero-medial (arrow in I) gradients of differentiation and expression in the mantle is restricted to the deep layers of the incipient cortical plate. (I±M) Set of coronal sections showing Tbr2 expression at E12.5. (N±R) Tbr2 expression in the neural retina at E16.5 (N), in the olfactory bulb at P0 (O), in the developing cerebellum (at E16.5 in P, and P0 in Q), and in the fore limb bud (R, arrow indicates expression in a posterior metacarpal pre-cartilage condensation). Other abbreviations: CGE, caudal ganglionic eminence; CGT, cerebellar germinal trigone; Cp, cerebellar plate; CP, cortical plate; CPU, caudo-putamen; Cx, cortex; DG, dentate gyrus; EGL, external granular layer; ET, eminentia thalami; H, hippocampus; INL, inner neuroblast layer; LGE, lateral ganglionic eminence; M, mesencephalon; MCL, mitral cell layer; MGE, medial ganglionic eminence; ONL, outer neuroblast layer; Rh, rhombencephalon; T, telencephalon; VZ, ventricular zone; WM, white matter.
caudal ganglionic eminence (CGE in Fig. 4C), anlage of the amygdala. At HH25, cTbr2 is expressed in scattered cells which appear to be in the proliferative zone and more diffusely in the MZ, whereas cTbr1 is solely expressed in the MZ (Fig. 4C±F). By HH stage 28, besides being expressed in the proliferative zone, strong cTbr2 expression is concentrated in a layer near the interface of the VZ and MZ (Fig. 4G,H).
3. Methods Mouse and chicken Tbr2 and Tbr1 cDNA clones were obtained by degenerate PCR and by screening embryonic libraries. In situ hybridization was performed according to published protocols (Riddle et al., 1993; Rugarli et al., 1993).
136
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
Fig. 3. Early chick embryonic expression of Tbr2. Whole-mount in situ hybridization of embryos at stages HH3 (A) and HH4 (B). Transverse sections of embryo 4A (C±H) and 4B (I±P) at levels indicated. The location of Hensen's node is indicated (arrowheads in A and B).
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
137
Fig. 4. (A,B) Tbr2 transcript distribution in E12.5 mouse and HH stage 25 chick embryos (sagital sections). (C±H) Comparison of chicken Tbr2 and Tbr1 pallial expression, in sagital sections, at HH stages 25 and 28. Other abbreviations: CGE, caudal ganglionic eminence; Cx, cortex; DVR, dorsal ventricular ridge; EGL, external granular layer; ET, eminentia thalami; Hy, hippocampus; Is, isthmus; M, mesencephalon; MGE, medial ganglionic eminence; MZ, mantle zone; OB, olfactory bulb; OS, optic stalk; P1-4, prosomeres 1-4; S, septum; VZ, ventricular zone; ZL, zona limitans.
Acknowledgements We thank Celia Pardini, Cristina Mocchetti and Micaela Quarto for technical assistance, Andreas Russ for valuable discussions, and Melissa Smith for preparation of this manu-
script. This work was supported by the Italian Telethon Foundation (Grant no. B.37), the Merck Genome Research Institute (Grant no. 37 to A.B.), the EC (Grant no. BMH4CT97-2341 to A.B.), and NINDS Grant no. NS34661-01A1 to J.L.R.R.
138
A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138
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. Basson, C.T., Bachinsky, D.R., Lin, R.C., Levi, T., Elkins, J.A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T.A., Leblanc-Straceski, J., Renault, B., Kucherlapati, R., Seidman, J.G., Seidman, C.E., 1997. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15, 30±35. Bulfone, A., Smiga, S.M., Shimamura, K., Peterson, A., Puelles, L., Rubenstein, J.L., 1995. T-brain-1: a homolog of Brachyury whose expression de®nes molecularly distinct domains within the cerebral cortex. Neuron 15, 63±78. Bulfone, A., Wang, F., Hevner, R., Anderson, S., Cutforth, T., Chen, S., Meneses, J., Pedersen, R., Axel, R., Rubenstein, J.L.R., 1998. An olfactory sensory map develops in the absence of normal projection neurons or gabaergic interneurons. Neuron 21, 1273±1282. Chapman, D.L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S.I., Gibson-Brown, J.J., Cebra-Thomas, J., Bollag, R.J., Silver, L.M., Papaioannou, V.E., 1996. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206, 379±390. Chapman, D.L., Papaioannou, V.E., 1998. Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391, 695±697. Gibson-Brown, J.J., Agulnik, S.I., Chapman, D.L., Alexiou, M., Garvey, N., Silver, L.M., Papaioannou, V.E., 1996. Evidence of a role for T-box genes in the evolution of limb morphogenesis and the speci®cation of forelimb/hindlimb identity. Mech. Dev. 56, 93±101. Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Niswander, L., Papaioannou, V.E., 1998. Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb speci®cation and development. Development 125, 2499±2509. Li, Q.Y., Newbury-Ecob, R.A., Terrett, J.A., Wilson, D.I., Curtis, A.R., Yi, C.H., Gebuhr, T., Bullen, P.J., Robson, S.C., Strachan, T., Bonnet, D.,
Lyonnet, S., Young, I.D., Raeburn, J.A., Buckler, A.J., Law, D.J., Brook, J.D., 1997. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15, 21±29. Logan, M., Simon, H.G., Tabin, C., 1998. Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity. Development 125, 2825±2835. Papaioannou, V.E., Silver, L.M., 1998. The T-box gene family. Bioessays 20, 9±19. P¯ugfelder, G.O., Heisenberg, M., 1995. Optomotor-blind of Drosophila melanogaster: a neurogenetic approach to optic lobe development and optomotor behaviour. Comp. Biochem. Physiol. A: Physiol. 110, 185± 202. Riddle, R.D., Johnson, R.L., Laufer, E., Tabin, C., 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401±1416. Rowe, L.B., Nadeau, J.H., Turner, R., Frankel, W.N., Letts, V.A., Eppig, J.T., Ko, M.S., Thurston, S.J., Birkenmeier, E.H., 1994. Maps from two interspeci®c backcross DNA panels available as a community genetic mapping resource. Mamm. Genome 5, 253±274. Rugarli, E.I., Lutz, B., Kuratani, S.C., Wawersik, S., Borsani, G., Ballabio, A., Eichele, G., 1993. Expression pattern of the Kallmann syndrome gene in the olfactory system suggests a role in neuronal targeting. Nat. Genet. 4, 19±26. Ryan, K., Garrett, N., Mitchell, A., Gurdon, J.B., 1996. Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell 87, 989± 1000. Ryan, K., Butler, K., Bellefroid, E., Gurdon, J.B., 1998. Xenopus eomesodermin is expressed in neural differentiation. Mech. Dev. 75, 167±170. Wattler, S., Russ, A., Evans, M., Nehls, M., 1998. A combined analysis of genomic and primary protein structure de®nes the phylogenetic relationship of new members of the T-box family. Genomics 48, 24±33. Wilkinson, D.G., Bhatt, S., Herrmann, B.G., 1990. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657± 659.