Gene expression pattern

Expression pattern of the Tbr2 (Eomesodermin) gene during mouseand chick brain development

Alessandro Bulfonea,*, Salvador Martinezb, Valeria Marigoa, Marilena Campanellaa,Andrea Basilea, Nandita Quaderia, Claudio Gattusoa, John L.R. Rubensteinc, Andrea Ballabioa

aTelethon Institute of Genetics and Medicine (TIGEM), San Raffaele Biomedical Science Park, Via Olgettina, 58, 20132 Milan, ItalybDepartment of Morphological Sciences, University of Murcia, 30071 Murcia, Spain

cNina 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 develop-

mental 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 T-

domain (Papaioannou and Silver, 1998). T-box genes have

important developmental functions during gastrulation (e.g.

Brachyury, Wilkinson et al., 1990), mesodermal speci®ca-

tion (Tbx6, Chapman and Papaioannou, 1998), limb pattern-

ing, (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 respon-

sible 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.,

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 conserva-

tion 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 interspe-

ci®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 chro-

mosome 9 (Fig. 1B), where three neurological mutants are

known to map: ducky, tippy and spinner.

Mechanisms of Development 84 (1999) 133±138

0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0925-4773(99)00053-2

* Corresponding author. Tel.: 1 39-02-21560234; fax: 1 39-02-

21560220.

E-mail address: bulfone@tigem.it (A. Bulfone)

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, hippo-

campus, 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 olfac-

tory 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 gastru-

lation, 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±138134

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.

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 diffu-

sely 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).

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.

A. Bulfone et al. / Mechanisms of Development 84 (1999) 133±138136

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).

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. BMH4-

CT97-2341 to A.B.), and NINDS Grant no. NS34661-01A1

to J.L.R.R.

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.

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