Developmental Biology 213, 85–100 (1999) Article ID dbio.1999.9330, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Interference with Brachyury Function Inhibits provided by Elsevier - Publisher Connector Convergent Extension, Causes Apoptosis, and Reveals Separate Requirements in the FGF and Activin Signalling Pathways Frank L. Conlon and J. C. Smith Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom Brachyury plays a key role in mesoderm formation during vertebrate development. Absence of the gene results in loss of posterior mesoderm and failure of the notochord to differentiate, while misexpression of Brachyury in the prospective ectoderm of Xenopus results in ectopic mesoderm formation. Brachyury is therefore both necessary and sufficient for posterior mesoderm formation. Here we present a detailed cellular and molecular analysis of the consequences of inhibiting Brachyury function during Xenopus development. Our results show that Brachyury is required for the convergent extension movements of gastrulation, for mesoderm differentiation in response to FGF, and for the survival of posterior mesodermal cells in both Xenopus and mouse. © 1999 Academic Press INTRODUCTION tion is essential for its biological function (Conlon et al., 1996). In order to understand how the gene exerts its effects Brachyury, or T, is required for the formation of posterior during early development, it is necessary to identify targets mesoderm and notochord in mouse (Herrmann et al., 1990), of Brachyury (Casey et al., 1998; Tada et al., 1998) as well as zebrafish (Halpern et al., 1993; Schulte-Merker et al., 1994), to understand how its expression is controlled (Clements et and Xenopus (Conlon et al., 1996) embryos. In all these al., 1996; Latinkic et al., 1997). In addition it is important to species, and in the chick (Kispert et al., 1995b), the gene is define the role of Brachyury at the molecular and cell expressed transiently throughout nascent mesoderm and biological levels, and these are the issues addressed in this transcripts persist in tailbud and notochord; Brachyury is paper. therefore essential for formation of the mesodermal tissues Our experiments concentrate on Xenopus laevis, because in which it is expressed. The importance of Brachyury in more is known about mesoderm formation in this species vertebrate mesoderm formation is emphasised by experi- than in any other vertebrate (Slack, 1994). The function of ments in which the gene is misexpressed in prospective Xenopus Brachyury (Xbra) can be inhibited by expressing a ectodermal cells of Xenopus; this causes the formation of construct in which the activation domain of Xbra is re- ectopic mesoderm, with low concentrations of Brachyury placed by the repressor domain from Drosophila engrailed inducing ventral cell types and high concentrations induc- (EnR), thus creating the fusion protein Xbra-EnR (Conlon et ing dorsal tissues (Cunliffe and Smith, 1992). Expression of al., 1996). Embryos injected with RNA encoding Xbra-EnR Brachyury alone is not sufficient to induce notochord, but do not gastrulate normally and fail to form tail structures coexpression of Brachyury with noggin (Smith and Harland, and (in many cases) a notochord (Conlon et al., 1996). They 1992) or with Pintallavis (Ruiz i Altaba and Jessell, 1992) therefore resemble mouse and zebrafish embryos which does cause notochord to form (Cunliffe and Smith, 1994; carry mutations in the Brachyury gene (Chesley, 1935; O’Reilly et al., 1995). Halpern et al., 1993; Herrmann et al., 1990; Schulte-Merker Brachyury encodes a sequence-specific DNA-binding pro- et al., 1994). The inhibitory effects of Xbra-EnR are specific tein which functions as a transcription activator (Conlon et to Xbra, because the phenotypes obtained when EnR is al., 1996; Kispert and Herrmann, 1993; Kispert et al., fused to the DNA-binding domains of the T-box proteins 1995a), and the ability of Brachyury to activate transcrip- eomesodermin and Brat differ from those obtained with 0012-1606/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 85 86 Conlon and Smith TABLE 1 Molecular Markers Used in This Study Stage(s) of Gene examination Expression pattern Reference Xbra 10, 13, 17 Pan mesodermal and axial mesoderm Smith et al., 1991 Mix.1 10, 13, 17 Pan mesodermal and endoderm Rosa, 1989 Goosecoid 10, 13, 17 Organiser Cho et al., 1991 Noggin 10, 13, 17 Organiser and axial mesoderm Smith and Harland, 1992 Chordin 10, 13, 17 Organiser, axial mesoderm, dorsal endoderm Sasai et al., 1994, 1996 Pintallavis 10, 13, 17 Dorsal and axial mesoderm Ruiz i Altaba and Jessell, 1992 Xnot 10, 13, 17 Dorsal and axial mesoderm von Dassow et al., 1993 Xwnt-8 10, 13, 17 Ventral and lateral mesoderm Christian et al., 1991; Smith and Harland, 1991 Endodermin 10, 13, 17 Notochord, prechordal plate, and endoderm Sasai et al., 1996 Otx-2 20 Anterior mesendoderm and neurectoderm Kablar et al., 1996; Pannese et al., 1995 HoxB9 20 Posterior mesoderm and neurectoderm Sharpe et al., 1987 Keratin 40 Epidermis Snape et al., 1990 Cardiac actin 40 Skeletal and cardiac muscle Mohun et al., 1984 aT4 globin 40 Blood Walmsley et al., 1994 N-CAM 40 Neural tissue Kintner and Melton, 1987 EF-1a All Ubiquitous Sargent and Bennett, 1990 Xbra-EnR (Horb and Thomsen, 1997; Ryan et al., 1996). tiation in response to FGF but not in response to activin. Furthermore, the effects of Brat-EnR can be rescued by However, the ability of activin to induce convergent exten- coexpression of Brat, but not by Xbra (Horb and Thomsen, sion movements in prospective ectodermal tissue (Symes 1997), and vice-versa (F.L.C. and J.C.S., unpublished). and Smith, 1987) is inhibited by Xbra-EnR, suggesting that In this paper we first present a detailed analysis of the Brachyury is required for normal gastrulation movements phenotype of Xenopus embryos in which Xbra function is to occur (see also Wilson et al., 1995). inhibited. Our results indicate that Xbra-EnR causes a decrease in expression of molecular markers associated with ventral and posterior tissue types and that after MATERIALS AND METHODS gastrulation cells expressing these genes are lost through programmed cell death. This suggests that Brachyury has a Xenopus Embryos, Microinjection, and Dissection trophic activity for posterior mesodermal cells, a conclu- Xenopus embryos were obtained by in vitro fertilisation (Smith sion strengthened by the observation that mouse embryos and Slack, 1983). They were maintained in 10% Normal Amphib- mutant for Brachyury also undergo apoptosis in posterior ian Medium (NAM; Slack, 1984) and staged according to Nieuw- structures. koop and Faber (1975). Xenopus embryos at the 1- to 2-cell stage or We have inferred additional functions for Brachyury in at the 32-cell stage were injected with RNA in 10 or 1 nl water, cell differentiation and cell movement from studies in respectively, as described (Smith, 1993). For animal cap assays, animal caps. The mesoderm of Xenopus is formed through embryos were dissected in 75% NAM, and caps were cultured in the same medium. Xenopus FGF-2 was prepared using an expres- an inductive interaction in which cells of the vegetal sion plasmid provided by David Kimelman and Marc Kirschner. hemisphere act on overlying equatorial cells (Sudarwati and Partially purified human activin A was prepared from the condi- Nieuwkoop, 1971). Two families of signalling molecules tioned medium of COS cells transfected with a human inhibin bA are thought to be involved in this interaction: members of cDNA. The cells were a gift from Dr. Gordon Wong (Genetics the fibroblast growth factor (FGF) family, such as FGF-2 Institute, Inc.). (Amaya et al., 1991; Isaacs et al., 1992; Kimelman and Kirschner, 1987; Slack et al., 1987), and members of the RNA Isolation and RNase Protection Assays transforming growth factor type b family, such as activin and Vg1 (Dale et al., 1993; Dyson and Gurdon, 1996; Smith RNase protection analysis was carried out as described (Jones et et al., 1990a; Thomsen et al., 1990; Thomsen and Melton, al., 1995). Probes used are listed in Table 1. The endodermin probe was constructed by excising an XhoI/SspI fragment corresponding 1993). Both FGF and activin induce expression of Xbra in to nucleotides 1928–2207 from a 5-kb endodermin cDNA kindly prospective ectodermal tissue in an immediate-early fash- provided by Dr. E. M. De Robertis. The fragment was cloned into ion (Smith et al., 1991), and in the second part of this paper pBSKS1. To make an RNase protection probe, the plasmid was R we use Xbra-En to investigate the role of Xbra in the digested with XhoI and transcribed with T7 RNA polymerase. response to these two factors. Our results indicate that RNase protection data shown in Figs. 2 and 7 are representative of Xbra function is essential for terminal mesoderm differen- at least two independent experiments. Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Inhibition of Brachyury during Xenopus Development 87 RNA Synthesis RNA encoding Xbra-EnR was prepared as described (Conlon et al., 1996). RNA synthesised from the following constructs was used as controls in various experiments; all gave results identical to those obtained with uninjected embryos (not shown). XbraDBD encodes the DNA-binding domain of Xbra (Cunliffe and Smith, 1992). XbraAD comprises amino acids 301 to 432 of Xbra preceded by an initiator methionine; it comprises the transcription activa- tion domain of the protein (Conlon et al., 1996). EnR encodes the R repressor domain of the Drosophila engrailed protein preceded by FIG. 1. Injection of RNA encoding Xbra-En inhibits posterior an initiator methionine (Conlon et al., 1996). Xbra-EnR(Mut) is mesoderm development.