Interference with Brachyury Function Inhibits Convergent Extension

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

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 ␣T4 globin 40 Blood Walmsley et al., 1994 N-CAM 40 Neural tissue Kintner and Melton, 1987 EF-1␣ 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 ␤A 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 ␤ 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 pBSKSϩ. 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.

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 activation 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. Uninjected control embryo (top) is indis- DBD identical to Xbra-EnR except that it contains a small internal tinguishable from embryos injected with RNA encoding Xbra , AD R R deletion within the Xbra DNA binding domain (amino acids 26 to Xbra ,En, or Xbra-En (Mut) (see Materials and Methods). Em- R 56) and does not bind DNA (F.L.C. and Elena Casey, unpublished bryo injected with 500 pg RNA encoding Xbra-En (bottom) lacks observations). posterior structures.

Mouse Strains and Embryos

The T mutation is maintained on the 129/Sv//Ev inbred back- RESULTS ground. Heterozygous animals were identified by vestigial tails. Embryos were staged according to morphological criteria (Downs Inhibition of Xbra Function Causes Down-regulation and Davies, 1993). The relative developmental stage of mutant of Ventral, Posterior, and (Some) Axial Markers embryos was assessed by comparison with heterozygous and wild Inhibition of Xbra function with Xbra-EnR results in the type-littermates. For whole-mount analyses, embryos were dis- formation of Xenopus embryos that lack posterior meso- sected free of maternal tissue in phosphate-buffered saline (PBS) containing 10% foetal calf serum and fixed overnight in 4% derm and a properly differentiated notochord (Fig. 1; see paraformaldehyde. Conlon et al., 1996). To characterise these embryos in more detail, we examined a panel of molecular markers whose expression is restricted to particular cell types during early Histology and Immunocytochemistry development (Table 1). Analyses were carried out at stages 10 (early gastrula), 13 (late gastrula), 17 (neurula), 20 (late For histological analysis, specimens were fixed, sectioned, and neurula), and 40 (tadpole), and the results are presented in stained as described (Smith, 1993). Whole-mount immunocyto- Fig. 2. Additional experiments in which embryos were chemistry with monoclonal antibody MZ15 (Smith and Watt, injected with RNA encoding XbraDBD, XbraAD,EnR, or Xbra- 1985), specific for notochord, was as described (Smith, 1993). EnR(Mut) (see Materials and Methods) gave results similar to those obtained with uninjected embryos (data not TUNEL Assays shown). At stages 10–17 (Fig. 2A), expression of most markers was Mouse and Xenopus embryos were fixed in 4% paraformalde- unaffected by inhibition of Xbra function, although, as hyde, washed in a graded series of methanol, and gradually rehy- previously reported (Conlon et al., 1996), levels of endoge- drated in methanol/PBT (PBS ϩ 0.1% Tween 20). Embryos were nous Xbra were reduced at the early gastrula stage. In situ then treated with proteinase K, postfixed in 4% paraformaldehyde, hybridisation analysis (Conlon et al., 1996) shows that this and washed three times in PBT. Next, embryos were incubated in decrease in Xbra is due to lack of expression in the prospec- Equilibration Buffer (Oncor) for1hatroom temperature and tive notochord. However, not all notochord markers are incubated overnight at 37°C in TdT enzyme diluted in Reaction R Buffer (Oncor). The reaction was terminated in Stop Buffer (Oncor) inhibited by Xbra-En ; Pintallavis expression is unchanged for3hat37°C, and embryos were then washed three times in PBT. (see also Conlon et al., 1996), and levels of noggin (Smith The embryos were blocked in 2 mg/ml BSA, 5% sheep serum in and Harland, 1992) and Xnot (von Dassow et al., 1993) are PBT for 2 h and then incubated overnight at 4°C in a 1/2000 only slightly reduced. The latter result is consistent with dilution of anti-digoxygenin–alkaline phosphatase or anti- the observation that levels of floating head, the zebrafish digoxygenin–peroxidase antibody (Boehringer Mannheim) diluted homologue of Xnot, are unaffected in Ntl mutant embryos in the blocking buffer. The embryos were then washed five times (Melby et al., 1997). Expression of Xwnt-8, which is ex- for 30 min in PBT and developed either using BM Purple AP pressed predominantly in ventral and posterior mesoderm substrate (Boehringer Mannheim) or DAB (Sigma) in PBS. (Christian et al., 1991; Smith and Harland, 1991), is very slightly reduced in Xbra-EnR-injected embryos by the late neurula stage, while goosecoid, which is expressed in the Cell Adhesion Assays organiser and subsequently in anterior mesoderm (Cho et Cell adhesion assays were carried out as described (Smith et al., al., 1991), is slightly elevated. 1990b). This analysis at stages 10–17 suggests that inhibition of

FIG. 2. Inhibition of Xbra function leads to a down-regulation of ventral and posterior markers. RNase protection analysis of uninjected control embryos and embryos injected with Xbra-EnR analysed at (A) stages 10 (early gastrula), 13 (late gastrula), and 17 (neurula); (B) stage 20 (late neurula); and (C) stage 40 (tadpole). In cases in which EF-1␣ lanes appear overexposed, shorter exposures conﬁrm approximately equal loading.

Xbra function causes slight down-regulation of at least By tadpole stage 40, levels of muscle-specific actin are some posterior- and notochord-specific markers and up- reduced in Xbra-EnR-injected embryos, consistent with the regulation of at least one anterior marker. This conclusion whole embryo phenotype. Similarly, levels of ␣T4-globin, a is confirmed at stage 20, at which levels of both chordin, posterior ventral mesoderm marker (Walmsley et al., 1994), which is expressed in notochord (Sasai et al., 1994), and and the endodermal marker IFABP (intestinal fatty acid HoxB9, a posterior marker (Sharpe et al., 1987), are reduced binding protein) (Shi and Hayes, 1994) are greatly reduced. by Xbra-EnR (Fig. 2B and data not shown). Accompanying Together, these data confirm that differentiation of noto- the down-regulation of these markers, the anterior marker chord and posterior/ventral cell types is greatly reduced in Otx-2 (Pannese et al., 1995) is up-regulated. embryos in which Xbra function is inhibited. We note,

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Inhibition of Brachyury during Xenopus Development 89 however, that most genes (with the exception of Xbra itself) with RNA encoding ␤-galactosidase were allowed to de- are expressed normally at early gastrula stages, and this velop to stage 30, when they were subjected to whole- suggests that the loss of the posterior and axial cell types mount TUNEL assays, which detect the 3Ј-OH ends gener- occurs during late gastrula and neurula stages. The in- ated by the DNA fragmentation which occurs during creased expression of Otx-2 in embryos in which Xbra apoptosis. The images labelled TUNEL in Fig. 3 show that function is inhibited suggests that the fates of posterior posteriorly located blastomeres expressing Xbra-EnR un- Xbra-EnR-injected cells are shifted anteriorly; this is inves- dergo apoptosis, while anteriorly positioned cells do not tigated below. (data not shown). The temporal pattern of apoptosis in embryos expressing Xbra-EnR was investigated by fixing injected and control Blastomeres Injected with RNA Encoding Xbra-EnR embryos at stages 10 to 28 (Fig. 4). Little apoptosis was Do Not Give Rise to More Anterior Structures but detected in control embryos before neurula stages and then, Are Excluded from the Notochord and Produce as reported by Hensey and Gautier (1998), programmed cell Fewer Progeny death was observed in the lateral and anterior margins of The elevated levels of Otx-2 in embryos injected with the neural plate. During gastrula stages, embryos injected RNA encoding Xbra-EnR (Fig. 2B) suggest that posterior with RNA encoding Xbra-EnR resembled controls (Figs. 4A tissues are converted to a more anterior fate. To address this and 4B), but apoptosis began at the early neurula stage and question we have injected individual cells of the 32-cell- persisted at least until stage 33 (Figs. 4D, 4F–4H, 5B and stage Xenopus embryo with RNA encoding Xbra-EnR to- 5C). Together, these results suggest that the loss of poste- gether with RNA encoding ␤-galactosidase as a cell lineage rior tissues in Xbra-EnR-injected embryos is due to pro- marker (Fig. 3). grammed cell death. Three conclusions derive from these studies. First, we To determine whether programmed cell death is an observe that Xbra-EnR-injected cells rarely contribute to evolutionarily conserved consequence of lack of Brachyury notochord. For example, blastomere B1 normally makes function, we examined apoptosis in mouse embryos which substantial contributions to axial structures, but inhibition were either homozygous or heterozygous for the T deletion. of Xbra function prevents B1 progeny from forming noto- Examination of mouse embryos at 9.5 days postcoitum by chord (compare B1 with B1 Xbra-EnR; MZ15 and MZ15 whole-mount TUNEL assays demonstrates that mice Xbra-EnR show similar embryos in which the monoclonal which lack a functional copy of T undergo significantly antibody MZ15 is used to mark notochord). Injection of more programmed cell death in the posterior portion of the RNA encoding Xbra-EnR into blastomere C1 provides an- embryo than do heterozygous embryos (Figs. 5D, 5E, and other example of this phenomenon, and the results were 5F). These results indicate that programmed cell death is an confirmed by injecting the Xbra-EnR construct at the 32- evolutionarily conserved feature of embryos which lack cell-stage and tracing the fate of the cells with an antibody Brachyury function. Interestingly, apoptosis was not ob- specific for the repressor domain of the Drosophila en- served in the notochord of mouse or Xenopus embryos grailed protein (not shown). lacking Brachyury function (data not shown), and it may be The second conclusion, discussed further below, is that significant that this tissue undergoes very little cell divi- Xbra-EnR-injected cells give rise to fewer progeny than sion (Bellomo et al., 1996, and F.L.C., unpublished). controls, particularly following injection into ventral/ posterior blastomeres (compare B3 with B3 Xbra-EnR and Xbra-EnR Induces Endoderm in Animal Caps and C1 with C1 Xbra-EnR of Fig. 3). Inhibits Mesoderm Differentiation Finally, our results indicate that posterior blastomeres R in Response to FGF injected with Xbra-En do not, at a gross level, adopt more anterior fates. The inhibition of notochord differentiation The experiments described above, and previous work and the decrease in cell number hinder illustration of this (Conlon et al., 1996), identify at least three defects in effect, but compare B3 with B3 Xbra-EnR (arrows in B3 Xenopus embryos in which Xbra function is inhibited: Xbra-EnR indicate labelled cells). gastrulation is impaired, embryos become anteriorised, and posterior and ventral mesoderm undergo apoptosis. In an effort to analyse these defects in more detail, we have used Inhibition of Xbra Function Causes Apoptosis animal cap assays in which FGF and activin function as of Posterior and Ventral Cell Types mesoderm-inducing factors. In all animal cap experiments, In the experiments described above we noted that blas- injection of RNA encoding XbraDBD, XbraAD,EnR, or Xbra- tomeres injected with RNA encoding Xbra-EnR produced EnR(Mut) (see Materials and Methods) gave results similar fewer progeny than control blastomeres (Fig. 3). To ask if to those obtained with uninjected samples (not shown). this decrease in cell number was due to programmed cell In the first series of experiments, animal caps derived death we investigated apoptosis in such embryos. from uninjected or from Xbra-EnR-injected embryos were In preliminary experiments, Xenopus embryos injected at cultured in the presence or absence of FGF-2. Gross mor- the 32-cell stage with RNA encoding Xbra-EnR together phology (Figs. 6A and 6B) and histological analysis (Figs. 6C

and 6D) of caps fixed at stage 28 suggest that the loss of Xbra function results in a failure to form mesoderm in response to FGF. These results were confirmed by RNase protection analysis (Fig. 7) using the markers listed in Table 1. At early gastrula stage 10 induction of most markers (including Pintallavis, which has not previously been shown to re- spond to FGF) was little affected, although induction of endogenous Xbra is greatly inhibited (Fig. 7A). Inhibition of Xbra expression may be due to interference with an indirect autoregulatory loop in which Xbra induces expression of eFGF and eFGF in turn maintains expression of Xbra (Casey et al., 1998; Isaacs et al., 1994; Schulte-Merker and Smith, 1995; Tada et al., 1997). Interestingly, chordin (Sasai et al., 1994), which is expressed in notochord and dorsal endoderm precursors (Sasai et al., 1996), and endodermin, most strongly expressed in endoderm but which is also present in notochord (Sasai et al., 1996), are induced by RNA encoding Xbra-EnR whether or not FGF is present (Fig. 7A). This may reflect a partial conversion of the animal cap to an endodermal fate but the apparent conversion is incomplete, since an epidermal marker (keratin) is also expressed. We also observe expression of the anterior marker Otx-2 in the absence of FGF induction, suggesting that Xbra-EnR-injected animal caps may be predisposed in some way to an anterior fate. This effect is specific because it can be reversed in a dose-dependent fashion by RNA encoding Xbra (data not shown). Analysis at the late neurula stage shows that induction of HoxB9 by FGF is reduced by Xbra-EnR (Fig. 7B), as is induction of muscle-specific actin at tadpole stages (Fig. 7C). These results are consistent with the histological analysis which indicates that Xbra-EnR inhibits mesodermal differentiation in response to FGF. Surprisingly, however, we note that induction of ␣T4-globin in response to FGF is greatly enhanced by Xbra-EnR; this contrasts with the effect of Xbra-EnR on whole embryos (Fig. 2C) and is discussed below.

Xbra Function Is Required for Convergent Extension Movements in Activin-Treated Animal Caps FIG. 3. Blastomeres injected with RNA encoding Xbra-EnR give R rise to posterior and ventral cell types but not notochord. The We next investigated the effects of Xbra-En on meso- indicated blastomeres of Xenopus embryos at the 32-cell stage were derm induction and cell movements induced by activin. injected with 100 pg RNA encoding Xbra-EnR together with 200 pg Treatment with activin causes animal caps to undergo RNA encoding ␤-galactosidase (right) or with 200 pg convergent extension movements resembling those occur- ␤-galactosidase RNA alone (left). Embryos were allowed to develop ring in the dorsal marginal zone during gastrulation (Symes to stage 28 or 40 (stage 40 data not shown) and were then stained and Smith, 1987). These cell movements are inhibited by with X-gal. Blastomeres injected with RNA encoding Xbra-EnR tend to adopt their normal fate except when injected into the B1 blastomere, when they are excluded from the notochord. These results were conﬁrmed by staining with the notochord-speciﬁc antibody MZ15, which stains the notochord sheath without ob- scuring the dense ␤-galactosidase labelling. Arrow in B3 Xbra-EnR images) demonstrates that apoptosis occurs when injected blas- indicates labelled cells partially obscured by pigment granules. tomeres occupy posterior positions in the embryo. Apoptotic cells TUNEL staining of control embryos and those injected in the appear dark blue and those expressing ␤-galactosidase appear tur- indicated blastomeres with RNA encoding Xbra-EnR (bottom four quoise.

FIG. 4. Apoptosis in control embryos and in embryos injected with RNA encoding Xbra-EnR begins during neurula stages. Apoptosis in embryos in which Xbra function is inhibited occurs in posterior structures. TUNEL assays were performed on uninjected embryos (A, C, E) and on embryos expressing Xbra-EnR (B, D, F, G, H). Embryos were ﬁxed at stages 10.5 (A, B), 18 (C, D, G), and 24 (E, F, H). High-power views of Xbra-EnR-injected embryos are shown in (G, H). Arrows in (C) and (E) indicate lack of apoptosis in the posterior regions of control embryos; those in (D) and (F) show apoptotic cells in embryos injected with RNA encoding Xbra-EnR.

Xbra-EnR (Figs. 8A–8C), consistent with the suggestion that chord (not shown). The appearance of notochord is, at ﬁrst Brachyury is required for convergent extension during gas- sight, surprising, but this may reﬂect the fact that the effect trulation. Histological analysis of activin-treated Xbra-EnR- of Brachyury absence on notochord formation is a late injected caps at stage 28 reveals that despite the lack of event and that levels of Xbra-EnR may have decreased by the convergent extension movements the caps still form meso- critical time. It is probable that the notochord that is derm, including muscle (Fig. 8F) and, in many cases, noto- formed is anterior in nature; notochord may be present in

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Inhibition of Brachyury during Xenopus Development 93 the caudal regions of mouse T/T mutant embryos (see Xbra-EnR and treated with activin (Fig. 10C) or FGF (Fig. Beddington et al., 1992). 10D). Such cells were only visible in the superficial layer of These results were confirmed by RNase protection anal- these explants; the reason for this is not known. ysis (Fig. 7), which indicates that expression of most markers is little affected by Xbra-EnR, although induction of endogenous Xbra by activin is, as expected, reduced (Fig. DISCUSSION 7A). Expression of muscle-specific actin is also reduced (Fig. 7C), sometimes significantly so (data not shown). This study concentrates on the cellular and molecular consequences of inhibiting Brachyury function during Xe- nopus development. We have previously shown that expres- Xbra Function Is Not Required for Adhesion sion of the dominant-negative Brachyury construct Xbra- or Migration on Fibronectin R En causes loss of posterior structures and impairment of The results described above (Figs. 8A–8C) demonstrate notochord formation (Conlon et al., 1996). In the present that Xbra function is required for convergent extension study we demonstrate that the loss of posterior mesoderm movements in response to activin. To investigate other in embryos in which Brachyury is inhibited is due to aspects of activin-induced cell motility we asked whether inhibition of convergent extension movements during gas- Xbra-EnR inhibits the ability of activin-treated animal pole trulation, repression of terminal mesodermal differentia- blastomeres to adhere to a fibronectin substrate (Smith et tion in response to FGF, and a progressive loss of posterior al., 1990b). No such inhibition could be detected (Figs. 9A and ventral cell types through apoptosis. and 9C), and formation of focal adhesions also occurred normally (data not shown). However, after culture to the Xbra and Gastrulation equivalent of neurula stage 17, blastomeres derived from embryos injected with RNA encoding Xbra-EnR began to One of the major consequences of inhibiting Brachyury round up and detach from the fibronectin-coated substrate, function in Xenopus is a disruption of gastrulation (Conlon while control cells formed a monolayer of healthy cells et al., 1996). At least two types of cell movement are (Figs. 9B and 9D). The timing of this apparent cell death is involved in gastrulation in Xenopus: migration and conver- similar to that revealed by TUNEL staining in intact gent extension (Gerhart and Keller, 1986). Convergent ex- embryos and is specific to mesoderm, since no detachment tension involves a rearrangement of cells of the deep of uninduced Xbra-EnR-expressing blastomeres was identi- mesoderm, involving both radial and tangential intercala- R fied when Xbra-En caps were cultured on poly-L-lysine or tion, and it causes elongation of the embryo along the purified ECM matrix (not shown). anteroposterior axis (Gerhart and Keller, 1986). The pheno- To investigate apoptosis in animal caps we performed type of Xenopus embryos injected with RNA encoding TUNEL assays. Animal pole regions were isolated from Xbra-EnR (Fig. 1) is similar to that observed when physical control embryos, or from embryos injected with RNA means are used to inhibit convergent extension: the blas- encoding Xbra-EnR, and were left untreated or exposed to topore fails to close and posterior structures fail to differ- FGF or activin. The caps were cultured to the equivalent of entiate (Keller et al., 1991). This loss of posterior structures stage 24–26, fixed, sectioned, and subjected to TUNEL is not a general consequence of inhibition of gastrulation, analysis. No apoptosis was detected in uninduced animal however, because interfering with gastrulation by other caps (not shown)nor in those that were derived from control means, such as treatment with suramin, has exactly the embryos and then treated with activin or FGF (Figs. 10A opposite effect and causes anterior truncations (Gerhart et and 10B). Apoptotic cells were present, however, in ex- al., 1989). Our results therefore indicate that the gastrula- plants derived from embryos injected with RNA encoding tion defect of Xbra-EnR-injected embryos is due to inhibi-

FIG. 5. Embryos lacking Brachyury function undergo apoptosis in posterior and ventral tissues. TUNEL assays were performed on uninjected control stage 33 Xenopus embryos (A) or embryos injected at the one-cell stage with RNA encoding Xbra-EnR (B, C). Uninjected embryos (A) show programmed cell death in anterior but not posterior tissues (arrow indicates absence of apoptotic cells posteriorly). Embryos injected with RNA encoding Xbra-EnR (B, C) display areas of programmed cell death in ventral and posterior tissues (arrows). In contrast to their wild-type siblings (D, embryo on left), mouse embryos homozygous for a null mutation in Brachyury display extensive programmed cell death in posterior and ventral cell types (D, embryo on right). (E) and (F) show higher magniﬁcations of the tail buds of wild type and homozygous null embryos, respectively. FIG. 6. Xbra-EnR inhibits mesoderm differentiation in response to FGF. (A) Morphology of FGF-treated animal caps derived from uninjected embryos. (B) Animal caps derived from embryos injected with 200 pg RNA encoding Xbra-EnR. (C) Histological section of an FGF-treated animal cap derived from an uninjected embryo. Mesenchyme and mesothelium are marked. (D) Section of an FGF-treated animal cap derived from an embryo injected with 500 pg Xbra-EnR RNA. Cells appear yolky. Caps were analysed at the equivalent of stage 28.

FIG. 8. Inhibition of Xbra function prevents gastrulation-like movements in response to activin but does not abolish mesodermal differentiation. (A) Control animal caps treated with activin undergo elongation. (B) Untreated animal caps form spheres. (C) Animal caps derived from embryos injected with Xbra-EnR RNA do not elongate in response to activin. (D) Histological section of a control animal cap treated with activin. Note formation of muscle and notochord. (E) Section of an untreated animal cap which forms atypical epidermis. (F) Section of an animal cap derived from an embryo injected with Xbra-EnR RNA and treated with activin. Note formation of muscle. Caps in A–C were analysed at stage 17 and those in D and E at stage 28.

tion of convergent extension, a conclusion supported by the animal pole cells from adhering to a fibronectin substrate. fact that activin-induced elongation of animal caps is inhib- We note, however, that blastomeres derived from embryos ited in Xbra-EnR-injected animal caps (Figs. 8A–8C). We injected with RNA encoding Xbra-EnR later detach from the note that the inhibition of convergent extension cannot be substrate, perhaps reflecting the phenomenon of anoikis due to death of gastrulating cells, because programmed cell (see below). death in embryos in which Xbra function has been inhib- Together, our results demonstrate that Brachyury func- ited does not begin until the end of gastrulation. tion in Xenopus is required for convergent extension during The second type of cell movement occurring during gastrulation but not for mesodermal migration, and we are Xenopus gastrulation involves the migration, as individu- now carrying out experiments to attempt to understand als, of prospective anterior mesendodermal cells (Gerhart why these two types of cell movement should differ in their and Keller, 1986). These cell movements can be reproduced requirement for Xbra activity. in vitro by culturing such cells on a fibronectin substrate, The conclusion that Brachyury is required for normal and animal pole cells can be induced to undergo this gastrulation is consistent with work in the mouse embryo behaviour by treatment with activin (Smith et al., 1990b). (Hashimoto et al., 1987; Wilson et al., 1995; Yanagisawa Since anterior structures are unaffected by inhibition of and Fujimoto, 1977; Yanagisawa et al., 1981), but it is Brachyury function it is likely that mesodermal migration difficult to extend the interspecies comparison because the occurs normally. Consistent with this proposal, inhibition requirement for Brachyury in the mouse is cell autono- of Brachyury function does not prevent activin-treated mous; when ES cells lacking a functional Brachyury gene

FIG. 7. Inhibition of Xbra function prevents mesoderm induction in response to FGF but not to activin. Animal caps were derived from control embryos or embryos injected with 500 pg Xbra-EnR RNA and treated with activin or FGF or left untreated. RNase protection analysis was carried out at (A) stages 10 (early gastrula), 13 (late gastrula), and 17 (neurula); (B) stage 20 (late neurula); and (C) stage 40 (tadpole). In cases in which EF-1␣ lanes appear overexposed, shorter exposures conﬁrm approximately equal loading.

FIG. 9. Xbra function is not required for adhesion on a ﬁbronectin substrate, but blastomeres subsequently detach at neurula stages. Animal pole blastomeres derived from control embryos (A, B) or embryos injected with 500 pg RNA encoding Xbra-EnR (C, D) were plated on a ﬁbronectin substrate and photographed at stage 11 (A, C) and stage 17 (B, D). Note detachment of blastomeres in (D).

are introduced into early mouse embryos, the mutant cells contrast, clones of Xenopus blastomeres in which fail to move away from the primitive streak and accumulate Brachyury function is inhibited gastrulate normally (Fig. 3). in the tail bud (Rashbass et al., 1991; Wilson et al., 1995). By It is likely that gastrulation in Xenopus is a more coherent

FIG. 10. Inhibition of Xbra function leads to programmed cell death in the superﬁcial layer of animal caps treated with FGF or activin. Animal caps derived from control embryos (A, B), or those injected with 500 pg RNA encoding Xbra-EnR (C, D), were treated with FGF (A, C) or activin (B, D). Caps were cultured to the equivalent of stage 25 when they were ﬁxed and sectioned and subjected to TUNEL analysis.

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Inhibition of Brachyury during Xenopus Development 97 process than in the mouse and that Xbra-EnR-injected cells detachment of cells from the extracellular matrix leads to can be more readily rescued by their wild-type neighbours apoptosis (Frisch and Francis, 1994), a process which may (Fig. 3). prevent them from occupying an inappropriate location in The results in Fig. 3 show that Xbra-EnR-injected cells do the body (Khwaja et al., 1997). It is possible, therefore, that not adopt a more anterior fate. Rather, as discussed below, although apoptosis is not a direct consequence of inhibition they undergo apoptosis. If this is so, why are levels of Otx-2 of Brachyury function, it does arise directly from changes in elevated in whole embryos in which Xbra function is cell adhesion that are associated with the disruption of inhibited? One possibility is that levels of this anterior gastrulation. Consistent with this idea, a recent study has neurectodermal marker are normally repressed by posterior shown that cell death is induced by directly inhibiting the tissue, and in the absence of such tissue, in Xbra-EnR- interaction of the cytoskeleton with the extracellular ma- injected embryos, levels of Otx-2 remain high (Nieuwkoop, trix (Buckley et al., 1999). 1952). Xbra and Mesoderm Induction Programmed Cell Death Previous work has demonstrated that Xbra and FGF are The first consequence of inhibition of Brachyury func- components of an indirect autoregulatory loop in which tion during Xenopus development is a disruption of conver- each gene product maintains expression of the other (Isaacs gent extension, and this is likely to be the immediate cause et al., 1994; Schulte-Merker and Smith, 1995). Recent of the truncated phenotype we observe. But what happens experiments show that the activation of eFGF expression by to the cells that would have formed posterior structures? Xbra is direct (Casey et al., 1998). The importance of this Two lines of evidence suggest that they undergo apoptosis. loop is demonstrated by the fact that inhibition of FGF First, injection of RNA encoding Xbra-EnR together with a signalling prevents Xbra from inducing animal pole tissue lineage label into individual blastomeres of the Xenopus to form mesoderm (Schulte-Merker and Smith, 1995), and embryo reveals that the progeny of these marked cells this paper describes the complementary experiment in decrease in number compared with controls (Fig. 3). Second, which inhibition of Xbra function prevents terminal differ- TUNEL labelling provides direct evidence that apoptosis entiation of mesoderm in response to FGF. According to occurs in posterior cells (Figs. 3–5), and analysis of mouse this view, the consequences of inhibiting FGF signalling embryos demonstrates that this is an evolutionarily con- should be similar in many respects to the consequences of served consequence of the loss of Brachyury function (Fig. inhibiting Xbra function, and indeed we note that the 5). These findings are consistent with earlier studies de- presence of a truncated FGF receptor prevents activin- scribing necrotic cells in the posterior mesoderm of mice induced elongation of Xenopus animal caps but has no homozygous for the T/T Brachyury mutation (Chesley, effect on the ability of those cells to spread on fibronectin 1935; Yanagisawa et al., 1981). They are also reminiscent of (Cornell and Kimelman, 1994). The phenotype of embryos work in Drosophila, in which embryos lacking the T-box in which FGF signalling is inhibited may not be identical in gene brachyenteron display ectopic expression of reaper all respects to that of embryos expressing Xbra-EnR, and and undergo apoptosis in the hindgut and anal pads (Singer indeed FGF may induce the expression of other genes et al., 1996). involved in mesodermal patterning (Griffin et al., 1995). It is likely, however, that apoptosis is an indirect conse- Analysis of gene expression in animal pole regions de- quence of loss of Brachyury function rather than a direct rived from embryos injected with RNA encoding Xbra-EnR effect. In both Xenopus and mouse embryos, for example, reveals, as expected, that induction of Xbra itself by FGF is apoptosis occurs later than the first observable phenotype, greatly reduced, but that expression of other early markers the disruption of gastrulation. This conclusion is supported is little affected. The early response to activin (again with by observation of FGF- or activin-treated animal caps, in the exception of Xbra) is also largely unaffected by Xbra- which apoptosis can be detected at late stages (Fig. 10), but EnR. These observations suggest that the Xbra/eFGF auto- for which there is no evidence for cell death during gastru- regulatory loop involves few, if any, additional genes and in lation. For example, we observe no decrease in size of this sense is highly specific. We note, however, that at later animal caps in response to RNA encoding Xbra-EnR (data stages Xbra-EnR has some effects, such as the induction of not shown) and no decrease in levels of ubiquitously ex- Otx-2 and the enhancement of ␣T4-globin expression in pressed RNAs, such as cytoskeletal actin or EF-1␣ (Fig. 7). response to FGF, which are rather unexpected. Thus, al- Furthermore, although inhibition of Brachyury function though terminal differentiation of mesodermal cell types in causes blastomeres to detach from a fibronectin-coated response to FGF is inhibited by Xbra-EnR, the activation of substrate, this does not occur until late neurula stages (Fig. some mesoderm-specific genes, such as of ␣T4-globin, is 10). These observations are again consistent with studies in actually increased. T/T mouse embryos, in which pyknosis was not observed The most surprising observation is that animal caps before day 9 (Chesley, 1935; Yanagisawa et al., 1981). derived from embryos injected with RNA encoding Xbra- The detachment of activin-treated blastomeres is remi- EnR activate expression of endodermin, chordin, and Otx2. niscent of the phenomenon known as anoikis, in which Consideration of the expression patterns of these genes

(Table 1) would suggest that interference with Xbra func- to mesoderm-inducing factors, may play a role in ventral meso- tion causes this animal pole tissue to differentiate as dermal patterning during embryogenesis. Development 111, anterior endoderm, and this conclusion is consistent with 1045–1055. the histological appearance of the explants and with the fact Clements, D., Taylor, H. C., Herrmann, B. G., and Stott, D. (1996). that globin expression in response to FGF is enhanced by Distinct regulatory control of the Brachyury gene in axial and non-axial mesoderm suggests separation of mesodermal lineages Xbra-EnR; blood cell differentiation is stimulated by caudal early in mouse gastrulation. Mech. Dev. 56, 139–149. endoderm (Tseng, 1958). We note that these effects of R Conlon, F. L., Sedgwick, S. G., Weston, K. M., and Smith, J. C. Xbra-En differ from those obtained using a truncated (1996). Inhibition of Xbra transcription activation causes defects version of Xbra, which causes activation of neural genes in mesodermal patterning and reveals autoregulation of Xbra in (Rao, 1994). The mode of action of the truncated Xbra dorsal mesoderm. Development 122, 2427–2435. construct is unknown, so this difference is difficult to Cornell, R. A., and Kimelman, D. (1994). Activin-mediated meso- interpret. derm induction requires FGF. Development 120, 453–462. But how can Xbra-EnR interfere with Xbra function in Cunliffe, V., and Smith, J. C. (1992). Ectopic mesoderm formation animal pole tissue, in which Xbra is not expressed? One in Xenopus embryos caused by widespread expression of a possibility is that there is a basal level of expression of Xbra Brachyury homologue. Nature 358, 427–430. target genes, perhaps activated in response to maternal Cunliffe, V., and Smith, J. C. (1994). Specification of mesodermal Xbra transcripts (Smith et al., 1991), and that the role of pattern in Xenopus laevis by interactions between Brachyury, noggin and Xwnt-8. EMBO J. 13, 349–359. these target genes is somehow to prevent anterior Dale, L., Matthews, G., and Colman, A. (1993). Secretion and endoderm formation. We plan to investigate this question mesoderm-inducing activity of the TGF-beta related domain of by isolating Xbra targets (Tada et al., 1998) and by observing Xenopus Vg1. EMBO J. 12, 4471–4480. the effects of ablating maternal Xbra transcripts. Downs, K. M., and Davies, T. (1993). Staging of gastrulating mouse embryos by morphological landmarks in the dissecting micro- scope. Development 118, 1255–1266. ACKNOWLEDGMENTS Dyson, S., and Gurdon, J. B. (1996). Activin signalling has a necessary function in Xenopus early development. Curr. Biol. 7, This work is supported by the Medical Research Council. 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