Cell,

Vol.

67, 79-87,

October

4, 1991,

Copyright

0 1991

by Cell

Press

Expression of a Xenopus Homolog Is an Immediate-Early Response to Mesoderm Induction J. C. Smith,* B. M. J. Price,’ J. B. A. Green,’ D. Weigel,t and B. G. Herrmannt *Laboratory of Developmental Biology National Institute for Medical Research The Ridgeway, Mill Hill London NW7 1AA England tMax-Planck lnstitut fur Entwicklungsbiologie Abteilung Biochemie Spemannstrasse 3.5 7400 Ttibingen Federal Republic of Germany

The Brachyury (T) gene is required for mesoderm formation in the mouse. In this paper we describe the cloning and expression of a Xenopus homolog of Brachyury, Xbra. As with Brachyury in the mouse, Xbra is expressed in presumptive mesodermal cells around the blastopore, and then in the notochord. We show that expression of Xbra occurs as a result of mesoderm induction in Xenopus, both in response to the natural signal and in response to the mesoderm-inducing factors activin A and basic FGF. Expression of Xbra in response to these factors is rapid, and will occur in dispersed cellsand in the presence of a protein synthesis inhibitor, indicating that this is an “immediateearly” response to mesoderm induction. Introduction Recently, great progress has been made in coming to understand the molecular basis of mesoderm formation in both amphibian and mammalian embryos. For the amphibia, it is well known that the mesoderm arises through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying animal pole cells (Nieuwkoop, 1969,1973). Candidates for the inducing factors made by the vegetal cells fall into two classes (see reviews by Smith, 1989; New and Smith, 1990). One consists of members of the fibroblast growth factor (FGF) family (Slack et al., 1987; Kimelman and Kirschner, 1987). These tend to induce posterior and ventral tissue types and genes. The other class comprises members of the transforming growth factor 6 (TGFP) family, of which the activins are the most potent (Asashimaet al., 1990; Albano et al., 1990; Smith et al., 1990; van den Eijnden-Van Raaij et al., 1990; Thomsen et al., 1990). The activins preferentially induce dorsal cell types and genes (Green et al., 1990) and also cause responding cells to act as Spemann’s organizer (Cooke et al., 1987; Cooke, 1989; Ruiz i Altaba and Melton, 1989). An important property of the activins is that they act as morphogens; that is, they induce different cell types and expression of different mesoderm-

of Brachyury

(T)

specific genes at different concentrations (Green and Smith, 1990). Despite their impressive properties, it is not yet clear which members of the FGF and TGF8 families, if any, are the natural mesoderm-inducing factors (MIFs). Basic FGF (bFGF) mRNA is expressed during oogenesis in Xenopus (Kimelman et al., 1988) and there is sufficient bFGF protein in the early embryo for the molecule to act as an inducer (Kimelman et al., 1988; Slack and Isaacs, 1989). However, bFGF lacks a classic secretory signal sequence and nothing is known about its spatial distribution in the early embryo. The situation with the activins is even more confused. Zygotic expression of activins A and B only commences at the neurula and late blastula stages, respectively (Thomsen et al., 1990), and even for activin B this is almost certainly too late for the molecule to be a natural primary inducer (Jones and Woodland, 1987). It is possible, however, that there is maternal activin protein present in the embryo (see van den Eijnden-Van Raaij et al., 1990) and this is currently under investigation. It is not yet known whether mesoderm in mammalian embryos arises through induction, but an important advance in the understanding of mesoderm formation in the mouse came through the cloning of the Brachyury (T) gene, which is located in the Tltcomplex on chromosome 17 (Herrmann et al., 1990). Loss of function of Brachywy leads to a disturbance of the primitive streak, owing to insufficient mesoderm being formed during gastrulation. The most strongly affected part of the mesoderm in Brachyury embryos is the notochord, a result consistent with the expression pattern of the gene. Brachyury transcripts are first observed in and near the primitive streak at 7 days of development. Expression is then downregulated in prospective somite and lateral plate mesoderm to undetectable levels, but it persists during and beyond gastrulation in the notochord (Wilkinson et al., 1990). The mechanism of action of Brachyury is not yet clear, but it has been suggested that the Brachyuryprotein acts intracellularly and has a general role in regulating the cellular changes associated with the formation of the mesoderm and the notochord (Herrmann et al., 1990; Wilkinson et al., 1990). The Bfachyury gene is highly conserved among vertebrates (Herrmann et al., 1990) suggesting that it plays an important role in mesoderm formation throughout the subphylum. In this paper we describe a Xenopus homolog of Brachyury (Xbra). The sequence of Xbra shows considerable homology with Brachyury. Transcripts first appear at the mid-blastula transition, but highest expression occurs, as in the mouse, during gastrulation. Like the mouse gene, Xbra is expressed in presumptive mesodermal cells around the blastopore lip. Transcript levels decline around the end of gastrulation, but persist longest in the notochord. Expression of Xbra can be controlled by MIFs: transcription is induced in animal pole ectoderm by bFGF and activin A. This may represent an immediate-early re-

Cdl 60

involved in mesoderm formation in all vertebrates, and the observation that Xenopus Brachyury is induced by bFGF and activin A suggests that regulation of mouse Brachyury may also occur in this way. Results Cloning and Sequencing of Xenopus Brachyury From previously published work we knew that the cDNA for the mouse Brachyury gene, me75, recognizes three BamHl restriction fragments in the genome of Xenopus laevis when analyzed at reduced stringency (Herrmann et al., 1990). We therefore utilized pme75 as a probe to screen a cDNA library prepared from RNA of developing neurulastageXenopusembryos. Alarge number of clones hybridized to the mouse probe. Several cDNAclones were purified and analyzed, and one, xt6, was sequenced completely (Figure 1). It contains an open reading frame coding for 432 amino acids (compared with the mouse protein of 436 amino acids). xt6 has no in-frame stop codon preceding the putative start codon for translation. Furthermore, there is significant similarity between me75 and xt6 in the region 3’ of the putative translation start codon at bases 138-140, while no similarity exists 5’ of the start codon. These data strongly suggest that the amino acid sequence indeed starts with the codon at bases 138-140. xt6 lacks a signal sequence for polyadenylation at its 3’ end. From the estimated size of 2.4 kb for the RNA of xt6 (Figure 3A), it appears that approximately 200 bases are missing from the 3’ end of the cDNA. Comparison of the amino acid sequences of the putative mouse and Xenopus Brachyury proteins allows two domains to be distinguished (Figure 2). The N-terminal half is highly conserved between both proteins, while the similarity decreases in the C-terminal region. The overall similarity is 750/o, not including conservative changes. In the N-terminal half the similarity exceeds 90%. Two functional domains have also been predicted previously for the mouse Brachyury gene from the analysis of the dominant negative mutation Twi’ (Herrmann et al., 1990). The high similarity between both proteins strongly suggests that xt6 encodes the Xenopus homolog of the mouse Brachyury gene, which we now refer to as Xbra. Further support comes from the data described below.

sponse to mesoderm induction; Xbra can be induced in dispersed cells and in the presence of a protein synthesis inhibitor (see Rosa, 1989). Our results provide further evidence that Brachyury is

Expression Pattern of Xenopus Erachyury We used the entire Xenopus Brachywy cDNA to probe Northern blots of RNA prepared from Xenopus embryos at different stages. A single transcript of 2.4 kb was observed, which was maximally expressed during gastrulation (Figure 3A). Maternal transcripts were not detected by this method of analysis, and expression of the gene was greatly reduced following neurula stages, although the stage at which Xbra transcripts became undetectable varied from egg batch to egg batch; prolonged exposure of some blots revealed that transcripts were stiRpresent at stage 37. The early expression of Xbra revealed by Northern blotting was further investigated by RNAase protection analysis using an antisense probe prepared from the 3’end of the cDNA (see Figure 1). Using

Mesoderm 81

Induction

Activates

Xenopus

Brachyury

5e 60 118 120 178

Figure 2. Comparison of the Predicted Acid Sequences of Xenopus and Brachyury The sequence of Xenopus mouse is on the bottom. of the sequence represent N-glycosylation sites.

Amino Mouse

is on the top; that of Underlined portions conserved potential

160 237 239 295 a97 355 356 356

409

357

414

410 415

NQYDVTAHSR III II I SQYD-TAOSL

432

LSSTdTP”AP PS” /I// I II I LIASWTPVSP PSM

436

this more sensitive technique, low maternal levels of Xbra mRNA were observed, while zygotic expression of the gene was coincident with that of EF-la, a gene that is strongly expressed at the mid-blastula transition in all embryonic cells (Figure 38; see Krieg et al., 1989). The spatial pattern of expression of Xbra was studied by in situ hybridization. At the early gastrula stage (Figures

4A-4D) transcripts were present in the marginal zone of the embryo, which contains cells destined to become mesoderm. In some sections silver grains were observed over cells at the surface of the embryo, which, in Xenopus, eventually form endoderm (Keller, 1975, 1976; Smith and Malacinski, 1983). It is not possible to say whether Xbra is expressed in all presumptive mesodermal cells at the early

B

A

HOUIS after fertilization

stage

stage Brachyuq

28s xbra (2.4 kb) 18s

EF-la

Figure

3. Temporal

Expression

Pattern

of Xenopus

Brachyury

(A) Northern blot using 30 wg of total RNA from Xenopus embryos at the indicated stages. Also included was 5 pg of poly(A)’ RNA from the XTC cell line (Pudney et al., 1973). Weak nonspecific hybridization to 18s and 28s RNA demonstrates equal loading of RNA, which was also confirmed by probing a parallel blot with aXenopus FGF receptor cDNA kindly provided by Dr. Mark Mercola (Department of Cellular and Molecular Physiology, Harvard Medical School). Levels of FGF receptor RNA remain approximately constant throughout early development (Musci et al., 1990). (6) RNAase protection showing onset of expression of Xbra at the mid-blastula transition. Xenopus embryos were cultured at 22°C. and groups of five embryos were frozen at the indicated times and developmental stages. RNA was extracted and analyzed using antisense probes for Xbra and EF-la. Xbra and EF-la transcripts visible from stages 2 to 9 are maternal. Zygotic expression of Xbra is first apparent at stage 9/10, and this coincides with a slight increase in EF-la levels, marking the mid-blastula transition.

Cell 82

Figure

4. In Situ

Hybridizations

Showing

the Spatial

Expression

of Xbra

(A) Vegetal view of an early gastrula (stage 10) Xenopus embryo (Nieuwkoop and Faber, 1967). (B) and (D) are vertical sections in a plane similar to the one indicated by the arrow (it is not possible to be more precise because it is difficult to orientate the albino embryos used to obtain [B-D]). (C) is a horizontal section in the plane of the drawing. (B) Vertical section of a Xenopus early gastrula hybridized with an antisense Xbra probe. Cells of the marginal zone, including some superficial cells, express high levels of Xbra. (C) Horizontal section of a Xenopus early gastrula hybridized with an antisense Xbra probe. Transcripts are present throughout the dorsoventral axis, but are lower in superficial cells. (D) Vertical section of a Xenopus early gastrula hybridized with a sense Xbra probe. Little nonspecific hybridization is visible. (E) Lateral view of an early neurula (stage 14) Xenopus embryo (Nieuwkoop and Faber, 1967). The vertical arrow indicates the plane of section in (F) and the horizontal arrow indicates the plane of section in (G) and (H). (F) Transverse section of an early neurula shows hybridization of an Xbra antisense probe to the notochord. (G) Horizontal section of an early neurula shows hybridization in the anterior (left) and posterior (right) notochord, as well as in involuting mesoderm (right). (H) Higher-power view of anterior notochord in (G). Scale bar in (F) is 100 urn and also applies to (B), (C), (D), and (G). Scale bar in (H) is 50 urn.

Mesoderm 83

Induction

Activates

Xenopus

Brachyury

Brachywy

EF-la

Figure 5. RNAase Protection Its induction in AnimaCVegetal

Showing Spatial Expression of Xbra and Combinations and by Activin and bFGF

Each lane consists of RNA derived from seven dissected regions after dissection at stage 8 and culture until the equivalent of stage 11 (except for WE, which consists of RNA derived from two intact stage 11 embryos). AP, animal pole regions; MZ, marginal zones; VP, vegetal poles; APNP, animal-vegetal pole combinations; AP/activin, animal pole regions treated with 20 U/ml activin A (see Cooke et al., 1987, for definition of a unit of mesoderm-inducing activity); APIFGF, animal pole regions treated with 80 nglml bFGF.

gastrula stage, but horizontal sections (Figure 4C) showed that transcripts are present throughout the dorso-ventral axis. At the mid-gastrula stage expression of Xbra still occurs in a ring around the yolk plug (not shown). With the exception of the notochord, mesodermal cells that have migrated anteriorly do not express the gene, and comparison with sections made at earlier stages (for example, Figure 4B) suggests that they have down-regulated transcription of Xbra. This expression pattern persists during neurula stages (Figures 4E-4H). In the trunk and anterior regions (Figures 4F-4H) only the notochord expresses Xbra, but in the region around the blastopore, where involution of mesodermal cells continues, Xbra transcripts are still visible. To confirm the in situ hybridization results, mid-blastula (stage 8) Xenopus embryos were dissected into animal, equatorial, and vegetal regions and cultured until the midgastrula stage (stage 11). Xbra expression was analyzed by RNAase protection (Figure 5, first three lanes). As expected, Xbra transcripts were not detected in animal or vegetal pole regions, but expression was high in equatorial explants. Xenopus Brachyury Is induced by FGF and Activin A The expression pattern of Xbra suggested that the gene might be activated by mesoderm induction. To investigate this, animal pole regions from mid-blastula (stage 8) embryos were cultured until the mid-gastrula stage (stage 11) either alone, in combination with vegetal pole regions, or in the presence of activin A or bFGF. In the absence of induction, Xbra was not expressed. However, expression was strongly induced by MlFs as well as by coculture with vegetal pole cells (Figure 5).

Brachyury Is an Immediate-Early Response to induction The observation that Xbra is first expressed at the midblastula transition (see Figure 38) suggested that it might, like Mix. 7 (Rosa, 1989) represent an immediate-early response to induction, that is, a rapid response that does not depend on protein synthesis and that can occur in dispersed cells. We investigated this question essentially as described by Rosa (1989). The first series of experiments investigated the time required for activation of Xbra. Animal pole regions were dissected from mid-blastula stage embryos and split into three groups. One of these groups was cultured in the absence of inducing factors, while the other two were exposed to activin A or bFGF. Samples of ten animal caps were removed from each group at different times and they were frozen immediately on dry ice for analysis of Xbra expression. Low levels of Xbra transcripts were detectable in uninduced animal caps immediately after dissection at the mid-blastula stage, although they declined thereafter and were not detectable 2.5 hr after dissection, at the equivalent of the early gastrula stage (Figure 6A). Some of these transcripts probably represent maternal Xbra mRNA, although it is also possible that zygotic transcription is activated in the animal pole region of the embryo as well as in the marginal zone, as has recently been reported by Rupp and Weintraub (1991) for other mesodermspecific genes such as MyoDa and MyoDb, and also for Mix. 7, which is expressed in presumptive mesoderm and endoderm (Rosa, 1989). The function, if any, of Xbra mRNA in the animal pole of the embryo is not yet clear, but it complicates analysis of the time required for activation of Xbra because it then becomes a quantitative, rather than a qualitative, question. However, Figure 6A shows clearly that levels of Xbra RNA are elevated within 65 min of treatment with bFGF and within 250 min of treatment with activin A. This difference between bFGF and activin was not consistent: in another experiment (not shown) both bFGF and activin increased Xbra levels within 90 min. This time is rapid compared with the 5-7 hr required for activation of the cardiac actin gene, for example (Gurdon et al., 1985) but is comparable with, albeit slightly longer than, that observed with Mix. 7 (Rosa, 1989). To study whether protein synthesis is required for activation of Xbra, animal pole regions were treated with inducing factors in the presence or absence of 5 pglml cycloheximide (CHI), as described in Experimental Procedures. In five independent experiments, CHI inhibited incorporation of [35S]methionine into acid-precipitable material during the course of the experiment by at least 80%, but did not prevent activation of Xbra (Figure 6B). Interestingly, low levels of Xbra transcripts were present in animal pole regions treated with CHI alone (not shown). This may reflect enhanced stability of maternal transcripts, loss of labile transcriptional repressors, or direct stimulation of second messenger pathways by CHI (Mahadevan and Edwards, 1991). It is important to discover whether Xbra can be activated in dispersed animal pole blastomeres, because this would imply that activation does not require intercellular signals.

Cell 84

Minutes

A 15

35

after

induction

65

105

250

g5

-----E

EF-la

Figure

6. Xbra

Expression

Is an Immediate-Early

Response

to Mesoderm

Induction

(A) Expression of Xenopus Brachyury is a rapid response to mesoderm induction; transcripts present in the animal cap at the mid-blastula stage are lost by the onset of gastrulation. Animal pole regions were frozen at the indicated times after treatment with 80 nglml bFGF, with 100 U/ml activin A, or with a control solution. Xbra transcripts were visible in uninduced animal caps from 15 to 105 min, but had declined by 250 min. Expression of Xbra was elevated in response to bFGF within 65 min, and in response to activin within 250 min. Whole embryo shows RNA extracted from two intact embryos at the 250 min time point. (B) Xbra can be activated to near normal levels in the presence of a protein synthesis inhibitor. Mid-blastula stage Xenopus animal caps were exposed to 20 U/ml activin A, 80 nglml bFGF, or a control solution after treatment with 5 pglml CHI as described in the Experimental Procedures. Additional animal caps were exposed to inducing factors without prior treatment with CHI. Animal caps were cultured at 23°C and analyzed for expression of Xbra after approximately 4 hr at the equivalent of stage 10.5. Although incorporation of [%]methionine into acid-precipitable material was reduced by 81% during the period of induction, and cell division was observed to be inhibited, expression of Xbra was induced to normal, or near normal, levels. Whole embryo shows RNA extracted from two intact embryos at stage 10.5. (C) Xbra can be activated in dispersed cells. Inner layer ceils from mid-blastula stage Xenopus animal pole regions were dispersed by culture in medium lacking calcium and magnesium. The cells were split into three groups, one of which was treated with 10 U/ml activin A, one with 40 ngl ml bFGF, and one with a control solution. Cells were cultured at 23% and kept dispersed until the equivalent of stage 10.5, when they were analyzed for expression of Xbra. Intact animal pole regions from the same egg batch were treated with the same concentrations of inducing factors as controls. Expression of Xbra is induced even if cells are dispersed during treatment with inducing factors.

Dispersed animal pole cells were exposed to activin or bFGF in three independent experiments. In each, Xbra was strongly activated both by activin and bFGF (Figure 6C). The level of activation differed from experiment to experiment and may depend on the exact conditions of treatment; preliminary experiments (J. B. A. G. and J. C. S., unpublished data) show that, like muscle-specific actin (Green and Smith, 1990) the level of Xbra activation declines at high concentrations of XTC-MIF (Xenopus activin A). Discussion Genetic evidence indicates that the product of the Brachyurygene is required for mesoderm formation in the mouse (reviewed by Willison, 1990); in embryos homozygous for mutant alleles of Bfachyufy, gastrulation movements are disturbed and the number of mesodermal cells is reduced, while the number of ectodermal cells is increased (Yanagisawa et al., 1981). Such embryos die at about 10 days of gestation, owing to poor development of the allantois

(Gluecksohn-Schoenheimer, 1944). The notochord does not form in homozygous mutant Brachyury embryos (Chesley, 1935) and this is consistent with the expression pattern of Brachyury in the mouse. Transcripts are first detected in early gastrula stage embryos in mesoderm and primitive ectoderm in and near the primitive streak. At later stages expression around the primitive streak continues, but anteriorly it declines in paraxial (future somitic) and lateral mesoderm and persists only in the notochord (Wilkinson et al., 1990). gene is There are few clues as to how the Brachyury activated and to how it exerts its effects. It is known that protein lacks a signal peptide sequence, so the Brachyury it is likely that it acts within the cell (Herrmann et al., 1990; Wilkinson et al., 1990) and recently it has been shown that its function is cell autonomous (Rashbass et al., 1991). is inYanagisawa (1990) has suggested that Bfachyury volved in determining the anteroposterior axis of the mouse mesoderm, with higher levels of gene product required for more posterior development. To investigate these questions further, we have turned to amphibian em-

Mesoderm 85

Induction

Acttvates

Xenopus

Brachyuv

bryos, where mesoderm is induced from equatorial ceils of the blastula under the influence of growth factor-like signals from the vegetal hemisphere (for reviews see Smith, 1989; New and Smith, 1990). We find that the putative protein encoded by Xenopus Brachyury (Xbra) is remarkably similar to its mouse homolog; in particular, the N-terminal half of the protein has changed little during evolution. Furthermore, in a manner exactly analogous to mouse Brachyury, Xbra is expressed during gastrula and neurula stages in involuting mesoderm and in the notochord. These observations suggest that Brachyury plays an important role in mesoderm formation throughout the vertebrates, and they raise the possibility that mesoderm formation occurs through similar mechanisms in all members of the subphylum. Expression of Xbra can be induced by vegetal pole cells of the Xenopus blastula as well as by MlFs bFGF and activin A. This induction is rapid, and will occur in dispersed cells in the presence of CHI, suggesting that Brachyury is an immediate-early gene that is a direct target of mesoderm induction. We have not yet performed a detailed dose-response analysis of the response of Xbra to bFGF and activin, but it is significant that both factors are efficient inducers of the gene (Figure 5). In this respect Xbra resembles Xenopus Snail (Sargent and Bennett, 1990), but differs from Xhox3, which is more strongly induced by bFGF (Ruiz i Altaba and Melton, 1989), and from Mix. 7, which is only induced by activin (Rosa, 1989). It is possible that bFGF is responsible for activation of Xbra in ventral marginal zone tissue, and activin for expression in the dorsal marginal zone, which eventually forms notochord (Keller, 1976); this is currently under investigation. Zygotic expression of Brachyury first occurs at the midblastula transition, although there are also low maternal levels of the mRNA. These maternal transcripts may account for the presence of Brachyury mRNA in animal caps isolated at the mid-blastula stage, although it is also possible that the zygotic activation of Brachyuty occurs in all regions of the embryo before becoming restricted to the prospective mesoderm. Rupp and Weintraub (1991) have observed this phenomenon for the muscle-specific genes MyoDa and MyoDb and also for the inducible gene Mix. 1, which is expressed in presumptive mesoderm and endoderm (Rosa, 1989). The significance of this observation is unclear. One suggestion for MyoD (Rupp and Weintraub, 1991) is that induction is selective rather than direct. After an initial general activation of MyoD, the induction process might lead to an accumulation of MyoD protein above a threshold level only in the marginal zone. This would then lead to a positive feedback loop and the stabilization of MyoD expression (see Thayer et al., 1989). If the same were true of Xbra, we would predict that this gene, like MyoD, shows positive autoregulation; this is now being tested. The discovery that Xenopus Brachyury shows an analogous expression pattern to its mouse homolog, and that it is activated in response to MIFs, makes several important experiments possible. For example, genetic studies indicate that Brachyury is required for mesoderm formation in the mouse, and we intend to interfere with Brachyury

expression in Xenopus to discover whether the same is true in amphibia. In addition, we intend to study the effects of overexpression of Brachyury in Xenopus to discover whether ectopic mesoderm formation occurs. It will be important to search for activin and bFGF response elements in Xenopus Brachyury and then to investigate whether these are conserved in the mouse gene. Finally, it will be interesting to compare the expression patterns of members of the FGF and TGFP families in the early mouse embryo with that of Brachyury in order to identify candidates for mammalian MlFs (see, for example, Haub and Goldfarb, 1991; H6bert et al., 1991; Slager et al., 1991). Experimental

Procedures

Library Screening Atotalof600,OOOpfu of aXenopus neurulaLgtlOcDNAlibrary(Kintner and Melton, 1987) was plated onto four 22 x 22 cm agar plates. Plaques were adsorbed to GeneScreen plus membrane, and duplicate filters were hybridized with a random hexamer-primed (Feinberg and Vogelstein, 1984) probe of the mouse Brachyury gene cDNA me75 (Herrmann et al., 1990). Filterswere hybridized in 7% SDS/O.5 M NaP, (pH 6.8) (Church and Gilbert, 1984) at 60°C and washed in 1% SDS, 0.04 M NaP, (pH 6.8) at 6OOC. Purified phage DNA was subcloned into a plasmid vector and sequenced from double-stranded templates using a T7 polymerase sequencing kit (Pharmacia) according to the supplier’s instructions. Overlapping templates were generated with the Erase-a-Base system (Promega) according to manufacturers’ instructions. Gaps in the sequence were filled in by sequencing from oligonucleotides synthesized on a gene assembler apparatus (Pharmacia). Embryos and Dissections Xenopus embryos were obtained by artificial fertilization as described by Smith and Slack (1983). They were dejellied with cysteine hydrochloride (pH 8.1) and staged according to Nieuwkoop and Faber (1967). Animal pole regions, and, where appropriate, equatorial and vegetal pole regions, were dissected from stage 8 embryo; and cultured in 75% normal amphibian medium (NAM; Slack, 1984) in petri dishes coated with 1% agarose. For experiments involving culture of explants in MIFs, the medium contained 0.1% bovine serum albumin. MlFs XTC-MIF (Xenopus activin A) was purified as described by Smith et al. (1990). Some experiments used recombinant bovine activin A (the kind gift of Drs. P. de Waele and D. Huylebroeck [Innogenetics, Ghent, Belgium; see van den Eijnden-Van Raaij et al., 19901) or recombinant human activin A (the kind gift of Dr. G. Wong, Genetics Institute, Massachusetts). Pure recombinant Xenopus bFGF was kindly provided by Dr. J. M. W. Slack (ICRF, Oxford), who produced it using a plasmid supplied by Drs. D. Kimelman and M. W. Kirschner (University of California, San Francisco; see Kimelman et al., 1988). RNA Isolation, Northern Blots, and RNAase Protections Embryosortissueexplantswerefrozenondryiceinaminimumvolume of 75% NAM and stored at -80°C. Samples were homogenized in 50 mM NaCI, 50 mM Tris-Cl (pH 7.5), 5 mM EDTA, 0.5% SDS, 200 pg/ ml proteinase K, and RNA was extracted as described by Sambrook et al. (1989). Northern blots were prepared by electrophoresing denatured RNA samples on 1% agarose-formaldehyde gels, and transferring them to nitrocellulose filters. The filters were baked at 80°C for 2 hr and probed with the entire Brachyury EcoRl fragment (Figure l), which was labeled with =P by random hexamer priming (Feinberg and Vogelstein, 1984). For RNAase protections, an antisense probe was prepared by cloning Xbra into the EcoRl site of pSP73, cutting the plasmid (pXbra) with Sspl (see Figure I), and transcribing with T7 RNA polymerase. This gives a probe size of 293 and a protected fragment of 214 nucleotides. As a loading control we used an antisense probe for EF-1 a, which is expressed in all embryonic cells (Krieg et al., 1989; see Sargent and Bennett, 1990). Probe production and RNAase pro-

Cell 86

tections (1990).

were

carried

out

essentially

as described

by Green

et al.

In Situ Hybridizations In situ hybridizations were carried out exactly as described by Wilkinson and Green (1990). %S-labeled probes in both the sense and the antisense orientations were derived by SP6 and T7 transcription, respectively, of pXbra, and they were hydrolyzed to an average length of approximately 150 nucleotides. Protein Synthesis Inhibition and Cell Dispersal Experiments To study whether protein synthesis is required for activation of Xbra, animal pole regions were dissected from mid-blastula (stage 8) Xenopus embryos and collected in 75% NAM, in which the divalent cation concentrations had been reduced to 0.1 mM. This prevents animal caps from “rounding up” and becoming refractory to induction (Cooke et al., 1987). Half the caps were then exposed to 5 pglml CHI in the same medium for 30 min and these were then further subdivided into three groups: one group was cultured in 75% NAM containing fresh CHI without inducing factors, and the other two were exposed to bFGF or activin A, also in the presence of CHI. After another 30 min the explants were transferred to the same solutions but in the absence of CHI. They were then cultured until control embryos reached the early gastrula stage (stage 10.5) when they were frozen and analyzed for expression of Xbra. Control explants were treated in the same way but without CHI. This CHI treatment regime was based on detailed experiments by Cascio and Gurdon (1987), and has also been used by Rosa (1989). Like these authors, we found that it reduced incorporation of [%]methionine into acid-precipitable material by 80%-90% during the courseof the experiment. The residual CHI-resistant protein synthesis has been suggested by Cascio and Gurdon (1987) to be mitochondrial. To discover whether Xbra can be activated in dispersed cells, midblastula (stage 8) animal caps were dissected directly into calciumand magnesium-free medium (Sargent et al., 1986). The inner layer of cells was dispersed and divided into three groups. One was exposed to bFGF, another to activin A, and another received no addition. The cells were kept dispersed by gently blowing them apart every 30 min with a Pasteur pipette, and they were frozen for analysis when control embryos reached stage 10.5. In the same experiments, intact animal caps were treated with bFGF or activin A as a positive control. Acknowledgments This work was supported in part by the Medical Research Council. We are grateful to Drs. P. de Waele and D. Huylebroeck, and to Dr. G. Wong for recombinant activin A. E3. G. H. thanks U. Schwartz for support and A. Kispert for determining part of the Xbra sequence. J. C. S. thanks Dr. Tony Mescher (Indiana University) for help with in situ hybridizations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

May 28, 1991;

revised

August

2. 1991

Albano, Ft. M., Godsave, S. F., Huylebroeck, D., van Nimmen, K., Isaacs, H. V., Slack, J. M. W., and Smith, J. C. (1990). A mesoderminducing factor produced by WEHImurine myelocytic leukaemia cells is activin A. Development 770, 435-443. Asashima, M., Nakano, H., Shimada, K., Kinoshita, K., Ishii, K., Shibai, H., and Ueno, N. (1990). Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). Roux’s Arch. Dev. Biol. 798, 330-335. S., and Gurdon, J. B. (1987). The initiation of new gene tranduring Xenopus gastrulation requires immediately preceding synthesis. Development 100, 297-305.

Chesley, P. (1935). Development of the short-tailed house mouse. J. Exp. 2001. 70, 429-459.

Cooke, ganiser 229-241.

J. (1989). phenomenon

Mesoderm-inducing in amphibian

Genomic

factors development.

sequencing.

Proc.

and Spemann’s Development

or707,

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Brachyurygene

and

mesoderm

forma-

Accession

The accession M77243.

number

H. (1981). movement

Effects in the

Number for the sequence

reported

in this

paper

is