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573 Development 115, 573-585 (1992) Printed in Great Britain © The Company of Biologists Limited 1992 Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development L. DALE1, G. HOWES2, B. M. J. PRICE2 and J. C. SMITH2 1 School of Biochemistry, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK ^Laboratory of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Summary The mesoderm of amphibian embryos such as Xenopus laevis arises through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial and animal pole cells. Three classes of 'mesoderm-inducing factor' (MIF) that might be responsible for this interaction in vivo have been discovered. These are members of the transforming growth factor type /J (TGF-/?), fibroblast growth factor (FGF) and Wnt families. Among the most potent MIFs are the activins, members of the TGF-/J family, but RNA for activin A and B is not detectable in the Xenopus embryo until neurula and late blastula stages, respectively, and this is probably too late for the molecules to act as natural inducers. In this paper, we use the polymerase chain reaction to clone additional members of the TGF-/3 family that might possess mesoderminducing activity. We show that transcripts encoding Xenopus bone morphogenetic protein 4 (XBMP-4) are detectable in the unfertilized egg, and that injection of XBMP-4 RNA into the animal hemisphere of Xenopus eggs causes animal caps isolated from the resulting blastulae to express mesoderm-specific markers. Surprisingly, however, XBMP-4 preferentially induces ventral mesoderm, whereas the closely related activin induces axial tissues. Furthermore, the action of XBMP4 is 'dominant' over that of activin. In this respect, XBMP-4 differs from basic FGF, another ventral inducer, where simultaneous treatment with FGF and activin results in activin-like responses. The dominance of XBMP-4 over activin may account for the ability of injected XBMP-4 RNA to 'ventralize' whole Xenopus embryos. It is interesting, however, that blastopore formation in such embryos can occur perfectly normally. This contrasts with embryos ventralized by UVirradiation and suggests that XBMP-4-induced ventralization occurs after the onset of gastrulation. Introduction Smith, 1990; and see Smith and Harland, 1991; Sokol et al., 1991). The first group consists of members of the fibroblast growth factor family, such as basic fibroblast growth factor (bFGF). These induce ventral mesoderm when added to isolated animal caps. A second group comprises members of the transforming growth factor-)? (TGF-/3) superfamily, of which the most potent are activins A and B (Asashima et al.. 1990; Smith et al., 1990; Sokol et al., 1990; Thomsen et al., 1990; van den Eijnden Van Raaij et al., 1990). These latter proteins induce dorsal mesoderm when added to isolated animal caps. The final group includes members of the Wnt family of proteins. These are not available in soluble form, but when synthetic RNA encoding one of the members of the family is injected into the ventralvegetal quadrant of an early Xenopus embryo, it induces a second dorsal axis (McMahon and Moon, 1989; Christian et al., 1991; Smith and Harland, 1991; Sokol et al., 1991). At present, it is not known which members of the FGF, TGF-/J and Wnt families, if any, are the natural The mesoderm in amphibian embryos such as Xenopus laevis arises through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial and animal pole cells (Nieuwkoop, 1969; reviewed by Smith 1989). Embryological experiments suggest that only two types of mesoderm are initially induced: the dorsalmost quadrant of the vegetal hemisphere induces dorsal 'organizer' mesoderm, while the remaining ventral and lateral quadrants induce ventral mesoderm (Dale et al., 1985; Dale and Slack, 1987). The dorsal organizer then acts upon the ventral mesoderm to generate the typical vertebrate mesodermal pattern, a process known as dorsalization (Smith and Slack, 1983; Dale and Slack, 1987; Stewart and Gerhart, 1990). Recently, progress has been made in identifying 'mesoderm-inducing factors' (MIFs), and the candidates fall into three groups (reviewed by Whitman and Melton 1989; Dawid and Sargent, 1990; New and Key words: mesoderm induction, Xenopus, bone morphogenetic protein-4, activin, FGF. 574 L. Dale and others endogenous inducing factors, although the evidence is good that an FGF-like molecule does play a role in mesoderm formation. bFGF mRNA and protein are present in the Xenopus egg and early embryo (Kimelman and Kirschner 1987; Kimelman et al., 1988; Slack and Isaacs 1989), and recent work by Shiurba et al. (1991) has shown that both basic and acidic FGF are concentrated in the vegetal hemisphere and marginal zone of the blastula. In addition, injection of RNA encoding dominant negative mutations of the FGF receptor into fertilized Xenopus eggs causes posterior defects in the resulting tadpoles (Amaya et al., 1991), a result consistent with the proposed role of FGF as an inducer of posterior mesoderm (Green et al., 1990; Ruiz i Altaba and Melton, 1989; Cho and De Robertis, 1990). In this paper we concentrate on members of the TGF-/S family. One strong candidate for a TGF-/S-like natural inducing factor is the protein product of Vgl, a vegetally localised maternal mRNA whose sequence is most closely related to the bone morphogenetic proteins (Rebagliati et al., 1985; Weeks and Melton 1987). However, there is no evidence that Vgl protein has mesoderm-inducing activity, and indeed very little, if any, of the 40 000 relative molecular mass (Mr) immature protein is processed into the mature 17 000 MT form (Dale et al., 1989; Tannahill and Melton 1989). Another candidate is activin B, transcripts for which are first detected at the mid-blastula transition in Xenopus, when zygotic transcription begins (Thomsen et al., 1990). However, work by Jones and Woodland (1987) suggests that mesoderm induction in vivo begins at the 64-cell stage, at least 3 hours before the mid-blastula transition, indicating that the natural mesoderm-inducing factor(s) should be maternally encoded. Consistent with this, Asashima et al. (1991) have recently shown that the Xenopus egg and early embryo contains activin-like inducing activity. Two approaches might be adopted to identify and characterize the endogenous activin-like mesoderminducing factors. The first involves purifying and sequencing the proteins, and work of this kind is under way in one of our laboratories (G.-D. Guex and J.C.S., unpublished data). The alternative is to clone members of the TGF-)3 family that are expressed during early development in Xenopus, to study their expression patterns, and then discover whether they have inducing activity. In this paper, which is an example of the second approach, we describe the cloning and expression pattern of Xenopus bone morphogenetic protein-4 (XBMP-4), a member of the TGF-0 family, and one which is particularly closely related to Drosophila decapentaplegic (dpp), Xenopus Vgl, mouse Vgr-1, and BMPs -2 and -3 (Padgett et al., 1987; Weeks and Melton, 1987; Wozney et al., 1988; Lyons et al., 1989). We also investigate the inducing activity of XBMP-4 by injecting mRNA into the animal hemisphere of Xenopus eggs and dissecting animal caps from the resulting blastulae. Using this technique, we find that XBMR-4 preferentially induces ventral mesoderm, unlike the closely related activin which induces axial tissues. This result confirms those of Koster et al. (1991), who treated animal caps with recombinant Xenopus BMP-4. We also find, however, that the action of XBMP-4 is 'dominant' over that of activin. In this respect, XBMP-4 differs from basic FGF, another ventral inducer, where simultaneous treatment with FGF and activin results in activin-like responses (Cooke, 1989). The dominance of XBMP-4 over activin may account for the ability of injected XBMP-4 RNA completely to 'ventralize' intact Xenopus embryos. It is interesting, however, that blastopore formation in such embryos can occur perfectly normally. This contrasts with embryos ventralized by UV irradiation and suggests that XBMP-4-induced ventralization occurs after the onset of gastrulation. Materials and methods Polymerase chain reaction To identify additional members of the TGF-/^activin family that have mesoderm-inducing activity, we designed degenerate primers based on the known sequences of mammalian activins A and B for use in the polymerase chain reaction (PCR). The sequences of the primers are shown below: A 5' TGGAACGACTGGATGATGGC 3' T T T T B 5' GCACCCACACTCCTCCACAATCAT G G 3' Thefirstprimer corresponds to the N-tenninal region of the mature activin A protein, and the second to the C-terminal region (Fig. 1C). DNA to be amplified was Xenopus genomic DNA (1 fA of 1 mg ml" 1 DNA) or cDNA made from poly(A)+ RNA derived from the Xenopus XTC cell line (5 /d of reaction product from a reverse transcription reaction using 3 ng poly(A)+ RNA in a volume of 30 fA). For each PCR reaction, we mixed DNA with 0.5 [A of 5 U ml" 1 Taq DNA polymerase, 10 /il of 5xTaq polymerase buffer (0.25 M TrisHC1 pH 8.3, 0.375 M KC1,15 mM MgCl2, 50 mM DTT, 0.625 mM dNTPs) and 1 fA of each primer at 1 mg ml"1. The final volume was adjusted to 50 u\ with water. Thirty cycles of amplification were performed, each consisting of denaturation at 94°C for 1 minute, annealing at 52°C for 1.5 minutes and elongation at 72°C for 1.5 minutes. The final elongation step was extended to 10 minutes. PCR products were approximately 260 nucleotides. They were blunt-end cloned into the vector pSP72 and sequenced. The 86 amino acids encoded by one cloned fragment differed by only one amino acid from human bone morphogenetic protein 4 (BMP-4), and this was used for screening an XTC cDNA library (see below). cDNA library screening A Xenopus XTC cell cDNA library prepared in AZAP (the kind gift of Dr I. B. Dawid, N1H, Bethesda) was plated on E. coli BB4 cells. 8 x 105 plaques were screened with the cloned PCR fragment. Three positive clones were isolated and sequenced, of which the longest was about 1.3 kb. This clone, pA, contained all but the first 50 amino acids of the coding region of Xenopus BMP-4 (XBMP-4). To obtain a full-length cDNA, we used this clone to screen a stage 15/16 Xenopus cDNA library prepared in AgtlO. 106 plaques were screened, and those with the longest inserts were selected as described BMP-4 and mesoderm induction by Elliott and Green (1989). Two cDNAs were obtained, one of which was 1.9 kb (XBMP-4.1) and the other 2.3 kb (XBMP-4.4). Both these cDNAs were subcloned into pBluescript KS+ and XBMP-4.1 was also cloned into pSP64T (Krieg and Melton, 1984) in both the sense and antisense orientations to yield, respectively, pSP64T-XBMP-4+ and pSP64T-XBMP-4-. Sequencing XBMP-4.1 was sequenced using a Pharmacia T7 sequencing kit. RNAase protections RNAase protections were carried out essentially as described by Green et al. (1990). An XBMP-4 antisense probe was prepared by digesting pA with Hpal, which was then transcribed with T7 RNA polymerase to give a probe of approximately 360 bases and a protected fragment of 300 bases, consisting of 200 bases of 3' untranslated region and 100 bases of the C terminus of XBMP-4 (see Fig. 1). Probes for Xhox3 (Ruiz i Altaba and Melton, 1989) and musclespecific actin (Mohun et al., 1984) were also used. As a loading control in RNAase protections, we used an antisense probe for EF-la, which is expressed in all embryonic cells in the absence of induction (Krieg et al., 1989; see Sargent and Bennett, 1990). Northern blots RNA was extracted from Xenopus embryos and analysed by northern blotting as described by Sambrook et al. (1989). For XBMP-4 the probe was a Sad/HinCH fragment containing most of the non-mature and about half of the mature region of the protein (see Fig. 1C). To detect actin, RNA blots were probed with an antisense RNA probe spanning the 3' UTR and the last protein coding exon of muscle-specific o^actin (Mohun et al., 1984). To detect globin transcripts, an antisense RNA probe spanning the 5' HaeUl fragment of the Xenopus tadpole /?Tl-globin gene was used (Banville et al., 1983). Embryos and inducing factors Embryos of Xenopus laevis were obtained by artificial fertilization as described by Smith and Slack (1983). They were chemically dejellied using 2% cysteine hydrochloride (pH 7.8-8.1), washed and transferred to Petri dishes containing 10% normal amphibian medium (NAM: Slack, 1984). The embryos were staged according to Nieuwkoop and Faber (1967). Embryos were dissected using sharpened forceps and electrolytically sharpened tungsten needles. Treatment of embryos with LiCl was as described by Cooke and Smith (1988). Recombinant human activin A was the generous gift of Dr G. Wong (Genetics Institute, Massachusetts). Pure recombinant Xenopus bFGF was prepared from an expression plasmid kindly provided by Drs D. Kimelman and M.W. Kirschner (University of California, San Francisco; see Kimelman et al., 1988). RNA injections Capped synthetic XBMP-4 mRNA was synthesized from pSP64T-XBMP-4+ and pSP64T-XBMP-4- to give both the sense and antisense transcripts. Polyacrylamide gel electrophoresis of the products of in vitro translation of sense RNA resulted in a band of the expected size (Afr 43 xlO3), while no band was visible with antisense RNA. Different concentrations of RNA were injected into Xenopus embryos 575 between the one-cell and four-cell stages. Some embryos were then allowed to develop until stage 38-40, while the animal pole regions of others were dissected and allowed to develop in the presence or the absence of inducing factors, as appropriate. Histology Embryos and explants were fixed and processed for histology as described by Green et al. (1990). 7 pm sections were stained by the Feulgen/Light Green/Orange G technique of Cooke (1979). Results Isolation and characterization of Xenopus BMP-4 cDNAs In an attempt to discover potential mesoderm-inducing factors, we used the polymerase chain reaction to search for activin-related molecules expressed during early Xenopus development. Our primers were based on the N- and C-terminal regions of mammalian activins, which also resemble members of the BMP family. PCR of both Xenopus genomic DNA and cDNA from the XTC cell line gave fragments which showed high homology to mammalian BMP-4, and we went on to screen two libraries in order to obtain fulllength cDNAs for Xenopus BMP-4 (see Materials and Methods). XBMP-4.1 was completely sequenced, and was found to be homologous, over most of its length, to the Xenopus BMP-4n of Koster et al. (1991). The sequences diverged, however, 5' of nucleotide 295 of our sequence (see Fig. 1A, showing the first 360 nucleotides of XBMP-4.1). This 5' sequence includes an in-frame ATG at nucleotide 267 (italics), which would add 12 amino acids to the proXBMP-4 of Koster et al. (1991) which begins at our nucleotide 303. We suggest, however, that XBMP-4.1 is derived from an unprocessed RNA. Firstly, there is a 3' splice-site consensus sequence 8 nucleotides upstream from nucleotide 303 (bold). Secondly, sequence analysis of XBMP-4.4, which is longer than XBMP-4.1, shows that the extreme 5' region of this cDNA is homologous to human BMP-4 but that it then diverges in the same way as XBMP-4.1 (data not shown). It is possible that XBMP-4.4 contains the entire intron; our method for selecting the largest inserts (see Materials and Methods) may inadvertently have led us to isolate cDNAs representing unprocessed transcripts. The amino acid sequence of XBMP-4 (Fig. IB) shows 80% similarity with the human protein (66 mismatches out of 398), with the highest homology in the C-terminal TGF-£-like domain (2 mismatches out of 102). Both XBMP-4 and its human homologue possess an Nterminal signal sequence, characteristic of secretory proteins, and all four potential asparagine-linked glycosylation sites are conserved, with an additional site in Xenopus at Asn 237. Consistent with the expected secretion of this protein, when synthetic XBMP-4 RNA is translated in a message-dependent Xenopus egg cellfree system (Matthews and Colman, 1991), the resulting 576 61 121 181 241 L. Dale and others EcoRI AaajlT TGCCGI^GACGCTCTCACTCAGATTAGCAACACGATGGCCCTGACTATCTOATWITCAGC TCTCCTTGTCTATTATTrorrTrCATCCCGCTCTATCACTT(lATTTTTCTCAGTCTAATC CCCCCACATCCTCCCTATTTATTTGCA TCTATTTTCTTTCTTCTGCACCCTCCAOAGACA TCATCATTCCTGffTAACCGAATCHrrCATCCTCATTrTATTATCCCAACTCCTCCTCOCAC M I P O N R H L M V I L L C Q V L L C C B i KIPCNRHLMV ILLCQVLLGG TNHASLIPDT CKKKVAADIQ GGRRSAQCHE 51 LLRDFEVTLL QMFGU3KRPQ PSKEWWPAY MRDLYRLQSA EEEDELHDIS 101 KEYPERPTSR AHTVRSFHHE EHLENLPSTA ENGNFRFVFH_L2S IPENEVI 151 GSAELRLYRE QIDHCPAWDE CFHRIN1YEV MKPIAANGLM INRLLDTRLI 201 HHimQWESF DVSPAIMRWT RDKQIHHGLA IEVIHLU2IK THQGKHVRIS 251 RSLLPQEDAD WSQMHPLLIT FBHDGRCHAL TRRSKRBPKQ QRPRKKNKHC 301 RRHSLYVDFS DVCWNDHIVA PPCYQAFYCH CDCPFPLADH LMSUOtAIVQ 351 TLVNSVH4SI PKACCVPTEL SAISHLYLDE YDKWLKHYQ EHWECCGCK SacI I HinCII/Hpal Splice site? Fig. 1. (A) The first 360 nucleotides of XBMP4.1. The first ATG in the open reading frame is in italics and the potential splice site mentioned in the text is in bold. (B) The amino acid sequence of Xenopus BMP-4. Potential Nlinked glycosylation sites are underlined. Asterisk marks the suggested cleavage site of the mature protein. (C) Map of XBMP-4.1. The protein-coding region is boxed, with the mature region of the protein shown in heavier stipple. The potential splice site is indicated. The northern blot probe was the indicated Sacl/HinCll fragment and an RNAase protection probe was made by transcribing in the antisense direction to the Hpal site. The PCR primers A and B are also shown. protein is both segregated within the endoplasmic reticulum and glycosylated (data not shown). Active human BMP-4 has been isolated and shown to comprise the C-terminal 116 amino acids (Hammonds et al., 1991), with cleavage from proBMP-4 occurring after the amino acid sequence RAKR. Since a similar sequence is found in our clone at amino acids 284-287 (RSKR), we expect mature XBMP-4 to comprise a dimer of the C-terminal 114 amino acids. Fig. 1C shows a map of XBMP-4 indicating the probes used for RNAase protection analyses and northern blots. Expression of Xenopus BMP-4 RNAase protection analysis (Fig. 2A) showed that zygotic expression of XBMP-4 begins at the midblastula transition and persists at least until stage 34. As expected from PCR analysis and screening of the XTC cDNA library, transcripts are also present in XTC cells. Further analyses showed that low levels of maternal XBMP-4 mRNA are also detectable (Fig. 2B). This expression pattern differs slightly from that of BMP-4r (Koster et al., 1991), where there is only a slight increase in RNA levels after the mid-blastula transition. XBMP-4 transcripts were studied by northern blot analysis of RNA samples from fertilized eggs and late gastrula-staged embryos (Fig. 2C). As expected from the RNAase protection analysis described above, we obtained a much stronger signal with late-gastrula RNA than with unfertilized egg RNA. Two transcripts were detected, one with an apparent size of 1.9 kb, and one of about 3.1 kb. This larger RNA may be the result of alternative splicing or may represent a close homologue of the 1.9 kb transcript. Such a homologue has been identified by Koster et al. (1991) and, from the sequence of this gene, we might anticipate hybridisation with our probe. However, K6ster et al. (1991) have provided evidence that the RNA for their clone is similarly represented in both the maternal and zygotic pool, while the larger transcript in Fig. 2C is clearly more abundant in the zygotic pool than in the maternal pool. The spatial expression of XBMP-4 at the early gastrula stage was studied by dissection of embryos into five regions (animal pole, vegetal pole, dorsal marginal zone, lateral marginal zone and ventral marginal zone) followed by RNAase protection analysis. Although in one experiment levels were lower in the animal pole, and in another in the lateral marginal zone, overall our results suggest that expression occurs throughout the early gastrula (Fig. 3). Injection of XBMP-4 'ventralizes' Xenopus embryos To investigate the function of XBMP-4 during Xenopus development, we injected 1.5 - 2.0 ng XBMP-4 RNA into the animal hemispheres of fertilized eggs. AH embryos injected in this way developed normally up to the early gastrula stage and formed a dorsal lip, but the development of some of those receiving the higher concentrations became slower at this stage, and the blastopore did not close (Fig. 4A, B). Sections of embryos (Fig. 4C, D) in which the progress of gastrulation was delayed resembled those of embryos in which ectopic mesoderm had formed in the animal cap, in response to injection of Brachyury RNA (V. T. Cunliffe and J.C.S., unpublished data), or to intrablastocoelic injection of inducing factors (Cooke et al., 1987; Cooke and Smith, 1989). Even when this did not happen, however, by stage 40 virtually all injected embryos appeared extremely abnormal (Fig. 4E, F). Superficially they resembled the 'grade 0' embryos caused by UV irradiation of the vegetal hemisphere of fertilized eggs, and we were able to use the 'Dorsoanterior Index' (DAI) of Kao and Elinson (1988) to reveal a graded response resulting from injections of different amounts of RNA (Table 1). In this index, '5' represents a normal embryo, '0' a completely ventralized case and '10' a completely dorsalized individual, and our results showed that a 100-fold dilution of injected RNA (1500 pg to 15 pg) increased the mean DAI of the resulting embryos from 0.39 to 3.32. Control injections of antisense XBMP-4 RNA (see Fig. 4 and Table 1), and RNA encoding Vgl (Dale et al., BMP-4 and mesoderm induction 2 N 577 3 13 is DC ^H ^^ "1 O H JO J J « QO &\ ^^ ^^ ^H f> 1^ ^rf s S £j ^N UU f XBMP-4 I 28S 18S EF-la B 00 fif XBMP-4 (W^ EF-la 00 «'•*•" « • • 'Mint Fig. 2. (A) RNAase protection analysis showing expression of XBMP-4 from the late blastula stage (stage 9) until the tailbud stage (stage 34). XTC cells also express XBMP-4. (B) RNAase protection analysis showing low maternal levels of XBMP-4 with zygotic expression beginning around the mid-blastula transition. (C) Northern blot analysis of 10 fig total RNA from unfertilized Xenopus eggs and stage 12 late gastrulae. Two transcripts (3.1 and 1.9 kb) are visible at the late gastrula stage. In the unfertilized egg sample only the smaller transcript is visible. 1989) and prolactin (Leaf et al., 1990) did not perturb development; in each case the resulting embryos had a mean DAI of greater than 4.75 (data not shown). More direct evidence that injection of XBMP-4 causes ventralization comes from histological examination of 'grade 0' injected embryos. In these cases neither notochord nor muscle could be identified; rather, they appear to produce an excess of ventral mesoderm including large numbers of cells resembling red blood cells (Fig. 4G, H). Lack of notochord and muscle was confirmed by whole-mount immunocytochemistry with the monoclonal antibodies MZ15 (Smith and Watt, 1985) and 12/101 (Kintner and Brockes, 1984) (data not shown), and a large reduction in muscle was also indicated by RNAase protection analysis of injected embryos at stage 20 (data not shown) as well as northern blot analysis performed at stage 40 (Fig. 8). To determine whether XBMP-4 RNA injection causes production of additional ventral mesoderm, as well as loss of dorsal tissue, we examined levels of Xhox3 (Ruiz i Altaba and Melton, 1989) and globin (Banville et al., 1983) mRNA in injected embryos. Expression of both these genes is restricted to posterior and/or ventral mesoderm in normal embryos and the expression of 578 L. Dale and others Table 2. Injection of 500 pg XBMP-4 RNA into dorsal blastomeres causes more extreme ventralization than injection into ventral blastomeres DAI XBMP-4 Injection position 0 1 2 3 4 5 Mean Dorsal Ventral 19 3 9 5 3 7 0 7 2 7 0 1 0.70 2.43 Numbers in Table indicate number of cases at each DAI. In the same experiment injection of 1500 pg sense XBMP-4 into 13 embryos gave a DAI of 0.00, while injection of 500 pg antisense RNA into dorsal and ventral blastomeres gave DAIs of 5.0. Prolactin and Vgl RNAs were similarly without effect. EF-la was greater than 4.75 irrespective of the site of injection. Fig. 3. Regional expression of XBMP-4. Xenopus early gastrulae were dissected into animal pole (AP), vegetal pole (VP), dorsal marginal zone (DMZ), lateral marginal zone (LMZ) and ventral marginal zone (VMZ) regions and analysed by RNAase protection for XBMP-4 and EF-la expression. Similar levels of XBMP-4 RNA are present in all regions. Table 1. Injection of different amounts of XBMP-4 RNA gives a graded series of defects DAI DUDJ RJJA pg 0 1 2 3 4 5 Mean 1500 300 75 15 77 41 9 9 10 5 7 4 2 7 9 12 5 3 14 7 2 3 0 0 9 31 0.39 0.68 2.67 3.32 12 8 Numbers in Table indicates number of cases at each DAI (see Kao and Elinson, 1988, for explanation of the dorsoanterior index). In the same experiment injection of 1500 pg antisense XBMP-4 into 39 embryos gave a DAI of 4.98. both is elevated in UV-irradiated embryos (Cooke and Smith, 1987; Ruiz i Altaba and Melton, 1989), and we find that the same is true in embryos injected with XBMP-4 RNA (Figs 5 and 8). These results indicate that injection of XBMP-4 RNA causes ventralization of Xenopus embryos, with a reduction in the amount of dorsal mesoderm being complemented by an increase in ventral mesoderm. One prediction from this is that injection of RNA into the dorsal side of the embryo should have a more dramatic effect than injection into the ventral side. We therefore injected 0.5 ng RNA into either a single dorsal or a single ventral blastomere at the 4-cell stage. Table 2 shows that injection into a dorsal blastomere did result in more extreme ventralization; whereas embryos resulting from dorsal injections had a mean DAI of 0.7, those resulting from ventral injections had a mean DAI of 2.67. Once again the mean DAI of control injections Injection of XBMP-4 RNA inhibits the response of animal caps to activin to a greater extent than to FGF In the experiments described above, XBMP-4 might cause ventralization of Xenopus embryos by interfering with the action of an endogenous dorsal mesoderminducing factor, and one plausible candidate for such a molecule is activin. When added to isolated animal pole cells in vitro activin induces a wide range of mesodermal tissues, including notochord and muscle, in a concentration-dependent manner (Green et al., 1990). By contrast, we would not expect XBMP-4 to inhibit the action of a ventral mesoderm-inducing factor such as bFGF (Slack et al., 1987; Green et al., 1990; Amaya et al., 1991). To examine this, animal caps derived from embryos injected with either sense or antisense XBMP4 RNA, or from uninjected embryos, were treated with activin, FGF, or a control solution. The first indication that injection of XBMP-4 RNA inhibits the response to activin was that the elongation of animal pole regions that is observed in response to this factor (Symes and Smith, 1987) was inhibited (not shown). When such animal caps were allowed to Fig. 4. Effects of injecting 2 ng XBMP-4 RNA into fertilized Xenopus eggs. (A) The blastopores of embryos injected with sense XBMP-4 RNA do not close normally, unlike those injected with antisense RNA (B). (C) Section of one of the embryos in A. This resembles sections of embryos that have been injected with Brachyury RNA (V.T. Cunliffe and J.C.S., unpublished data) or which have received intrablastocoelic injections of mesoderm-inducing factors (Cooke and Smith, 1989). There is a thick pad of mesoderm-like tissue at the animal pole to which the endodermal mass adheres strongly. This obliterates the blastocoel and creates a compensatory cavity at the vegetal hemisphere. (D) Section of an embryo that received an injection of antisense XBMP-4 RNA. The blastopore has closed normally. (E) Embryos injected with sense XBMP-4 allowed to develop to the equivalent of stage 39 appear ventralized compared with controls, which received injections of antisense RNA (F). (G) Sections of embryos shown in E reveal no notochord or muscle, unlike (H) sections of those in F. Scale bars in C and D are 100 /m\, and also apply to G and H. Scale bar in F is 1 mm, and also applies to A, B and E. BMP-4 and mesoderm induction 579 580 L. Dale and others ise c B pM < t EF-la • Fig. 5. Levels of Xhox3 are elevated in embryos injected with sense XBMP-4 RNA. Embryos injected with 1.5 ng sense or antisense XBMP-4 RNA were allowed to develop to stage 12 (late gastrula). Xhox3 expression was analysed by RNAase protection. develop further, histological analysis showed that they did not form significant amounts of muscle or notochord but either appeared uninduced or resembled the weak 'ventral' inductions formed in response to low concentrations of FGF (Fig. 6 and Table 3). By contrast, injection of XBMP-4 RNA appeared to have B little effect on induction by FGF, where the animal caps formed structures similar to those differentiating in response to FGF alone. Animal caps derived from embryos injected with XBMP-4 RNA and then allowed to develop in the absence of inducing factors appeared uninduced or showed weak ventral-like inductions, which were distinct from those obtained with FGF alone (Fig. 6 and Table 3). As controls, animal caps derived from uninjected embryos or embryos receiving antisense XBMP-4 RNA were treated with activin, FGF or control solutions; the results were similar to those reported by Green et al. (1990) (see Fig. 6 and Table 3). The above data were confirmed by molecular analysis of treated animal caps. Induction of muscle-specific actin in response to activin was greatly reduced in animal caps derived from embryos receiving injections of XBMP-4 RNA (Fig. 7A). Animal caps derived from embryos injected with XBMP-4 RNA, but not exposed to exogenous growth factors, express very low levels of muscle-specific actin (Fig. 7A). As with the histological data, this suggests that XBMP-4 is itself an inducer of mesoderm. This is confirmed by data presented in Fig. 7B. Xhox3 is most strongly expressed in ventral and Svv.: Fig. 6. Histological analysis of animal pole regions from uninjected embryos (C), (F), (I), or embryos injected with sense (A), (D), (G), or antisense (B), (E), (H) XBMP-4 RNA. The animal caps were cultured in the absence of inducing factors (G), (H), (I), or treated with 10 units ml" 1 recombinant activin A (A), (B), (C) or 50 units ml" 1 recombinant Xenopus basic FGF (D), (E), (F) (see Cooke et al., 1987, for definition of units of inducing activity). They were cultured until the equivalent of stage 41 and then processed for histology. XBMP-4 RNA has little effect on induction by FGF, but significantly inhibits the response to activin. XBMP-4 RNA itself produces only a weak response at the histological level. Scale bar in I is 100 fan, and applies to all other frames. BMP-4 and mesoderm induction Activin Table 3. Injection of XBMP-4 RNA inhibits the response of animal pole regions to activin, but has little effect on induction by FGF Treatment Axial Ventral Con 11 Response Uninduced B c < O 1/3 Sense XBMP-4 RNA Activin 0 FGF 0 No inducer 0 8 22 9 Antisense XBMP-4 RNA Activin 16 FGF 0 No inducer 0 0 11 0 No RNA injection Activin 13 FGF 0 No inducer 0 1 16 0 581 © U 8 •a us P Muscle actin •« 0 If 1 1IS Table indicates numbers of explants falling into each category. Xenopus eggs were injected with 1.5 ng sense or antisense XBMP-4 RNA or were left uninjected. At stage 8 (mid-blastula) animal caps were dissected and cultured until the equivalent of stage 40 in the absence of inducing factors or in the presence of 10 units ml" 1 activin A or 50 units ml" 1 FGF. After fixation and sectioning they were classified as 'uninduced', 'ventral mesoderm' (lacking muscle and notochord) or 'axial mesoderm' (containing muscle and/or notochord). posterior mesoderm, and animal caps from embryos injected with XBMP-4, but not exposed to exogenous growth factors, express levels of Xhox3 similar to those occurring in response to bFGF. This suggests that XBMP-4 is an inducer of ventral mesoderm. These experiments also confirmed that XBMP-4 mRNA injection 'ventralizes' the response to activin, because such animal caps express high levels of Xhox3. Ventralization caused by BMP-4 cannot be rescued by LiCl Xenopus embryos can also be ventralized by UVirradiation of the vegetal hemisphere shortly after fertilization (Scharf and Gerhart, 1983), a procedure that blocks the formation of the dorsal mesoderminducing centre in the vegetal hemisphere (Gimlich and Gerhart, 1984). This block can be 'reversed' by exposing UV-irradiated embryos to solutions of lithium chloride during early cleavage stages (Kao et al., 1986). As a result, embryos become 'hyperdorsal', with dorsal mesoderm differentiating from all sectors of the marginal zone (Kao and Elinson, 1988). The first signs of hyperdorsalization are observed at the early gastrula stage, when the blastoporal lip of the invaginating mesoderm is found all round the vegetal hemisphere of LiCl-treated embryos but localized to the dorsal quadrant in normal embryos. LiCl is believed to act by sensitizing the response of animal cap cells to ventral mesoderm-inducing signals (e.g. FGF or low concentrations of activin), such that they respond as though they were exposed to a dorsal signal (e.g. a high concentration of activin: Kao and Elinson, 1988; Slack et al., 1988; Cooke et al., 1989). Cytoskeletol actin tit Activin B FGF < Xhox3 # Con i on •i t f EF-la Fig. 7. XBMP-4 RNA inhibits muscle-specific actin expression in response to mesoderm induction but enhances Xhox3 expression. (A) Mid-blastula animal pole regions derived from control embryos or embryos injected with sense or antisense XBMP-4 RNA were exposed to 10 units ml" 1 activin A, 50 units ml" 1 Xenopus recombinant FGF, or a control solution. They were cultured until the equivalent of stage 20 and actin gene expression was analysed by RNAase protection. XBMP-4 RNA itself is a weak inducer of muscle-specific actin gene expression, but it reduces muscle-specific actin expression in animal caps treated with FGF and apparently abolishes expression in response to activin. (B) Mid-blastula animal pole regions derived from embryos injected with sense or antisense XBMP-4 RNA were exposed to 10 units ml" 1 activin A, 50 units ml" 1 Xenopus recombinant FGF, or a control solution. They were cultured until the equivalent of stage 12 and Xhox3 gene expression was then analysed by RNAase protection. XBMP-4 RNA itself, like FGF, is a strong inducerNof Xhox3 gene expression, and it elevates the expression seen in response to activin. 582 L. Dale and others Table 4. Lithium chloride treatment does not significantly 'rescue' embryos injected with 1500 pg XBMP-4 RNA DAI Treatment 0 l 2 3 4 5 6 1 8 9 10 Mean -LiCl Sense RNA Antisense 54 0 9 0 14 1 7 0 0 1 0 62 0 0 0 0 0 0 0 0 0 0 0.69 4.94 +LiCI Sense RNA Antisense 35 0 9 0 5 0 0 0 0 0 0 3 0 0 0 5 0 11 0 0 0 0.39 7.78 8 Numbers in Table indicate number of cases at each DAI. Control Antisense Sense Cytoskeletal Actin Muscle Actin Globin Fig. 8. Globin expression is elevated, and muscle-specific actin expression reduced, in embryos receiving injections of sense XBMP-4 RNA. This 'ventralization' is not rescued by LiCl treatment of embryos. Fertilized eggs received injections of 1.5 ng of sense, or antisense, XBMP-4 RNA, and about half of these, together with uninjected controls, were subsequently exposed to LiCl at the 64- to 128-cell stages. The embryos were allowed to develop to stage 40, when RNA was extracted and analysed by northern blotting. This figure also illustrates the effect of LiCl on globin gene expression. Cooke and Smith (1988) showed that LiCl-treated embryos contain 'substantial blood', but that this is immature compared with untreated controls. Consistent with this, comparison of lanes 1 and 2 suggests that LiCl treatment reduces globin expression somewhat, but this is not observed in lanes 3 and 4. The reason for this difference is not clear. We examined whether embryos ventralized by injection of BMP-4 RNA could be rescued in this way by exposing them to 0.3 M LiCl at the 64- to 128-cell stage. The results were assessed at stage 38 by the DAI index (Table 4) and by northern blot analysis using probes for actin and globin (Fig. 8). Both methods of analysis showed that LiCl treatment cannot rescue BMP-4 injected embryos. Table 4 shows that antisense-injected embryos were hyperdorsalized by exposure to LiCl, with a DAI of 7.78 compared with a control value of 4.94. Histological examination showed that these embryos differentiated more notochord at the expense of muscle ( data mnt shown\. and1 a sevp.;re. reduction in muscle differentiation is reflected in the reduced levels of muscle-specific actin expression shown in Fig. 8. By contrast, exposure of XBMP-4 injected embryos to LiCl had no effect on the DAI index (0.69 in the absence of LiCl compared to 0.39 in the presence of LiCl), and histological examination confirmed that the embryos remained ventralized, differentiating blood and mesothelium at the expense of muscle and notochord (data not shown). This is also confirmed in Fig. 8, which shows that exposure to LiCl has no effect on the enhanced expression of a tadpole /J-globin in XBMP-4 injected embryos. Interestingly, however, in embryos injected with both sense and antisense XBMP-4 RNA, the blastopore Up appeared around all regions of the marginal zone as if presaging hyperdorsal development. Like our earlier observation that XBMP-4 injection can cause ventralization even though the blastopore lip forms normally (see above), this result suggests that XBMP-4 acts late to produce ventralized embryos. Discussion The results described in this paper add a further level of complexity to the analysis of mesoderm induction in Xenopus. In a search for additional mesoderm-inducing factors, we have used the polymerase chain reaction followed by cDNA library screening to obtain a fulllength cDNA clone for Xenopus bone morphogenetic protein 4 (XBMP-4). Like Koster et al. (1991) we observe low levels of maternal mRNA for XBMP-4, and this is followed by a dramatic increase in RNA at the late blastula stage. The increase reported by Koster et al. (1991) was less dramatic than that seen in our Fig. 2, and this may be because their expression study concentrated on BMP-4r, while our sequence resembles their BMP-4n. At the early gastrula stage XBMP-4 is expressed at similar levels in all regions of the embryo. Bone morphogenetic proteins were originally identified in bone extracts by their ability to induce the formation of ectopic cartilage and bone following implantation in rats (Wang et al., 1988). BMP-4 (formerly called BMP-2b) was cloned because of its homology to the closely related BMP-2, a component of bone extracts (Wozney et al., 1988), and was subsequently shown to have similar activity (Hammonds et BMP-4 and mesoderm induction al., 1991). A recent analysis using in situ hybridization has implicated BMP-4 in diverse processes during murine embryogenesis, including development of the limbs, heart, face and pituitary gland (Jones et al., 1991). Although expression of this RNA in pregastrula mouse embryos was not identified, in early neurulae it was localized to the' posterior and ventral mesoderm suggesting a role for BMP-4 in the development of mesoderm in vertebrates. The results we have presented for Xenopus suggest that this is indeed the case. Using morphological, histological and molecular criteria, we show that overexpression of XBMP-4 by microinjection of transcripts into the animal hemisphere of the fertilized egg causes 'ventralization' of the resulting embryo. In these embryos, dorsal mesodermal tissues such as notochord and muscle are severely reduced and replaced by ventral mesodermal tissues such as blood. Consistent with this, injection of transcripts into the dorsal half of the early embryo has a more dramatic effect than injection into the ventral half. Furthermore, like Koster et al. (1991), we show that XBMP-4 can act as a ventral mesoderm-inducing factor. Thus, injection of XBMP-4 mRNA into the animal hemisphere of the Xenopus embryo followed by dissection of the animal cap at the mid-blastula stage results in the formation of ventral mesodermal cell types, as assayed both by conventional histology and by the level of activation of Xhox3, which in normal embryos is most highly expressed in posterior and ventral mesoderm (Ruiz i Altaba and Melton, 1989). In this respect XBMP-4 differs from the activins, to which it is closely related, because the activins preferentially induce anterior and dorsal mesoderm (Green et al., 1990). Rather, the action of XBMP-4 more resembles that of members of the FGF family (Slack et al., 1987; Kimelman and Kirschner, 1987). It is possible that the ability of XBMP-4 to induce ventral mesoderm is responsible for its ability to ventralize whole embryos. In order to cause ventralization in this way it is necessary that the ventralizing effect of XBMP-4 is 'dominant' over treatment with dorsal mesoderm-inducing factors such as activin. This is observed in experiments in which animal caps derived from embryos receiving injections of XBMP-4 RNA are treated with activin, and the effect is demonstrated most dramatically by the observation that injection of XBMP-4 RNA can even ventralize embryos that subsequently are treated with the dorsalizing agent LiCl. This dominant effect of a ventral mesoderminducing factor contrasts with work in which activin and FGF are applied simultaneously to animal cap tissue; here, the ability of activin to induce animal pole cells to form Spemann's organizer is not compromised by the ventral mesoderm-inducing factor bFGF (Cooke, 1989). Superficially, XBMP-4-injected embryos resemble those resulting from UV irradiation of the vegetal hemisphere soon after fertilization (Scharf and Gerhart, 1983). UV irradiation blocks the cortical rotation which is responsible for the formation of a dorsovegetal inducing centre, which in turn induces dorsal organizer 583 mesoderm from the overlying marginal zone (Gimlich and Gerhart, 1984). However, there are clear differences in the gastrulation movements of UV-irradiated and XBMP-4-injected embryos. UV-irradiated embryos form a 'ventral-type' blastoporal lip which appears late and synchronously around the whole circumference of the embryo (Scharf and Gerhart, 1983). By contrast, XBMP-4 injected embryos form a dorsal lip at the same time as controls. Although in some cases gastrulation then arrests, due to formation of ectopic mesoderm in the animal hemisphere of the embryo (see Cooke et al., 1987; Cooke and Smith, 1989; V. T. Cunliffe and J.C.S., unpublished data), in most cases the blastopore closes on a time scale similar to that of embryos injected with antisense RNA. XBMP-4 injected embryos also differ from UVirradiated embryos in that they cannot be 'rescued' by exposure to LiCl, even though in both types of embryo LiCl causes the formation of a precociously complete blastoporal lip around the circumference of the entire vegetal hemisphere. Together, these results suggest that the ventralization caused by XBMP-4 is a late event, occurring after the onset of gastrulation. The mechanism by which this occurs is unclear, although one possibility is that the action of XBMP-4 resembles that of polysulphonated compounds such as trypan blue and suramin. If injected into the blastocoels of embryos when the dorsal lip first appears, these compounds inhibit convergent extension movements while allowing blastopore closure. The resulting embryos fail to differentiate dorsal mesodermal tissues and become ventralized (Gerhart et al., 1989). If, however, these agents are injected after the end of gastrulation, when convergent extension has been completed, they have no effect on the differentiation of dorsal mesoderm. Like XBMP-4, but unlike UV-irradiation, both trypan blue and suramin can reverse the hyperdorsalizing effects of LiCl. It is not clear how trypan blue and suramin inhibit convergent extension and cause ventralization, although suramin is known to disrupt the binding of some growth factors to their receptors. These include members of all three families of mesoderm-inducing factor such as FGF and TGF-0 (Coffey et al., 1987), and members of the Wnt family (Papkoff and Schryver, 1990; Chakrabarti et al., 1992). It is possible that the continued presence of a member of one of these families is required for convergent extension and dorsal development and that an excess of exogenous XBMP-4 competes for the receptor for such a protein. This is under investigation. The role of XBMP-4 in Xenopus development will remain unknown until it becomes possible to eliminate the protein or its receptor from the embryo. It is clear, however, that the action of XBMP-4 must be considered in the context of the other inducing factors known to be present in the embryo, such as members of the activin, FGF and Wnt families as well as other bone morphogenetic proteins. It will be a formidable task to understand how the functions of all these factors are coordinated to form the normal embryo. 584 L. Dale and others This work is supported by the Medical Research Council. L.D. thanks Professor Alan Colman for support. J.C.S. thanks Dr Igor Dawid for the XTC cDNA library. 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