Bone Morphogenetic Protein-4 (BMP-4) Acts During Gastrula Stages to Cause Ventralization of Xenopus Embryos

Development 122, 1545-1554 (1996) 1545 Printed in Great Britain © The Company of Biologists Limited 1996 DEV1074

Bone morphogenetic protein-4 (BMP-4) acts during gastrula stages to cause ventralization of Xenopus embryos

C. Michael Jones1,3, Leslie Dale2, Brigid L. M. Hogan3, Christopher V. E. Wright4 and J. C. Smith1 1Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK 2Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK 3Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA 4Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA

SUMMARY

Injection of RNA encoding BMP-4 into the early Xenopus RNA cannot be rescued by grafts of Spemann’s organizer embryo suppresses formation of dorsal and anterior cell at gastrula stages. Such embryos therefore differ from types. To understand this phenomenon, it is necessary to those made ventral by UV-irradiation, where the defect know the stage at which BMP-4 acts. In this paper, we occurs early and rescue can be effected by the organizer. present three lines of evidence showing that BMP-4 misex- Finally, the dorsalizing effects of the organizer, and of the pression has no effect on the initial steps of mesoderm candidate dorsalizing signal noggin, both of which exert induction, either dorsal or ventral, but instead causes ven- their effects during gastrulation, can be counteracted by tralization during gastrulation. Firstly, activation of BMP-4. Together, these experiments demonstrate that organizer-speciﬁc genes such as goosecoid, Xnot, pintallavis BMP-4 can act during gastrulation both to promote ventral and noggin occurs normally in embryos injected with mesoderm differentiation and to attenuate dorsalizing BMP-4 RNA, but transcript levels are then rapidly down- signals derived from the organizer. regulated as gastrulation proceeds. Similarly, BMP-4 does not affect the initial activation of goosecoid by activin in animal caps, but expression then declines precipitously. Key words: BMP-4, ventralization, Xenopus, mesoderm induction, Secondly, embryos made ventral by injection with BMP-4 gastrulation

INTRODUCTION zone in the absence of dorsalizing signals. However, we have previously shown that injection of mRNA encoding BMP-4 Fate maps of the early Xenopus embryo show that much of the promotes the development of ventral mesoderm in Xenopus somitic muscle is derived from the ventral half of the blastula embryos (Dale et al., 1992; Jones et al., 1992), and that these (Dale and Slack, 1987a; Moody, 1987); yet, if this tissue is ventralizing effects override both endogenous dorsal explanted prior to gastrulation, it gives rise only to blood, mesoderm-inducing signals and those produced by activin. mesothelium and mesenchyme (Dale and Slack, 1987b). This These results suggest that ventral mesoderm formation requires tissue specification within the blastula reflects the inductive active signalling and is not simply a passive process (for potency of the vegetal hemisphere, in which only the extreme review see Sive, 1993). A role for BMP-4 in ‘ventralization’ dorsovegetal cells are capable of inducing dorsal mesoderm is supported by the observation that BMP-4 is expressed in the (notochord and muscle), while ventral and lateral cells induce ventral and lateral marginal zones (VMZ and LMZ) of the ventral mesoderm (Boterenbrood and Nieuwkoop, 1973; Dale early gastrula and is absent from the organizer (Fainsod et al., et al., 1985; Dale and Slack, 1987b). The inductive interaction 1994; Schmidt et al., 1995) and by experiments in which that diverts the fate of ventrally induced mesoderm towards embryos are injected with RNA encoding a truncated BMP-4 more dorsally located tissues, such as striated muscle, is known receptor: this causes loss of ventral structures and an increase as ‘dorsalization’ (Slack and Forman, 1980; Smith and Slack, in dorsal tissue (Graff et al., 1994; Maeno et al., 1994; Suzuki 1983). Candidates for the dorsalizing signal(s) include noggin, et al., 1994). chordin and the recently reported Xenopus nodal-related genes, Although these results are consistent with the idea that Xnr-1, Xnr-2 and Xnr-3, all of which are capable of promoting BMP-4 acts as a ventralizing agent, no reports have directly muscle differentiation in explanted ventral mesoderm (Smith addressed the stage at which BMP-4 functions; it is possible, and Harland, 1992; Smith et al., 1993, 1995; Sasai et al., 1994; for example, that it causes ventralization at blastula stages, by Jones et al., 1995). inhibiting a dorsovegetal organizer-inducing signal, rather than This model regards ventral mesoderm as a ‘ground state’ by acting during gastrulation. In this paper, we show that over- that is adopted by lateral and ventral sectors of the marginal expression of BMP-4 does not affect the initial response to 1546 C. M. Jones and others mesoderm induction, but does cause a rapid down-regulation antibody MZ15 (Smith and Watt, 1985), which is specific for of organizer-specific genes immediately after the onset of gas- notochord. trulation. This, together with the observations that embryos injected with BMP-4 RNA cannot be rescued by grafts of Spemann’s organizer, and that the dorsalizing effects of the RESULTS organizer and of noggin can be counteracted by BMP-4, provides strong evidence that BMP-4 acts during gastrulation BMP-4 represses expression of dorsally expressed to promote ventral mesoderm differentiation and to attenuate genes during gastrulation dorsalizing signals derived from the organizer. Xenopus embryos injected with RNA encoding BMP-4 become ventralized, failing to differentiate dorsal structures such as the neural tube, notochord and somitic muscle (Dale et MATERIALS AND METHODS al., 1992; Jones et al., 1992). At the beginning of gastrulation, however, formation of the dorsal blastopore lip in BMP-4- Xenopus embryos injected embryos is indistinguishable from that of controls, and Xenopus embryos were obtained by in vitro fertilisation (Smith and it extends and closes in an apparently normal fashion (Jones et Slack, 1983). They were maintained in 10% Normal Amphibian al., 1992). This suggests that BMP-4 might promote ventral- Medium (NAM; Slack, 1984) and staged according to Nieuwkoop and ization during gastrulation, after formation of the dorsal Faber (1967). Manipulation of intact embryos was performed in 75% organizer. If this were the case one would anticipate that genes NAM and, after healing, they were returned to 10% NAM. Animal expressed exclusively at the dorsal blastopore lip of the early caps and marginal zone explants were cultured in 75% NAM. gastrula would initially be expressed normally in BMP-4- Microinjection injected embryos and levels of expression would only decline Capped synthetic mRNA encoding human BMP-4 was prepared as later, as gastrulation proceeds. Previous studies, utilising previously described (Jones et al., 1992). Embryos at the 1-cell stage whole-mount in situ hybridisation, suggest this might not be were injected with 2 ng of BMP-4 RNA in a volume of 10 nl. For the case; expression of both goosecoid and Xnot were not DNA injections, hBMP-4 was cloned into the CSKA expression detectable in the dorsal lip of gastrulae previously injected with vector (Condie et al., 1990), thus placing the BMP-4 coding region BMP-4 RNA (von Dassow et al., 1993; Fainsod et al., 1994; under the control of a Xenopus cytoskeletal actin promoter. This Schmidt et al., 1995). To clarify this point, and to extend promoter is activated at the midblastula transition, resulting in over- previous findings (Hogan et al., 1994), we have used RNAase expression of BMP-4 during gastrula stages. 50-100 pg of supercoiled protection to analyse the expression levels of goosecoid, Xnot, CSKA:BMP-4 DNA was injected into each embryo at the 1-cell stage, noggin and pintallavis from blastula to late gastrula stages in either alone or in combination with CSKA:Noggin (20-50 pg). For Xenopus embryos injected with BMP-4 RNA (Fig. 1A). The lineage tracing, 10 nl of 5 mg/ml FLDx (fluorescein-lysine-dextran, results show that all four genes are expressed at near-normal M 10,000: Molecular Probes) was injected into donor embryos at the r levels in early gastrulae but, as gastrulation proceeds, 1-cell stage. All microinjections were performed in 75% NAM containing 5% Ficoll. When embryos were required to develop beyond expression of goosecoid, noggin and Xnot declines when stage 10, they were transferred to 10% NAM. compared to controls. In the experiment reported, noggin transcript levels fall dramatically during midgastrula stages but, in RNAase protection other experiments, the decrease was less pronounced. In RNAase protection analyses were performed as previously described contrast, although pintallavis expression is slightly reduced (Cho and De Robertis, 1990). Samples were analysed with antisense when compared to controls at the beginning of gastrulation, probes specific for goosecoid (Blumberg et al., 1991), Xhox3 (Ruiz i transcripts never disappear and are detected at comparable Altaba and Melton, 1989), noggin (Smith and Harland, 1992), pin- levels in both control and BMP-4-injected embryos at late tallavis (Ruiz i Altaba and Jessell, 1992) Xnot (von Dassow et al., gastrula stages (Fig. 1A). α α 1993) and -actin (Mohun et al., 1984). EF1 , which is expressed in In contrast to organizer-specific transcripts, Xhox3, which is all embryonic cells (Krieg et al., 1989; Sargent and Bennett, 1990), most strongly expressed in lateral and ventral mesodermal was used as a loading control. RNA samples from a single injection experiment were analysed with all the relevant probes and assays were tissues (Ruiz i Altaba and Melton, 1989), is upregulated during performed at least twice on RNA from separate injection experiments. gastrulation (Fig. 1A). Similar results are obtained if levels of Digestions were performed with both RNAase A (80 µg/µl) and goosecoid and of Xhox3 are monitored in dorsal marginal RNAase T1 (67 Units/ml) at room temperature for 10 minutes, or with zones (DMZs) explanted from BMP-4-injected embryos; RNAase T1 alone for 30 minutes at 37¡C. Protected fragments were goosecoid is initially expressed normally and is then down- separated by electrophoresis through 8% sequencing gels and regulated, while levels of Xhox3 are simultaneously upregu- analysed by autoradiography using two intensifying screens. lated (data not shown). These results add to those obtained previously (see Introduction) by providing evidence that BMP-4 Histological examination acts during gastrulation to cause ventralization of the Xenopus Embryos injected with FLDx were fixed overnight in 4% embryo. paraformaldehyde in PBS at 4¡C. Following dehydration, clearing and µ embedding in wax, tissues were sectioned at 7-10 m, mounted on Overexpression of BMP-4 during gastrulation TESPA-coated slides and dried overnight at 50¡C. Sections were then causes ventralization dewaxed, hydrated, rinsed in PBS and stained with 0.1% eriochrome black (to quench autofluorescence of yolk granules) and 0.1% DAPI To confirm that BMP-4 can act during gastrulation, expression (to stain nuclei). Immunofluorescence was according to O’Reilly et of the gene was placed under the control of a Xenopus al. (1995) and used antibody 12/101, which is specific for muscle cytoskeletal actin promoter (CSKA; Condie et al., 1990). Tran- (Kintner and Brockes, 1984). Whole-mount immunolocalization used scription of BMP-4 under control of this promoter begins at BMP-4 ventralizes during gastrula stages 1547

effect of CSKA:BMP-4 is therefore weaker than that of BMP- 4 RNA, where in a typical experiment the average DAI is close to zero (data not shown, but see Dale et al., 1992). This dif- ference may be due to a lower level of expression obtained with CSKA:BMP-4, or to the mosaic expression typically observed after DNA injections (Christian and Moon, 1993). Neverthe- less, the results conﬁrm that BMP-4 can exert its ventralizing effect on embryonic mesoderm after zygotic transcription begins at the mid blastula transition. Consistent with the weaker effect of the CSKA construct, RNAase protection analysis shows that CSKA:BMP-4 DNA has no effect on the expression of goosecoid during gastrulation and that up-regulation of Xhox3 expression is delayed compared with embryos that have been injected with BMP-4 RNA (Fig. 1B). These observations indicate that BMP-4 can cause ventralization without affecting early expression of goosecoid.

BMP-4 counteracts activin-induced dorsal inductions during gastrula stages The above findings suggest that BMP-4 countermands the earlier-acting effects of endogenous dorsal mesoderm- inducing signals. To test this idea directly, we have studied the effects of BMP-4 on expression levels of goosecoid and of Xhox3 at different times after activin treatment of Xenopus animal caps. Previous results demonstrate that BMP-4 ventralizes the response to activin (Dale et al., 1992; Jones et al., 1992), but it was not clear at what stage this occurred. Fig. 2 shows that activin induces strong expression of goosecoid in animal caps and that this expression persists to stage 11.5, but is weaker by stage 16. The presence of BMP-4 has little effect on the initial activation of goosecoid by activin, but by stage 11.5 transcript levels are greatly reduced when compared with animal caps treated with activin alone. Again, this decrease in goosecoid expression is accompanied by a precocious increase in Xhox3 transcript levels, which are strongly induced by BMP-4, but not by activin. The results of this induction assay support the idea that the effects of BMP-4 occur during Fig. 1. BMP-4 represses dorsal-specific gene expression during gastrula stages. (A) Embryos were injected at the 1-cell stage with gastrula stages and that it has little effect on initial induction RNA encoding BMP-4. RNA was prepared from a large batch of of dorsal mesoderm. embryos at each of the indicated stages and analysed for expression of the various markers by RNAase protection. The organizer-specific BMP-4-injected embryos cannot be rescued by genes goosecoid, Xnot, noggin and pintallavis are all induced in transplanted Spemann organisers BMP-4-injected embryos, but expression of goosecoid, Xnot and noggin is subsequently suppressed. In contrast, Xhox-3 expression is We next investigated whether embryos ventralized by injection precociously increased under the influence of BMP-4 in the same of BMP-4, like those ventralized by UV-irradiation, can be embryos. C, control injected embryos; B, BMP-4 RNA injected rescued by Spemann’s organizer. Dorsal marginal zones were embryos. (B) Overexpression of BMP-4 under the control of the isolated from lineage-labelled, but otherwise normal, early cytoskeletal actin promoter has no effect on the expression of gastrulae (stage 10) and transplanted into the blastocoels of goosecoid, but Xhox-3 levels are increased in late gastrulae. See early gastrulae that had previously been injected with BMP-4 Table 1 for details of CSKA:BMP-4 injections. C, control injected RNA (see Slack and Isaacs, 1994, for a discussion of this α embryos; B, CSKA:BMP-4-injected embryos. EF1- serves as a ‘Einsteck’ method). All the resulting embryos (n=56) were control for RNA integrity in all lanes. indistinguishable from BMP-4-injected embryos that had not received an implant (Fig. 3A,C). As a control, organizer tissue the midblastula transition and peaks during gastrula stages was transplanted into the blastocoels of uninjected early (data not shown). Table 1 shows that injection of a gastrulae and all of the resulting embryos (n=18) developed a CSKA:BMP-4 DNA construct at the 1-cell stage causes sig- secondary dorsal axis (Fig. 3B). The results show that trans- nificant ventralization of Xenopus embryos. According to the plants of Spemann’s organizer cannot rescue dorsal axial dorsoanterior index (DAI) of Kao and Elinson (1988), in which development in embryos ventralized by injection of BMP-4 normal embryos score 5 and fully ventralized embryos score RNA, demonstrating directly that BMP-4 acts during gastrula- 0, injected embryos scored an average of 1.7. The ventralizing tion. 1548 C. M. Jones and others

Table 1. Effects of zygotically expressed BMP-4 on Xenopus development DorsoAnterior Index Average Sample Total 5 4 3 2 1 0 DAI Control 50 49 1 −−−−4.9 CSKA: BMP-4 102 3 11 18 22 17 31 1.7

100 pg of supercoiled plasmid DNA expressing BMP-4 under the control of the cytoskeletal actin promoter (CSKA) was injected into Xenopus embryos at the 1-cell stage. Quantitation of the ventralizing effects used the DorsoAnterior Index (DAI) of Kao and Elinson (1988). Zygotically expressed BMP-4 causes signiﬁcant ventralization of Xenopus embryos, which develop with an average DAI of 1.7 compared to control embryos which are normal (DAI=5).

To conﬁrm that the host embryos had formed no dorsal Ventralization of the organizer by BMP-4-injected mesoderm, embryos were sectioned. Since the transplanted embryos organizer was isolated from embryos previously injected The above results show that organizer tissue is ventralized by with the lineage label ﬂuorescein-lysine-dextran (FLDx), we transplantation into BMP-4-injected host embryos. Although were able to distinguish between host and donor tissues. ventralization of the organizer may not take place during When grafted to normal embryos, the organizer contributes predominantly to the notochord of the secondary axis, while additional somitic tissue is mostly host-derived (Fig. 4C; see Smith and Slack, 1983; Slack and Isaacs, 1994). In contrast, when organizer tissue is grafted into BMP-4-injected embryos, it becomes incorporated into the ventralized embryo and forms tissues indistinguishable from those present in the BMP-4-injected hosts (Fig. 4B,D). Neither notochord nor muscle was formed by the organizer in these embryos. These observations demonstrate both that an organizer cannot rescue a BMP-4-injected embryo and, perhaps more surprisingly, that signals derived from the BMP-4-injected host ventralize the transplanted dorsal tissue.

Fig. 2. BMP-4 mediated repression of goosecoid in activin-induced animal caps. Embryos were injected with BMP-4 RNA and caps were isolated from injected or control embryos at blastula stages and Fig. 3. Effects of a wild-type organizer grafted into BMP-4-injected treated with activin, or used as controls. Stage 10 samples embryos. (A) Morphological appearance of BMP-4-injected demonstrate that in animal caps, initial induction of goosecoid by embryos. (B) Normal embryo following implantation of an organizer activin is largely unaffected by the presence of BMP-4. By stage at the early gastrula stage. Note the primary (1¡) and secondary (2¡) 11.5, the actions of BMP-4 repress goosecoid expression, which axes. (C) BMP-4-injected embryos following implantation of a becomes completely eliminated by stage 16. Simultaneously, BMP-4 normal organizer. Control grafted embryos are induced to form induces a dramatic increase in the levels of Xhox-3, which is secondary dorsal axes, while grafted embryos expressing BMP-4 are maintained well into neurula stages (stage 16). EF1-α demonstrates indistinguishable from BMP-4-injected embryos that have not equal RNA loading among samples at each stage. received a transplant. BMP-4 ventralizes during gastrula stages 1549

Fig. 4. Histological analysis of control (A,C,E) and BMP-4-injected (B,D,F) embryos that have received fluorescently labelled organizer transplants. Differential interference contrast optics reveals the general architecture of representative samples of control (A) and BMP-4-injected (B) host embryos. Lineage tracing analysis of grafted organizers demonstrates that, in control embryos (C), the fluorescent organizer tissue contributes mostly to axial mesoderm in the secondary axis. In contrast, no axial mesoderm is formed in BMP-4- injected hosts (D) and the organizer itself becomes ventralized and differentiates into tissues indistinguishable from those forming in the ventralized host. DAPI staining shows overall nuclear staining in each section (E,F). normal development, this observation provides a convenient assay for determining the developmental stages at which ventralization may occur. Organisers were transplanted into BMP- 4-injected hosts either immediately after dissection at the early gastrula stage (stage 10) or after different periods of culture in vitro (equivalent to stages 11 and 12). In all cases, host embryos were at stage 10. Embryos were then cultured to tadpole stages and subjected to whole-mount immunocyto- chemistry using the notochord-specific monoclonal antibody MZ15. Notochord, a dorsal cell type, was not formed by transplants made at stage 10 or 11 (n=38) and only rarely differen- tiated (2/20) when transplants were made at stage 12 (Fig. 5). These results demonstrate that embryonic tissues remain competent to respond to the ventralizing effects of BMP-4 even at late gastrula stages.

Fig. 5. Immunolocalization of notochord using antibody MZ15 in control embryos (A) and BMP-4-injected embryos into which a wild- type organizer has been grafted (B). Grafted embryos (B) are representative cases in which organizer tissue was isolated at stage 10, cultured to stage 12 and then transplanted into a stage 10 BMP-4- injected host. Approximately 95% (n=56) of all organizers transplanted at either stage 10, 11 or 12 become ventralized and contain no MZ15 immunopositive cells. The positive case displaying weak notochord staining was one of two such embryos from twenty hosts receiving transplants after culture to stage 12. not, notochord. 1550 C. M. Jones and others Ventral marginal zone tissue expressing BMP-4 DISCUSSION cannot be dorsalized by signals from the organizer The results described above indicate that BMP-4 can act In previous work, we have shown that BMP-4 inhibits dorsal during gastrula stages to ventralize mesodermal tissues. We mesoderm development, both in whole embryos and in animal next asked the reciprocal question of whether overexpression caps treated with the mesoderm-inducing factor, activin (Dale of BMP-4 in ventral marginal zone tissue might make those et al., 1992; Jones et al., 1992). The studies reported here cells refractory to dorsalizing signals derived from the address for the first time the stage at which BMP-4-mediated organizer. This point was not addressed in the experiments ventralization occurs and we also demonstrate that overex- described above because transplantation of the organizer into pression of BMP-4 makes ventral cells refractory to the dorsalizing effects of the organizer and of noggin. whole BMP-4-injected embryos caused the organizer to become ventralized and presumably to lose its dorsalizing BMP-4 does not block induction of organizer- effects. To overcome this problem, ventral marginal zone specific genes but instead causes their rapid down- tissue was explanted from BMP-4-injected early gastrulae, regulation juxtaposed with lineage-labelled dorsal marginal zone tissue Dorsal mesoderm is induced by dorsovegetal blastomeres from sibling control embryos and cultured to tadpole stages. (Gimlich and Gerhart, 1984; Dale et al., 1985), an activity that Muscle was detected by immunohistochemistry with the can be mimicked by factors such as activin (Smith et al., 1990; antibody 12/101. In control experiments VMZ tissue from Thomsen et al., 1990), Vg1 (Thomsen and Melton, 1993; uninjected embryos was induced to form substantial amounts Kessler and Melton, 1995), and two Xenopus nodal-related of striated muscle under the influence of signals from the genes (Jones et al., 1995). One explanation for the ventraliz- DMZ (Fig. 6A-C; see Dale and Slack, 1987b; Lettice and ing effects of BMP-4 overexpression is that it antagonises this Slack, 1993). In contrast, if the VMZ is derived from embryos dorsovegetal signal, thus blocking formation of the organizer. injected with BMP-4 RNA, juxtaposition with dorsal marginal In this paper, however, we show that four organizer-specific zone tissue does not induce muscle differentiation and tissues genes, goosecoid, Xnot, pintallavis and noggin, each of which within the juxtaposed VMZ are indistinguishable from those is activated in the blastula as an early response to dorsal observed when two wild type VMZs are explanted and mesoderm-inducing signals (Cho et al., 1991; Smith and cultured (Fig. 6D-F, and data not shown). The dorsal marginal Harland, 1992; Ruiz i Altaba and Jessell, 1992; von Dassow et zone tissue in these combinations, like that in control combi- al., 1993), are activated in embryos injected with BMP-4 RNA. nations, forms notochord, muscle and neural tissue, suggest- Similar results are obtained when goosecoid expression is ing that it is still capable of dorsalizing VMZ tissue. These analysed in animal caps treated with high concentrations of results indicate that ventral marginal zone tissue expressing activin. These results are consistent with our earlier observa- elevated levels of BMP-4 cannot be dorsalized by signals from tion that the dorsal blastopore lip forms normally in BMP-4- the organizer. injected embryos (Dale et al., 1992; Jones et al., 1992). They show that BMP-4 has little or no effect on early dorsovegetal Zygotic expression of BMP-4 attenuates noggin- signalling, nor on the ability of cells to respond to such signals. induced dorsalization An alternative explanation for the ventralizing effects of The data presented above indicates that BMP-4 causes ven- BMP-4 is that it promotes the down-regulation of dorsal- tralization during gastrulation, when dorsalizing signals specific gene expression during gastrulation. Analysis of the establish distinct mesodermal cell types. These dorsalizing longer term effects of BMP-4 on the expression of goosecoid, signals can be mimicked by secreted molecules such as noggin Xnot and noggin shows that this is the case. Although (Smith and Harland, 1992; Smith et al., 1993) and it is possible goosecoid and Xnot are expressed at near-normal levels in that BMP-4 counteracts the effects of such factors. To test this early gastrulae, they are then rapidly down-regulated and transcripts are undetectable by mid-gastrulation. This is consistent possibility, we have co-expressed elevated levels of noggin and with previous reports that goosecoid and Xnot are not BMP-4 under the control of the CSKA promoter, which gives expressed in the organizer region of BMP-4-injected mid- maximal expression during gastrula stages. Injections were gastrulae (von Dassow et al., 1993; Fainsod et al., 1994; targeted to presumptive ventral blastomeres at the 4-cell stage Schmidt et al., 1995; Re’em-Kalma et al., 1995). We also and ventral marginal zones were explanted at the early gastrula observe a BMP-4-induced reduction in levels of noggin during stage. In confirmation of previous work (Smith et al., 1993), gastrulation stages but, in contrast to goosecoid and Xnot, noggin promoted muscle differentiation in isolated VMZs (Fig. noggin transcripts reappear at near-normal levels in early 7E), which otherwise form loosely packed mesenchyme and neurulae. This later expression is maintained until tailbud blood cells (Fig. 7D). These noggin-induced changes are seen stages (Dale and Jones, unpublished), even though notochord, at the morphological level, where explants become elongated where noggin is most highly expressed at this time (Smith and (Fig. 7A,B) and are confirmed by the detection of muscle- Harland, 1992), is absent. A similar phenomenon is observed specific actin transcripts (Fig. 8). When BMP-4 is co-expressed with pintallavis (Fig. 1A). At present we do not know which with noggin, however, dorsalization is inhibited: elongation cells in BMP-4-injected embryos are expressing these genes. does not occur (Fig. 7C) and muscle differentiation is not These experiments emphasize the importance of studying detected either by histology (Fig. 7F) or by RNAase protection levels of gene expression at different times during gastrulation. (Fig. 8). These results demonstrate that BMP-4 signalling Experiments by Schmidt et al. (1995) and Re’em-Kalma et al. attenuates the effects of a specific dorsalization signal during (1995) have recently shown that BMP-4 inhibits dorsal- gastrula stages. specific gene expression at gastrula stages, but since they did BMP-4 ventralizes during gastrula stages 1551 not carry out a time-course, the two groups reached opposite process of dorsalization is thought to create a smooth conclusions as to when BMP-4 acts. Schmidt et al. (1995) aver continuum of cell types between these two extremes (Smith that BMP-4 exerts its effects after primary mesoderm and Slack, 1983). This might occur in three ways. induction, while Re’em-Kalma et al. (1995) are of the opinion 1. A signal from the organizer might diffuse into adjacent that BMP-4 primarily suppresses the initial induction of dorsal BMP-4-expressing ventral tissue and re-specify that ventral mesoderm. tissue as dorsal. In such a model, the effects of the dorsalizing Further evidence that BMP-4 ventralizes embryos during signal would not involve any interaction with BMP-4 or other gastrulation is provided by experiments in which BMP-4 is ventralizing agents such as Xwnt-8 (Christian and Moon, placed under the control of a cytoskeletal actin (CSKA) 1993), whose sole function is to define cells as ventral. promoter. Injection of CSKA:BMP-4 gives maximal 2. In a refinement of (1), ventralizing agents such as BMP- expression during gastrula stages, rather than blastula stages as 4 and Xwnt-8 might simultaneously inhibit dorsalizing signals, with RNA injection, and therefore reflects more accurately thus preventing them from spreading too far. expression of the endogenous gene (Dale et al., 1992; Nishi- 3. Finally, and in contrast to (2), the function of dorsalizing matsu et al., 1992). Injection of this construct also suppresses signals might not be actively to specify dorsal development, development of dorsal mesoderm. but rather to inhibit the function of ventralizing signals like Finally, we show that transplantation of dorsal marginal BMP-4 and Xwnt-8. In this case the ‘default’ pathway for zone tissue from control early and late gastrula-staged embryos mesoderm development, the pathway adopted by cells in the into the blastocoels of BMP-4-injected hosts at the early absence of any signal, would be dorsal (see Dale et al., 1992; gastrula stage causes the transplanted tissue to form ventral cell Jones et al., 1992; Graff et al., 1994). types. This provides direct evidence that BMP-4-mediated ven- Work with BMP-4 supports the third possibility. The results tralization can occur during gastrula stages. The expression presented in this paper show that overexpression of BMP-4 in pattern of BMP-4 is also consistent with the idea that this gene ventral tissues makes cells refractory to the effects of dorsal- product acts during gastrulation, although we cannot, of izing signals. This rules out the first possibility mentioned course, rule out the possibility that it might also function during above. Evidence in favour of the third alternative, rather than blastula stages (see Köster et al., 1991). the second, comes from two experiments. First, the Xenopus dorsalizing gene chordin (Sasai et al., 1994) has recently been Ventralization? shown to encode a structural and functional homologue of the Of all the candidates thought to play a role in inducing and Drosophila gene short gastrulation (sog), which inhibits the patterning the mesoderm of Xenopus, the case for BMP-4 is function of decapentaplegic (dpp), a homologue of BMP-4 the strongest. BMP-4 is expressed at the right place and time (François et al., 1994; François and Bier, 1995; Holley et al., to act as a ventralizing factor (Dale et al., 1992; Fainsod et 1995; see Jones and Smith, 1995). This suggests that at least al., 1994; Schmidt et al., 1995; Hemmati-Brivanlou and one dorsalizing factor acts by inhibiting the ventralizing Thomsen, 1995), it has the appropriate effects on isolated activity of BMP-4 in lateral regions of the mesoderm, thus responding tissue (Dale et al., 1992; Jones et al., 1992) and promoting the formation of more dorsal tissues such as muscle inhibition of BMP-4 signalling, by overexpression of a and pronephros. Secondly, overexpression of a truncated truncated BMP-4 receptor, causes loss of ventral structures BMP-4 receptor in lateral and ventral sectors of the marginal and an increase in dorsal tissue (Graff et al., 1994; Maeno et zone causes formation of additional dorsal structures (Graff et al., 1994; Suzuki et al., 1994). It is ironic, therefore, that the al., 1994; Maeno et al., 1994; Suzuki et al., 1994; 1995; techniques of experimental embryology have yet to reveal the Schmidt et al., 1995; Ishikawa et al., 1995). This demonstrates existence of ventralization (see Smith and Slack, 1983). As directly that inhibition of BMP-4 function is sufficient to we discuss below, it seems likely that this is because the dorsalize ventral tissue. dorsal marginal zone (which Smith and Slack used as the ‘test The ability of the organizer to inhibit the function of ven- tissue’ for ventralization) expresses molecules that inhibit the tralizing agents such as BMP-4 may explain why ‘ventraliza- function of BMP-4. tion’ is difficult to achieve in embryological assays (Smith and Slack, 1983) and can only be obtained by strong misexpres- Interactions between dorsalizing and ventralizing sion of BMP-4 in dorsal marginal zone tissue. signals Not all dorsalizing and ventralizing signals necessarily Our results show that BMP-4 does more than just promote function in the manner described above. It is not known, for ventral development in Xenopus embryos; it also makes ventral example, whether other dorsalizing agents, such as noggin marginal zone tissue refractory to the dorsalizing effects of (Smith et al., 1993), the nodal-related genes (Jones et al., 1995; both the organizer and of the secreted molecule noggin. This Smith et al., 1995) and follistatin (Hemmati-Brivanlou et al., observation provides clues as to what happens during ‘dorsal- 1994), act by directly inhibiting the function of BMP-4, or ization’ and (if it occurs) ‘ventralization’. whether other ventralizing agents, such as Xwnt-8, are Experimental embryological studies (Gimlich and Gerhart, inhibited by the organizer. Recent evidence does suggest, 1984; Dale and Slack, 1987b) and indirect evidence from the however, that follistatin inhibits the function of BMP-4 as well expression patterns of many genes, such as goosecoid (Cho et as that of activin (Sasai et al., 1995), and we are currently al., 1991), pintallavis (Ruiz i Altaba and Jessell, 1992), chordin investigating the modes of action of other dorsalizing signals. (Sasai et al., 1994), Xwnt-8 (see Christian and Moon, 1993) Nevertheless, one outcome of the interaction between dorsal- and BMP-4 itself (Fainsod et al., 1994; Schmidt et al., 1995), izing and ventralizing signals is almost certainly to generate an suggest that at the early gastrula stage there are two types of effective gradient of BMP-4 activity, and it is possible that this mesoderm in the Xenopus embryo: dorsal and ventral. The provides positional information along the dorsoventral axis of 1552 C. M. Jones and others

Fig. 6. Muscle differentiation in control and BMP-4-injected VMZ explants juxtaposed to wild-type DMZs. VMZs derived from control (A-C) or from BMP-4-injected (D-F) early gastrulae were juxtaposed with fluorescently labelled DMZs from wild-type embryos. In each case, the DMZ tissue is marked by fluorescence (A,D) and forms characteristic tissues which include notochord (arrow, use as a reference point in each panel) and muscle. 12/101 antibody staining localises muscle tissue in each explant conjugate (B,E). In control explants (B), substantial muscle differentiation is detected in tissues arising from the VMZ (not marked by fluorescence). In the case of recombinants where the VMZ over- expresses BMP-4 (E), muscle is only formed in the DMZ itself, and never in the BMP-4-injected VMZ. DAPI staining (C,F) illuminates nuclei throughout each section.

Fig. 7. Zygotically expressed BMP-4 attenuates noggin-induced dorsalization of ventral mesoderm. Ventral marginal zones derived from control (A,D), CSKA:Noggin (B,E), or CSKA:Noggin/CSKA:BMP-4 injected (C,F) embryos were cultured to stage 17 (A-C) or stage 35 (D-F). Noggin induces extensive morphogenetic movements (B), compared with controls (A) or with tissues expressing both noggin and BMP- 4 (C). Histological analysis (D-F) demonstrates that noggin (E) induces large muscle blocks (mus) while controls (D) form loosely packed mesenchyme (mes). When BMP-4 is over-expressed simultaneously with noggin (F), no muscle differentiation is observed and the tissue resembles controls, although they always remain more compact. ym, yolk mass. BMP-4 ventralizes during gastrula stages 1553

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