Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12141-12145, December 1995 Developmental Biology

Competition between and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development YAEL RE'EM KALMA*, TERESA LAMBt, AND DALE FRANK*t *Department of Biochemistry, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel; and tDepartment of Molecular and Cellular Biology, Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720 Communicated by John Gerhart, University of California, Berkeley, CA, September 15, 1995

ABSTRACT Bone morphogenetic protein 4 (BMP-4) in- dorsalization signal (6-9), it is not clear how distinct axial borders duces ventral mesoderm but represses dorsal mesoderm for- are defined within the developing . It is possible that cell mation in Xenopus . We show that BMP-4 inhibits two borders are defined by a combination of positive and negative signaling pathways regulating dorsal mesoderm formation, pathways which demarcate the dorsal/ventral border. the induction of dorsal mesoderm (Spemann organizer) and Bone morphogenetic protein 4 (BMP-4) is a member of the the dorsalization of ventral mesoderm. Ectopic expression of transforming growth factor ( family (11). The has been BMP-4 RNA reduces goosecoid and forkhead-l transcription isolated from Xenopus laevis, and ectopic expression of in vitro in whole embryos and in activin-treated animal cap explants. synthesized BMP-4 RNA in developing embryos causes de- Embryos and animal caps overexpressing BMP-4 transcribe velopmental changes which suggest that BMP-4 protein in- high levels of expressed in ventral mesoderm (Xbra, duces ventroposterior mesoderm. Late-gastrula-stage animal Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ven- caps isolated from BMP-4-injected embryos transcribe Xhox-3 tralized in these embryos; abnormally high levels of Xwnt-8 mRNA, a ventroposterior mesoderm marker (12, 13). Histo- mRNA and low levels of goosecoid mRNA are detected in the logical analysis of animal cap explants treated with soluble organizer. In addition, the organizer loses the ability to dorsalize BMP-4 protein as well as animal cap explants derived from neighboring ventral marginal zone to muscle. Overexpression of BMP-4-injected embryos suggests that these cells resemble BMP-4 in ventral mesoderm inhibits its response to dorsaliza- ventral mesoderm (12-14). BMP-4-injected animal caps also tion signals. Ventral marginal zone explants ectopically express- form posterior mesoderm in Einstecken experiments (13). ing BMP-4 form less muscle when treated with soluble noggin Basic fibroblast growth factor (bFGF) also induces ventropos- protein or when juxtaposed to a normal Spemann organizer in terior mesoderm in animal cap explants (15, 16), but the mode comparison to control explants. Endogenous BMP-4 transcripts of action of bFGF differs from that of BMP-4. BMP-4 inhibits are downregulated in ventral marginal zone explants dorsalized dorsal induction by activin in a dominant manner; animal caps by noggin, in contrast to untreated explants. Thus, while BMP-4 injected with BMP-4 and treated with activin do not make muscle inhibits noggin protein activity, noggin downregulates BMP-4 (12, 13). In contrast, bFGF does not antagonize activin's induc- expression by dorsalizing ventral marginal zone to muscle. tion of dorsal mesoderm in animal caps (17). Ectopic expression Noggin and BMP-4 activities may control the lateral extent of of BMP-4 RNA also inhibits dorsal axis formation in whole dorsalization within the marginal zone. Competition between embryos; they fail to elongate and do not make detectable these two molecules may determine the final degree of muscle notochord or muscle (12, 13). In addition, ectopic expression of formation in the marginal zone, thus defining the border between a dominant negative Xenopus BMP-4 receptor in embryos con- dorsolateral and ventral mesoderm. verts ventral mesoderm to dorsal mesoderm (18). Thus, while BMP-4 acts as a ventroposterior mesoderm inducer, it also InXenopus embryos, mesoderm forms by a multistep inductive inhibits dorsal mesoderm formation. process (reviewed in refs. 1-3). During early development, Previous studies did not determine by which signaling pathway vegetal pole cells exist in two states, either dorsal or ventral (4, BMP-4 inhibited dorsal mesoderm formation. We show that 5). The most dorsal mesoderm (Spemann organizer) is induced overexpression of BMP-4 inhibits two signaling pathways regu- in the subequatorial region (marginal zone, MZ) by underlying lating dorsal mesoderm formation. Overexpression of BMP-4 dorsal vegetal pole cells (Nieuwkoop center). Ventral meso- RNA suppresses the initial induction of dorsal mesoderm in derm (blood, mesenchyme) is induced in the ventral MZ whole embryos and in activin-treated animal caps, thus inhibiting (VMZ) by underlying ventral vegetal pole cells. During gas- formation of the Spemann organizer. BMP-4 also strongly inhib- trulation, a dorsalizing signal emanating from the Spemann its dorsalization of ventral mesoderm by the Spemann organizer organizer converts adjacent lateral mesoderm from ventral to or soluble noggin protein. Our results suggest that competition more dorsal fates such as muscle, heart, and pronephros (6-8). between noggin and BMP-4 activities may determine the eventual Since noggin is expressed in the Spemann organizer during limit of muscle formation in the MZ. and secreted noggin protein dorsalizes ventral mesoderm to muscle, noggin is a potential candidate molecule MATERIALS AND METHODS for acting as the endogenous dorsalizing signal (9, 10). Little Xenopus Embryos and Inducing Factors. Ovulation, in vitro is known about the regulation of the dorsalization process; it fertilization, embryo culture, and dissections were carried out is not clear what factors limit the extent of dorsalization within as described (19). Embryos were staged according to Nieuw- the marginal zone, thus demarcating which cells will eventually koop and Faber (20). PIF/activin was prepared from the make muscle and which cells will maintain a ventral fate. While cell line Activin-treated animal cap explants it is clear that cells fated for ventral mesoderm have the potential P338D1 (21). to differentiate into muscle when placed near the source of the Abbreviations: BMP-4, bone morphogenetic protein 4; MZ, marginal zone; VMZ, ventral MZ; DMZ, dorsal MZ; LMZ, lateral MZ; bFGF, The publication costs of this article were defrayed in part by page charge basic fibroblast growth factor; EF1a, elongation factor la; LCMR, payment. This article must therefore be hereby marked "advertisement" in low-Ca2+/Mg2+ modified Ringer's solution; DAI, dorsal-anterior index. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 12141 Downloaded by guest on September 27, 2021 12142 Developmental Biology: Re'em-Kalma et al. Proc. Natl. Acad. Sci. USA 92 (1995) were grown in 0.5 x modified Ringer's solution with gentami- RESULTS cin from stage 8 until stage 11. Noggin was prepared from CHO-B3 cell conditioned medium (22). Noggin-treated BMP-4 Induces Ventroposterior Mesoderm but Inhibits (0.25 x CHO-B3 conditioned medium) VMZ explants were Formation of Dorsal Mesoderm. We examined the inhibitory grown in 1 x low-Ca2+/Mg2+ modified Ringer's solution role of BMP-4 on activin induction of dorsal mesoderm. ref. 7) with from Previous studies showed that BMP-4 inhibited the ability of (LCMR; gentamicin stage 10-10.25 until activin to induce muscle in animal cap explants (12, 13). It is stage 13; at this stage, explants were transferred for further not clear whether BMP-4 mediated this effect by inhibiting growth in fresh lx LCMR lacking noggin. Control explants initial dorsal mesoderm induction or by inhibiting the dorsal- were grown in lx LCMR with 0.25 x conditioned medium ization of ventral mesoderm to muscle. Experiments were from the non-noggin-producing parental CHO cell line. Re- carried out to elucidate whether BMP-4 inhibits initial forma- combinant explants were dissected and grown in 1 x LCMR. tion of dorsal mesoderm (Spemann organizer). MZ explants were removed at stage 10-10.25, a stage at which We injected 2 ng of in vitro synthesized BMP-4 RNA into a pigment line is formed at the dorsal lip by involuting bottle one-cell fertilized Xenopus embryos. Animal caps were re- cells, thus enabling identification of presumptive dorsal and moved from blastulae (stage 8) and grown until the mid- ventral sides. A 45°-60° region of VMZ or dorsal MZ (DMZ) gastrula stage (stage 11). RNA was isolated for Northern was dissected with an eyebrow knife. Lateral MZ (LMZ) analysis to determine whether BMP-4 prevented induction of represents the remainder ofthe MZ after removal of DMZ and markers characteristic of dorsal mesoderm. The Xenopus VMZ. Recombinants were juxtaposed to one another with forkhead-1 (XFKH-1), goosecoid (gcd), and Xlim-1 genes are watchmaker's forceps. expressed in the Spemann organizer and are induced by activin RNA Injections. Capped synthetic Xenopus BMP-4 RNA in animal caps (26-28). These markers were used to measure was prepared from the pSP64T-XBMP-4+ vector (12) accord- the formation of dorsal mesoderm. XFKH-1, gcd, and Xlim-1 ing to Smith and Harland (23). Various concentrations of RNA (not shown) mRNAs were significantly reduced in BMP-4- (0.5-3.0 ng) were injected into embryos at the one-cell stage. injected, activin-treated gastrula-stage animal caps in compar- In each experiment, 25-50 embryos were allowed to develop ison to uninjected, activin-treated controls (Fig. 1A). These until stage 40. At this stage the dorsal-anterior index (DAI) effects were specific for BMP-4 RNA, since animal caps taken was determined (24). from embryos injected with 2 ng of prolactin RNA behaved Northern Blot Analysis. RNA was extracted from embryos like the uninjected controls (data not shown). At gastrula and prepared for Northern blot analysis (19). Electrophoresis, stages, BMP-4-injected whole embryos also expressed less gcd probe preparation, filter hybridization, and exposure were as mRNA (Fig. 1B), as well as less XFKH-1 and Xlim-1 mRNA described (19). Quantitation was performed with a Fuji phos- (data not shown), in comparison to control embryos. Analysis phor-imaging system. Elongation factor la (EFla) was used as of dissected DMZs from stage 10-10.25 BMP-4-injected em- a positive standard for comparing amounts of RNA loaded per bryos showed a >10-fold reduction in gcd mRNA versus well at any given stage (25). controls (Fig. 1B). In situ hybridization has also shown that gcd A Stage 1 1 Emb Animal Caps

Activin - + - + BMP-4 - -- + +

1 0+ Xbra a "* B Stage _ . Xwnt-8 W _ _Emb D V Emb D V C Emb Explants BMP-4 - - - + + + I - - --I Xpo - _l .ai BMP-4 (ng injected) - 1 2 3 - - - 1 2 3 Mix. - EE E E V D D-V D-V D-V D-V Xwnt-8 L _L gcd _* gcd m-actin II XFKH-1 - 3NW Xbra __4 i iNW

cx EF1 a _ _ EFla " "_- _ EFl a @ i 4 t

FIG. 1. Ectopic expression of BMP-4 inhibits dorsal mesoderm formation and induces ventral mesoderm in gastrula- and neurula-stage embryos and embryonic explants. (A) Embryos at the one-cell stage were injected with 2 ng ofin vitro synthesizedXenopus BMP-4 RNA. Animal cap explants were removed at stage 8 and treated with PIF/activin until stage 11. Total RNA was isolated from pools of five embryos (Emb) and 16 animal caps from each treatment at stage 11. One embryo equivalent of RNA was loaded per well for Northern analysis; all animal cap RNA was loaded per well. The filter was sequentially hybridized with Xenopus cDNA probes for goosecoid (gcd), forkhead-1 (XFKH-1), Brachyury (Xbra), Xwnt-8, Xpo, Mix.1, and EFla. (B) Embryos at the one-cell stage were injected with 2 ng of BMP-4 RNA. Total RNAwas isolated from pools offive injected (+) and uninjected (-) early gastrula (stage 10-10.25) embryos (Emb). Nine VMZ (V) and DMZ (D) regions were dissected and total RNA was isolated. One embryo equivalent of RNA was loaded per well for Northern analysis; all of the RNA from the dissected regions was loaded. The filter was sequentially hybridized with cDNA probes for gcd, Xwnt-8, Xbra, and EFla. (C) Embryos at the one-cell stage were injected with 1, 2, or 3 ng of BMP-4 RNA. At stage 10+, DMZs were removed from injected and noninjected embryos. DMZ explants were recombined with VMZ explants taken from uninjected embryos at the same stage. Ten recombinant explants from each of the three injection and control groups were grown in 1 x LCMR until stage 20 and total RNA was isolated for Northern analysis. As controls, nine DMZ explants from each ofthe three injection groups as well as nine VMZ and DMZ explants from the noninjection group were grown to stage 20 for RNA isolation. Total RNA was also isolated from pools of five control and BMP-4-injected embryos (Emb, E). For Northern analysis, 1 embryo equivalent of RNA was loaded per well and 7 explant equivalents was loaded. Filters were sequentially hybridized with Xenopus cDNA probes for muscle-specific actin (m-actin) and EFla. On all filters, muscle actin mRNA is the lower band; the upper band is cross-hybridization to cytoskeletal actin mRNA. Only explants from the uninjected DMZs are shown; the BMP-4-injected DMZs did not express significant levels of muscle actin. Embryos injected with 1, 2, and 3 ng had a DAI of 0.1; muscle actin expression was inhibited by a factor of 25-50. Downloaded by guest on September 27, 2021 Developmental Biology: Re'em-Kalma et al. Proc. Natl. Acad. Sci. USA 92 (1995) 12143 expression is inhibited in BMP-4-injected embryos (29). Initial could possibly act as an inhibitor of the dorsalization process. activation of gcd transcription in BMP-4-injected embryos was To address BMP-4's role as a repressor of dorsalization, we similar to that reported by Hogan et al. (30) examined whether noggin activity was inhibited by BMP-4. The Spemann organizer induces muscle in the VMZ region VMZ explants taken from BMP-4-injected embryos were (6). To address the effect of BMP-4 on organizer dorsalization removed at stage 10-10.25 and treated with noggin. VMZs activity, recombinant explants were made between stage 10+ were grown until stage 20 and RNA was isolated for Northern Spemann organizer regions (DMZs) from BMP-4-injected analysis. Dorsalization activity of noggin was completely in- embryos and VMZs from noninjected control embryos. Ex- hibited in VMZ explants taken from embryos injected with 2 plants were grown to stage 20 and RNA was isolated for ng of BMP-4 RNA; muscle actin transcripts were not detected Northern analysis (Fig. 1C). In recombinant DMZ-VMZ (decrease by a factor .25) in comparison to VMZs dorsalized explants from normal embryos, high levels of muscle actin by noggin (Fig. 2A). Noggin and BMP-4 appear to compete expression were observed (Fig. 1C). In recombinant explants with one another; by titration of injected BMP-4 RNA between comprising a DMZ from BMP-4-injected embryos (1-3 ng) 0.5 to 2.0 ng, it was shown that noggin could overcome BMP-4 and a VMZ from control embryos, muscle actin expression was inhibition and rescue dorsalization in VMZ explants (Fig. 2A). reduced by a factor of 6-50 (Fig. 1C). In addition, BMP-4- Noggin partially rescued dorsalization in VMZs taken from the injected DMZ-VMZ explants did not elongate in comparison 0.5- and 1.0-ng groups; muscle actin expression was inhibited to controls (data not shown). These results suggest that ectopic by a factor of 7 in the 1.0-ng group and by a factor of only 2 expression of BMP-4 inhibits the functional ability of a Spe- in the 0.5-ng group in comparison to control VMZs treated mann organizer to dorsalize ventral mesoderm to muscle. with noggin (Fig. 2A). BMP-4's inhibitory effect in VMZs is Previous studies have suggested that BMP-4 can induce not simply repression of muscle actin transcription, since ventroposterior mesoderm, since Xhox-3 mRNA was ex- pressed in BMP-4-injected neurula-stage animal caps (12, 13). Embryos VMZ We have extended these findings by examining a battery of A inducible mesoderm markers expressed in gastrula-stage ani- noggin I - + + + + mal cap explants removed from BMP-4-injected embryos. By BMP-4 (ng injected) - 2.0 1.0 0.5 - - 2.0 1.0 0.5 Northern analysis, we assayed expression of four additional I I mesoderm markers, the Brachyury (Xbra), Xwnt-8, Xpo, and

Mix.1 genes (31-34). Xbra is expressed in all gastrula meso- m-actin .5 : -, 5 derm, including the Spemann organizer region. Xwnt-8 and Xpo are expressed mainly in ventroposterior/nonorganizer Ef 1 a -_ m__*r _ _ mesoderm (23, 32, 35). Mix.1 is an early inductive response gene expressed in the vegetal pole and marginal zone of Emb Explants gastrulae (34). BMP-4 induced expression of high levels of the B Imb Xbra, Xwnt-8, Xpo, and Mix.1 transcripts in animal caps (Fig. 1A). Xenopus MyoD (XMyoD), which is expressed in ventral BMP-4 injected - + - + - + - + mesoderm at gastrula stages (19), was also transiently tran- E E VV D D D-V D-V scribed in BMP-4-injected animal caps (data not shown). I I 1 I In BMP-4-injected activin-treated animal caps, Xbra, Xwnt-8, Xpo, and Mix.1 were expressed at the same levels as m-actin ** in BMP-4-injected caps (Fig. 1A), suggesting that BMP-4 induces ventral mesoderm in a dominant manner. This effect EF1 . _ was striking when Xwnt-8 and Xpo expression was examined; activin induced low Xwnt-8 and Xpo expression in animal caps; however, BMP-4 and BMP-4/activin-treated animal caps ex- FIG. 2. Ectopic expression of BMP-4 inhibits dorsalization of VMZ pressed high levels of Xwnt-8 and Xpo RNA (Fig. 1A). by noggin and Spemann organizer. (A) Embryos at the one-cell stage BMP-4-injected whole embryos expressed mesodermal were injected with 2.0, 1.0, or 0.5 ng of BMP-4 RNA. Eighteen VMZ explants were removed from uninjected and injected groups of em- markers in an analogous pattern. In gastrula embryos, gcd, bryos (stage 10-10.25). Nine explants from each group were treated XFKH-1, and Xlim-1 transcripts were decreased by BMP-4 with noggin until stage 12.5-13, and nine untreated explants served as whereas Xbra and Xwnt-8 transcripts (Fig. 1B) as well as Xpo, controls. Explants from each group were grown to stage 20 and total Mix.1, and XMyoD transcripts (data not shown) were in- RNA was isolated. Total RNA was also isolated from pools of five creased. DMZs isolated from stage 10-10.25 BMP-4-injected whole embryos from the control and the three BMP-4 injected groups. embryos were ventralized; they expressed high levels of Xwnt-8 For Northern analysis, 1 embryo equivalent of RNA was loaded per mRNA instead of gcd mRNA; control DMZs expressed no well and 5 explant equivalents was loaded. Filters were sequentially detectable Xwnt-8 mRNA (Fig. 1B). Xbra expression was hybridized with Xenopus cDNA probes for muscle actin (m-actin) and about 50% higher in the DMZ and VMZ of injected embryos EFla. Of the untreated VMZs (- noggin) only the noninjected group in DMZ is shown; the BMP-4-injected VMZ (- noggin) groups also did not than controls (Fig. 1B). BMP-4-injected explants also express m-actin. In whole embryos injected with 1-2 ng of BMP-4 failed to elongate after growth in culture to neurula stages in RNA, m-actin levels were suppressed by a factor of 25-50 (DAI = 0.1). comparison to control explants, thus resembling VMZ ex- In embryos injected with 0.5 ng of BMP-4 RNA, m-actin expression plants (data not shown). These results suggest that BMP-4 can was inhibited by a factor of 6 (DAI = 0.4). (B) Embryos at the one-cell induce ventroposterior mesoderm while simultaneously inhib- stage were injected with 2 ng of BMP-4 RNA. At stage 10+, VMZs iting dorsal mesoderm formation (12, 13). were removed from injected and noninjected embryos. These VMZ BMP-4 Inhibits Dorsalization of Ventral Mesoderm to explants were recombined with DMZ explants taken from uninjected Muscle. Embryonic muscle forms by the dorsalization of embryos at the same stage. Twelve recombinant explants from BMP-4 ventral mesoderm to a more dorsal fate by the Spemann injected and noninjected control VMZs were grown in 1 x LCMR until stage 20 and total RNA was isolated. As controls, nine explants each organizer (6, 7). The noggin molecule mimics this signal; of BMP-4 injected VMZ, control VMZ, and control DMZ were also gastrula-stage VMZ explants differentiate to muscle after grown until stage 20 and total RNAwas isolated. Total RNAwas isolated noggin treatment (9). The dorsal inhibitory activity of BMP-4 from pools of five control and BMP-4-injected embryos (Emb, E). For may not be exclusively restricted to induction of dorsal meso- Northern analysis, 1 embryo equivalent of RNA was loaded per well and derm. BMP-4 mRNA is expressed throughout the MZ at the 9 explant equivalents of RNA was loaded. Filters were sequentially onset of gastrulation (see Fig. 3); thus during gastrulation it hybridized with cDNA probes for muscle actin (m-actin) and EFla. Downloaded by guest on September 27, 2021 12144 Developmental Biology: Re'em-Kalma et al. Proc. Natl. Acad. Sci. USA 92 (1995)

XMyoD expression was also inhibited in BMP-4/noggin- Stage 10+ Stage 20 treated VMZs in comparison to noggin-treated controls (data not shown). In addition, BMP-4-injected VMZ explants did Emb D L V Emb V I X 3X 8X not extend to the same extent as dorsalized VMZ explants (data not shown). Inhibition of noggin by BMP-4 RNA is a BMP-4 BMP-4 specific effect; noggin-treated VMZs removed from embryos l. injected with other translatable RNAs, encoding a truncated FGF receptor (dS0) or prolactin, underwent normal dorsal- Xwnt-8 m-actin *i urn ization after noggin treatment (data not shown). 6 w Inhibitory effects were *n also observed in embryos zygotically t*o 0 expressing BMP-4 from the cytomegalovirus (CMV) promoter. gcd EF1xl_a DNA (100-150 pg per cell) was injected into the MZ of both blastomeres at the two-cell stage. Muscle actin expression in EFla uu _ noggin-treated VMZs from CMV-BMP-4-injected embryos was reduced by a factor of 2 in comparison to embryos injected with FIG. 3. Dorsalization of the VMZ lowers BMP-4 expression. At plasmid DNA containing only the CMV promoter (data not stage 10+, nine VMZs, LMZs, and DMZs (D, L, and V) were shown). This result resembles effects observed when lower levels dissected and total RNA was immediately isolated from these three regions. In addition, nine VMZ explants were treated with increasing of BMP-4 RNA (0.5 ng) were injected, and it is most likely the concentrations of noggin protein: 1 x, 3 x, and 8 x. The noggin result of mosaic expression from the CMV promoter. preparation was concentrated 6.7-fold by Centricon filtration (Ami- To additionally address the effect of BMP-4 on endogenous con). The 1 x concentration is comparable to the amount of noggin dorsalization of ventral mesoderm within the embryo, recom- used in the standard dorsalization assay. In this modified dorsalization binant explants were made between stage 10+ Spemann assay the ratio of noggin and/or control supernatant was raised to 30% organizer regions (DMZ) from normal embryos and VMZs of the total volume in comparison to 25% in the standard assay. from BMP-4-injected and control embryos (Fig. 2B). Explants Noggin-treated and control VMZs were grown to stage 20 and total were grown until stage 20 and total RNA was isolated for RNA was isolated. Total RNA was also isolated from pools of five Northern analysis. Neither organizer nor VMZ was specified control embryos at stage 10+ and stage 20. For Northern blot analysis, to was 1 embryo equivalent of RNA was loaded per well and 9 explant differentiate into muscle, since muscle actin expression equivalents of RNA was loaded. The stage 10+ filter was sequentially not observed in these explants (Fig. 2B). In recombinant hybridized with cDNA probes for BMP-4, Xwnt-8, gcd, and EF1 . The DMZ-VMZ explants from normal embryos, high levels of stage 20 filter was sequentially hybridized with cDNA probes for muscle actin expression were observed (Fig. 2B). In recom- BMP-4, muscle actin (m-actin), and EFla. There are two BMP-4 binant explants comprising a VMZ from a BMP-4-injected transcripts, of 1.9 and 3.1 kb (12, 14). embryo and a DMZ from a control embryo, muscle actin expression was reduced by a factor of 6 (Fig. 2B). These results riched expression of BMP-4 in ventral mesoderm suggests that suggest that ectopic expression of BMP-4 in the VMZ inhibits BMP-4 may play a role in defining the dorsal/ventral border its ability to respond to the natural dorsalizing signal. When the within the embryo. DMZ used in the recombinant explant was specified to make muscle, the dorsalization inhibitory effect of BMP-4-injected DISCUSSION VMZs was decreased; muscle actin expression was reduced by a factor of only 3 (data not shown). This phenomenon may be We have shown that ectopic expression of BMP-4 mRNA related to subtle variations in the time of DMZ removal or disrupts two signaling pathways regulating dorsal induction. perhaps to differing sizes of organizer tissue. The Spemann organizer is inhibited by BMP-4, losing the BMP-4 Expression Is Lowered in VMZ Explants Dorsalized ability to dorsalize ventral mesoderm to muscle (Fig. 1C). by Noggin. BMP-4 is equally expressed throughout the whole Goosecoid, XFKH-1, and Xlim-1 mRNAs are reduced in MZ at stage 10.5 (12) but isventrally localized by stage 11-11.5 either whole embryos or activin-treated animal caps which (29, 36). We extended these findings by examining the speci- express ectopic levels of BMP-4 (Fig. 1 A and B). In whole fication of BMP-4 expression in dorsalized explants grown in embryos, the DMZ is ventralized, since it expresses high levels culture from stage 10-10.25 until stage 20. By dissection of the of Xwnt-8 mRNA, but remarkably low levels of goosecoid marginal zone and Northern analysis, we found that BMP-4 mRNA (Fig. 1B). BMP-4 appears to act as a strong inducer of was expressed equally throughout the marginal zone at the ventral mesoderm; we observed high Xbra, Xwnt-8, Xpo, onset of gastrulation (Fig. 3). Expression of Xwnt-8 and gcd Mix.1, and XMyoD expression in gastrula-stage animal caps and served as controls for dissection accuracy (Fig. 3). VMZs whole embryos (Fig. 1 A and B). Thus, formation of a functional removed from early gastrula and grown to late neurula ex- Spemann organizer is severely impaired by overexpression of pressed higher levels of BMP-4 mRNA relative to VMZ BMP-4. These results suggest that BMP-4 activity may regulate explants dorsalized by noggin. Dorsalized VMZs downregulate the extent of organizer formation in the embryo (Fig. 4). BMP-4 mRNA in a noggin concentration-dependent manner; We also have shown that dorsalization of ventral mesoderm over an 8-fold noggin concentration range, BMP-4 expression to muscle by a functional Spemann organizer is inhibited by was downregulated by a factor of -25 (Fig. 3). In noggin- ectopic BMP-4 expression in the VMZ. BMP-4-injected VMZs dorsalized VMZs, a correlation was seen between increases in treated with noggin or recombined with DMZ had highly muscle actin mRNA and decreases in BMP-4 transcripts (Fig. reduced expression of muscle actin (Fig. 2). 3). Thus while BMP-4 inhibited noggin protein activity, noggin Noggin could be the target of BMP-4 in both of these downregulated expression of BMP-4 mRNA by dorsalizing inductive pathways (Fig. 4), since noggin appears to have both VMZ to muscle. In situ hybridization of mid-gastrula and maternal and zygotic functions (9, 10). Noggin can rescue neurula embryos with BMP-4 shows enriched expression in Nieuwkoop center formation in UV-ventralized embryos, ventral versus dorsoanterior regions of the embryo (29, 36). suggesting that maternal noggin is involved in the dorsal/ Thus while BMP-4 expression starts out equally throughout the ventral polarity established during the first cell cycle (10). In MZ at the onset of gastrulation, the dorsalization process addition, zygotically acting noggin can function as the dorsal- restricts transcription to ventral areas. During normal devel- izing activity of the Spemann organizer (9). opment, competition between noggin and BMP-4 may define Our results support a model in which BMP-4 and noggin which cells eventually end up with ventral fates; noggin down- compete to regulate dorsalization of ventral mesoderm (Fig. regulates BMP-4 expression in LMZ fated for muscle. En- 4). Ventral mesoderm has a potential to be dorsalized to Downloaded by guest on September 27, 2021 Developmental Biology: Re'em-Kalma et aL Proc. Natl. Acad. Sci. USA 92 (1995) 12145

Animal Pole state of inhibition by BMP-4. The major role of dorsalization may be to unmask this repressed state by turning off BMP-4 thus enabling the VMZ to differentiate muscle. Graff et al. (18) suggest that while BMP-4 makes ventral mesoderm in a dominant manner, there must be an endogenous signal which can override its signaling. We suggest that the ability of noggin to shut down BMP-4 expression enables dorsalization in dorsolateral meso- Ventral Spemann Organizer derm adjacent to the organizer. In contrast, the maintenance of Marginal Zone high levels of BMP-4 in more ventrolateral regions farther away from the organizer suffices to produce ventral mesoderm while simultaneously inhibiting the dorsalization process. Nieuwkoop Center We thank Drs. R. Harland, J. Smith, I. Dawid, W. Smith, A. Hemmati-Brivanlou, M. Jamrich, E. Amaya, A. Fainsod, and J. Yisraeli for plasmids. We thank Dr. A. Fainsod for sharing unpub- lished data. This research was supported by a grant from the Israel Vegetal Pole Science Foundation (Israel Academy of Sciences and Humanities) and by Bruce Rappaport and Technion research funds. FIG. 4. Model of the interaction between BMP-4 and noggin activi- ties. Zygotic emanates 1. New, H. V., Howes, G. & Smith, J. C. (1991) Curr. Opin. Genet. Dev. 1, function. Noggin activity (N) from the Spemann 196-203. organizer and dorsalizes the ventrally fated LMZ. Noggin inhibits BMP-4 2. Kimelman, D., Christian, J. L. & Moon, R. T. (1992) Development (Cam- expression in dorsalized marginal zone, thus BMP-4 (B) expression is bridge, U.K) 116, 1-9. restricted to the ventral marginal zone. Noggin signaling weakening across 3. Sive, H. (1993) Genes Dev. 7, 1-12. the LMZ and the maintenance of high BMP-4 levels in VMZ cells will 4. Nieuwkoop, P. D. (1969) Roux's Arch. Dev. Bio. 162, 341-373. cause an equilibrium in which spreading of the dorsalization signal is 5. Sudwarti, S. & Nieuwkoop, P. D. (1971) Roux's Arch. Dev. Bio. 166, stopped and the lateral/ventral mesoderm junction is formed. Maternal 189-204. function. Noggin activity (N) and Vg-1/activin activity (Vgl) function as 6. Dale, L. & Slack, J. M. (1987) Development (Cambridge, U.K) 100, 279- the Nieuwkoop center. Maternal BMP-4 (B) activity could inhibit spread- 295. ing of the Nieuwkoop signal to the presumptive 7. Stewart, R. M. & Gerhart, J. C. (1990) Development (Cambridge, U.K) 109, ventral side, thus 363-372. regulating formation of the Spemann organizer. 8. Lettice, L. A. & Slack, J. M. (1993) Development (Cambridge, U.K) 117, 263-271. muscle if it is close to the noggin source; competition between 9. Smith, W. C., Knecht, A. K., Wu, M. & Harland, R. M. (1993) Nature noggin and BMP-4 activities determines the final extent of (London) 361, 547-549. muscle fomtnation in the LMZ/VMZ. High levels of BMP-4 10. Smith, W. C. & Harland, R. M. (1992) Cell 70, 829-840. 11. Lyons, K. M., Jones, C. M. & Hogan, B. L. M. (1991) Trends Genet. 7, inhibit muscle formation in whole embryos and noggin-treated 408-412. VMZ explants. When levels of injected BMP-4 RNA in 12. Dale, L., Howes, G., Price, M. J. & Smith, J. C. (1992) Development explants were decreased from 2 ng to 0.5 ng, noggin-treated (Cambridge, UK) 115, 573-585. explants underwent significant dorsalization rescue (Fig. 2A). 13. Jones, C. M., Lyons, K. M., Lapan, P. M., Wright, C.V.E. & Hogan, B. L. M. (1992) Development (Cambridge, UK) 115, 639-647. We have demonstrated that noggin downregulates expression 14. Koster, M., Plessow, S., Clement, J. H., Lorenz, A., Tiedmann, H. & of endogenous BMP-4 mRNA in a concentration-dependent Knochel, W. (1991) Mech. Dev. 33, 191-200. manner in dorsalized VMZ explants, thus potentially lowering the 15. Kimelman, D. & Kirschner, M. (1987) Cell 51, 869-877. VMZ's dorsalization inhibition capacity (Fig. 3). Over an 8-fold 16. Green, J. B. A., Howes, G., Symes, K., Cooke, J. & Smith, J. C. (1990) noggin concentration range, BMP-4 expression dropped to Development (Cambridge, U.K) 108,173-183. nearly 17. Cooke, J. (1989) Development (Cambridge, UK) 107, 229-241. undetectable levels in VMZs; a concurrent increase in muscle 18. Graff, J. M., Thies, R. S., Song, J. J., Celeste, A. J. & Melton, D. A. (1994) actin expression was observed in the same explants. These results Cell 79, 169-179. suggest that cells maintain a certain plasticity when responding to 19. Frank, D. & Harland, R. M. (1991) Development (Cambridge, UK) 113, BMP-4 and noggin activities. Cells farther away from the orga- 1387-1393. 20. Nieuwkoop, P. & Faber, J. (1967) Normal Table of Xenopus laevis Am- nizer receive less noggin signaling activity and maintain higher sterdam (Daudin) (North-Holland). levels of BMP-4 expression. At some critical point, an equilibrium 21. Sokol, S., Wong, G. G. & Melton, D. A. (1990) Science 249, 561-564. is reached in which noggin signaling activity is relatively low and 22. Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A., the expression of BMP-4 is high enough so that the spreading of Stahl, N., Yancopolous, G. D. & Harland, R. M. (1994) Science 262, 713-718. dorsalization is stopped. Cells expressing relatively high levels of 23. Smith, W. C. & Harland, R. M. (1991) Cell 67, 753-765. BMP-4 and receiving relatively low levels of noggin signaling will 24. Kao, K. & Elinson, R. (1988) Dev. Biol. 127, 64-77. not form muscle, and the dorsolateral-ventral mesoderm junction 25. Krieg, P. A., Vamum, S. M., Wormington, W. M. & Melton, D. A. (1989) is formed (Fig. 4). Dev. Biol. 133, 93-100. 26. Blumberg, B., Wright, C. V. E., De Robertis, E. M. & Cho, K. W. Y. (1991) A three-signal model has been suggested to describe induc- Science 253, 194-196. tion of mesoderm in Xenopus embryos (1). In this model, the 27. Dirksen, M. L. & Jamrich, M. (1992) Genes Dev. 6, 599-608. dorsalizing signal is defined as a dominant signal emanating 28. Taira, M., Jamrich, M., Good, P. J. & Dawid, I. B. (1992) Genes Dev. 6, from the organizer which converts ventrally fated cells in the 356-366. dorsolateral MZ to a more dorsal suggest 29. Fainsod, A., Steinbeisser, H. & De Robertis, E. M. (1994) EMBO J. 21, fate. Our data that 5015-5025. regulation of dorsalization is more complex than was previ- 30. Hogan, B. L., Blessing, M., Winnier, G. E., Suzuki, N. & Jones, C. M. ously shown. It is likely that negative signaling molecules such (1994) Development (Cambridge, UK), Suppl., 53-60. as BMP-4 participate in the dorsalization process in conjunc- 31. Smith, J. C., Price, B. M. J., Green, J. B. A., Weigel, D. & Herrmann, B. G. tion with positive regulating molecules such as noggin. Only by (1991) Cell 67, 79-87. 32. Christian, J. L., McMahon, J. A., McMahon, A. P. & Moon, R. T. (1991) the proper combination of positive and negative signaling Development (Cambridge, UK) 111, 1045-1055. pathways is the correct pattern established in the mesoderm. 33. Sato, S. M. & Sargent, T. D. (1991) Development (Cambridge, UK) 112, VMZs removed from embryos injected with a truncated 747-753. Xenopus BMP-4 receptor differentiate and elongate as muscle 34. Rosa, F. M. (1989) Cell 57, 965-974. 35. Amaya, E., Stein, P. A., Musci, T. J. & Kirschner, M. W. (1993) Develop- (18). This result suggests that elimination of BMP-4 signaling is ment (Cambridge, U.K) 118, 477-487. sufficient to dorsalize VMZ to muscle. It is possible that the VMZ 36. Schmidt, J. E., Suzuki, A., Ueno, N. & Kimelman, D. (1995) Dev. Biol. 196. has a latent potential to differentiate muscle which is in a constant 37-50. Downloaded by guest on September 27, 2021