Development 125, 431-442 (1998) 431 Printed in Great Britain © The Company of Biologists Limited 1998 DEV3723

XBMPRII, a novel Xenopus type II mediating BMP signaling in embryonic tissues

Amanda Frisch and Christopher V. E. Wright* Department of Cell Biology, Vanderbilt University Medical School, 1161 21st Avenue South, Nashville, TN 37232-2175, USA *Author for correspondence (e-mail: [email protected])

Accepted 17 November 1997: published on WWW 13 January 1998

SUMMARY

Bone Morphogenetic Proteins (BMPs) are potent tBR can induce partial secondary axes, marker analysis regulators of embryonic cell fate that are presumed to shows that tBRII-induced axes are more anteriorly initiate in recipient cells through extended. Additionally, coinjection of tBRII and tBR multimeric, transmembrane, serine/threonine kinase synergistically increases the incidence of secondary axis complexes made up of type I and type II receptors. formation. BMPRII was identified previously in mammals as the only A truncated activin type II receptor (∆XAR1) is known type II receptor that binds BMPs, but not activin or TGFβ, to block both activin and BMP signaling in vivo. Here we in vitro. We report the cloning and functional analysis in show that such crossreactivity does not occur for tBRII, in vivo of its Xenopus homolog, XBMPRII. XBMPRII is that it does not affect activin signaling. Furthermore, our expressed maternally and zygotically in an initially studies indicate that the full-length activin type II receptor unrestricted manner. Strikingly, XBMPRII transcripts then (XAR1) overcomes a block in BMP4 signaling imposed by become restricted to the mesodermal precursors during tBRII, implicating XAR1 as a common component of BMP gastrulation. Expression is lower in the dorsal organizer and activin signaling pathways in vivo. These data region, potentially providing a mechanism to suppress the implicate XBMPRII as a type II receptor with high actions of BMP4 on dorsally fated tissues. Similar to the selectivity for BMP signaling, and therefore as a critical results seen for a truncated type I BMP receptor (tBR), a mediator of the effects of BMPs as mesodermal patterning dominant-negative form of XBMPRII (tBRII) can dorsalize agents and suppressors of neural fate during ventral mesoderm, induce extensive secondary body axes, embryogenesis. block mesoderm induction by BMP4 and directly neuralize ectoderm, strongly suggesting that XBMPRII mediates Key words: Bone morphogenetic protein (BMP), Type II BMP BMP signals in vivo. However, although both tBRII and receptor, Activin, Mesoderm induction, Neural induction, Xenopus

INTRODUCTION a close linkage with ventral specification. During gastrulation, for example, transcripts become restricted from the Determination of mesodermal cell fates in the marginal zone presumptive neural plate and dorsal marginal zone, while being of the early Xenopus laevis embryo is thought to be specified maintained in ventral regions of the embryo (Fainsod et al., by their location within a dorsoventral gradient of instructive 1994; Hemmati-Brivanlou and Thomsen 1995; Schmidt et al., signals (for review see Slack, 1994). Early models postulated 1995). Third, the secreted organizer molecules and that the signals establishing this instructive gradient emanated can functionally inhibit BMP4 through direct binding from the dorsally located Spemann’s Organizer. However, (Piccolo et al., 1996; Zimmerman et al., 1996). These latter recent evidence suggests that at least some dorsally derived findings suggest a mechanism for generating a BMP4 activity signals are diffusible long-range inhibitors of ventralizing gradient in the marginal zone despite the initially uniform signals, and that it is their degree of attenuation of a dominant distribution of BMP4 RNA. In this model, the local ventralizing influence that instructs marginal zone fate (e.g. concentration of active BMP4 in the marginal zone is regulated Piccolo et al., 1996; Zimmerman et al., 1996). by the ratio of BMP4 to noggin/chordin and this serves to Many lines of evidence implicate BMP4 as an instructive instruct the different dorsoventral mesodermal cell fates. ventral signal. First, BMP4 is a ventral mesoderm inducer Future modifications to this model may arise from the finding (Dale et al., 1992; Jones et al., 1992; Koster et al., 1991) that that BMP4/7 heterodimers are more active than either can suppress the dorsal mesoderm-inducing activities of activin homodimer (Suzuki et al., 1997), although the existence of and noggin (Dale et al., 1992; Jones et al., 1992, 1996; Re’em- endogenously occurring heterodimers is under investigation. Kalma et al., 1995). Second, BMP4 expression patterns suggest Nevertheless, strong genetic evidence for an early requirement 432 A. Frisch and C. V. E. Wright for BMP4 signaling in vertebrate embryogenesis comes from stages when dorsal and ventral signals are being actively the observation that most homozygous null BMP4 mouse interpreted to determine mesodermal cell fate. We describe embryos arrest development during gastrulation and lack experiments indicating that XBMPRII mediates the effects of embryonic mesoderm (Winnier et al., 1995). BMP4 on mesodermal patterning and suppression of neural BMP4 signals are also thought to be important in fate. Our data suggest substantial differences between the in maintaining an epidermal cell fate within uninduced ectoderm vivo effects of a dominant-negative isoform of XBMPRII, (for review see Hemmati-Brivanlou and Melton, 1997). tBRII, and a dominant-negative type I BMP receptor, tBR Prolonged dissociation of Xenopus ectodermal cells causes (Graff et al., 1994; Suzuki et al., 1994). Moreover, we show their neuralization, but an epidermal fate is maintained when that XBMPRII differs significantly from the activin type II sufficient BMP4 protein is added to the medium bathing the receptor XAR1, since XAR1 can mediate both activin and dispersed cells (Wilson and Hemmati-Brivanlou, 1995). BMP signaling in vivo, whereas XBMPRII appears to be Furthermore, disrupting BMP signaling within intact animal specific for BMPs. caps by several methods, i.e. inhibiting receptor activity (Wilson and Hemmati-Brivanlou, 1995; Xu et al., 1995), adding BMP4 inhibitory proteins (Piccolo et al., 1996), or MATERIALS AND METHODS overexpressing dominant-negative BMP4 isoforms (Hawley et al., 1995), results in direct neural induction. The clearing of Isolation of XBMPRII BMP4 RNA from the presumptive neural plate (Hemmati- A BamHI-EcoRI cDNA fragment encoding C-terminal sequences of Brivanlou and Thomsen, 1995; Schmidt et al., 1995) is mouse BMPRII (gift from M. Kawabata and H. Moses) was used to consistent with these findings. screen ~2 million pfu of a dorsal lip cDNA library (Blumberg et al., As TGFβ-related molecules, BMP signals are presumably 1991) at a stringency giving single-copy bands on Xenopus genomic transduced through receptor complexes consisting of type I and Southern blots. Clone ADL12 encodes amino acids 392 to 1020 at the C terminus of XBMPRII. An ADL12-based screen of a stage 28-30 type II transmembrane serine/threonine kinases (for review see λ ten Dijke et al., 1996, Hogan, 1996). In general, type II ZAPII cDNA head library (Hemmati-Brivanlou et al., 1991) gave an additional ~400 bp of 5′ sequence. PCR amplification of this region receptors primarily bind the ligand, while type I receptors, after and rescreening the head library led to two independent cDNAs. Clone cross-phosphorylation by ligand-bound type II receptors, C6 was sequenced on both strands using the Sequenase II (USB) initiate nuclear signal transduction by activating downstream and shown to contain a 3.4 kb open reading frame representing the targets like the Smad proteins (Massague, 1996). BMP entire protein coding sequence, with approximately 150 bp and 350 receptors differ somewhat from this paradigm in that both type bp of 3′ and 5′ untranslated sequence, respectively. I and type II receptors can apparently bind ligand independently, although high affinity binding and signal Dominant negative receptors transduction require both receptor subtypes (Liu et al., 1995; A dominant negative human BMPRII (hDNTALK) was generated by Nohno et al., 1995; Rosenzweig et al., 1995). Additional PCR amplifying the human BMPRII clone CL4-1-1 (Kawabata et al., complexity may be superimposed on this type I/type II 1995), truncating the receptor 10 amino acids after the transmembrane domain. This fragment was MscI/BstEII-digested and exchanged for heteromer model based on the findings of fairly promiscuous the β-globin insert in pSP64TXβm (Krieg and Melton, 1984). A associations amongst several different type I and type II similarly truncated fragment (tBRII) was made from XBMPRII clone receptors, and that some receptors bind multiple ligands (Liu C6. The amplified fragment was EcoRI/XbaI-digested and ligated into et al., 1995; Rosenzweig et al., 1995). pCS2+ (Turner and Weintraub, 1994). While screening for XBMPRII, Although past studies on TGFβ/BMP signaling pathways initial experiments, including generation of secondary axes, rescue of during Xenopus embryogenesis have focused mainly on the UV ventralization, and neural induction in animal caps, were done ligands, a complete understanding of how these molecules with hDNTALK. After cloning XBMPRII, experiments were repeated exert their effects in vivo will require a similar analysis of their several times with tBRII with similar results; subsequent experiments receptors, including details of their expression patterns during used only the Xenopus construct. Figs 3 and 5A represent results relevant embryonic processes, and their ligand-binding and generated with hDNTALK constructs. All experiments were repeated signaling specificity. For many TGFβ-related molecules likely at least twice with similar results, except for those shown in Fig. 7, which gave the same result twice. to be key developmental regulators (e.g. Vg1 and -related factors), specific receptors have not been identified. Moreover, Whole-mount in situ hybridization for the identified receptors, only limited information exists on In situ hybridization was performed as described previously (Harland, their spatiotemporal expression patterns, particularly during 1991) with improvements communicated by the author. Antisense those stages of development when basic decisions regarding XBMPRII probe was made from EcoRI-linearized ADL12 plasmid cell fate and axial specification are being made. and T7 RNA polymerase; sense probes were made from XhoI-digested Recently, a novel type II receptor that binds BMP2, BMP4 ADL12 and T3 RNA polymerase. Probes for HoxB9, BMP-4, nrp-1, and BMP7, but not activin or TGFβ, was identified from Xotx2, Krox20, XEn2, Xbra and goosecoid were prepared as described mammalian cells (Kawabata et al., 1995; Liu et al., 1995; previously; specific details may be obtained upon request. Nohno et al., 1995; Rosenzweig et al., 1995). This receptor, Fertilization and manipulation of embryos known as BMPRII, T-ALK, or BRK3, has until now only been Xenopus eggs were fertilized in vitro, injected and manipulated as characterized in vitro. Given the particular suitability of the described (Kay and Peng, 1991), and staged according to Nieuwkoop frog embryo system for functional analyses in vivo, we have and Faber (1967). Animal caps were explanted at stage 8-9 in 1× RSB cloned and characterized the Xenopus homolog, XBMPRII. We and cultured on agarose in 0.75× NAM until collection, when tissues report its expression pattern during embryogenesis, including were either frozen (RNA analysis) or fixed (histology). Vegetal the first published description of any BMP receptor during explants were isolated at stage 8 as described (Gamer and Wright, BMP type II receptor signaling 433

1995). Dorsal and ventral marginal zones (DMZs and VMZs) were number U81958). Northern blot analysis with a C6 cDNA probe isolated at stage 10.25-10.5, with the blastopore lip marking the dorsal on poly(A)+ RNA from embryonic stages 9, 15 and 22 detected side. DMZs corresponded to an ~60° arc of marginal zone tissue transcripts of ~11 kb (data not shown), similar to the size of centered on the dorsal lip midline, VMZs were cut analogously from human BMPRII mRNA (Kawabata et al., 1995). the region opposite the DMZ and both were cultured in 0.75× NAM until collection. XBMPRII expression Preparation of mRNA and microinjections Fig. 1B shows that XBMPRII is expressed both maternally and Capped mRNA was made with the mMessage mMachine kit zygotically. Transcript levels decreases during early (Ambion). hDNTALK and tBRII plasmids were linearized with gastrulation (stage 10.25), but then increase from the gastrula BamHI or XhoI, respectively, and transcribed with SP6 RNA through late tadpole stages. The spatial expression pattern of polymerase. During injection, embryos were immersed in 5% Ficoll XBMPRII (Fig. 2) was determined by whole-mount in situ dissolved in 0.1× RSB. For animal cap experiments, 10 nl was injected hybridization. At stage 9, XBMPRII signal is stronger over the at the 1- to 2-cell stage. For injections at the 2- to 8-cell stage, 5 animal hemisphere (Fig. 2A), but since detection of transcripts nl/blastomere was injected. Dorsally or ventrally targeted injections by whole-mount in situ hybridization is often difficult in at the 4- to 8-cell stage used pigmentation differences to determine vegetal cells, we used RNAse protection (Fig. 2C) to detect the appropriate site of injection. XBMPRII RNA in dissected animal and vegetal explants. This Histological analysis analysis confirmed a higher level of XBMPRII RNA in animal Embryos were fixed in 70% ethanol overnight, dehydrated in an relative to vegetal cells. ethanol series, washed in xylene, soaked overnight in xylene:paraplast By pregastrula stages, XBMPRII expression is no longer (Oxford Labware; 1:1 ratio) and paraplast embedded. Sections (5-10 widespread but becomes localized to the torus of prospective µm) were stained with hematoxylin/eosin (Sigma/Surgipath). mesodermal cells at the equatorial region of the embryo. Some stage 10− embryos display a uniformly intense ring of staining, Immunohistochemistry but in slightly more advanced embryos (stage 10+), staining is Embryos were fixed in Dent’s solution (Kay and Peng, 1991) substantially reduced in the dorsalmost region of the marginal overnight in a Petri dish, or in MEMFA (Harland, 1991) for 2 hours zone, the site of the future Organizer (Fig. 2D). At early on a nutator. Pigmented embryos were bleached in 10% H2O2/70% gastrula, XBMPRII signal is detected in preinvoluting methanol under fluorescent light. Immunohistochemistry was performed as described (Hemmati-Brivanlou et al., 1991). The 12/101 mesodermal cells, being excluded from the superficial layer. and MZ15 antibodies were used at dilutions of 1:500 and 1:750, Expression does not extend to the involuting vegetalmost respectively, and the secondary goat anti-mouse/HRP-linked antibody margin of the dorsal lip (Fig. 2E), in contrast to the pan- (Jackson Immunoresearch) was used at a dilution of 1:1000. In some mesodermal marker, Xbra, whose expression in the cases, color reactions were enhanced by adding 0.04% NiCl2. mesodermal precursors at this stage extends to the dorsal lip (data not shown; Smith et al., 1991). RNAse protection At neurula stages, XBMPRII expression continues in A 190 bp HindIII fragment from clone C6 was subcloned into prospective mesoderm (Fig. 2G,H) but not the superficial pBluescript, XhoI-linearized and antisense RNA made with T7 RNA polymerase. Digestion after hybridization was with RNAses A and T1. Probes for EF1α, NCAM and Xbra were synthesized as described previously (information available upon request). RNA was isolated by SDS/proteinase K digestion and LiCl precipitation. RT-PCR RNA was isolated from embryonic tissues and RT-PCR performed as described (Chang et al., 1997). Specific primers for FGFR, goosecoid, NCAM, Xbra, Xwnt-8, muscle actin, Krox20, HoxB9 and Xotx2 were synthesized according to sequences reported (references available upon request). Primer pairs were retested to determine conditions under which transcript detection was in the linear range of amplification.

RESULTS

Xenopus BMPRII Overlapping cDNAs encoding XBMPRII were isolated by screening Xenopus libraries with a human BMPRII probe, of Fig. 1. Structure and expression of XBMPRII. (A) Functional which clone C6 contained a 3.4 kb open reading frame with short domains of XBMPRII are defined by percentage amino acid 5′ and 3′ untranslated regions. Based on the similarity in size and similarity to human BMPRII (Kawabata et al., 1995). The dominant negative XBMPRII (tBRII) is truncated 10 amino acids after the structure of the protein conceptually translated from C6 (Fig. ′ ′ putative transmembrane domain (arrow). PM, plasma membrane. 1A), the in-frame stop codons at the 5 and 3 ends of its open- (B) RNAse protection analysis during development (stages indicated reading frame, and the biological activities described below, we above the lanes) shows maternal and zygotic XBMPRII transcription. conclude that C6 contains the full protein-coding sequence of RT-PCR detection of the constitutive fibroblast growth factor XBMPRII (Xenopus BMP Receptor type II; GenBank accession receptor (FGFR) RNA was used as a loading control. 434 A. Frisch and C. V. E. Wright ectoderm (Fig. 2L). Weak staining along the dorsal midline is locally blocks the function of BMP4 (and/or related ligands), visible at this stage (data not shown), although the loss of signal with ventral delivery causing secondary axis induction and following sectioning prevented attributing this to notochordal or dorsal injection resulting in hyperdorsalization. We tested neural tube expression. By early tailbud, XBMPRII is primarily expressed in two ventrolateral mesodermal regions: one surrounding the future proctodeum (Fig. 2J,K) and extending dorsally towards the tail bud (Fig. 2J), and an anterior focus around the stomodeum/heart anlage (Fig. 2J). Weaker expression is apparent along the entire neural tube (Fig. 2K), with higher levels in the developing brain and around the eye (Fig. 2J). At the late tadpole stages (Fig. 2N,O), XBMPRII expression expands to include the neural tube, branchial arches, tail bud, eye, olfactory placode, otic vesicles, head mesenchyme and brain, but is absent from the notochord and somitic derivatives (data not shown). Fig. 2F,I,M,P shows that XBMPRII expression is reminiscent of that of BMP4 (Fainsod et al., 1994; Hemmati-Brivanlou and Thomsen, 1995; Schmidt et al., 1995), a finding consistent with the hypothesis that XBMPRII mediates BMP4 signaling during mesodermal and neural development in Xenopus embryogenesis. However, while BMP4 is expressed in the ventral blood islands (Fig. 2P) and migrating neural crest (Fainsod et al., 1994), XBMPRII was not detected in these areas (Fig. 2N). Secondary axis induction by dominant- negative XBMPRII Dominant-negative isoforms of TGFβ, BMP type I and activin receptors, which prevent the reciprocal receptor phosphorylation that initiates signal transduction, are generated by deleting the intracellular kinase domain (Chen et al., 1993; Graff et al., 1994; Hemmati-Brivanlou and Melton, 1992;). Previously, it had been reported that ventral Fig. 2. Spatiotemporal expression pattern of XBMPRII by whole-mount in situ expression of a human dominant-negative BMPRII hybridization. (A) Side view of a stage 9 blastula showing enhanced signal in resulted in secondary axes that lacked anterior the animal region. (B) Sense probe control – neurula stage. (C) The pattern in A structures (Ishikawa et al., 1995). Thus, to explore was confirmed by RNAse protection analysis of XBMPRII in animal cap (AC) the function of the frog cognate in vivo, we injected and vegetal pole (VP) explants from stage 9 embryos. EF1α was used as a Xenopus embryos with RNA encoding a similarly loading control (four AC or ten VP explants/lane). (D) Vegetal and dorsovegetal truncated XBMPRII isoform (Fig. 1A), referred to views of stage 10 (left) and 10.5 (right) embryos show expression restricted to as tBRII, and analyzed the resulting effects in more the marginal zone, with substantially less expression adjacent to the forming detail. Injecting tBRII RNA into the two dorsal dorsal lip (arrowheads). (E) Eosin-counterstained sections of stage 10.5 embryos blastomeres at the 4-cell stage resulted in show expression in preinvoluting mesodermal precursors (bracketed), but not extending to the dorsal lip margin (arrowhead). (G) At the neurula stage, anterodorsalized embryos with enlarged heads and expression is in the posterior, preinvoluting mesoderm. (H) Sectional analysis at diminished or absent posterior structures (data not this stage (plane indicated by bars in G) demonstrates a ring of signal shown), while ventral injections induced partial surrounding the yolk plug (YP), and at higher magnification (L – area boxed in secondary axes that could be distinguished from the H), the subepithelial localization of expression. (J) At late neurula stages, primary axis by the absence of a visible floorplate expression is concentrated in two areas of ventral mesoderm with fainter (Fig. 3B). The secondary axis resulted from a expression in the brain and eye (e). (K) Sections through the posterior focus bifurcation of the neural tube, but contained no (bars in J indicate plane) demonstrate subepithelially localized expression notochord or longitudinally arrayed somite blocks, extending from the proctodeum (PR) to midway up the lateral wall (bars although they did contain muscle tissue (Fig. 4D). indicate dorsal limit). Faint neural tube staining is seen (arrowhead). (N) and (O) While the secondary axes did not have proper heads, A complex pattern of expression is seen at the tailbud stage, including expression in neural tube (NT), brain (BR) and around the otic vesicle (OV). anterior structures were often seen, including otic CG, cement gland. F, I, M and P show BMP4 expression at similar stages vesicles, mucus-secreting cement glands (Fig. 4A) (photographs kindly provided by E. M. De Robertis), demonstrating a high and the occasional dense focus of pigmented tissue degree of overlap with XBMPRII expression. In B,G,I,J and M-P, anterior is to probably representing melanized retinal epithelium the left, and dorsal uppermost. Arrowheads in F and P indicate dorsal lip and (data not shown). We infer that tBRII overexpression ventral blood islands, respectively. BMP type II receptor signaling 435

Fig. 3. Secondary axis induction by ventral tBRII injection. (A) Wild-type and (B) tBRII-injected embryos at the neurula stage. The arrowhead indicates the secondary axis, which lacks a visible floorplate. (C) Wild-type embryo at the tadpole stage. (D) Embryo injected ventrally with tBRII exhibits a secondary axis (arrows) lacking normal head morphology. (E) H&E-stained section of a tadpole stage tBRII RNA-injected, secondary axis embryo. The endogenous axis contains organized somites (SO), neural tube (NT), notochord (NO) and gut (G), while the ectopic axis (boxed area) comprises highly disorganized tissues. (F) Higher magnification of area boxed in E. Arrowheads indicate representative melanocytes scattered throughout the ectopic tissue.

Fig. 4. Whole-mount analysis of secondary axes. (A) tBRII RNA- whether secondary axis induction was a specific effect of tBRII injected double-axis embryo. Black arrows and white arrowheads activity by coinjecting mRNA encoding the normal (wild-type) denote primary and secondary axes, respectively. Cement glands XBMPRII receptor and tBRII. As shown in Table 1, increasing (black arrowheads) are present in both axes, but the secondary axis the dose of wild-type receptor progressively reversed, and lacks recognizable head structures. tBRII injected embryos were eventually completely abrogated, the effects of tBRII. analyzed by in situ hybridization (B,C, E-H) or immunohistochemistry Members of the Smad family of intracellular effectors (D) to determine the tissue types present in the secondary axis. Arrows appear relatively specific for distinct signal transduction and arrowheads (B-H) indicate primary and secondary axes, pathways (for review, see Massague, 1996). Smad1 respectively. (B) General neural marker, nrp-1. (C) Xbra, notochord overexpression in Xenopus embryonic cells mimics and tailbud marker. The secondary axis lacks expression. (D) 12/101 muscle-specific antibody. (E) HoxB9, spinal cord marker. (F) En2, ventralizing signals such as those provided by BMPs, while midbrain/hindbrain boundary marker. (G) Krox20, marker of Smad2 mimics dorsal induction such as that caused by activin. rhombomeres 3 and 5. (H) Otx2, anterior neural marker. Embryos are It has recently been found that Smad1 or Smad2 activity is oriented with anterior to the left and dorsal up. greatly increased on coexpression with their common heteromeric partner, Smad4 (Candia et al., 1997). Therefore, to test whether tBRII-induced secondary axes were caused by rhombomeres 3 and 5 (Bradley et al., 1992), and Otx2, a interference with BMP signaling, we coinjected tBRII RNA marker of anterior neural structures including the forebrain and together with Smad1/Smad4 RNAs on the ventral side of the midbrain (Blitz and Cho, 1995). Each of these markers was embryo. With 1.25 ng of each RNA per embryo, tBRII alone expressed in approximately the correct position relative to each induced secondary axes in 88% (n=26) of the embryos, while other along the secondary axis (Fig. 4E,F,G,H). 89% (n=36) of those coinjected with Smad1/Smad4 contained In contrast to these results for tBRII, it has been reported only one axis (data not shown). Together with the failure of that a truncated BMP type I receptor, tBR, has no effect when Smad2/Smad4 to cause this reversal when coinjected with injected dorsally into the 4-cell embryo and that secondary tBRII, this strongly suggests that tBRII specifically interferes axes induced by ventral injections lack anterior structures with BMP-like ventralizing signals. (Graff et al., 1994; Suzuki et al., 1994). We therefore compared The tBRII-induced secondary axes are highly disorganized, directly the in vivo effects of these similarly truncated and contain abundant neural crest-derived melanocytes and receptors. In agreement with the previous reports, our tBR- multiple tubules resembling floorplate-less neural tubes (Fig. induced secondary axes lacked anterior structures (cement 3E,F). The presence of extensive neural tissue throughout the glands or otic vesicles) and notochord, although muscle tissue secondary axes was confirmed molecularly by whole-mount was present (data not shown). However, while tBRII-induced analysis (Fig. 4B) for the pan-neural marker nrp-1 (Richter et axes expressed a broad range of A/P neural markers including al., 1990). We characterized the neural tissues present in the the anterior markers Otx2/Krox20/En2, tBR-induced axes induced axes more precisely using markers specific for expressed only the posterior neural marker, HoxB9 (n=9 per different anteroposterior (A/P) domains of the neural tube. marker), even at the highest RNA doses tested (1 ng/embryo; These were: HoxB9, a spinal cord marker (Wright et al., 1990), data not shown). Therefore, although these two components are En2, a marker of the midbrain/hindbrain junction (Hemmati- presumed to function in the same signaling complex in vivo, Brivanlou et al., 1991), Krox20, a marker of hindbrain we conclude that there is a substantial difference in the A/P 436 A. Frisch and C. V. E. Wright

Table 1. Inhibition of tBRII-induced secondary axis formation by wild-type XBMPRII phenotype %% % % RNA injected n (inj.) n (surv.) dead 2°axis normal defective 1ng tBRII 90 62 31 72 19 8 1ng tBRII + wt 500pg 85 57 33 16 78 6 1ng tBRII + wt 1ng 105 46 56 6 43 51 1ng tBRII + wt 2ng 105 51 51 0 51 49

Embryos were injected ventrally at the 4-cell stage and scored for secondary axes at the late tadpole stage. Embryos in the 2° axis, normal and defective columns are expressed as a percentage of surviving embryos [n(surv.)]. Embryos counted as ‘defective’ are those exhibiting non-specific, RNA toxicity-induced gastrulation defects whose morphology was so abnormal as to prevent scoring for secondary axes. Thus, the number of normal embryos only returns to ~50%, while the proportion of secondary axis embryos decreases. extent of the neural tissue induced in the secondary axes by We conclude that inhibiting BMP signaling via tBRII in similarly truncated type I and type II BMP receptors. ventral regions of the embryo induces dorsolateral mesoderm Non-neural tissues were also detected in the ectopic axes (muscle), but not more dorsal mesoderm (notochord). The induced by either tBRII or tBR. The muscle-specific 12/101 presence of extensively patterned neural tissue in the secondary antibody (Kintner and Brockes, 1984) showed the presence of axis, in the absence of dorsal mesoderm, suggests either its disorganized muscle tissue located in the posterior regions of direct induction, as seen in other examples of disrupted BMP all secondary axes tested (Fig. 4D; n=7 for each RNA). These signaling (Hawley et al., 1995, Wilson and Hemmati- axes lacked notochordal tissue (Fig. 4C; n=15), as defined by Brivanlou, 1995, Xu et al., 1995, Piccolo et al., 1996), or its the absence of Xbra expression, which marks notochord and induction by the dorsolateral mesoderm of the secondary axis. tail organizer (Smith et al., 1991), and the lack of immunostaining with the MZ15 antibody (data not shown), Direct neuralization by tBRII which is specific for an extracellular matrix protein on the To test whether tBRII blocks BMP signaling in animal cap notochord surface (Smith and Watt, 1985). ectoderm and causes its direct neuralization, we explanted caps from embryos injected at the 1-cell stage with 1 ng of tBRII RNA and analyzed them at late gastrula (stage 10.5-11) for expression of the pan-mesodermal marker Xbra, or tailbud (stage 25) for the pan-neural marker NCAM (Kintner and Melton, 1987). NCAM was induced in tBRII-loaded animal caps without coincident mesoderm induction (Fig. 5A). Caps loaded with RNA encoding a dominant-negative activin type II receptor (∆XAR1; Hemmati-Brivanlou and Melton, 1992) and activin- treated uninjected caps served as positive controls for neural and mesodermal induction, respectively (Fig. 5A; lanes 4 and 5). The NCAM expressed in activin-treated caps is caused by secondary induction by the activin-induced dorsal mesoderm. The A/P character of the neural tissue induced in the animal caps by tBRII was tested using the region-specific markers described above. We detected expression of Otx2, but not the more posterior markers Krox20 (Fig. 5B) or HoxB9 (data not shown). Thus, we conclude that tBRII blocks BMP signaling in animal cap ectoderm, switching it to an anterior neural fate, consistent with previous reports using different agents to block BMP signaling (Hawley et al., 1995; Sasai et al., 1995; Wilson and Hemmati-Brivanlou, 1995; Xu et al., 1995). We also found that coinjection of Smad1 RNA reversed the tBRII-induced neuralization of the explant, while Smad2 induced dorsal mesodermal markers in tBRII-loaded explants (data not shown). This is consistent with the reversal of tBRII- Fig. 5. Direct anterior neural induction in animal caps by tBRII. (A) induced secondary axis induction by Smad1, in showing that RNAse protection analysis of animal cap RNA (10 caps/lane) for tBRII specifically interferes with BMP-related signaling neural (NCAM) and mesodermal (Xbra) tissues at stages 25 (tailbud) pathways. and 11 (gastrula) respectively. tBRII induces neural tissue without α mesoderm induction. EF1 is the loading control. WE, whole tBRII dorsalizes ventral mesoderm embryo control; uninj., control caps from uninjected embryos (B) RT-PCR analysis of animal cap RNA for region-specific neural The morphologically incomplete secondary axes suggested that marker expression. tBRII induces the anterior neural marker, Otx2, ventral expression of tBRII cannot induce a fully functional but not the hindbrain marker, Krox20. FGFR expression was used as ectopic Spemann organizer. In agreement with this hypothesis, a loading control. −RT, no reverse transcriptase control. gastrulation-stage embryos that were ventrally injected with BMP type II receptor signaling 437 tBRII RNA developed neither a secondary dorsal lip, nor ectopic expression of the organizer marker goosecoid (Blumberg et al., 1991; Cho et al., 1991) as assayed by whole-mount in situ analysis (data not shown). We tested whether partial dorsalization of the ventral mesoderm by tBRII might contribute to the non-neural tissues found in the secondary axis. Gastrula- stage dorsal and ventral marginal zones (DMZ and VMZ) were explanted from embryos that were ventrally injected at the 4-cell stage with 3 ng of tBRII RNA (1.5 ng/blastomere) and analyzed at stage 12.5 (early) or stage 25 (late) for expression of goosecoid, the late dorsolateral mesodermal marker actin (Mohun et al., 1984), or for Xwnt8, a ventrolateral mesodermal marker with much lower expression in the DMZ (Christian et al., 1991; Lemaire and Gurdon, 1994). We compared these to explants from embryos injected similarly with tBR RNA, which was previously shown to dorsalize VMZs (Graff et al., 1994; Maeno et al., 1994). As shown in Fig. 6, gsc and actin were induced in VMZs by either tBR or tBRII, coincident with Fig. 6. tBRII dorsalizes ventral marginal zones. Dorsal and ventral decreased Xwnt8 expression. Thus, truncated type I or type II marginal zones (DMZ and VMZ) were analyzed at stage 12.5 (early) receptors induce similar DMZ marker expression in VMZ or stage 25 (late) by RT-PCR for expression of the mesodermal explants, consistent with the appearance of ectopic muscle, but markers gsc, Xwnt8 and actin. As expected, DMZs show similar not notochord, in tBRII-induced secondary axes. The failure to marker expression profiles among the three experimental groups. detect ectopic gsc expression in whole embryos injected tBRII or tBR-loaded VMZs express the dorsal markers gsc and actin, ventrally with tBRII RNA, while gsc was detected in tBRII- and the ventral marker, Xwnt8, is suppressed. FGFR is the loading loaded VMZs, could reflect the different sensitivities of the two control. WE, RNA from whole embryos; uninj, DMZs or VMZs from uninjected embryos; −RT, no reverse transcriptase control. assays, but may also be related to the repressive actions of endogenous ventralizing signals present in the whole embryo.

Synergistic action of tBR and tBRII tBRII RNA, alone or in combination, at doses from 20 to 1000 Because tBRII-induced secondary axes extended more pg/embryo for each receptor (total combined doses of 40 to anteriorly than those induced by tBR, we tested whether 2000 pg; Table 2). While the secondary axes induced by coexpressing both truncated receptors might affect the coexpressing tBR and tBRII were essentially indistinguishable completeness of the induced secondary axis. Embryos were morphologically from those induced by tBRII alone (data not injected into two ventral cells at the 4-cell stage with tBR or shown), tBR/tBRII did synergistically increase the incidence of

Table 2. Synergy between truncated type I (tBR) and type II (tBRII) BMP receptors in secondary axis induction phenotype %%%% RNA injected (pg) n (inj.) n (surv.) dead 2° axis normal defective tBRII 20 45 41 9 0 95 5 tBRII 50 46 45 2 18 82 0 tBRII 100 45 40 11 18 72 0 tBRII 250 118 111 6 27 59 14 tBRII 750 97 94 3 54 37 9 tBRII 1000 185 139 25 82 10 8 tBR 20 45 42 7 0 98 2 tBR 50 45 42 9 2 95 3 tBR 100 45 36 20 14 69 17 tBR 250 65 46 29 48 33 19 tBR 1000 85 47 45 57 13 30 tBRII+tBR 20+20 60 37 38 49 35 16 tBRII+tBR 50+50 75 51 32 53 18 29 tBRII+tBR 250+250 40 26 35 58 4 38 tBRII+tBR 1000 +1000 40 13 68 46 8 46

Embryos were injected ventrally at the 4-cell stage and analyzed at the late tadpole stage for secondary axes. The percentage of embryos in the ‘2° axis’, ‘normal’ and ‘defective’ columns are calculated from the number of surviving embryos [n(surv.)]. Embryos with gastrulation defects arising from RNA toxicity at the higher doses were categorized as ‘defective’ and had such abnormal morphology as to prevent scoring for secondary axes. The number of such embryos increased progressively with total RNA dose. When expressed as a percentage of the total number of surviving embryos, the incidence of secondary axis induction resulting from co-injection of tBR/tBRII did not rise above 50%. However, if the percent secondary axis is calculated from the number of scorable embryos [i.e. n (surv.) - n (defective)], the incidence of ectopic axis induction caused by coinjection of tBR/tBRII approaches 100%. For example, for ‘tBRII + tBR 250 pg ea.’, if calculated from the number of scorable embryos, the percent of embryos with secondary axes equals 58/(58+4) or 94%. 438 A. Frisch and C. V. E. Wright

1 2 3 4 5 6 7 8 9 10 11 Fig. 7. tBRII blocks BMP4-mediated mesoderm induction. RT-PCR analysis of animal cap RNA at stage 16 for expression of the pan- mesodermal marker, Xbra, or ventrolateral mesodermal marker, Xhox3. When compared to control caps (lane 3), incubation of uninjected animal caps with rhBMP4 induces mesodermal markers (lane 5). The induction is blocked by tBRII (lane 6) or ∆XAR1 (lane 9). The inhibition is reversed by coexpressing the wild-type BMP type II (lanes 7 and 11) or activin type II (lanes 8 and 10) receptor. FGFR expression was used as the loading control. WE, RNA from whole embryos; uninj, animal caps from uninjected embryos; −RT, no reverse transcriptase control. axis induction relative to injection dose. For example, co- injecting 20 pg of each receptor RNA (40 pg total) induced secondary axes in ~50% of the surviving embryos. In contrast, 20 pg of tBR or tBRII RNA alone did not induce secondary axes, while doses of 50 pg of tBR or tBRII RNA caused axial induction in only 2% or 18% of the embryos, respectively. In contrast, the degree of neuralization of animal cap ectoderm effected by coexpressing both truncated receptors was similar to that caused by each truncated receptor alone (data not shown. Rescue of tBRII-induced block in BMP signaling by type II activin receptor We then tested whether tBRII could block mesoderm induction by BMP4, and asked whether full-length versions of two 1 2 3 4 5 6 7 8 9 10 11 12 different type II receptors, XBMPRII and the activin XAR1 receptor, could reverse this block. Embryos were injected at the Fig. 8. tBRII does not block activin signaling. (A) Morphological 1-cell stage with 2 ng of RNA encoding either tBRII or the analysis of animal caps at stage 22. Uninjected animal caps elongate truncated activin type II receptor ∆XAR1. Animal caps were in response to 5 ng/ml activin (top two panels). This elongation is not affected by tBRII (next four panels) but is diminished greatly by then explanted and incubated with or without recombinant ∆ human BMP4 (rhBMP4) at 50 ng/ml, and analyzed by RT-PCR XAR1 (bottom four panels). (B) RT-PCR analysis of animal cap RNA at sibling stage 11 (early) or 25 (late) for expression of the pan- at stage 14 for expression of the Xbra, and the ventrolateral mesodermal marker, Xbra, or the dorsolateral mesodermal marker, mesodermal marker, Xhox3 (Ruiz i Altaba and Melton, 1989), actin. Neither marker is significantly expressed in control or tBRII which are both induced in animal caps by BMP4 (Dale et al., injected caps (lanes 3-5), but both are induced by treating uninjected 1992; Jones et al., 1992). As shown in Fig. 7, rhBMP4 induced caps with activin (lane 6). Marker expression is not inhibited by tBRII Xbra and Xhox3 (lane 5), and this induction was completely (lanes 7 and 8), but ∆XAR1 dose-dependently blocks expression abrogated (Xbra), or substantially diminished (Xhox3), by (lanes 9 and 10). FGFR expression was used as a loading control. tBRII, indicating an efficient block to ventral mesoderm WE, RNA from whole embryos; uninj, animal caps from uninjected induction by rhBMP4. This block was reversed by coinjecting embryos; act, activin; −RT, no reverse transcriptase control. RNA encoding full-length XBMPRII (lane 7) or activin type II receptor (lane 8). ∆XAR1 also eliminated mesoderm induction induced block in Xbra and Xhox3 expression (lanes 10 and 11). by rhBMP4 (lane 9), as shown previously (Chang et al., 1997; The finding that both type II receptors can restore functional Hemmati-Brivanlou and Thomsen, 1995). However, both the BMP4 signaling indicates that these activin and BMP type II wild-type activin or BMP type II receptors rescued the ∆XAR1- receptors are both able to mediate BMP4 signaling in vivo. BMP type II receptor signaling 439

Thus, XAR1 has the potential to be a shared component in 1995; Rosenzweig et al., 1995), although the strength of ventralizing and dorsalizing pathways. binding and signal transduction depends upon the ligand (see below). The 63% identity (~81% similarity) between human Specificity of tBRII for BMP signaling and frog BMPRII in the region between the signal sequence The selectivity of XBMPRII for BMP versus activin signaling and transmembrane domains predicts a similar ligand pathways in vivo was tested by challenging activin-induced specificity in vivo. In the Xenopus embryo, BMP2, BMP4 and mesoderm induction for its sensitivity to tBRII (Fig. 8). BMP7 are expressed in somewhat overlapping domains in the Animal caps from embryos injected with 1 or 2 ng of tBRII animal hemisphere and marginal zone during blastula and RNA at the 1-cell stage were cultured with or without 5 ng/ml gastrula stages, but their expression domains become more activin and compared to animal caps loaded with RNA distinct as development proceeds (Clement et al., 1995; encoding ∆XAR1. As expected, activin induced profound Fainsod et al., 1994; Hawley et al., 1995; Hemmati-Brivanlou convergent extension in uninjected caps (Fig. 8A) and induced and Thomsen, 1995). From the early gastrula stage, XBMPRII Xbra and actin (Fig. 8B), while ∆XAR1 dose-dependently expression appears most similar to that of BMP4, which is blocked extension and marker induction. In contrast, tBRII did especially apparent in the premesodermal region in gastrula not block induction by activin, regardless of the amount embryos where both XBMPRII (Fig. 2) and BMP4 transcripts injected. By both morphological and molecular criteria, we (Fainsod et al., 1994; Hemmati-Brivanlou and Thomsen, 1995; conclude that tBRII does not crossreact with activin signaling Schmidt et al., 1995) start to become restricted from the pathways. dorsalmost regions. We also tested whether tBRII could interfere with signaling by Although the activin type II receptors have been implicated in an unrelated ligand, bFGF. Our results were similar to those BMP signaling (Yamashita et al., 1995), BMPRII/XBMPRII is reported for the truncated activin type II receptor and truncated the only known type II receptor that preferentially binds BMP BMP type I receptor (Hemmati-Brivanlou and Melton, 1992; ligands in vitro. Thus, the heteromer model predicts that Suzuki et al., 1995) in that, instead of abrogating bFGF activity, BMPRII/XBMPRII, together with type I receptors, would injection of tBRII RNA altered the response of animal caps to provide the most productive signaling by BMPs. The absence of bFGF. Caps treated with bFGF (50 ng/ml) normally form XBMPRII expression from the animal region of gastrula-stage vesicular, ovoid structures containing mostly ventral mesoderm. embryos is consistent with the hypothesis that removing BMP However, the majority of caps receiving both tBRII RNA and signals, which leads to a loss of epidermalizing signals, is a bFGF treatment became substantially elongated with the major component of the neural induction process (Hemmati- elaboration of large cement glands at either end (data not shown). Brivanlou and Melton, 1997). By in situ hybridization analysis, From histological analysis, we surmised that these caps contained XBMPRII RNA is also absent from the ventral ectoderm at early both anterior and posterior neural tissues, the latter being likely gastrula stages, which argues against its involvement in responsible for the explant extension. This situation would be maintaining the epidermal fate of this region. However, it is explained by a combination of anterior neural induction by tBRII possible that the wider expression of XBMPRII RNA throughout together with partial posteriorization by bFGF, consistent with the animal region at earlier stages (Fig. 2) might provide these previous findings (see above mentioned references). embryonic cells with a pool of stable type II receptors, giving them the potential to respond to BMP ligands that are first DISCUSSION secreted after the disappearance of XBMPRII transcripts. In principle, these ligands would include BMP2 and BMP7, for While TGFβ/BMP-related factors are crucial to embryogenesis which transcripts are expressed maternally (and thus stand in (reviews by Hogan, 1996; Kingsley, 1994), only a subgroup of contrast to BMP4), and are enriched in the animal hemisphere receptors mediating their effects have been identified. In addition, during blastula stages (Clement et al., 1995; Hawley et al., 1995; the potential for receptor crossreactivity both in vitro and in vivo, Hemmati-Brivanlou and Thomsen, 1995). The generation of and the lack of data on spatial expression at both the RNA and specific antibodies against different BMPs and XBMPRII would protein level currently make it difficult to assign specific ligand allow direct testing of these possibilities. effects in vivo to particular receptor combinations. Our studies Although XBMPRII distribution has not been determined at here address these issues by providing the first detailed the protein level, the finding that most XBMPRII RNA is expression analysis of any BMP receptor, XBMPRII, at early localized to the marginal zone in gastrula-stage embryos stages of embryogenesis when critical patterning decisions are suggests that its main function at this time of development lies being made. Our data suggest that XBMPRII can mediate the in transducing BMP signals as part of the mesodermal patterning mesodermal patterning and neural suppressing effects of BMP4 process. BMP4 signaling is particularly important during this in the embryo, and we have demonstrated a qualitative difference window of embryogenesis, as demonstrated by several findings. between XBMPRII, the only type II BMP-specific receptor Maternally derived BMP4 transcripts are expressed at very low known to date, and the type II activin receptor, XAR1, in their levels (Dale et al., 1992), and zygotic BMP4 transcription occurs crossreaction between dorsalizing (activin) and ventralizing mostly after stage 9/10, during which its expression becomes (BMP) signaling pathways. Furthermore, we have shown that rapidly restricted to the ventral and lateral regions of the similarly truncated BMP type I and type II receptors have marginal zone (Fainsod et al., 1994; Hemmati-Brivanlou et al., recognizably different effects in vivo. 1995; Schmidt et al., 1995). In addition, careful temporal analyses indicate that the ventralizing effects of BMP4 impact Implications of XBMPRII-II expression dorsoventral mesodermal patterning during and just after Mammalian BMPRII selectively binds BMP2, BMP4 or BMP7 gastrulation, but not earlier (Jones et al., 1992, 1996). in vitro (Kawabata et al., 1995; Liu et al., 1995; Nohno et al., Since exogenous BMP4 increases endogenous BMP4 440 A. Frisch and C. V. E. Wright expression (Jones et al., 1992), it has been proposed that BMP4 and signal transduction arise when mammalian transcription is under positive autoregulatory control. If BMPRII/BMPRIB are coexpressed with BMP7, or when XBMPRII expression is also upregulated by ventralizing BMP BMPRII/ActRI are coexpressed with BMP4 (Liu et al., 1995; signals, then blocking functional BMP4, for example, through Rosenzweig et al., 1995). In contrast, BMPRII/ActRI the actions of noggin and/or chordin (Piccolo et al., 1996; coexpression elicits high affinity binding and strong signal Zimmerman et al., 1996), would also lead to decreased transduction by BMP7. Together with similar findings for other transcription of XBMPRII in dorsal regions. Such a scenario TGFβ-related receptors (e.g. Attisano et al., 1993), this has led might locally reduce the potential for BMP signaling and to the idea that independent assortment of type I/type II facilitate the action of other dorsalizing molecules. Alternatively, receptors might produce a multitude of different receptor the lower levels of XBMPRII expression in dorsal regions may pairings that mediate distinct downstream effects of a directly result from the earlier actions of the Nieuwkoop particular ligand. Some support for this idea has come from in center/Spemann organizer in dorsoventral specification. In either vivo studies in Xenopus embryos showing that a truncated case, it is possible that the decreased XBMPRII expression in the activin type I receptor can block the activity of BMP4 as a DMZ protects these regions against the ventralizing effects of mesoderm-inducing signal, but not as an epidermalizing factor overly strong or residual BMP4 ligands. (Chang et al., 1997). It is clear that information on the preferred type I/type II pairings and their activities in different cell types Dorsal-ventral mesodermal patterning will further enhance our understanding of the molecular Consistent with its expected role as a dominant negative mechanisms for patterning by BMPs. inhibitor of XBMPRII-mediated signal transduction, we found Our results demonstrate that tBRII expression is sufficient to that tBRII induced dorsal marker expression in ventral block BMP4-induced mesoderm induction, dorsalize ventral marginal zone explants. On the contrary, ventral injection of mesoderm and neuralize ectoderm. Together with the overlapping tBRII RNA induced only partial secondary axes and, by this expression patterns of XBMPRII and BMP4, the preferred criterion, did not induce an ectopic Spemann organizer. It is binding of BMPRII to BMPs in vitro and the ability of the likely that the removal of ventralizing signals induces only coinjected BMP-specific ventralizing factor, Smad1, to reverse partial dorsal specification, while complete axial development the tBRII effects, the studies presented here imply strongly that requires a full complement of Organizer activities, normally XBMPRII mediates BMP signals in vivo. Moreover, we have arising from Nieuwkoop center signaling. This is supported by also tested whether the BMPRII crossreactivity shown by the observation that injection of RNA encoding the organizer overexpression studies in cultured cells occurs in vivo. ∆XAR1 molecules noggin or gsc induces partial secondary axes (Cho blocks BMP signaling in animal caps, but tBRII does not cause et al., 1991; Smith and Harland, 1992), while Xwnt8 or siamois a reciprocal block in activin signaling, indicating that XBMPRII RNA mimic Nieuwkoop center activity and cause complete is relatively specific for ventralizing BMP signals. Furthermore, axial duplications (Lemaire et al., 1995; Sokol et al., 1991; the ability of both the full-length activin or BMP type II receptors Smith and Harland, 1991). Moreover, recent studies on to rescue a block in BMP-mediated mesoderm induction imposed and lim-1/Xlim-1 indicate that subregions of the either by ∆XAR1 or tBRII strongly suggests that this type IIB Organizer direct the formation of dorsoanterior structures ‘activin receptor’ can, under appropriate circumstances, function through the activity of a ‘head organizer’ (Bouwmeester et al., as a BMP receptor in vivo. This finding would argue against the 1996; Shawlot and Behringer, 1995). The failure to generate hypothesis suggested by New et al. (1997) in which different such head organizer tissue would explain the finding that receptor/ligand affinities result in the transduction of ventral localized injection of tBR or tBRII RNA into UV-ventralized (BMP-like) or dorsal (activin-like) signals specifically through embryos causes only a partial axial rescue, with the resulting type II or type IIB activin receptors, respectively. However, the embryos having only posterior tissues and rudimentary anterior activities of different receptor complexes in transducing BMP structures (Graff et al., 1994; and our unpublished results). signals during embryogenesis will depend not only upon their ligand-binding preferences, but also on the expression patterns of BMP signaling and crossreactivity of dorsal/ventral the ligands and receptors. The finding that both BMPRII and signaling pathways XAR1 can interact with multiple type I receptors, and that XAR1 Significant crossreaction has been demonstrated amongst can function as a BMP receptor, makes it important to determine several type I and type II receptors. For example, a truncated the relative amounts and expression patterns of these type II type II activin receptor, ∆XAR1, also interferes with signaling receptors during critical periods of BMP signaling and to by BMPs (Hemmati-Brivanlou and Thomsen, 1995; Wilson determine the effect of different type I/type II pairings on ligand and Hemmati-Brivanlou, 1995; Yamashita et al., 1995) and binding and signal transduction in vivo. Vg1 (Schulte-Merker et al., 1994). Furthermore, XAR1 can be functionally replaced by the type II TGFβ receptor in activin- Synergy and differential activities of tBR and tBRII mediated mesoderm induction (Bhushan et al., 1994). Although type I and type II BMP receptors are presumed to Similarly, the BMPRIA, BMPRIB and ActRI type I receptors function in the same signaling complex, similarly truncated can all form complexes with BMPRII in vitro (Liu et al., 1995; versions of each receptor have different effects on secondary axis Rosenzweig et al., 1995). induction. Ectopic axes induced by tBRII extend much more Despite the promiscuity in the assembly of signaling anteriorly, and contain Otx2-expressing neural tissues, cement complexes and their potential for interacting with different glands and otic vesicles, whereas those induced by tBR do not, ligands, not all ligand-receptor combinations cause equivalent even at the highest doses (Suzuki et al., 1994; Graff et al., 1994, signal transduction. Of specific relevance to our studies on and our results). Several not necessarily mutually exclusive XBMPRII, in vitro studies show that only low levels of binding possibilities could explain this. First, since the primary function BMP type II receptor signaling 441 of TGFβ-like type II receptors is to bind ligand and initiate REFERENCES complex formation (ten Dijke, 1996), signaling specificity may Attisano, L., Carcamo, J., Ventura, F., Weis, F. M. B., Massague, J. and be regulated more through type II receptor/ligand recognition Wrana, J. L. (1993). 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