Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10255-10259, October 1994 Developmental Biology A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo ATSUSHI SUZUKI*, R. ScoTT THIESt, NOBORU YAMAJIt*, JEFFREY J. SONGt, JOHN M. WOZNEYt, KAZUO MURAKAMI§, AND NAOTO UENO*1 *Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan; tGenetics Institute Inc., 87 Cambridge Park Drive, Cambridge, MA 02140; tYamanouchi Pharmaceutical Co., Ltd., Tokyo 103, Japan; and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Communicated by Igor B. Dawid, July 13, 1994 ABSTRACT Bone morphogenetic proteins (BMPs), which corresponding proteins are present in developing Xenopus are members of the trnsming growth factor 13 (TGF-I) embryos, and overexpression of BMP4 in the embryos superfamily, have been implicated in bone formation and the enhances the formation of ventral mesoderm (8-11). Animal regulation ofearly development. To better understand the roles cap ectoderm treated with a combination of BMP4 and of BMPs in Xenopus laevis embryogenesis, we have cloned a activin also results in the formation of ventral mesoderm, cDNA coding for a serine/threonine kinase receptor that binds suggesting that BMP-4 is a ventralizing factor that acts by BMP-2 and BMP-4. To analyze its function, we attempted to overriding the dorsalizing signal provided by activin (8, 9). block the BMP signaling pathway in Xenopus embryos by using Therefore, activin and BMP-4 are thought to play important a domint-negative mutant of the BMP receptor. When the roles in the dorsal-ventral patterning of embryonic meso- mutant receptor lacking the putative serine/threonine kinase derm. Although the exogenously applied BMP has been used domain was expressed in ventral blastomeres of Xenopus to address the functions of the ligand in the embryo, little is embryos, these blastomeres were respecifted to dorsal meso- known about the involvement of endogenous BMP ligands in derm, eventually resulting in the formation ofa secondary body dorsal-ventral patterning of the mesoderm. axis. These findings suggest that endogenous BMP-2 and Here we report the isolation of a vertebrate serine/ BMP-4 are involved in the dorsal-ventral specification in the threonine kinase receptor cDNA whose product displays embryo and that ventral fate requires induction rather than specific binding to BMP-2 and BMP-4. 11 In addition, we were resulting from an absence of dorsal specification. able to block the BMP signaling pathway in early Xenopus embryos by using a functionally negative mutant ofthe BMP Pattern formation in vertebrate embryos involves the spec- receptor. Overexpression of the negative receptor in ventral ification ofregional cell fates along the two major body axes, blastomeres of Xenopus embryos resulted in duplication of the anterior-posterior and dorsal-ventral axes. The estab- the body axis by respecifying the presumptive ventral me- lishment of these body axes is believed to require various soderm to dorsal mesoderm. The data presented here signaling molecules that regulate inductive cell-cell interac- strongly suggest that specification of the dorsal-ventral axis tion as well as autonomous cell differentiation. Recent stud- of Xenopus embryo is controlled by endogenous BMP pre- ies in the African clawed frog, Xenopus laevis, have sug- sent in the embryo. gested that diffusible polypeptide growth factors play funda- MATERIALS AND mental roles in dorsal-ventral patterning ofthe early embryo. METHODS Activin, a member of the transforming growth factor ,3 Cloning of a Mouse BMP Receptor. A set of degenerate (TGF-(3) superfamily, has been studied intensely since it was oligonucleotide primers was designed in serine/threonine ki- found to induce dorsal mesoderm in animal cap ectoderm nase subdomains VIII and XI (12), based on amino acid explants (1). An important issue regarding the function of sequences of mouse activin type II receptor (13) and human growth factors in early development is whether the protein is TGF-13 type II receptor (14). The forward polymerase chain present at the relevant tissue and place. The mRNA for reaction (PCR) primer (domain VIII) was 5'-TA(TC)ATGGC- activin and an activin-like biological activity have been (TCAG)CC(TCAG)GA(AG)GT-3'; the reverse PCR primer detected during Xenopus embryogenesis (2, 3). Xenopus (domain XI) was 5'-(AG)TC(AG)TG(AG)TCCCA(AG)- activin receptor genes, which encode transmembrane pro- CA(TC)TC-3'. Total RNA was purified from the mouse teins possessing the intracellular serine/threonine kinase MC3T3-E1 cells and subjected to reverse transcription PCR domains, have been cloned recently, and activin signaling (RT-PCR) as described (15). Forty cycles of amplification pathways via these receptors have been implicated in meso- were performed, each consisting ofdenaturation at 940C for 1 derm induction and dorsal axis formation (4, 5). In addition, min, annealing at 450C for 1 min, and extension at 720C for 2 the function of endogenous activin was addressed by using a min. The amplified PCR products were subcloned into pBlue- dominant-negative form of activin receptor that lacks the script II KS (-) and sequenced using Sequenase (United serine/threonine kinase domain. Overexpression of the mu- States Biochemical). One of the clones, designated as tant activin receptor in the early embryo blocked the forma- mTFR11, was used as aprobe for screening the MC3T3-E1 cell tion of mesoderm, suggesting that endogenous activin is cDNA library and mouse brain cDNA library, resulting in the essential for mesoderm induction (6). isolation of a clone that encoded a 532-amino acid protein. In addition to activin, bone morphogenetic proteins Binding Assay and Affinity Cross-Linking. Human TGF-p1 (BMPs), originally identified in bovine bone, have been was purchased from R&D Systems. Human activin A is a gift implicated in various morphogenetic processes such as bone from Y. Eto and H. Shibai (Ajinomoto). Human BMP-2 and formation, neurogenesis, and epithelial-mesenchymal inter- actions (7). The transcripts for BMP-2 and -4 as well as the Abbreviations: TGF-3, transforming growth factor P; BMP, bone morphogenetic protein; RT-PCR, reverse transcription polymerase chain reaction; DAI, dorsoanterior index. The publication costs ofthis article were defrayed in part by page charge ITo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" lThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. D16250). 10255 Downloaded by guest on September 23, 2021 10256 Developmental Biology: Suzuki et al. Proc. Nati. Acad. Sci. USA 91 (1994) -4 were purified from Chinese hamster ovary cell lines (16). To express mTFR11 in COS cells, full-length cDNA of mTFR11 was subcloned into pMV2, which is a derivative of pMT2 (17). Human recombinant BMP-4 was iodinated by the methods of Frolik et al. (18). The iodinated BMP-4 exhibited biological activity similar to that of unlabeled BMP-4 (data not shown) in a W-20-17 cell (16). Transfected COS cells were incubated with BMP-4 and/or 125I-labeled BMP-4 (125I_ BMP-4) at 370C formin.60 Following binding, the cells were washed twice and radioactivity was determined in a 'y counter. In the competition assay, COS cells transfected with mTFR11 were incubated with 1251-BMP-4 with or without increasing concentrations of unlabeled BMP-2, BMP-4, ac- tivin A, or TGF-#1. Results are expressed as means for B mT .-I A triplicate wells from one experiment and were confirmed in lx-I. a second independent experiment. For cross-linking studies, B~~~~~~~~~~~~~~~~~~mT,hAK- the cells were incubated with 500 p.M disuccinimidyl suberate for 20 min at 40C following binding. Cell extracts were L. analyzed on SDS/PAGE under reducing conditions. x T TFR1I L Microinjection of Synthetic mRNAs and Histolgcal Anal- 65 ysis. A dominant-negative mutant of mTFR11 was con- structed by using one of3'-nested deletion clones of mTFR11 mALK-6 ~~1LiI~~~~~i7........ cDNA. This3'-deleted cDNA has a termination codon in- 40r stead of Tyr at amino acid 176 and codes for the extracellular rMTGF-3RII hALK-5 L U L _ domain and transmembrane domain of mTFR11. The trun- cated and a full-length cDNAs of mTFR11 were subcloned mActR -uKhI-l ii into pSP64T (19). Synthesis and microinjection of capped mRNA were performed essentially as described by Moon and ct hALK-5 nmAmTGF->5RIRI! ~~~~~~~~~~~~~~~~~~~~~~~~~LAd.mrGF-L711117111i1|_Ar!-' Christian (20). Embryos were staged according to Nieuw- koop and Faber (21). The injected embryos were allowed to develop until stages 34-40 for the observation of external - appearance and then subjected to histological analysis. dafG-4 C - - U-- Whole-mount in situ hybridization was performed as de- dat- L e .Iii~7 scribed by Harland (22). RESULTS FIG. 1. Amino acid sequence and homology of mTFR11. (A) Identification of a Vertebrate BMP-2/-4 Receptor. To bet- Translated amino acid sequence of the mTFR11 cDNA. The signal ter understand the molecular basis of BMP functions in early peptide and transmembrane domains are indicated by a single Xenopus embryos, we isolated cDNA encoding a BMP underline. Potential sites of N-linked glycosylation are indicated by receptor. Since recent isolation of type I and type II activin asterisks. The ends of the kinase domain are indicated by arrows. and has revealed a conserved intracellular Ten of the conserved cysteine residues in the extracellular domain TGF-p receptors are in reverse print. Type I box is indicated by a double underline. serine/threonine kinase domain (23), we employed the RT- An arrowhead is under the tyrosine residue that changes to stop PCR using degenerate oligonucleotide primers in serine/ codon in the dominant negative BMP receptor.