Xenopus Mothers Against Decapentaplegic Is an Embryonic Ventralizing Agent That Acts Downstream of the BMP-2/4 Receptor

Development 122, 2359-2366 (1996) 2359 Printed in Great Britain © The Company of Biologists Limited 1996 DEV3476

Xenopus mothers against decapentaplegic is an embryonic ventralizing agent that acts downstream of the BMP-2/4 receptor

Gerald H. Thomsen Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215, USA

SUMMARY Dorsal-ventral patterning in vertebrate embryos is ing no readily identiﬁed functional motifs. This report regulated by members of the TGF-β family of growth and demonstrates that a gene closely related to Drosophila Mad differentiation factors. In Xenopus the activins and Vg1 are exists in Xenopus (called XMad) and it exhibits activities potent dorsal mesoderm inducers while members of the consistent with a role in BMP signaling. XMad protein bone morphogenetic protein (BMP) subclass pattern induces ventral mesoderm when overexpressed in isolated ventral mesoderm and regulate ectodermal cell fates. animal caps and it ventralizes embryos. Furthermore, Receptors for ligands in the TGF-β superfamily are serine- XMad rescues phenotypes generated by a signaling- threonine kinases, but little is known about the components defective, dominant-negative, BMP-2/4 receptor. These of the signal transduction pathway leading away from these results furnish evidence that XMad protein participates in receptors. In Drosophila the decapentaplegic protein (dpp), vertebrate embryonic dorsal-ventral patterning by func- a homolog of vertebrate BMP-2 and BMP-4, functions in tioning in BMP-2/4 receptor signal transduction. dorsal-ventral axial patterning, and a genetic screen for components involved in signaling by dpp has identiﬁed a gene named mothers against decapentaplegic (Mad). Mad Key words: mesoderm, pattern formation, signal transduction, Mad, encodes a unique, predicted cytoplasmic, protein contain- BMP, Xenopus, Drosophila

INTRODUCTION homologs since the signaling systems in which they function have been conserved in evolution as a mechanism to generate Bone morphogenetic proteins (BMPs) are members of the dorso-ventral polarity in insect and vertebrate embryos. The transforming growth factor β (TGF-β) superfamily, and in axial polarity of these organisms is, however, reversed relative Xenopus embryos they participate in patterning the ventral to one another (Hogan, 1995; Holley et al., 1995; Winnier et mesoderm and the ectoderm. BMP-2 and BMP-4 are capable al., 1995). This conservation of molecular players in dorsal- of inducing ventral mesoderm and re-specifying prospective ventral patterning across a wide evolutionary distance is under- dorsal mesoderm to differentiate into ventral tissues in scored by the capacity of BMP-4 and dpp to functionally sub- Xenopus embryos (Koster et al., 1991; Dale et al., 1992; Jones stitute for one another in vertebrate and Drosophila embryos et al., 1992; Fainsod et al., 1994; Clement et al., 1995; (Padgett et al., 1993; Holley et al., 1995). Thus it is anticipated Hemmati-Brivanlou and Thomsen, 1995). BMP signals are that vertebrate homologs of molecules involved in transmitting required in the embryo to form ventral mesoderm because or receiving dpp signals may participate in inductive signaling blocking BMP-2/4 receptor activity in the ventral part of the by BMPs in vertebrate embryos. embryo eliminates blood formation and dorsalizes the Receptors for TGF-β-related ligands consist of heterodimers mesoderm (Graf et al., 1994; Suzuki et al., 1994). BMP-4 of single transmembrane serine/threonine kinases that are clas- protein also promotes the differentiation of epidermis from sified as type I or type II according to structural and functional Xenopus ectoderm (Wilson and Hemmati-Brivanlou, 1995), features (Massague et al., 1994). Types I and II BMP receptor and if BMP signals in the ectoderm are blocked by a dominant- subunits have been cloned from animals as diverse as negative BMP-2/4 receptor (Sasai et al., 1995), or if ectoder- nematodes and mammals (Estevez et al., 1993; Graf et al., mal cells are disaggregated (Grunz and Tacke, 1989), they dif- 1994; Suzuki et al., 1994; ten Dijke et al., 1994; Liu et al., ferentiate into neural tissue. 1995), including a receptor in Xenopus that binds BMP-2 and Embryonic dorsal-ventral patterning in Drosophila is BMP-4 (BMP-2/4 receptor; Graf et al., 1994; Suzuki et al., accomplished partly through the action of decapentaplegic 1994). Type II receptor subunits can bind ligand and are con- (dpp), which is a TGF-β member most closely related to ver- stitutively phosphorylated, but receptor signals are not trans- tebrate BMP-2 and BMP-4. These factors are probably true mitted until a type II receptor pairs with a type I receptor in a 2360 G. H. Thomsen ligand-dependent step. This results in phosphorylation of the probed in 50% formamide, 5% SDS, 1× SSC, 1× Denhardt’s and 0.1 type I subunit and activation of the receptor complex (Wrana mg/ml sonicated salmon sperm DNA. The probe was a 32P-labeled, et al., 1994; Liu et al., 1995). Little is known, however, about random-primed fragment of the noncoding 3′ end of XMad. Final blot the signal transduction components downstream of TGF-β- wash conditions were 60¡C, 0.2× SSC, 0.2% SDS. In situ hybridiz- related receptors. Studies in Xenopus suggest that crosstalk can ation was performed with an antisense, digoxygenin-labeled RNA occur between the signal transduction pathway of activin probe (Harland, 1991) using BMPurple colorimetric substrate (Boehringer-Mannheim). receptors and receptor tyrosine kinases (Cornell and Kimelman, 1994; LaBonne and Whitman, 1994; Umbhauer et Embryonic ventralization and mesoderm induction assays al., 1995), but signal transduction molecules for tyrosine kinase BMP-4 (Nishimatsu et al., 1992) and XMad were cloned into the CS2+ receptors probably do not directly function in signaling by vector (Rupp et al., 1994), and DMad was cloned into pSP64T (Krieg TGF-β-related receptors. and Melton, 1987). Capped mRNA was synthesized from linearized A genetic screen for enhancers of weak dpp alleles vectors using mMessage Machine kits (Ambion). In ventralization uncovered a maternal-effect gene named mothers against dpp, and animal cap assays, 1.5 ng of mRNA were injected per blastomere. or Mad (Raftery et al., 1995; Sekelsky et al., 1995), which Animal caps were injected at the 2-cell stage and cut at blastula stages plays a role in dpp signaling. Drosophila Mad (herein referred 8 to early 9. RNA was prepared from whole embryos and embryonic to as DMad) encodes a unique protein, predicted to be intra- explants and northern blots were performed as described (Thomsen cellular but containing no recognizable functional motifs, and and Melton, 1993). VMZs were isolated as described (Graf et al., 1994; Hemmati-Brivanlou and Thomsen, 1995) RT-PCR assays and it is a member of a new family of proteins (Sekelsky et al., primer sequences were as published (Wilson and Melton, 1994; 1995). Mad-related genes, dubbed dwarfins, have also been Wilson and Hemmati-Brivanlou, 1995), except for αT1 globin identified in C. elegans and they correspond to the small (accession no. J00976), which were: upstream, 5′-TTGCTGTCTCA- alleles, sma-2, sma-3 and sma-4 (Savage et al., 1996). A pan- CACCATC-3′ (bases 30-37); downstream, 5′-TCTGTACTTGGAG- creatic tumor suppressor gene, DPC4, has also been identified GTGAG-3′ (bases 198-215). Some primer sequences were obtained that is related to Mad and the dwarfins (Hahn et al., 1995). A from the Xenopus Molecular Marker Resource on the internet (http//: prediction from genetic studies in the fly and nematode is that vize222.zo.utexas.edu/). The NCAM primers span a putative intron Mad proteins may function in TGF-β receptor signal trans- (unpublished observations), and the sequences are: upstream primer, 5′-CACAGTTCCACCAAATGCCG-3′ (bases 2817-2836); down- duction. ′ ′ This paper reports on a Xenopus homolog of Drosophila stream primer, 5 -GCTGGGGTGCCCTTGACATC-3 (bases 3230- 3211). Amplification with NCAM primers was performed by adding Mad that functions in the signal transduction pathway from cDNA template to the PCR reaction during the first 94°C denatura- BMP receptors. The Xenopus Mad gene (XMad) is closely tion step. related to DMad, and both encode factors that induce and pattern ventral mesoderm in Xenopus embryos. Significantly, XMad rescues mesodermal and ectodermal patterning defects RESULTS generated by a dominant-negative BMP-2/4 receptor, placing XMad downstream of the BMP-2/4 receptor. The evidence I Sequence and embryonic expression of Xenopus present strongly supports a function for Mad proteins in signal Mothers against dpp transduction from TGF-β-related receptors. This study also In preliminary studies DMad was tested for activity in Xenopus provides additional support for the hypothesis that components (by microinjecting synthetic mRNA) and was found to ven- of the vertebrate BMP and Drosophila dpp signaling systems tralize embryos (Fig. 3E) in a fashion similar to the BMP-4 are homologous, and constitute a mechanism to generate ligand. Therefore it was anticipated that a Xenopus homolog dorsal-ventral pattern that has been conserved during of DMad might exist and function in inductive signaling by evolution. BMP ligands. A full-length Xenopus Mad gene (XMad) was isolated from a blastula stage cDNA library using DMad as a probe, and XMad encodes a protein of 464 amino acids that is MATERIALS AND METHODS 75% identical to DMad protein and 85% similar when conser- Cloning of Xmad vative substitutions are considered (Fig. 1). This remarkable level of conservation between XMad and DMad proteins is A Xenopus blastula (stage 9) cDNA library in Lambda Zap was screened with a random-primed 32P-labeled DMad probe. About especially striking within the N- and C-terminal regions. 2×107 cpm were hybridized at 42¡C to 4×105 plaques on nylon filters, Within the N-terminal portion of XMad, 133 of 145 residues in 50% formamide, 5× Denhardt’s, 5× SSC and 0.1 mg/ml denatured (92%) are identical to DMad. At the C terminus, 112 of the salmon sperm DNA. Filters were washed in 0.5× SSC, 0.5% SDS, last 120 amino acids (93%) are identical to DMad, and each 55¡C. More than 25 cDNAs were initially isolated and among the difference represents a conservative substitution, with the purified clones a full-length cDNA was identified and sequenced exception of residue 339. Several mutations in DMad alleles (Sanger et al., 1977) on both strands by primer walking. DNA and map to the C-terminal 50 amino acids, highlighting the func- protein sequences were analyzed with Lasergene software (DNAStar tional importance of this very conserved domain (Sekelsky et Inc.). al., 1995). The final four amino acids at the C terminus of Xmad expression studies DMad and XMad comprise a serine tail (SSVS), and XMad In the developmental northern blot, 20 µg of total RNA were loaded contains 47 serine, 21 threonine and 16 tyrosine residues, alto- per lane on the gel, which was run and transferred to nylon membrane gether constituting 18% of the protein. These represent an (Micron Separations, Inc.) in 10× SSC according to standard proce- abundance of potential target sites for phosphorylation (or dures (Sambrook et al., 1990). The blot was pre-hybridized and other modifications), which might be important in regulating Xenopus mothers against dpp 2361

Mad activity given that TGF-β-related receptors are Perhaps this reflects an increase in posterior mesoderm. Red serine/threonine kinases. Surprisingly, XMad and other Mad- blood differentiation is also slightly higher in these embryos related proteins contain no conserved protein domains that compared to controls (compare lanes b and d, Fig. 3F). would suggest particular functions for these factors, other than XMad and DMad induce ventral-posterior mesoderm in predicting that they will be cytoplasmic. animal caps when expressed from microinjected mRNA (Fig. Analysis by northern blot revealed that transcripts of XMad 4). Injection of 3 ng of mRNA for either factor induces are stored maternally and that the gene is expressed through- mesoderm-specific genes that mark ventral (globin, Xwnt-8), out development through swimming tadpole stages (Fig. 2A). posterior (Xhox-3 and XlHbox-6) and lateral plate (Xtwist) ter- The most abundant transcript is 3.4 kb in size, but minor tran- ritories, but markers of dorsal mesoderm such as goosecoid and scripts of 3.0 and 4.3 kb are also detected. The full-length cardiac (muscle) actin are not induced (data not shown). XMad XMad cDNA cloned in this study is 3.2 kb long, closely cor- and DMad do not induce neural tissue (see Fig. 6). The fact relating with these transcript sizes, but it is not known whether that mesoderm induction by XMad and DMad is essentially these transcripts derive from several related XMad genes or are identical to that of BMP-4 suggests that Mad activates the alternative splicing products of a single gene. BMP-4 signaling pathway. By whole mount in situ hybridization in embryos (Fig. 2B- E) XMad mRNA is not localized in the blastula and early XMad functions downstream of the BMP-2/4 gastrula stages, a result confirmed by a northern blot on RNA receptor of dissected embryonic regions (data not shown). XMad tran- If XMad operates in inductive signaling by BMP-2 or BMP-4, scripts begin to show localized expression at mid-gastrula does it act before or after the reception of a BMP ligand by its stages, at which time they become enriched within involuting receptor? If XMad functions within cells that receive BMP mesoderm around the yolk plug, in addition to being abun- signals, elevated levels of XMad protein might compensate for dantly expressed in the ectoderm (Fig. 2C). At the end of neu- a block in the activity of BMP-2/4 receptors. If, however, rulation XMad is localized to the nervous system (not shown), XMad functions upstream of the receptor, for example in steps and by early tadpole stages XMad transcripts are restricted to the central 1 nervous system, eye and head neural XMad MN crest cell populations (Fig. 2D,E). XMad DMad MDTDDVESNTSSAMST transcripts are located in areas adjacent 1 10 to, or overlapping with, regions where 10 20 30 40 50 60 70 80 BMP-2 and BMP-4 transcripts are found VTSLFSFTSPAVKRLLGWKQGDEEEKWAEEAVDALVKKLKKKKGAIQELEKALTCPGQPSNCVTIPRSLDGRLQVSHRKG :.|||||||||||:|||||||||||||||.|||:|||||||:||||:|||:||:||||||:||||||||||||||||||| (Fainsod et al., 1994; Hemmati- LGSLFSFTSPAVKKLLGWKQGDEEEKWAEKAVDSLVKKLKKRKGAIEELERALSCPGQPSKCVTIPRSLDGRLQVSHRKG Brivanlou and Thomsen, 1995; Schmidt 20 30 40 50 60 70 80 90 et al., 1995), consistent with expecta- 90 100 110 120 130 140 150 160 tions that a molecule involved in BMP LPHVIYCRVWRWPDLQSHHELKPLECCEYPFGSKQKEVCINPYNYKRVESPVLPPVLVPRHSEYNPQHSLLAQFRNLEPS receptor signaling would be expressed ||||||||||||||||||||||||| |:|||::||||||||||:|||||||||||||||||||:.| ||:| ||.:: : LPHVIYCRVWRWPDLQSHHELKPLELCQYPFSAKQKEVCINPYHYKRVESPVLPPVLVPRHSEFAPGHSML-QFNHV--A in cells near a source of BMP ligands. 100 110 120 130 140 150 160 170 Ventral mesodermal patterning 170 180 190 200 210 220 230 240 EPHMPHNATFPDSFQQPNSHPFPHSPNSSYPNSPGSSSTYPHSPASSDPGSPFQIPADTPPPAYMPPEDQMTQDNSQPMD by Xenopus Mad || ||||.:::: | :|.: |.:::|. :.|:.:|:|.||:: |:|||||| |:|| ::::| | In Xenopus embryos and animal cap EPSMPHNVSYSN------SGF-----NSHSLSTSNTSVGSPSSVNSNPNSPYDSLAGTPPPAYSPSED---GNSNNPND assays XMad and DMad display similar 180 190 200 210 220 230 activities when expressed from microin- 250 260 270 280 290 300 310 320 jected mRNA. Expression of 3 ng of TNMMVPNISQDINRADVQAVAYEEPKHWCSIVYYELNNRVGEAFHASSTSVLVDGFTDPSNNRNRFCLGLLSNVNRNSTI .. :: ..:: :||..|:|.|| | ||.||||| ||||.|| ::.||:|||||:||||.:| ||| |||||||||| XMad mRNA in the dorsal marginal -GGQLL--DAQM--GDVAQVSYSEPAFWASIAYYELNCRVGEVFHCNNNSVIVDGFTNPSNNSDRCCLGQLSNVNRNSTI zone completely ventralizes the 240 250 260 270 280 290 300 310 embryos (Fig. 3C), yielding a dorso- 330 340 350 360 370 380 390 400 anterior index (Kao and Elinson, 1989), ENTRRHIGKGVHLYYVGGEVYAECLSDSSIFVQSRNCNFHHGFHPTTVCKIPSGCSLKIFNNQEFAQLLAQSVNHGFETV or DAI of 0 (n=18). This is also reflected ||||||||||||||||.|||||||||||:|||||||||:||||||:||||||:||||||||||||||||:||||:|||:| ENTRRHIGKGVHLYYVTGEVYAECLSDSAIFVQSRNCNYHHGFHPSTVCKIPPGCSLKIFNNQEFAQLLSQSVNNGFEAV by the highly elevated levels of red 320 330 340 350 360 370 380 390 blood cell differentiation (a ventral mesodermal derivative) and a complete 410 420 430 440 450 460 YELTKMCTIRMSFVKGWGAEYHRQDVTSTPCWIEIHLHGPLQWLDKVLTQMGSPHNPISSVS loss of muscle (a dorsal mesodermal ||||||||||||||||||||||||||||||||||||||||||||||||||||||||:||||| tissue) in those embryos (compare lanes YELTKMCTIRMSFVKGWGAEYHRQDVTSTPCWIEIHLHGPLQWLDKVLTQMGSPHNAISSVS a and c in Fig. 3F). DMad also ventral- 400 410 420 430 440 450 izes Xenopus embryos, albeit less effec- tively than XMad (DAI=1.9, n=20; Fig. Fig. 1. Protein sequence comparison of Xenopus Mad with Drosophila Mad. The predicted Xenopus Mad (XMad) protein sequence is aligned with Drosophila Mad protein (DMad). The 3E). Over-expression of XMad on the XMad cDNA encodes a predicted protein of 464 amino acids. XMad protein is 75% identical ventral side results in embryos with a to DMad at the amino acid level (identical residues are indicated by vertical bars) and 85% slightly enlarged posterior-ventral similar when conservative amino acid substitutions are considered (double dots). Note the high region, but which otherwise appear degree of conservation in the N-terminal and C-terminal portions of the proteins. The normal (DAI=5, n=20; Fig. 3D). GenBank accession number for XMad is U58834. 2362 G. H. Thomsen

Fig. 3. XMad and DMad ventralize Xenopus embryos. Synthetic mRNAs encoding control (pGem vector) or Mad sequences were injected into the equatorial region of two dorsal or two ventral blastomeres at the 4-cell blastula stage, and phenotypes were scored at tadpole stage 40. Dorsal (A) or ventral (B) injections of control (pGem vector) mRNA resulted in normal embryos. (C) Dorsal injection of XMad mRNA caused severe ventralization. (D) Ventral Fig. 2. Developmental expression of Xenopus Mad. (A) A injection of XMad mRNA caused posterior thickening and a slight developmental northern blot of total embryonic RNA shows that reduction in the tail. The average dorso-anterior index, a measure of XMad is maternal and expressed at all stages of early development. the degree of dorsal and anterior mesodermal patterning (Kao and Three transcripts are detected with the sizes (in kb) indicated on the Elinson, 1989), was for each group: A, DAI=5 (n=18); B, DAI=5 left. The most abundant mRNA is 3.4 kb, and minor transcripts of (n=18); C, DAI=0 (n=18); D, DAI=5 (n=20). (E) Expression of 4.3 kb and 3.0 kb are also detected. It is not known whether these DMad in dorsal blastomeres ventralized the embryos (DAI=1.9, transcripts derive from closely related genes or splicing variants. n=20), while ventral expression yielded relatively normal embryos Lane numbers correspond to developmental stages (Nieuwkoop and (DAI=5, n=21; not shown). (F) Northern blot analysis of muscle Faber, 1967): (7, 9) blastula, (11) gastrula, (15, 18) neurula, (26) actin (a dorsal mesodermal marker) and αT1 globin (a ventral tailbud tadpole and (38) swimming tadpole. (B) In situ hybridization mesodermal marker) gene expression in the control and XMad- shows that XMad transcripts are uniformly distributed in the early injected embryos shown in A-D. Lanes a-d correspond to embryos in gastrula (stage 10). Dense staining covers the entire prospective A-D. Note the loss of muscle actin and the significant increase in ectoderm and marginal zone, but vegetal cells (the lighter region) do globin expression in embryos injected dorsally with XMad mRNA not stain efficiently by this procedure (Harland, 1991). A northern (lane c). This indicates that overexpression of XMad protein in the blot on isolated dorsal, ventral, animal and vegetal regions confirmed presumptive dorsal mesoderm respecified its fate to that of ventral the uniformity of XMad expression at early stages (not shown). mesoderm. Ventral expression of XMad boosted globin expression (C) A mid-gastrula embryo (stage 12) split sagitally reveals that slightly (lane d), but dorsal or ventral injections of vector mRNA had XMad is expressed in the ectoderm and neurectoderm (arrows mark no effect (lanes a and b). Histone H4 mRNA (Perry et al., 1985) was the ectodermal-mesodermal boundary), and expression in the scored as a control for RNA loading. underlying mesoderm is greater in the posterior, adjacent to the yolk plug (YP). This embryo is lightly pigmented and the brown line anterior to the yolk corresponds to involuted bottle cells. The embryo embryos (Graf et al., 1994; Maeno et al., 1994; Suzuki et al., is positioned with the anterior to the left and dorsal at the top. (D) At 1994). Ectopic expression of dominant-negative BMP tailbud tadpole stage 26 XMad expression is high in the central receptors in the ventral side of Xenopus embryos causes nervous system and head. (E) A close-up of the head of the embryo prospective ventral mesoderm to differentiate into dorsal in D, highlighting XMad expression in the brain (b), eye (e) and head mesoderm, resulting in development of ectopic dorsal axial neural crest derivatives (mc, mandibular crest; hc, hyoid crest; abc, structures. Cultured VMZ explants expressing a dominant- anterior branchial crest; pbc, posterior branchial crest). Expression in negative BMP receptor are similarly dorsalized, do not form the otic vesicle, between the hyoid crest and anterior branchial crest, is also visible. Scale bars, 0.1 mm. blood and instead develop muscle (Graf et al., 1994; Suzuki et al., 1994; and see Fig. 5). In Xenopus ectoderm expression of a dominant-negative associated with ligand synthesis, processing or secretion, BMP-2/4 receptor triggers neural differentiation by blocking XMad would not be expected to complement defective receptor activation by endogenous BMP-2 or BMP-4 (Sasai et receptors. Truncated BMP-2/4 receptors lacking an intracellu- al., 1995). The likelihood that BMP-4 signals specify lar ser/thr kinase domain act as dominant-negative inhibitors epidermal rather than neural differentiation was demonstrated of cellular responses to BMP-2 and BMP-4 ligands in Xenopus by exposing dissociated animal cap cells to BMP-4 protein Xenopus mothers against dpp 2363 followed by reaggregation (Wilson and Hemmati-Brivanlou, receptors is blocked (Sasai et al., 1995; Xu et al., 1995). Fig. 1995). This treatment induces epidermis in the animal cap 6 demonstrates that XMad can suppress neural differentiation cells, which otherwise differentiate into neurons (Grunz and triggered by the dominant-negative BMP-2/4 receptor, tBR. Tacke, 1989; Wilson and Hemmati-Brivanlou, 1995). Neural Expression of tBR alone induces N-Cam and NRP-1 (a ribonu- differentiation can be considered the ‘default’ program that cleoprotein gene enriched in neural tissue), but induction of ectodermal cells follow in the absence of BMP-4 signals. these markers is reversed by expressing XMad (XM) together If XMad functions downstream of the BMP-2/4 receptor in with tBR. XMad alone does not induce neural tissue. The Xenopus, over-expression of XMad might rescue phenotypes results of this dominant-negative BMP receptor rescue exper- generated by interfering with BMP-2/4 receptor activity. This iment, like those done in the marginal zone (Fig. 5), demon- possibility was tested by co-expressing XMad together with a strate that XMad functions downstream of the BMP-2/4 dominant-negative BMP-2/4 receptor (tBR, (Graf et al., 1994). receptor. The results in animal caps also suggest that control Expression of XMad together with tBR reverses the formation over the activity of XMad could be a mechanism to regulate of ectopic axial structures caused by expression of tBR alone the decision between epidermal or neural differentiation. in the ventral side of whole embryos (compare Fig. 5A with It is curious that it takes much less XMad mRNA to rescue 5B). This rescue was 100% effective at a dose of XMad mRNA the dominant-negative-BMP receptor than it does to induce (50 pg) equivalent to that of tBR mRNA needed to generate a mesoderm or ventralize embryos. An interpretation of these secondary axis. Defects caused by higher doses of tBR (up to results is that a relatively low level of BMP signaling is suffi- 200 pg mRNA) are also rescued by an equivalent amount of cient to suppress both the neuralization of the ectoderm and the XMad mRNA (data not shown). Cultured VMZ explants latent capacity of the presumptive ventral mesoderm to develop injected with tBR mRNA elongate and produce melanocytes into dorsal, trunk-type mesoderm when BMP signals are indicating neural induction (Fig. 5D), and they form muscle blocked. Mesoderm induction in the animal cap, and ventral- but not erythrocytes, as demonstrated by a northern blot on the VMZ RNA (Fig. 5G, lane d). Co-expression of XMad and tBR (each at 50 pg mRNA) in VMZ explants completely reverses these effects and the rescued VMZs form blood, lack muscle, and look like control VMZ explants (Fig. 5E-G). Intact animal cap explants form epidermis in culture, but they undergo neural differentiation if signaling by BMP-2/4

Fig. 5. XMad rescues dominant-negative BMP2/4 receptor phenotypes. The panels display phenotypes of whole embryos (A-C) and isolated ventral marginal zones (VMZs, D-E) expressing the dominant-negative BMP-2/4 receptor (Graf et al., 1994), alone or in combination with XMad. 50 pg of each mRNA were injected into the marginal zone of two ventral blastomeres at the 4-cell stage. VMZs were explanted at stage 10.5, and embryos and VMZ explants were scored at stage 40. (A) Expression of the dominant-negative BMP- 2/4 receptor (tBR) from injected mRNA resulted in tadpoles with secondary axial structures in 33% of the cases (n=18). A typical secondary axis is indicated by the arrows. (B) Co-expression of XMad together with tBR resulted in 100% normal embryos (n=18). (C) Injection of control (pGem) mRNA resulted in 100% normal Fig. 4. XMad and DMad induce mesoderm in animal caps. The panel embryos (n=21). (D) VMZs expressing tBR elongated and developed shows an RT-PCR analysis of mesodermal marker gene expression pigmented melanocytes, a neural derivative. (E) VMZs from in animal caps injected with 3.0 ng of mRNA for pGem (C), DMad embryos co-expressing tBR and XMad were rescued to normalcy (DM), XMad (XM) or Xenopus BMP-4 (B4). The last two lanes and formed oblong ‘belly pieces’ like control VMZs (F). show RT-PCR products from stage-18 embryonic cDNA synthesized (F) Injection of control (pGem) mRNA into VMZs resulted in a in the presence (emb RT+) or absence (emb RT−) of reverse typical wild-type VMZ morphology. (G) A northern blot on RNA transcriptase to control for cDNA synthesis and DNA contamination, from the VMZs shown in D-F. VMZs expressing tBR (lane d) respectively. Note that expression of DMad, XMad and BMP-4 developed dorsal mesoderm, as revealed by the expression of muscle induced each of the ventro-posterior mesoderm markers assayed. actin, and they lacked ventral mesoderm (red blood) as reﬂected by EF1-α was scored as a positive control for cDNA synthesis. Animal the absence of αT1 globin expression. When XMad was co-expressed caps were harvested at stage 30 (tadpole) to score αT1 globin with tBR (lane e) dorsalization of the VMZ was reversed; the explants expression, stage 11 (mid-gastrula) to score Xwnt 8 and Xtwist, and lacked muscle and expressed globin, similar to control VMZs (lane f). stage 18 (neurula) to score Xhox-3 and Xlhbox6. The eF1-α signal Cytoplasmic actin mRNAs cross-hybridize with the muscle actin shown corresponds to that of stage 11 cDNA, but all other cDNA probe and migrate as two bands above the muscle-speciﬁc message, samples treated with reverse transcriptase were positive. providing a positive control for RNA loading in the gel. 2364 G. H. Thomsen

do not induce blood (Kessler and Melton, 1994). Conversely, activin and Vg1 induce primarily dorsal mesoderm, but maximum doses of XMad, BMP-2 and BMP-4 do not, arguing that XMad does not function in signal transduction pathways for activin, Vg1 or indeed other factors capable of inducing or patterning dorsal mesoderm such as noggin (Smith et al., 1993), chordin (Sasai et al., 1994), or nodal-related factors (Jones et al., 1995). In the ectoderm BMP-4 protein, like XMad, inhibits neural differentiation, but activin does not Fig. 6. Neural induction by tBR expression in animal cap ectoderm is (Wilson and Hemmati-Brivanlou, 1995). In addition, FGF inhibited by co-expression of XMad. Lanes 1-4 correspond to an RT- induces and patterns neural tissue (Doniach, 1995), which is a PCR analysis of cDNA from animal caps injected at the 2-cell stage property opposite to that of XMad. Thus I conclude that XMad with 2 ng pGem RNA (lane C),1.0 ng XMad mRNA (lane XM), 50 functions in signal transduction from BMP-2/4 receptors. pg tBR mRNA (lane tBR), and 50 pg tBR mRNA plus 50 pg XMad XMad may also function in BMP-7 signal transduction, since mRNA (lane tBR + XM). Caps were cut at stage 8 and harvested at this BMP can also specify ventral mesoderm like BMP-2 and neurula stage 18. Note the induction of N-CAM and NRP1 (a neural- BMP-4 (S. Nishimatsu and G. H. Thomsen, unpublished obser- enriched ribonucleoprotein; Richter et al., 1990) mRNAs when BMP vations). It is, of course, formally possible that XMad operates signals were blocked by tBR. This effect was suppressed by co- in another, perhaps parallel, signal transduction pathway that expression of XMad. Some background expression of NRP1 is triggers biological responses similar to those of BMPs. normal. Furthermore, XMad alone did not induce neural tissue (lane XM). Lanes 5 and 6 are positive and negative controls for RT-PCR Generally speaking the sum of the data predict that various as in Fig. 2. EF1-α is a positive control for cDNA synthesis. Mad proteins will function in signal transduction for the family of TGF-β receptors, but direct biochemical tests will be required to firmly establish the link between TGF-β-related receptors and Mad proteins. ization of the dorsal/anterior mesoderm of the organizer, In this study overexpression of XMad was shown to induce however, appear to require a much higher level of XMad (or and pattern ventral but not dorsal mesoderm, which prompts BMP) activity. In fact, there is evidence that Xenopus animal the suggestion that other Mad-related factors may transduce cap ectoderm has low and high response thresholds for BMP- signals for TGF-β-related ligands involved in dorsal mesoderm 4 ligand. Wilson and Hemmati-Brivanlou (1995) showed that induction or patterning, such as activin, Vg1 or nodal-related it takes only 0.3 ng/ml of BMP-4 to switch ectodermal cells proteins. Similarly, the TGF-βs proper are principally involved from a neural to epidermal fate, yet it takes a much higher dose, in the control of cell growth, so it is tempting to suggest that 1000 ng/ml, to induce mesoderm. DPC4, a Mad-related tumor suppressor gene, operates in TGF- β receptor signaling (Hahn et al., 1995), but that remains to be established. The conclusion that Mad proteins function in DISCUSSION signal transduction for members of the TGF-β family is also supported by genetic experiments in C. elegans, which demon- Drosophila mothers against dpp was isolated as an enhancer strate that mutations in the Mad-related small genes (or of dpp alleles, placing it somewhere in the pathway of Dpp dwarfins) phenocopy mutations in the C. elegans BMP-2/4 signaling. The Drosophila genetic studies did not, however, receptor, daf-4, and are not rescued by daf-4 overexpression establish whether Mad protein acts in cells that deliver or (Savage et al., 1996). respond to dpp ligand. The present study establishes that a ver- It is interesting that wild-type Xenopus and Drosophila Mad tebrate homolog of Drosophila Mad exists and functions in the display activity (mesoderm induction, BMP receptor rescue) specification of embryonic ventral mesodermal cell fates. when overexpressed as wild-type proteins, in contrast to other XMad may also function in the decision between neural and signal transduction molecules such as wild-type ras and MAP epidermal cell fates in the ectoderm. XMad mimics all the kinase, which do not induce mesoderm (Whitman and Melton, activities of the BMP-4 ligand: it can induce mesoderm, 1992). Instead only mutant, constitutively activated, forms of respecify presumptive dorsal mesoderm to form ventral ras and MAP-kinase induce mesoderm when overexpressed in mesoderm and suppress neural differentiation. Most impor- animal caps (Whitman and Melton, 1992; Cornell and tantly, this study demonstrates that XMad can rescue defects Kimelman, 1994; LaBonne and Whitman, 1994; Schmidt et al., caused by blocking signals from a BMP-2/4 receptor, 1995). My results imply that XMad protein has some level of providing strong evidence that XMad functions in BMP signal intrinsic activity when expressed in animal cap and marginal transduction. zone cells, even in the absence of BMP-2/4 receptor activity. The possibility that XMad is a component of signal trans- This behavior of XMad is similar to that seen with components duction pathways for receptors other than the BMPs in in the wnt signal transduction pathway, such as disheveled Xenopus seems unlikely. Among the known mesoderm- (Sokol et al., 1995) and GSK-3 (Dominguez et al., 1995; He inducing factors in Xenopus only BMP-4 is capable of inducing et al., 1995), which are also active as wild-type forms in ventral mesoderm, ventralizing dorsal mesoderm and inhibit- Xenopus mesodermal patterning assays. ing neural differentiation, each of which I have shown in the A molecular explanation for why XMad is active in over- present study to be properties of XMad. Of the known expression and dominant-negative receptor assays may relate mesoderm inducers, activin, Vg1 and fibroblast growth factor to its level of phosphorylation. Recently, Hoodless et al. (1996) (FGF) each induce some types of ventral mesoderm, but they found that the human equivalent of XMad (hXMADR1) is Xenopus mothers against dpp 2365 partially phosphorylated in the absence of BMP signals, and it (1993). The daf-4 gene encodes a bone morphogenetic protein receptor becomes hyper-phosphorylated upon BMP-2 stimulation. This controlling C. elegans larval development. Nature 365, 644-649. might explain why XMad is active in embryonic cells lacking Fainsod, A. Steinbeisser, H. and De Robertis, E.M. (1994). On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. BMP signals, and why relatively high amounts of injected 13, 5015-5025. XMad mRNA are required to induce mesoderm. It will be Graf, J. Theis, R. Song, J. Celeste, A. and Melton, D. (1994). Studies with a informative to determine whether region-specific differences in Xenopus BMP receptor suggest that ventral mesoderm-inducing signals XMad phosphorylation exist in Xenopus embryos, and whether override dorsal signals in vivo. Cell 79, 169-179. Grunz, H. and Tacke, L. (1989). Neural differentiation of Xenopus laevis phosphorylation differences correlate with activity. ectoderm takes place after dissagregation and delayed reaggregation without The regional expression of XMad mRNA correlates with the inducer. 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