NORMAL GENOMIC

• Oelgeschlager M, Kuroda H, Reversade B, De Robertis EM
• Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.
• Dev Cell. 2003; 4: 219-30
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• We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.

• Garcia Abreu J, Coffinier C, Larrain J, Oelgeschlager M, De Robertis EM
• Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems.
• Gene. 2002; 287: 39-47
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• In fruit flies as well as in humans the Short gastrulation (Sog)/Chordin protein functions as an antagonist of the signaling of decapentaplegic (Dpp)/bone morphogenetic protein (BMP) in the extracellular space. Such antagonism inhibits Dpp/BMP signaling by blocking its binding to the receptor. Modulation of Dpp/BMP signaling is phylogenetically conserved and is a key step for the establishment of the dorso-ventral axis in vertebrates and invertebrates. Molecular studies have shown that the inhibitory activity of Chordin on BMP resides in specific cysteine-rich (CR) domains. Interestingly, Chordin-like CR domains are present in a growing number of extracellular proteins, several of which appear to be involved in BMP signaling regulation. We review here the conservation of the Chordin and Sog proteins, and in particular their functional domain, the CR domain. We discuss how the study of CR domains may provide a general mechanism for the regulation of growth factor signaling in the extracellular space.

• Abreu JG, Ketpura NI, Reversade B, De Robertis EM
• Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta.
• Nat Cell Biol. 2002; 4: 599-604
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• Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-beta binding domains. Here we show that CTGF directly binds BMP4 and TGF-beta 1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-beta 1. These results show that CTGF inhibits BMP and activates TGF-beta signals by direct binding in the extracellular space.

• Millet C, Lemaire P, Orsetti B, Guglielmi P, Francois V
• The human chordin gene encodes several differentially expressed spliced variants with distinct BMP opposing activities.
• Mech Dev. 2001; 106: 85-96
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• During early embryogenesis of both vertebrates and invertebrates, antagonism between bone morphogenetic proteins (BMPs) and several unrelated secreted factors including Chordin (Chd) is a general mechanism by which the dorso-ventral axis is established. High affinity binding of Chd sequesters the BMP ligands in the extracellular space, preventing interactions with their membrane receptors. Another level of regulation consists in processing of vertebrate Chd or its Drosophila counterpart Sog by astacine metalloproteases like Xolloid-BMP-1/Tolloid, respectively, which releases an active BMP. Recently, it was shown that cleavage of Sog by Tolloid could generate novel BMP inhibitory activity and that sog is also capable of stimulation of BMP activity in a tolloid-dependant way. Activity and/or cleavage of Chd/Sog are influenced by other secreted factors like twisted gastrulation. In this study, we have cloned cDNAs of the human chordin gene (CHRD) and characterized alternative splice variants that code for C-truncated forms of the protein. We have found that CHRD is expressed in fetal as well as in adult tissues with relatively high levels in liver, cerebellum and female genital tract, suggesting functions in late embryogenesis and adult physiology. We also show that spliced variants are present with specific patterns in various tissues. When tested in an axis-duplication assay in Xenopus, we find that these variants can antagonize BMP activity. Altogether, these results suggest that, in addition to processing by metalloproteases, alternative splicing (AS) is another mechanism by which sub-products of CHRD can be generated to influence BMP activity in different developmental and physiological situations.

• Nakayama N et al.
• A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages.
• Dev Biol. 2001; 232: 372-87
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• Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.

• De Robertis EM et al.
• Molecular mechanisms of cell-cell signaling by the Spemann-Mangold organizer.
• Int J Dev Biol. 2001; 45: 189-97
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• We review how studies on the first Spemann-Mangold organizer marker, the homeobox gene goosecoid, led to the discovery of secreted factors that pattern the vertebrate embryo. Microinjection of goosecoid mRNA formed secondary axes and recruited neighboring cells. These non-cell autonomous effects are mediated in part by the expression of secreted factors such as chordin, cerberus and Frzb-1. Unexpectedly, many of the molecules secreted by the Spemann-Mangold organizer turned out to be antagonists that bind growth factors in the extracellular space and prevent them from binding to their receptors. The case of chordin is reviewed in detail, for this molecule has provided biochemical insights into how patterning by Spemann's organizer can be regulated by diffusion and proteolytic control. The study of the BMP-binding repeats of Chordin, which are present in many extracellular proteins, may provide a new paradigm for how cell-cell signaling is regulated in the extracellular space not only in embryos, but also in adult tissues.

• Larrain J, Bachiller D, Lu B, Agius E, Piccolo S, De Robertis EM
• BMP-binding modules in chordin: a model for signalling regulation in the extracellular space.
• Development. 2000; 127: 821-30
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• A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.

• Dosch R, Niehrs C
• Requirement for anti-dorsalizing morphogenetic protein in organizer patterning.
• Mech Dev. 2000; 90: 195-203
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• The amphibian Spemann organizer is subdivided in trunk and head organizer and it is unclear how this division is regulated. The Xenopus trunk organizer expresses anti-dorsalizing morphogenetic protein (ADMP), a potent organizer antagonist. We show that ADMP represses head formation during gastrulation and that its expression is activated by BMP antagonists. A specifically acting dominant-negative ADMP anteriorizes embryos and its coexpression with BMP antagonists induces secondary embryonic axes with heads as well as expression of head inducers. Unlike other BMPs, ADMP is not inhibited by a dominant-negative BMP type I receptor, Noggin, Cerberus and Chordin but by Follistatin, suggesting that it utilizes a distinct TGF-beta receptor pathway and displays differential sensitivity to BMP antagonists. The results indicate that ADMP functions in the trunk organizer to antagonize head formation, thereby regulating organizer patterning.

• Iemura S, Yamamoto TS, Takagi C, Kobayashi H, Ueno N
• Isolation and characterization of bone morphogenetic protein-binding proteins from the early Xenopus embryo.
• J Biol Chem. 1999; 274: 26843-9
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• Using a surface plasmon resonance biosensor as a sensitive and specific monitor, we have isolated two distinct bone morphogenetic protein (BMP)-binding proteins, and identified them as lipovitellin 1 and Ep45, respectively. Lipovitellin 1 is an egg yolk protein that is processed from vitellogenin. Both vitellogenin and Ep45 are synthesized under estrogen control in the liver, secreted, and taken up by developing oocytes. In this paper, we have shown that of the TGF-beta family members tested, Ep45 can bind only to BMP-4, whereas lipovitellin 1 can bind to both BMP-4 and activin A. Because of this difference in specificity, we have focused on and further studied Ep45. Kinetic parameters were determined by surface plasmon resonance studies and showed that Ep45 associated rapidly with BMP-4 (k(a) = 1.06 x 10(4) M(-1)s(-1)) and dissociated slowly (k(d) = 1.6 x 10(-4) s(-1)). In Xenopus embryos microinjected with Ep45 mRNA, Ep45 blocked the ability of follistatin to inhibit BMP activity and to induce a secondary body axis in a dose-dependent manner, whereas it had no effect on other BMP antagonists, chordin and noggin. These results support the possibility that Ep45 interacts with BMP to modulate its activities in vivo.

• de Souza FS, Gawantka V, Gomez AP, Delius H, Ang SL, Niehrs C
• The zinc finger gene Xblimp1 controls anterior endomesodermal cell fate in Spemann's organizer.
• EMBO J. 1999; 18: 6062-72
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• The anterior endomesoderm of the early Xenopus gastrula is a part of Spemann's organizer and is important for head induction. Here we describe Xblimp1, which encodes a zinc finger transcriptional repressor expressed in the anterior endomesoderm. Xblimp1 represses trunk mesoderm and induces anterior endomesoderm in a cooperative manner with the pan-endodermal gene Mix.1. Furthermore, Xblimp1 can cooperate with the BMP inhibitor chordin to induce ectopic heads, while a dominant-negative Xblimp1 inhibits head formation. The head inducer cerberus is positively regulated by Xblimp1 and is able to rescue microcephalic embryos caused by dominant-negative Xblimp1. Our results indicate that Xblimp1 is required for anterior endomesodermal cell fate and head induction.

• Streit A, Lee KJ, Woo I, Roberts C, Jessell TM, Stern CD
• Chordin regulates primitive streak development and the stability of induced neural cells, but is not sufficient for neural induction in the chick embryo.
• Development. 1998; 125: 507-19
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• We have investigated the role of Bone Morphogenetic Protein 4 (BMP-4) and a BMP antagonist, chordin, in primitive streak formation and neural induction in amniote embryos. We show that both BMP-4 and chordin are expressed before primitive streak formation, and that BMP-4 expression is downregulated as the streak starts to form. When BMP-4 is misexpressed in the posterior area pellucida, primitive streak formation is inhibited. Misexpression of BMP-4 also arrests further development of Hensen's node and axial structures. In contrast, misexpression of chordin in the anterior area pellucida generates an ectopic primitive streak that expresses mesoderm and organizer markers. We also provide evidence that chordin is not sufficient to induce neural tissue in the chick. Misexpression of chordin in regions outside the future neural plate does not induce the early neural markers L5, Sox-3 or Sox-2. Furthermore, neither BMP-4 nor BMP-7 interfere with neural induction when misexpressed in the presumptive neural plate before or after primitive streak formation. However, chordin can stabilise the expression of early neural markers in cells that have already received neural inducing signals. These results suggest that the regulation of BMP signalling by chordin plays a role in primitive streak formation and that chordin is not sufficient to induce neural tissue.

• Hsu DR, Economides AN, Wang X, Eimon PM, Harland RM
• The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.
• Mol Cell. 1998; 1: 673-83
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• Using a Xenopus expression-cloning screen, we have isolated Gremlin, a novel antagonist of bone morphogenetic protein (BMP) signaling that is expressed in the neural crest. Gremlin belongs to a novel gene family that includes the head-inducing factor Cerberus and the tumor suppressor DAN. We show that all family members are secreted proteins and that they act as BMP antagonists in embryonic explants. We also provide support for the model that Gremlin, Cerberus, and DAN block BMP signaling by binding BMPs, preventing them from interacting with their receptors. In addition, Cerberus alone blocks signaling by Activin- and Nodal-like members of the TGF beta superfamily. Therefore, we propose that Gremlin, Cerberus, and DAN control diverse processes in growth and development by selectively antagonizing the activities of different subsets of the TGF beta ligands.

• Nishimatsu S, Thomsen GH
• Ventral mesoderm induction and patterning by bone morphogenetic protein heterodimers in Xenopus embryos.
• Mech Dev. 1998; 74: 75-88
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• Bone morphogenetic proteins (BMPs) perform diverse functions in vertebrate development. Here we demonstrate that the heterodimeric BMP-4/7 protein directly induces ventral mesoderm and blood in Xenopus animal caps, and BMP-2/7 heterodimers may function similarly. We also provide indirect evidence that BMP heterodimers function in embryos, using assays with dominant-negative BMP ligands. Homodimeric BMP-2 and BMP-4 proteins do not induce mesoderm, but they ventralize mesoderm induction by activin. In contrast, BMP-7 protein interferes with mesoderm induction by activin, but BMP-7 stimulates ventral mesoderm induction by the heterodimer, BMP-4/7. This novel property of BMP-7 distinguishes it from other BMPs. BMP-7 may therefore function in early embryogenesis to antagonize activin signals and potentiate BMP signals. We propose that BMP heterodimers convey signals for ventral mesoderm induction and patterning in Xenopus development.

• Bhushan A, Chen Y, Vale W
• Smad7 inhibits mesoderm formation and promotes neural cell fate in Xenopus embryos.
• Dev Biol. 1998; 200: 260-8
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• We report the isolation and characterization of a new inhibitory Smad in Xenopus, which we have designated as Xenopus Smad7. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-beta-like signals is mediated by receptor ALK-4 and we show that Smad7 blocks signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway.

• Leyns L, Bouwmeester T, Kim SH, Piccolo S, De Robertis EM
• Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.
• Cell. 1997; 88: 747-56
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• Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

• Piccolo S, Agius E, Lu B, Goodman S, Dale L, De Robertis EM
• Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity.
• Cell. 1997; 91: 407-16
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• The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites in biochemical experiments Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein was unable to antagonize BMP activity. Furthermore, Xolloid digestion released biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicated that the in vivo function of Xolloid is to limit the extent of Spemann's organizer field. We propose that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis: [formula in text].

• Miller-Bertoglio VE, Fisher S, Sanchez A, Mullins MC, Halpern ME
• Differential regulation of chordin expression domains in mutant zebrafish.
• Dev Biol. 1997; 192: 537-50
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• Patterning along the dorsal-ventral (D-V) axis of Xenopus and Drosophila embryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous to chordin and sog within cysteine-rich domains and exhibits conserved aspects of expression and function. As in Xenopus embryos, zebrafish chordin is expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafish chordin mRNA to the ventral side of Xenopus embryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullins et al., 1996). In accordance with the proposed function of chordin in D-V patterning, dorsalized zebrafish mutants showed expanded domains of chordin expression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast to Xenopus, zebrafish chordin is also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles for chordin in zebrafish embryonic development. Surprisingly, paraxial mesodermal expression of chordin appeared unaltered in spadetail mutants that later lack trunk muscle (Kimmel et al., 1989), while axial mesodermal expression was affected. This finding reveals an unexpected function for spadetail in midline mesoderm and in differential regulation of chordin expression during gastrulation.

• Onichtchouk D et al.
• The Xvent-2 homeobox gene is part of the BMP-4 signalling pathway controlling [correction of controling] dorsoventral patterning of Xenopus mesoderm.
• Development. 1996; 122: 3045-53
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• We describe a novel Xenopus homeobox gene, Xvent-2, which together with the previously identified homeobox gene Xvent-1, defines a novel class of homeobox genes. vent genes are related by sequence homology, expression pattern and gain-of-function phenotype. Evidence is presented for a role of Xvent-2 in the BMP-4 pathway involved in dorsoventral patterning of mesoderm. (1) Xvent-2 is expressed in regions that also express BMP-4. (2) Xvent-2 and BMP-4 interact in a positive feedback loop. (3) Xvent-2 ventralizes dorsal mesoderm in a dose-dependent manner resulting in phenoytpes ranging from microcephaly to Bauchstuck pieces, as does BMP-4. (4) Like BMP-4 and gsc, Xvent-2 and gsc are able to interact in a crossregulatory loop to suppress each other. (5) Microinjection of Xvent-2 mRNA can rescue dorsalization by a dominant-negative BMP-4 receptor. The results suggest that Xvent-2 functions in the BMP-4 signalling pathway that antagonizes the Spemann organizer.

• Ault KT, Dirksen ML, Jamrich M
• A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.
• Proc Natl Acad Sci U S A. 1996; 93: 6415-20
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• The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.

• Sasai Y, Lu B, Piccolo S, De Robertis EM
• Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps.
• EMBO J. 1996; 15: 4547-55
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• Spemann's organizer has potent neural inducing and mesoderm dorsalizing activities in the Xenopus gastrula. A third activity, the organizer's ability to induce a secondary gut, has been difficult to analyze experimentally due to the lack of early gene markers. Here we introduce endodermin, a pan-endodermal gene marker, and use it to demonstrate that chordin (Chd), a protein secreted by the organizer region, is able to induce endodermal differentiation in Xenopus. The ability of chd, as well as that of noggin, to induce endoderm in animal cap explants is repressed by the ventralizing factor BMP-4. When FGF signaling is blocked by a dominant-negative FGF receptor in chd-injected animal caps, neural induction is inhibited and most of the explant is induced to become endoderm. The results suggest that proteins secreted by the organizer, acting together with known peptide growth factors, regulate differentiation of the endodermal germ layer.

• Smith WC, McKendry R, Ribisi S Jr, Harland RM
• A nodal-related gene defines a physical and functional domain within the Spemann organizer.
• Cell. 1995; 82: 37-46
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• A functional screen for gene products that rescue dorsal development in ventralized Xenopus embryos has yielded Xenopus nodal-related 3 (Xnr3), a diverged member of the TGF beta superfamily. Xnr3 is specifically expressed in the Spemann organizer and is only expressed in the epithelial layer of the organizer immediately preceding and extending through gastrulation. Like noggin, Xnr3 can induce muscle in ventral mesoderm explants, consistent with a role in patterning the gastrula. In other ways, the activity of Xnr3 is different from noggin. Embryos receiving injections of Xnr3, particularly in the animal pole, send out tube-like extensions of tissue from the site of injection. These protrusions usually contain no axial mesoderm and only occasionally are positive for neural markers. It has previously been proposed that the epithelial layer of the organizer initiates and coordinates the morphogenetic movements at gastrulation. The protrusions observed may reflect an activity of Xnr3 in promoting morphogenesis.

• Moos M Jr, Wang S, Krinks M
• Anti-dorsalizing morphogenetic protein is a novel TGF-beta homolog expressed in the Spemann organizer.
• Development. 1995; 121: 4293-301
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• We have identified a novel growth factor in Xenopus, which is most closely related to human Bone Morphogenetic Protein-3. Its expression peaks during gastrulation, most prominently in the Spemann organizer, and persists in the posterior neural floor plate and prechordal plate during neurulation. Injection of the corresponding mRNA into dorsal blastomeres results in dose-dependent suppression of dorsal and anterior structures, even in the presence of lithium chloride. Overexpression of the gene downregulates the dorsalizing factors noggin, goosecoid and follistatin, as well as the dorsal markers NCAM, muscle actin and MyoD; conversely, ventral markers are induced. We therefore designate this gene product Anti-Dorsalizing Morphogenetic Protein (ADMP). Though development of dorsoanterior structures is suppressed when exogenous ADMP is injected, the gene is induced by lithium chloride treatment or activin, both of which are known to produce the opposite effect. Thus, the expression of ADMP resembles that of several dorsalizing signals, but its product exerts dorsal-suppressing activity. This suggests that ADMP may moderate organizer-associated dorsalizing influences. These findings are also consistent with the recently advanced proposal of dorsally expressed inhibitory activin-like signals.

• Re'em-Kalma Y, Lamb T, Frank D
• Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.
• Proc Natl Acad Sci U S A. 1995; 92: 12141-5
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• Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

• Hemmati-Brivanlou A, Kelly OG, Melton DA
• Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity.
• Cell. 1994; 77: 283-95
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• In the accompanying paper, we show that the expression of a dominant negative activin receptor can convert prospective ectoderm into neural tissue, which suggests that activin is an inhibitor of neuralization. Here we report the isolation and characterization of an activin antagonist, follistatin, that can induce neural tissue directly in vivo. Follistatin RNA is localized in the Spemann organizer and notochord, tissues known to be potent neural inducers. We demonstrate that follistatin RNA and protein are able to block the activity of activin in embryonic explants. Furthermore, we show that follistatin RNA directly neuralizes ectodermal explants in the absence of detectable mesoderm. Thus, follistatin is present at the correct time and location to play a role in neural induction in vivo.

• Taira M, Otani H, Saint-Jeannet JP, Dawid IB
• Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus.
• Nature. 1994; 372: 677-9
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• Like all known LIM class homeobox genes, Xlim-1 encodes a protein with two tandemly repeated cysteine-rich LIM domains upstream of the homeodomain. In Xenopus laevis, Xlim-1 is specifically expressed in the Spemann organizer, whose major functions include neural induction and dorsalization of ventral mesoderm. From RNA injection experiments we conclude here that: (1) the LIM domains behave as negative regulatory domains; (2) LIM domain mutants of Xlim-1 elicited neural differentiation in animal explants; (3) mutant, and to a lesser extent wild-type, Xlim-1 enhanced muscle formation after coinjection with Xbra; (4) both of these activities are mediated by extracellular signals as seen in combined explant experiments; (5) Xlim-1 mutants activated goosecoid (gsc) expression in animal explants, but not expression of noggin or follistatin; (6) mutant Xlim-1 elicited formation of partial secondary axes, and cooperated with gsc in notochord formation. Thus Xlim-1 has latent activities, implicating it in organizer functions.

• Dohrmann CE, Hemmati-Brivanlou A, Thomsen GH, Fields A, Woolf TM, Melton DA
• Expression of activin mRNA during early development in Xenopus laevis.
• Dev Biol. 1993; 157: 474-83
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• Activins are members of the transforming growth factor-beta superfamily, a class of peptide growth factors that can regulate the growth and differentiation of a variety of cell types. In mesoderm induction assays, activins A and B were shown to be very potent inducers and it was only recently demonstrated that they are crucial for initial mesoderm induction in Xenopus embryos. To determine the source of activin protein for initial mesoderm induction and to investigate whether activins may play further roles in embryonic development we have examined the localization of the mRNAs encoding the activin beta A and beta B subunits in Xenopus embryos. Activin beta A and beta B mRNAs are found in the follicle cells surrounding oocytes but not in oocytes themselves or fertilized eggs. During embryogenesis activin mRNA is first detected after the midblastula transition and expression increases as development proceeds. Activin beta B mRNA is homogeneously distributed during blastula and early gastrula stages but restricted to the dorso-anterior region in neurula stage embryos. At the early tailbud stage activin expression becomes confined to the brain, eye analgen, visceral pouches, otic vesicles, and the anterior notochord.

• Niehrs C, Keller R, Cho KW, De Robertis EM
• The homeobox gene goosecoid controls cell migration in Xenopus embryos.
• Cell. 1993; 72: 491-503
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• Goosecoid (gsc), a homeobox gene expressed specifically in the dorsal blastopore lip of the Xenopus gastrula, is considered to play an important role in Spemann's organizer phenomenon. Lineage tracing and time-lapse microscopy were used to follow the fate of embryonic cells microinjected with gsc mRNA. Microinjected gsc has non-cell autonomous effects, recruiting neighboring uninjected cells into a twinned dorsal axis. Ectopic expression of gsc mRNA in ventral blastomeres as well as overexpression of gsc in dorsal blastomeres leads to cell movement toward the anterior of the embryo. The results suggest a function for gsc in the control of gastrulation movements in groups of cells, but not in dissociated cells, and demonstrate that a vertebrate homeobox gene can regulate region-specific cell migration.

• Christian JL, Moon RT
• Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.
• Genes Dev. 1993; 7: 13-28
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• This study analyzes the hierarchy of signals that spatially restrict expression of Xenopus Xwnt-8 to mesodermal cells outside of the Spemann organizer field and examines the potential role that endogenous Xwnt-8 may play in dorsoventral patterning of the embryonic mesoderm. The effects of ectopic introduction of a Nieuwkoop center-like activity or of ectopic expression of goosecoid, on the distribution of endogenous Xwnt-8 transcripts were analyzed. The results of these studies are consistent with the hypothesis that maternally derived signals from the Nieuwkoop center function to positively regulate expression of the homeo box gene goosecoid in Spemann organizer cells, leading to a subsequent repression of Xwnt-8 expression in these cells. This exclusion of Xwnt-8 from cells of the organizer field may be important for normal dorsal development, in that ectopic expression of Xwnt-8 in organizer cells after the midblastula stage, by injection of plasmid DNA, ventralizes the fate of these cells. This is distinct from the previously observed dorsalizing effect of Xwnt-8 when expressed prior to the midblastula stage by injection of RNA. The effects of plasmid-derived Xwnt-8 on isolated blastula animal cap ectoderm were also analyzed. Expression of Xwnt-8 in animal pole ectoderm after the midblastula stage ventralizes the response of dorsal animal pole cells to activin and allows naive ectodermal cells to differentiate as ventral mesoderm in the absence of added growth factors. Collectively, these data are consistent with the hypothesis that Xwnt-8 plays a role in the mesodermal differentiation of ventral marginal zone cells during normal development. Furthermore, endogenous Xwnt-8 may ventralize the response of lateral mesodermal cells to dorsalizing signals from the organizer, thus contributing to the graded nature of the final body pattern.

• Smith WC, Knecht AK, Wu M, Harland RM
• Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm.
• Nature. 1993; 361: 547-9
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• A dorsalizing signal acts during gastrulation to change the specification of lateral mesodermal tissues from ventral (blood, mesenchyme) to more dorsal fates (muscle, heart, pronephros). This signal, from Spemann's organizer, cannot be mimicked by the mesoderm inducers activin and fibroblast growth factor. The gene noggin is expressed in the organizer, and could be the dorsalizing signal. Here we show that soluble noggin protein added to ventral marginal zones during gastrulation induces muscle, but that activin does not. Dorsal pattern can be partially rescued in ventralized embryos by injection of a plasmid that expresses noggin during gastrulation. The results suggest that the noggin product may be the dorsalizing signal from the organizer.

• Jones CM, Lyons KM, Lapan PM, Wright CV, Hogan BL
• DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction.
• Development. 1992; 115: 639-47
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• Establishment of mesodermal tissues in the amphibian body involves a series of inductive interactions probably elicited by a variety of peptide growth factors. Results reported here suggest that mesodermal patterning involves an array of signalling molecules including DVR-4, a TGF-beta-like molecule. We show that ectopic expression of DVR-4 causes embryos to develop with an overall posterior and/or ventral character, and that DVR-4 induces ventral types of mesoderm in animal cap explants. Moreover, DVR-4 overrides the dorsalizing effects of activin. DVR-4 is therefore the first molecule reported both to induce posteroventral mesoderm and to counteract dorsalizing signals such as activin. Possible interactions between these molecules resulting in establishment of the embryonic body plan are discussed.

• Cho KW, Blumberg B, Steinbeisser H, De Robertis EM
• Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid.
• Cell. 1991; 67: 1111-20
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• This study analyzes the function of the homeobox gene goosecoid in Xenopus development. First, we find that goosecoid mRNA distribution closely mimics the expected localization of organizer tissue in normal embryos as well as in those treated with LiCl and UV light. Second, goosecoid mRNA accumulation is induced by activin, even in the absence of protein synthesis. It is not affected by bFGF and is repressed by retinoic acid. Lastly, microinjection of goosecoid mRNA into the ventral side of Xenopus embryos, where goosecoid is normally absent, leads to the formation of an additional complete body axis, including head structures and abundant notochordal tissue. The results suggest that the goosecoid homeodomain protein plays a central role in executing Spemann's organizer phenomenon.