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. Author manuscript; available in PMC: 2008 Apr 28.
Published in final edited form as: Int J Dev Biol. 2001;45(1):189–197.

Molecular mechanisms of cell-cell signaling by the Spemann-Mangold organizer

EDDY M DE ROBERTIS 1,*, OLIVER WESSELY 1, MICHAEL OELGESCHLÄGER 1, BRENDA BRIZUELA 1, EDGAR PERA 1, JUAN LARRAÍN 1, JOSÉ ABREU 1, DANIEL BACHILLER 1
PMCID: PMC2354921  NIHMSID: NIHMS43279  PMID: 11291846

Abstract

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.

Keywords: Spemann-Mangold organizer, chordin, cerberus, Frzb-1, tolloid, collagen, BMP, TGFβ

Introduction

Since the original experiment of Spemann and Mangold (1924), isolating the molecules responsible for the inductive activities of organizer grafts has been the Holy Grail of vertebrate embryologists. After a number of premature attempts (related in Holtfreter and Hamburger, 1955; Nakamura and Toivonen, 1978), the advent of recombinant DNA technology opened the organizer problem to experimentation. The molecular exploration of the Spemann-Mangold organizer proved a gold mine for new genes and included unexpected surprises. The main surprise was that the organizer is a source of secreted antagonists of growth factors, which they bind in the extracellular space. These secreted antagonists can act as inhibitors that prevent binding to the cognate growth factor receptors, as well as modulators of signaling, in which a growth factor that was inactive can be brought back to signaling by the action of specific proteases that degrade the inhibitor. The principles of signaling regulation in the extracellular space learned from studies on the vertebrate Spemann-Mangold organizer may serve as a useful paradigm for understanding homeostasis of adult tissues and organs as well. This review is concerned with the homeobox gene goosecoid, which provided the first organizer gene marker, and with chordin and other secreted antagonists isolated in the course of efforts to identify genes transcriptionally activated by goosecoid.

Goosecoid and the isolation of organizer specific genes

The isolation of the homeobox gene goosecoid in 1991 provided the first organizer-specific gene (Cho et al., 1991). This was an important landmark, because before it had not been possible to visualize Spemann-Mangold organizer tissue, and its existence could only be inferred from the results of transplantation experiments. goosecoid is a homeobox-containing gene, and the fact that overexpression of its mRNA in ventral cells led to the formation of secondary axes implicated, right from the outset, homeobox transcription factors in the execution of Spemann-Mangold organizer activity (Cho et al., 1991). Microinjection of goosecoid mRNA was able to recruit neighboring cells into a secondary body axis and to trigger anteriorward cell movements in the injected cells (Niehrs et al., 1992). As this single mRNA could mimic many of the properties of organizer cells, it came as a surprise that later on it was found that a great many other transcription factors have similar or even more potent activities than goosecoid (Fig. 1). In time it became clear that the Spemann-Mangold organizer is composed by multiple populations of cells that are controlled by a plethora of transcription factors. These transcription factors in turn regulate the production of downstream secreted factors that mediate the inducing activities of the organizer (Fig. 1; reviewed in De Robertis et al., 1997; Harland and Gerhart, 1997; Nieto, 1999).

Fig. 1. Organizer-specific genes that pattern the early Xenopus embryo.

Fig. 1

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Schematic representation of a gastrula stage embryo showing the localization of the organizer in the dorsal marginal zone and its effect on patterning of all three germ layers, the endo-, meso- and ectoderm. The boxes show secreted and nuclear factors that are expressed in this region and that have been suggested to contribute to the function of the organizer.

Studies on goosecoid expression helped identify the corresponding homologous regions of the Spemann-Mangold organizer in other vertebrates, such as mouse, chick and zebrafish (reviewed in De Robertis et al., 1993). Together with Brachyury – which provides a marker of all trunk mesoderm - goosecoid, which marks the Spemann-Mangold organizer and subsequently the prechordal plate, is widely used to define anatomical points of reference of the various vertebrate embryos during gastrulation. Studies in the mouse showed that organizer formation starts with the initial appearance of primitive streak cells in the posterior of the embryo. Organizer cells are then found in the anterior primitive streak (Blum et al., 1992) before becoming located in the definitive node (more properly called the Hensen's node of the mouse), head process and prechordal plate. Transplantation experiments in mouse have confirmed this allocation of organizer tissue to the anterior primitive streak (Beddington, 1994; Tam and Steiner, 1999), which was initially supported only by crude Einsteck experiments (transplantation into the blastocoel cavity) using mouse day 6.5 embryo fragments transplanted into Xenopus gastrulae (Blum et al., 1992).

Studies using chick goosecoid showed, in addition to expression in the tip of the progressing primitive streak and Hensen's node, a much earlier phase of expression in the posterior of the embryo (Izpisúa-Belmonte et al., 1993). This expression is seen in the recently laid chicken egg, in cells located just beneath the epiblast in a structure called Koller's sickle. Although Koller's sickle had been known for over a hundred years, its role could only be analyzed when a molecular marker became available. As has proven often the case, the availability of novel markers coupled to lineage tracing of cell fates can lead to new insights. In the case of the goosecoid positive cells of Koller's sickle, they were shown to mark the initial precursors of what will become, after extensive cell migrations, the organizer region in the anterior of the primitive streak (Izpisúa-Belmonte et al., 1993).

The study of goosecoid provided the first visualization of Spemann-Mangold organizer cells and of their dynamic changes during gastrulation. A disappointment was that the knockout of the goosecoid gene was lethal but lacked severe effects on gastrulation (Yamada et al., 1995; Rivera-Pérez et al., 1995). Subsequent studies showed that the region that develops in association with the prechordal plate is indeed affected, reflecting early defects in the formation of the midline of the base of the cranium (Belo et al., 1997). Transplantation experiments of mouse node into chick gastrulae indicate that gsc−/− nodes are severely reduced in their neural-inducing strength (Zhu et al., 1999a). Importantly, Filosa et al. (1997) have shown that in gsc−/−;HNF-3β+/− compound mutants, dorso-ventral patterning of the CNS is severely disrupted in day 8.5 embryos. This is a recurring theme in organizer studies; with so many genes involved, double mutant studies are required to uncover redundant compensatory functions. We also now know that vertebrates contain at least three genes of the goosecoid family (reviewed in Belo et al., 1997), which could contribute to the relatively weak phenotypes observed in homozygous mutant mice.

Mouse homologues of the Xenopus organizer specific-genes Siamois and Xtwn (Lemaire et al., 1995; Laurent et al., 1997) have not yet been cloned in the mouse and therefore have not been mutated. Another homeobox gene in zebrafish (variously designated dharma or nieuwkoid; Yamanaka et al., 1998; Koos and Ho, 1998 and 1999) has a strong mutant phenotype, leading to cyclopia in zebrafish mutants known as bozozok (Fekany et al., 1999). Interestingly, the dharma/bozozok gene is more related in sequence to goosecoid than to Siamois or Xtwn, and functions upstream of goosecoid, whose expression is severely inhibited in zebrafish bozozok mutants (Fekany et al., 1999). From these studies we now know that goosecoid is not the initial organizer homeobox gene to be activated, but rather part of a second wave of gene expression that takes place in dorsal mesoderm once the gastrula organizer is induced. Unraveling the respective contributions of the many genes involved in Spemann-Mangold organizer activity (Fig. 1) will undoubtedly require detailed genetic analyses in the future.

Secreted antagonists as patterning molecules

During the course of differential screens of a cDNA library prepared from Spemann-Mangold organizer tissue, we have identified several secreted factors (Sasai et al., 1994; Bouwmeester et al., 1996). As shown in Fig. 2, these include Frzb-1, a Wnt inhibitor (Leyns et al., 1997; Wang et al., 1997; Mayr et al., 1997), cerberus, a multivalent inhibitor of Nodal, Wnt and BMP signals (Bouwmeester et al., 1996; Piccolo et al., 1999), and chordin, a BMP inhibitor thought to play a central role in organizer function (Sasai et al., 1994; 1995; Piccolo et al., 1996). Other groups used different methods to isolate additional organizer-specific secreted factors such as the BMP inhibitor Noggin (Smith et al., 1992; Lamb et al., 1993; Zimmerman et al., 1996); the Activin and BMP inhibitor Follistatin (Hemmati-Brivanlou and Melton, 1994; Sasai et al., 1995; Fainsod et al., 1997) and the Wnt inhibitor Dickkopf (Glinka et al., 1998).

Fig. 2. The organizer is a source of secreted antagonists that bind growth factors in the extracellular space.

Fig. 2

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Three different types of extracellular modulators were been discovered in the organizer. All three have been shown to encode secreted inhibitors. Chordin, Noggin and Follistatin bind to BMPs and thereby inhibit them from activating their receptor. Frzb-1 and Dkk-1 antagonize Xwnt-8 in the extracellular space. Cerberus is a multivalent inhibitor of three different signals, BMPs, Xwnt-8 and the Xenopus Nodal-related mesoderm-inducing molecules (Xnr1, 2 and 4).

In overexpression experiments, molecules such as chordin and noggin can induce neural tissue in ectodermal explants and dorsal mesoderm in ventral mesoderm explants (Lamb et al., 1993; Sasai et al., 1995; Piccolo et al., 1996). In addition to their neural inducing activity, chordin and noggin can induce endoderm, in particular dorsal endoderm, in animal cap explants (Sasai et al., 1996). This lead to the current model by which BMP antagonists emanating from the organizer would pattern all three germ layers of the embryo (Fig. 3). This view is in agreement with the result of injecting chordin mRNA into a ventral blastomere of the Xenopus embryo. As can be seen in Fig. 4, secondary axes are induced that contain, as in Spemann and Mangold's original experiment, a secondary neural tube, dorsal mesoderm (somites) and a secondary gut. Thus, the organizer phenomenon can be reproduced by the injection of a single molecule. Similar results can be obtained with noggin, another BMP antagonist, or with short-gastrulation mRNA. Short-gastrulation is the Drosophila homologue of chordin (Holley et al., 1996; Schmidt et al., 1995) and was known to act as a genetic antagonist of decapentaplegic (Dpp, a BMP homologue) signaling in Drosophila (Ferguson and Anderson, 1992). This gave us the clue that chordin might function as a BMP antagonist (reviewed in De Robertis and Sasai, 1996).

Fig. 3. Spemann-Mangold organizer secreted factors antagonize ventral signals provided by BMPs.

Fig. 3

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BMPs are secreted by a wide region at the ventral side of the embryo and are antagonized by organizer secreted factors such as Chordin, Noggin and Follistatin (blue oval). These factors directly bind to BMPs in the extracellular space of ectoderm, mesoderm and endoderm and thereby pattern these three germ layers.

Fig. 4. Chordin mRNA induces secondary axes.

Fig. 4

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(Upper panel) Ventral injection at the 8-cell stage lead to the formation of a twinned embryo, which contains eyes and cement gland. (Lower panel) A sagittal histological section of this embryo shows that it contains two notochords (ntc), neural tubes (ne) and gut (1° G and 2° G). Thus, injection of a single molecule can recapitulate the inductions mediated by organizer grafts.

Unraveling the mechanism of action of Chordin

In microinjection experiments, the neural inducing activity of chordin could be inhibited by co-injection of BMP-4 mRNA (Sasai et al., 1995). In what came initially as a surprise, the neuralizing activities of noggin and follistatin could also be blocked by BMP-4 (Sasai et al., 1995). In the converse experiment, blocking BMP signaling with a dominant-negative BMP receptor (tBR, Suzuki et al., 1994; Graff and Melton, 1994) or with antisense BMP-4 RNA (Steinbeisser et al., 1995) resulted in the induction of anterior neural tissue in animal cap ectodermal explants in this groundbreaking study by Sasai et al. (1995).

The molecular mechanism by which these neural inducers work was resolved with the purification of Chordin (Piccolo et al., 1996) and Noggin (Zimmerman et al., 1996) proteins, and the demonstration that they bind BMPs, preventing binding to BMP receptors (Fig. 5). The equilibrium dissociation constant (KD) of Chordin for BMP-4 is 3 × 10−10 M, which is about the same as the affinity of BMP for its cognate receptors on cell membranes (Piccolo et al., 1996). The affinity of Noggin for BMP is higher, of the order of 1.5 × 10−11 M (Zimmerman et al., 1996). A concentration of 1 nM Chordin protein is sufficient to induce NCAM in ectodermal animal cap explants or α-actin in ventral marginal zone explants. In the case of Noggin, 1 nM will dorsalize mesoderm, but concentrations in the 10 nM range are required to induce neural tissue (Harland and Gerhart, 1997). Thus, embryonic cells have biological responses to the various neural inducers that are not solely dependent on in vitro affinities.

Fig. 5. Model of BMP signal re-activation by Xolloid cleavage.

Fig. 5

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BMP-4 binds to BMP receptors inducing the ventral pathway. Binding to Chordin blocks this signaling, whereas cleavage of Chordin by the Xolloid metalloprotease at two specific sites releases active BMP-4, re-establishing the ventralizing signal.

Chordin inhibition can be reversed by the Xolloid metalloprotease

In Drosophila, the gene tolloid functions genetically to increase dpp (BMP) signaling (Ferguson and Anderson, 1992). In Xenopus, a related gene called xolloid was isolated by Leslie Dale's laboratory (Goodman et al., 1998; Piccolo et al., 1997). Microinjection of xolloid mRNA causes an almost textbook-like ventralization of Xenopus embryos (loss of notochord, decrease in somite, increase in blood islands) as shown in Fig. 6. This phenotype is similar to what one observes by injecting intermediate doses of BMPs, or would expect to find from a partial inhibition of Spemann-Mangold organizer activity.

Fig. 6. xolloid mRNA ventralizes mesodermal pattern.

Fig. 6

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Sagittal sections of tail bud stage embryos at the trunk level. The various dorso-ventral tissues are indicated in the lower panel. (A) Control. (B) Embryo after radial injection of xolloid mRNA at the four cell stage. Note the ventralization of the entire mesodermal layer in embryos expressing ectopic xolloid metalloprotease.

Using a biochemical approach it was shown that the xolloid zinc metalloprotease can cleave Chordin, but not Noggin, at two specific sites (Piccolo et al., 1997). Furthermore, it was demonstrated that the cleavage of Chordin is able to reactivate previously inactive BMP, which is once again able to signal (Fig. 5) in Xenopus assays (Piccolo et al., 1997). Thus, the way in which xolloid works is through the specific inactivation of Chordin and the ensuing release of reactivated BMPs. In parallel studies in Drosophila, Marqués et al. (1997) found that Drosophila Tolloid cleaves Short-gastrulation at three sites, two of which correspond to the cleavage sites of xolloid on Chordin.

More recent studies have revealed additional vertebrate homologues of Tolloid and that some of them, in particular BMP-1 and mouse tolloid-like-1, are effective at inactivating Chordin as well (Wardle et al., 1999; Scott et al., 1999). The exact location of the two sites at which Chordin is cleaved by Tolloid metalloprotesases (Piccolo et al., 1997; Scott et al., 1999) is of interest. As shown in Fig. 7,Chordin is a large protein containing four cysteine-rich domains (CRs) of about 70 amino acids each. xolloid cleaves Chordin just downstream of CR1 and CR3 at conserved aspartic acid residues. It has recently been shown that the BMP-binding activity of Chordin resides in the CR repeats and that CR1 and CR3 bind BMP much better than CR2 and CR4 (Larraín et al., 2000). The binding affinity of CR1 or CR3 repeats is about 10 times lower (KD of 3 × 10−9 M) than that of intact Chordin. Thus, a possible model of how Chordin might work is by binding a BMP dimer to each Chordin monomer (in agreement with results from crosslinking studies, Piccolo et al., 1996) via CR1 and CR3 (Fig. 8). Once xolloid cleaves Chordin, CRs still bound to BMP would be released. Since the affinity of this interaction is 10 times lower, this decrease in affinity might suffice to allow binding of BMP to its receptors (Fig. 8), or perhaps additional components may be required to liberate BMP from the CRs.

Fig. 7. Chordin is cleaved by Tolloid/xolloid/mTII-1 at two specific sites.

Fig. 7

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Schematic drawing of Xenopus Chordin showing its signal peptide (gray box) and the BMP binding modules, the four cysteine-rich domains (CR1−4). The metalloprotease Tolloid/xolloid/mTII-1 cleaves the mature protein at two specific sites 29 amino acids downstream of CR1 and 16 amino acids downstream of the third CR repeat and thereby inactivates the protein. The recognition sequences for the protease and the relative positions of the cleavage sites are indicated. After experiments of Piccolo et al., 1997 and Scott et al., 1999.

Fig. 8. Hypothetical model showing that Chordin binds BMP with higher affinity than CR1.

Fig. 8

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Chordin blocks BMP signaling efficiently (KD 3 × 10−10 M) probably because the presence of CR1 and CR3 provide a Chordin monomer with two high affinity sites for each BMP dimer. In contrast, the CR1 fragment produced by xolloid digestion (cleavage sites on Chordin are indicated by arrows) binds BMP-4 with a 10-fold lower affinity (KD 3 × 10−9 M) and is less efficient in blocking BMP signaling.

Genetic studies discussed below indicate that the interaction of Chordin with BMPs is of central importance for generating dorso-ventral pattern. The binding of BMP to Chordin could permit the diffusion of BMPs to distant sites without being sequestered by BMP receptors that are present at high concentrations in adjoining cell membranes. Once the Chordin-BMP complex meets the metalloprotease, active BMPs can be released at a distance (reviewed by Weinmaster, 1998). In Drosophila, it is known from genetic mosaic and other studies that Short-gastrulation can affect Dpp/Screw signaling many cell diameters away (Zusman, 1988; Ashe and Levine, 1999). In conclusion, mechanistic studies on the Chordin protein have provided important insights into how gradients of dorso-ventral positional information are generated by protein interactions in the extracellular space.

Many proteins contain CR domains

The Chordin CR domains contain a series of conserved cysteines and hydrophobic residues that are present in a number of other proteins (Fig. 9A). CR repeats are present in Thrombospondin, von Willebrand factor and fibrillar procollagens (Bornstein, 1992; Sasai et al., 1994; François et al., 1994). Other proteins with such repeats include Nel-like proteins with four CRs and six EGF repeats (Watanabe et al., 1996), a C. elegans EST (Larraín et al., 2000) homologous to a chick transmembrane protein called CRIM1 with five CRs in the extracellular portion (Kolle et al., 2000), and Drosophila peroxidasin (Nelson et al., 1994).

Fig. 9. CR domains present in procollagen IIA modulate BMP signaling. (A).

Fig. 9

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Sequence comparison of CR domains contained in different extracellular matrix proteins. Coll-CR, type IIA Xenopus procollagen; CR2, murine Chordin second repeat; Nel, rat nel; Pxdasin, Drosophila peroxidasin; C. eleg. EST, C. elegans hypothetical protein containing five procollagen-like domains (accession No. CAA94866). (B) Ventral injection of Xenopus procollagen IIA mRNA induces secondary axes. A construct encoding the splice variant Coll IIB lacking the CR domains is inactive in this assay (after Larraín et al., 2000). (C) Hypothetical model for the binding of BMP-4 to procollagen IIA triple helix. The cartoon shows how the presence of multiple CR domains in the procollagen triple helix could bind BMP-4 with high affinity, as is the case for Chordin. The question mark indicates the proposed protease that, like Tolloid, would release active BMPs from this extracellular reservoir of growth factors (see text) when required for tissue homeostasis.

The recent demonstration that at least some of these CRs are also implicated in BMP/TGFβ signaling, may have important consequences for the regulation of extracellular signals not only in the embryo, but also in adult tissues and organs. Collagen IIA mRNA is able to induce secondary axes in microinjected Xenopus embryos (Larraín et al., 2000) and this dorsalizing activity requires the CR repeats (Fig. 9B). The isolated CR repeat of Collagen II has BMP and TGFβ binding capacity (Zhu et al., 1999b; Larraín et al., 2000) but is devoid of biological activity. Presumably this is because, as in the case of Chordin, multiple CRs are required for high affinity binding. As shown in Fig. 9C, three CRs can come into close proximity in the NH2-propeptide of procollagen trimers. This could provide binding sites for BMPs or other growth factors of the TGFβ superfamily at places where they could be most required, such as developing prerichondrium, tendon or bone, all of them rich in fibrillar collagens (Sandell et al., 1991; Su et al., 1991; Cheah et al., 1991; Zhu et al., 1999b). In order for these growth factors to become active, one would have to propose the existence of a protease that, like Tolloid, would cleave the complexes close downstream of the CRs (indicated in Fig. 9C). Thus, Chordin has provided a paradigm for understanding extracellular signaling regulation in adult tissues. This model may perhaps also apply to other Spemann-Mangold organizer secreted factors that are members of large multigene families also expressed in adult tissues (Rattner et al., 1997; Hsu et al., 1998; Pearse et al., 1999).

Chordin mediates dorso-ventral patterning

Since multiple BMP inhibitors are secreted by the Spemann-Mangold organizer (Fig. 3), loss-of-function studies are required to determine their individual functions and whether they compensate for each other. In zebrafish extensive genetic screens have been carried out, and two ventralized (as one would expect from loss or reduction of the Spemann-Mangold organizer) mutants have been isolated (Hammerschmidt et al., 1996a). The strongest mutation, chordino, was a loss-of-function of chordin (Schulte-Merker et al., 1997). As indicated in Fig. 10, at the gastrula stage neural (fkh) and dorsal mesodermal (shh) markers are reduced, and ventral mesodermal markers (eve-1) are expanded in chordino mutants (Hammerschmidt et al., 1996b). Chordino embryos regulate, and despite the much reduced initial neural plate, eventually a CNS develops, but the embryos have smaller heads and for the most part die at the stage shown in Fig. 10. These loss-of-function studies showed that the organizer-specific gene chordin is required for patterning both the ectodermal and mesodermal germ layers, as had been predicted from Xenopus overexpression studies.

Fig. 10. Chordin is required for dorsal-ventral patterning in zebrafish.

Fig. 10

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The Chordino mutant phenotype is due to a loss-of-function of zebrafish chordin. In mutant embryos the tail is enlarged at the expense of head and anterior trunk. In situ analyses of mutant embryos demonstrated a reduction of the neural plate (marked by fkd3) and dorsal mesoderm (marked by shh), and an expansion of ventral mesoderm (marked by eve1) (Hammerschmidt et al., 1996). Therefore the antagonism between the Spemann-Mangold organizer secreted protein Chordin and BMP is required for the establishment of the dorsal-ventral polarity of ectoderm and mesoderm in zebrafish. Zebrafish photographs courtesy of Dr. Stefan Schulte-Merker (Tübingen).

The opposite class of mutations, the dorsalized mutants, are also of interest in the context of organizer function. The strongest mutant, swirl, is a mutation in BMP-2 (Mullins et al., 1996; Kishimoto et al., 1997; Nguyen et al., 1998). BMP-2 has a strong maternal component, and in its absence transcription of zygotic BMP-4 is also prevented in the ventral side (Kishimoto et al., 1997). Double mutants of swirl;chordino have a swirl phenotype (Hammerschmidt et al., 1996b); these epistatic studies show that chordin is a dedicated BMP antagonist. Other dorsalized mutations affect additional components of the BMP signaling pathway such as Smad-5 and BMP-7 (Hild et al., 1999; Dick et al., 2000; Schmid et al., 2000).

These genetic results in zebrafish highlight that a large part of the dorso-ventral patterning by the Spemann-Mangold organizer is effected through BMP signaling (Fig. 3). Last but not least, the most frequently isolated dorsalized mutation, mini-fin, has been identified as the zebrafish homologue of tolloid/xolloid (Connors et al., 1999), confirming the importance of proteolytic control in establishing gradients of dorso-ventral polarity.

In the case of mouse embryos, mutation of chordin results in milder phenotypes and neural induction is normal (Bachiller et al., 2000, and unpublished results). Similarly, in noggin mutants development is relatively normal until embryonic day 8.5, although strong posterior axial deficits are seen at later stages (McMahon et al., 1998; Brunet et al., 1998). Double mutant studies have shown that these two BMP antagonists, which are co-expressed in the mouse Hensen's node, compensate for each other. In chordin−/−;noggin−/− embryos, the prosencephalic vesicle is essentially absent at day 8.5 (Fig. 11, note the lack of expression of the Six-3 forebrain marker). This antero-posterior deficit can be traced back to the early neural plate stages (Bachiller et al., 2000). In addition, dorso-ventral phenotypes are observed such as the lack of notochord and sonic hedgehog expression in the anterior of the embryo (Fig. 11B, arrowhead). Finally, the left-right axis is also affected, with randomization of the heart situs (Bachiller et al., 2000). Therefore, genetic studies in the mouse demonstrate that the BMP antagonists Chordin and Noggin are required for correct patterning of the three main body axes of the mammalian embryo.

Fig. 11. Loss of prosencephalon in chordin and noggin double mutants.

Fig. 11

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(AB) [illegible] almost completely absent, whereas Krox-20 is expressed in the double mutant at the expected position. The white arrowhead in B marks the level of anterior-most expression of Shh. These double mutant mouse embryos lose the prosencephalon as well as the anterior notochord.

Conserved patterning mechanisms in evolution

Our interest in homeobox genes started with the isolation of the first Hox gene from vertebrates (Carrasco et al., 1984). As is now clear, the antero-posterior patterning system uses an intricate system of conserved genes in all bilateral animals (e.g., de Rosa et al., 1999). The isolation of a homeobox gene from a Spemann-Mangold organizer cDNA library, goosecoid, opened a new avenue of exploration for dorso-ventral patterning (Cho et al., 1991). Since microinjection of goosecoid mRNA had non-cell autonomous effects recruiting neighboring cells into secondary axes, this led to the search for downstream targets of goosecoid that could mediate these inductive activities. One such secreted target gene activated at the transcriptional level by injection of goosecoid mRNA was identified with the cloning of chordin, a gene that can mimic Spemann-Mangold organizer transplantation when overexpressed (Sasai et al., 1994). As shown in Figure 12, Chordin is part of a conserved dorsal-ventral patterning system involving dpp/sog/tolloid in Drosophila and BMPs/chd/tolloid in vertebrates (Sasai et al., 1994; François et al., 1994; Holley et al., 1995; Schmidt et al., 1995; Piccolo et al., 1997; Marqués et al., 1997). The molecular machinery required for the generation of gradients of BMP signaling is intricate and involves activation of BMP signaling via a double inhibition mechanism requiring a novel proteolytic control step in the extracellular space. There is one important difference, however, and that is that the dorsal-ventral axis has been reversed during the course of evolution (reviewed in De Robertis and Sasai, 1996; De Robertis, 1997). The last common ancestor of protostomes and deuterostomes, called the Urbilateria, had both a dorsal-ventral and an antero-posterior patterning system in place. Current phylogenetic analyses place Urbilateria very deep in evolutionary times, even before the split of molting animals (such as arthropods and nematodes) from the other protostomes (such as mollusks, and annelids, Aguinaldo et al., 1997; de Rosa et al., 1999). Therefore, the body patterns of all bilateral animals are constructed using conserved dorso-ventral and antero-posterior patterning systems, raising the question of whether this placed any constraints in the evolution of body plans.

Fig. 12. Vertebrates and invertebrates share a common dorsal-ventral patterning system.

Fig. 12

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Vertebrate xolloid blocks Chordin suppression of BMP-4 allowing ventralization (antineural); likewise in Drosophila, Tolloid blocks short-gastrulation suppression of Dpp allowing dorsalization (antineural) differentiation. This is a powerful argument in favor of the last common ancestor of protostomes and deuterostomes, the Urbilateria, having this dorso-ventral patterning system in place. During the course of evolution the dorsal axis has become inverted, as first proposed by French Zoologist Etienne Geoffroy Saint-Hilaire (1822).

The Spemann and Mangold experiment provided embryologists with an intellectual framework for asking questions about pattern development in vertebrates. It is remarkable that more than 75 years later a simple transplantation experiment still continues to stimulate new research, as can be seen throughout this volume.

Acknowledgements

We would like to express our gratitude to the talented postdocs that participated in these studies on the Spemann-Mangold organizer. The organizer has been not only a fertile fishing ground but also a training ground for new generations of independent investigators: Eric Agius (CNRS, Toulouse), José A. Belo (Gulbenkian Institute, Lisbon), Martin Blum (University of Karlsruhe), Bruce Blumberg (UC Irvine), Ken W.Y. Cho (UC Irvine), J.C. Izpisúa-Belmonte (Salk Institute), Stefano Piccolo (U. of Padova), Tewis Bouwmeester (EMBL Heidelberg), Herbert Steinbeisser (Max Planck Tübingen), Luc Leyns (Free University of Brussels), Yoshiki Sasai (Kyoto University), Christof Niehrs (German Cancer Center, Heidelberg), C.V.E. Wright (Vanderbilt University) and Ahikito Yamamoto (Kumamoto University, Japan). Linda Gont, Bin Lu and Sung Kim were graduate students and are at earlier stages of their careers. O.W., M.O. and E.P. were HFSPO fellows and J. L. and J.A. are PEW fellows. This work was made possible by long-term support from the NIH (R37 HD21502-15), the Norman Sprague Endowment and the Howard Hughes Medical Institute.

Abbreviations used in this paper

BMP

Bone Morphogenetic Protein

gsc

goosecoid

Xtwn

Xenopus Twin homeobox gene

Dpp

Decapantaplegic

CR

cysteine rich domain of the chordin type

fkh

forked-head gene.

References

  1. AGUINALDO AMA, TURBEVILLE JM, LINFORD LS, RIVERA MC, GAREY JR, RAFF RA, LAKE JA. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature. 1997;387:489–492. doi: 10.1038/387489a0. [DOI] [PubMed] [Google Scholar]
  2. ASHE HL, LEVINE M. Local inhibition and long-range enhancement of Dpp signal transduction by Sog. Nature. 1999;398:427–431. doi: 10.1038/18892. [DOI] [PubMed] [Google Scholar]
  3. BACHILLER D, KLINGENSMITH J, KEMP C, BELO JA, ANDERSON RM, MAY SR, McMAHON JA, McMAHON AP, HARLAND R, ROSSANT J, DE ROBERTIS EM. The organizer secreted factors Chordin and Noggin are required for forebrain development in the mouse. Nature. 2000;403:658–661. doi: 10.1038/35001072. [DOI] [PubMed] [Google Scholar]
  4. BEDDINGTON RSP. Induction of a second neural axis by the mouse node. Development. 1994;120:613–620. doi: 10.1242/dev.120.3.613. [DOI] [PubMed] [Google Scholar]
  5. BELO JA, LEYNS L, YAMADA G, DE ROBERTIS EM. The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants. Mech. Dev. 1998;72:15–26. doi: 10.1016/s0925-4773(97)00204-9. [DOI] [PubMed] [Google Scholar]
  6. BLUM M, GAUNT SJ, CHO KWY, STEINBEISSER H, BLUMBERG B, BITTNER D, DE ROBERTIS EM. Gastrulation in the mouse: The role of the homeobox gene goosecoid. Cell. 1992;69:1097–1106. doi: 10.1016/0092-8674(92)90632-m. [DOI] [PubMed] [Google Scholar]
  7. BORNSTEIN P. Thrombospondins: structure and regulation of expression. FASEB J. 1992;6:3290–3299. doi: 10.1096/fasebj.6.14.1426766. [DOI] [PubMed] [Google Scholar]
  8. BOUWMEESTER T, KIM SH, SASAI Y, LU B, DE ROBERTIS EM. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature. 1996;382:595–601. doi: 10.1038/382595a0. [DOI] [PubMed] [Google Scholar]
  9. BRUNET LJ, McMAHON JA, McMAHON AP, HARLAND RM. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science. 1998;280:1455–1457. doi: 10.1126/science.280.5368.1455. [DOI] [PubMed] [Google Scholar]
  10. CARRASCO AE, MCGINNIS W, GEHRING WJ, ROBERTIS EM. Cloning of an X. laevis gene expressed during early embryogenesis coding for a peptide region homologous to Drosophila homeotic genes. Cell. 1984;37:409–414. doi: 10.1016/0092-8674(84)90371-4. [DOI] [PubMed] [Google Scholar]
  11. CHEAH KS, LAU ET, AU PKC, TAM PPL. Expression of the mouse α1(II) collagen gene is not restricted to cartilage during development. Development. 1991;111:945–953. doi: 10.1242/dev.111.4.945. [DOI] [PubMed] [Google Scholar]
  12. CHO KWY, BLUMBERG B, STEINBEISSER H, DE ROBERTIS EM. Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid. Cell. 1991;67:1111–1120. doi: 10.1016/0092-8674(91)90288-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. CONNORS SA, TROUT J, EKKER M, MULLINS MC. The role of tolloid/minifin in dorsoventral pattern formation of the zebrafish embryo. Development. 1999;126:3119–3130. doi: 10.1242/dev.126.14.3119. [DOI] [PubMed] [Google Scholar]
  14. DE ROBERTIS EM. The ancestry of segmentation. Nature. 1997;387:25–26. doi: 10.1038/387025a0. [DOI] [PubMed] [Google Scholar]
  15. DE ROBERTIS EM, SASAI Y. A common plan for dorsoventral patterning in Bilateria. Nature. 1996;380:37–40. doi: 10.1038/380037a0. [DOI] [PubMed] [Google Scholar]
  16. DE ROBERTIS EM, FAINSOD A, GONT LK, STEINBEISSER H. The evolution of vertebrate gastrulation. Development. 1994;(Suppl):117–124. [PubMed] [Google Scholar]
  17. DE ROBERTIS EM, KIM SH, LEYNS L, PICCOLO S, BACHILLER D, AGIUS E, BELO JA, YAMAMOTO A, HAINSKI-BROSSEAU A, BRIZUELA B, WESSELY O, LU B, BOUWMEESTER T. Patterning by genes expressed in Spemann's organizer. Cold Spring Harbor Symp. Quant. Biol. 1997;62:169–175. [PubMed] [Google Scholar]
  18. DE ROSA R, GRENIER JK, ANDREEVA T, COOK CE, ADOUTTE A, AKAM M, CARROLL SB, BALAVOINE G. Hox genes in brachiopods and priapulids and protostome evolution. Nature. 1999;399:772–776. doi: 10.1038/21631. [DOI] [PubMed] [Google Scholar]
  19. DICK A, HILD M, BAUER H, IMAI Y, MAIFELD H, SCHIER AF, TALBOT WS, BOUWMEESTER T, HAMMERSCHMIDT M. Essential role of Bmp7 (snailhouse) and its prodomain in dorsoventral patterning of the zebrafish embryo. Development. 2000;127:343–354. doi: 10.1242/dev.127.2.343. [DOI] [PubMed] [Google Scholar]
  20. FAINSOD A, DEISSLER K, YELIN R, MAROM K, EPSTEIN M, PILLEMER G, STEINBEISSER H, BLUM M. The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4. Mech. Dev. 1997;63:39–50. doi: 10.1016/s0925-4773(97)00673-4. [DOI] [PubMed] [Google Scholar]
  21. FEKANY K, YAMANAKA Y, LEUNG TC, SIROTKIN HI, TOPCZEWSKI J, GATES MA, HIBI M, RENUCCI A, STEMPLE D, RADBILL A, SCHIER AF, DRIEVER W, HIRANO T, TALBOT WS, SOLNICA-KREZEL L. The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures. Development. 1999;126:1427–1438. doi: 10.1242/dev.126.7.1427. [DOI] [PubMed] [Google Scholar]
  22. FERGUSON EL, ANDERSON KV. Localized enhancement and repression of the activity of the TGF-β family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. Development. 1992;114:583–597. doi: 10.1242/dev.114.3.583. [DOI] [PubMed] [Google Scholar]
  23. FILOSA S, RIVERA-PÉREZ JA, GÓMEZ AP, GANSMULLER A, SASAKI H, BEHRINGER RR, ANG S-L. goosecoid and HNF-3β genetically interact to regulate neural tube patterning during mouse embryogenesis. Development. 1997;124:2843–2854. doi: 10.1242/dev.124.14.2843. [DOI] [PubMed] [Google Scholar]
  24. FRANÇOIS V, SOLLOWAY M, O'NEILL JW, EMERY J, BIER E. Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene. Genes Dev. 1994;8:2602–2616. doi: 10.1101/gad.8.21.2602. [DOI] [PubMed] [Google Scholar]
  25. GEOFFROY SAINT-HILAIRE E. Considérations générales sur la vertèbre. Mém. Mus. Hist. Nat. 1822;9:89–119. [Google Scholar]
  26. GLINKA A, WU W, DELIUS H, MONAGHAN AP, BLUMESTOCK C, NIEHRS C. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature. 1998;391:357–362. doi: 10.1038/34848. [DOI] [PubMed] [Google Scholar]
  27. GOODMAN SA, ALBANO R, WARDLE FC, MATTHEWS G, TANNAHILL D, DALE L. BMP1-related metalloproteinases promote the development of ventral mesoderm in early Xenopus embryos. Dev. Biol. 1998;195:144–157. doi: 10.1006/dbio.1997.8840. [DOI] [PubMed] [Google Scholar]
  28. GRAFF JM, SCOTT THIES R, SONG JJ, CELESTE AJ, MELTON DA. Studies with a Xenopus BMP receptor suggest that ventral mesoderm-inducing signals override dorsal signals in vivo. Cell. 1994;79:169–179. doi: 10.1016/0092-8674(94)90409-x. [DOI] [PubMed] [Google Scholar]
  29. HAMMERSCHMIDT M, PELEGRI F, MULLINS MC, KANE DA, VAN EEDEN FJM, GRANATO M, BRAND M, FURUTANI-SEIKI M, HAFFTER P, HEISENBERG C-P, JIANG Y-J, KELSH RN, ODENTHAL J, WARGA RM, NÜSSLEIN-VOLHARD C. dino and mercedes, two genes regulating dorsal development in the zebrafish embryo. Development. 1996a;123:95–102. doi: 10.1242/dev.123.1.95. [DOI] [PubMed] [Google Scholar]
  30. HAMMERSCHMIDT M, SERBEDZIJA GN, McMAHON AP. Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor. Genes Dev. 1996b;10:2452–2461. doi: 10.1101/gad.10.19.2452. [DOI] [PubMed] [Google Scholar]
  31. HARLAND R, GERHART J. Formation and function of Spemann's Organizer. Ann. Rev. Cell Dev. Biol. 1997;13:611–667. doi: 10.1146/annurev.cellbio.13.1.611. [DOI] [PubMed] [Google Scholar]
  32. HEMMATI-BRIVANLOU A, MELTON DA. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell. 1994;77:273–281. doi: 10.1016/0092-8674(94)90319-0. [DOI] [PubMed] [Google Scholar]
  33. HILD M, DICK A, RAUCH JG, MEIER A, BOUWMEESTER T, HAFFTER P, HAMMERSCHMIDT M. The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development. 1999;126:2149–2159. doi: 10.1242/dev.126.10.2149. [DOI] [PubMed] [Google Scholar]
  34. HOLLEY SA, JACKSON PD, SASAI Y, LU B, DE ROBERTIS EM, HOFFMAN FM, FERGUSON EL. A conserved system for dorsal-ventral patterning in insects and vertebrates involving short gastrulation and chordin. Nature. 1995;376:249–253. doi: 10.1038/376249a0. [DOI] [PubMed] [Google Scholar]
  35. HOLTFRETER J, HAMBURGER V. Embryogenesis: progressive differentiation. In: Willier BH, Weiss PA, Hamburger V, editors. Analysis of Development. W.B. Saunders; Philadelphia: 1955. [Google Scholar]
  36. 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;1:673–683. doi: 10.1016/s1097-2765(00)80067-2. [DOI] [PubMed] [Google Scholar]
  37. IZPISÚA-BELMONTE JC, DE ROBERTIS EM, STOREY KG, STERN CD. The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm. Cell. 1993;74:645–659. doi: 10.1016/0092-8674(93)90512-o. [DOI] [PubMed] [Google Scholar]
  38. KISHIMOTO Y, LEE K-H, ZON L, HAMMERSCHMIDT M, SCHULTEMERKER S. The molecular nature of swirl: BMP2 function is essential during early dorsoventral patterning. Development. 1997;124:4457–4466. doi: 10.1242/dev.124.22.4457. [DOI] [PubMed] [Google Scholar]
  39. KOLLE G, GEORGAS K, HOLMES GP, LITTLE MH, YAMADA T. CRIM1, a novel gene encoding a cysteine-rich repeat protein, is developmentally regulated and implicated in vertebrate CNS development and organogenesis. Mech. Dev. 2000;90:181–193. doi: 10.1016/s0925-4773(99)00248-8. [DOI] [PubMed] [Google Scholar]
  40. KOOS DS, HO RK. The nieuwkoid gene characterizes and mediates Nieuwkoop center-like activity in the zebrafish. Curr. Biol. 1998;8:1199–1206. doi: 10.1016/s0960-9822(07)00509-x. [DOI] [PubMed] [Google Scholar]
  41. KOOS DS, HO RK. The nieuwkoid/dharma homeobox gene is essential for bmp2b repression in the zebrafish pregastrula. Dev. Biol. 1999;215:190–207. doi: 10.1006/dbio.1999.9479. [DOI] [PubMed] [Google Scholar]
  42. LAMB TM, KNECHT AK, SMITH WC, STACHEL SE, ECONOMIDES AN, STAHL N, YANCOPOLOUS GD, HARLAND RM. Neural induction by secreted polypeptide noggin. Science. 1993;262:713–178. doi: 10.1126/science.8235591. [DOI] [PubMed] [Google Scholar]
  43. LARRAÍN J, BACHILLER D, LU B, AGIUS E, PICCOLO S, DE ROBERTIS EM. BMP-binding modules in chordin: a model for signaling regulation in the extracellular space. Development. 2000;127:821–830. doi: 10.1242/dev.127.4.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. LAURENT MN, BLITZ IL, HASHIMOTO C, ROTHBÄCHER U, CHO KWY. The Xenopus homeobox gene Twin mediates Wnt induction of Goosecoid in establishment of Spemann's organizer. Development. 1997;124:4905–4916. doi: 10.1242/dev.124.23.4905. [DOI] [PubMed] [Google Scholar]
  45. LEMAIRE P, GARRETT N, GURDON JB. Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell. 1995;81:85–94. doi: 10.1016/0092-8674(95)90373-9. [DOI] [PubMed] [Google Scholar]
  46. LEYNS L, BOUWMEESTER T, KIM S-H, PICCOLO S, DE ROBERTIS EM. Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann Organizer. Cell. 1997;88:747–756. doi: 10.1016/s0092-8674(00)81921-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. MARQUÉS G, MUSACCHIO M, SHIMELL MJ, WÜNNENBERG-STAPLETON K, CHO KWY, O'CONNOR MB. Production of DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins. Cell. 1997;91:417–426. doi: 10.1016/s0092-8674(00)80425-0. [DOI] [PubMed] [Google Scholar]
  48. MAYR T, DEUTSCH U, KÜHL M, DREXLER HCA, LOTTSPEICH F, DEUTZMANN R, WEDLICH D, RISAU W. Fritz: a secreted frizzled-related protein that inhibits Wnt activity. Mech. Dev. 1997;63:109–125. doi: 10.1016/s0925-4773(97)00035-x. [DOI] [PubMed] [Google Scholar]
  49. McMAHON JA, TAKADA S, ZIMMERMAN LB, FAN CM, HARLAND RM, McMAHON AP. Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev. 1998;12:1438–1452. doi: 10.1101/gad.12.10.1438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. MULLINS MC, HAMMERSCHMIDT M, KANE DA, ODENTHAL J, BRAND M, VAN EEDEN FJM, FURUTANI-SEIKI M, GRANATO M, HAFFTER P, HEISENBERG C-P, JIANG Y-J, KELSH RN, NÜSSLEIN-VOLHARD C. Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes. Development. 1996;123:81–93. doi: 10.1242/dev.123.1.81. [DOI] [PubMed] [Google Scholar]
  51. NAKAMURA O, TOIVONEN S. Organizer – a Milestone of Half-Century from Spemann. Elsevier/North-Holland Biomedical Press; Amsterdam: 1978. [Google Scholar]
  52. NELSON RE, FESSLER LI, TAKAGI Y, BLUMBERG B, KEENE DR, OLSON PF, PARKER CG, FESSLER JH. Peroxidasin: a novel enzyme-matrix protein of Drosophila development. EMBO J. 1994;13:3438–3447. doi: 10.1002/j.1460-2075.1994.tb06649.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. NGUYEN VH, SCHMID B, TROUT J, CONNERS SA, EKKER M, MULLINS MC. Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2/swirl pathway of genes. Dev. Biol. 1998;199:93–110. doi: 10.1006/dbio.1998.8927. [DOI] [PubMed] [Google Scholar]
  54. NIEHRS C, KELLER R, CHO KWY, DE ROBERTIS E. The homeobox gene goosecoid controls cell migration in Xenopus embryos. Cell. 1993;72:491–503. doi: 10.1016/0092-8674(93)90069-3. [DOI] [PubMed] [Google Scholar]
  55. NIETO MA. Reorganizing the organizer 75 years on. Cell. 1999;98:417–425. doi: 10.1016/s0092-8674(00)81971-6. [DOI] [PubMed] [Google Scholar]
  56. PEARCE JJH, PENNY G, ROSSANT J. A mouse cerberus/Dan-related gene family. Dev. Biol. 1999;209:98–110. doi: 10.1006/dbio.1999.9240. [DOI] [PubMed] [Google Scholar]
  57. PICCOLO S, AGIUS E, LEYNS L, BATTACHARYYA S, GRUNZ H, BOUWMEESTER T, DE ROBERTIS EM. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature. 1999;397:707–710. doi: 10.1038/17820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. PICCOLO S, AGIUS E, LU B, GOODMAN S, DALE L, DE ROBERTIS EM. Cleavage of Chordin by the Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. Cell. 1997;91:407–416. doi: 10.1016/s0092-8674(00)80424-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. PICCOLO S, SASAI Y, LU B, DE ROBERTIS EM. Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of Chordin to BMP-4. Cell. 1996;86:589–598. doi: 10.1016/s0092-8674(00)80132-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. RATTNER A, HSIEH JC, SMALLWOOD PM, GILBERT DJ, COPELAND NG, JENKINS NA, NATHANS J. A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proc. Natl. Acad. Sci. USA. 1997;94:2859–2863. doi: 10.1073/pnas.94.7.2859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. RIVERA-PÉREZ JA, MALLO M, GENDRON-MAGUIRE M, GRIDLEY T, BEHRINGER RR. goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development. Development. 1995;121:3005–3012. doi: 10.1242/dev.121.9.3005. [DOI] [PubMed] [Google Scholar]
  62. SANDELL LJ, MORRIS N, ROBBINS JR, GOLDRING MB. Alternatively spliced Type II Procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide. J. Cell Biol. 1991;114:1307–1319. doi: 10.1083/jcb.114.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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;15:4547–4555. [PMC free article] [PubMed] [Google Scholar]
  64. SASAI Y, LU B, STEINBEISSER H, DE ROBERTIS EM. Regulation of neural induction by the chd and BMP-4 antagonistic patterning signals in Xenopus. Nature. 1995;376:333–336. doi: 10.1038/376333a0. [DOI] [PubMed] [Google Scholar]
  65. SASAI Y, LU B, STEINBEISSER H, GEISSERT D, GONT LK, DE ROBERTIS EM. Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell. 1994;79:779–790. doi: 10.1016/0092-8674(94)90068-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. SCHMID B, FÜRTHAUER M, CONNORS SA, TROUT J, THISSE B, THISSE C, MULLINS MC. Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation. Development. 2000;127:957–967. doi: 10.1242/dev.127.5.957. [DOI] [PubMed] [Google Scholar]
  67. SCHMIDT JE, FRANÇOIS V, BIER E, KIMELMAN D. Drosophila short gastrulation induces an ectopic axis in Xenopus: evidence for conserved mechanisms of dorsal-ventral patterning. Development. 1995;121:4319–4328. doi: 10.1242/dev.121.12.4319. [DOI] [PubMed] [Google Scholar]
  68. SCHULTE-MERKER S, LEE KJ, McMAHON AP, HAMMERSCHMIDT M. The zebrafish organizer requires chordino. Nature. 1997;387:862–863. doi: 10.1038/43092. [DOI] [PubMed] [Google Scholar]
  69. SCOTT IC, BLITZ IL, PAPPANO WN, IMAMURA Y, CLARK TG, STEIGLITZ BM, THOMAS CL, MAAS SA, TAKAHARA K, CHO KWY, GREENSPAN DS. Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 1999;213:283–300. doi: 10.1006/dbio.1999.9383. [DOI] [PubMed] [Google Scholar]
  70. SMITH JC, PRICE BMJ, GREEN JBA, WEIGEL D, HERRMANN BG. Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell. 1991;67:79–87. doi: 10.1016/0092-8674(91)90573-h. [DOI] [PubMed] [Google Scholar]
  71. SMITH WC, HARLAND RM. Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell. 1992;70:829–840. doi: 10.1016/0092-8674(92)90316-5. [DOI] [PubMed] [Google Scholar]
  72. SPEMANN H, MANGOLD H. Über Induktion von Embryonalanlagen durch Implantation Artfremder Organisatoren. Roux’ Arch. Entw. Mech. 1924;100:599–638. [Google Scholar]
  73. STEINBEISSER H, FAINSOD A, NIEHRS C, SASAI Y, DE ROBERTIS EM. The role of gsc and BMP-4 in dorsal-ventral patterning of the marginal zone in Xenopus: a loss of function study using antisense RNA. EMBO J. 1995;14:5230–5243. doi: 10.1002/j.1460-2075.1995.tb00208.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. SU MW, SUZUKI HR, BIEKER JJ, SOLURSH M, RAMIREZ F. Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts. J. Cell Biol. 1991;115:565–575. doi: 10.1083/jcb.115.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. SUZUKI A, THIES R, YAMAJI N, SONG JJ, WOZNEY JM, MURAKAMI K, UENO N. A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo. Proc. Natl. Acad. Sci. USA. 1994;91:10255–10259. doi: 10.1073/pnas.91.22.10255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. TAM PPL, STEINER KA. Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo. Development. 1999;126:5171–5179. doi: 10.1242/dev.126.22.5171. [DOI] [PubMed] [Google Scholar]
  77. WANG S, KRINKS M, LIN K, LUYTEN FP, MOOS M. Frzb, a secreted protein expressed in the Spemann Organizer, binds and inhibits Wnt-8. Cell. 1997;88:757–766. doi: 10.1016/s0092-8674(00)81922-4. [DOI] [PubMed] [Google Scholar]
  78. WARDLE FC, WELCH JV, DALE L. Bone morphogenetic protein 1 regulates dorsal-ventral patterning in early Xenopus embryos by degrading chordin, a BMP4 antagonist. Mech. Dev. 1999;86:75–85. doi: 10.1016/s0925-4773(99)00114-8. [DOI] [PubMed] [Google Scholar]
  79. WATANABE TK, KATAGIRI T, SUZUKI M, SHIMIZU F, FUJIWARA T, KANEMOTO N, NAKAMURA Y, HIRAI Y, MAEKAWA H, TAKAHASHI E. Cloning and characterization of two novel human cDNAs (NELL1 and NELL2) encoding proteins with six EGF-like repeats. Genomics. 1996;38:273–276. doi: 10.1006/geno.1996.0628. [DOI] [PubMed] [Google Scholar]
  80. WEINMASTER G. Reprolysins and astacins. Science. 1998;279:336–337. doi: 10.1126/science.279.5349.336. [DOI] [PubMed] [Google Scholar]
  81. YAMADA G, MANSOURI A, TORRES M, STUART ET, BLUM M, SCHULTZ M, DE ROBERTIS EM, GRUSS P. Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death. Development. 1995;121:2917–2922. doi: 10.1242/dev.121.9.2917. [DOI] [PubMed] [Google Scholar]
  82. YAMANAKA Y, MIZUNO T, SASAI Y, KISHI M, TAKEDA H, KIM CH, HIBI M, HIRANO T. A novel homeobox gene, dharma, can induce the organizer in a non-cell-autonomous manner. Genes Dev. 1998;12:2345–2353. doi: 10.1101/gad.12.15.2345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. ZHU L, BELO JA, DE ROBERTIS EM, STERN CD. Goosecoid regulates the neural inducing strength of the mouse node. Dev. Biol. 1999a;216:276–281. doi: 10.1006/dbio.1999.9508. [DOI] [PubMed] [Google Scholar]
  84. ZHU Y, OGANESIAN A, KEENE DR, SANDELL LJ. Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-β1 and BMP-2. J. Cell Biol. 1999b;144:1069–1080. doi: 10.1083/jcb.144.5.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. ZIMMERMAN LB, DE JESÚS-ESCOBAR JM, HARLAND RM. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell. 1996;86:599–606. doi: 10.1016/s0092-8674(00)80133-6. [DOI] [PubMed] [Google Scholar]
  86. ZUSMAN SB, SWEETON D, WIESCHAUS EF. short-gastrulation, a mutation causing delays in stage-specific cell shape changes during gastrulation in Drosophila melanogaster. Dev. Biol. 1988;129:417–427. doi: 10.1016/0012-1606(88)90389-2. [DOI] [PubMed] [Google Scholar]

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