The Chordin Page

Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes

Yoshiki Sasai, Bin Lu, Herbert Steinbeisser, Douglas Geissert, Linda K. Gont and E.M. De Robertis

Cell 79, 779-790 (1994)

A Xenopus gene whose expression can be activated by the organizer-specific homeobox genes goosecoid and Xnot2 was isolated by differential screening. The chordin gene encodes a novel protein of 941 amino acids that has a signal sequence and four Cys-rich domains. The expression of chordin starts in Spemann's organizer subsequent to that of goosecoid, and its induction by activin requires de novo protein synthesis. Microinjection of chordin mRNA induces twinned axes and can completely rescue axial development in ventralized embryos. This molecule is a potent dorsalizing factor that is expressed at the right time and in the right place to regulate cell-cell interactions in the organizing centers of head, trunk, and tail development.

Expression pattern of chordin in the Xenopus

Stages 9.5, 10.25, 10.75, 11.5, 13, 26, 33.

Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus

Yoshiki Sasai, Bin Lu, Herbert Steinbeisser and E.M. De Robertis

Nature 376, 333-336 (1995)

In Drosophila the amount of neurogenic ectoderm, from which the central nervous system (CNS) derives, is regulated by a dorsal-ventral system of positional information in which two secreted molecules of antagonistic functions, decapentaplegic (dpp) and short-gastrulation (sog), play fundamental roles. The vertebrate homologue of dpp is either bmp-4 or bmp-2, and the homologue of sog is chd (s-chordin). In Xenopus the CNS is induced by signals emanating from the organizer, and two proteins secreted by the organizer, noggin and follistatin, have been shown to induce neural tissue in animal-cap assays. Here we report that Chd, another organizer-specific secreted factor, has neuralizing activity and that this activity can be antagonized by Bmp-4. Inhibition of the function of the endogenous Bmp-4 present in the animal cap also leads to neural differentiation. We suggest that conserved molecular mechanisms involving chd/sog and bmp-4/dpp gene products pattern the ecdoderm in Xenopus and in Drosophila.

Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4

Stefano Piccolo, Yoshiki Sasai, Bin Lu, and E.M. De Robertis

Cell 86, 589-598 (1996)

Chordin (Chd) is an abundant protein secreted by Spemann organizer tissue during gastrulation. Chd antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Recombinant Xenopus Chd binds to BMP-4 with high affinity (KD 3 x 10-10 M), binding specifically to BMPs but not to activin or TGF-b1. Chd protein is able to dorsalize mesoderm and to neuralize ectoderm in Xenopus gastrula explants at 1 nM. We propose that the noncell-autonomous effects of Spemann's organizer on dorsoventral patterning are executed in part by diffusible signals that directly bind to and neutralize ventral BMPs during gastrulation.

BMP-binding modules in chordin: a model for signalling regulation in the extracellular space

Juan Larrain, Daniel Bachiller, Bin Lu, Eric Agius, Stefano Piccolo and E. M. De Robertis

Development 127, 821-830 (2000)

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.

The organizer factors Chordin and Noggin are required for mouse forebrain development.

Daniel Bachiller, John Klingensmith, Caroline Kemp, Jose A. Belo, R .M. Anderson, S.R. May, J. A. McMahon, A. P. McMahon, Richard Harland, Janet Rossant, and E. M. De Robertis, E.M.

Nature 403, 658-661 (2000)

In mice, there is evidence suggesting that the development of head and trunk structures is organized by distinctly separated cell populations. The head organizer is located in the anterior visceral endoderm (AVE) and the trunk organizer in the node and anterior primitive streak. In amphibians, Spemann's organizer, which is homologous to the node, partially overlaps with anterior endoderm cells expressing homologues of the AVE markers cerberus, Hex and Hesx1. For mice, this raises the question of whether the AVE and node are independent of each other, as suggested by their anatomical separation, or functionally interdependent as is the case in amphibians. Chordin and Noggin are secreted bone morphogenetic protein (BMP) antagonists expressed in the mouse node, but not in the AVE. Here we show that mice double-homozygous mutants that are for chordin and noggin display severe defects in the development of the prosencephalon. The results show that BMP antagonists in the node and its derivatives are required for head development.

The establishment of Spemann's organizer and patterning of the vertebrate embryo

De Robertis, E.M., Larrain, J., Oelgeschlager, M. and Wessely, O.

Nature Reviews Genetics 1, 171-181 (2000)

Molecular studies have begun to unravel the sequential cell-cell signalling events that establish the dorsal-ventral, or 'back-to-belly', axis of vertebrate animals. In Xenopus and zebrafish, these events start with the movement of membrane vesicles associated with dorsal determinants. This mediates the induction of mesoderm by generating gradients of growth factors. Dorsal mesoderm then becomes a signalling centre, the Spemann's organizer, which secretes several antagonists of growth-factor signalling. Recent studies have led to new models for the regulation of cell-cell signalling during development, which may also apply to the homeostasis of adult tissues.

Neural induction in the absence of mesoderm: beta-catenin-dependent expression of secreted BMP antagonists at the blastula stage in Xenopus

Oliver Wessely, Eric Agius, Michael Oelgeschlager, Edgar M. Pera and E. M. De Robertis

Developmental Biology 234, 161-173 (2001)

A growing body of work indicates that neural induction may be initiated prior to the establishment of the gastrula mesodermal organizer. Here, we examine neural induction in Xenopus embryos in which mesoderm induction has been blocked by Cerberus-short, a reagent that specifically inhibits Nodal-related (Xnr) signals. We find that extensive neural structures with cyclopic eyes and brain tissue are formed despite the absence of mesoderm. This neural induction correlates with the expression of chordin and other BMP inhibitors-such as noggin, follistatin, and Xnr3-at the blastula stage, and requires beta-Catenin signaling. Activation of the beta-Catenin pathway by mRNA microinjections or by treatment with LiCl leads to differentiation of neurons, as well as neural crest, in ectodermal explants. Xnr signals are required for the maintenance, but not for the initiation, of BMP antagonist expression. Recent work has demonstrated a role for beta-Catenin signaling in neural induction mediated by the transcriptional down-regulation of BMP-4 expression. The present results suggest an additional function for beta-Catenin, the early activation of expression of secreted BMP antagonists, such as Chordin, in a preorganizer region in the dorsal side of the Xenopus blastula.

Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling

Juan Larrain, Michael Oelgeschlager, Nan L. Ketpura, N.I., Bruno Reversade, Lise Zakin, and E. M. De Robertis

Development 128, 4439-4447 (2001)

Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). We have analyzed the molecular mechanism by which Tsg regulates BMP signaling. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, as they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay revealed that Tsg has two distinct and sequential activities on BMP signaling. First, Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. Second, after cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated.

Cerberus-like is a secreted BMP and nodal antagonist not essential for mouse development

José António Belo, Daniel Bachiller, Eric Agius, Caroline Kemp, A.C. Borges, S. Marques, S. Piccolo S and E.M. De Robertis

Genesis 26, 265-270 (2000)

Mouse cerberus-like (cer-l) is a member of the Cerberus/Dan family of secreted factors. As other members of this family of proteins, Cer-l functions in the extracellular space, inhibiting signaling molecules. Here we show that the neural-inducing and mesoderm-inhibiting activities of Cer-l result from specific binding to BMP and Nodal molecules, respectively. These properties resemble the ones from the related factor Xenopus Cerberus. However, Xenopus Cerberus in addition to BMP4 and Nodal also binds to and inhibits Wnt proteins. We show that Cer-l does not directly inhibit Wnt signals. A null allele of the mouse Cer-l gene was generated by targeted inactivation in ES cells. Homozygous embryos show no anterior patterning defects, are born alive, and are fertile. Since mouse Cer-l and Xenopus Cerberus differ in biochemical activities, we propose the existence of additional members of this family of inhibitors, which may compensate for the loss of cer-l..

The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome

Daniel Bachiller, John Klingensmith, N. Shneyder, Uyen Tran, R. Anderson, Janet Rossant, and E. M. De Robertis

Development 130, 3567-3578 (2003)

The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. We present the phenotype produced by the targeted inactivation of the chordin gene in mouse. Chordin homozygous mutant mice show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally, displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis.

Integrin-a3 mediates binding of Chordin to the cell surface and promotes its entocytosis

Juan Larrain, Carlos Brown and E. M. De Robertis

EMBO Reports 4, 813-818 (2003)

Dorsoventral patterning in animal development is regulated by a morphogenetic gradient of Bone morphogenetic protein signalling, which is established by a set of proteins that are conserved from Drosophila to vertebrates. These include Chordin (Chd)/Short gastrulation, Xolloid/Tolloid and Twisted gastrulation. Here, we report the identification of a cell-surface component of this morphogenetic pathway. Prompted by the observation that Chd protein bound to the surface of certain cell lines with subnanomolar affinity, we identified two cell-surface proteins that bind to Chd, one of which corresponds to Integrin-alpha3. Integrin-alpha3 and Chd are co-expressed in the Xenopus embryo. Transfection of Integrin-alpha3 increased the binding of Chd to the cell surface, which was competed by an excess of soluble Integrin-alpha3. After binding to the cell surface, Chd was translocated into intracellular endocytic compartments in a temperature-dependent manner. We propose that Integrin-alpha3 may regulate the concentration of Chd protein in the extracellular space by endocytosis.

Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos

Michael Oelgeschlager, Hiroki Kuroda, Bruno Reversade and E. M. De Robertis

Developmental Cell 4, 219-230 (2003)

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.

Analysis of Spemann organizer formation in Xenopus embryos by cDNA macroarrays

Oliver Wessely, James I. Kim, Douglas Geissert, Uyen Tran and E.M. De Robertis

Developmental Biology 269, 552-566 (2004)

The understanding of vertebrate development has greatly benefited from the study of gastrulation in the Xenopus embryo. Over the years, the molecular dissection of the Spemann organizer has proven to be a very fruitful source for gene discovery. Here, we report a comprehensive screen of gene expression in the Xenopus gastrula using cDNA macroarrays. Nylon filters containing more than 72000 cDNAs from a gastrula stage library were hybridized with differential probes from embryos in which organizer induction had been inhibited by reducing Nodal-related or maternal beta-Catenin signaling. Combining the changes in gene expression levels caused by these two major signaling pathways in a single graph identified both known and novel dorsoventral regulated genes. The most highly enriched organizer-specific genes were the secreted molecules chordin and Xnr-3, followed by the transmembrane protein paraxial protocadherin (PAPC). Ventral-specific abundant cDNAs included S10-40-H5, members of the Hyaluronan synthase family, Xvent-2 and XFD2/FoxI1. A differential probe of dorsal and ventral lips identified many more organizer-specific cDNAs than the screens inhibiting Nodal-related and beta-Catenin signaling, suggesting that additional, as yet uncharacterized signaling pathways, contribute to organizer formation. Finally, extension of this approach to the blastula preorganizer signaling center identified the transcription factor pintallavis/FoxA2 as a new preorganizer component.

Neural Induction in Xenopus: Requirement for Ectodermal and Endomesodermal Signals via Chordin, Noggin, ß-Catenin, and Cerberus

Hiroki Kuroda, Oliver Wessely and E. M. De Robertis

PLoS Biology 2, 623-634 (2004)

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early ß-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator ß-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula ß-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.

Dorsal-ventral patterning and neural induction in Xenopus embryos

E. M. De Robertis and Hiroki Kuroda

Annu. Rev. Cell Dev. Biol. 20, 285-308 (2004)

We review the current status of research in dorsal-ventral (D-V) patterning in vertebrates. Emphasis is placed on recent work on Xenopus, which provides a paradigm for vertebrate development based on a rich heritage of experimental embryology. D-V patterning starts much earlier than previously thought, under the influence of a dorsal nuclear -Catenin signal. At mid-blastula two signaling centers are present on the dorsal side: The prospective neuroectoderm expresses bone morphogenetic protein (BMP) antagonists, and the future dorsal endoderm secretes Nodal-related mesoderm-inducing factors. When dorsal mesoderm is formed at gastrula, a cocktail of growth factor antagonists is secreted by the Spemann organizer and further patterns the embryo. A ventral gastrula signaling center opposes the actions of the dorsal organizer, and another set of secreted antagonists is produced ventrally under the control of BMP4. The early dorsal -Catenin signal inhibits BMP expression at the transcriptional level and promotes expression of secreted BMP antagonists in the prospective central nervous system (CNS). In the absence of mesoderm, expression of Chordin and Noggin in ectoderm is required for anterior CNS formation. FGF (fibroblast growth factor) and IGF (insulin-like growth factor) signals are also potent neural inducers. Neural induction by anti-BMPs such as Chordin requires mitogen-activated protein kinase (MAPK) activation mediated by FGF and IGF. These multiple signals can be integrated at the level of Smad1. Phosphorylation by BMP receptor stimulates Smad1 transcriptional activity, whereas phosphorylation by MAPK has the opposite effect. Neural tissue is formed only at very low levels of activity of BMP-transducing Smads, which require the combination of both low BMP levels and high MAPK signals. Many of the molecular players that regulate D-V patterning via regulation of BMP signaling have been conserved between Drosophila and the vertebrates.

	Figure 1. The Spemann-Mangold organizer experiment repeated in Xenopus laevis.
	Figure 2. Organizer formation in Xenopus laevis.
	Figure 3. Proteins secreted by dorsal (Spemann organizer) or ventral gastrula signaling centers.
	Figure 4. Cysteine-rich (CR) BMP-binding modules in Chordin and Crossveinless-2.
	Figure 5. Integration of multiple signaling pathways at the level of Smad1 phosphorylation during neural induction.
	Figure 6. Signaling centers at blastula and gastrula that have critical roles for body plan formation in Xenopus.
	Figure 7. Inversion in the course of evolution of a conserved network of extracellular regulators of BMP/Dpp signaling involved in D-V patterning.