cDNAs of the human chordin gene (CHRD) have been cloned and alternative splice variants have been characterized that code for C-truncated forms of the protein. 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. Spliced variants are present with specific patterns in various tissues. When tested in an axis-duplication assay in Xenopus, it has been found that these variants can antagonize BMP activity. Altogether, these results suggest that, in addition to processing by metalloproteases, alternative splicing is another mechanism by which sub-products of CHRD can be generated to influence BMP activity in different developmental and physiological situations (Millet, 2001).

Chordin is a key developmental protein that dorsalizes early vertebrate embryonic tissues by binding to ventralizing TGF-beta-like bone morphogenetic proteins and sequestering them in latent complexes. This study reports the first characterization of mammalian chordin. The full-length cDNA sequence for mouse chordin is given, and RNA blot analysis shows the murine chordin gene Chrd is expressed at relatively high levels in 7-day postcoitum mouse embryos and at much decreased levels at later developmental times and in adult tissues. These results imply a major role for chordin during gastrulation of the mammalian embryo. Nevertheless, both murine and human chordin genes are shown to be expressed at readily detectable levels in several fetal and adult tissues, most notably liver and cerebellum, suggesting additional roles in organogenesis and homeostasis. Chrd was mapped to mouse chromosome 16 using interspecific crosses, and the cognate human gene CHRD was localized to human chromosome 3q27 by radiation hybrid mapping (Pappano, 1998).

Fate-mapping experiments in the mouse have revealed that the primitive streak can be divided into three functional regions: the proximal region gives rise to germ cells and the extra-embryonic mesoderm of the yolk sac; the distal region generates cardiac mesoderm and node-derived axial mesendoderm; and the middle streak region produces the paraxial, intermediate and lateral plate mesoderm of the trunk. To gain insight into the mechanisms that mediate the assembly of the primitive streak into these functional regions, the gene has been cloned and functionally identified that is disrupted in the amnionless (amn) mouse, which has a recessive, embryonic lethal mutation that interferes specifically with the formation and/or specification of the middle primitive streak region during gastrulation. The gene Amn encodes a novel type I transmembrane protein that is expressed exclusively in the extra-embryonic visceral endoderm layer during gastrulation. The extracellular region of the Amn protein contains a cysteine-rich domain with similarity to bone morphogenetic protein (BMP)-binding cysteine-rich domains in chordin, its Drosophila melanogaster homolog (Short gastrulation) and procollagen IIA. These findings indicate that Amn may direct the production of trunk mesoderm derived from the middle streak by acting in the underlying visceral endoderm to modulate a BMP signaling pathway (Kalantry, 2001).

cyr61 was first identified as a growth factor-inducible immediate-early gene in mouse fibroblasts. The encoded Cyr61 protein is a secreted, cysteine-rich heparin-binding protein that associates with the cell surface and the extracellular matrix, and in these aspects it resembles the Wnt-1 protein and a number of known growth factors. During embryogenesis, cyr61 is expressed most notably in mesenchymal cells that are differentiating into chondrocytes and in the vessel walls of the developing circulatory system. cyr61 is a member of an emerging gene family that encodes growth regulators, including the connective tissue growth factor and an avian proto-oncoprotein, Nov. cyr61 also shares sequence similarity with two Drosophila genes, twisted gastrulation and short gastrulation, which interact with decapentaplegic to regulate dorsal-ventral patterning. Purified Cyr61 has the following effects: (1) it promotes the attachment and spreading of endothelial cells in a manner similar to that of fibronectin; (2) it enhances the effects of basic fibroblast growth factor and platelet-derived growth factor on the rate of DNA synthesis of fibroblasts and vascular endothelial cells, although it has no detectable mitogenic activity by itself; and (3) it acts as a chemotactic factor for fibroblasts. Taken together, these activities indicate that Cyr61 is likely to function as an extracellular matrix signaling molecule rather than as a classical growth factor and may regulate processes of cell proliferation, migration, adhesion, and differentiation during development (Kireeva, 1996).

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 mRNA into Xenopus embryos induces a secondary axis. Recombinant CHL protein inhibits the BMP4-dependent differentiation of embryonic stem cells in vitro and interacts directly with BMPs, similar to chordin. However, CHL also weakly binds to TGFbetas. In situ hybridization has revealed that the mouse CHL gene, located on the X chromosome, is 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 is expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL are 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 (Nakayama, 2001).

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. The expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud and muscular Hox genes are activated by signals from the limb mesenchyme. This study examines the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein is detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme is dependent on the apical ectodermal ridge (AER), but later expression is independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme is induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintains HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses is restricted to the posterior region. HOXA-13 expression in the autopodal mesenchyme is dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses is dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induces ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulate the expression of HOXA-13 in the posterior region of the muscle masses and inhibit posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme is affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression is independent of BMP-2 from the polarizing region, but is under the control of as yet unidentified signals from the AER. These results show that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells (Hashimoto, 1999).

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. A previously unrecognized regulator of mammalian chondrogenesis is encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The Cyr61 protein is a member of an emerging family of extracellular proteins characterized by sequence and by conservation of all 38 cysteine residues in their secreted portions. This protein family, called the CNN family (Cyr61/CEF10, CTGF/Fisp12 and Neuroblastoma overexpressed), includes three distinct members to date: (1) the murine Cyr61 and its chicken homolog, CEF-10; (2) the human connective tissue growth factor and its murine homolog, Fisp12; and (3) Nov, whose gene was identified as aberrantly expressed in retrovirus-induced avian nephroblastomas. Drosophila Twisted gastrulation and Short gastrulation also share sequence similarities with members of this gene family. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins that undergo chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo, based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis, both in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities, which are otherwise unable to support differentiation, and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis, which contributes to the development of the mammalian embryonic skeleton (Wong, 1997).

Twisted gastrulation gene products have been identified from human, mouse, Xenopus, zebrafish and chick. Expression patterns in mouse and Xenopus embryos are consistent with in vivo interactions between Tsg, BMPs and the vertebrate SOG ortholog, chordin. Tsg binds both the vertebrate Decapentaplegic ortholog BMP4 and chordin, and these interactions have multiple effects. Tsg increases chordin's binding of BMP4, potentiates chordin's ability to induce secondary axes in Xenopus embryos, and enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence; also, the presence of Tsg enhances the secondary axis-inducing activity of two products of chordin cleavage. It is concluded that Tsg acts as a cofactor in chordin's antagonism of BMP signaling (Scott, 2001).

Tsg is coexpressed with chordin and various BMPs in vertebrate development. In Xenopus, maternal Tsg RNA was detected in eggs by RT (reverse transcription)-PCR, while whole-mount in situ hybridization showed uniform Tsg expression across the entire animal hemisphere and marginal zone of the early gastrula. At tailbud stage, Tsg, chordin and BMP4 expression domains partially overlap in the developing tail, anterior brain, eye and heart. In mouse, Tsg is broadly expressed throughout the 7.5-days-post-coitus (7.5-d.p.c.) gastrula and in extraembryonic tissues. Chordin, Tsg and BMPs 2, 4 and 7 are highly expressed in the digital rays of 15.5- and 17.5-d.p.c. embryo hindlimbs. Strong chordin expression in the interzone of the joint cavity is juxtaposed with strong Tsg expression at the joint articular surfaces and the interzone. Thus, Tsg is properly situated for potential interactions with chordin and BMPs during various stages of vertebrate embryogenesis (Scott, 2001).

In Drosophila, TSG influences cleavage of the chordin ortholog Short gastrulation (SOG) by Tolloid, altering the pattern of SOG cleavage products. There are four mammalian tolloid-related proteases. Two of these, BMP1 and mammalian tolloid-like 1 (mTll1), each cleave chordin at two specific sites, yielding fragments of relative molecular mass (Mr) 15K, 13K and 83K, corresponding to the amino-terminal, carboxy-terminal, and internal portions of chordin, respectively. Murine Tsg appears to enhance cleavage of mouse chordin and to influence the relative abundance of cleavage products, such that fragments of Mr 65K and 29K, minor forms in the absence of Tsg, become major products in the presence of Tsg. A third related protease, mammalian tolloid (mTld), which has little detectable chordin-cleaving activity, has significant activity in the presence of Tsg, also producing the 65K and 29K fragments as major forms. The fourth mammalian tolloid-like protease, mTll2, lacks chordin-processing activity in the presence or absence of Tsg (Scott, 2001).

The 65K and 29K chordin cleavage products preferentially produced in the presence of Tsg are subfragments of the 83K internal chordin fragment, as established by N-terminal amino-acid sequencing, and result from cleavage at a previously unmapped site between Ala 670 and Thr 671. Thus, the 29K form contains chordin cysteine-rich repeats (CRs) 2 and 3, whereas the 65K form contains no CR domains (Scott, 2001).

To determine how Tsg affects chordin cleavage, Tsg's ability to physically interact with tolloid-like proteases was examined. Co-immunoprecipitation of Tsg with BMP1 or mTll1 fails to detect physical interactions. However, co-immunoprecipitations show that Tsg binds chordin. Whether Tsg/chordin interactions might influence chordin's ability to bind BMP4 was examined. Co-immunoprecipitation of chordin and BMP4 is greatly enhanced in the presence of Tsg. It was also found that Tsg binds BMP4. In summary, Tsg's interactions with chordin and/or BMP4 enhance chordin/BMP4 complex formation, suggesting that Tsg might enhance chordin's antagonism of BMP signaling (Scott, 2001).

Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). The molecular mechanism by which Tsg regulates BMP signaling has been analyzed. 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, since they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay has revealed that Tsg has two distinct and sequential activities on BMP signaling. (1) Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. (2) 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 (Larraín, 2001).

The opposing activities of Tsg on BMP binding to its receptor suggest a sequential molecular mechanism that may help reconcile disparate observations in the literature. (1) Tsg forms a ternary complex with Chordin and BMP, which is a potent inhibitor of BMP signaling. This antagonist function must be the predominant one in zebrafish, because loss-of-function of Tsg and Chordin using antisense morpholinos ventralizes the embryo. (2) After cleavage of Chordin by Xolloid, Tsg competes the residual activity of Chordin fragments, providing a permissive signal that promotes BMP binding to its cognate receptor. This function is consistent with injection experiments in Xenopus embryos, in which reduction of endogenous Xenopus Tsg activity enhances the anti-BMP activity of CR1 fragments. (3) Overexpression of Tsg facilitates the degradation of endogenous Chordin in Xenopus. This activity may help explain why Tsg can ventralize the embryo and inhibit axis duplication by Chordin in a Xolloid-dependent manner. It is proposed that in overexpression experiments, an excess of Tsg protein displaces the equilibrium in the reaction, so that after cleavage of Chordin by Xolloid, Tsg dislodges BMP from the proteolytic products and facilitates their degradation in vivo. The Tsg/BMP binary complex acts as a permissive signal, because at physiological concentrations Tsg does not interfere with BMP binding to its receptor. Finally, at high concentrations, Tsg can also act as a BMP antagonist in the absence of Chordin, inducing in animal cap explants the cement gland marker XAG-1, but not the neural marker NCAM, by partially inhibiting BMP activity (Larraín, 2001).

The present results provide mechanistic insights into how sharp borders may be generated in embryos. In Drosophila, Tsg is required for the peak BMP signaling that induces a sharp band of Mad phosphorylation in the dorsal-most tissue. In lateral regions of the Xenopus embryo, where free full-length Chordin is still present, Tsg/BMP binary complexes released by Xolloid will have a higher affinity for Chordin than for the BMP receptor promoting the re-formation of inhibitory ternary complexes that can diffuse further. However, once all Chd is proteolytically cleaved by Xolloid, the function of Tsg switches from an inhibitory to a permissive signal that increases binding of BMPs to their cognate receptors. This switch in activity would facilitate the formation of sharp boundary differences. In lateral regions, where ternary complexes are constantly re-formed and re-cleaved as diffusion takes place, the situation is conceptually analogous to that occurring in an organic chemistry fractional distillation column. Although much remains to be learned about this interesting patterning system, the opposing functions of Tsg suggest a novel molecular mechanism for the establishment of cell differentiation territories in the embryo (Larraín, 2001).

The roles of the BMP antagonists Chordin and Noggin in development of the mandible, which is derived from the first branchial arch (BA1) were examined. Both genes are expressed in the pharynx during early mandibular outgrowth and later in the mandibular process. Mice mutant for either Nog or Chd have only mild mandibular defects; however, double mutant pups exhibit a range of mandibular truncation phenotypes, from normal to agnathia. A few embryos homozygous null for both genes survive to late gestation; many are agnathic, though a few have significant mandibular outgrowth. In mandibular explants, ectopic BMP4 rapidly induces expression of both Chd and Nog, consistent with results obtained in vivo with mutant embryos. FGF8 is a survival factor for cells populating the mandibular bud. Excess BMP4 represses Fgf8 transcription in mandibular explants. Embryos lacking these BMP antagonists often show a strong reduction in Fgf8 expression in the pharyngeal ectoderm, and increased cell death in the mandibular bud. It is suggested that the variable mandibular hypoplasia in double mutants involves increased BMP activity downregulating Fgf8 expression in the pharynx, decreasing cell survival during mandibular outgrowth (Stottmann, 2001).

The roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins (BMPs), were investigated in formation of the mammalian head. The mouse chordin and noggin genes (Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, embryos of the genotype Chrd^-/-;Nog^+/- were characterized. These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia, holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. Defective Chrd^-/-;Nog^+/- embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, it is hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head (Anderson, 2002).

The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. Chordin homozygous mutant mice, generated by targeted mutagenesis, 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 (Bachiller, 2003).

To study the interaction of Chrd with genes known to cause DiGeorge or DiGeorge-like phenotypes in mice, the expression of Tbx1 and Fgf8 was analyzed in Chrd mutant embryos. Tbx1 is a member of the T-box family of transcription factors. It maps within the DGS/VCFS 22q11 microdeletion in humans and has been shown to cause DiGeorge-like phenotype upon inactivation in mice. Expression of Tbx1 is altered in Chrd^-/- embryos. In wild-type E7.5 animals, Tbx1 is expressed in the foregut (future pharyngeal endoderm) and head mesoderm. At this stage, mutant littermates showed a clear reduction in the levels of Tbx1 expression in the same areas. The reduction in Tbx1 mRNA is equally clear in the pharyngeal region of Chrd homozygous embryos at E8.0, E8.5 and E9.0. Transverse histological sections show that at the cellular level the abundance of Tbx1 transcripts is drastically reduced in endoderm, both in the pharynx and foregut up to the level of the hepatic diverticulum. Diminution in the concentration of Tbx1 mRNA is also evident in mesoderm, including head, splanchnic and somatic mesoderm in the peripharyngeal region. In addition, Tbx1 expression at E9 in the mesodermal core of the first pharyngeal arch is diffuse, extending to most of the arch, and Tbx1 transcripts are absent from the otic vesicle (Bachiller, 2003).

Fgf8 is a secreted growth factor expressed in a variety of tissues, including the pharyngeal endoderm and neighboring mesoderm. During early development, Fgf8 is required for gastrulation and the establishment of the left/right axis of symmetry. At later stages of Fgf8 is required for limb and craniofacial development. Recent experiments have shown that mice with reduced Fgf8 activity present a spectrum of cardiovascular and pharyngeal defects that closely mimic DiGeorge syndrome. In addition, Fgf8 expression is abolished in the pharyngeal endoderm of Tbx1^-/- mutants and both genes interact genetically during the differentiation of the pharyngeal arch arteries. At E9, Fgf8 expression in Chrd mutants is normal in the mid-hindbrain isthmus, frontonasal prominence and tail. However, in pharyngeal endoderm, Fgf8 transcript levels are drastically reduced. The reduction of Tbx1 and Fgf8 expression in Chrd^-/- embryos suggest that both genes act downstream of Chrd in the same regulatory pathway. These experiments do not determine whether Chrd is required for the maintenance or for the induction of Tbx1 and Fgf8 in the pharynx and neighboring tissues. To test whether Chrd can induce Tbx1 and Fgf8, Chrd mRNA (50 pg) was injected into the ventral region of Xenopus embryos at the four-cell stage. Ventral marginal zone (VMZ) explants were dissected at early gastrula, cultured until sibling embryos reached early neurula stage, and analyzed by RTPCR. Tbx1 and Fgf8 mRNAs are expressed at high levels in whole embryos and dorsal marginal zone (DMZ) explants at this stage, and at low levels in VMZ explants. Upon microinjection, Chrd mRNA increases the levels of Tbx1 and Fgf8 in VMZ. In situ hybridization of microinjected Xenopus embryos confirmed that the Tbx1 transcripts induced by Chrd mRNA are located in pharyngeal endoderm. It is concluded that Chrd, a Bmp antagonist, can induce Tbx1 and Fgf8 expression in Xenopus embryos, and is required for full expression of these genes in the pharyngeal region of the mouse embryo (Bachiller, 2003).