Table of contents

BMP interaction with Chordin and Twisted gastrulation

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin (homolog of Drosophila Short gastrulation), a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. 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 signaling (Larrain, 2000).

The Chordin/BMP pathway is regulated by the zinc metalloprotease Xolloid, a homolog of Drosophila Tolloid that regulates the activity of Sog. The observations made in this study begin to provide a molecular explanation for how Xolloid may regulate Chordin. Xolloid cleaves Chordin at two sites, which had been roughly mapped close to a region downstream of CR1 and CR3. Recently, the cleavage sites have been sequenced and found to correspond to conserved aspartic residues. The CR1 protein used in this study is very similar in length (only 8 amino acids shorter) to the fragment generated by metalloprotease cleavage in the N-terminal site of Chordin. CR1 binds BMP4 with a lower affinity (8-fold lower), is less efficient in competing BMP4 binding to BMPR (10 times lower), and has less biological activity (5- to 10-fold lower) than full-length Chordin. It is conceivable that the Xolloid protease inactivates Chordin by the generation of smaller fragments that can still bind BMP and perhaps transport it. However, each of these binding modules alone would not have high enough affinity to compete with the higher affinity of BMP for its cognate receptors, which is in the same range as that of full-length Chordin for BMP4 (Larrain, 2000 and references therein).

In Drosophila, Sog not only inhibits Dpp signaling but is also able to enhance it at a distance. This enhancement of BMP signals requires Sog diffusion (presumably carrying bound Dpp or Screw) and the activity of the Tolloid protease. It has been suggested that the cleavage products of Sog, or Sog fragments complexed with Dpp, could augment the binding of Dpp/Screw to its receptors. None of the Chordin constructs used in the current study, including a series of carboxy-terminal protein truncations, display ventralizing effects as would be expected if there were increased binding to receptors. Rather, the observation that Chordin fragments are either weakly dorsalizing or inactive in Xenopus assays tends to support the proposal that diffusion of Chd/Sog complexed with BMP/Dpp contributes to the formation of morphogen gradients in which maximal levels of signaling are achieved by cleavage of the inhibitor and release of the active BMP signal (Larrain, 2000 and references therein).

During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensen's node. A unique secreted protein, Tsukushi (TSK), was identified which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensen's node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensen's node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensen's node (Ohta, 2004).

Chick TSK is a unique member of the Small Leucine-Rich Proteoglycan family (SLRPs), which comprises 11 members by virtue of the LRR motifs and sugar modification. DNA database search and general screening have identified C-TSK orthologs in Xenopus laevis (X-TSK), zebrafish (Z-TSK), mouse, and human. All TSK orthologs have 12 LRRs, which are located between the two cysteine clusters at the N and C termini. An individual LRR of C-TSK consists of 21-26 amino acid residues with the consensus sequence. The N-terminal cysteine cluster has the C-X3-C-X-C-X17-C pattern. Secretion of C-TSK is confirmed by its localization in the cell supernatant when C-TSK cDNA is transfected into COS-7 cells. There are some potential sites of glycosaminoglycan (GAG) attachment (Ser-Gly) and N-glycosylation (Asn-X-Ser/Thr), and N-glycosidase F treatment confirms the existence of N-glycosylation (Ohta, 2004)

BMP interaction with a vertebrate Crossveinless 2 homologue

Proteins that bind to bone morphogenetic proteins (BMPs) and inhibit their signalling have a crucial role in the spatial and temporal regulation of cell differentiation and cell migration by BMPs. A chick homologue of crossveinless 2, a Drosophila gene that was identified in genetic studies as a promoter of BMP-like signalling, has been identified. Chick Cv-2 has a conserved structure of five cysteine-rich repeats similar to those found in several BMP antagonists, and a C-terminal Von Willebrand type D domain. Cv-2 is expressed in the chick embryo in a number of tissues at sites at which elevated BMP signalling is required. One such site of expression is premigratory neural crest, in which at trunk levels threshold levels of BMP activity are required to initiate cell migration. When overexpressed, Cv-2 can weakly antagonise BMP4 activity in Xenopus embryos, but in other in vitro assays Cv-2 can increase the activity of co-expressed BMP4. Furthermore, increased expression of Cv-2 causes premature onset of trunk neural crest cell migration in the chick embryo, indicative of Cv-2 acting to promote BMP activity at an endogenous site of expression. It is therefore proposed that BMP signalling is modulated both by antagonists and by Cv-2 that acts to elevate BMP activity (Coles, 2004).

Drosophila Crossveinless-2 (dCV-2) is required for local activation of Mad phosphorylation in the fruit fly wing and has been postulated to be a positive regulator of BMP-mediated signaling. In contrast, the presence of 5 Chordin-like cysteine-rich domains in the CV-2 protein suggests that CV-2 belongs to a family of well-established inhibitors of BMP function that includes Chordin and Sog. A human homolog of Drosophila CV-2 (hCV-2) has been identified. Purified recombinant hCV-2 protein inhibits BMP-2 and BMP-4 dependent osteogenic differentiation of W-20-17 cells, as well as BMP dependent chondrogenic differentiation of ATDC5 cells. Interestingly, hCV-2 messenger RNA is expressed at high levels in human primary chondrocytes, whereas expression in primary human osteoblasts is low. These results suggest that hCV-2 may regulate BMP responsiveness of osteoblasts and chondrocytes in vivo. Taken together this study shows that contrary to the function predicted from the fruit fly, Crossveinless-2 is a novel inhibitor of BMP function (Binnerts, 2004).

One extracellular regulatory molecule is the Chordin/Short gastrulation protein (Chordin/Sog), a secreted protein that acts as an antagonist to BMP/Dpp. Chordin/Sog contains four cysteine-rich (CR) domains that bind to and inactivate BMP/Dpp. In contrast, a positive regulator has been identified in Drosophila. Named crossveinless 2 (cv-2), this molecule contains five CR domains at the N-terminal half and a von Willebrand factor D domain at the C-terminal part. Genetic data suggest that Cv-2 potentiates Dpp signaling. Chick and mouse CV-2 genes have been isolated and found to be secreted and enhance BMP signaling. Expression patterns were closely related to those of BMPs, supporting the likelihood of a tight link. These data show that CV-2 is a conserved, positive regulator of BMP signaling and that CR domain proteins act as both positive and negative modulators of BMP signaling (Kamimura, 2004).

Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. It was found, through embryological and biochemical experiments, that (1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo, (2) CV2 complexes with Twisted gastrulation and BMP4, (3) CV2 is not a substrate for tolloid proteinases, (4) CV2 binds to purified Chordin protein with high affinity (KD in the 1 nM range), (5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes, and (6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. It is proposed that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels (Ambrosio, 2008).

Crossveinless 2 (CV-2) is an extracellular BMP modulator protein belonging to the Chordin family. During development it is expressed at sites of high BMP signaling and like Chordin CV-2 can either enhance or inhibit BMP activity. CV-2 binds to BMP-2 via its N-terminal Von Willebrand factor type C (VWC) domain 1. This study reports the structure of the complex between CV-2 VWC1 and BMP-2. The tripartite VWC1 binds BMP-2 only through a short N-terminal segment, called clip, and subdomain (SD) 1. Mutational analysis establishes that the clip segment and SD1 together create high-affinity BMP-2 binding. All four receptor-binding sites of BMP-2 are blocked in the complex, demonstrating that VWC1 acts as competitive inhibitor for all receptor types. In vivo experiments reveal that the BMP-enhancing (pro-BMP) activity of CV-2 is independent of BMP-2 binding by VWC1, showing that pro- and anti-BMP activities are structurally separated in CV-2 (Zhang, 2008).

Bmper, an ortholog of Drosophila Crossveinless 2, is a secreted factor that regulates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been postulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. This demonstrates that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mechanism leads to the efficient degradation of Bmper and Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4-dependent Smad signaling. Noggin and Gremlin, but not Chordin, trigger endocytosis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implications for Bmp signal regulation by secreted mediators (Kelley, 2009).

DPP homologs: Interactions with HtrA1, a serine protease

HtrA1, a member of the mammalian HtrA serine protease family, has a highly conserved protease domain followed by a PDZ domain. Because HtrA1 is a secretory protein and has another functional domain with homology to follistatin, whether HtrA1 functions as an antagonist of Tgfß family proteins was investigated. During embryo development, mouse HtrA1 is expressed in specific areas where signaling by Tgfß family proteins plays important regulatory roles. GST-pulldown assay shows that HtrA1 binds to a broad range of Tgfß family proteins, including Bmp4, Gdf5, Tgfßs and activin. HtrA1 inhibits signaling by Bmp4, Bmp2, and Tgfß1 in C2C12 cells, presumably by preventing receptor activation. Experiments using a series of deletion mutants indicate that the binding activity of HtrA1 requires the protease domain and a small linker region preceding it, and that inhibition of Tgfß signaling is dependent on the proteolytic activity of HtrA1. Misexpression of HtrA1 near the developing chick eye led to suppression of eye development that was indistinguishable from the effects of noggin. Taken together, these data indicate that HtrA1 protease is a novel inhibitor of Tgfß family members (Oka, 2004).

Evolutionary conserved interaction between BMP and the BMP-binding protein Twisted gastrulation promotes BMP signaling

Dorsal-ventral patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional information gradient. The components involved include bone morphogenetic proteins (BMP/Dpp), a BMP antagonist (Chordin/Short gastrulation; Chd/Sog) and a secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. Xenopus Twisted gastrulation (xTsg), another member of this signaling pathway, is described. xTsg is expressed ventrally as part of the BMP-4 synexpression group and encodes a secreted BMP-binding protein that is a BMP signaling agonist. The Xenopus homolog of Tsg shares sequence similarities with the cysteine-rich domains of Chordin, and is part of the BMP synexpression group. Biochemical studies show that xTsg and Drosophila Twisted gastrulation (dTsg) directly bind BMPs with dissociation constants in the low nanomolar range. In microinjection experiments, xTsg mRNA behaves as an agonist of BMP signaling, ventralizing the Xenopus embryo. xTsg competes efficiently with CR1 for binding to BMP and can bind full-length Chordin, forming a ternary complex containing Chordin, BMP and xTsg in vitro. The dorsalizing activity of CR1 is readily competed by wild-type xTsg, and is greatly potentiated by reducing endogenous xTsg activity. The results indicate that xTsg is involved in dorsal-ventral patterning, permitting peak BMP signaling by antagonizing the residual anti-BMP activity of the cleavage products of Chordin. The data suggest a molecular mechanism by which xTsg dislodges latent BMPs bound to Chordin BMP-binding fragments generated by Xolloid cleavage, providing a permissive signal that allows high BMP signaling in the embryo (Oelgeschlager, 2000).

In Drosophila, the phenotype of sog loss-of-function mutants is intriguing: as expected for a Dpp/Scw antagonist, ventral structures are lost but, in addition, the amnioserosa is reduced. This result is paradoxical, as the amnioserosa is the dorsal-most tissue and therefore Sog, a BMP antagonist, is required for maximal BMP signaling. A model proposed to explain the role of Sog in promoting peak Dpp activity suggests that Sog-BMP complexes may permit the diffusion of BMPs originating from more ventral regions, which are then released dorsally by the proteolytic activity of Tolloid. The recent demonstration that BMPs remain bound to individual cysteine-rich domains, which remain intact in the Chordin proteolytic products, makes this interpretation unlikely, unless an additional factor that releases BMP from the cysteine-rich modules is proposed. The twisted gastrulation gene encodes a secreted protein that is specifically required for the differentiation of amnioserosa cells in Drosophila and is a candidate for such a factor (Oelgeschlager, 2000 and references therein).

A full-length xTsg complementary DNA was isolated by using a human expressed sequence tag (EST) to probe a Xenopus gastrula library. The cDNA encodes a protein sharing 41% amino-acid identity with Drosophila Tsg (dTsg), 89% identity with the partial human Tsg sequence and 94% identity with a mouse EST. The xTsg sequence contains a signal peptide, as expected for a secreted protein, and two conserved domains containing multiple cysteines at its amino and carboxy termini. Whole-mount in situ hybridization and polymerase chain reaction with reverse transcription (RT-PCR) show that abundant xTsg maternal transcripts are distributed throughout the animal half of the embryo during cleavage stages. At the late gastrula stage, maternal transcripts decrease and zygotic transcripts appear specifically in the ventral region of the embryo. After neurulation, xTsg transcripts surround ventrally the closed blastopore slit and the neural tube. At the tailbud stage, xTsg transcripts are detected in the postanal region, heart and dorsal eye and closely mimic the expression patterns of BMP-4 and BAMBI, a transmembrane protein that is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. The postanal region of xTsg expression derives from the ventral-most tissue of the gastrula embryo. It is concluded that xTsg is part of the BMP-4 synexpression group and is expressed in the ventral pole of the embryo (Oelgeschlager, 2000).

Microinjection of xTsg mRNA into each blastomere of the four-cell embryo results in the reduction of dorso-anterior structures. This phenotype is reminiscent of the chordin zebrafish mutant, and of Xenopus embryos microinjected with Xolloid mRNA or with low doses of BMP-4 mRNA. Molecular marker analyses showed that dorsal ectoderm (Sox-2) and dorsal mesoderm (MyoD and Shh) are reduced and that ventral tissues (BMP-4) are expanded in xTsg-injected embryos. It is concluded from these results that xTsg has ventralizing activity (Oelgeschlager, 2000).

To determine whether xTsg functions in the BMP pathway, epistatic analyses were performed by injecting xTsg mRNA into a ventral blastomere together with a dominant-negative BMP receptor (tBR) or extracellular BMP antagonists (noggin, chordin and CR1). xTsg had no effect on the formation of secondary axes by tBR, indicating that it may ventralize the embryo upstream of the BMP receptor. To test whether xTsg could antagonize the dorsalizing activity of the proteolytic fragments of Chordin, a construct was generated containing the N-terminal CR1 domain and terminating at the Xolloid cleavage site. CR1 mRNA induces secondary axes (although at 32-fold higher molar concentrations than those required for full-length chordin mRNA): this induction is blocked by coinjection of xTsg. Xolloid mRNA is able to block the activity of full-length chordin but has no effect on secondary axes induced by the CR1 construct. This indicates that xTsg efficiently antagonizes high doses of CR1 downstream of Xolloid cleavage. The ability of xTsg to inhibit full-length Chordin mRNA presumably results from the activity of uniformly expressed Xolloid proteases in the early embryo. That the ventralizing activity of xTsg requires Xolloid cleavage is confirmed by the observation that a CR1 construct containing 80 additional amino acids downstream of the first Xolloid site could not be antagonized by co-injection of either xTsg or Xolloid mRNAs. The ventralizing activity of xTsg appears to be specific for the Chordin/BMP pathway, since inhibition of BMP by noggin is not affected by co-injection of xTsg mRNA. These epistatic studies are consistent with a model in which xTsg would ventralize the embryo by antagonizing the residual anti-BMP activity of Chordin proteolytic cleavage products (Oelgeschlager, 2000).

When the N-terminal domain of xTsg is compared with the cysteine-rich domains of Chordin, sequence similarities are apparent. Because cysteine-rich domains are BMP-binding modules, a test was performed to see whether epitope-tagged xTsg secreted by transfected 293T cells could bind BMP-4. xTsg does bind to BMP-4 in solution, and this interaction is specific as it can be competed by BMP-2 but not by a 10-fold excess of platelet-derived growth factor, epidermal growth factor, Activin or transforming growth factor (TGF)-beta1. Chemical crosslinking was used to determine whether this molecular interaction is direct. The BMP-binding activity resides in the N-terminal region of xTsg and dTsg, that is, in the domain that shares sequence similarities with the BMP-binding modules of Chordin. It has been concluded that xTsg is a secreted BMP-binding protein that functions in embryos as an agonist of BMP signaling. The other secreted BMP-binding proteins identified, such as Chordin, Noggin, Follistatin and members of the Cerberus family, are antagonists of BMP activity (Oelgeschlager, 2000).

It was next tested whether Chordin and xTsg could compete for BMP binding. Full-length Chordin was immunoprecipitated in the presence of 0.5 nM BMP-4 and increasing amounts of xTsg protein. Instead of competing for the limited amounts of BMP-4, xTsg stimulates binding of BMP-4 to full-length Chordin. Further analyses have showen that xTsg itself can bind to Chordin even in the absence of added BMP. This binding has been confirmed using a cell-culture assay in which permeabilized cells transfected with xTsg cDNA were stained with a Chordin-alkaline phosphatase fusion protein. The binding of xTsg to Chordin requires intact xTsg, since neither the N- nor the C-terminal fragments suffice for the interaction. The observation that the N terminus is unable to bind Chordin but can still bind BMP-4 suggests that these two interactions occur through different sites (Oelgeschlager, 2000).

Crosslinking experiments have showen that xTsg stimulates the binding of BMP-4 to full-length Chordin by forming a trimolecular complex with a relative molecular mass of about 220,000. This shift is consistent with the crosslinking of one dimer of BMP-4 and one dimer of xTsg per Chordin monomer. To investigate the molecular mechanism by which xTsg can antagonize the activity of CR1, equimolar amounts (1 nM) of BMP-4, CR1 and xTsg were incubated for 1 h at room temperature before crosslinking. When CR1 and BMP-4 are incubated together, a complex of Mr 50K is formed, corresponding to the binding of a CR1 monomer to a BMP dimer. Incubation of xTsg and BMP-4 result in the formation of a complex of about 100K, corresponding to a dimer of xTsg bound to a dimer of BMP-4. Remarkably, when CR1, BMP-4 and xTsg are incubated together, only the [xTsg]2–[BMP]2 complex is formed. Furthermore, when BMP-4 and CR1 are preincubated for 1 h, BMP-4 can still be dislodged from these preformed complexes by addition of xTsg. It is concluded that, in contrast to full-length Chordin, CR1 bound to BMP-4 is released in the presence of xTsg, resulting in a binary complex of BMP-4 and xTsg (Oelgeschlager, 2000).

To investigate the effect of loss of function of endogenous xTsg, two complementary approaches were used. A construct secreting the C-terminal conserved domain (dn-xTsg) was found to have dominant-negative effects and double-stranded xTsg RNA (RNAi) was also found to interfere with endogenous xTsg function. xTsg RNAi was injected into each animal blastomere at the eight-cell stage together with epitope-tagged xTsg and CR1 mRNAs. RNAi inhibits the expression of secreted xTsg but not of CR1 protein, indicating that RNAi is effective and specific in Xenopus. Microinjection of dn-xTsg or of RNAi results in abnormal development of the postanal region, in particular loss of the ventral fin and shortening of the tail; this is only evident at swimming tadpole stages. At earlier stages of development, only mild defects in the perianal region are observed. However, both dn-xTsg mRNA and RNAi greatly potentiate the dorsalizing effects of low doses of CR1 mRNA injected into all blastomeres of four-cell embryos. These effects could be partially rescued by co-injection of wild-type xTsg mRNA, although the rescue is more effective for dn-xTsg than for xTsg RNAi (Oelgeschlager, 2000).

The Xenopus homolog of Drosophila Tsg is transcribed in the ventral-most region of the embryo. As described, overexpression of this secreted protein ventralizes the embryo through a molecular mechanism functioning upstream of the BMP receptor. Epistasis experiments support a model in which xTsg promotes BMP signaling downstream of Chordin cleavage by the metalloproteinase Xolloid. Chordin is abundantly secreted by the dorsal pole of the embryo, reaching concentrations of 6-12 nM in the extracellular space of Spemann's organizer. Chordin binds BMPs through cysteine-rich modules. Xolloid cleaves Chordin downstream of the two cysteine-rich domains that have the highest affinity for BMP binding. These proteolytic digestion products retain residual BMP-binding activity and can still inhibit the interaction between BMP and its cognate receptor. The results of this study show that xTsg binds BMP directly in the low nanomolar range and will compete effectively with a cysteine-rich module for binding to BMP, leading to the formation of xTsg–BMP complexes that permit BMP signaling. In this way, xTsg would promote BMP activity by dislodging the growth factor from an inhibitor in the extracellular space (Oelgeschlager, 2000).

The initial impetus to investigate whether xTsg binds BMP was provided by the observation that the N-terminal conserved domain shares amino-acid similarities with Chordin cysteine-rich repeats. This similarity was found to be functionally significant, since this domain contains the BMP-binding activity of xTsg. Cysteine-rich modules of the Chordin type are found in many extracellular proteins such as von Willebrand factor, thrombospondin, Nel and fibrillar procollagens. The cysteine-rich domain in procollagen IIA binds BMP and TGF-beta1 and is responsible for the dorsalizing activity of procollagen IIA in Xenopus. It is therefore possible that the molecular mechanism described here for xTsg may provide a more general paradigm for signaling in the extracellular space. Similar functions in the release of latent growth factors bound to cysteine-rich domains could be executed by other secreted proteins that share structural similarities with the N-terminal repeat of xTsg, such as members of the connective tissue growth factor (CTGF) and insulin-like growth factor-binding protein (IGFBP) families. In addition, the Cripto and Oep (one-eyed pinhead) proteins, which provide a permissive function for signaling by nodal TGF-beta family members in mouse and zebrafish, share sequence similarities to xTsg in their conserved EGF-like domains (Oelgeschlager, 2000).

In Drosophila, loss of function of Tsg results in the loss of amnioserosa, which requires maximal amounts of Dpp/Scw signaling, but does not affect the rest of the dorsal-ventral pattern. Overexpression of Drosophila Tsg driven by a promoter expressed in the ventral-most region of the embryo rescues dorsal amnioserosa formation in a Drosophila Tsg mutant background, indicating that the protein can diffuse throughout the embryo. Since Drosophila Tsg does not affect other dorsal-ventral fates in Drosophila, it was thought to provide a permissive signal required for amnioserosa differentiation after cells have been exposed to peak levels of Dpp/Scw signaling (Oelgeschlager, 2000 and references therein).

Drosophila Tsg can bind BMPs through its N-terminal domain. Drosophila Sog, although not yet demonstrated to bind Dpp/Scw/BMP in biochemical studies, is cleaved by Tolloid at three sites in the presence of BMP. Two of these cleavage sites are located at similar positions to those in Xenopus Chordin. It is proposed that Drosophila Tsg can act only in the amnioserosa because its function is to release active Dpp/Scw from the cleavage fragments of Sog by Tolloid. Peak signaling would be mediated by Dpp/Scw originating in more ventral regions and reaching the dorsal region by diffusion as a complex with Sog. Release would occur only in the dorsal-most regions in which maximal levels of Tsg, Tolloid, and fragments of Sog bound to BMP are prevalent. In more ventral regions, full-length free Sog would be in excess and would inhibit any released BMPs. This would be particularly true in the presence of Tsg in dorso-lateral regions, in view of the observation that xTsg increases the binding of BMP to full-length Chordin. This model has the attraction of explaining the paradoxical effects of Sog, which inhibits Dpp/Scw signaling in ventral ectoderm but promotes it in the most dorsal regions of the embryo (Oelgeschlager, 2000 and references therein).

By introducing Drosophila Tsg as an additional factor that permits the release of Dpp/Scw from inactive complexes in the amnioserosa, it is not necessary to invoke positive (that is, non-inhibitory) signaling effects of Sog proteolytic cleavage products to account for the Tolloid-dependent long-range effects of Sog diffusion on peak Dpp signaling (Oelgeschlager, 2000 and references therein).

One of the great surprises of developmental genetics has been the evolutionary conservation of molecular mechanisms. The best known example is the discovery that conserved sets of Hox genes determine the anterior–posterior axis in all bilateral animals. The activities and expression patterns of Dpp/BMP-4, Sog/Chordin and Tolloid/Xolloid have led to the view that the dorsal–ventral axis has been inverted in the course of evolution. Drosophila Tsg, a gene expressed in the dorsal-most blastoderm, has a vertebrate homolog expressed in the ventral-most tissues of the Xenopus embryo. xTsg is part of an intricate extracellular signaling pathway in which the two poles of the dorsal-ventral pattern are defined by sources of secreted Sog/Chordin and dTsg/xTsg on opposite sides of the embryo. The results are consistent with the idea that the ventral side of the arthropod is homologous to the dorsal side of the vertebrate (Oelgeschlager, 2000 and references therein).

BMP activity is controlled by several secreted factors including the antagonists chordin and Short gastrulation (Sog). A second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signaling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, Tsg and Sog together make a more effective inhibitor of BMP signaling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. Tsgs from different species are functionally equivalent, and it has been concluded that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signaling (Ross, 2001).

Since the phenotypes of tsg and sog mutants are similar, attempts were made to determine whether Tsg can enhance the binding of Sog to ligand. Co-immunoprecipitation of DPP by Sog is greatly enhanced when these two factors are coexpressed in S2 cells along with Tsg. To test whether the combination of Sog and Tsg blocks Dpp signaling better than Sog alone, an S2 cell-culture assay was developed for Dpp signaling. At high concentration Tsg alone can block Dpp signaling; however, at lower concentration, the combination of Tsg and Sog together dramatically reduces the Dpp-dependent accumulation of P-MAD much more efficiently than either can alone. In vivo overexpression of sog and tsg together can completely reverse the phenotype of ectopic dpp expression in the wing, whereas the expression of either alone has no effect. It is concluded that a complex of Tsg and Sog is an efficient antagonist of Dpp signaling (Ross, 2001).

To determine whether Tsg is conserved among other species, genes in the database related to Drosophila Tsg were sought and found in human, mouse, zebrafish and Xenopus. In addition, a second tsg-related sequence was found in Drosophila (tsg2) and a second zebrafish tsg (tsg1) was obtained using degenerate polymerase chain reaction (PCR) methods. The protein products show extensive similarity with about 50% of 202 amino-acid residues matching in all four species. The pairs of tsg genes in fly and fish are closer to each other than to tsg in any other species, suggesting independent gene-duplication events in these two species. The human, mouse and zebrafish (tsg1) genes were mapped by a combination of fluorescence in situ hybridization (FISH) or radiation hybrid mapping. The mouse gene maps to 17E1.3–E2, a region that is syntenic to 18p11.2–3 where the human homologue resides. In zebrafish, tsg1 is located at linkage group 24-74.5, which is syntenic to the human locus and indicates that all three genes are probably functional orthologues (Ross, 2001).

The zebrafish tsg1 gene is expressed uniformly in early embryos, whereas zebrafish tsg2 is only expressed at later stages. Hence, the analysis was focused on zebrafish tsg1 and morpholino oligonucleotides were used to reduce the function of this gene in early zebrafish development. Injection of a tsg1 morpholino oligonucleotide (ztsg-MO: see Taylor, 1996 for a description of the morpholino modification) produces a phenotype characteristic of expanded BMP signaling. Using morphological criteria and fluorescent red blood cells, it was found that embryos develop expansions of the ventral fin region that correspond to ectopic blood islands, a tissue derived from ventral mesoderm. Injected embryos also show an expansion of GATA2, loss of paraxial mesoderm (visualized with the marker myoD), and a mild reduction of anterior ectodermal tissues (detected by staining for krox20). Caudal expression of bmp4 is also expanded in these embryos, while the anterior ectodermal marker otx2 is reduced. Treated embryos also exhibit an expansion in apoptotic cells ventral to the yolk extension, similar to dino and mercedes mutants. Overall, this phenotype is very similar to that of ogon/mercedes mutants and moderate chordin loss-of-function mutants, and represents a modest ventralized phenotype (Ross, 2001).

Since Drosophila data suggest that one function of Tsg is to cooperate with Sog to inhibit BMP signaling, it was asked whether the same relationship is true in vertebrates by determining whether a modest reduction of zebrafish chordin activity can enhance the effect of a moderate reduction in tsg1 activity. Sub-inhibitory levels of a zebrafish chordin morpholino oligonucleotide and tsg1-MO were injected into wild-type embryos, and the effect on ectopic blood island development was scored. These two morpholino oligonucleotides synergistically enhance blood island expansion, supporting the view that both of these gene products co-operatively inhibit BMP signaling. As with the Drosophila components, it was found that the combination of purified mouse chordin and Tsg is better able to inhibit mouse BMP-stimulated phosphorylation of Mad in S2 cells than either can alone (Ross, 2001).

A test was performed for synergy between Tsg and chordin mRNA in Xenopus embryos by co-injecting their mRNAs and scoring for enhancement of secondary axis formation. Co-injection of Xenopus Tsg and chordin reveals a dose-response optimum. When a sub-inhibitory dose of chordin mRNA is supplemented with increasing levels of Tsg mRNA, the fraction of embryos exhibiting a secondary axis increases up to 4.5-fold over chordin alone at a 1/5 ratio of Tsg/chordin mRNA. However, if the Tsg/chordin ratio is increased to 1:1 or higher, the number of secondary axes is reduced to basal levels and the resulting tadpoles have normal morphology. Injection of 150 pg Tsg alone (the highest concentration of Tsg mRNA used in these experiments) has no effect on embryonic development. Notably, if the level of Tsg relative to chordin is increased in the S2 experiments, no reversal of the inhibition phenotype is seen, suggesting that additional factors probably modulate the in vivo response. Taken together, it is concluded that, like Drosophila Tsg, vertebrate Tsg can co-operate with chordin to inhibit BMP signaling (Ross, 2001).

As a final test of the functional equivalence of the vertebrate and invertebrate tsg genes, the human and mouse genes were expressed under the control of the UAS promoter in flies, and Drosophila Tsg mRNA was injected into zebrafish embryos. The phenotype of animals expressing human Tsg and Drosophila sog in wing discs resembles that of dpp shortvein alleles and is very similar to that produced by coexpression of the Drosophila tsg and sog genes. When injected into zebrafish, Drosophila tsg produces a dorsalized phenotype equivalent to that produced by zebrafish tsg1, which includes reduced axial length and expansion of krox20 (Ross, 2001).

These experiments suggest that Tsg has three molecular functions. (1) It can synergistically inhibit Dpp/BMP action in both Drosophila and vertebrates by forming a tripartite complex between itself, Sog/chordin and a BMP ligand. (2) Tsg seems to enhance the Tld/BMP-1-mediated cleavage rate of Sog/chordin and may change the preference of site utilization. (3) Tsg can promote the dissociation of chordin cysteine-rich (CR)-containing fragments from the ligand. Different organisms may exploit each of these properties to different degrees during development depending on the relative in vivo concentrations of each molecule. It is proposed that in Drosophila and zebrafish the primary function of Tsg is to form a tripartite complex between itself, Sog/chordin and a BMP ligand. In Drosophila, this complex acts to redistribute a limiting amount of Dpp, such that activity is elevated dorsally at the expense of being lowered laterally. The net driving force for this redistribution is likely to be diffusion of Sog from its ventral source of synthesis. This is consistent with the finding that Sog diffusion is essential for activation of genes such as race that require high levels of Dpp/SCW signaling. In this model Tld would serve to modulate both the net movement of Dpp and its release from the inhibitory complex by cleaving Sog. The ability of Tsg to enhance the rate of Sog cleavage may also be an important aspect of this model in that it helps ensure the proper timing of these rapid developmental events. It seems unlikely that Tsg is needed to remove an inhibitory CR-containing fragment from Dpp, since the affinity of full-length Sog for Dpp in the absence of Tsg seems to be low. Likewise, in zebrafish the phenotype of reduced Tsg function is ventralized and not dorsalized as would be predicted if Tsg were primarily needed to release inhibitory CR fragments from ligand. In Xenopus, however, perhaps the endogenous levels of full-length chordin and CR fragments are higher than in zebrafish, thereby making the CR displacement activity of Tsg the more important biological function. Determination of the in vivo levels of these proteins, along with a more careful analysis of the concentration optima for each type of reaction involving Tsg function, will be required before all of its in vivo activities can be understood (Ross, 2001).

The characterization of the vertebrate Tsg homologs is reported. Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. These data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis (Chang, 2001).

Human Twisted gastrulation (TSG) was isolated in a screen for secreted factors, and mouse and Xenopus Tsg were isolated by low-stringency hybridization using human TSG as the probe. These vertebrate Tsgs have a high sequence homology to one another (more than 80% identical) and are about 30% identical to Drosophila Tsg at the amino-acid level. Tsg is expressed maternally and in all developmental stages in Xenopus, and at least from gastrula stages onward in mouse. Expression of Tsg is also detected in a variety of adult tissues in both mouse and human (Chang, 2001).

To study the function of Tsgs, their activities were examined in Xenopus ectodermal explants (animal caps). Human, mouse and Xenopus Tsg induce the cement gland and the neural markers XAG-1, OtxA and NRP-1 with comparable efficiency, suggesting that these vertebrate Tsgs function similarly in Xenopus. The induction of cement gland and neural markers in animal caps in the absence of mesoderm is normally associated with inhibition of the BMP signaling, so whether Tsg could directly block the activity of BMP was addressed. The effect of Tsg on ventralization of the ectodermal cells by BMPs was examined. Intact animal caps express high levels of epidermal keratin. This expression is suppressed when caps from blastula stages are dissociated for 4 h. Cement gland and neural markers are turned on in these dissociated samples. While Bmp2 restores the transcription of epidermal keratin and inhibits the expression of neural markers, it cannot do so in the presence of Tsg. These results indicate that Tsg directly antagonizes the neural inhibition and epidermal induction activity of BMPs in dissociated animal caps (Chang, 2001).

The effect of Tsg on ventralization of the mesodermal explants by BMPs was examined. Bmp4 inhibits dorsal and induces ventral gene expression in dorsal marginal zone (DMZ) explants. Coexpression of Xenopus Tsg with Bmp4 re-establishes dorsal marker expression and reduces the ventral gene induction in these explants, indicating that it also inhibits BMP activity in the mesodermal cells. Notably, a reduction in the level of OtxA (a marker for anterior neural tissue at tailbud stages) was detected when Tsg alone was expressed in the DMZ. This phenotype has not been observed with other BMP antagonists, and suggests that Tsg may be involved in processes other than inhibition of BMP signaling. To determine whether Tsg also blocks other signal transduction pathways, the effect of Xenopus Tsg on marker induction by activin, basic fibroblast growth factor (FGF) and Wnt8 was examined. Tsg specifically inhibits BMP-dependent mesoderm induction, but does not interfere with the expression of the markers induced by the other signaling molecules. These results demonstrate that Tsg is specific for a BMP pathway and does not block activin, FGF or Wnt signaling (Chang, 2001).

The in vivo function of Tsg was examined by gain-of-function studies. Injection of Tsg RNAs from different vertebrates in early embryos induces a similar phenotype, indicating that they have similar activities in vivo. The embryos injected with Tsg RNA exhibit several developmental defects at tadpole stages, such as an enlargement of the dorsal fin, malformation of the proctodeum, and a reduction of the head. Lineage tracing with nuclear ß-gal shows that the effect induced by Tsg is non-cell autonomous. The phenotype of Tsg-expressing embryos is unique and does not resemble the phenotypes induced by overexpression of other BMP antagonists. Therefore, whether Tsg affects the cells and tissues that rely on the BMP signaling was addressed. The formation of the blood cells, which are derived from ventral tissues and require active BMP signals, was examined. In control embryos, the blood cells stained with benzidine were localized at the ventral side of the embryos; however, this staining was not observed in embryos injected with Xenopus Tsg RNA. Furthermore, in situ hybridization with a blood-specific anti-T1-globin probe reveals that blood cells are absent in the Tsg-expressing embryos. The formation of the heart, which has been reported in both the chick and the frog to require BMP signaling, was examined. In situ hybridization with a heart marker, Nkx2.5, shows that the Tsg-expressing embryos have reduced transcription of Nkx2.5. In contrast, the expression of a dorsal mesoderm marker in muscle is not decreased by Tsg. These data show that Tsg interferes with the development of several tissues that require BMP signaling (Chang, 2001).

To further analyse the effects of Tsg, expression of both dorsal and ventral genes was assayed in Tsg-injected embryos at different developmental stages. In gastrula embryos, several ventral markers, such as Xhox3, Vent1 and Msx1, are downregulated by Xenopus Tsg, whereas dorsal markers, including Sox2, goosecoid and Hex, are not much affected. The expression of the dorsal gene chordin also remains the same in most cases, although a slight reduction of its level was occasionally seen. The pattern of gene expression changes during development, so that at later stages, Xhox3 and Msx1 are also expressed in the neural tissue and neural crest cells. The reduction of their expression is less profound, and at tailbud stages Xhox3 expression is normal. In agreement with whole-mount staining results, the transcripts of both globin and Nkx2.5 are reduced by Tsg, whereas the muscle actin remains intact at tailbud stages. Unexpectedly, the reduction of several anterior genes, such as Hex (a marker for anterior endoderm) and goosecoid (a prechordal mesoderm gene), by Xenopus Tsg is seen from neurula stages onward. Sox2, whose expression domain includes the anterior neural region, is also slightly downregulated. These data demonstrate that Tsg reduces BMP signaling at early developmental stages; furthermore, Tsg may have one or more additional functions in anterior embryonic development at a stage after the onset of gastrulation (Chang, 2001).

Although Tsg can inhibit BMP function, it does not induce a partial secondary axis when injected into the ventral side of early Xenopus embryos. One possible explanation for this is that Tsg may be a highly diffusible molecule, as has been observed in Drosophila, and does not accumulate to high concentrations at the ventral side to establish an organizer-like activity. To test this hypothesis, a membrane-tethered form of Tsg was constructed by fusing the full-length Xenopus Tsg in frame with an integral membrane protein CD2. The chimaeric protein, Tsg-CD2, induces cement gland and neural markers in animal caps to the same extent as wild-type Xenopus Tsg, suggesting that the two proteins have similar activities. Injection of the CD2 RNA into early frog embryos does not induce any phenotype, but ventral injection of Tsg-CD2 RNA leads to embryos with partial secondary axes. These results indicate that localized Tsg behaves like other BMP antagonists and can induce ectopic dorsal structures on the ventral side of frog embryos (Chang, 2001).

To investigate whether Tsg modifies the BMP inhibitory activity of chordin, the effect of coexpression of chordin and Xenopus Tsg in both ectodermal explants and whole embryos was examined. In animal caps, a low dose of either chordin or Tsg does not efficiently block Brachyury (Bra) induction by Bmp4; coexpression of both genes leads to a more effective inhibition of the BMP activity. In whole embryos, chordin induces a partial secondary axis when expressed at the ventral side; coexpression with Xenopus Tsg at a ratio of Tsg/chordin below 1/1 does not prevent the ectopic axis-induction by chordin. Instead, many embryos show a dorsalized phenotype with reduced trunk and tail. Notably, with an increasing Tsg/chordin ratio (2/1 or higher), the secondary axis disappears in an increasing number of embryos, and these embryos resume a typical Tsg phenotype. Tsg and chordin still inhibit Bra induction by Bmp4 in animal caps at these high ratios. The data suggest that although Tsg and chordin expression together lead to a more efficient BMP inhibition in ectodermal explants regardless of the amounts of RNA injected, the in vivo phenotypes depend on the relative abundance of the two genes. The exact mechanism underlying this observation remains unclear. There are at least two possibilities that are not mutually exclusive. The highly diffusible Tsg may help to diffuse the chordin protein through complex formation, which may result in dilution of the chordin function at the ventral side and inhibition of the secondary axis induction by chordin. Alternatively, other endogenous factors may interact with chordin and/or Tsg and modify their activities and the phenotypes of coexpression of chordin and Tsg (Chang, 2001).

Tsg, first identified in Drosophila and shown to have a function in development of the dorsal midline, was originally proposed to potentiate DPP activity. This hypothesis, however, has recently been challenged by the observation that a processed SOG product, which has a broader inhibitory spectrum towards BMP members, rescues the tsg mutant phenotype, whereas Dpp does not. This result indicates that Tsg may participate in alternative processing of Sog to generate a 'supersog' to block the DPP signaling. Tsg may therefore be involved in inhibiting, rather than enhancing, the Dpp activity. A similar mechanism suggesting that Tsg may participate in inhibition of the BMP signaling has now been proposed to also work in vertebrates. Tsg stimulates chordin cleavage at a unique site, and Tsg enhances chordin activity in both Xenopus and zebrafish. This study shows that Tsg can function as a BMP antagonist in embryonic explants and interfere with BMP-dependent tissue formation in frog embryos. The activity of Tsg, however, is different from that of other BMP antagonists, since Tsg also induces defects in the anterior tissues, such as the anterior endoderm expressing the Hex gene and the prechordal mesoderm that expresses goosecoid. This phenotype may underlie the recent interpretation that vertebrate Tsg acts to promote BMP signaling, since it has been reported that elevated BMP expression leads to reduction of dorsal markers at the gastrula stages, which results in truncation of anterior and dorsal tissues at late stages. It is currently unclear, however, whether the defects induced by TSG truly reflect an enhancement of BMP signaling. Although the possibility cannot be ruled out that Tsg can function both as a BMP agonist and antagonist depending on the presence or absence of other factors in specific regions of the embryos at particular developmental stages, it is also possible that the late effects are still mediated by inhibition of a BMP pathway. Several BMPs are expressed in the anterior endoderm and/or prechordal mesoderm in chick and fish, and in chick, BMPs may be involved in specification of prechordal mesoderm. It is possible that Tsg inhibits a different spectrum of BMPs from other BMP antagonists, either alone or by alteration of chordin specificity, thereby leading to the unique phenotype in the anterior tissues. Another possibility is that Tsg may have one or more additional functions independent of BMP signaling. Further investigation is required to understand the in vivo function of Tsg (Chang, 2001).

Twisted gastrulation (Tsg) is a secreted protein that regulates Bmp signaling in the extracellular space through its direct interaction with Bmp/Dpp and Chordin (Chd)/Short gastrulation (Sog). The ternary complex of Tsg/Chd/Bmp is cleaved by the metalloprotease Tolloid (Tld)/Xolloid (Xld). Studies in Drosophila, Xenopus and zebrafish suggest that Tsg can act both as an anti-Bmp and as a pro-Bmp. Tsg loss-of-function was analyzed in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. Evidence is provided that Tsg interacts genetically with Bmp4. When only one copy of Bmp4 is present, a requirement of Tsg for embryonic development is revealed. Tsg-/-;Bmp4+/- compound mutants die at birth and display holoprosencephaly, first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development (Zakin, 2004).

DPP homologs: Transcriptional regulation

Recent experiments in the Xenopus embryo suggest that proper regulation of BMP-4 signaling is critical to the dorsal ventral specification of both mesoderm and ectoderm. Regulation of BMP-4 signaling is known to occur extracellularly by direct binding with chordin, noggin, and follistatin, and intracellularly through the antagonistic signal interaction with dorsalizing TGF-beta family member activin. However, tight repressional regulation of BMP transcription may also be required to sustain the dorsal and neural status of the induced cells. Both a dominant negative mutant of the BMP receptor (DN-BR) or application of BMP-4 antagonizers, chordin and noggin, negatively regulate BMP-4 transcription in animal cap explants. It is suggested that repression of BMP-4 transcription is important in the maintenance of dorsal fate and that continuous input of BMP-4 signaling is required to sustain the expression of BMP-4 transcription in the maintenance of epidermal/ventral fate. Consistent with this postulation, the promoter region of the isolated BMP-4 genomic DNA is found to include several consensus binding sites for transcriptional regulators functioning under BMP-4 signaling, such as GATA binding and ventralizing homeobox genes. In a functional assay the GATA binding and ventral homeobox proteins were found to positively modulate BMP-4 promoter activity. DN-BR decreases BMP-4 promoter activity. This is likely due to a repression of the above-mentioned transcription factors (Kim, 1998).

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, the mechanism underlying the induction of Bmp2 was investigated. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicate that RA stimulates the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicate that Bmp2 transcription starts 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation is detected between base pairs -2,373 and -2,316 relative to the translation start site. A required Sp1 binding site was identified between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1 (Heller, 1999).

Dorsoventral specification of the zebrafish gastrula is governed by the functions of the dorsal shield, a region of the embryo functionally analogous to the amphibian Spemann organizer. The bozozok locus encodes the transcription factor nieuwkoid/dharma, a homeobox gene with non-cell-autonomous organizer-inducing activity. The nieuwkoid/dharma gene, most closely related to Drosophila Gooseberry distal (56% homology throughout the homeodomain), is expressed prior to the onset of gastrulation in a restricted region of an extraembryonic tissue, the yolk syncytial layer, that directly underlies the presumptive organizer cells. A single base-pair substitution in the nieuwkoid/dharma gene results in a premature stop codon in boz(m168) mutants, leading to the generation of a truncated protein product that lacks the homeodomain and fails to induce a functional organizer in misexpression assays. Embryos homozygous for the boz(m168) mutation exhibit impaired dorsal shield specification often leading to the loss of shield derivatives, such as prechordal plate in the anterior and notochord in the posterior, along the entire anteroposterior axis. Furthermore, boz homozygotes feature a loss of neural fates anterior to the midbrain/hindbrain boundary. Characterization of homozygous mutant embryos using molecular markers indicates that the boz ventralized phenotype may be due, in part, to the derepression of a secreted antagonizer of dorsal fates, zbmp2b, on the dorsal side of the embryo prior to the onset of gastrulation. Furthermore, ectopic expression of nieuwkoid/dharma RNA is sufficient to lead to the down regulation of zbmp2b expression in the pregastrula. Based on these results, it is proposed that gastrula organizer specification requires the Nieuwkoop center-like activity mediated by the nieuwkoid/dharma/bozozok homeobox gene and that this activity reveals the role of a much earlier than previously suspected inhibition of ventral determinants prior to dorsal shield formation (Koos, 1999).

The Cubitus interruptus (Ci) and Gli proteins are transcription factors that mediate responses to Hedgehog proteins (Hh) in flies and vertebrates, respectively. During development of the Drosophila wing, Ci transduces the Hh signal and regulates transcription of different target genes at different locations. In vertebrates, the three Gli proteins are expressed in overlapping domains and are partially redundant. To assess how the vertebrate Glis correlate with Drosophila Ci, each was expressed in Drosophila and their behaviors and activities were monitored. Each Gli has distinct activities that are equivalent to portions of the regulatory arsenal of Ci. Gli2 and Gli1 have activator functions that depend on Hh. Gli2 and Gli3 are proteolyzed to produce a repressor form able to inhibit hh expression. However, while Gli3 repressor activity is regulated by Hh, Gli2 repressor activity is not. These observations suggest that the separate activator and repressor functions of Ci are unevenly partitioned among the three Glis, yielding proteins with related yet distinct properties (Aza-Blanc, 2000).

Although in aggregate the Gli proteins appear to embody the many different attributes of Ci, only some of the Ci activities are in each. Most intriguing, perhaps, is the differential activation of ptc and dpp expression by Gli1 and Gli2, respectively. The basis for the selectivity of Gli1 for ptc and Gli2 for dpp is not understood, but it has many conceivable causes. One is that Gli2 interacts with proteins known to associate with Ci, such as CBP, but that Gli1 does not. Alternatively, the ability of Gli2 to activate dpp more strongly could be related to the conversion of Gli2 to a repressor form. It is formally possible that the activator and repressor forms can cooperate in some manner to enhance dpp transcription, or that the repressor form competes with the activator for binding sites at the ptc promoter. Consistent with this latter proposal, the level of ptc induction in wing discs is inversely related to the level of Gli2 expression: higher levels of expression produce lower levels of ptc. Since Ci75 is abundant in A cells that express high levels of dpp, but it is not in cells closer to the compartment border where ptc is expressed, this model may be relevant to Ci. Perhaps the most interesting possibility to consider is that the reason for the differential activation of dpp and ptc may be that Gli1 and Gli2 represent different forms of Ci Act, one with a preference for ptc and the other for dpp (Aza-Blanc, 2000).

The Snail family of genes comprises a group of transcription factors with characteristic zinc finger motifs. One of the members of this family is the Slug gene. Slug has been implicated in the development of neural crest in chick and Xenopus by antisense loss of function experiments. Functional derivatives of Xslug have been generated by constructing cDNAs that encode the Xslug protein fused with the transactivation domain of the virus-derived VP16 activator or with the repressor domain of the Drosophila Engrailed protein. The results suggest that Xslug normally functions as a transcriptional repressor and that Xslug-VP16 behaves as a dominant negative of Xslug. Xslug functions by controlling its own transcription. Xslug is expressed in the dorsal mesendoderm at the beginning of gastrulation, where is it able to upregulate the expression of dorsal genes. When Xslug is expressed outside of the organizer it represses the expression of ventral genes. These results indicate that this effect on mesodermal patterning depends on BMP activity, showing that Xslug can directly control the transcription of BMP-4 (Mayor, 2000).

The most novel finding of this study is the early function that Xslug has in mesendoderm development. Xslug is expressed in the organizer region and is able to control the expression of organizer genes such as chordin, cerberus and other dorsal mesodermal genes such as goosecoid, pintallavis and Frzb. That Xslug is required for the normal development of the embryo is shown by the injection of Xslug-VP16; these embryos lack a head. This phenotype is probably explained by the inhibition of the expression of some dorsal genes, particularly cerberus, which is known to be involved in head development. The lack of ectopic head structures and a proper secondary axis after Xslug injection could be explained by the fact that the level of cerberus induced ectopically by Xslug overexpression is not sufficient to activate the cascade required by the organizer activity (Mayor, 2000).

When Xslug is expressed in ventral mesoderm it induces dorsal genes and represses ventral genes, BMP-4 being one of these ventral genes. However, when Xslug-VP16 RNA is injected in dorsal blastomeres, dorsal genes are inhibited while no expression of ventral mesodermal genes is observed. This can be explained by the requirement of BMP-4 for the expression of ventral markers (Xvent-1, Xwnt-8), and as BMP-4 is not expressed in the dorsal mesoderm no ventral marker can be induced there. Interestingly, these results show that Xslug is able to block BMP transcription, and as a consequence, Xvent-1, a gene downstream of BMP, is blocked by Xslug mRNA injection. In addition, the effect of Xslug-VP16 can be rescued only by co-injection of BMP mRNA but not with Xwnt-8. The inhibition of Xwnt-8 caused by Xslug overexpression could also be a consequence of the interference with BMP-4 transcription, since a dominant negative BMP-4 receptor suppresses Xwnt-8 expression. Taken together these results indicate that Xslug could be a repressor of BMP in dorsal mesoderm. Thus, when it is ectopically expressed in ventral mesoderm the downregulation of BMP leads to an upregulation of dorsal genes such as chordin or cerberus (Mayor, 2000).

Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-beta genes, the potential of Xenopus c-Jun (AP-1) as a mediator of BMP-4 expression during Xenopus development was analyzed. RNA injection experiments reveal that both heteromeric c-Fos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling activates the Xenopus c-Jun gene only to a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP-1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, a spatial overlap of c-Jun and BMP-4 transcripts was found which suggests that AP-1 might serve as an additional activatory component for the auto-regulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5' flanking region and two in the second intron of the BMP-4 gene. AP-1 acts independently of Xvent-2, which mediates the early expression of BMP-4 in gastrula stage embryos. In summary, it has been shown both for the wild type gene and for promoter/reporter constructs that the Xenopus BMP-4 gene is activated by c-Jun (AP-1). Corresponding target sites are localized in the 5' flanking region and in the second intron. Results obtained from biological and molecular investigations lead to the conclusion that c-Jun (AP-1) is a transcriptional activator of the BMP-4 gene and, since the c-Jun gene is weakly activated by BMP signaling, c-Jun is another potential mediator of the BMP-4 auto-regulatory loop (Knochel, 2000).

In the early Xenopus embryo, the Xiro homeodomain proteins of the Iroquois (Iro) family control the expression of proneural genes and the size of the neural plate. Xiro1 functions as a repressor that is strictly required for neural differentiation, even when the BMP4 pathway is impaired. Xiro1 and Bmp4 repress each other. Consistently, Xiro1 and Bmp4 have complementary patterns of expression during gastrulation. The expression of Xiro1 requires Wnt signaling. Thus, Xiro1 is probably a mediator of the known downregulation of Bmp4 by Wnt signaling (Gomez-Skarmeta, 2001).

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein kinases is stimulated in response to a wide array of cellular stresses and proinflammatory cytokines. Mice lacking individual members of the Jnk family (Jnk1, Jnk2, and Jnk3) are viable and survive without overt structural abnormalities. Mice with a compound deficiency in Jnk expression can survive to birth, but fail to close the optic fissure (retinal coloboma). JNK initiates a cytokine cascade of bone morphogenetic protein-4 (BMP4) and sonic hedgehog (Shh) that induces the expression of the paired-like homeobox transcription factor Pax2 and closure of the optic fissure. BMP4 is under the control of JNK. In vitro studies using retinal explant cultures indicate that the function of BMP4, in part, is to induce the expression of Shh in a cytokine cascade that leads to the expression of the paired-like homeobox transcription factor Pax2. Interestingly, the role of JNK to regulate BMP4 expression during optic fissure closure is conserved in Drosophila during dorsal closure, a related morphogenetic process that requires JNK-regulated expression of the BMP4 ortholog Decapentaplegic (Weston, 2003).

BMP4 has been implicated in the regulation of Shh expression in the mouse. To test whether BMP4 regulates the expression of Shh and Pax2 in the eye, retinal explant cultures were examined. Consistent with in vivo results, strong expression of both Shh and Pax2 was detected in control retinas, but not in mutant retinas. When the mutant retinas were cultured in the presence of BMP4 for 48 h, induced expression of both Shh and Pax2 was detected, indicating that BMP4 is sufficient to cause expression of Shh and Pax2, and that the BMP4-Shh-Pax2 pathway is intact in the JNK-deficient mutant embryonic eyes. In contrast, no BMP4-stimulated expression of Shh or Pax2 in the mutant retinas was detected in the absence or presence of an antagonistic antibody to Shh. Similarly, when control retinas were cultured in the presence of BMP4 plus the antagonistic antibody to Shh, there was a dramatic decrease in both Shh and Pax2 expression. These data imply that BMP4 induces the expression of Shh and Pax2 in mutant retinas, and that Shh is upstream of Pax2 expression. This signaling cascade is initiated by JNK and is absent in JNK-deficient retinas (Weston, 2003).

It is striking that the effects of Pax2 deficiency are similar to those caused by JNK deficiency. For example, both of these mutations cause failure of optic fissure closure (coloboma) and renal epithelial cell necrosis. Furthermore, both mutations alter the expression of Shh at the basis of the diencephalon in E9.5 embryos. These similar phenotypes are most likely accounted for by the observation that Pax2 expression is markedly reduced in the eyes and kidney epithelium of JNK-deficient mice. A further contributing factor may be that JNK can phosphorylate Pax2. However, because the level of Pax2 mRNA and protein in JNK-deficient eyes is extremely low, the role of altered Pax2 phosphorylation is unclear (Weston, 2003).

Although it is well established that Six3 is a crucial regulator of vertebrate eye and forebrain development, it is unknown whether this homeodomain protein has a role in the initial specification of the anterior neural plate. Exogenous Six3 can expand the anterior neural plate in both Xenopus and zebrafish, and this occurs in part through Six3-dependent transcriptional regulation of the cell cycle regulators cyclinD1 and p27Xic1, as well as the anti-neurogenic genes Zic2 and Xhairy2. However, Six3 can still expand the neural plate in the presence of cell cycle inhibitors and this is likely to be due to its ability to repress the expression of Bmp4 in ectoderm adjacent to the anterior neural plate. Furthermore, exogenous Six3 is able to restore the size of the anterior neural plate in chordino mutant zebrafish, indicating that it has the ability to promote anterior neural development by antagonising the activity of the BMP pathway. On its own, Six3 is unable to induce neural tissue in animal caps, but it can do so in combination with Otx2. These results suggest a very early role for Six3 in specification of the anterior neural plate, through the regulation of cell proliferation and the inhibition of BMP signalling (Gestri, 2005).

To elucidate whether Bmp4 and Xsix3 might antagonise each other, the effects that the overexpression of each of these genes exert on the other were analyzed. Bmp4 overexpression leads to a strong reduction of Xsix3 expression. Conversely, interfering with BMP signalling by injection of either tBR, a dominant-negative BMP receptor, or chordin mRNA induces a strong activation of Xsix3 both in animal caps and in the anterior neural plate of the embryo. Conversely, both VP16-Xsix3 and MoXsix3 injection leads to expansion of Bmp4 expression in the presumptive anterior neural plate. Additionally, TUNEL analysis shows that both Bmp4- and VP16-Xsix3-injected embryos display an anterior accumulation of apoptotic nuclei (Gestri, 2005).

To analyse whether the effects of Xsix3 loss of function are a consequence of BMP4 expansion in the anterior neural plate, whether interfering with BMP signalling can counteract the reduction of the anterior neural plate in MoXsix3-injected embryos was examined. To achieve this, the expression of Zic2 (a gene expressed both in the anterior and posterior neural plate that is strongly modulated by Xsix3), was examined in MoXsix3/tBR co-injected embryos. Injection of MoXsix3 alone represses anterior Zic2 expression. Conversely, MoXsix3/tBR co-injected embryos showed a complete or partial rescue of the Zic2 expression domain. None of the co-injected embryos showed the strong expansion of Zic2 seen for tBR alone. As a control, a similar rescue is observed when MoXsix3 is co-injected with Xsix3. Taken together, these results indicate a mutual antagonism between Xsix3 and Bmp4 (Gestri, 2005).

Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. It was hypothesized that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. The present study shows that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation (Zhao, 2006; full text of article).

The first vasculature of the developing vertebrate embryo forms by assembly of endothelial cells into simple tubes from clusters of mesodermal angioblasts. Maturation of this vasculature involves remodeling, pruning and investment with mural cells. Hedgehog proteins are part of the instructive endodermal signal that triggers the assembly of the first primitive vessels in the mesoderm. A combination of genetic and in vitro culture methods was used to investigate the role of hedgehogs and their targets in murine extraembryonic vasculogenesis. Bmps, in particular Bmp4, are crucial for vascular tube formation, Bmp4 expression in extraembryonic tissues requires the forkhead transcription factor Foxf1 (Drosophila homolog: Biniou), and the role of hedgehog proteins in this process is to activate Foxf1 expression in the mesoderm. In the allantois. genetic disruption of hedgehog signaling (Smo-/-) has no effect on Foxf1 expression, and neither Bmp4 expression nor vasculogenesis are disturbed. By contrast, targeted inactivation of Foxf1 leads to loss of allantoic Bmp4 and vasculature. In vitro, the avascular Foxf1-/- phenotype can be rescued by exogenous Bmp4, and vasculogenesis in wild-type tissue can be blocked by the Bmp antagonist noggin. Hedgehogs are required for activation of Foxf1, Bmp4 expression and vasculogenesis in the yolk sac. However, vasculogenesis in Smo-/- yolk sacs can be rescued by exogenous Bmp4, consistent with the notion that the role of hedgehog signaling in primary vascular tube formation is as an activator of Bmp4, via Foxf1 (Astorga, 2006).

Translational regulation of BMPs

Activation of the Xenopus bone morphogenetic protein (BMP) pathway is coincident with the onset of zygotic transcription but requires maternal signaling proteins. The mechanisms controlling the translation of mRNAs that encode proteins of the BMP pathway were investigated by using polysome association as an assay for translational activity. Five different mRNAs encoding proteins of the BMP pathway are translationally regulated during Xenopus development. These mRNAs are either not associated or inefficiently associated with polysomes in oocytes, and each is recruited to polysomes at a different developmental stage. The Smad1 and ALK-2 mRNAs are recruited to polysomes during oocyte maturation, whereas the BMP-7 and XSTK9 mRNAs are recruited during the early stages of embryogenesis. The ALK-3 mRNA is not efficiently associated with polysomes during any maternal stage of development and is efficiently recruited to polysomes only after the onset of zygotic transcription. In general, for all stages except oocytes, polysome recruitment is associated with the presence of a 3' poly(A) tail. However, there is not an obvious correlation between the absolute length of poly(A) and the efficiency of polysome recruitment, indicating that the relationship between poly(A) tail length and translation during early frog embryogenesis is complex. Sequence elements within the 3'UTR of BMP-7 mRNA are necessary for recruitment to polysomes and sufficient to direct the addition of poly(A) and activate translation of a reporter during embryogenesis. Interestingly, the BMP-7 mRNA lacks the previously defined eCPE sequences proposed to direct poly(A) addition and translational activation during embryogenesis (Fritz, 2001).

BMPs in C. elegans

The dbl-1 gene, a C. elegans homolog of Drosophila decapentaplegic and vertebrate BMP genes, has been cloned. Loss-of-function mutations in dbl-1 cause markedly reduced body size and defective male copulatory structures. Conversely, dbl-1 overexpression causes markedly increased body size and partly complementary male tail phenotypes, indicating that DBL-1 acts as a dose-dependent regulator of these processes. Evidence from genetic interactions indicates that these effects are mediated by a Smad signaling pathway, for which DBL-1 is a previously unidentified ligand. Expressing cells in the ventral cord appear to be of the DA, DB, VA and VB classes, throughout the entire length of the cord. Although several pharyngeal neurons express dbl-1 reporters, there is so far no phenotypic indication of a role for dbl-1 in the pharynx. In the male tail, the reporters are expressed, as in the hermaphrodite, in the DVA neuron and also in the glial cells that form the sockets for the spicules starting from the L4 stage. This study of the dbl-1 expression pattern suggests a role for neuronal cells in global size regulation as well as male tail patterning. In the male tail, although DBL-1 seems unlikely to act as a morphogen, its apparent activity as a general dorsalizing factor would be consistent with the role of its homolog Dpp in Drosophila embryogenesis and leg development. Regarding body size, DBL-1 acts in a dose dependent manner to control the size of the animal as a whole, probably by influencing cell size. Its mechanism may relate to the role of Dpp in Drosophila wing size control. The wing phenotypes resulting from changes in dpp expression are associated with changes in cell number. In C. elegans, where the number of cell divisions appears to be strictly controlled, the result of inhibiting or stimulating cell growth may be to produce smaller or large cells, respectively, with concomitant effects on body size (Suzuki, 1999).

KIN-8 in C. elegans is highly homologous to human ROR-1 and 2 receptor tyrosine kinases of unknown functions. These kinases belong to a new subfamily related to the Trk subfamily. A kin-8 promoter::gfp fusion gene was expressed in ASI and many other neurons, as well as in pharyngeal and head muscles. A kin-8 deletion mutant has been isolated that shows constitutive dauer larva formation (Daf-c) phenotype: about half of the F1 progeny became dauer larvae when they are cultivated on an old lawn of E. coli as food. Among the cells expressing kin-8::gfp, only ASI sensory neurons are known to express DAF-7 TGF-beta, a key molecule preventing dauer larva formation. In the kin-8 deletion mutant, expression of daf-7::gfp in ASI is greatly reduced; dye-filling in ASI is specifically lost, and ASI sensory processes do not completely extend into the amphid pore. The Daf-c phenotype is suppressed by daf-7 cDNA expression or a daf-3 null mutation. In the kin-8 mutant, ASI-directed expression of kin-8 cDNA under the daf-7 promoter or expression by a heat shock promoter rescues the dye-filling defect, but not the Daf-c phenotype. These results show that the kin-8 mutation causes the Daf-c phenotype through reduction of the daf-7 gene expression, and that KIN-8 function is cell-autonomous for the dye-filling in ASI. KIN-8 is required for the process development of ASI, and also involved in promotion of daf-7 expression through a physiological or developmental function (Koga, 1999).

Alternative models (termed cases A, a, B and b) are considered for KIN-8 function. KIN-8 may be a component in a signal transduction pathway directly controlling daf-7 expression (case A); KIN-8 acts in another pathway that influences or modifies the direct pathway (case a) and the activation of KIN-8 is dynamically regulated by the amount of its ligand, which probably responds to an environmental condition (case B); KIN-8 is constitutively activated (case b). Although no evidence clearly supports any one of these four cases, the B cases (AB or aB) seem interesting. Transcription of the daf-7 gene is controlled by environmental stimuli; the expression is high under non-dauer-inducing conditions (abundant food, a low concentration of dauer pheromone and a low temperature), whereas it is suppressed under dauer-inducing conditions, and these stimuli are sensed by sensory neurons in the amphid. Therefore, in the B cases, a possible ligand for KIN-8 should be secreted under the non-dauer inducing conditions and not under the dauer inducing conditions. Sensory neurons in the amphid may be plausible candidates producing the KIN-8 ligand, because sensing of the environmental stimuli and secretion of a ligand can be simply linked within a single cell and because secretion of an endocrinologic factor is a fairly common property of neurons. This implies a neuroendocrinological signaling pathway in which a sensory neuron perceives environmental information and transforms this information into an amount of the KIN-8 ligand to be secreted. The ligand may be received by KIN-8 in ASI or related cells. The Daf-c phenotype of kin-8 is not temperature-sensitive and similar to that of daf-7;ttx-3 double mutant, which might imply that environmental temperature information could be transduced to ASI through such a KIN-8 pathway (Koga, 1999).

As is KIN-8, the DAF-1/DAF-4 receptor for DAF-7 TGF-beta is expressed in amphid sensory neurons, including ASI and many interneurons. DAF-2 insulin receptor-like molecules may also work in neurons, because a downstream factor, DAF-16 fork head transcription factor, is expressed in the neurons, ectoderm, muscles and intestine. Although a ligand for DAF-2, a presumed insulin-like molecule has not yet been identified: this ligand might also be produced in a sensory neuron. These arguments have led to the speculation that the neurons sensing environmental signals secrete factors such as DAF-7, a DAF-2 ligand and a KIN-8 ligand; then the information represented by these factors is processed and integrated through paracrine, endocrine or autocrine mechanisms among the neurons expressing the corresponding receptors. This type of signaling may be advantageous in a process such as decision between normal development and dauer formation, in which integration of a variety of environmental information over a period of time is probably required. The kin-8 mutant phenotypes in the posterior body part are similar to the phenotypes called withered tails (Wit) in C. elegans that are caused by defects in migration or elongation of the canal-associated-neurons. There may be such defects in the kin-8 mutant. The kin-8 mutant shows some defects in development of ASI sensory process and in migration of DTCs. These results suggest that KIN-8 may be involved in migration of those cells and processes, although specific mechanisms are unknown (Koga, 1999 and references therein).

What are the molecular mechanisms of KIN-8 function? kin-8(ks52) deletes most of the cytoplasmic region, including the kinase domain; it is null for dauer formation and a kinase-negative KIN-8 is functional. Given that little or no KIN-8 truncated protein is present in the kin-8(ks52) mutant, it may be possible that only the extracellular region of KIN-8 is required as a scaffold for other proteins in the wild type. But if kin-8(ks52) is null in spite of the presence of truncated KIN-8, the cytoplasmic region of wild-type KIN-8 is thought to have a function. In this case, a protein is expected to be associated with the KIN-8 cytoplasmic region. This presumed associated protein is possibly activated without the kinase activity of KIN-8 when a ligand binds to KIN-8. A possible mechanism for activation is suggested: Dimerization of KIN-8 upon ligand binding would bring the associated proteins in close contact with one another and would enhance activation. The Unc phenotype of kinase-negative KIN-8 suggests that the kinase activity has another role than dauer formation, if the Unc phenotype represents an authentic KIN-8 function (Koga, 1999).

Specification of cell fate is key to understanding the development and function of a nervous system. In C. elegans, where cell lineages are reproducible and gene expression programs can be studied with single-cell resolution, it is possible to explore the progression of cell-state changes that lead to the generation of cells with individual identities. Studies were carried out to take advantage of the neurons of male sensory rays; these studies have addressed how neurons are programmed to adopt a particular neurotransmitter identity. There are nine similar bilateral pairs of rays extending out of the body on each side of the male tail. Each ray comprises two sensory neurons (denoted A-type and B-type) and a support cell surrounded by a hypodermal sheath. Each ray consists of similar cells and is generated by repetition of a stereotyped cell sublineage. Yet each ray also has individual characteristics. These include position within the genital specialization, morphology, neurotransmitter usage, expression of a sensory receptor, transcription factor expression profile and functional role in mating behavior. Further individual characteristics may encompass expression of other sensory receptors and modalities, axon pathfinding and synaptic targets. Thus, two developmental processes involved in the generation of each ray may be distinguished: a program of neurogenesis that results in the generation of three differentiated cell types and a pattern-formation process that assigns the characteristics that differ among the rays. The existence of two processes is supported by the identification both of genes required in all the rays and genes involved in specifying ray-specific properties (Lints, 1999).

Dopamine (DA) is expressed by R5A, R7A and R9A, the A-type sensory neurons present respectively in rays 5, 7 and 9. A TGFbeta-family signaling pathway and a Hox gene have been identified that are involved in directing the dopaminergic fate specifically to these three neurons. C. elegans genes encoding components of TGFbeta-like signaling cascades have been defined by genome sequence analysis and by genetic studies. A TGFbeta pathway involved in defining male ray morphology, which is referred to as the DBL-1 pathway, has been defined by mutations in six genes. The pathway ligand, encoded by a gene variously named dbl-1 and cet-1, is a member of the Vg1/Dpp/BMP subfamily of TGFbeta molecules, most closely resembling Nodal. sma-6 and daf-4 encode type I and type II receptors, respectively. sma-2, sma-3 and sma-4 encode SMAD proteins likely to act as downstream transducers. The DAF-4 receptor and the signal transduction pathway have been shown to act cell autonomously in specification of ray morphology. dbl-1 is expressed in several neurons within the tail, but the source important for ray morphology has not been identified (Lints, 1999).

Expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine were used to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFbeta family signaling pathway (the DBL-1 pathway), dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability (Lints, 1999).

One question of interest is whether pathways leading to the adoption of the neuronal cell fate are entirely independent of those that pattern individual neuronal properties. Several lines of evidence indicate that egl-5 acts in specification of the ray neuroblast fate, not only in ray 6, but also redundantly with mab-5 in other V-rays. egl-5 also acts in the specification of ray morphology and, as shown here, in the specification of DA expression. Since a single regulatory factor acts both to specify the neuronal cell fate and also the differentiated properties of individual neurons, this demonstrates that there are steps in common between the developmental pathways leading to expression of pan-neural genes and pathways leading to expression of neuron-specific genes. Similar multiple roles and times of action have been demonstrated for mab-5 in posterior hypodermal cell lineages and for the Hox gene lin-39 in vulva development. In its late action, EGL-5 could act directly on the cat-2 (tyrosine hydroxylase) promoter or alternatively on the promoter of another transcription factor at some level in the hierarchy upstream of cat-2. Additional intermediate transcription factors might include those of the LIM and POU families, which play widespread roles in C. elegans and in other organisms in specifying the properties of individual neurons. A transcription factor of the POU family regulates dopamine decarboxylase gene expression in Drosophila. Since EGL-5 is expressed in all branches of the ray sublineages of rays 3 to 6, additional factors must intervene to direct the action of EGL-5 in the dopaminergic pathway exclusively to the lineage branch leading to the A-type neuron. Likewise, turn-on of cat-2 appears to be delayed until the ray sublineage is completed. Whether lineage-branch-specific and timing cues are integrated by the promoter of the cat-2 gene itself, or by the promoters of intermediate transcription factors acting between egl-5 and cat-2, is an interesting question for future studies (Lints, 1999).

Dpp homologs in other invertebrates

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. In the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. A compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. It is concluded that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. It is suggested that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains (Nederbragt, 2002).

In early embryogenesis of spiders, the cumulus is characteristically observed as a cellular thickening that arises from the center of the germ disc and moves centrifugally. This cumulus movement breaks the radial symmetry of the germ disc morphology, correlating with the development of the dorsal region of the embryo. Classical experiments on spider embryos have shown that a cumulus has the capacity to induce a secondary axis when transplanted ectopically. The house spider, Achaearanea tepidariorum, has been studied to characterize the cumulus at the cellular and molecular level. In the cumulus, a cluster of about 10 mesenchymal cells, designated the cumulus mesenchymal (CM) cells, is situated beneath the epithelium, where the CM cells migrate to the rim of the germ disc. Germ disc epithelial cells near the migrating CM cells extend cytoneme-like projections from their basal side onto the surface of the CM cells. Molecular cloning and whole-mount in situ hybridization show that the CM cells express a spider homolog of Drosophila decapentaplegic, which encodes a secreted protein that functions as a dorsal morphogen in the Drosophila embryo. Furthermore, the spider Dpp signal appears to induce graded levels of the phosphorylated Mothers against dpp (Mad) protein in the nuclei of germ disc epithelial cells. Adding data from spider homologs of fork head, orthodenticle and caudal, it is suggested that, in contrast to the Drosophila embryo, the progressive mesenchymal-epithelial cell interactions involving the Dpp-Mad signaling cascade generate dorsoventral polarity in accordance with the anteroposterior axis formation in the spider embryo. These findings support the idea that the cumulus plays a central role in the axial pattern formation of the spider embryo (Akiyama-Oda, 2003).

Expression patterns of spider homologs of Drosophila region-specific genes, shown in this study, offer molecular clues for determining homologous domains in the early spider and Drosophila embryos. In the Drosophila cellular blastoderm, otd is expressed at a region close to the anterior end, whereas At.otd is expressed at a peripheral region of the germ disc encircled along the equator of the spider. The At.otd-expressing cells likely migrate circumferentially during stage 6 and settled in the anterior region of the germ band. Drosophila cad is expressed at a region close to the posterior end in the cellular blastoderm, whereas At.cad is expressed in the caudal lobe, which is derived from the central area of the germ disc. These results strongly suggest that the peripheral region of the germ disc corresponds to the anterior end of the Drosophila embryo, and the central region corresponds to the posterior end. It is possible that in the spider germ disc, the AP positional information pre-exists as a series of concentric circles. Based on this topology, At.fkh-expressing cells were located at the future anterior and posterior ends of the stage 5 embryo, similar to the pattern of fkh expression in the Drosophila cellular blastoderm and in the early embryo of other insects, Bombyx and Tribolium. Since the fkh-expressing cells become foregut and hindgut in these insects, the two populations of At.fkh-expressing cells are probably fated to be gut precursors (Akiyama-Oda, 2003).

One possibly important difference in the mode of AP axis formation is that in the spider embryo, it appears to occur in accordance with DV axis formation, whereas in the Drosophila embryo, the AP and DV axes are formed independently. Although a concentric series of AP positional information may pre-exist on the spider germ disc, the expression patterns of the AP patterning genes do not allow one to find the AP axis in the early germ disc. The anterior pole of the spider embryo can be defined only after the onset of the cumulus movement (Akiyama-Oda, 2003).

Drosophila dpp and probably other insect dpp homologs are involved in DV patterning of the embryo. dpp is expressed in the dorsal ectoderm during the germband extending stages, and this expression probably contributes to pattern formation within segments, such as positioning of the limb buds. This role of dpp is probably conserved between the insects and spiders, as suggested by the later expression of At.dpp. At least in Drosophila, however, the earliest function of dpp is the specification of the DV pattern in the cellular blastoderm, not within segments of the germband. The most dorsal region of the cellular blastoderm, where dpp is expressed at the strongest level, becomes extra-embryonic tissue (the amnioserosa), and the next regions become dorsal ectoderm in Drosophila. In the spider, the area of the germ disc that is directly influenced by At.dpp-expressing CM cells during their migration develops into dorsal structures including the extra-embryonic tissue. This suggests a conserved function of dpp for dorsal fate specification of the early embryo. The expression of At.dpp in the CM cells is probably comparable with the dorsal expression of dpp in the Drosophila cellular blastoderm, and might be related to the expression of dpp in an early cell population fated to be extra-embryonic tissue in more basal insect embryos (Akiyama-Oda, 2003).

In the Drosophila blastoderm the expression of dpp, as well as some other zygotic genes involving DV patterning, is initiated in an asymmetric manner according to the gradient of the nuclear Dorsal protein peaking at the most ventral region. In the spider embryo, however, the early detectable asymmetry is in the migration of the At.dpp-expressing CM cells rather than the expression of At.dpp itself. What mechanisms regulate the CM cell migration? The answer to this question may be the key to the developmental origin of the DV axis of the spider. A localized cue(s) that attracts or repulses the CM cells might pre-exist on the germ disc. Alternatively, the CM cells might sense only the AP positional information. In the latter case, the direction of the cell migration is determined at the start point randomly, or according to a local unevenness. Further molecular investigations are needed to find the earliest asymmetries potentially present in the germ disc and primary thickening (Akiyama-Oda, 2003).

Two important differences concern the Dpp signal between Drosophila and the spider: (1) the Dpp signal is produced and transduced within the surface epithelial cells in the Drosophila embryo, in contrast to the spider embryo, in which the Dpp signal is produced by the mesenchymal cells (the CM cells), and is transmitted to the surface epithelial cells; (2) the activation of the Dpp-Mad signaling pathway takes place simultaneously along the AP axis in the Drosophila embryo, but takes place progressively from the center to the rim of the germ disc in the spider embryo. These differences have implications for evolutionary change in the mechanism governing DV axis formation (Akiyama-Oda, 2003).

The evolutionary origin of the anterior–posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. This study cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral–aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral–aboral axis. It is concluded that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral–aboral body axis of Nematostella and one particular body axis of Bilateria (Rentzsch, 2006a).

Nearly all metazoans show signs of bilaterality, yet it is believed the bilaterians arose from radially symmetric forms hundreds of millions of years ago. Cnidarians (corals, sea anemones, and 'jellyfish') diverged from other animals before the radiation of the Bilateria. They are diploblastic and are often characterized as being radially symmetrical around their longitudinal (oral-aboral) axis. The deployment of orthologs of a number of family members of developmental regulatory genes that are expressed asymmetrically during bilaterian embryogenesis from the sea anemone, Nematostella vectensis, have been studied. The secreted TGF-beta genes Nv-dpp, Nv-BMP5-8, six TGF-beta antagonists (NvChordin, NvNoggin1, NvNoggin2, NvGremlin, NvFollistatin, and NvFollistatin-like), the homeodomain proteins NvGoosecoid (NvGsc) and NvGbx, and the secreted guidance factor, NvNetrin, were studied. NvDpp, NvChordin, NvNoggin1, NvGsc, and NvNetrin are expressed asymmetrically along the axis perpendicular to the oral-aboral axis, the directive axis. Furthermore, NvGbx, and NvChordin are expressed in restricted domains on the left and right sides of the body, suggesting that the directive axis is homologous with the bilaterian dorsal-ventral axis. The asymmetric expression of NvNoggin1 and NvGsc appear to be maintained by the canonical Wnt signaling pathway. The asymmetric expression of NvNoggin1, NvNetrin, and Hox orthologs NvAnthox7, NvAnthox8, NvAnthox1a, and NvAnthox6, in conjunction with the observation that NvNoggin1 is able to induce a secondary axis in Xenopus embryos argues that N. vectensis could possess antecedents of the organization of the bilaterian central nervous system (Matus, 2006).

Planarians can be cut into irregularly shaped fragments capable of regenerating new and complete organisms. Such regenerative capacities involve a robust ability to restore bilateral symmetry. Three genes needed for bilaterally asymmetric fragments to regenerate missing body parts have been identified. These genes are candidate components of a signaling pathway that controls the dorsal-ventral patterning of many animal embryos: a BMP1/Tolloid-like gene (smedolloid-1), a SMAD4-like gene (smedsmad4-1), and a BMP2/4/DPP-like gene (smedbmp4-1). BMP signaling is involved in the formation of new tissues at the midline of regeneration, the dorsal-ventral patterning of new tissues, and the maintenance of the dorsal-ventral pattern of existing adult tissue in homeostasis. smedbmp4-1 is normally expressed at the dorsal midline. Asymmetric fragments lacking a midline display new smedbmp4-1 expression prior to formation of a regenerative outgrowth (blastema). Asymmetric fragments containing the midline display expanded smedbmp4-1 expression towards the wound. It is suggested injured animals that lack left-right symmetry reset their midline through modulation of BMP activity as an early and necessary event in regeneration (Reddien, 2007; full text of article).

Animal embryos have diverse anatomy and vary greatly in size. It is therefore remarkable that a common signaling pathway, BMP signaling, controls development of the dorsoventral (DV) axis throughout the Bilateria. In vertebrates, spatially opposed expression of the BMP family proteins Bmp4 and Admp (antidorsalizing morphogenetic protein) can promote restoration of DV pattern following tissue removal. bmp4 orthologs have been identified in all three groups of the Bilateria (deuterostomes, ecdysozoans, and lophotrochozoans). By contrast, the absence of admp orthologs in ecdysozoans such as Drosophila and C. elegans has suggested that a regulatory circuit of oppositely expressed bmp4 and admp genes represents a deuterostome-specific innovation. This study describes the existence of spatially opposed bmp and admp expression in a protostome. An admp ortholog (Smed-admp) is expressed ventrally and laterally in adult Schmidtea mediterranea planarians, opposing the dorsal-pole expression of Smed-bmp4. Smed-admp is required for regeneration following parasagittal amputation. Furthermore, Smed-admp promotes Smed-bmp4 expression and Smed-bmp4 inhibits Smed-admp expression, generating a regulatory circuit that buffers against perturbations of Bmp signaling. These results suggest that a Bmp/Admp regulatory circuit is a central feature of the Bilateria, used broadly for the establishment, maintenance, and regeneration of the DV axis (Gaviño, 2011).

Because Smed-admp represents the first admp ortholog characterized in a protostome, the genomes of other lophotrochozoans were searched to determine whether admp orthologs are widespread in protostomes. Indeed, predicted admp orthologs were identified in the genomes of the snail Lottia gigantis, the leech Helobdella robusta, and the polychaete annelid Capitella teleta. The presence of putative admp orthologs in these species, coupled with the expression pattern and functional properties of Smed-admp, suggest that a Bmp/Admp regulatory circuit is an ancestral and central feature of the DV axis. This model predicts that an admp gene was present in the ancestor of C. elegans and Drosophila but was subsequently lost in the evolution of these species; consequently, the potential widespread significance of admp genes for the DV axis of Bilaterians was previously unknown (Gaviño, 2011).

Table of contents

decapentaplegic: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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